SUPERCRITICAL FLUID
CHROMATOGRAPHY
PrimerSUPERCRITICAL FLUID
CHROMATOGRAPHY
PrimerTerry A. Berger

This information is subject to change without notice.
© Agilent Technologies, Inc., 2015
Printed in the USA, July 1, 2015
5991-5509EN

CONTENTS
Contents 
Foreword 
About the Author Introduction 
Symbols Abbreviations 

III
VI
XI
XIII
XIV
XIV

1 Introduction to Supercritical
Fluid Chromatography 

1

1.1 What is SFC? 
1.2 Why deploy SFC? 
1.3 What can SFC separate? 
1.4 What’s in a name? 

1
2
12
16

2 The Mobile Phase 

19

2.1 Why use CO 2? 
2.2 Using 100 % CO 2 
2.3 Modifiers or cosolvents 
2.4 Additives 
2.5 Extending solute polarity with added water 

19
21
23
31
34

3 The Stationary Phase 35

3.1 Materials 
3.2 Achiral bonded phases 
3.3 Stationary phase comparisons 
3.4	Relationship between particle diameter andcolumn dimensions 
3.5 Recommended column dimensions 
3.6 Columns for chiral separations 

35
35
40
41
45
48

III

4 E ffect of Mobile Phase Variables
on Retention and Selectivity 

IV49

4.1 Modifier concentration 
4.2 Temperature 
4.3 Pressure 
4.4 Flow 
4.5	Generalizations on effects of control variables on
retention and selectivity 

49
50
53
55

5 Method Development 58

5.1 Matching solute and phase polarity 
5.2 Polarity windows 
5.3 Getting started 
5.4 Polar solutes 
5.5 Low polarity solutes 
5.6 Multivariate methods 

58
60
60
61
64
65

6 Achiral Separations 66

6.1 Case Study 1 – A typical low-polarity sample 
6.2 Case study 2 – A moderately polar sample 
6.3 Case study 3 – Sulfonamides 
6.4 Other observations 

66
71
83
89

7 Chiral Separations 90

7.1 Background 
7.2 Enantiomeric excess determinations 
7.3 Normal phase for chiral separations 
7.4 Effect of control variables on chiral separations 
7.5 Developing a chiral method 

90
91
91
93
98

57

8 Quantification in SFC 

103

8.1 Stages of Validation 
8.2	Method development for quantification of sorbate,benzoate, and caffeine in beverages and foods 
8.3 Calibration 
8.4	Summary of the quantification of benzoate, sorbate
and caffeine 
8.5 Chiral separations 103

9 Instrumental Considerations 121

9.1 Pumping 
9.2 UV detector optimization 
9.3 The doubling gradient 
9.4 Autosampler considerations 
9.5 Miscellaneous 
9.6 Ultrahigh performance with SFC 
9.7 Hybrid systems switching between SFC and HPLC 
9.8 Interfacing to mass spectrometers 
9.9 Other Detectors 

121
124
137
138
146
148
150
154
156

References 

159

105
108
116
116

V

FOREWORD
The author, Terry Berger has a unique, convoluted history with what we
now call supercritical fluid chromatography (SFC), and is uniquely qualified
to write this primer. Many of the early interactions were incidental andunfocused, but later meaningful. He brings nearly 40 years of relationships
with people associated with SFC, along with over 35 years of direct
experience. SFC had first been proposed nearly 60 years ago, in 1958, by
Jim Lovelock, who later became a personal friend of the author throughthe 1980s and 1990s with numerous discussions about SFC.
Ernst Klesper demonstrated SFC experimentally in 1962 when he separated
metal porphorins with dense clorofluorocarbons as mobile phase. The author
later interacted with Professor Klesper on numerous occasions, throughthe late 1970s to mid-1980s in both the USA and Germany, and again had
extensive discussions about SFC.
Through the 1960s and 1970s, the technique largely languished with,at most, a dozen groups worldwide making a total of a few sporadic
scientific contributions per year. Applications tended to be extensionsof gas chromatography (GC) to higher molecular weight, higher boiling,
relatively nonpolar solutes.
In the early 1970s, the author was a graduate student at Purdue University
in Indiana. The adjacent laboratory was that of L.B. “Buck” Rodgers,a highly respected chromatographer at the time. After attending ascientific symposium in 1971, he came back determined to explore thisnew form of chromatography. Controlling the pressure of the mobile
phase was the primary control variable, and pressure programmingfrom low to high pressures was then the norm. All equipment in thisperiod was homemade, with poor instrumental control. The physicaland chemical characteristics of the mobile phases were primitively
understood, at best. Rodgers rescued ultrahigh pressure hydrogenation
pumps from storage, built an oven roughly 4-feet long that looked likea small coffin using 2-inch thick household foam insulation. Columns
were 30 inches long or longer, and packed with irregular particles witha wide particle-size distribution. The author watched the progressionof these developments and recalls that, at the time, being skeptical that
this odd form of chromatography was useful. Rodgers results suggested
there was a serious problem with density gradients along the length ofVIthe column. This, and later work by Milos Novotney, seemed to indicatethat the technique was unlikely to produce results as good as the current
high-performance liquid chromatography (HPLC). This tended to suppress
further work on packed-column SFC.
The author was trained in HPLC at Imperial College in London where hedeveloped an amperometric electrochemical detector for HPLC, in the early
1970s. After subsequently teaching for a year in Brazil, he worked on anumber of projects for instrumentation for spacecraft in Cleveland, Ohio.
The author was then hired by Hewlett-Packard in 1979, and joined
the research group at the Avondale Division. Dennis Gere, Henk Lauer,
Doug McManigill, and Harry Weaver, all scientists or engineers at
Hewlett-Packard Labs or the Avondale Division, presented a series oftalks on dense gas chromatography at that year’s Pittsburg Conference(where the author was actually hired). Some of these talks were later
published in such journals as Analytical Chemistry, and Science, as
well as related application notes. These works resulted in a customer
demand that led to the creation of a commercial kit that converted a
Hewlett‑Packard model HP 1084 HPLC instrument into an SFC system.
Thus, commercial SFC was born.
This version of SFC used a mechanical backpressure regulator, a binary
pump and composition programming, much like HPLC. Flow, composition,column temperature, and column outlet pressure were all independently
controlled. Unlike previously home-built instruments, there was no pressure
programming. Columns were more modern with much of the work done on
spherical silica as small as 3 µm. The author’s group produced the designfor this kit.
About the same time that a capillary version of SFC, using pressure
programming of pure CO2, was introduced, Hewlett-Packard made theHP 1084 instrument obsolete, replacing it with the SFC-incompatible
HP 1090 HPLC instrument. Milos Novotny and Milton Lee were thedrivers behind the capillary SFC development. This author’s researchgroup at Hewlett-Packard had just invented fused-silica capillarycolumns, capable of withstanding the high pressures (400 to 600 bar)necessary for capillary SFC. Milton Lee was an academic leader in the
new field of bonding the stationary phase to capillary columns.
VIIPacked-column SFC largely disappeared, due to lack of commerciallyavailable instrumentation, and capillary SFC was seen as the wave of the
future. At this time, the physical chemistry of the fluids and their interaction
with the stationary phase were largely unknown. Many controversies
erupted pitting the few remaining packed column users against thecapillary proponents. The author became the senior research scientist
working with SFC at Hewlett-Packard in 1985, and was assigned to unravel
these controversies. In the process, he visited every European user (about16) for extensive discussions about the controversies and potential.
At the time, it was widely believed that density was the primary control
variable, and that changing the mobile phase density had the biggest
effect on retention. It was thought that dense CO2 was as polar as
isopropanol, due to an unfortunate misunderstanding by Giddings1. If it
were true, adding an alcohol modifier could change density significantly,
but only insignificantly change solvent strength. This misinterpretation
was not challenged for decades. At the time, there were no direct
density measurements of methanol/CO2 (or any other) mixtures used in
SFC. Equations of state for binary mixtures were inaccurate. The author
subsequently borrowed a densitometer from the University of Delawareand made some of the first density measurements of methanol/CO2 inthe literature. He then measured the effect of methanol concentration
on retention at constant density, showing that the modifier was indeed
responsible for dramatically increasing the elutropic strength of themobile phase.
Not long afterwards, solvatochromic dyes were shown to offer a simple way
to measure the strength and polarity of the solvation sphere surrounding
the dye molecules in HPLC2. The author then applied the same principle to
SFC45. The results indicated that the solvent strength of binary fluids was
highly nonlinear, with the first small additions having an inordinate effect.
CO2 was shown to be similar to pentane in polarity and solvent strength.
With density and solvatochromaic dye measurements, the Giddings’
elutropic series1 was conclusively revised, although it still has followers.
The result is that changing modifier concentration was shown to have amuch larger impact on retention than pressure, temperature, and density,
at least for modified fluids.
It was widely demonstrated during the late 1980s that many polar solutes
such as organic acids and bases eluted with poor peak shapes or did
not elute at all when using binary fluids consisting of CO2 modified withan alcohol such as methanol or ethanol. It was generally thought that
this indicated that the stationary phase was too active or containedVIIIactive sites that interacted too strongly with these polar solutes. The
proposed solution was to use less-polar stationary phases, with further
deactivation. This approach simply did not work. Polar molecules typicallywent unretained and often still tailed badly. The author introduced the useof a polar additive such as a strong acid or base, or both, mixed with theorganic modifier, along with the use of more polar columns. This approach
was shown to dramatically improve peak shapes. The use of such additives
opened up the possibility for what is modern SFC, with the elution andseparation of polar solutes that now includes peptides up to 40’mers.
Going back to at least “Buck” Rodgers papers 3 there was a perceptionthat pressure drops across the column resulted in density gradients,
causing serious efficiency losses. This was reinforced by Novotnyin the mid-1970s 4. By the mid-1980s there were multiple competingtheories that at least one of which5 suggested that packed-column SFC
was incapable of generating more than about 20,000 plates, due to thedensity gradients accompanying the pressure drops across the column.
The author connected eleven 20-cm long columns with 5-µm packing inseries, and demonstrated 220,000 plates with pressure drops of up to
250 bar, conclusively refuting most of the arguments. The effect of density
gradients continues to be an area of interest and the topic of numerous
recent publications.
The author convinced Jon Parcher at the University of Mississippi toperform tracer-pulse MS experiments to measure the adsorption ofmobile‑phase components onto the stationary phase. The results indicatedthat the extensive adsorbed films had a dramatic effect on the nature of
the stationary phase, and subsequently, on the chromatography.
With most of the misconceptions disproved, the author convinced
Hewlett-Packard management to fund a next-generation SFC project.
The resulting product was released in 1992, and was capable ofboth capillary and packed-column performance. This instrument, theHP G1205A, could simultaneously and independently control flow,
composition, pressure, and temperature. It could also perform density
programming using an internal equation of state.
With the improved understanding of the mobile-phase characteristics
by the early to mid-1990s, it was widely recognized that capillary SFC
had been greatly oversold, particularly for polar solutes. This recognition
was largely due to the revision of Giddings’ elutropic series. Sadly thisresulted in the death of capillary SFC, even though it had demonstrated
numerous advantages over other techniques with less polar solutes.

IX

In 1995, the author had the opportunity to buy the SFC business from
Hewlett-Packard, and, along with another former employee and an
outside investor, created Berger Instruments (BI). BI refocused the design
to primarily perform packed-column SFC, and reduce cost. BI introduced
the first chiral method development system, the first successful
semipreparative instruments for both multigram (mostly chiral) andhigh-throughput library purification, the first commercial SFC-MS system,
the first successful gas-delivery system, and a number of other minor
products. The company was sold to Mettler Toledo in late 2000, where
the author served as Chief Technical Officer until 2003.
In 2008 the author started Aurora SFC Systems, which produced amodule that converted a more or less standard HPLC instrument intoa state-of-the-art SFC system. Aurora was sold to Agilent Technologies
in 2012, and the upgraded module is now at the heart of the Agilent
analytical SFC system. The author’s R&D group has been awarded
two different R&D 100 awards, one at BI and one at Aurora, each fordesigning one of the 100 top inventions in 2004 and 2008.
The author currently has an SFC consulting business (SFC Solutions, Inc.),with a state-of-the-art SFC laboratory. Freed from the necessity of runninga business, he is actively producing research and is publishing original
research on instrumentation, columns, and applications, including this primer.
The author’s long-term, deep understanding of the physical chemistryof the fluids, applications and instrumentation of SFC, is unique in theindustry. He has seen many fads come and go, and many negative
theories fade away. He is extremely optimistic about the future of SFC.
It is hoped that this deep understanding can be transferred to thereader of this primer in such a way that they can enjoy great successin implementing SFC in their laboratory.

XABOUT THE AUTHOR
Terry A. Berger received his PhD in analytical chemistry from Imperial
College of Science and Technology, University of London, UK, in 1976,
following graduate work at Florida, Wisconsin and Perdue. Dr. Berger
is considered by many to be the father of modern supercritical fluid
chromatography (SFC). In 2004, Dr. Berger was awarded the Martin
Gold Medal by the Chromatographic Society of Great Britain, one of thepremier awards in chromatography.
Terry A. Berger
PhD, DIC
President, SFC Solutions, Inc.

Starting in 1985, at Hewlett-Packard (now Agilent Technologies), he spent
more than a decade systematically undoing many of the misconceptions
about packed column SFC that were then prevalent. In the process, heuntangled the effects of density and solvent strength, which had been major
points of contention. He showed that, contrary to several proposed theories,
large pressure drops did not result in serious losses in efficiency (a point stillof active interest). He introduced the use of additives and systematicallystudied their effect on peak shape and retention. He demonstrated thefirst separations of broad classes of compounds and, against the common
perception, showed that packed-column SFC was broadly applicableto many more polar solutes than previously thought possible. Examples
included small drug-like molecules such as primary amines.
Dr. Berger’s book Packed Column SFC, published by the Royal Societyof Chemistry in 1995, fundamentally changed the perception of SFC inseparation science, making modern SFC more similar to HPLC than GC.
This book has remained the primary source for understanding of thefundamentals of the technique, up to the publication of this primer, whichis intended to supersede it. Other books on SFC, published before thisprimer, are compendia by numerous authors of specific topics.
Dr. Berger has also published about 75 peer-reviewed scientific papers,
7 book chapters, 13 encyclopedia and reference work entries, and dozens
of application notes. He has taught more than 20 short courses on SFC,
given hundreds of oral presentations and posters, and has received orapplied for about 25 patents in GC, SFC, and HPLC.

XI

In 1995, he formed Berger Instruments, which introduced the first SFC
chiral method systems, the first successful semipreparative SFC system forboth library purification and multigram separations, the first commercial
SFC-MS, as well as the first gas-delivery systems. Later he founded Aurora
SFC, which built a module to convert analytical-scale HPLC instruments
into SFC systems. This module became the basis for the Agilent analytical
SFC system and the Agilent analytical SFC/UHPLC hybrid system.
His technical teams have won two R&D 100 Awards over the last
12 years. They have also won a NICE III environmental award from the
Department of Energy. Dr Berger continues to be an editor and reviewer
for chromatographic publications and societies.

XII

INTRODUCTION
This primer is intended to supersede the author’s book Packed Column
SFC, which was first published in 1995 by the Royal Society of Chemistry.
This original work fundamentally changed the perception of SFC inseparation science making modern SFC more similar to HPLC than GC.
This perception is continued and expanded upon in this primer.
While the first chapter describes some of the basic principles of SFC,
it also discusses the rationale, motivation and benefits of deploying thetechnique in the modern analytical laboratory. The following chaptersprovide a more detailed review of the mobile and stationary phases aswell as the effects of these phases on the important chromatographic
characteristics of retention and selectivity.
A primer on separation science would not be complete without a thoroughdiscussion on the practical implementation of the technique of interest,
including the fields of application. This is covered in chapters on method
development, achiral and chiral separation, and on quantification.
Finally, instrumental considerations are discussed, including detailed
explanations of critical aspects of mobile-phase delivery, sample injection,and detection.

XIIISymbolsA

contribution of eddy diffusion in van Deemter equationB

contribution of axial diffusion in van Deemter equationC

contribution of radial diffusion in van Deemter equationDsolute binary diffusion coefficient in mobile phasedp

particle diameter

H

plate heightkpartition ratioµlinear velocity of mobile phaseP

partition coefficientP’

elution strength (according to Snyder)

ΔP

pressure drop across separation column

Abbreviations

XIV

ACN

acetonitrile

BPR

backpressure regulator

CZE

capillary zone electrophoresis

EtOH

ethanol

FID

flame ionization detectorGC

gas chromatography

HPLChigh performance liquid chromatography

idinside diameter

IPA

isopropanol (isopropyl alcohol)

IPAm

isopropylamine

MeOH

methanolMS

mass spectrometer

RI

refractive index

RSD

relative standard deviation

SFC

supercritical fluid chromatography

TEA

triethylamine

TFA

trifluoroacetic acid

THF

tetrahydrofuran

UHPLC

ultrahigh pressure liquid chromatography

UV

ultraviolet

INTRODUCTION TO SUPERCRITICAL FLUID
CHROMATOGRAPHY
1.1
What is SFC?

1

As supercritical fluid chromatography (SFC) is a separation technique,which uses instrumentation that is almost identical to that used in highperformance liquid chromatography (HPLC). Complex mixtures can beseparated and the amount, and sometimes the identity, of the individualcomponents in the mixture can be determined. A solution of the sample
is injected into a high-pressure flow stream that sweeps the sample into
a tube or column filled with fine particles. The individual components
in the sample interact differently with the surface of the particles, andare separated in time and space as they pass through the column. The
components emerge from the column at different times, as Gaussian or
pseudo-Gaussian peaks, and pass through a detector.
The most significant difference from HPLC is the replacement of mostof the liquid mobile phase with a dense compressed gas, almost always
carbon dioxide (CO 2). At high pressures such as greater than 80 bar,
CO 2 acts as a solvent. Because it is a compressed gas, a backpressure
regulator is required on the system outlet to ensure the mobile phaseremains a single dense phase throughout the chromatograph. This, inturn, requires some detectors, such as an ultraviolet (UV) detector, tobe operated at elevated pressures.
CO 2 is a highly nonpolar solvent, similar to a hydrocarbon but in a different
solvent family. Consequently, for more polar solutes, an organic modifier,sometimes called a cosolvent and most often an alcohol, is added to themobile phase. Gradient elution from low to high modifier concentration is
the norm. Peaks elute from lower to higher polarity.
For many highly polar solutes, the interactions with the stationary phase
are too intense, and the solutes often fail to elute, or elute with poor peak
shapes. This problem can usually be solved by including a highly polar
additive in the mobile phase such as a strong acid or base dissolved in
the modifier.

1

SFC is usually a normal phase technique because composition isprogrammed from low to high polarity. However, SFC has significant
advantages compared to normal phase HPLC. Equilibration is extremely
fast, reproducibility is excellent, and even aqueous-based samples canbe injected.
For polar solutes, polar stationary phases are used. Classical polar phases
included bare silica, cyano, diol and amino. In the last few years, a numberof new stationary phases have been developed specifically for SFC. These
phases include several ethylpyridines and a number of proprietary phases.
For low polarity solutes, reversed phase columns such as C18, C8, C4, andmethyl are sometimes used.
Over the last few years, the use of sub-2-µm particles has become fairly
common. However, the dominant use of SFC in the past several decades
has been in chiral separations6, 7. The same columns used in HPLC are usedin SFC. Although the most effective older chiral columns were coated andnot bonded, the newer bonded phases have not yet displaced the coatedcolumns due to superior selectivity and less complex optimization.
SFC is also useful for the separation of much less polar compounds suchas many natural products, including fat soluble vitamins, carotenoids, andlipids. With such samples the stationary phase is usually C18.

1.2
Why deploy SFC?
1.2.1
Faster analysis times

The van Deemter (Knox) equation describes the kinetic performance ofa chromatographic column. In its simplest form, this equation comprises
three terms, each describing a different form of diffusion, see Equation 1.1.

H=A

0.33

+

B D1,2μ+C dp2 μD1,2

Equation 1.1 The van Deemter (Knox) equation.

The terms B and C indicate the contribution of axial and radial diffusion,and contain the ratio of the solute binary diffusion coefficient in themobile phase, D1,2, to the mobile phase linear velocity, µ. The term B
indicates that higher diffusion coefficients result in higher optimum linear
velocities. In term C, a higher diffusion coefficient results in less loss of
efficiency at higher flow rates.

2

Diffusion coefficients in pure CO 2 are in the order of 10 to 15-times fasterthan in water or aqueous mixtures. For example, the diffusion coefficientof benzoic acid in water is 1.0 x 10 -5 cm2s-1 at 20 °C 8. In 100 % CO 2,
the diffusion coefficient of benzoic acid is 16 x 10-5 cm2s-1 at 100 bar and
40 °C, dropping to about 9.5 x 10-5 cm2s-1 at 300 bar 9. Similarly, several
slightly larger dimethyl anilines exhibited diffusion coefficients between
7 and 12.5 x 10-5 cm2s-1 between 150 and 350 bar, and 40 to 60 °C 10.
The intermolecular interactions between CO 2 molecules are weak.
Consequently, it is a gas at room temperature and atmospheric pressure.
When such molecules are forced close together (compressed), theresulting dense fluid acts as a solvent. Nevertheless, the intermolecularforces are still weak even at the higher density. Consequently, othercompounds dissolved in the CO2 diffuse rapidly through it.
In recent years, SFC has seldom been performed using pure CO 2.
The addition of a polar modifier, for example, an alcohol such as methanol,
ethanol or isopropanol, decreases diffusion coefficients significantly.
The addition of only 5.5 % methanol decreases diffusion coefficientsby nearly half11. Higher concentrations have an even larger effect.
For example, malvidin-3,5-diglucoside12 has a molecular weight of
670, making it fairly large, with a number of polar functional groups,
compared to many small drug-like molecules. With 20 % methanol in
CO 2 , and at 200 bar, the diffusion coefficient was still 4.7 to 5.1-times
higher than the diffusion coefficient in pure methanol at atmosphericpressure and at 40 to 60 °C, as shown in Figure 1.1. It has been shownthat polar solutes are surrounded by a cluster of the polar modifier, andthis larger solvated entity diffuses more slowly11, 13, due to the increased
cross-sectional area of the cluster.

3

D1,2 x10-5 [cm2s-1]

O

CIO+

HO

4.0

HO

3.0

O
HO

2.0

O

O

OH
OH

OH

OH
O
OH
OH
O

OH

1.0
0

20

0
60 °C

50 °C

40

60

80
100
Mole fraction [%]40 °CFigure 1.1 Diffusion coefficient of malvidin-3,5-diglucoside, which has a molecular weightof 670, in CO2 /MeOH mixtures. The CO2 /MeOH data was collected at 200 bar. The pure
MeOH data was collected at atmospheric pressure. At 20 % modifier, the diffusion coefficientswere 5.08, 4.50, and 4.70-times higher than in pure MeOH at the same temperatures.

Since SFC is usually performed with between 5 and 50 % modifier, it
is typically stated that SFC, with modified mobile phases, is three tofive‑times faster than HPLC, with the same chromatographic efficiency, on
the same sized particles. This is still true when using columns packed with
sub-2-µm particles. This means that run times are one-third to one-fifth aslong and throughput is three to five-times greater than in HPLC (or UHPLC),
on the same sized column. Re-equilibration is fast, resulting in short cycle
times for gradient analysis.
Smaller molecules have higher diffusion coefficients. Unfortunately,
there is little data in the literature about the diffusion coefficients ofsmall molecules in modified CO 2. Most of the values in the literature arepresented in Table 1.1.

4

SoluteTemperature

PressureD1,2 [cm2s-1]
5 % MeOH

2-nitroanisol

40 °C
50 °C

Dichlorobenzene

40 °C

200 bar

7.8 x 10 -5

5.5 % MeOH

10 % MeOH
7.22 x 10 -5300 bar6.85 x 10-56.25 x 10 -5200 bar8.36 x 10 -5

7.56 x 10 -5300 bar7.52 x 10-56.76 x 10 -5200 bar9.36 x10 -5

8.47 x 10 -5300 bar7.98 x 10

7.6 x 10 -5

8.33 x 10 -5-5t-butyl benzene

50 °C

200 bar7.57 x 10 -5

Acridine

55 °C

173 bar7.78 x 10

Benzoic acid55 °C

173 bar6.69 x 10 -5

Phenanthrene

55 °C

173 bar10.0 x 10 -5-5

Table 1.1 Diffusion coefficients of a few small molecules with small additions of methanol
to CO2 10, 11.

1.2.2
Lower pressure drops

The same low intermolecular interactions that result in higher diffusion
coefficients also result in low viscosity of both pure and modified CO 2.
If we ignore 100 % CO 2 for the moment and concentrate on modified
CO 2, Figure 1.2 shows literature14 viscosity values for mixtures ofwater/methanol, at 40 and 60 °C, and water/acetonitrile at 60 °C.
These HPLC-like conditions are compared to values for CO2 /methanol
mixtures estimated from measured pressure drops (ΔP) across an SFC
column, at 50 °C, extrapolated to 100 % methanol (dashed line). The
aqueous-based measurements were obtained at constant pressure.
The CO 2 /methanol measurements were obtained from measurements
between about 200 and 400 bar at constant flow.

5Viscosity [mPa·s]
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

0

0.1

0.2

H2O/MeOH, 40 °C
H2O/ACN, 60 °C

0.3

0.4

0.5

0.6

0.7
0.8
0.9
1.0
Mole fraction of modifier

H2O/MeOH, 60 °C
CO2/MeOH, 50 °C

Figure 1.2 Viscosity of H2O/MeOH, H2O/ACN, and CO2 /MeOH mixtures as a function of themole fraction of the modifier. The pressure drop across the column is directly related to theviscosity.

In the late 1990s there was a revival of the use of sub-2-µm particles,
but these were mostly pellicular and were often packed in capillaries.
Small diameter, superficially porous particles then appeared. In 2003,
Agilent introduced the first totally porous, sub-2-µm particles. Such
particles offered the possibility of increasing analysis speed nine- or
tenfold, compared to 5-µm particles, with the same efficiency,
according to the van Deemter equation.
These smaller particles generated much higher pressure drops (ΔP) thanthe older, larger particles, since ΔP is proportional to dp2 (at constantefficiency). The use of sub-2-micron particles in HPLC sometimes requires
pumps capable of greater than 1000 bar. In SFC, the viscosity of
CO 2 /modifier mixtures is dramatically lower than the aqueous-based
fluids, as shown at the bottom of Figure 1.2. Subsequently, ΔP values
are much lower than in HPLC, even at higher flow rates. It is actually
unusual to exceed 400 bar in SFC, even using 1.8-µm particles in columnsof 100 mm in length. The ΔP of several columns packed with 3.5 and1.8‑µm particles was plotted against percent of modifier at constant outletpressure, and various flow rates. The results are presented in Figure 1.3
and Figure 1.4, respectively.

6

Column pressure drop [bar]
240
200
160
120
80
40
0

0

10

20

30

40

50

60

70

80
90 100Methanol content [%]4.6 by 50 mm, 1.8 µm, 5 mL/min, 150-bar outlet pressure
4.6 by 50 mm, 1.8 µm, 5 mL/min, 100-bar outlet pressure
4.6 by 150 mm, 3.5 µm, 3 mL/min, 150-bar outlet pressure
4.6 by 250 mm, 5 µm, 2 mL/min, 150-bar outlet pressure
Figure 1.3 Pressure drop across several ZORBAX RX-Sil columns at 50 °C, with various
concentrations of MeOH in CO 2 , with the ΔP due to tubing subtracted.

Column head pressure [bar]
450
400
350
300
∆P

250
200
150
100
50
0

0

1

2

3

4
Flow rate [mL/min]Figure 1.4 Column head pressure using a 3 by 100 mm, 1.8 µm ZORBAX RX-Sil column,
with 22.5 % methanol in CO2, 150-bar outlet pressure, 50 °C. The optimum flow rate isapproximately 1.8 mL/min.

7

Simply stated, modern HPLC with sub-2-µm particles is often performed
at elevated temperatures from 40 up to 70 °C, in order to decrease theviscosity and pressure drop. SFC is generally performed between 40
and 60 °C, but for completely different reasons. It is a general rule ofthumb that ΔP in SFC is one third to one fifth of the ΔP in HPLC at three
to five‑times higher flow rates. There is no real requirement for ultrahighpressure pumping in SFC, unless you use long columns packed with
sub‑2‑µm particles or are operating at exceedingly high flow rates, eitherof which can be desirable.

1.2.3
Orthogonal to reversed
phase HPLCA minor component is often chemically similar to, and elutes near or evenunder a major component using any one technique. Several methods
with different selectivity may be required for a single sample. However,two different reversed phase methods are often used, both using a
C18 stationary phase to try to resolve such coelution. An orthogonaltechnique, like normal phase HPLC or SFC provides a superior alternative
for significantly reducing the risk of missing such coeluting compounds.
SFC should be preferred since it is faster, uses less hazardous modifiers,and generates much less toxic waste.
The retention order of any group of peaks in SFC is vaguely opposite
to the retention order in reversed phase HPLC. A typical example15
is presented in Figure 1.5. The solutes represent a diverse group offunctionalities, including sulfonamides, corticosteroids and xanthenes.
Such major changes in selectivity should be highly desirable. Two
separation mechanisms with different chemical interactions have positive
consequences. Major components often have a tail where the signal does
not return to the baseline in a timely manner. If the minor component
coelutes, or elutes on the tail of the major component, it may be difficult,or impossible, to quantify the minor component with any precision.
If, however, the minor component elutes before the major component,
where the baseline is flat, it might be more easily quantified with higher
precision and accuracy.

8

[mAU]
120
SFC
100
12 overlapped
80
injections
60
40
20
0
0
0.5
1.0

7
1

1.5

4
2

5

3

8

6

2.0

2.5

3.0

3, 4, 5, 6

700
600

HPLC

10 overlapped
injections

500
400

7

8

300

1

200

2

100
0

0

1.0

2.0

3.0

4.0

5.0
Time [min]Figure 1.5 SFC is orthogonal to reversed phase HPLC. Comparison of SFC and HPLC
chromatograms for a mixture of: 1. caffeine, 2. theophyline, 3. cortisone, 4. prednisone,
5. hydrocortisone, 6. prednisilone, 7. sulfamerazine, 8. sulfaquinoxaline. SFC Conditions:
4 mL/min of 5 to 25 % methanol in CO2 in 3 minutes. 150-bar outlet pressure with a
4.6 by 150 mm, 5 µm, RX-SIL column. HPLC conditions: 1.5 mL/min of 10 to 90 % methanol
in water in 4.5 minutes, 40 °C, on a 4.6 by 150 mm, 2.7 µm, Poroshell C18 column15.

The Agilent analytical SFC/UHPLC hybrid system provides for rapid
switching back and forth between reversed phase HPLC and SFC in
minutes. Sequences can be programmed to perform such comparisonsautomatically. This facilitates sequential running of two methods with thesame sample, using dramatically different selectivity, one reversed phasethe other normal phase, in a short period of time. The chromatograms
presented in Figure 1.5 are just such an example. Six SFC runs were
performed followed by six reversed phase HPLC runs, followed by four
SFC runs, then four reversed phase HPLC runs and finally two SFC runs.
All the SFC and reversed phase HPLC runs were then superimposed.

1.2.4
Separation of chiral and otherisomers

Reversed phase HPLC relies mostly on differences in hydrophobic
interactions to separate compounds. Normal phase techniques rely
mostly on polar‑polar interactions, and can better differentiate subtle
differences in shape, particularly around polar functional groups. As aconsequence, a typical application area of normal phase HPLC is in
the separation of isomers, where the separation is based on shapeof the molecule.
9

Most drugs are chiral, meaning they have pairs of isomers that are mirror
images of each other called enantiomers. Mixtures containing equal
concentrations of the enantiomers are called racemic. Older synthetic
approaches almost always made racemic mixtures. However, eachenantiomer often has unique biological activity. Consequently, the US Foodand Drug Administration and many other regulatory agencies require fulltesting of each pure enantiomer. It has also been possible to repatent a
drug as a pure enantiomer since the pure enantiomer is often faster acting
and less toxic than the older racemic form. Because of these factors, thedemand for chromatographic separation of enantiomers has increased.
Over the last 20 years SFC has been proven to be superior to normal
phase HPLC for the separation of enantiomers and other isomers. In fact,many larger pharmaceutical companies have focused on SFC for theseapplications. For example, Craig White6, who, at the time ran an analyticaland semipreparative purification service laboratory, analyzed all chiralsamples submitted to his laboratory for one year, which amounted tohundreds of samples. In 96 % of the separations, SFC was found superior,in terms of speed and resolution. In fact, SFC was subsequently designatedas the primary technique for both analytical, and semipreparative chiralseparations and HPLC was only used to evaluate the SFC problem samples.
Similarly, Mohammed Maftouh7, evaluated 500 proprietary drugs and had
a 95 % success rate by SFC using only four older (coated, not bonded)chiral stationary phases. With a set of 98 marketed drugs they had a 98 %success rate with the same system. Since 2006 they have been using SFC
as the primary screen, with capillary zone electrophoresis (CZE) as theirback-up. Both Pfizer and Merck have also published similar but slightly
less definitive statements about the value of SFC, for chiral separations.

1.2.5
Higher efficiency

10

The low ΔP values in SFC make it easy to generate high efficiency.
Columns can be, and often are, linked in series. Either the same or different
stationary phases have been coupled in this manner. Both chiral and achiralcolumns have been coupled. In the earliest example15, eleven columns,
200-mm long each, were coupled to produce a column 2.2-m long (5-µm
particles) producing over 220,000 plates, in a 400-bar system. In anotherexample, up to five different chiral columns have been coupled to make a
pseudo-universal phase16.

Alternately, long columns packed with sub-2-µm particles can be used togenerate reasonably high efficiencies because the pressure drops are low.
The Agilent analytical SFC system is capable of 600-bar operation, which
would support such a column about 0.5-m long, theoretically producing
up to 139,000 plates.

1.2.6
Lower operating costs

Most of the CO 2 used in SFC is food or beverage grade. Since it is meant
for human consumption, its purity is regulated. Carbonated beverages
are ubiquitous, the distribution infrastructure is in place, more or less,worldwide, and huge volumes are consumed. Consequently, CO 2 is
inexpensive. In high-pressure (50 to 70 bar) steel cylinders (25 kg),
liquid CO 2 can be as inexpensive as US$ 1 per kilogram. Typical usage
in SFC is 1 to 3 mL/min, meaning a cylinder lasts about 200 hours.
CO 2 is also available in Dewar flasks and bulk tanks both of which operatecryogenically at –30 to –40 °C, and 20 to 30 bar. Dewar flasks generally
hold about 150 kg, but are less convenient than either cylinders or bulk
tanks. The CO 2 in Dewar flasks generally cost about the same as in steel
cylinders, but last six times longer. Bulk tanks are usually installed only
when the organization commits to large-scale use of SFC. The cost of
the CO 2 from a bulk tank can drop an order of magnitude to US$ 0.10 per
liter. This compares favorably with the cost of heptane, which can exceed
US$ 70 per liter when purchased in cases of four 4-liter bottles.
Both Dewar flasks and bulk tanks require a booster pump or gas delivery
system to increase the pressure from about 20 to above 70 bar in order
for a reciprocating pump to be able to pump it. With bulk tanks, the CO 2 isgenerally plumbed to multiple locations within a facility.
In SFC, expensive, toxic acetonitrile is seldom used. Instead, an inexpensive
alcohol is used. Further, typical operation starts at a low %B such as 5 % but
seldom exceeds 40 %. Thus, most of the mobile phase is inexpensive CO2.
At the end of the chromatograph, the mobile phase decompresses andforms two phases, one gaseous, and the other a liquid. The gaseous CO 2
is vented, while the liquid modifier is collected in a trap. Since most of themobile phase evaporates, the volume of liquid waste is greatly reduced.
What waste that is generated is also less toxic.
Labor costs are one of the single largest expenses when operating achromatograph. Since higher diffusion coefficients dictate higher flow
rates and shorter run times in SFC compared to HPLC, on the same-sizedcolumn, a single operator produces three to five-times more work in thesame time frame.
111.2.7
Environmentally friendly

Despite using CO 2 as the main fluid, SFC is considered environmentallyfriendly or green because the CO 2 is recycled from other industries.
In HPLC, the mobile phase is often burned after use, generating new CO2.
Toxicity can be decreased by using small concentrations of alcohols
instead of acetonitrile. Since the CO2 evaporates at the end of the system,
the volumes of toxic liquid waste requiring expensive disposal is greatly
reduced.

1.3
What can SFC separate?

As a rule of thumb, any compound soluble in methanol or a less polar
solvent, is a good candidate for separation by SFC17. Conversely, compounds
that require a completely aqueous, buffered solution to dissolve areprobably poor candidates. This should not be interpreted as SFC being
incompatible with water in the mobile phase, aqueous samples, or many
biological samples. There is a review of the use of water as part of the
SFC mobile phase18.
Although SFC is not thought of as appropriate for large biomolecules suchas proteins, peptides up to 40’mers have been eluted19–21. Further, peptide
isomers varying only in the position of one amino acid have been well
separated in short times22, 23. This is an area of active research. If a peptide
can be eluted, it is likely to have significantly enhanced chromatographic
speed under the conditions used compared to HPLC.
In the early days of SFC most applications involved the separation ofrelatively nonpolar solutes, often homologous series, such as silicone oils,
surfactants, waxes, lipids, and the like that were too high in molecularweight, or too thermally labile for high-temperature gas chromatography(GC). Most of this work was performed using pure CO 2, pressure
programming, and a flame ionization detector (FID). This slowly started tochange in the late 1980s, particularly after the first chiral SFC separations
were published24–26.
Over the last 15 years, SFC has been used largely in the pharmaceuticalindustry for the rapid elution of small drug-like molecules, particularly
for chiral separations. With recent improvements in robustness andparticularly in UV sensitivity, SFC has started to re-expand into a muchwider range of applications.
The application areas of SFC are compared to the application areas of thevarious forms of liquid chromatography in Figure 1.6. As shown, SFC withvarious mobile phase combinations covers nearly the same application
space as HPLC in its various forms. The only area not significantly covered
is ion chromatography.

12Where Does SFC Fit Relative to HPLC?
SFC: pure CO2
SFC: CO2, modifiers,
additives and water

SFC: CO2 and organic modifiers

SFC: CO2, modifiers
and additives
Normal phase HPLC

HILIC
Ion chromatography

Reversed phase HPLCIon pairing
INCREASING POL ARIT Y
Hydrocarbons
fatty acids
and waxes
Ethers andestersA ldehydes
and ketones
Analine andbenzoic acids

Hydroxy acids
and poly acids

PeptidesPrimary amines
Zwitter ions

Ions
Proteins
DNA/RNA

Figure 1.6 Comparison of various forms of SFC to those used in the subsets of HPLC,
showing that SFC largely covers the same application space as HPLC.

1.3.1
Specific aspects ofretention in SFC

Normal phase SFC is about polar-polar interactions between thesolutes and a polar stationary phase. Steric hindrance is important in
SFC. A polar functional group, largely surrounded by nonpolar moieties
within the same molecule, tends to result in much less retention of themolecule on polar stationary phases, compared to a molecule with thepolar functionality unhindered. For example, in the series mono-, di-, andtribenzylamine the basicity increases with the degree of substitution on
the nitrogen27. Thus, tribenzylamine is a stronger base and is more polar
than dibenzylamine, which, in turn, is a stronger base and is more polar
than monobenzylamine. In this series, the molecular weight also increases
with the number of phenyl substitutions. Larger molecules tend to be moreretained. If polarity and molecular weight were the determining factors,
the elution order on a polar column ought to be benzylamine followed bydibenzylamine and then tribenzylamine. However, tribenzylamine is almost
unretained, dibenzylamine elutes second, and monobenzylamine as thesmallest and weakest base is the most retained. Clearly, each additional
substitution of a phenyl group on the nitrogen progressively hinders thelone pair on the nitrogen, getting close to and interacting with the polar
13

stationary phase. For similar reasons, nitrogen atoms in rings tend tohave weak interactions with polar stationary phases and seldom needan additive to achieve good peak shapes.
The choice of stationary phase can have a large impact on the speed ofanalysis. Some stationary phases are much more retentive than others.
If two different stationary phases provide reasonable solutions to an
analytical problem, the one using the lower modifier concentrations
should generally be preferred since the lower the modifier concentration,
the lower the viscosity and column pressure drop.

1.3.2
LogP

A widely used measure of lipophicity in drug development is logP, whichis the logarithm of the partition coefficient (P) of the compound between
octanol and water. Octanol is an alcohol, making the hydroxyl headmoderately polar, while the C8 backbone is nonpolar. Octanol and water
are not miscible and form two distinct phases when brought in contact
with each other, giving the molecules of the compound of interest the
choice of which phase to be in.
A positive logP means the drug partitions preferentially into the octanol.
A negative logP means it preferentially partitions into the water.
Lapinski created a set of rules he called the rules-of-5 for effective drug
development 29. One of the rules stated that to be an effective oral drug,
the compounds logP should be between 0 and 5. With a logP of 2.5,
more than 300 molecules would partition into the lower polarity octanol,for each molecule that partitions into the water.
The distribution of actual logP values of 5000 commercial drugs is plottedin Figure 1.7, and shows the largest drop between –1 and 6, with anaverage of about 3, just as Lipinski suggested. Solutes with a range of
logP from less than –2 to greater than 7 have been eluted and separatedusing CO 2 -based mobile phases. Thus, SFC is ideal for the elution andseparation of small drug-like molecules.

14

Demonstrated SFC region

Virtually ALL
small, drug-likemolecules
-10

-8

-6

-4

-2

0

2

4

6

8

10

12
LogP

Injectables, peptides and proteins
Figure 1.7 A plot of the log of the partition coefficient between octanol and water (logP)of 5000 commercially available drugs, which shows that the vast majority is only moderately
polar and fit well with SFC.

The rule of thumb suggesting SFC is not appropriate for compounds
requiring an aqueous buffered environment has, in the past, been
misinterpreted as meaning SFC was only appropriate for nonpolar solutes,
which is untrue. Many organics are very poorly soluble in water (logPmuch greater than 5). This makes them poor candidates as drugs, since
they would be permanently adsorbed into fatty tissues and would neverinteract with the appropriate biological receptors. A major effort indrug development is often to increase water solubility, to decrease thelikelihood of the compound being stored in fatty tissues. This can also be
misinterpreted to suggest that drug-like molecules must be highly soluble
in water (and biological fluids). This is also not true. If compounds are toowater-soluble, they would be poor drug candidates, since they would bequickly eliminated from the body. Increasing water solubility often meansincreasing from insoluble to only slightly soluble. Such compounds arelikely to spend a significant amount of time in the cells, interacting withthe appropriate receptors. Thus, such compounds are considered to bemoderately fat loving, or lipophylic.
Another rule-of-5 states that ideal oral drugs should have a molecularweight no greater than 50028. In Figure 1.7, there are several drugs with
logP of –2 and below. These are mostly proteins and large peptides, which
are too large to pass through the gut intact, and are destroyed making
them inappropriate for oral administration. Instead, they are injectable.
Such compounds are mostly not amenable to separation by SFC.

15

1.4
What’s in a name?

Too much is usually read into the prefix super in supercritical because it
seems to suggest that something special happens when you cross theborder. However, super simply means above. Similarly, critical is alsoominous sounding and is often misunderstood. The critical point is thetemperature and pressure above which only a single phase can exist.
Supercritical simply means above the critical point.
Supercritical is not recognized as a separate state of matter. There are
only gases, liquids, solids, and plasmas. The supercritical region is atransitional region, in which a gas can be converted to a liquid, and viceversa, by changing temperature and pressure, without a phase transition.
The choice of the name supercritical fluid chromatography was
unfortunate because it does not cover all the fluid characteristics ofinterest. The conditions used are often subcritical, usually meaning thepressure is above the critical pressure but the temperature is below thecritical temperature. Generally, you cannot tell the difference. There is nosudden change in retention, density, efficiency, or linear velocity when thedefinition of the fluid changes.
Obviously an author would rather not use a technically incorrect nameand call something supercritical when it is not. However, stressing the factthat the fluid is not supercritical implies there is something fundamentallydifferent about the two conditions, which is not correct. This confusion
has resulted in the creation of many different names for what is essentiallythe same thing.
During an open discussion at a GC conference in 1957, Jim Lovelock
suggested using compressed inorganic gases such as SO 2 as the mobile
phase to chromatographically separate highly polar substances. He
suggested the name critical state chromatography, but never performedexperiments. He had a letter notarized in 1958 that still survives, which
described what he meant.
Ernst Klesper was the first to actually use a fluid at high pressures andabove its critical temperature as a chromatographic mobile phase30.
He used fluorocarbon mobile phases to separate porphorins with a packed
column. In the first ever SFC paper, he described it as high-pressure
gas chromatography above critical temperatures. There are a number of
papers where the temperature was above the critical temperature, but thepressure, even though high, was below the critical pressure. Fluids undersuch conditions are by definition a gas, and the technique could be called(high-pressure) gas chromatography. However, this name fails to convey
the nature of the technique. The fluid acted as a solvent, which is counter16

to the general understanding in GC, where the mobile phase is an inert
carrier (that is, not a solvent).
In 1966 Giddings published papers on turbulent gas chromatography andultrahigh pressure gas chromatography31, 32. In another paper, several
years later, he changed the name to dense gas chromatography to try
to differentiate the technique from regular GC, since the mobile phase
was acting as a solvent, and the solvent strength was proportional todensity33. Interestingly, the last two papers both used pressures up to2000 atmospheres. Substantially higher than what currently is consideredultra-performance in HPLC.
In 1967 Sie and Rijnders 34 were the first to call the technique supercriticalfluid chromatography34. This is better than calling the fluid a gas, but
implies the fluid needs to always be supercritical in order to display thecharacteristics of interest, which is incorrect.
In 1985 Caude appears to have coined the name subcritical fluid
chromatography35. By definition, operating at higher than the criticalpressure but lower than the critical temperature, the fluid is technicallya liquid. However, they rightly concluded that calling it a liquid would also
be confusing, since the fluid remained highly compressible and needed
special hardware such as pumps, and a means to significantly elevate andcontrol the outlet pressure. Nevertheless, the term subcritical continues tobe widely used and many people appear to think there is truly a significant
difference between sub- and supercritical fluid characteristics.
Olesik coined the name enhanced fluidity chromatography, starting in
1991 for a unique variant36. She has also called it high performance liquid
chromatography with enhanced fluidity. She used CO 2 as a significant
fraction of the mobile phase, but at typically 10 to 25 %. She operated athigh pressures but below the critical temperature. The fluids retain someof the higher diffusion coefficients, lower viscosity inherent in SFC, but
retain more of the polarity inherent in aqueous based fluids. Enhanced
fluidity chromatography is a continuum to what others call subcritical fluid
chromatography. Both groups recognize that by definition, if the fluid is
not supercritical it must be a liquid, or subcritical.
As is evident from the above, the name of the technique has evolved
over time in efforts to try to clarify what the essence of the processes
involved is. However, at some point these new names created completely
artificial borders or barriers that have no real chemical or chromatographic
significance. Calling the fluid a liquid, or a gas, does not capture thefull essence of the technique. Calling the fluid supercritical also fails to17

describe the processes because, by definition, the fluids with essentiallythe same properties are subcritical some of the time.
There are several unifying aspects all these names are trying to describe.
First, the fluid acts as a solvent, and the solvent strength can be adjusted
by changing the composition, or density (temperature and pressure), or
both. Second, the fluids remain highly compressible, requiring special
pumps, and a means of maintaining high outlet pressures. These two
aspects are true of all the fluids of interest, regardless of definition of thefluid at any particular temperature or pressure.
I would like to suggest that all these names describe the same technique,and use the same hardware. A better name might have been compressible
solvent chromatography but it is much too late now. We have too many
names already. Simply allowing SFC to describe all these techniques would
be a helpful development.

18

THE MOBILE PHASE
2.1
Why use CO2?

2

Today, almost all applications in SFC use CO 2 modified with an organic
solvent and sometimes a highly polar additive. CO 2 is the preferred fluid
because it is:• readily available,• inexpensive,• has an accessible critical point,• relatively safe,• considered green since it has been recycled, and• miscible with a wide range of highly polar modifiers.

2.1.1
Availability

Many fluids have been used as the primary fluid in SFC 37. These fluids
include nitrous oxide (N 2 O), halocarbons, ammonia (NH 3), sulfur dioxide(SO 2), and supercritical water. Some continue to be used to this day.
Some are dangerous (nitrous oxide, ammonia). Nothing else comes close to
the overall performance of CO2 although a few fluids have specialty niches.
Massive amounts of CO2 are used in the beverage industry worldwide.
The distribution infrastructure is in place even in relatively remote places.
The raw material is a waste product from other industries. These
circumstances combined ensure a ready supply at a remarkably low cost.

2.1.2
Cost

Organizations with many SFC instruments, or who perform significant
semipreparative SFC, usually install a bulk tank. Such tanks can hold many
tons of CO 2 and are usually filled from a large tank truck. At this scale CO 2
costs about US$ 0.10 per liter. In Dewar flasks and steel cylinders, costs
are in the order of US$ 1.00 per liter. Almost all the increased cost is due
to extra handling. Nevertheless, the cost is remarkably lower than mostcommon solvents.

2.1.3
PuritySFC-grade CO 2 was developed in the late 1980s through early 1990s toprovide pure mobile phase for the nascent SFC business. Its introduction
was needed at that time, since ordering a cylinder of commercial
industrial-grade CO 2 could result in the delivery of a cylinder partly filledwith corn oil (author’s own experience!). Further, steel CO 2 cylinders used19

in the beverage industry were sometimes (inadvertently) backfilled withwater or with stale beer, which oxidized the steel, producing colloidal
iron particles that were detrimental to the performance, and difficult
to clean out. At that time, dip tubes were used to take the liquid CO 2from the bottom of the cylinder to minimize the chilling power needed.
This meant that anything dissolved in the CO 2 also wound up in thechromatograph. These negative experiences in the late 1980s resulted in
the development of an ASTM-certified, guaranteed-purity SFC grade CO2.
It is still available. It is typically shipped in 30-pound aluminum cylinders
and costs around US$ 20 per liter, which negates the operational costadvantage of SFC. The primary difference from other grades is thata purity test is run on each batch and a certificate of analysis issued
showing it meets certain criteria. There appears to be no reason to buy
this grade presently, except perhaps for ultratrace work.
Bulk CO 2 is typically of high purity. Most contamination occurs when
the fluid is repackaged in smaller containers. The supply situation has
improved dramatically and high-purity industrial grades are usuallysatisfactory. Many grades are more than 99.99 % pure CO 2. Beverageor food grade is a convenient, safe choice.
It is useful to point out that the Agilent analytical SFC system has apowerful chiller that uniquely liquefies the vapor phase from the cylinder.
Thus, an eductor or dip tube, extending from the valve to just above
the bottom of the cylinder is not required. By using the vapor phase andnot the liquid phase, the fluid is distilled just before use, leaving any
nonvolatile contaminants behind in the cylinder.

2.1.4
Accessible critical point

The critical point of pure CO 2 is readily accessible at just over 31 °C
and 70 bar. This simply means that CO 2 can be compressed to a dense
fluid at relatively low temperatures and pressures. A dense solvent at
relatively low temperature is unlikely to damage temperature sensitive,
labile compounds. The need for only modest pressures to achieve a dense
solvent is convenient and does not impose a significant technical orenergy penalty.

2.1.5
SafetyCO 2 is a product of human respiration and as such is not toxic at low
concentrations. Nevertheless, at high concentrations it can be lethal.
People should not be alarmed about the presence of CO2 in the laboratory.
Most convenience stores, restaurants, and cafeterias have cylinders ofhigh-pressure CO 2 associated with their soft drink machines. Many fire
extinguishers also contain high-pressure CO 2 and are widely distributed

2.1.5.1
Toxicity of CO2
20

throughout factories and office buildings. The concentration of CO 2 inproperly designed SFC laboratories is much lower than is typical in occupied
conference rooms or theaters. Large quantities of CO 2 are seldom stored
directly in the laboratory, so large-scale escapes are unlikely.
CO 2 is denser than air and can tend to accumulate near the floor of poorly
ventilated spaces. Sensors and alarms should be mounted near waist level.
Oxygen sensors do not provide needed information, since in any potentially
dangerous situation the oxygen level is likely to be near normal, even when
there is a dangerous level of CO2.

2.1.5.2
Fire and explosion hazard

In normal phase HPLC, the mobile phase often consists of heptane mixed
with another organic modifier such as isopropanol. Heptane, in particular,
is highly flammable and the vapors can be explosive. In many jurisdictions,the amount of such solvents in a laboratory is restricted. In SFC, using CO 2
as the primary component in the mobile phase, it is almost impossible toget the mobile phase to burn.

2.2
Using 100 % CO2

The solvent strength of pure (100 %) CO 2 is a function of its density.
Much of the work in SFC between about 1984 and 1995 employedcapillary columns, a flame ionization detector (FID), with isocratic orpressure or density programming with 100 % CO 2 as the mobile phase38.
Several applications were also developed using packed columns 37.
These applications were done with pressure or density programming ofpure (100 %) CO 2 and with flame ionization detection, particularly forseparation of relatively low-polarity homologous series such as silicone oils
and surfactants. Pressure or density programming has largely faded away.
The density of CO2 changes over a wide range with changes in temperatureand pressure as shown in Figure 2.1. At 40 °C, most of the change indensity occurs over only a narrow range of pressure between about 70
and 110 bar. Operation in this region means that small changes in pressure
produce large changes in density and retention. As the temperature isincreased, the curves tend to flatten out, creating a shallower gradient ofdensity against pressure. Most pressure programming has been done atelevated temperatures since, as is obvious from Figure 2.1, it is easier tomake small changes in density on a shallower slope.

21

Density [g/cm3]
1.2
1.0
0.8
0.6
0.4
0.2
0

0

100
40 °C

60 °C

200

300

80 °C

100 °C

400

500
600
Pressure [bar]Figure 2.1 Density of pure CO2 as a function of pressure at four different temperatures.
From top to bottom: 40, 60, 80 and 100 °C.

In the petroleum industry, pure CO 2 is still used with packed columns.
At low temperatures, such as 28 to 40 °C, with an appropriate silica
column, petroleum group separations are performed to determine thearomatic content in diesel fuels for environmental reasons. The method
can also be used to determine the aromatics content in crude oil.
Under the appropriate conditions, all the aliphatics will elute as a single
narrow peak. Olefins also tend to elute as a single peak but often forma tail on the aliphatics. Aromatics elute as a series of peaks, each one
representing an increase in ring number. This application has been an
ASTM method (D5186) for aromatics in diesel fuel since the mid-1990s
and is one of a few techniques that can accurately quantify low levelsof aromatics.
A related method (ASTM 7347) uses both the silica column and a silver
loaded column with valve switching to better separate the olefins fromthe aliphatics. The aliphatics and olefins are partially separated on thesilica column and allowed to enter the silver column. The aliphatics passthrough the silver column, to the FID. The olefins are highly retained
and must be back-flushed off the silver column. The aromatics are neverallowed to enter the silver column and are directly quantified with the FID.
This has become an important method for the determination of olefins ingasoline. Most present applications are not amenable to using pure CO 2
as the mobile phase.

22

2.3
Modifiers or cosolvents

Today, most SFC applications are performed on relatively polar stationary
phases with CO 2 modified with an organic solvent and sometimes otherhighly polar components, such as acids and bases, called additives.

2.3.1
Methanol

Methanol is by far the most widely used modifier and among the most polar
modifiers completely miscible with CO2. Advantages of methanol include:• availability,• inexpensiveness,• complete miscibility with CO 2,• low UV cut-off (about 205 nm),• relatively low toxicity.

2.3.1.1
Density of methanol/CO2
mixtures

The density of pure CO2 was the primary means of changing retention of
low polarity solutes in the past, so changes in density of mixtures should
also have some effect on retention. The densities of a few ­methanol/CO2
mixtures have been measured39, 40. The measured densities were found toincrease both with increasing methanol concentration, and with increasingpressure, as shown in Figure 2.2. These values can be used to deconvolutethe effects of density and composition on retention by adjusting pressure
to maintain constant density, while composition is changed. Such work has
shown that there is a small density effect but once a modifier is added, themodifier concentration produces most of the retention change. However,the less polar the modifier, the more pronounced the dependence ofretention on density becomes.

23

50°C

Density [g/cm3]
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50

80

100
2%

120
4%

8%

140

160

16 %

5.63 %

180

200

10.35 %

220
240
Pressure [bar]15.04 %

0%

Figure 2.2 Density of MeOH/CO2 mixtures as a function of pressure at 50 °C. The densityof pure methanol is 0.791 g/cm3. The open symbols are from reference 39. The solid symbols
are from reference 40.

When changing a method from 5-µm to sub-2-µm particles, the pressure
drop across the column increases substantially. Thus, the average density inthe column is changed. Both retention and selectivity can change noticeably.
However, this is usually a secondary effect, as shown in a later section.

2.3.2
Modifier solvent
strength scales

24

Almost any organic solvent can be used as a modifier. Their elution
strength roughly follows Snyder’s P’ elution strength scale41, 42 or the
Hildebrand solvent strength scale for silica43, each developed many
years ago for elution from silica in normal phase HPLC. Values forseveral common organic modifiers are listed in Table 1.1. The actual
values have been changed over time. For example, today, methanol ismuch closer to water than originally thought. The modern P’ values formethanol, acetonitrile, ethanol, and isopropanol are 6.6, 6.2, 5.2, and4.3 respectively. If the same concentration of modifier is used, relative
retention tends to follow the solvent strength scale in Table 2.1, butthere are exceptions.

Modifier

P’

Water

9

Methanol

6.6

0.73

Acetonitrile

6.2

0.5

Methoxyethanol

5.7

Acetone5.4

0.47 to 0.53

Ethanol

5.2

0.68

Isopropanol

4.3

0.62

Ethyl acetate

4.3

0.38 to 0.48

Tetrahydrofuran

4.2

0.53

Dichloromethane

3.4

0.32

Methylene chloride

3.4

0.32

Carbon tetrachloride

1.7

Heptane, CO20

Acetic acid

6.2

Octanol

3.4

Hildebrand elution strength (on silica)

0.01

Table 2.1 Snyder’s P’ values and Hildebrand values for some of the modifiers completely
miscible with CO2.

With a chiral separation44, the retention time of the second eluting peakincreased from 7.98, to 8.32, to 10.48 minutes when the modifier waschanged from methanol, to ethanol, to isopropanol, as expected and as
shown in Figure 2.3. The resolution actually decreased with increasing
retention times. The same pair of enantiomers was separated using
acetonitrile, as modifier, at the same concentration. The retention of thelast peak increased substantially to 16.13 minutes, but the separation
noticeably improved. It is often the case because acetonitrile is a much
weaker solvent than most of the alcohols. On the Hildebrand scale all
three alcohols have higher values than acetonitrile, which is consistent
with the results in Figure 2.3. Apparently hydrogen bonding is inordinately
important in SFC.

25

6.936
7.982
7.655

8.315
9.814

10.47511.45016.131

Pola

rity

Time [min]Methanol
α = 1.151

Ethanol
α = 1.086

Isopropanol
α = 1.067

Acetonitrile
α = 1.409

Figure 2.3 A chiral separation showing the increase in retention time as a function of
decreasing modifier polarity according to both the P‘ and Hildebrand solvent strength scales.
Acetonitrile is much less polar than expected by P’ but not by Hildebrand. All conditions
except the identity of the modifier were held constant.

2.3.3
Nonlinear solvent strength

Snyder’s P’ scale41, 42 assumes that mobile phase solvent strength (asmeasured with P’) is a linear function of the mole fractions of the twopure liquids, see Equation 2.1 where x is the mole fraction of A.

P’AB = cP’A + (1 – c)P’B
Equation 2.1 Snyders calculation of mobile phase solvent strength. Does NOT apply to SFC.

Thus, progressively varying the composition of the mobile phase from one
pure solvent to the other should produce a linear relationship in solvent
strength. However, retention (elution strength) is a nonlinear function of %B.
In normal phase HPLC this nonlinearity is explained as due to competitive
adsorption between the modifier (B) and the solutes.
In SFC, plots of log k against %B are also nonlinear, as shown in Chapter 4“Effect of Mobile Phase Variables on Retention and Selectivity”. However,
the explanation used in SFC is different.

26

2.3.3.1
Solvatochromic dyes

A way to measure actual mobile phase solvent strength (as opposed
to elution strength) involves the use of solvatochromic dyes. The UV
maxima in the absorbance spectra of such dyes changes, depending on
the polarity of the solvent the dye is dissolved in. Nile Red is such a dye.
The Nile Red energy scale (ENR), calculated from the wavelength of theabsorbance maximum, can be plotted against P’, correlating ENR to P’.
Using Nile Red as the solvatochromic dye produced the data presented in
Figure 2.4, for pure solvents45. The relative order of ENR solvent strengths
is consistent with the P’ values. Nile Red can also be used to measure
the polarity of mixtures of these same modifiers with CO 245, 46. A few
such results are presented in Figure 2.5. Unlike Snyder’s assumed linear
relationship, the measured solvent strength is a nonlinear function ofmodifier concentration. Since there is no stationary phase, a competitive
adsorption mechanism cannot explain the nonlinearity. Further, substituting
the ENR energy scale for %B produces linear plots of log k, at least above
about 1 % modifier46.

27

P`
12

10
Water

Formamide

8
Dimethylformamide

6

Acetonitrile

Acetic acid
Methanol4

Ethanol
1-propanol

THF
Methylene
chloride2

1-octanol
2-propanol

Carbon tetrachloride

Pentane

0
60

56

52

48
ENR [Kcal/mole]

Figure 2.4 Relationship between Snyder’s P’ scale to the Nile Red solvent strength scale forpure solvents. Note that ACN appears to be more polar than MeOH on the P’ scale but muchless polar on the Nile Red scale, consistent with the results in Figure 2.3.

28

Nonlinear Solvent Strength as Measured with Nile Red
5 10

20

5

10

40 60 80 100%

0 5 10

20

20

40 60

CO2
0

40

60 80 100%

1

2

Acetonitrile in CO2
Methylene chloride in CO2

80 100%Octanol

3

Methanol in CO2

Methanol

4

5

Water

6

7

8

9

10
P`

Figure 2.5 Nile Red can also be used to measure the solvent strength of mixtures of CO2
with mixtures of organic solvents. Again, ACN is shown to be a weaker solvent than MeOH.
Note the major nonlinear nature of solvent strength against modifier concentration.

2.3.3.2
Clustering

There is widespread consensus that polar modifier molecules form locallyinhomogeneous mixtures with CO 2. It appears that when the modifier issubstantially more polar than the CO2, the polar modifier molecules cluster
together, and in particular, cluster around polar solute molecules47. Thissurrounds the solute particles with a solvation sphere with a higher local
modifier concentration than in the bulk of the fluid. These clusters are notas well defined as a micelle.

2.3.3.3
Adsorption on thestationary phase

Using tracer-pulse MS experiments it has been shown that the first small
additions of polar modifier to CO2 results in extensive adsorption of modifier
molecules onto polar stationary phase. In fact, about 1 % methanol was
shown to produce several monolayers of modifier coverage on a diolcolumn48. At higher concentrations only a relatively small additional amount
was adsorbed. This coverage by the modifier tends to change the chemical
nature of the stationary phase.
At the same time, multiple monolayers of CO 2 can also be adsorbed49.
Thus, there is a deep pool of high-density modified fluid adsorbed on polar
stationary phases, the composition of which depends on temperature,pressure and composition50. These effects have not been studied in detail.

29

2.3.3.4
Changing selectivity withthe modifiers

The absolute value of the elution strengths listed in Table 2.2 is only partof the solvent strength picture. Snyder also developed a solvent selectivity
classification scheme starting with Rohrschneider’s gas-liquid partition
coefficients for ethanol, dioxane, and nitromethane with 82 solvents 41, 42.
It was assumed that Van der Waal forces were universal. Consequently,
he plotted the data on a triangular graph, each side representing theability of the solvent to interact with proton donors, proton acceptors ora dipole. A simplified version is represented in Figure 2.6. The solvents
tended to clump together in groups that Snyder characterized as eightsolvent families, the members of each group having similar selectivity.
Changing modifier from one selectivity family, keeping the P’of themodifier constant, tends to not change absolute retention significantly but
does change selectivity. In other words, if one modifier fails to provide
adequate separation of closely related compounds, try a modifier from adifferent solvent family, instead of another from the same family.
H-acceptors

0.20

0.60
0.55

II
Alcohols

0.30

III

cd
Acetic acid

VIII

Acetonitrile
Acetone VI
Ethyl acetate

Chloroform
Water

0.45

THF
DMF
DMSO

IV

0.40

0.50

I

EthersAmines0.35
VMethylene
chloride

VII

Nitrobenzene
Toluene0.20
H-donors

0.30

0.40

cn

0.50

ce

0.60

0.25

0.70
DipoleFigure 2.6 Snyder’s solvent triangle, breaking down solvents into eight families. To change
selectivity significantly, switch to a modifier in a different solvent family.

30

Additive

Comment

Isopropylamine

Reacts with some ketones

Dimethylethylamine

Volatile but stinks

Triethylamine

Sometimes less effective due to steric hindrance

Many other amines
Ammonia

Relatively recent

Tert-alkylammonium salts
Alkylsulfonic acids
Ttrifluoroacetic acid
Formic acidAcetic acid
CItric acid
Ammonium acetate
Ammonium formate
Water
Table 2.2 Additives widely used in SFC.

2.4
Additives

Many strong acids and bases, and most amphoteric compounds do notelute or elute with poor peak shapes when eluted with binary mixturesof CO 2 and a modifier such as methanol or ethanol. The addition ofa small amount of a highly polar additive51, 52, dissolved in the modifier
usually dramatically improves peak shapes, as shown in Figure 2.7. The
concentration of additives is usually between 0.1 and 2 % in the modifier.

31

[mAU]
120
100
80
60
40
20
0
60
50
40
30
20
10
0
-10

0

1

2

3

4

5

6
7
8
Retention time [min]Figure 2.7 The effect of additive on peak shape and elution using the drug Adderall, as an
example. Adderall is a nonracemic mixture of the enantiomers of amphetamine with excessof the D enantiomer. The lower chromatogram represents elution without additive; the upper
chromatogram with triethylamine. All other conditions unchanged.

Many times a strong acid improves the peak shapes of strong acids, whilestrong bases usually improve the peak shapes of other strong bases.
However, this is only a generalization and the opposite is sometimes true53(acid for a base and vice versa), probably as a result of something likeion pairing in HPLC. Amphoteric solutes are often the most difficult andmay require multiple ions or counter ions. A list of common additives is
included in Table 2.2. Additives can:• suppress solute ionization,• cover active sites on the stationary phase, and• form neutral ion pairs with the solutes.
Additives strongly adsorb onto polar stationary phases54 sometimes
forming near mono-layers, even at concentrations well below 1 % in themodifier (much, much less than 1 % in the total mobile phase). Often no
additive elutes for some minutes after the additive is first introduced into
the mobile phase, particularly at low modifier concentrations. Care should
be taken to wait long enough for the additive concentration to equilibrate.
32

Additives are not always washed off after they are removed fromthe mobile phase. The use of additives can permanently 55, or at least
temporarily changes column characteristics. It is often true that evenafter removal of the additive from the mobile phase, peak shapes tend toremain improved for some time, although there may be a slow degradationof performance back toward the performance initially observed without
additive. Such apparently irreproducible but predictable behavior cancomplicate screening, or require the use of several sets of identicalcolumns, one for use with an acidic additive and another for use with abasic additive, for example. Switching back and forth between acidic andbasic additives without an intermediate wash should be avoided.
Columns should never be stored without a careful wash after the use ofan additive, particularly amines. In the author’s lab, after using a basic
additive, the column is washed for about 10 column volumes, with 40 %methanol in CO 2, followed by 40 % (methanol + 0.2 % acetic acid) in CO 2,followed by a rinse with 40 % methanol in CO 2. The acetic acid, being a
relatively weak acid, can displace or neutralize strongly adsorbed basic
additives from the surface of the stationary phase, yet can usually itself be
washed off with CO 2 /methanol leaving a relatively bare stationary phasewithout adsorbed polar additives.
Other additives sometimes desorb with pure organic solvents. In extreme
cases, isopropanol has been used followed by an isopropanol -water
mixture, followed by isopropanol, in order to remove polar additives.
Columns should, at the very least, be rinsed with 40 % modifier in CO 2
for greater than 3 to 10 column volumes before storage. After washing,
the columns can be stored wet or dry. In the author’s lab, the columns are
vented to atmospheric pressure, meaning the columns are not filled witha liquid solvent. Then, the columns are stored without plugs inserted in thecolumn end fittings, after the column is removed from the system.
In recent years, there have been concerted attempts to develop stationary
phases that do not require additives in the mobile phase. These phases will
be discussed in more detail in Chapter 3 “The Stationary Phase”.
Additives can interfere with mass spectrometric (MS) detection, particularly
if they have poor volatility. When using a mass spectrometer for detection,
it is important to choose an additive that is quite volatile, such as ammonium
acetate or ammonium formate, or even ammonium hydroxide.

33

2.5
Extending solute polarity
with added water

Normal phase HPLC is not ideal for use with aqueous samples, particularly
when the mobile phase is nonpolar. Small amounts of water, from tracesin the modifier or from an aqueous sample, tend to preferentially adsorb
onto the polar stationary phase. Re-equilibration tends to be slow. Someolder reports suggest it takes up to 1000 column volumes to re‑equilibrate.
Consequently, aqueous samples are usually avoided. SFC has none ofthese issues, since the low polarity CO 2 is miscible with much more polar
solvents, compared to hexane or heptane. These more polar modifiers canhelp solvate small amounts of water.
Water is only soluble to a few tenths of 1 % in pure CO 2. However, whenmethanol is mixed with CO 2, at least 10 % water can be added to themethanol without significantly distorting peaks of very polar compounds.
Additives can still be added. Nucleic acids and nucleosides were rapidly
separated56 on an amino column using 10 % water in methanol withammonium acetate and formic acid added. Equilibration time remains fast.
The addition of water enables the separation of solutes that are morepolar than possible using just methanol. Further, aqueous samples, canbe diluted with methanol 3:1 or 4:1 and injected directly, without loss ofretention time or area reproducibility.

34

THE STATIONARY PHASE
3.1
Materials

3

The vast majority of SFC applications have used totally porous, very highpurity, bare silica or bonded phases on the same totally porous silica. There
are no issues with particle strength, since the particles are not subjected
to ultrahigh pressures or high pressure drops. There are only a few reportson the use of other materials such as porous shell, monolithic columns or
zirconia based particles.
Today totally porous particles are almost always spherical with diameters
between 1.7 and 5 µm. Larger particles are in decreasing use, even for
semipreparative scale, with up to 5-cm id columns. The particles that givethe best retention and peak shape, at least for polar solutes, are usually
totally porous, smaller pore, higher surface area particles with pore sizes
between 60 and 120 Angstroms, and surface areas greater than 350 m2 /g.

3.2
Achiral bonded phases
3.2.1
Nonpolar phases

Much of the early work in SFC involved relatively non-polar solutes,
which were best separated on a nonpolar column such as C18. A few
workers characterize this work as reversed phase SFC, specifically when
the separation involved a C18 or similar stationary phase and moderately
polar modifiers. However, the mobile phase usually consisted mostly of
CO 2 and a highly nonpolar solvent similar to heptane in solvent strength,
which were mixed with a more polar modifier. The modifier concentration
was almost always programmed with increasing modifier concentration.
Thus, the mobile phase was being programmed from lower to higher
polarity and hence definitely not reversed phase.
Reversed phase HPLC, clearly one of the most dominant chromatographic
techniques with hundreds of thousands of practitioners, is most often
used with C18. Consequently, there are many such high-quality columns
available. These columns can also be used for SFC. The C18 phases
work well with triglycerides, surfactants, silicone oils, carotenoids, otherterpenoids, and many compounds with a long aliphatic tail. With polar
solutes there is generally little retention and often poor peak shapes.

35

3.2.2
Polar phases

SFC is generally performed as a normal phase technique, where polar
stationary phases are used and the mobile phase is programmed from
low to higher polarity. Although HPLC started out as a normal phasetechnique, it was long ago supplanted by reversed phase in just about
every application area except for chiral separations and older Pharmacopeia
methods. Subsequently, until recently there has been little effort bycolumn manufacturers toward making newer polar stationary phases.
Traditional polar normal phase stationary phases include several formsof bare silica, cyano, amino, and classic diol. Chromatograms of a mix ofcaffeine, theophyline and theobromine demonstrate the differences inselectivity between C18, cyano, bare silica, amino and diol, as shown in
Figure 3.1. These moderately polar compounds were virtually unretainedon the nonpolar C18 even at a low (8 %) methanol concentration.
In contrast, they were well retained and separated on a cyano column
under the same conditions. The bare silica column was more retentive,
requiring higher modifier concentrations (18 %) to achieve similar retention
times. Selectivity was similar to cyano. The amino column actuallycreated a reversal in elution order (1, 3, 2 instead of 1, 2, 3) but theresolution between 2 and 3 was still not ideal. The diol phase was a bit
more retentive than the amino phase, without the peak reversal, but thepeaks were slightly better resolved due to differences in selectivity. Thesechromatograms indicate a significant variation in relative retention based
solely on the identity of the bonded phase. The chromatograms presented
were collected some years ago, using the same high-purity, silica-basedmaterial. Nevertheless, the results are representative of modern results,
although modern diol columns are more like bare silica.

36

1, 2, 3

C18 8 %
2

1

3

Cyano 8 %1

2

3

Silica 18 %1

3

2

Amino 20 %1

Diol 20 %1 = Caffeine

2 = Theophylline

23

3 = Theobromine

Figure 3.1 Retention and selectivity of caffeine (1), theophyline (2) and theobromine (3) onvarious stationary phases.

Both SFC and hydrophylic interaction liquid chromatography (HILIC) haverenewed the interest of column manufactures for creating new polarphases. The HILIC columns appear to be similar to bare silica but are
sometimes called diol.
Most of the innovation has come from smaller column manufacturers
trying to meet the niche needs of SFC users. As indicated in Chapter 2“The Mobile Phase”, it is often necessary to add an additive to the mobile
phase, particularly with aliphatic amine solutes. Several ethylpyridine
phases have been developed specifically for separating amines, which
often minimize, or eliminate, the need for an additive. Other proprietaryphases have also been developed for separating acids without an additive.
These newer phases tend to be more retentive than the classic polarphases, requiring a higher modifier concentration, which makes themslightly less desirable. A partial list of the more common phases ispresented in Table 3.1.

37

Agilent

Other suppliers

RX-SIL (5, 3.5, 1.8 µm)

Ethylpyridine (5, 3, 1.7 µm)

SB-CN (5, 3.5, 1.8 µm)

Premier (5, 3 µm)

Hexylphenyl (5, 3.5, 1.8 µm)

Diol (5 ,3, 1.7 µm)

HILIC (5, 3.5, 1.8 µm)

Many others

SB-C18 (5, 3.5, 1.8 µm)
Eclipse C18 (5, 3.5, 1.8 µm)
XDB C18 (5, 3.5, 1.8 µm)
Amino (5 µm)
Table 3.1 Common stationary phases.

It is worth mentioning that it is relatively difficult to get more than one
version of some of these polar phases with smaller particle sizes. These
include diol and amino. However, HILIC, bare silica, cyano, hexaphenyl
and C18 are all readily available in 1.8 and other sub-2-µm particle sizes.

3.2.3
Too many phases?

38

In SFC, we have a wide range of good stationary phases with variableselectivity to try to resolve difficult peak pairs, although it usually is notnecessary. It is fairly simple to match a stationary phase to the solutes, assuggested by Figure 3.2. There are usually a number of different stationary
phases that could be used for any specific separation, any one of which
could be chosen, and which will probably yield a viable separation.

Proteins
DNA/RNA
Inorganic ionsZwitter ions
PeptidesBenzoic acids
Hydroxy acids
Polyacids
Aliphatic amines

AmidesSteroids
Ring nitrogens
Alcohols
SugarsAnalines

Hydrocarbons
Waxes
Esters/ethers
Fatty acids
Terpenoids
Ketones/aldehydes

INCREASING POL ARIT Y
MOBILEPHASE

CO2Ethyl acetate/Acetone/Aetonitrile/CO2
Isopropanol/CO2

Ethanol/CO2

Methanol/CO2
Methanol/
Water

Additives
STATIONARY
PHASE

C18, C4, C1
Hexaphenyl
Cyano
SilicaDiol
AminoEthylpyridine

Figure 3.2 Specific mobile and stationary phases are matched with solute families.
Progressively more polar solutes require progressively more polar mobile phases andstationary phases.

For polar solutes, just about any of the polar phases will do. A method
can generally be optimized by changing mobile phase composition,
temperature and pressure as outlined in Chapter 4 “Effect of Mobile
Phase Variables on Retention and Selectivity”. The only time you needto change the stationary phase is in situations when changing the mobile
phase parameters fails to produce adequate selectivity.

39

3.3
Stationary phasecomparisonsLesellier57 developed a method for visualizing differences in column
selectivity based on a solvation parameter model, vaguely akin to Snyder’ssolvent triangle, except it applies to stationary phases, not the mobile
phase. In this case, five parameters were used to generate a spidergram,in which columns were evaluated and compared. A simplified version
is presented in Figure 3.3. As can be seen, all the classic polar phasessuch bare silica, cyano, amino, and diol cluster fairly closely together
across the axis of proton donors. Diol has a tendency to also be a proton
acceptor more than the others, while cyano and amino tend toward
dipole‑polarizability characteristics. The relatively new pentafluorophenyl(PFP) has not been widely used in SFC. In this model, it represents
dipole-polarizability, something akin to some of the chlorinated solventsin Snyder’s triangle.
Excess mole fraction
polarizability
C8

C18
PFP

McGowancharacteristic
volumeC4

Dipolarity/
polarizability

OPHE

Diol
Proton acceptor

SilicaCyano
Ethylpyridine
Amino
Proton donor

Figure 3.3 Column selectivity using Lesellier’s solvation parameter model.
OPHE: SynergiPolar RP (phenyl-propyl) Phenomenix, EP: 2-ethyl-ethylpyridine,
PFP: pentafluorophenyl-propyl (Supelco).

40

The hydrocarbons C8 and C18 are clustered together, more or less directly
opposite to the classic polar phases, as one might expect (reversed phaseagainst normal phase). The relatively new phenyl-propyl phase appears to
be relatively neutral, placing it near the center of the plot directly between
the nonpolar aliphatic hydrocarbons (C8, C18) and the polar classic columns(bare silica, cyano, amino or diol). C4 is removed from the other aliphatichydrocarbons. Again, if one stationary phase provides poor separation, it isprobably appropriate to switch to a different stationary phase group.

3.4
Relationship between
particle diameter andcolumn dimensions

Older SFC applications were largely performed using 4.6 by 250-mm
columns packed with 5-µm totally porous particles. Instruments capable ofproducing the full efficiency of columns packed with 3 or 3.5-µm particles
have been available for several decades, but such particles were not often
used in SFC. Even 10 years ago, a large part of SFC achiral applications
involved rapid screening, with 4.6 by 50 to 150-mm columns, but still
mostly with 5-µm particles.
Sub-2-µm particles have been around for over a decade but mostly inthe form of C18 for use in UHPLC. Such particles offer a three to four-foldimprovement in speed, compared to 3-µm particles, or about a nine-foldadvantage over 5-µm columns, at the same efficiency, when each isoperated at optimum velocity. Decreasing the particle diameter decreases
the run time in two ways. The optimum flow rate and the efficiency areboth inversely proportional to d p. Decreasing the particle size by a factorof two requires twice the flow rate, and generates twice the efficiency inthe same length. Cutting the length in half generates the same efficiency
as the column with larger particles but in one-quarter of the run time.
Since the C term in the van Deemter Equation is rather flat, much of
the early UHPLC literature stressed using even shorter (30 to 50-mm)
columns operated substantially above optimum velocity to increase
speed even further.
There is no reason why such particles could not also be used in SFC,
provided appropriate phases are available, and the instrumentation has
appropriately low dispersion. Although used for a decade in UHPLC,
sub-2-µm particles have only started to be used in SFC, over the last few
years58. Initially only bare silica was available. However, in recent years
several of the classic polar phases, and 2-ethylpyridine have become
available in this format. Both the column id and the particle diameter (dp)have been decreasing rapidly. Columns with 3-mm id and 100-mm length,
packed with sub-2-µm particles are becoming a de facto standard.

41

3.4.1Pressure drops, thermal
gradients and loss of
efficiency

The pressure drop across a column is a function of dp2, at constant efficiency.
Thus, the pressure drop using 1.8-µm particles is about 7.7-times higher
than the pressure drop using 5-µm particles, with equal efficiencies, atthe optimum velocity for each. Operating at double the optimum velocity
further increases ΔP to greater than 15 times. The resulting high pressuresrequired the redesign of many pumping systems to perform UHPLC.

3.4.1.1
Thermal gradients in UHPLCHigh pressure-drops were predicted to cause axial and radial temperature
gradients from the earliest days of HPLC 59–61, and were found to be a
problem62 when using sub-2-µm packing under extreme conditions.
The mobile phase heats up as it expands, and temperature at the column
outlet can be significantly above the column compartment set temperature.
The gradient along the axis is not considered a problem. However, if
there is significant heat transfer from the mobile phase into the column
compartment, radial thermal gradients can form along the column. Such
radial gradients can result in lower viscosity and higher velocity at thecolumn axis, compared to higher viscosity and lower mobile velocity near
the wall. Thus, different parts of the same peak could travel down thecolumn at different speeds, broadening the peak, and causing a loss of
efficiency. The existence of such radial gradients is a major justification for
the use of 2.1 or even 1-mm id columns, since the shorter distance between
the column axis and the wall should decrease the amplitude of the radial
temperature of the gradient.
There are major instrumental consequences and considerable debate overthis topic. Others have used 4.6-mm columns with sub-2-µm particles at
up to 600 bar without significant efficiency losses. This may be due to theway column temperature is controlled. The mobile phase can be heatedto the working temperature before entering the column, with the column
in a stagnant air bath oven and insulated from the heat exchanger. This
minimizes heat transfer out of the column and along the column, since boththe stainless steel hardware and stagnant air are poor heat conductors.
Under such conditions, the column acts largely adiabatically. This enables
the unimportant axial gradients to form but minimizes any problematicradial gradients.

42

3.4.1.2
Thermal gradients in SFC

Similar arguments about efficiency losses due to thermal gradients havealso been made in SFC 63, 65 . If the column is placed in a water bath,the full length of the stainless steel hardware can be maintained at thesame temperature. The axial gradient will form, but now the differencein temperature between the axis, caused by expansion, and wall will
result in a radial gradient, and serious losses in efficiency have been
demonstrated. Therefore, just as in UHPLC, it is often recommended that
2.1 or even 1-mm id columns be used.
However, most of the relevant experiments were done using pure CO 2,
or 5 % methanol in CO 2. Under those conditions, the mobile phase cools
down significantly on expansion. Unlike UHPLC, temperature gradientsin SFC, due to Joule-Thomson (J-T) expansion, are strongly dependent
on the composition of the mobile phase. For pure CO 2, expansion from
600 to 100 bar would result in a temperature drop of 16 degrees acrossthe length of the column, as noted in those reports. At 5 % methanol, themaximum temperature drop would still be about 10 degrees. However,at 10 % methanol, the fluid first warms 2 to 3 degrees before falling5 degrees. Above about 18 % the fluid actually heats up slightly, whileat 50 % methanol the fluid never cools and heats up by 10 degrees.
Methanol concentration [%]Tout [°C]

DT

0

40

-10

5

44

-6

10

47

-3

20

52

+2

50

60

+10

100

65

+15

Table 3.2 Theoretical adiabatic temperature drop across a column, assuming the inlet was
at 600 bar and 50 °C, and the outlet was at 150 bar.

43Remember that the pressure drops in SFC are one-third to one-fifth aslarge as in UHPLC. Although some instruments are capable of 600-bar
operation, such high pressures are almost never required. More typicaloperation is with an outlet pressure at 150 bar and with an inlet pressure
between 200 and 350 bar. Under these conditions, an axial gradientwould be no more than a few degrees. The Agilent thermostatted column
compartment is a still-air column compartment, in which a heat exchanger
is used to preheat the mobile phase before entering the column and thecolumn can easily be insulated from the heat exchanger. As a result,
there is little radial gradient formed and little efficiency loss due to thisphenomenon, under practical conditions. This creates little incentive touse columns 2.1-mm or 1-mm id in SFC, as is sometimes recommendedin UHPLC and SFC.
Using sub-2-µm particles in 2.1 or even 1-mm id columns puts severe
constraints on the design of the chromatograph in terms of extracolumn
band broadening. For example, a 2.1-mm column packed with sub-2-µm
particles requires a detector cell with a volume of less than 1 µL, alongwith short (less than 60 cm) connector tubing of 125 or even 100-µm id.
Currently there is no commercially available instrument that can produce
more than about half the theoretical efficiency of a 2.1 by 100-mm column
packed with sub-2-µm particles, using standard configurations. Using suchcolumns requires a major redesign or replumbing, or both, of all existing
SFC systems. However, it is both feasible and desirable to use larger idcolumns that decrease extracolumn band broadening requirements,
making it possible to see most of the efficiency of sub-2-µm particles withexisting hardware with only modest modifications.
In the other direction, there is also little incentive to use 4.6-mm id
columns with sub-2-µm particles, since, in SFC, the optimum flow rate on
a 4.6-mm column approaches 5 mL/min. This is the maximum flow rateof the Agilent analytical SFC system. Some other SFC systems have alower maximum flow rate, particularly at higher column head pressures,and would need to operate suboptimally much of the time. Thus, column
inside diameters significantly smaller than 4.6 mm are desirable, and arenecessary for operating above the optimum velocity or flow rate with
sub-2-µm particles. Since both 2.1 and 4.6-mm id columns have issues
with either extracolumn band-broadening or with flow rate limitations,a reasonable compromise seems to be a 3-mm id column size. With a
3 by 100-mm column packed with 1.8-µm particles, the Agilent analytical
SFC system can be easily reconfigured to allow about 90 % (or more andat least 96 %) of the theoretical efficiency of the column to be observed,
as demonstrated in Section 3.5.2 “High-speed work”.
44

3.5
Recommended column
dimensions
3.5.1
Routine work

For new methods, there is no longer any obvious advantage to using totally
porous 5-µm particles in SFC for analysis. There is no aspect of speed,
resolution or sensitivity that is not exceeded by using smaller particles inshorter, faster columns. Instrumentation and columns are readily available
that produce equivalent performance, but much faster, and potentially withhigher sensitivity, on smaller diameter particles. For validated methods,
5-µm particles, in 250-mm columns, will continue to be used.
Columns of 4.6 mm in diameter, packed with 3 or 3.5-µm particles
are compatible with larger UV detector flow cells, such as those with
10-mm path length and 13-µL cell volume that are widely used in Agilent’s
multiple-wavelength and diode-array detectors. The long path length of
the UV detector cell retains or improves on the sensitivity observed with
5-µm particles, since, with the smaller particles and higher flow, the peaks
are narrower in time with greater height.
The tubing connecting the injection valve to the column, and the column
to the detector, can collectively be over a meter long with 175-µm id. With
such a system, nearly 100 % of the theoretical efficiency of the column
can be observed. The 175-µm tubing minimizes the extracolumn pressure
drop across the system.
For routine work, a column of 4.6 by 150 mm, packed with 3 or 3.5-µm
particles is recommended. Methods developed on such columns should berobust and yield results similar to the best achieved with HPLC. Retention
time reproducibility should be much less than 1.0 % RSD (n=10) in all but
the most extreme cases, and it is often true that RSDs less than or equal
to 0.1 % are achieved. Area reproducibility depends on signal-to-noise.
For a peak with signal-to-noise greater than about 100, area RSDs should
be below 1 %. For much larger peaks, RSDs should be well below 1 %,sometimes approaching 0.1 %. Linearity in calibration curves generally
exceeds 0.999, as shown in later sections (see, for example, in Chapter 8“Quantification in SFC”).

45

3.5.2High-speed work

For high-speed work, sub-2-µm particles can be used. Agilent’s sub-2-µm
packing is 1.8 µm. As mentioned previously, 4.6-mm id columns packed
with 1.8-µm particles have an optimum flow rate of about 5 mL/min,
which is the maximum flow rate of the Agilent analytical SFC system,
leaving little or no margin for adjustment. Decreasing the column inside
diameter to 3 mm drops the optimum flow rate to about 1.7 to 2 mL/min,leaving plenty of margin for super- (above)-optimum flows. This lower
flow rate (compared to the use with 4.6-mm id columns) significantly
decreases the pressure drop in the extracolumn components of thesystem, such as the connector tubing. However, using both the smaller
particles and a smaller inside diameter requires significant decreases inextracolumn dispersion. Much of the standard 175-µm id connector tubing
in the Agilent analytical SFC system must be replaced with the shortest
possible lengths of 125-µm id tubing. Further, the standard 10-mm, 13-µL
flow cell must be replaced with a new 3-mm, 2-µL high-pressure flow cell.
These rather simple changes enable about 90 % of the theoretical
efficiency of the column to be observed, even at k equal to about 2 to 3.
A column of 3 by 100 mm packed with 1.8-µm RX-SIL has been shown
to produce 120 % of the efficiency of a 4.6 by 150-mm column packed
with 3.5-µm RX-SIL at three times the speed, as shown in Figure 3.4.
Efficiency, N
30,00025,00020,00015,00010,000
5,000
0

0

1

2

3

4

5

6
7
Partition ratio, k

3.0 by 100 mm, 1.8-µL column; 3-mm path length, 2-µL flow cell;37.5 cm of 0.007-inch tubing
4.6 by 150 mm, 3.5-µL column; 13-µL flow cell; 1.175 cm of 0.007-inch tubing
Figure 3.4 Measured column efficiencies. The upper curve was obtained using a
3 by 100 mm, 1.8-µL column, with a 3-mm path length, 2-µL flow cell, 37.5 cm of 0.007-inchtubing (standard heat exchangers) and 80 cm of 0.005-inch connector tubing. The lower
curve was obtained with 4.6 by 150 mm, 3.5-µm column with a 13-µL flow cell and 1.175 cm
of 0.007-inch tubing. Although the smaller column exhibits poorer peak fidelities, it produced
about 20 % higher absolute efficiencies in less than one-third of the time.

46

3.5.3
Columns of less utility

Porous shell particles have not yet been widely used in SFC, althoughseveral reports have appeared66, 68. It has been demonstrated that somelarger, superficially porous particles, can generate significantly higher
efficiencies at higher speeds than totally porous particles of the same
diameter 66. Such larger particles can rival sub-2-µm totally porous particles
for efficiency and speed, but with a fraction of the pressure drop. Suchcolumns have been shown to produce sub-2-µm-like performance in HPLC
when using 400 to 600-bar chromatographs. This is of great interest in
HPLC, but not important for SFC, since the pressure drops with totally
porous sub-2-µm particles are already similarly low.
One aspect of porous shell particles that needs further investigation is
sample capacity. In one study 66, using various polar solutes and polarphases, reduced plate heights to about 1.6 dp, but only with small injection
volumes less than 0.5 µL and on a 4.6-mm id column. The sample solvent
was methanol. In another report 67, using a C18 phase with nonpolar
solutes, higher injection volumes (up to 3 µL on 4.6-mm columns) could
be used before there was any significant loss of efficiency. The sample
solvent was, in this case, methanol/methylene chloride, but the solutes
were highly nonpolar. Another paper 68 by the same authors showed asteady decrease in efficiency with increasing injection volume, again using
C18 porous shell columns.
These anomalies appear to be related to the strong solvent effect, where
the sample solvent is stronger than the mobile phase. Injecting larger
volumes of such a strong solvent is known to distort early eluting peaks,
particularly on columns with low surface area.
A relatively small number of SFC papers have been published using theseparticles with similar results. No useful generalization about injection
volume against column inside diameter, modifier concentration, or sample
solvent can be made. However, it is fairly simple to recognize the problem
and possibly correct it by using a less-polar sample solvent. Any form ofsolventless injection, where the sample solvent is removed and the solutes
are then redissolved in the mobile phase, eliminates this problem.

47

3.6
Columns for chiralseparations

Virtually all the types of chiral stationary phases have been usedeffectively in SFC. Pirkle-type, cyclodextrin-based and amino acid-based
stationary phases have all been used fairly extensively. However, by
far the most nearly universal phases have been made with derivatizedmacrocrystaline cellulose or amylose coated on silica. Patents on severalof these highly successful phases have expired and there are now several
manufacturers producing them. This has spurred the development ofbonded versions that enables the use of a wider choice of modifiers.
The coated phases, because they were macrocrystalline, were usually coatedon particles with large pores (1500 Angstroms or more). This yielded low
surface areas and relatively poor sample capacity. Some of the other types
such as Pirkle-type phases could be bonded to silica with much smaller pores,
yielding significantly higher surface area and sample capacity. This is mostimportant in semipreparative work where throughput is the highest priority.
Analytical SFC was largely responsible for the shift from 10-µm particles
as standard to the ubiquitous use of 5-µm particles, starting 10 to 15 years
ago. In the last few years, 3-µm particles have become common. Column
length, and to some extent inside diameter, have both decreased as a result.
There are only a few reports65, 67 of the use of sub-2-µm particles for chiralseparations in UHPLC, and none in SFC. This will likely change in the future.
As in achiral analysis, the tendency in SFC is toward rapid screening,sacrificing efficiency for speed. The current tendency is to use 50 to 100‑mm
columns with 3-µm particles. Columns 150 to 250-mm long, with 3 to 5-µm
packing are more often used for trace analysis (enantiomeric excess, EE,
determinations). For more in-depth discussions, refer to Chapter 7 “Chiral
Separations”.

48

EFFECT OF MOBILE PHASE VARIABLES ON
RETENTION AND SELECTIVITY

4

SFC software provides the analyst with four mobile phase control
variables:
1. modifier concentration,
2. temperature,
3. pressure, and4. flow.
Each variable affects retention and selectivity to a different extent.

4.1
Modifier concentration

Among the mobile phase control variables, adjustment of the modifier
concentration provides the most powerful control over retention, but lessover selectivity.
As a rule-of-thumb, doubling modifier concentration halves retention.
Two examples69, 70 from the literature of the elution of families of relatively
polar compounds, hydroxysteroids and tricyclic antidepressants, are shownin panels A and B of Figure 4.1. As predicted from the solvatochromic
dye studies in Chapter 2 “The Mobile Phase”, the first small additions ofmodifier can have a large effect on retention. In the first example, thechange in retention of the hydroxysteroids above 5 % methanol is minor,
as shown in panel A of Figure 4.1. However, between the first few tenthsof 1 to 2 %, the partition ratio, k, changed from nearly 40 to less than 5.
The plot is a logarithmic plot of k against percent modifier and is nonlinear.
Such large shifts in retention with small changes in modifier concentration
make reproducibility, over time and between labs, difficult. Consequently,
such conditions are avoided. Nevertheless, such an example shows thepower of changing modifier concentration.

49

A

Partition ratio, k

14

10

8

12
10

5
4
3
2
1

B

Retention time [min]

40
30
20

6
4
2
0

2
Androsterone

4

6Cortisone81012Modifier [%]

1

0

5

10

15
20
25
Methanol concentration [%]Figure 4.1 A: Change in retention as a function of modifier concentration for cortisone
and androsterone, showing large changes with small changes in concentration at low
concentrations. B: Change in retention of five tricyclic antidepressants as a function ofmodifier concentration at higher modifier concentrations.

A more typical example is shown in panel B of Figure 4.1, where tricyclic
antidepressants were separated. In this example, the retention of all fivecompounds changes significantly between 5 and 20 % modifier. In themost extreme case, k changed from about 14 at 5 % to about 2 at 20 %.
All the curves are nearly parallel over this fairly wide range of modifier
concentration. The fact that the curves do not cross each other indicatesthat no peak reversals occurred, so changing modifier concentration tends
to have a large effect on retention but much less effect on selectivity.

4.2
Temperature

Changes in temperature tend to have less effect of retention than modifier
concentration, but can sometimes result in significant shifts in selectivity.
After the modifier concentration is varied to get all the solutes into a
reasonably short retention window, the temperature can be varied topotentially change selectivity.
Chromatograms of two sulfonamides71 at various temperatures, but withall other variables held constant, show baseline separation with theelution order 1, 2 at 28 °C, but coelution with a decrease in retention at
40 °C, then increasing retention and increasing resolution with elution
order 2, 1 above 60 °C as shown in Figure 4.2.

50

Absorbance [mAU]
0.50

1
2

0.25

80 °C

0.00
0.50
2

0.25

1

60 °C

0.00
0.50

2, 1

0.25

50 °C
0.00
0.50

1, 2

0.25

40 °C

0.00
0.50

1
2

0.25

28 °C
0.00

0

2

4

6

8

10

12

14
Time [min]Figure 4.2 Peak reversal of two sulfonamides caused by small changes in temperature.

Three additional graphical examples70, 72, 73 are shown in Figure 4.3. In thefirst example, panel A, several stimulants were separated at constant flow,
mobile phase composition, and column outlet pressure, but the temperature
was changed from 30 to 90 °C. The retention of some of the compounds
increased, some decreased and some stayed nearly the same when thetemperature was increased. Crossing curves indicate peak reversals. Note,however, that the overall change in retention was modest, particularly whencompared to the effect of modifier concentration on retention.
51

Similarly, 10 antidepressants were separated under identical conditions,
except for temperature (panel B). Again, some of the compounds increased
retention, some decreased, while others hardly changed between 30 and
60 °C. The curves are nearly horizontal, indicating only modest changes inretention, but several cross indicating peak reversals.
A third example, panel C, showing the separation of antipsychotics
demonstrates behavior similar to the other two families of compounds.
There is a telling detail. At about 6 minutes, three of the solutes coelute at
35 °C, producing a single symmetrical peak. Two other peaks also coelutenear 7 minutes. However, at 40 °C, only 5 degrees warmer, the coeluting
three peaks near 6 minutes, and the two peaks near 7 minutes were allbaseline resolved! These compounds all have the same central three-ring
structure, and differ by only a few substitutions on the rings.
Unfortunately, at 40 °C, peaks near 3 minutes and at 8.5 minutes coelute.
Raising the temperature further to 45 °C results in both the first triplet
and the doublet remaining baseline resolved. In fact, almost all thesolutes are nearly evenly spaced and well-resolved, at 45 °C, except forthe second and third least retained peaks, which merged at 40 °C andremained coeluting at 45 °C and above. Under a few conditions like this,a temperature program might separate all the compounds.

52

A

Retention time [min]
6

10

5

8

4

6

3

4

2

2

1

30

40

50

60

70

80
90
Temperature [°C]

B

Retention time [min]

0

30

40

50
60
Temperature [°C]CRetention time [min]
10
9
8
7
6
5
4
3
30

40

50

60
70
Temperature [°C]
Figure 4.3 Effect of temperature on retention and selectivity. Some compounds
increase retention while others decrease retention, causing large changes in selectivity.
A: stimulants; B: antidepressants; C: antipsycotics.

4.3
PressureWith pure CO 2, pressure is a primary control parameter. However,with modified fluids pressure tends to have much less effect on either
retention or selectivity, particularly with higher concentrations of morepolar modifiers. Again, three different families of compounds70, 73, 74,
including phenylurea pesticides, tricyclic antidepressants and stimulants
were separated at various column outlet pressures with the rest of theconditions held constant. The curves, shown in panels A, B and C of
Figure 4.4, for the various members of each family are nearly parallel andrather flat, except for the stimulants. There are a few curves that cross
but nothing like what occurs when temperature is the variable.

53

A

Retention time [min]7

B

Retention time [min]
7

6

6

5

5
4

4

3

3

2

2

1

1
100

150

200
Outlet pressure [bar]

0

100

150

200
Outlet pressure [bar]CRetention time [min]
9
8
7
6
5
4
3
2

100

200

300
Outlet pressure [bar]Figure 4.4 Effect of column outlet pressure on retention and selectivity.
A: phenylurea herbicides, note the peak reversal between peaks 8 and 9;
B: tricyclic antidepressants; C: stimulants.

Translating a method from 5-µm particles to sub-2-µm particles results
in a significant increase in column head pressure when using the smaller
particles. The pressure drop is proportional to the inverse square of theparticle diameter. Thus, at constant efficiency (from a decrease in column
length), a 1.8-µm column will have 7.7-times higher pressure drop thana column with 5-µm particles. This means that the average pressureand density of the mobile phase in the two columns will be significantly
different. This can have subtle effects on retention and selectivity as
outlined above. This is most obvious at low modifier concentrations andespecially with low column outlet pressures (less than 120 bar) andtemperatures (less than 50 °C), as shown previously for the density ofpure CO 2 in Figure 2.1, and for CO 2 and methanol in Figure 2.3. The slopesof the density against pressure curves tend to be less steep at higher
pressures, minimizing such effects.

54

The difference in average density on the two columns has been recently
introduced as a new controversy, but the concept has been well understoodfor decades. If the original work on the larger particles was performed well
above 100 bar (that is, 150 to 200 bar), the outlet pressure with the smaller
particles could be reduced to make the average pressure (and density) on
the two columns the same or at least similar.
If a problem occurs in translating a method from a large-particle column
to a smaller-particle column, take the pressure drop across the column
with the smaller particles and divide by two to yield the average pressure
drop. Increase the column outlet pressure of the column with the larger
particles until the average pressure across that column is the same as
the average pressure across the column with the smaller particles. The
density effects are nonlinear, so small additional changes in column outletpressure may be required to achieve similar selectivity.
Alternately, simply raising the outlet pressure on the column with thelarger particles tends to normalize or minimize the effect.

4.3.1
Average column density

It is difficult to calculate the average density in a column with binary fluids.
Remember that there are few systems where the density of binary fluids
is even marginally known or can be calculated. The best example ismethanol/CO 2. There is a database and an equation of state that works
over a narrow range of conditions75–77.
While the lack of data may change in the future, past history suggests thegeneration and collection of such data is unlikely with the present lack offunding for basic research in SFC.

4.4
Flow

In HPLC, changing the flow rate tends to have little effect on selectivity, but
can shorten run times provided that resolution can be traded for speed. SFC isa little more complicated, since increasing the flow rate increases the pressure
drop both in the column and in the connector tubing. Since the backpressure
regulator holds the system outlet pressure constant, the increased pressure
drop results in the pump pressure increasing. Higher pressure results inmodest increases in density and decreases in retention (lower k) beyond that
expected due simply to the higher flow rate as outlined in a previous section.
The effect of flow rate on the retention of caffeine, theophyline, theobromine
and uracil is shown in Figure 4.5. The pressure drop increased from 33 barat 2 mL/min to 147 bar at 5 mL/min. Retention decreased about 2.5-times
when flow was increased by the same amount. Since the change in retention
was proportional to the change in flow rate, the results indicate that the55

effect of the pressure drop is minimal. However, normalizing retention(actually k) to the values at 2 mL/min results in the values shown in Figure
4.6. Retention (k) decreased as much as 13 %. While this is a rather mild
effect, you should be aware of this form of behavior.
Retention time [min]
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

1

2
Caffeine

3
Theophylline

4
Theobromine

56Flow [mL/min]UracilFigure 4.5 Effect of flow rate on the retention of caffeine, theophyline, theobromine anduracil. The curves are nonlinear due to the change in density of the mobile phase, causedby the increased column pressure drop at higher flow rates. The pressure drop increased
from 33 to 147 bar (188 to 297-bar inlet pressure). Conditions 15 % methanol at 50 °C and150-bar outlet pressure. Column: 4.6 by 150 mm, 3.5 µm RX-SIL.
Normalized retention time [min]
1.2
1.0
0.8
0.6
0.4
0.2
0

2

3
Caffeine

Theophylline

4
Theobromine

56Flow [mL/min]UracilFigure 4.6 Normalized retention times from Figure 4.2 showing that retention decreased
as much as 13 % when the flow rate was increased from 2 to 5 mL/min due to the increasein the density of the mobile phase through the column. The pressure drop increased from33 to 147 bar (188 to 297-bar inlet pressure). Other conditions were held constant at 15 %methanol, 50 °C, and 150-bar outlet pressure. Column: 4.6 by 150 mm, 3.5 µm, RX-SIL.

56

This data was collected using a 4.6 by 150-mm column, packed with
3.5-µm RX-SIL particles (Pin = 188 to 297 bar). Columns packed with
sub-2-µm particles are likely to have even larger deviations, due to changes
in pressure drops. However, at near optimum flow rates, column pressure
drops seldom exceed 250 bar (less than 400-bar outlet pressure) and, atmodest modifier concentrations ΔP values are often about 50 to 100 bar,in a similar range to those in Figure 4.4.

4.5
Generalizations on effectsof control variables on
retention and selectivity

Modifier concentration is the most effective way change the retentionof peaks substantially without changing columns. Doubling modifier
concentration roughly halves retention. Modifier concentration should be
the first variable to use to move peaks into an appropriate retention window.
If peaks are inadequately resolved, a small change in temperature canoften improve resolution between specific pairs of peaks. A change of± 10 degrees usually indicates whether selectivity improves. If it does,
try additional increments of 5 to 10 degrees in the best direction, for
optimization.
Pressure should be considered as a secondary control variable, but asignificant pressure step (that is, 25 to 50 bar) might produce subtle
changes in retention or selectivity, or both. Flow rate should be set tonear the optimum until resolution can be optimized or maximized for all
the solutes. The flow could then be increased until the resolution is justacceptable.

57

5

METHOD DEVELOPMENT
Chapter 1 “Introduction to Supercritical Fluid Chromatography” wasintended to give you a broad overview of what SFC is and what SFC isnot. Chapter 2 “The Mobile Phase” familiarized you with the various
aspects of the mobile phase characteristics such as the low polarity ofpure CO 2, the large impact on solvent strength when polar modifiers areadded, how the solvent strength is a nonlinear function of concentration,and how many solutes require highly polar additives in the mobile phaseto elute. Chapter 3 “The Stationary Phase” explained the size and shapeof the appropriate column hardware, as well as commented on therelative polarity and interactions of the actual stationary phases available.
Chapter 4 “Effect of Mobile Phase Variables on Retention and Selectivity”
described the effect of the various mobile phase variables on retention
and selectivity, showing that modifier concentration has, by far, thebiggest effect on retention but much less effect on selectivity comparedto the other instrumental mobile phase set points. Temperature has muchless effect on retention but can have a surprisingly significant effect on
selectivity. Pressure is simply a secondary control variable, having only aminor impact on either retention or selectivity. Flow has even less effecton selectivity and only through changing the pressure drop across thecolumn. It is now time to combine all this background information into arational approach to method development.

5.1
Matching soluteand phase polarity

The old adage of like-dissolves-like, meaning a solvent with similarfunctionality to a solute is likely to be a good solvent, while a solventwith different chemical characteristics is likely to be a poor solvent.
For example, salt and water compared to salt and heptane. This adage
can be extended to chromatography by choosing stationary and mobile
phases that have similar chemical characteristics to the solutes of interest.
CO 2 in some ways can be viewed as a relatively inert diluent, when used
to separate relatively polar solutes. The modifiers polarity and chemicalfunctionality primarily dictates the characteristics of the mobile phase.
Specifically, there needs to be a strong interaction between the solutes
and the stationary phase, and between the solutes and the modifier.
Such strong competing interactions result in rapid equilibration. Weakinteractions are ill-defined, and as a consequence, result in longequilibration times and poor retention time reproducibility.

58

Various solute functionalities are listed across the top of Figure 3.2.
Below the solutes are several mobile and stationary phases, with arrows
indicating the appropriate ranges of polarities for each phase. These areapproximations, but provide good starting points. Finding an appropriate
solute family, then dropping a vertical line indicates several possiblecombinations of mobile and stationary phases likely to produce a viable
separation.
Acids and bases will require polar stationary and mobile phases, suchas bare silica, diol or amino. Solutes that are capable of strong hydrogenbonding should be separated using hydrogen-bonding stationary andmobile phases. Solutes that can ionize in aqueous media, depending on
pH, will likely need a polar modifier, and an even more polar additive.
The additive should be chosen to suppress such potential ionization.
At the other extreme, solutes with minimal polar functionality and longhydrocarbon chains, or both, may be best separated with a nonpolar
stationary phase such as C18, and a relatively non-polar modifier. For suchseparations, it is unlikely that an additive will be required.

5.1.1
Additives

In order to choose an additive, another analogy that may be helpful isthe acid-base shake-out test. Bases partition into aqueous media when incontact with a nonmiscible organic solvent, under conditions where thepH in the aqueous phase allows the base to ionize. However, at pHs in the
aqueous phase that suppress ionization, the same base will likely partitionout of the aqueous phase into the organic phase. Think of the stationary
phase as the aqueous phase, and the mobile phase as the organic phase.
The additive should be such that it suppresses ionization of the basic solute,
keeping it from adsorbing onto the stationary phase.
True optimization may require trying several different additives. However,most similar additives will produce similar effects, and it is usually not
worth the extra effort, except in extreme circumstances where nothingmuch seems to work.

59

One of the most obvious cases for the need for an additive involves primaryor secondary aliphatic amines. In such cases, it may be worthwhile tostart with an ethylpyridine column to attempt to avoid the use of a basic
additive, particularly when using a detector like a mass spectrometer.
However, ethylpyridine columns tend to be much more retentive than theclassic phases (cyano, silica, diol, amino) and often require a relatively
higher modifier concentration. This decreases the diffusion coefficientsof the solutes, slowing the optimum linear velocity, and increases
viscosity, increasing pressure drop. Alternately, a strong base, such as
triethylamine, added at about 0.1 % in the modifier, will probably yieldfull column efficiency while allowing a much lower modifier concentration(with higher speed and efficiency).
Amphoteric compounds are usually best dealt with using an additive suchas ammonium formate or ammonium acetate, often supplemented witha much stronger acid or base. Such additives tend to suppress both acidic
and basic ionization of such solutes, or form neutral ion pairs.

5.2
Polarity windows

The phases should create a polarity window bracketing the solutes.
If the sample consists of a broad range of polarity, size and functionality,
the difference in polarity of the phases should be wider, creating awide window of polarity that can be narrowed through composition
programming, over a wide range of composition. If the sample consistsof a family of closely related compounds with similar polarity, the phases
should be closer together and similar to the solutes.

5.3
Getting started

It is important to define the goals of the separation at the beginning.
Optimization may take different paths if, on the one hand, the goal is thefastest separation with some minimal resolution between peaks, or, on
the other, trace analysis. One goal should always be to develop a method
wasting as little time as possible. Toward that end, always try to start withhigh concentrations of appropriate standards and strong elution conditions
to avoid as much uncertainty as possible. If a specialty detector is required,such as an evaporative light scattering (ELS) detector, try to separate
developing the separation method, and optimizing the detector. The ELS
detector is usually used when the solutes have poor chromophores.
Use solutes as near as possible to the solutes of interest, but that do havea chromophore. You can then use a UV detector to help optimize the
ELS detector using the solute with a chromophore, which should produce
operating conditions nearly appropriate for the solutes of interest.

60

5.4
Polar solutes

The next sections describe a step-by-step process toward developing an
optimized method. In Figure 5.1 shows a decision tree for polar solutes
that follows these guidelines.
Initial conditions

Try additives
Acid for acids
Base for bases
AmphotericIon pairing
Change stationary phase
Add 10 % water to modifier
Lower surface areaNO

PEAKS?

YESRETAINED?

NO

YES
Increase modifier concentration
Increase program rate
Increase modifier polarity
Increase pressure
Decrease pressure
Change stationary phaseNO

Decrease modifier concentration
Decrease program rate
Decrease modifier polarity(for example, MeOH to IPA)
Drop pressure
Increase temperature
Change stationary phase

FAST RUN
TIME?

YES

ADEQUATE
SELECTIVITY?

NO

Change temperature 10 degrees
Increase pressure 50 bar
Change modifier class
Change stationary phase

YES
Longer column
Smaller particles
Decrease modifier concentrationNO

ADEQUATERESOLUTION?

YES
Final method
Figure 5.1 Decision tree for method development using polar solutes.

61

Highly polar solutes such as polyfunctional acids or aliphatic amines, arefound in the upper right of Figure 3.2. Dropping a vertical line indicatesthat any of the polar stationary phases could be used. The line alsosuggests methanol and an appropriate additive will probably be required.

5.4.1
Is an additive needed?

The first question to be addressed with polar solutes is whether themobile phase needs to include an additive. Choose a polar stationary
phase such as bare silica. Use 40 % methanol in CO 2 at 50 °C, 150-bar
outlet pressure at 1.5-times the optimum flow rate. Most solutes should
elute within a few minutes under these strong conditions. After the first
injection, are there symmetrical, high-efficiency peaks?

5.4.1.1
Peaks?

If there are appropriately shaped peaks, without tailing, no additive isneeded and you should proceed to optimize the modifier concentration.

5.4.1.2
No peaks?

If nothing elutes or peaks tail badly, an additive is needed. Usually 0.1 to
0.2 % added to the modifier is adequate. If the solutes are bases, add astrong base such as ammonia or triethylamine. If the solutes are acids, add
a strong acid such as trifluoroacetic acid. Many solutes are multifunctional.
For such solutes, an amphoteric additive such as ammonium formate
or ammonium acetate may be appropriate. On the second injection, withadditive included, do symmetrical, high-efficiency peaks emerge?

5.4.1.3
Peaks!

In more than 90 % of the cases, the addition of an additive produces
appropriately shaped peaks. If, with the additive, peaks emerge, proceed
to optimizing the modifier concentration in exactly the same was as if noadditive were required. It only took a few fast isocratic runs to answer oneof the more important questions about polar solutes.

5.4.1.4
Still no peaks?

If no peaks emerge or those that do have poor peak shapes, try ion pairing(acidic additive for basic solutes, and vice versa). If still no peaks emerge
or peak shapes are poor, the success or failure of the separation will dependon finding the right additive or combination of additives. The last thing to
try is to add 10 % water to the methanol, along with additives.
An alternative is to switch to one of the newer phases such as ethylpyridine
for bases, or Premier for acids. These stationary phases may enable theelution of the solutes even without additives.

625.4.2
Optimizing modifier
concentration – methanol

Methanol is one of the strongest solvents that is completely misciblewith CO 2. As such it is often too strong a solvent for many solutes, causing
them to coelute near the column hold-up time, at 40 % methanol.
Using the rule of thumb that halving modifier concentration roughly doubles
retention times, progressively decrease the modifier concentration bya factor of two (to 20 %, then 10 %, then 5 %) and observe the retention
times of all the peaks. By slowly changing the concentration of modifier, it
is fairly clear if peaks reverse elution order. On the other hand, it only takes
a few isocratic runs to characterize what further steps might be necessary.
If the solutes display a wide range of retention, a composition gradient isin order. If the peaks are significantly retained but tend to bunch together
with similar retention times, an isocratic method is probably in order.
Develop either, such that the run time is reasonably short, but do not expect
to adequately resolve all the peaks using modifier concentration alone.
When the modifier composition is near optimum and all the peaks areat least partially resolved, proceed to using the column temperature tomodify selectivity.

5.4.2.1
Methanol still too strong

If, at 5 % methanol, some of the peaks are still nearly unretained withinadequate resolution, switch to a weaker solvent such as either ethanol
or isopropanol. In most cases, one of the alcohols will produce a viable
separation. In extreme cases, switching to a different solvent family may
be appropriate. If the alcohols are inappropriate, the initial evaluation of
the sample was probably wrong.

5.4.3
Using temperature tochange selectivity

As pointed out in Chapter 4 “Effect of Mobile Phase Variables on
Retention and Selectivity”, temperature tends to have only modest effecton retention but can have significant effect on selectivity. Once the modifier
concentration has been optimized to produce a reasonably short run time,changing the temperature by ±5 to 10 degrees often changes the relative
retention of difficult pairs of peaks. If such a change in temperature
improves the resolution of difficult pairs, try a larger change in temperaturein the same direction.

5.4.4
PressureWith polar solutes, pressure is a secondary control variable and isunlikely to change either retention or selectivity significantly, particularly
at higher modifier concentrations. However, it is always worth changing
the pressure in 25 or 50-bar increments, if a separation is not optimized.
It is simple to try, takes little time, and may actually be useful. Changing
63

the outlet pressure changes the density of the fluid, which can havesome desirable effects. UV detector noise tends to be lower at higher
pressures. Therefore, it is usually advisable to perform trace analysis at
higher pressures.

5.5
Low polarity solutes

Many low polarity solutes are amenable to SFC. Many are complex natural
products, or homologous series. A few typical lower polarity solute families
are listed on the top left of Figure 3.2. Choosing one of the solute types
and dropping a vertical line indicates appropriate phases. For compoundswith long hydrocarbon chains, the most appropriate stationary phaseis probably C18, but others such as C8, phenyl and cyano, may also be
useful. The polar stationary phases are likely to produce little retention
and distorted peaks, since there will be weak interactions between thepolar stationary phases and the nonpolar solutes.
The use of pure CO 2 is unlikely to produce good peak shapes at low
temperatures and pressures. The most reliable way to develop arobust method for nonpolar but heavy solutes is to use a modified fluid
either isocratically or with composition programming. Gradients are still
programmed from low to high modifier concentrations.
A relatively lower polarity modifier such as isopropanol is a good starting
point. Since low polarity solutes tend to not hydrogen bond, a nonalcohol
for modifier may be better. It is more common to use acetonitrile withlower polarity solutes, compared to higher polarity solutes. Chlorinatedsolvents are avoided, although they can be used. With such solutes, it isunlikely that an additive would be needed.

5.5.1
Optimizing modifier
concentration

With complex samples, it is likely that a gradient will be needed. However,
it is always good to minimize confusion by trying a quick run with a highisocratic modifier concentration to make sure that everything elutes. As
with the polar solutes, start with 40 % modifier, 50 °C and 150 bar.
It is highly likely that everything will elute rapidly. Halving modifier
concentration should double retention. If 5 % isopropanol is still too
strong a solvent, 10 % isopropanol can be added to heptane and the
mixture used as the modifier.

64

5.5.2Temperature and pressureWith polar solutes, small changes in temperature can have significant
impact on polar-polar interactions between the solutes and the phases,
causing selectivity changes. With much less polar solutes, interactions arelikely to be dominated by Van der Waals forces and temperature tends tohave less impact on selectivity.
The biggest effect of temperature and pressure will probably be throughchanging the density of the mobile phase. Retention is likely to increase at
higher temperature and lower pressures (lower densities). Retention is also
likely to be more a function of density, especially at low concentrations ofa lower polarity modifier.

5.6
Multivariate methods

The method development approach outlined above probably seems
old‑fashioned since it follows a univariate approach. Further, you need tomake some near real-time decisions, or waste time and materials. As will
be seen with the optimized separations in later chapters, multivariate
methods will have a difficult time finding the optimum conditions for many
separations. Such multivariate approaches are much less likely to produce
an optimized separation, compared to a more detailed univariate method
development optimization. This is true with changes in temperature, which
often causes peak reversals. However, the high speed of SFC makes it
possible to rapidly develop such methods, in a much shorter time comparedto HPLC.

65

6

ACHIRAL SEPARATIONSIn this chapter, several case studies are presented on how specific methods
were developed. The case studies chosen were all rather difficult, withunusual peak reversals, with odd responses to changes in mobile phaseparameters. It is hoped that the straightforward resolution of these difficult
mixtures, by following the recommendations in the previous chapters of
this monograph, will help you in developing your own methods. It should
be stressed that these examples are not typical, and do not represent
the norm. Most separations are much easier to optimize, for the specificanalytical requirement. You should also remember that there are different
criteria for what represents an optimized separation. All separations area trade-off between speed, resolution and sensitivity. In the following
examples, all these issues are addressed.

6.1
Case Study 1 – A typical
low-polarity sample

This first example is fairly atypical of the sorts of samples commonly
separated using SFC over the last 20 years. However, such applications weremuch more common in the earlier days of SFC. This application hints at thegreat potential for the technique in fields other than pharmaceutical research.
The separation of paprika oleoresin is a good example of a relatively
nonpolar family of compounds78. This commercial product is an extract from
paprika pepper, which in recent years has replaced most artificial red dyes
in processed foods, and is important economically.
There are only two primary colored pigments present: capsanthin, andcapsorubin. Each has two hydroxyl groups, one on a ring at each end of
a terpenoid chain, as shown in Figure 6.1. These hydroxyls form a large
number of mono- and di-esters with a range of fatty acids. Samples tend
to contain about 70 compounds, mostly the di-esters. The esters do notsignificantly change the color of the parent compound, but may help tostabilize it.

66Capsanthin
OH
HO

O

Capsorubin
O
3

6
5

OH

O

OH
Figure 6.1 The red color found in paprika comes directly from two ocompounds capsanthin,and capsorubin. They are mostly present as mono- and di-esters formed with various fatty
acids. Only a relatively few more polar mono- and di-alcohols exist in the peppers.

These esters are nonpolar, due to the long hydrocarbon tails on thefatty acids. You can find esters at the top and to the left of Figure 6.2.
The large vertical arrow indicates appropriate mobile and stationary
phases. The structure of the solutes suggests the use of a similar longhydrocarbon tail for the stationary phase, such as on a C18 phase, alongwith a relatively nonpolar modifier in CO 2, such as isopropanol. Threedifferent C18 columns with slightly different selectivity (Agilent) wereused, each with 1.8-µm particles.

67

Proteins
DNA/RNA
Inorganic ionsZwitter ions
PeptidesBenzoic acids
Hydroxy acids
Polyacids
Aliphatic amines

AmidesSteroids
Ring nitrogens
Alcohols
SugarsAnalines

Hydrocarbons
Waxes
Esters/ethers
Fatty acids
Terpenoids
Ketones/aldehydes

INCREASING POL ARIT Y
MOBILEPHASE

CO2Ethyl acetate/Acetone/Aetonitrile/CO2
Isopropanol/CO2

Ethanol/CO2

Methanol/CO2
Methanol/
Water

Additives
STATIONARY
PHASE

C18, C4, C1
Hexaphenyl
Cyano
SilicaDiol
AminoEthylpyridine

Figure 6.2 Paprika oleoresin contains a large number of mono- and di-esters of capsanthinand capsorubin. The arrow indicates appropriate stationary and mobile phases for esters.

Separations of the same sample using an identical gradient of 5 to 20 %isopropanol in CO2 on the three C18 columns produced similar but not
identical chromatograms with subtle differences in resolution, as shownin Figure 6.3. The peaks were well retained and reasonably spaced.
In contrast, the same sample separated on more polar stationary phases,
but using the identical gradient and conditions produced mostly poor
peak shapes with retention shifted to shorter times, as shown in Figure
6.4. Clearly the interactions between the solutes and these more polar
stationary phases are weaker than the interactions with the C18 phases.

68Absorbance [mAU]
300
200
100
0

2

4

6

8

2

4

6

8

2

4

6

300
200
100
0

400
300
200
100
0

8
Retention time [min]Figure 6.3 Separation of paprika oleoresin on three different C18 columns, all 3 by 100 mm,
1.8 µm (all from Agilent), using a gradient of 5 to 20 % IPA in 6.5 minutes.

69

SB-C18

Absorbance [mAU]
400
300
200
100
0
2

4

6

8

400

SB-CN

300
200
100
0
2

4

6

8

RX-Sil

400
300
200
100
0

2

4

6

8

Epic Diol HILIC

400
300
200
100
0

2

4

6

8
Retention time [min]Figure 6.4 The same paprika oleoresin and condition but separated on more polar columns,
compared to an SB-C18 column at the top. All the columns were 3 by 100 mm, 1.8 µm.
The nonpolar analytes are poorly retained, particularly on the bare silica and diol phases.

Other modifiers were substituted for isopropanol, using the same gradientand conditions with a ZORBAX SB-C18 column, as shown in Figure 6.5.
Acetonitrile, and acetone, are more polar, while ethyl acetate is equally
polar compared to isopropanol, according to Snyder’s P’ scale outlinedin Table 2.1, and all three produced shorter retention of the sample.
The observed retention order was; acetonitrile (6.2), ethyl acetate (4.3),
acetone (5.4), isopropanol (4.3). The numbers in parenthesis are the P’solvent strength values of each solvent from the table. Obviously, theP’ values do not correctly predict the retention order for this type ofsample. The Hildebrand scale was even worse with the retention order;acetonitrile (0.5), ethyl acetate (0.38–0.48), acetone (0.47–0.53),
isopropanol (0.62).

70

Absorbance [mAU]
400

Isopropanol

300
200
100
0

0

2

4

6

8

10

Acetonitrile

400
300
200
100
0

0

2

4

6

8

10

Ethyl acetate

400
300
200
100
0

0

2

4

6

8

10

Acetone

400
300
200
100
0

0

2

4

6

8
10
Retention time [min]Figure 6.5 Separation of the same paprika oleoresin with the same conditions except
substituting other modifiers. All the higher polarity modifiers produced shorter retention thanthe more polar IPA.

In some ways, this separation resembles reversed phase HPLC. Thereis greater retention on the less polar stationary phases, and the morepolar modifiers decrease retention (more or less). However, in all cases a
gradient of increasing concentration of the polar modifier in the less polar
CO 2 was used. Thus, the mobile phase polarity increased throughout thegradient, in contrast to reversed phase HPLC, where the mobile phasepolarity decreases throughout a gradient.

71

6.2
Most separations performed by SFC over the last decade have not been
Case study 2 – A moderately performed with relatively nonpolar solutes and C18 columns, such as in
the previous example. Instead, most of the work with SFC has been donepolar sample
on small drug-like molecules.

While the generalizations outlined in Chapter 4 “Effect of Mobile Phase
Variables on Retention and Selectivity” are a good guide as to what
to expect, they are only generalizations. Caffeine, theophylline andtheobromine have often been used as test solutes in SFC since they are
relatively safe and require no additive in the mobile phase. They all havethe same backbone with four-ring nitrogens. They differ from one anotherby the position of single methyl substitutions on the nitrogens, as shownin Figure 6.6. Since they are similar in structure, they are good test probes
for testing the generalizations. Uracil was added since it has a relatively
different structure and may behave differently to changes in conditions.
The initial goal was to develop the fastest separation with a resolution
greater than three between all the pairs.
H 3C
O

O
N

CH3
N

N
N
CH3

Caffeine

H 3C
O

O
N

H
N

N
N
CH3

Theophylline

O
HN
O

CH3
N

N
N
CH3

Theobromine

O
NH
N
H

O

UracilFigure 6.6 Structures of caffeine, theophylline theobromine and uracil.

The solutes were all moderately polar, requiring more polar mobile phaseand stationary phases, compared to the previous example. The nitrogens
are only present in the rings and unlikely to require an additive. Compounds
containing conjugated ring nitrogens are located approximately in the
middle of the polarity scale, at the top of Figure 6.7. A bare silica column,
and methanol as modifier were selected from the options available. The
column used for the experiments was 4.6 by 150 mm, packed with 3.5-µm
RX-SIL. Silica usually yields high efficiency, with minimal complications.

72

Proteins
DNA/RNA
Inorganic ionsZwitter ions
PeptidesBenzoic acids
Hydroxy acids
Polyacids
Aliphatic amines

AmidesSteroids
Ring nitrogens
Alcohols
SugarsAnalines

Hydrocarbons
Waxes
Esters/ethers
Fatty acids
Terpenoids
Ketones/aldehydes

INCREASING POL ARIT Y
MOBILEPHASE

CO2Ethyl acetate/Acetone/Aetonitrile/CO2
Isopropanol/CO2

Ethanol/CO2

Methanol/CO2
Methanol/
Water

Additives
STATIONARY
PHASE

C18, C4, C1
Hexaphenyl
Cyano
SilicaDiol
AminoEthylpyridine

Figure 6.7 The nitrogens in the xanthenes and uracil are all in rings, making them only
moderately polar. They are unlikely to require an additive.

Methanol is the most polar modifier that is completely miscible with CO 2.
If there is any question about whether a solute will elute, the obvious first
choice for modifier is methanol. Further, it is inexpensive, only moderately
toxic, and has low viscosity, as outlined previously.
A generic temperature (50 °C) and pressure (150 bar) were also arbitrarily
chosen. The flow was set to about 1.5-times optimum (at 4 mL/min) to
speed up method development.

73

6.2.1
Fastest chromatogram withresolution greater than 374

As outlined previously, the modifier concentration is the most important
mobile phase parameter for changing retention. Therefore, the first variableevaluated was the methanol concentration. The first run was made using ahigh isocratic concentration of methanol, without additive, primarily to see
if an additive was needed. If peaks did not elute or eluted with a poor peak
shape, an additive would be required. In this case, 30 % methanol eluted
all the peaks with high efficiency in a short time (about 1.25 minutes), so
no additive was needed. However, one of the peaks was a shoulder onanother, as shown in the top chromatogram of Figure 6.8, indicating themodifier concentration should be lowered.

Absorbance [mAU]
160

160

120

80

80

0

40
0
160

0

2

0

0.5

4

0

40
0
0

2

1.5

6

0

0.5

4

2

8
2 1

80

80

30 %

1

160

120

160

2 1
4 3

4

1.5

2

8

10

80

12

15 %

2 1

120

12

20 %

3

1

6

10

4 3

40
0
160

0

1

2

3

4

5

6

10 %

1,2

120
80

43

40
0
0

1

120

2

3

12

80

4

5

6

9%

3,4

40
0
120

0

1

2

3
1

80

4

5

6

7.5 %

2

40

3

4

0
0

1

2

40

1

3

4

5

5%

2
3

20
0
0

2

4

6

6

8

4

10
12
Retention time [min]Figure 6.8 The effect of modifier concentration on the separation of caffeine, ttheophylline
theobromine and uracil at 4 mL/min, 50 °C, 150-bar outlet pressure. , RX-SIL column, 4.6 by
150 mm, 3.5 µm. Note the unusual multiple peak reversals.

75

Progressively decreasing the modifier concentration resulted initially inslightly better resolution (at 20 %), but then a loss in baseline resolution
between the first two peaks (at 15 %), followed by coelution of the firstpair (at 10 %), with loss of resolution between the last pair. This wasfollowed (9 %) by a peak reversal of the first pair, and coelution of thelast pair. At lower concentrations (7.5 and 5 %, the resolution within boththe first and last pairs progressively increased. All the chromatograms in
Figure 6.8 had a time base of 6 minutes, except at 5 % where the timebase was 12 minutes.
Superficially, the separations at the lowest modifier concentration (5 %)appeared to give the maximum resolution and could form the basis offurther optimization. However, if the goal of the separation was thefastest separation, the time between the two pairs was excessive.
The chromatogram collected with 7.5 % methanol was only half as longwhile maintaining better than baseline resolution. Therefore, 7.5 %methanol at 4 mL/min, 50 °C and 150-bar outlet pressure was initiallychosen for further optimization.
Generally, changing temperature tends to have only modest effecton retention, but can often cause significant changes in selectivity.
Attempts to improve resolution through changing selectivity, by changing
column temperature were unsuccessful in this case, as shown in Figure
6.9. Increasing the temperature from 50 to 60 °C resulted in improved
resolution, but only at significantly longer retention times. At 10-degrees
lower temperature (40 °C), the baseline resolution of both the first andlast pairs was lost. Decreasing temperature a further 10 to 30 degrees
resulted in both pairs coeluting as shown in the top chromatogram. Clearly,
the different compounds are responding differently to these small changes
in temperature. Selectivity is changing, but, in this case, not in a helpful
way for method development.

76

Absorbance [mAU]
100
80
60
40
20
0
-20
1
0
80
60
40
20
0
-20
80
60
40
20
0
-20
80
60
40
20
0
-20

30 °C
1,2
3,4

2

3

4

5

6

7

40 °C

1 2
3 4

0

1

2

3
1

4

5

6

7

50 °C

2
3 4

0

1

2

3

4

5

6

60 °C

1

2
3

0

1

2

7

3

4

5

4

6
7
Retention time [min]1 =6.9Caffeine
Theophyllineon retention
3 =Theobromine
4 =forUracilFigure
The effect2of= temperatureand selectivity
caffeine, ttheophylline
theobromine and uracil, at 4 mL/min of 7.5 % MeOH, 150-bar outlet pressure.

Pressure is considered to be a secondary control variable, but can havemodest effects on retention and selectivity. Returning to 50 °C and150 bar, the column outlet pressure was varied to observe the effectof column outlet pressure on retention and selectivity. At progressively
lower outlet pressures, resolution degraded. At 120 bar, the last two
peaks coeluted, but at 100 bar they reversed elution order, as shown in
Figure 6.10.

77

Absorbance [mAU]
80
60
40
20
0
0

1

250 bar
1

2
4

2

80
60
40
20
0

3
1

3
4

5

1

2

80
60
40
20
0

7

200 bar

2
4

0

6

3

4

3
5

6

7

150 bar

1 2
4 3
0

1

2

80
60
40
20
0

3
1

4

5

6

7

120 bar

2
3,4

0

1

2

60
40
20
0

3

4

5

6

100 bar

1 2
3
0

1

2

3

7

4

5

4

6
7
Retention time [min]1 = Caffeine 2 = Theophylline 3 = Theobromine 4 = Uracil
Figure 6.10 Effect of pressure on the retention and selectivity of caffeine, ttheophylline
theobromine and uracil. Conditions: 4 mL/min of 7.5 % MeOH at 50 °C.

At higher outlet pressures, resolution within the two pairs improved
significantly, while retention decreased; both desirable characteristics.
The result using 4 mL/min of 7.5 % methanol at 50 °C and 250 bar
appeared to be near optimal with a run time of about 5 minutes.
This is an unsatisfactory optimization since there is still a rather large
time gap between the second and third eluting peaks. Re-evaluating
Figure 6.8, notice that at 20 % methanol, all the peaks were nearlybaseline resolved in just over 1.25 minutes. This is substantially faster78

than the optimized result with 7.5 % methanol. However, this set of
conditions was not initially selected for further optimization, since it isfairly obvious that subtle changes in modifier concentration are unlikely
to result in significant improvement in resolution. This is because theresults at both lower (15 %) and higher (30 %) concentrations produce
worse resolution due to the reversal in elution order of two of the peaks
over this range. Thus, you must rely on temperature and pressure to try
to further improve the separation, using this starting point.
It was found that when using this higher modifier concentration (20 %),the effect of both outlet pressure and temperature changed, probably
because the modifier concentration becomes even more dominant overthe density of the mobile phase. Resolution was improved by decreasing
the column outlet pressure (not shown). Decreasing flow rate also
improved resolution, particularly of the first pair, probably by decreasing
the pressure drop and density gradient across the column.
Surprisingly, temperature had almost no effect on either retention or
selectivity under these conditions. Chromatograms collected at 2 mL/minof 20 % methanol at 100-bar outlet pressure, between 30 and 60 °C, arepresented in Figure 6.11. Compared to Figure 6.9, they show remarkably
little change in either retention or selectivity between 30 and 60 °C. Overall
resolution is better, with a run time reduced to 3.25 minutes. The optimum
temperature was the lowest tried, whereas the previous optimization foundthe highest temperature was best. Similarly the lowest pressure gave
the best separation whereas, in the previous optimization, the highest
pressure gave the best result.

79

Absorbance [mAU]
160

30 °C

2

1

120
3

80

4

40
0
0

1

160

2

3

5

40 °C

2

1

120

4

80

3

4

40
0
0

1

2

160

3
1

120
80

4

5

50 °C

2
3

4

40
0
0

1

2

160

3

4

5

60 °C

1 2

120
80

3

4

40
0
0

1

1 = Caffeine

2 = Theophylline

23
3 = Theobromine

45
Retention time [min]4 = Uracil

Figure 6.11 The effect of temperature at 2 mL/min of 20 % MeOH and 100-bar outletpressure. Compared to Figure 6.9, the lack of change in selectivity is surprising.

A few minor adjustments of conditions produced a separation optimized
for speed with resolution greater than 3, with a run time of less than
2 minutes, using 3.5 mL/min of 20 % methanol at 60 °C and 100 bar, as
shown in Figure 6.12.
80

Absorbance [mAU]
160
140

Theophyline

120

Caffeine

100
80

Uracil60
40

Theobromine

20
0
-20
-40

Rs = 3.16 2.97
0

0.5

1

6.85
1.5
2
Retention time [min]Figure 6.12 Fastest separation with resolution near 3 for all pairs. The numbers under the
peaks are the resolutions between the peaks. Conditions: 3.5 mL/min of 20 % MeOH, 30 °C
and 100 bar.

6.2.2
Trace analysis

Since all separations involve a trade-off between speed, resolution, andsensitivity, it is important to define the objective of the analysis. The fastest
separation with resolution about or greater than 3 was obtained at
3.5 mL/min with 20 % methanol in CO2 at 30 °C and 100 bar, as shown in
Figure 6.12. However, if the goal is trace analysis, the primary requirement
is the highest sensitivity with adequate resolution, making speed a secondary
consideration.
The minimum resolution for trace analysis is usually considered to begreater than 6. UV noise is typically highest at the lowest pressures.
The chromatogram in Figure 6.12 was collected at the lowest pressure,with the highest noise. Further, from the method development experience,
it appears that it is unlikely resolution can be increased to greater than 6from this starting point. These two facts suggest that the chromatogramwith the highest speed is unlikely to be the best for trace analysis.
Another standard of the same solutes was prepared, in which the second
peak was at the trace level while the first peak was a major component,
to demonstrate a typical scenario in trace analysis. In this situation, theminor component is likely to be in the tail of the main component (tailingis failure to properly return to the baseline, as with a Gaussian peak).
The potential presence of a tail is the basis for requiring such high resolution
for trace analysis. So far, the greatest resolution between the first two
peaks was obtained using 5 % methanol, at 50 °C and 250 bar, as seen in
the lower chromatogram in Figure 6.8. These conditions were used as thestarting point for optimization for trace analysis. Decreasing the modifier
81concentration increases retention time, but also increases resolution,
which is in the desired direction. It was also found that a slight increasein temperature to 60 °C, further increased resolution between the firsttwo peaks. The best separation for trace analysis, related to the first two
peaks, was found at 5.5 % methanol at 4 mL/min, 60 °C and 250-bar
outlet pressure, as shown in Figure 6.13.
Absorbance [mAU]
1.0
0.8
0.6
0.4
0.2
0
0

2

4

6

8

10
12
Retention time [min]Figure 6.13 Optimization for trace analysis. The resolution between the first 2 peaks wasincreased to 8.68. The first peak is about 800 mAU while the second trace peak is about
0.8 mAU. Thus, the smaller peak is about 0.1 % of the larger peak, and has an S/N of about
60–80. The LOQ with S/N greater than 10 is estimated at 0.013–0.017 %. Conditions:
4 mL/min, 5.5 % MeOH, 60 °C, 250 bar.

The primary component was the first peak, which was about 800 mAU
high. The second peak elutes in the tail of the main peak, which is usually
undesirable. However, the resolution between the main peak and thefollowing trace component was 8.68, which enables easy quantification.
The trace component is about 0.8 mAU high or 0.1 % of the main peakyet it has signal-to-noise between 60 and 80.The limit of quantification(with signal-to-noise greater than 10) is estimated at 0.013 to 0.017 % of
the main component, which is nearly an order of magnitude better thantypically required for trace analysis.

82

In this case, resolution between the first two peaks is probably excessive
to requirements. Increasing the initial modifier concentration slightly
should decrease resolution from 8.68 to closer to 6, while decreasing run
time. Further, after the critical pair elute, the composition could then be
programmed to higher values, to more rapidly elute the well-resolved
other pair.
Separations can be optimized in several ways. The final conditions dependvery much on where you start. At low modifier concentration, changes inboth temperature and pressure created multiple peak reversals. At higher
modifier concentrations, no peak reversals were observed with changes
in temperature or pressure. Programming from low to high modifier
concentrations at any fixed temperature and outlet pressure is likely toresult in mixed results (variable peak reversals) depending on the rate ofprogramming. The number of peak reversals, observed with these solutes,
is rather unusual, but it is important for the user to understand what is
possible. Most separations are much more straightforward.

6.3
Case study 3 –Sulfonamides

The sulfonamides are an older family of antibiotics, still widely usedin humans. However, most are used today in veterinary medicine.
Many different sulfonamides continue to be used, but they have beenbanned, as a class, as residues in food products. The structures of ninesuch compounds are presented in Figure 6.14. While one side of allthese compounds is identical, the other side contains a wide range offunctionalities.

83

O

H O
N
S
N O

NH2

H2 N

Sulfamethoxazole (SMO)

H2N

CI

N
N

H2N

Sulfamethazine (SMZ)
H2N

O
O O
S
N
H

O O N
S
N N
H

N
N

Sulfaquinoxaline sodium salt (SQX)

O

O O
S

N N

NH

NH2

Sulfachloropyridazine (SCP)

H2N

O
O N
S
HN
N

H2N

Sulfadimethoxine (SDX)

O S

N N
N
H

S

N

Sulfapyridine (SPD)

Na
S N
O O

O

O O
S
N
H

Sulfadiazine (SDZ)

O O
S

CH3

Sulfamethizole (SMT)

H2N

N
N
H

N

CH3

Sulfamerazine (SMR)

Figure 6.14 Structures of the nine sulfonamides.

This was a challenging sample. The polarity of these compounds ismodest, as suggested by Figure 6.15, and again, may not require an
additive. However, the similarity of the molecules has traditionally madethem difficult to separate. The minimum acceptable resolution between
any two peaks should always be greater than 1.5, which became thede facto goal of this method development.

84

Proteins
DNA/RNA
Inorganic ionsZwitter ions
PeptidesBenzoic acids
Hydroxy acids
Polyacids
Aliphatic amines

AmidesSteroids
Ring nitrogens
Alcohols
SugarsAnalines

Hydrocarbons
Waxes
Esters/ethers
Fatty acids
Terpenoids
Ketones/aldehydes

INCREASING POL ARIT Y
MOBILEPHASE

CO2Ethyl acetate/Acetone/Aetonitrile/CO2
Isopropanol/CO2

Ethanol/CO2

Methanol/CO2
Methanol/
Water

Additives
STATIONARY
PHASE

C18, C4, C1
Hexaphenyl
Cyano
SilicaDiol
AminoEthylpyridine

Figure 6.15 Relative polarity of sulfonamides showing options for mobile and stationary
phases.

Initial conditions79 for the separation of these nine similar compounds
were: 2 mL/min of methanol modified CO 2 , with a gradient from 5 to
30 %, at 50 °C and 150 bar, using a 3 by 100 mm, 1.8-µm RX-SIL column.
Preliminary experiments showed that all the compounds in the mix eluted
at fairly modest methanol concentrations, but several coeluted. Peak
shapes were reasonable, so no additive was required. Peaks for severalof the compounds fronted slightly. Changing the sample solvent frommethanol to isopropanol significantly improved peak shapes.
All the compounds eluted, but closely spaced together, and responded
differently to changes in temperature and pressure. Changing one
parameter tended to improve the resolution between some pairs of peaks,
but degraded the resolution between others. An alternate form of data
presentation was used here, where the resolution between the four mostdifficult pairs of peaks was plotted vs. each of the mobile phase variables.

85

The mix could not be completely separated at the initial temperature andpressure tried, regardless of the percentage modifier used. A modifier
concentration of 13 % appeared to give the best, but inadequately
resolved, results. The temperature was increased, and then decreasedby 10 °C, to determine whether selectivity could be changed. It was
found that lowering the temperature tended to improve peak shapes andresolution. The temperature was further lowered to 30 °C, where at least
partial resolution of all the solutes was achieved.
The modifier concentration was again optimized. The resolution between
the four most difficult to separate pairs of peaks was plotted as a functionof modifier concentration over a narrow range of concentrations, from 8.5
to 11 % at 30 °C and 150 bar. As can be seen in Figure 6.16, the resolutionof both pairs 4–5 and 5–6 improved as the modifier concentration
was decreased. The resolution between pair 6–7 degraded, while theresolution of pair 3–4 stayed about the same. Only between 8.8 and
9.8 % modifier was the resolution between all the pairs greater than 1.5.
The vertical dashed line in Figure 6.16 indicates that the maximum
resolution between all the pairs that was obtained at 9.2 % methanol.
Resolution
3.00
2.75
2.50
2.25
2.00
1.75
1.50
1.25
1.00

8.5

8.0
6-7

9.0
4-5

9.5
5-6

10.0

10.5

11.0
11.5
Modifier [%]3-4

Figure 6.16 The effect of MeOH concentration on the resolution of four critical pairs in thenine-component sulfonamide mix. The highest resolution of all the pairs was achieved at
9.2 % MeOH. Resolution of some pairs increased while others decreased on over the narrow
range of modifier concentration between 8.5 to 11 %.

86The temperature was then varied between 25 and 40 °C, at 9.2 %methanol and 150 bar, and once again the resolution between pairs wasplotted. The results are shown in Figure 6.17. The results show that peaks
6 and 7 coelute at 40 °C and that the best overall temperature was 30 °C.
Resolution
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

20

25
6-7

4-5

30
5-6

35

40
45
Temperature [°C]3-4

Figure 6.17 The effect of column temperature on the resolution of 4 critical pairs of the
9-component sulfonamide mix. The resolution between some pairs increased while others
dramatically decreased over the 15-degree range studied. The highest overall resolution
was achieved at 30 °C.

The pressure was then varied between 100 and 180 bar, and the resolution
between the pairs was plotted as a function of pressure. The results, shownin Figure 6.18, indicate improving resolution at lower pressures down to
110 bar, but then degrading at lower pressures. Thus, the best overall
resolution of about 2 or better for all the pairs was obtained at 110 bar.
It is unusual for pressure to have such a strong impact on selectivity, but
the modifier concentration is fairly low and the modifier is only modestly
polar. The final optimum conditions found for maximum overall resolution
were 1.7 mL/min of 9.2 % methanol at 30 °C and 110 bar.

87

Resolution
3.00
2.75
2.50
2.25
2.00
1.75
1.50
1.25
1.00

80

100
6-7

4-5

120

140

160

180
200
Outlet pressure [bar]

3-4

5-6

Figure 6.18 The effect of column outlet pressure on the resolution of four pairs of thenine-component sulfonamide mix. The resolution of some pairs increased, while others
decreased between 100 and 180 bar. The highest overall resolution was achieved at 110 bar.

One further optimization was performed. The flow rate was also varied.
Since resolution was greater than 1.5 and resolution equal to 1.5 wasdeemed to be the goal of the analysis, some excess resolution could betraded for speed to yield a shorter run time. The resolution of the four
pairs was plotted as a function of flow rate and the results presented
in Figure 6.19. Flow could be increased to 2.9 mL/min before resolution
degraded to 1.5, which would decrease analysis time by 40 %. A separation
optimized for speed is presented in Figure 6.20.
Resolution
2.4
2.2
2.0
1.8
1.6
1.4
1.2

1.4

1.2
6-7

1.6
4-5

1.8
5-6

2.0

2.2

2.4

2.6

2.8
3.0 3.2
Flow [mL/min]

3-4

Figure 6.19 The effect of flow rate on the resolution between the four critical pairs.
While the best overall separation (Rs > 2 for all) occurred at 1.7 mL/min, an acceptable
separation (Rs > 1.5) was obtained at 2.9 mL/min with a run time reduced by 40 %.

88

Absorbance [mAU]
60
50
40
30
20
0.190
0.262

10
0
0

1

2

3

4

5
Retention time [min]Figure 6.20 Optimized separation of nine sulfonamides with Rs > 1.5, for all pairs. Column:
3 by 100 mm, 1.8 µm, RX-SIL. Conditions: 2.9 mL/min of 9.2 % MeOH in CO 2 at 30 °C and
110 bar. Flow cell: 3 mm, 2 µL tapered. Injection loop: 1.25 µL. Detection: signal 260 nmwith 16-nm bandwidth, reference 360 nm with 40-nm bandwidth, slit 16 nm, filter >0.05 minutes.

6.4
Other observations

None of the above case studies used an additive. However, the need,or lack of need for an additive is usually immediately obvious. The first
injection of any sample typically yields either relatively symmetrical peaks,
requiring no additive, or badly distorted peaks, or no elution, requiring an
additive. Once an additive is included in the mobile phase, it tends to havelittle impact on either retention or selectivity.

89

7
7.1
Background

CHIRAL SEPARATIONS
The predominant use of SFC over the last 15 years has been for chiralanalysis and semipreparative separations in the pharmaceutical industry,
although there has been minor interest in the petrochemical, polymer,
surfactant, pesticide and food industries. Most small drug-like molecules
are chiral, meaning they exist in several forms that are mirror images of
one another, called enantiomers. In nature, many compounds occur only
as one of the forms. However, when synthesized in the laboratory, they
typically occur with equal concentrations of the various forms. Mixtureswith equal concentrations of enantiomers are called racemates.
It is well known that the individual enantiomers of many compounds havedifferent physiological effects. For example, one of the enantiomers of
ibuprofen is much less effective as an anti-inflammatory than the other.
A pure version of the good enantiomer would be more effective since it
might be taken at twice the dose of the racemate and, subsequently, act
faster and more effectively. Alternately, the pure enantiomer could betaken as half the dose of the racemate, with the same effectiveness, butwith fewer toxic side effects.
Some enantiomers can be dangerous as is the case with thalidomide,
where one of the enantiomers causes birth defects. As a consequence theUnited States Food and Drug Administration (FDA) and other regulatory
bodies have required complete testing of each individual enantiomer inany racemate.
It is possible to repatent older drugs, originally sold as racemates, but
reintroduced as the pure enantiomer. This provides another 20 yearsof patent protection for a drug that has already survived clinical trials
and therefore should be much easier and faster to commercialize.
Pharmaceutical companies immediately recognized the economic
incentives and began developing drugs that were pure enantiomers.
Today, most new chiral drugs are sold as pure enantiomers.
Pure enantiomers are usually developed using a series of asymmetricsyntheses, where each step is intended to produce only one of theenantiomers. The syntheses start with small building blocks, andprogressively increase in both size and functionality, in a series ofsteps, until the final product is created. At each stage of synthesis, theenantiomeric purity of the intermediate product needs to be monitored90

to verify that the synthesis is producing appropriately pure educt for
the next synthesis step. This has led to an explosion of the use chiralchromatography at both analytical and semipreparative scales.

7.2
Enantiomeric excess
determinations

If the goal is to develop a pure enantiomer, the amount of the otherenantiomer needs to be quantified. Consequently, such trace analysis iscommon. The enantiomeric excess is used to a measure the success ofan asymmetric synthesis or chiral separation. The most commonly useddefinition of enantiomeric excess, ee, is given in Equation 7.1, where R
and S are the two enantiomers.

ee = 100

R–S
   
R+S

Equation 7.1 Calculation of enantiomeric excess.

7.3
Normal phase for chiralseparations

Normal phase HPLC is more effective in separating enantiomers andother isomers compared to reversed phase, and in the past was theworkhorse method in most laboratories doing chiral analyses. However,many normal phase separations use heptane mixed with other organics
such as isopropanol as the mobile phase. Heptane can be a significant fire
hazard, especially when used in large volumes. It is also expensive. Normal
phase HPLC retention time reproducibility is highly susceptible to small
amounts of water. Equilibration can take a long time. Gradients are seldom
used. SFC on the other hand is at least three- to five-times faster thaneven reversed phase HPLC, has none of the issues with traces of water,or re‑equilibration, and the CO 2 is inexpensive, as pointed out previously.
Further, it is almost impossible to burn modifiers dissolved in CO 2.
A major advantage in drug discovery is that SFC can be scaled to thesemipreparative level where each injection can contain as much as 100 mgof the racemic mixture, retaining the speed and pressure drop advantages
at the analytical scale, as well as the low flammability, compared to normal
phase HPLC. At the end of the SFC separation most of the mobile phaseinherently vaporizes, leaving behind small volumes of modifier that containthe pure enantiomers, which greatly speeds up and simplifies ­dry-down.
91

The pure enantiomers obtained need first to be shown to be pure withanalytical-scale chiral chromatography, and can then be used as a feed
for the next stage of synthesis. At some point, each pure enantiomer
must be studied in its pure form for absorption, metabolism and toxicity(ADMET) studies. Since nine out of 10 potential drugs fail, this approach
is faster and less expensive than developing a series of asymmetricsynthesis steps to make each pure enantiomer. Instead, a small amountof the racemic mixtures can be made and the enantiomers separated
chromatographically. Thus, only the successful compounds need the timeand expense to develop the series of asymmetric syntheses.
The advantages of SFC for chiral separations should be obvious. Manylarger pharmaceutical companies have recognized this and many haveswitched most chiral separations from normal phase HPLC to SFC.
For example, in 2005, White80 used four short 4.6 by 100 mm columns(ChiralPak AD-H, and AS-H, and ChiralCell OD-H and OJ-H) with a
2.5‑minute, 15 to 55 % gradient (cycle time 6 minutes), at 5 mL/min,
120 bar and 35 °C. He preselected the modifier and additives to minimize
the exposure of the chiral stationary phases to those combinations unlikely
to be effective. For basic solutes, he used a basic additive in methanol,
ethanol or isopropanol. However, he only ran the isopropanol when
the first two failed to produce a viable separation. For acidic solutes, hesubstituted 0.1 % trifluoroacetic acid for the base. He compared SFC
to HPLC on the hundreds of samples submitted to his analytical service
laboratory for a full year to determine which technique should be scaled
up for purification. At the end of the year, SFC was found to be superiorin 96 % of the separations, and was subsequently adopted as the primary
screening technique in all future work. As a further consequence, thelargest accepted purification jobs jumped from 5 to 60 grams (fasterchromatography, much faster dry-down, safer).
Of the compounds evaluated, 64 % were baseline resolved using
ChiralPac AD-H, 10 % by ChiralCell OD-H, 17 % by OJ-H, and 9 % by
AS-H (all from Chiral Technologies). He used a 1-minute equilibration
time when the column was switched, and a 5-minute equilibration when
the solvent was switched. Thus, a single solute could be screened onfour columns, with one modifier-additive combination in approximately
19 minutes. The solute could be screened on all four columns, with thethree different modifier-additive combinations in 54-minutes.
Maftouh81 (2005) also screened with four columns and up to six different
solvents. The columns were 4.6 by 250 mm, with 10-µm particles from
Chiral Technologies, and with the phases ChiralPac AD, and AS, and92

ChiralCel OD, and OJ, the same stationary phases used by White. He, like
White, also preselected the mobile-phase combinations for each solute.
Methanol and isopropanol were used pure, and with trifluoroacetic acid or
isopropylamine. The bases, neutrals and bifunctional solutes were eluted
using CO 2 with modifier and isopropylamine. The acids were eluted using
CO 2 with modifier and trifluoroacetic acid.
With 500 proprietary chiral compounds the success rate was; AD 60 %,OD 31 %, OJ 8 %, and AS 2 %. The overall success rate with these fourcolumns was 95 %. With another large set of marketed drugs, the successrate was 98 %. Conditions were 3 mL/min at 200 bar and 30 °C, with amodifier gradient. SFC was adopted as the primary analytical tool while
capillary zone electrophoresis (CZE) was adopted as the backup technique.

7.4
Effect of control variables
on chiral separations

Chiral separations are performed more or less the same as achiralseparations. However, in most cases the solutes are present in simple
mixtures, often containing only the enantiomers of interest.

7.4.1
Relative effects of eachvariable

Warfarin was separated on a Lux Celulose-1 column using methanol in
CO 2, and the effect of modifier concentration, temperature and pressure
were evaluated. The results, all shown in Figure 7.1 indicate that once
again the modifier concentration is the primary means of adjusting
retention. Neither temperature nor pressure had much effect on either
selectivity or retention. There is a slight trend toward higher resolution
at lower temperatures.
3 mL/min, 30 °C, 150 bar
Retention time [min]
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50
Modifier [%]Peak 1

3 mL/min, 20 %, 30 °C

3 mL/min, 20 %, 150 bar
16
14
12
10
8
6
4
2
0

10

30
50 70
Temperature [°C]

16
14
12
10
8
6
4
2
0

50 100 150 200 250 300
Pressure [bar]Peak 2

Figure 7.1 The effect of mobile phase parameters on the separation of warfarin enantiomers.

93

7.4.2
Conventional modifiers

As with achiral separations, methanol is the most polar solvent that iscompletely miscible with CO 2. As such, it is usually the first modifier
used when trying to ensure that all sample components elute. Manychiral compounds, particularly starting materials and intermediates, are
not highly polar and tend to elute near the void volume, even with low
methanol concentrations. The obvious choice under these circumstances
is to decrease modifier polarity. The usual approach is to try ethanol andthen isopropanol, if the ethanol is too strong, even at 5 %. For reachingeven lower polarity solutes, a relatively small concentration of isopropanol(5 to 10 %) can be added to heptane and the mixture pumped as themodifier. In this way relatively low concentrations of polar modifiers canbe delivered reproducibly.

7.4.3
Bonded phases andnonconventional modifiers

In the past, many solvents could not be used as modifier, since many ofthe more successful older stationary phases were coated, not bonded,with cellulose or amylose derivatives. Many interesting solvents suchas dichloromethane and tetrahydrofuran, could, unfortunately, wash off
the stationary phase. Further, some solutes have poor solubility in the
traditional solvents. There has been a significant effort over the last few
years to produce bonded (immobilized) phases 82–85, which can be usedwith a wider range of solvents.
Manufacturers initially attempted to duplicate the selectivity of the older
phases, but this was not successful. However, bonding the phases has
enabled the use of a wider range of solvents, which gives you much more
control over separation and can produce significantly improved solubility.
Nevertheless, these newer bonded phases have not significantly replaced
the older phases in screening, but, instead, have largely been used foralternate secondary screens84, particularly with solutes poorly soluble in
the traditional solvents.

7.4.4
The need for additives

94

As in achiral SFC, many chiral compounds, but particularly aliphatic
amines, need a highly polar additive in the mobile phase to improve
peak shapes. Adderall is a commercial product used for attention deficit
disorders, consisting mostly of D-amphetamine with a small amount of the
L- enantiomer. These are primary aliphatic amines, as shown in Figure 7.2.
The top chromatogram, acquired using 30 % methanol with no additive,
shows no elution. However, the addition of 0.2 % triethylamine to thesame 30 % methanol, produced a rapid, high-efficiency separation. Soluteretention is highly sensitive to the concentration of modifier. It is often
true in chiral SFC that doubling the modifier concentration shifts retention
much more than a factor of two.

30 % MeOH,
No additive

CH3
NH2

Adderall(mostly D-amphetamine)

30 % MeOH+ 0.2 % TEA

20 % MeOH+ 0.2 % TEA

D

10 % MeOH+ 0.2 % TEA
0

1

L
2

3

4

5

6

7
Time [min]Figure 7.2 Separation of a nonracemic mixture of amphetamine, a primary aliphatic amine.
Without the additive, nothing was eluted.

It was briefly mentioned in Chapter 2 “The Mobile Phase” that sterichindrance can have a significant effect on retention. Metoprolol is asecondary amine as shown in Figure 7.3. The isopropyl group tends tohinder access of the lone pair of electrons on the nitrogen to the stationary
phase. The result is that the enantiomers elute with 30 % methanol and are
rapidly separated, but with severe tailing. The addition of 0.1 % acetic acidto the methanol produced no change in peak shape or retention. However,the substitution of 0.1 % triethylamine resulted in symmetrical peaks and asignificant shortening of the run time without loss of resolution.

95

Metoprolol

O

OH3

H3CO

No additive

CH3
CH3

0.1 % Acetic acid

Absorbance [mAU]

Absorbance [mAU]
1.358

20

H
N

1.826

0.1 % TEA

Absorbance [mAU]

1.352
1.834

20

0.930

100

1.192

80

15
15

60

10
40

10
5

20

5

0

0
0

0.5

1.0
1.5
2.0
Retention time [min]

0

0.5

1.0
1.5
2.0
Retention time [min]0

0.2 0.4 0.6 0.8 1.0 1.2 1.4
Retention time [min]Figure 7.3 Effect of additives on the peak shape of metoprolol, a secondary amine.
Left: pure MeOH at 3 mL/min, 30 %, 30 °C, 120 bar; Center: same conditions but with 0.1 %glacial acetic acid added to the MeOH. Right: same conditions but with 0.1 % triethylamine
added to the MeOH. Column: 4.6 by 250 mm, 5 µm Regiscell.

The less hindered tertiary aliphatic amine, mirazapine, did not elute with
40 % methanol without additive, see Figure 7.4. However, the addition oftriethylamine resulted in the elution of sharp peaks, with little retention(k about equal to 1), or resolution. Decreasing the modifier concentration
resulted in significant increases in both retention and resolution.

96

N N

Remeron(mirtazapine)

H3C

N

40 %,
no additive

1.001

1.765

1.895

40 %,
with additive

1.004

2.080

2.285

30 %,
with additive

0.989

2.797

0.737

20 %,
with additive

0.977

3.345

4.047

3.176
0

1

2

3

4

5

6

7

10 %,
with additive
8
9Time [min]Figure 7.4 Separations of the tertiary amine remron (mirtazapine) as a function of modifier
concentration and the presence or absence of additive. Without additive nothing eluted.
3 mL/min of various percentages of MeOH + 0.1 % TEA, 40 °C, 150 bar. 4.6 by 250 mm,
5 µm Chirapak AD-H.

97

It is interesting to note that the need for additives in these three
examples is very different from the behavior of the compounds used togenerate Figure 6.11, where the nitrogens were in rings but the lonepairs of electrons were conjugated with either a carbon-carbon, or acarbon‑oxygen double bond. In those cases, no additive was required.
It should be remembered that additives strongly adsorb onto stationary
phases and may be difficult or even impossible to wash off. For additives
that can be removed, care should be taken not to switch directly froman acid to a base or vice versa, since this may generate nonreproducible
results. There are also claims of irreversible changes to some chiralstationary phases after an additive was used. Many analysts keep twosets of columns, one for acids and the other for bases.

7.4.5
Temperature

Enantiomers are chemically identical and differ from each other onlyin shape. Lower temperatures tend to improve chiral recognition by
decreasing kinetic energy. Gasparinni86 performed chiral separations
as low as –50 °C with increasing enantiomeric resolution, but at such
low temperatures the mobile phase tends to become highly viscous.
It is generally agreed that low temperatures do not increase throughput
significantly. Most standard ovens have a lower temperature limit of 20
to 30 °C. Further, at substantially lower temperatures condensationof water from the lab air can be a problem. Consequently, most chiralseparations in SFC have been performed between 30 and 40 °C.

7.5
Developing a chiral method

It is still true that you cannot predict which stationary phases will separate
a specific set of enantiomers. Consequently, selection of an appropriatechiral stationary phase is reduced to trial and error. Screening systems
have been developed to try to automatically find the best combinationof mobile and stationary phases. The most common approach is to use
automated column and solvent-selection valves, along with a software
wizard that allows you to choose which columns and mobile phases to try
for each specific compound.

7.5.1
Choosing a stationary phase

Although there are many chiral stationary phases available, both White80
and Maftouh81 had over a 95 % success rate with only four of the older
coated stationary phases. Many other workers have had similar success.
This suggests that any initial screen can start with a relatively small numberof stationary phases, to minimize wasted time and materials. It is fairly
common to have four to six different chiral stationary phases on a single
valve, or two matched valves that are switched simultaneously. The chiralstationary phases are usually tried serially, using the same mobile phase.
98

Others have used valves with more ports, or several valves in series.
For example, Welch87 described an SFC system using two 14-port valves.
Five columns and a bypass were mounted on each valve, which allowed
evaluation of 10 individual columns or 25 combinations of two columns inseries. Others have developed similar noncommercial systems with up to
19 different columns.
Faster screening methods continue to be developed with shorter columns
and smaller particles. Hamman88 recently (2011) compared six different 4.6
by 50 mm columns packed with 3-µm particles, using a 1.5-minute gradientfrom 10 to 55 % modifier, with a 1-minute hold at 55 %. The columns were
AD, Cellulose-1, OJ, IC, AS, and Cellulose-4. Three different modifiers wereavailable: methanol, ethanol, and isopropanol. If the solutes were a base,
0.1 % diethylamine, or triethylamine was added to the modifier. Again, theauthors preselected the modifier and additive combinations. The authors
state a six-column screen took 20 minutes (six columns, single modifier).
There are also several parallel systems where flow from a large pump is
split into up to eight streams, each directed through a different column.
Each column sees the same mobile phase at the same time. The injectionis also split. There are specialty detectors with up to eight multiplexed flow
cells. Alternately, individual detectors for each channel have also beenused. Thus, each solute is evaluated on up to eight different columns in asingle run time. One problem with such systems is the lack of versatility.

7.5.2
Choosing a mobile phase

In the choice of mobile phase there are two questions: First and mostimportantly, is an additive needed? Second, how polar does the modifier
need to be?
Regarding additives, it is advisable to group solutes into acids, neutrals,
bases, and bifunctional groups, such as White80, Maftouh81, Hamman88
and many others have done. If an additive is required on one chiralstationary phase, the additive will likely be required on all similar chiralstationary phases, regardless of how polar or nonpolar the solute is.
It makes little sense to automatically run pure modifiers, plus bothacidic and basic additives on every solute as part of an initial screen,
particularly when the pure modifier is run first. Use of a pure modifier
may not elute all the solutes, which could then build up on the column,
potentially changing selectivity.

99

Repeatedly switching back and forth between acidic and basic additives
for each solute is likely to cause damage to the columns, and potentiallyyield nonreproducible results, particularly when careful change-overprocedures are not followed. The most conservative approach is touse a strong base as additive for basic solutes, and a strong acid foracidic solutes, with careful preconditioning during change-over fromone additive to the other. Alternately, you can use two different setsof columns, one for acidic additives, and one for basic additives.

7.5.3
Other variations

Medvedovici89 screened 44 chiral compounds with a broad range offunctional groups using six different chiral stationary phases, of threedifferent types, and a 5 to 30 % methanol gradient containing either
trifluoroacetic acid or triethylamine. Chiralcel OD resolved 66 % of thepairs. Chiralpak AD resolved 70 % of the compounds. A Chirobiotic V
column resolved 48 %, while Chirobiotic T resolved 50 %. The brush-typephases did not perform as well, with the Chirex 3022 resolving 34 %, but
the Chirex 3005 resolving only 20 %.
Welch90 created a diverse library of 48 racemates, which he stored in
96- well microtiter plates specifically to evaluate new chiral stationary
phases. A universal method, of CO 2 with 4 to 40 % (methanol andisobutylamine) was used. Only a basic additive was used. ChiralPak
AD-H baseline resolved 60.4% of the solutes, while OD resolved 30%,
almost identical to Maftou’s findings 81, with different solutes.
In an alternate approach, Zhao91 in 2003 pooled up to 12 racemic mixturesand used SFC-MS to identify the peaks, based on mass. Acids, bases andneutral compounds were segregated. The Acids were separated using
methanol and trifluoroacetic acid. The bases and neutrals were separatedusing methanol and isopropylamine. Four different columns (Chiralpak AD,
AS, and Chiralcell OD, and OJ) were sequentially screened. The mixed
sample was separated using eight different concentrations of modifier
over 20 minutes, starting at 40 %, then decreasing by 5 % per run. Flowwas 2.5 mL/min, temperature was 35 °C, with 110-bar outlet pressure.
Success was reported in the order AD—AS—OJ—OD.
Also in 2003, Garzotti92 used electrospray MS in conjunction with SFC
to separate a broad range of racemates and also differentiated thepeaks based on mass. He used Chiralpak-AD, and Chiralcell-OD, andChiralcell‑OJ at 35 °C, 180 bar and 2.5 mL/min. He claimed AD resolved
80 %, OD resolved 45 % and OJ resolved 50 % of the racemates studied.
He mostly used ethanol with isopropylamine as additive. He injected
mixed standards of up to four racemic mixtures. He stated there was a100

significant increase in stereoselectivity with decreasing temperature, but
pressure had almost no effect on resolution.
The most effective chiral stationary phases described above arenonbonded, which limits the range of modifiers that can be used withoutwashing the phase off the column. New bonded phases attempting to
reproduce the selectivity of the nonbonded phases have resulted invariable results. There is no one-to-one replacement. However, thesenewer bonded phases enable the use of most organic solvents as
modifier and sample solvent and are becoming popular.

7.5.4
A method development
systemThe Agilent method development system uses two or three thermostatted
column compartments with four or six independent heat exchangers,
capable of controlling temperatures between 10 degrees below ambientand 100 °C. Two of the column compartments contain an 8-position/9- port
switching valve for column selection. Up to eight columns can be fitted.
Each column inlet is connected to one of the ports through one of the heatexchangers. All the column inlets are connected to the same valve, while
all the column outlets are connected to the other valve. The valves are
switched in unison to place successive columns in the flow path.
Several low dispersion heat exchangers can be mounted on each of thestandard heat exchangers, significantly decreasing the lengths of tubing
required to connect the columns to the valves. For rapid screening up to
four low dispersion heat exchangers, and four 100-mm long columns, canbe mounted in a single column compartment. Longer columns can also be
mounted in each column compartment. Combinations of both short andlong columns can be installed. The shorter columns can be used for rapid
screening to identify the best stationary phase for a separation, and thenthe appropriate longer column can be used for higher resolution work.
Recently, an alternative approach has been offered by Agilent thatuses a single column compartment equipped with a valve that enables
five columns and a bypass to be connected in parallel. A single valve
with small ports is used to connect both column inlets and outlets.
This significantly shortens the lengths of tubing required to connect thecolumns to the valve, which decreases extracolumn band broadening.
Solvent selection can also be automated with a 12-position/13-port valve,meaning up to 12 different modifiers could be tried. Thus, three modifiers
such as methanol, ethanol and isopropanol could each be tried pure,with a basic additive, or with an acidic additive. Both column and solvent
selection valves can be controlled through software, which facilitates101rapid development of sequences. Setup is simple. Column identities arelisted in one table. Modifier identity is listed in another.
It is often true that specific, well-established methods are used routinely,
but not on a continuous basis. A method development system can be usedin support of a group of medicinal chemists each primarily performing
asymmetric syntheses. Instead of having a large number of intermittently
used machines, or continuously changing columns for each different
method, the system can be configured with the most common stationary
and mobile phases. Each user can then submit a sample and the name of
their method (column and modifier combination) for automated analysis.

102

QUANTIFICATION IN SFC
8.1
Stages of Validation

8

In the past, SFC was seldom used in routine QA/QC applications because
the dynamic range of UV detectors was inadequate to monitor both major,
minor and trace components in the same run, and since UV noise was on
the order of 0.5 mAU with a filter setting of greater than 0.1 minutes. Thiswas up to 50-times worse than HPLC. This meant that a minor component,
representing 0.1 % of a major component having an absorbance less thanor equal to 1000 mAU, could not be quantified with a signal-to-noise ratioof greater than 10. Such quantification requires a noise level less than
0.1 mAU. In the recent past, such problems have been largely solved bysignificantly decreasing UV detector noise by as much as a factor of 50,
as described in detail in Chapter 9 “Instrumental Considerations”. Routine
analysis accounts for up to 10-times more users compared to method
developers. This is potentially a large and new application area for SFC.
In order to perform routine analysis, both the equipment and the method
must be shown to be robust. That is, they must be shown to adequately
perform the tasks demanded of them, over multiple days, months or years,
from lab to lab, instrument to instrument, and so on. This includes dynamic
range, sensitivity, linearity, accuracy, precision and repeatability.
There are multiple texts devoted exclusively to validation, which means
any detailed discussion here, in a short chapter, can only be inadequate.
However, an overview is provided, with the suggestion that you should
consult such texts.

8.1.1
Design qualification

Design qualification (DQ) is about whether the manufacturer understoodthe needs of the customer and designed the equipment to meet thoseneeds. This is largely about comparing specification sheets describing
the performance of the hardware, written by the manufacturer, to thecustomers’ needs, as perceived by the customer.

8.1.2
Installation qualification

Installation qualification (IQ) is usually performed by the instrument
manufacturer, where, after they perform the installation, they demonstrate
the hardware at least initially meets the design specifications as statedby the manufacturer. The end user and the manufacturer need to agree,
a priori, to how to describe success. Flow, pressure, temperature, ranges,
precision, accuracy, detector characteristics, and so on, were previously
103specified in the DQ process, and the means to measure these parameters
also needed to have been agreed to at the DQ level. Remember that SFC
differs from HPLC in that the SFC mobile phase remains highly compressible.
Terms such as flow rate and % modifier need to be carefully defined.
Preferably, a chromatographic test is used to confirm performance basedon retention times and resolution between the components of a standard
sample on a standard column, under a test protocol. If properly designed,
such a test confirms flow accuracy, composition accuracy, temperatureaccuracy, pressure control accuracy, and so on.

8.1.3
Operational qualification

Operational qualification (OQ) is closely associated with routine maintenanceand is often repeated regularly. Here, the primary question is: is the
instrument operating within the agreed upon specifications, at this time,and for this method? This is both an initial and an ongoing question.
Once again, a standard test sample along with a standard test column(only used for this purpose), can show if the system is performing properly.
Qualification teams sometimes wish to use various meters to directly
measure temperature, pressure, flow, composition, or other parameters.
Such meters need to be calibrated at least annually using traceable
standards. However, it is often true that the accuracy of available meters isworse than the accuracy and repeatability achievable by using a standard
sample and standard column and viewing the chromatographic results.

8.1.4
Performance qualification

Performance qualification (PQ), or performance validation (PV) (whichusually means the same thing) is more about the quality of the current
instrumental method and usage of the instrument, although there is someargument whether this actually constitutes several substeps.
To validate a method, the user needs to demonstrate that the accuracy,
precision, resolution, linearity range and robustness of the method canrepeatedly produce the desired result.
There are internationally agreed standards as to what an analyticalmethod must achieve in order for the results to be recognized as valid.
In the following exercise, a method is developed and validated for thequantification of sorbate and benzoate (widely used food preservatives)and caffeine in various beverages and a few viscous foods, to at leastvaguely address the needed steps.
Later, a trace method for the chiral separation of flurbiprofen isdemonstrated.

104

8.2
Method development forquantification of sorbate,benzoate, and caffeine inbeverages and foods
8.2.1
A statement of the problem

In order to demonstrate a typical routine quantification of major and minor
components, several preservatives and plus caffeine were determinedin a number of beverages and foods. Benzoic and sorbic acids, and their
sodium, potassium and calcium salts are used as preservatives in acidic
foods. They are widely used93–95 in soft drinks, fruit juices, other beverages,
soy sauce, catsup, salad dressing and many other processed foods.
They are banned for use in fresh meats but are used in some sausages.
They inhibit the growth of molds and yeasts, and are also effective against
a wide range of bacteria, which explains their widespread use and major
health benefits.
Both sorbate and benzoate are regarded as safe. Sorbate has anadditional advantage since it has no taste, and has shown less toxic
side effects than benzoate, since it is present in human metabolism.
Nevertheless, some adverse reactions have been noted, which haveresulted in limitations on the concentrations that can be used in foods.
In the USA, the limit for benzoic acid is less than 0.1 %, expressed onfood labels as less than 1/10 of 1 % sodium benzoate. There is no legal
limit on sorbate in the USA, but one international agreement sets a
maximum level of 0.3 %. European Union Directive 95/2/CE is much
more restrictive, having established the limit for benzoic acid at 150 mg/L
(0.015 %), and sorbic acid at 300 mg/L (0.03 %), if used separately, and
150 and 250 mg/L (0.015 and 0.025 %) if used together. These limits are
expressed as benzoic and sorbic acids (not the salts).

8.2.2
Some background (an aside)

As a result of public concern and the near ubiquitous use of these
additives, a group of countries, including the USA, UK, and China haveestablished a level of maximum annual exposure. This results in huge
numbers of analyses per year. As part of this program, the actual levelsof these additives in various foods are routinely measured, so that total
annual dietary intake can be estimated. Several other countries, such as
Brazil94, Portugal96, and New Zealand97, have performed similar studies.
Several techniques have been used, including gas chromatography 98, 99
with derivatization, various spectroscopic methods100, and capillaryelectrophoresis101. However, HPLC with ion pairing is most widelyused102, 103, and appears to be the method of choice. Nevertheless, peak
shapes and resolution have tended to be poor, with 10 to 20-minuteanalysis times104–107. Acidified acetonitrile is still used with a C18 column.
New HPLC methods continue to be developed.

105Surprisingly, there is no reference in the literature to the use of SFC for
the analysis of preservatives such as benzoate or sorbate in foods andbeverages, although benzoic acid was often used as a test probe in
the early days of SFC, and several papers have appeared related to theseparation of hydroxyacids108 and polycarboxylic aromatic acids109. SFC
has seldom been used for the analysis of aqueous samples, which may
explain this lack of references. However, many samples can be diluted
with methanol, filtered or centrifuged, or both, and then injected without
further pretreatment.
Benzoic acid has a UV wavelength maximum near 227 nm with a small
lobe near 280 nm. Sorbic acid has a UV maximum at 255 nm, while
caffeine has a maximum at 280 nm. Most studies have used a single
wavelength near 235 or 280 nm when all three are present. However,for optimum detection it is relatively straightforward to measure eachcompound present using its optimum wavelength.
Other food additives are sometimes present, which can complicate
the analysis110–113. The most common are caffeine, saccharin, and one
or more of the parabenes. They could be regarded as interferencesin the benzoate‑sorbate acid assay, but are usually included in themeasurements.

8.2.3
PurposeThe purpose of the present work is to develop a method and demonstrate
many of the steps required to validate it for the quantification of benzoate,sorbate and caffeine in several aqueous-based beverages and foods
using SFC.

8.2.4
Development of thechromatographic method

Since there were no reports in the literature on the separation of benzoic
and sorbic acid, as well as caffeine, it was necessary to develop amethod. Preliminary work showed that the analytes could be eluted using
relatively low concentrations of methanol as modifier, although benzoic
acid tailed severely without an acid additive. Both trifluoroacetic acidand acetic acid were shown to improve the peak shapes of both benzoic
and sorbic acids. Acetic acid was chosen for further work, since it is lessexpensive, more common, greener, and safer to work with. The column
temperature was arbitrarily set at 50 °C. The outlet pressure was chosen
as 150 bar to avoid any noise issues associated with significantly lower
pressures. The autosampler was set to overload the 5-µL loop with 15 µL
of standard or sample.

106

Initial experiments using a ZORBAX RX-SIL column showed that resolution
between sorbate and benzoate was only about 1.0 at 7.5 % (methanoland 0.3 % acetic acid), with little retention, while at the same time, there
was excessive time between benzoate and caffeine.
A Premier column, specifically designed for separation of acids in SFC,
produced exceptional selectivity, with high resolution as shown in the top
chromatogram in Figure 8.1. However, caffeine was almost unretained,
and the separation of the preservatives required 40 % organic modifier.
Methods where components are essentially in the solvent front need to
be avoided. A diol column produced a significantly improved separation,
eluting all three analytes in less than 2 minutes with resolution of greaterthan 4, and 2.64, between the two pairs, while requiring only 8.5 %(methanol and acetic acid), as shown in the bottom chromatogram in
Figure 8.1.
Premier with 40 % modifier

Absorbance [mAU]
500

Sorbate

400
300
200

Caffeine

Benzoate

100
0
0

0.5

1.0

1.5

2.0

3.0

3.5

4.0

Diol with 8.5 % modifier

Sorbate

1000
800
600
400
200
0

2.5

Benzoate
Caffeine
0

0.5

1.0

1.5

2.0

2.5

3.0

3.5
4.0
Time [min]Figure 8.1 Separation of benzoate, caffeine and sorbate at high concentrations.
Top: Separation using a Premier 4.6 by 250 mm, 5-µm SFC column at 3.5 mL/min of
40 % MeOH containing 0.3 % acetic acid in CO2, at 50 °C with 150-bar outlet pressure.
Bottom: Same separation on a 4.6 by 250 mm, 5-µm diol column with the same
conditions except at 8.5 % modifier. 5 µL injected, 235 nm, 16 nm, slit filter greaterthan 0.03 minutes. Resolution was greater than 4 and 2.64 on a diol column.

107

The retention order on the Premier column was different from the retention
order on the diol column. Such large differences in selectivity can be used
to help ensure that no interference peaks are coeluting with any of thesolutes since the methods are orthogonal.
Composition, temperature and pressure were changed to slightly higher
and lower values, which demonstrated robustness and reproducibility inretention times and resolution. Since the resolution with 8.5 % modifier
was greater than 1.5, the method was deemed acceptable for major andminor component analysis.

8.3
Calibration
8.3.1
Designing the calibration
curves to meet the analyticalrequirements

In order to validate this method, a calibration curve needs to be generated.
Details such as the number of concentrations of standards, the numberof injections per standard, the precision of the analysis with each of thestandards, the linearity of the calibration curve and the linear range allneed to be evaluated and shown to be adequate.
Calibration curves should include standards that bracket the expected
results by at least ±30 %. There should be several standards both higher
and lower in concentration compared to the expected result. With theseboundary conditions, it is feasible to try to make appropriate calibration
curves for the chosen analytes in this example.
The legal limit on benzoate in the USA is less than 1/10 of 1 %, or lessthan 0.1 %. Here, the aqueous samples were diluted 3:1 to avoid any
miscibility issues between the water and the mobile phase, so the upper
limit found in the samples should be less than 0.25 mg/mL. The highest
concentration used to generate the calibration curve was 0.5 mg/min
or twice the expected maximum. Standards were also prepared at 0.35,
0.25, 0.15, 0.075, 0.05 and 0.025 mg/mL with multiple standards bothabove and below the expected value.
There is no legal limit on sorbate but one international agreement
indicates the maximum permitted should be less than 0.3 % (3 mg/mL).
As with benzoate, the samples were diluted 3:1 so the upper limit of
the calibration curve ought to be 1.3 to 1.5-times 0.75 mg/mL.

108

There is a requirement that the amount of caffeine added to many beverages
must be listed on the label. The labels generally list the caffeine content as
the total weight of caffeine in the container. For the samples tested that listed
their caffeine content, the values ranged between 0.0529 to 0.336 mg/mL,
making 0.5 mg/mL adequate for use as the upper limit of the calibration
curve, but also required an extended calibration curve to lower values since
beverages contain a wide range of concentrations.
Each of the beverages and foods had 1, 2 or 3 of the analytes present,
meaning one or more were often absent. Thus, it was interesting but notnecessary to estimate the limit of detection and the limit of quantificationof each of the analytes in each sample. Most of the samples contained
either sorbate or benzoate but there is little interest in sorbate or benzoate
concentrations significantly below the legal or recommended limits.
However, since there were several beverages with reportedly a wide rangeof caffeine concentrations, the calibration curves were extended to cover
the lowest concentration expected.

8.3.2
Creating the calibration curves

A total of seven standards and a blank, bracketing the concentrations
found, were used. The standards contained all three analytes at equal
concentrations. Each analyte was monitored at its wavelength of maximum
absorbance. The standards covered a dynamic range of 20 between 0.025
and 0.500 mg/mL.

8.3.2.1
Precision

The precision of analysis of each standard was determined by making six
injections at each concentration, and the RSDs for both retention timeand area counts were determined. The RSDs for retention time for all three
analytes averaged 0.048 % with a maximum value of 0.08 %, as shownin Table 8.1.

109

Concentration

SorbateMean

BenzoateSD

RSD [%]

Mean

CaffeineSD

RSD [%]MeanSD

RSD [%]

0.5 mg/mLRT

1.37680.000373

0.03

1.62150.000548

0.03

1.79980.001169

0.06

Area

3337.04.3191

0.13

855.2

0.7183

0.08

707.3

0.92080.13

RT1.37670.000470.03

1.62220.000400.03

1.79820.00098

0.05

Area

2408.25.56940.23

605.272.0519

0.34

497.9

1.41250.28

RT

1.37630.00047

0.03

1.623

0.000810.05

1.79550.00104

0.08

Area

1757.51.53020.09

439.230.54650.12

360.030.53160.15

0.35 mg/mL

0.25 mg/mL

0.15 mg/mL
RT

1.376

0.00057

0.04

1.62340.00051

0.03

1.794

0.00063

0.04

Area

1051.130.69186

0.07

261.9

0.54406

0.21

213.730.413120.19

RT

1.37520.00068

0.05

1.624

0.000630.04

1.79230.01033

0.06

Area

513.230.57500.11

128.120.2562

0.20

104.4

0.22800.22

RT1.37370.000940.07

1.62420.00075

0.05

1.78950.01049

0.06

Area

322.770.1505

0.05

80.38

0.3600

0.45

65.33

1.10320.16

0.075 mg/mL

0.05 mg/mL

0.025 mg/mL
RT

1.37380.00098

0.07

1.62420.00098

0.06

1.78880.00098

0.05

Area

174.950.2429

0.14

43.75

308.22

0.07

35.67

0.1663

0.46

Blank

All 000
Table 8.1 The mean values, standard deviations, and RSDs for retention times and area
counts, with six injections of each, for the sorbate, benzoate and caffeine standards atconcentrations between 0.025 and 0.5 mg/mL used to generate the calibration curves.
Due to the short run times, the calibration curve, with seven standards and a blank,
consisting of 48 runs could be generated in approximately 2.4 hours with an isocratic
run time of 3 minutes.

110

The RSDs for area were all found to be under 1 % as also shown in
Table 8.1. As expected the RSDs for the higher concentration standards
were relatively low, typically less than 0.25 %. At the bottom end of theconcentration range, the RSDs became larger but still under 1 %. Bothsets of measurements were found to be acceptable for quantifying theanalytes over the ranges expected.

8.3.2.2
Linearity

The mean of the area counts at each concentration was plotted for eachanalyte, and the data was also subjected to linear regression. The plots ofarea against concentration showed straight lines with different slopes asshown in Figure 8.2. The regression showed linear correlation coefficientsof greater than 0.999 in all three instances, as shown in Table 8.2. Linear
correlations greater than 0.990 are deemed acceptable. Intercepts wererelatively small.
Mean area [counts]
4000
3000
2000
1000
0

0

0.1
Sodium benzoate

0.2

0.3
Caffeine

0.4

0.5
0.6
Concentration [mg/mL]

Potassium sorbate

Figure 8.2 Calibration curves for potassium sorbate, sodium benzoate, and caffeine
collected at 255, 235 and 280 nm respectively.

111

Diol, 5 µm, 4.6 by 250 mm
Sorbater

Benzoate

Caffeine

20.999574

0.999876

0.999925

Slope

6750.761721.29

1422.41

Intercept

14.23

0.51

–0.88

Table 8.2 The linear correlation coefficients, slopes and intercepts for the three calibration
curves for benzoate, caffeine and sorbate used in this analysis. The linearity was exceptional.
The intercepts were acceptable. The data for each analyte was collected at different
wavelengths: sorbate at 255 nm, benzoate at 235 nm, and caffeine at 280 nm.

8.3.3
Samples
8.3.3.1
Beverages

15 commercially bottled beverages were randomly purchased as local
markets for analysis. Approximately 20 mL of each sample were placed ina 40-mL scintillation vial. Carbonated beverages were vigorously shaken
not stirred with the cap tightly closed to partially degas. The caps wereloosened slightly to allow the gas to escape. The caps were retightened,and shaken again, for a total of three times. All the samples were thensonicated for 12 minutes.
A 1-mL aliquot of each sample was transferred into a 25-mL plastic
beaker, using an Eppendorf pipette, then 3 mL of methanol were added.
The beakers were covered with a small watch glass and sonicated for
12 minutes. Approximately 1.5 mL of each sample was then transferred
into an amber-colored 2-mL autosampler vial and closed with a PTFE‑lined
crimp cap for analysis.

8.3.3.2
Foods

Many of the food samples were viscous (catsup for example) or contained
significant solids (Italian salad dressing). For these samples, 1 g wasweighed into a 40-mL scintillation vial and 10 mL of acidified methanol wasadded. The samples were shaken thoroughly for 30 seconds and sonicatedfor 12 minutes. The solutions were allowed to settle and the supernatantliquid was transferred to 15-mL centrifuge tubes with closures. The tubes
were centrifuged for 10 minutes at 3200 rpm. Approximately 1.5 mL of theresulting supernatant liquid was transferred to amber vials, in the samemanner as the other samples. All the standards and samples were analyzed
within a few hours of preparation.

8.3.4
Analysis of samples

Similar to the standards, six injections of each sample were also madeand the RSDs determined. The standards and samples were run as partof a single sequence. Each of the samples was typically present as one ofseveral salts. Accurate quantitative analysis depended on which salt waslisted on the label with an appropriate correction.

112

8.3.4.1
Beverages

A typical chromatogram representing a tea is presented in Figure 8.3.
There was little caffeine present. Noise was of the order of 0.05 mAU
with a filter setting of greater than 0.03 minutes. The concentration ofcaffeine was found to be 0.035 mg/mL. Thus, the limit of quantification for
caffeine using this method (with signal-to-noise greater than 10) would be
about 0.0105 mg/mL. The limit of detection with signal-to-noise greaterthan 3, would be 0.0032 mg/mL.
Absorbance [mAU]
40
35
30
25
20
15
10
5
0

0

0.25

0.50

0.75

1.00

1.25

1.50

1.75
2.00 2.25
Retention time [min]Figure 8.3 Chromatogram of a tea containing both sorbate and benzoate with a low
concentration of caffeine. The caffeine was present at about 3.33-times the LOQ(S/N greater than 10) at 0.0105 mg/mL.

The results for the beverages suggest there might be greater regulatory
oversight compared to some of the prepared foods. Of the 15 beverages
analyzed, all were properly labeled, in terms of which of the preservatives
was present, or not, as shown in Table 8.3. Further, none of the samples
exceeded any legal or agreed-to limit regarding benzoate or sorbate.
However, some had RSDs as high as 2.99 % probably due to matrix
effects. While such RSDs are relatively high, in terms of the relatively
high concentrations of the preservatives, it is probably acceptable.
As usual, samples with higher concentrations had lower RSDs withrespect to area count.

113

SorbateCola

Benzoate

Concentration[mg/mL]

RSD

NF

NF

On labelCaffeine

Concentration[mg/mL]

RSD

NF

NF

On label

Concentration[mg/mL]RSD

On label

0.094

2.99

Yes

Cola diet

NF

NF

0.193

1.15

Yes

0.131

0.95

Yes

Cola zero

NF

NF

0.191

1.58

Yes

0.098

1.18

Yes

No caffeine

NF

NF

0.044

0.52

Yes

NF

NF

Decaffeinated zero

NF

NF

0.056

0.59

Yes

NF

NF

Soda #1

NF

NF

0.387

0.21

Yes

0.153

0.20

Yes

0.456

0.13

Yes

Yes

NF

NF

Soda #1 diet

NF

NF

Tonic

0.114

0.55

Yes

0.11
NF

Tonic diet

NF

NF

0.384

0.09

NF

NF

Energy diet

NF

NF

NF

NF

0.324

0.20

NF

NF

NF

NF

NF

NF

NF

NF

Punch

0.355

0.18

Apple

NF

NF

Yes

Yes

0.161
NF

Tea diet

0.267

0.14

Yes

NF

NF

0.049

0.45

Tea #1

0.303

0.08

Yes

0.212

0.48

Yes

0.035

0.64

Tea #2

0.130

0.32

Yes

0.247

0.23

Yes

0.024

1.89

Yes

Yes
Yes

Table 8.3 Sorbate, benzoate and caffeine content in various beverages. The preservative
content of all the beverages was found to be consistent with their labels. The US limit for
benzoate is 1.0 mg/mL.

With caffeine, there was a large range of concentrations present. For thesesamples, the concentration found was compared to the label claim, and theresults are presented in Table 8.4. The concentration of caffeine in all thesamples was fairly close to the label claims, with a range of values from
93.2 % (D = –6.8 %) of the label value to 106.8 % (D = +6.8 %) of thelabel value.

114

ProductLabel specification

Amount found [mg]Amount found [%]Cola

57 mg/20 oz

53.1 ±1.5993.2 ±3.0

Diet cola

76 mg/20 oz

74.0 ±0.70

97.4 ±0.09

Cola zero

57 mg/20 oz

55.7 ±0.66

97.7 ±0.12

Soda

91 mg/20 oz

86.8 ±1.74

95.4 ±2.0

Diet soda

91 mg/20 oz

91.2 ±0.10

100.2 ±0.11

Diet energy

80 mg/8.4 oz

77.1 ±1.5496.4 ±2.0

Diet tea

12 mg/8 oz

11.2 ±0.05

93.3 ±0.45

Tea #1

Not given

8.02 ±0.05

Not applicable

Tea #25 mg/8 oz

5.34 ±0.10

106.80 ±1.9

Table 8.4 Caffeine content found in several the beverages analyzed, compared toconcentrations on the label. The average found was 97.6 % of the given specification.

8.3.4.2
Foods

Several semiliquid foods such salad dressings and condiments werealso evaluated for their content of the preservatives. Only one contained
caffeine and it was most likely not added intentionally, since it was
chocolate syrup and chocolate naturally contains caffeine.
Unlike the beverages, many of these condiments were grossly mislabeled
in terms of which preservative were present, as shown in Table 8.5.
Perhaps the most egregious miss-labeling occurred with a soy sauce,
duck sauce and a Chinese mustard from a Chinese take-out restaurant.
All were labeled as containing less than 1/10 of 1 % sodium benzoate,
which to a point was true, since they all contained no detectable traceof benzoate, but all were found to have large concentrations of sorbate,
which was not listed on their labels.

115

Sorbate

Lime juice

Benzoate

Caffeine

Concentration[mg/g]RSD

On label

Concentration[mg/g]RSD

On label

Concentration[mg/g]

RSD

NF

NF

No

1.251

0.29

Yes

NF

NF

Chocolate syrup

0.941

0.70

Yes

NF

NF

No

0.246

18.85

Italian

0.665

0.72

No

0.479

1.11

Yes

NF

NF

Balsamic

0.841

0.92

No

0.910

1.46

Yes

NF

NF

Ranch

1.411

0.89

No

1.052

0.49

Yes

NF

NF

Catsup

NF

NF

No

0.387

0.21

No

NF

NF

Brand soy

NF

NF

No

0.456

0.13

Yes

NF

0.11

Soy

0.837

1.15

No

NF

NF

Yes

NF

NF

Duck sauce

0.911

1.11

No

NF

NF

NF

NF

NF

Mustard

0.745

32.14

No

NF

NF

Yes

NF

NF

On label

NaturalTable 8.5 Concentration of sorbate, benzoate and caffeine in various foods. The results arereported as the actual content found in the product. The agreed limit on sorbate is less than
3 mg/g. The legal limit of benzoate was less than 1 mg/g. The shaded entries are mislabeled.
The levels shown in red are above the legal limit.

Similarly, several salad dressings listed the presence of benzoate, which
was found to be present, but also significant levels of sorbate were found,which were not listed on the label. Both the lime juice and the ranch
dressing slightly exceeded the legal limit for benzoate.

8.4
Summary of thequantification of benzoate,sorbate and caffeine

The calibration curve was generated with multiple standards at least±30 % both above and below the expected value using seven standards
and a blank. The large number of standards was needed due to thewide range of concentrations expected. A total of six injections of eachstandard, produced RSDs of both retention time and area counts less than
1 % with linear correlation coefficients of greater than 0.999 %, withsmall relative intercepts.

8.5
Chiral separations

When developing chiral compounds as potential drugs, each enantiomer
needs to be synthesized or purified to contain less than 0.1 % of eachof the other enantiomer, to enable testing of the effects of each pureenantiomer, independently. Preferably, the major enantiomer is within thelinear dynamic range (typically less than 2000 mAU) of the UV detector,
while the minor contaminant (less than 0.1 %) can be simultaneously
measured with signal-to-noise greater than 10. If the major component

116

has a maximum absorbance of 1000 mAU (1 AU), then the minor
component (0.1 %) should have a maximum absorbance of 1 mAU,
and the noise should be less than 0.1 mAU peak-to-peak.
Chromatographic resolution should be at least 1.5 although significantly
larger values are highly desirable, particularly if the minor component
elutes after the major component. For such trace analysis, many consider aresolution of 3 to 6 as necessary, in order to quantify the minor component.
In a chiral separation of a racemate, the first peak is typically significantly
higher and narrower than the second peak, although in terms of area, thepeaks should be identical. The major and minor components each requiredevelopment of separate calibration curves with multiple standards thatbracket the expected results by at least ±20 %, and preferably ±50 %.
At least five bracketing standards should be used for each curve. Eachstandard and sample should be injected at least six times. The calibration
curves should be linear to at least 0.99. When signal-to-noise is greaterthan 100, the RSDs should approach or be better than 1 %.
In this example, the separation and quantification of the enantiomersof flurbiprofen over almost four orders of magnitude is demonstrated.
Conditions were adjusted such that a 1 mg/mL solution produced two
peaks, each approximately 1000 mAU high. Thus, a minor component
representing 0.1 % of either enantiomer should be about 1 mAU high.
The method consisted of 3 mL/min of 20 % methanol at 40 °C and 150
bar that separated the enantiomers of flurbiprofen using a 4.6 by 250 mm
column, packed with 5-µm ChiralPak ADH. An excellent separation, withresolution greater than 5.5 and selectivity greater than 1.5, was obtainedin less than 3 minutes.
The detector’s signal beam was set to 280 nm with a bandwidth of 16 nm,
and the reference beam was set to 360 nm with a bandwidth of 40 nm.
The slit width was set to 8 nm. The filter was set to either 0.01 minutes(20 Hz) or 0.025 min (10 Hz).
The standard was progressively diluted to create additional standards forthe calibration curves. Five bracketing standards (150, 125, 100, 75 and50 %) were made for the high end calibration. For the low end calibration,
five standards (0.015, 0.05, 0.1, 0.5 and 5 %) and a blank were used.
Each standard was injected six times. Representative chromatograms of
the six overlaid runs for 100, 0.05 and 0.015 % are presented in Figure 8.4.

117

100 % (10 µL of 1 mg/mL)

Absorbance [mAU]
1200
1000
800
600
400
200
0
0

0.5

1.0

1.5

2.0

3.0

2.5

0.05 % (10 µL of 0.0005 mg/mL)
S/N = 10.6 to 15.5

0.06
0.04
0.02
0.00
0

0.5

1.0

1.5

2.0

2.5

3.0

0.015 % (10 µL of 0.0002 mg/mL)
S/N = 3.5 to 4.8

0.06
0.04
0.02
0.00
-0.02
-0.04

0

0.5

1.0

1.5

2.0

2.5

3.0
Time [min]Figure 8.4 Chromatograms of racemic flurbiprofen at 100%, 0.05%, and 0.015% of theoriginal value, separated on a 4.6 by 250 mm column packed with ChiraPak ADH, with
20% MeOH, 40 °C, 150 bar.

A similar set of separations was conducted by a different organization
several thousand miles away, with a different instrument (a current Agilent
HPLC instrument and an earlier Agilent HPLC instrument, both modified forSFC), and different operator, column, and chemicals. The only significant
difference was the choice of 4 mL/min (against 3 mL/min) as the flow rate.
Nevertheless, the quantitative results were similar, as indicated in Table 8.6.

118

Flurbiprofen
User 1

User 2

Peak 1

Peak 2

Peak 1

Peak 2

Warfarin

ICH

Area RSD at 100 %

–

–

0.9 %

0.9 %

0.27 %¤ 1.0 %

Area RSD at 0.1 %

–

–

–

–

2.07 %¤ 20 %

Area RSD at 0.05 %3.36 %

3.87 %

4.6 %

5.0 %

–

None

Area RSD at 0.015 %

–

–

6.5 %

6.5 %

–

None

Area RSD at 0.01 %

–

–

–

–

14. 2 %None

RT RSD at 100 %

–

–

0.1 %

0.1 %

0.46 %¤ 1.0%

RT RSD at 0.05 %

–

–

0.1 %

0.1 %

–

–

RT RSD at 0.01 %

–

–

–

–

0.027 %–

S/N at 0.05 %

17.7

18.2

15.9

10.6

57.6

> 10

S/N at 0.015 %

–

–

4.7

3.5

–

None

S/N at 0.01 %

–

–

–

–

7.9

None

LOQ (S/N = 10)

0.027 %

0.029 %

0.03 %

0.047 %

0.013 %

¤ 0.05 %LOD (S/N = 3)

0.0084 %0.0073 %0.0095 %0.0128 %0.0038 %¤ 0.017 %S/N at 0.05 %

17.7

18.2

15.9

10.6

57.6

> 10

80 to 120 %0.99900.9991–––¤ 0.998

50 to 150 %––0.9985

0.998

–

–

0.05 to 5 %1.00001.00000.9998–––

LOQ 120 %

–

–

–

–

0.99999¤ 0.99Area reproducibility

Retention time reproducibility

Sensitivity

Linearity

Table 8.6 The chiral separation of flurbiprofen on a 4.6 by 250 mm, 5-µm ChiraPak ADH,
3 mL/min, 20 % MeOH, 40 °C, 150 bar. 13-µL, 10-mm flow cell, 0.01-minute filter;
10-µL injection.

119

The approximate limit of quantification (defined as the minimum signal
retaining a signal-to-noise equal to 10), and limit of detection (definedas the minimum signal with signal-to-noise equal to 3), were found tobe just under 0.015 and 0.005 %, as shown in Table 8.6. Both users
obtained results within the guidelines of the International Committee
for Harmonization (ICH), in terms of RSDs for area and retention time,
linearity, signal-to-noise, limit of detection and limit of quantification,
also shown in Table 8.6.
Also included in Table 8.6 are similar results from a separation of theenantiomers of warfarin on a ChiraCell ODH column with 30 % methanol
containing dimethyl ethylamine as modifier, at 30 °C and 200 bar, indicating
similar quantitative performance. Run time was under 4 minutes. Resolution
was 3.8, and selectivity (α) was 2.1.

120

INSTRUMENTAL CONSIDERATIONS
9.1
Pumping
9.1.1
Most SFC systems

9

In SFC, the CO 2 is usually supplied from steel cylinders containingliquefied CO 2 in contact with a gaseous headspace. The cylinder pressure
is typically 55 to 85 bar near room temperature. Since the modifier isa normal liquid at room temperature and pressure, the mismatch inpressures requires separate pumps for the CO 2 and for the modifier.
As a consequence, binary pumping systems are the norm.
Reciprocating pumps of the type used in HPLC and SFC have low
compression ratios and cannot compress a low density gas. To pump CO 2
with such a pump, it must be liquefied. Most SFC systems use the liquid
phase from the bottom of the cylinder to minimize power requirements.
The liquid in the cylinder is in contact with a gaseous headspace. Any
increase in temperature or decrease in pressure during a standard HPLC
pump refill stroke would cause part of the liquid to vaporize, and the pumpwould create gaseous cavities in the liquid flow. Consequently, both the
CO 2 and the CO 2 pump head are prechilled to 4 or 5 °C. Most SFC systems
first use that pump to precompress the fluid from cylinder pressure to thecolumn head pressure, and then accurately meter the CO 2 flow.
Chilling the pump head usually requires a bulky heat exchanger being
bolted onto the front of the pump, making maintenance cumbersome.
Further, a circulating chiller is often used to pump a cold glycol solution
through the pump head heat exchanger. The tubing used to connect thechiller to the pump head heat exchanger requires extensive insulation toprevent water from the lab air condensing on the cold surfaces, depriving
the chiller of much of its power.
Some pumps have a Peltier device mounted on the pump head, with alarge finned heat exchanger with a fan. The fan draws lab air through theheat exchanger to carry away the heat. The cold side and the pump headneed to be insulated to prevent excessive condensation of moisture out of
the lab air.
CO 2 is far more compressible than normal liquids, even when chilled andliquefied. At supply cylinder pressures and typical chiller temperatures,
adiabatic compressibility can exceed 1200 x 10 -6 /bar, which is 8 to
27-times the compressibility of normal liquids. Fortunately, as thepressure is increased, the compressibility decreases, and by 400 bar,
the compressibility of CO 2 is similar to some normal liquids. As a121

consequence of this much higher compressibility, a much larger portionof the total available stroke length is used up simply compressing the fluid.
This puts substantial demands on the pump and has often resulted insignificant perturbations in pressure during compression.

9.1.2
Agilent analytical SFC system

The Agilent analytical SFC system is rather unusual compared to other
SFC systems, as shown in Figure 9.1. All the aspects that make it an
SFC system are contained in a separate conversion module. A largely
unmodified HPLC makes up the rest of the system. In fact, the hardware
can be used to rapidly switch back and forth between SFC and either
normal or reversed phase HPLC in only a few minutes, as outlined in alater section. This rather detailed description of a specific SFC system isincluded since many of the comments below are relevant to this system,
although in general they apply to most SFC systems.

Degasser
CO2

Modifier
Binary pump

SFC conversion
module

CO2

P

Mixer

External-loop
autosampler

Booster
ChillerBack-pressure
regulator

Waste

L

R

Modifier
trap

Figure 9.1 Schematic diagram of the Agilent analytical SFC system.

122

Columncompartment(oven)

Detector

The vapor phase of CO 2 is supplied from a steel cylinder at room
temperature, at a pressure of about 55 bar. The SFC conversion module
contains a chiller that cools both the incoming fluid and the pump headof the booster pump. The booster pump increases the pressure of the
CO 2 to 8 bar below the column head pressure. A binary pump meters andmixes the CO 2 and modifier with separate pumps. The autosampler is anexternal loop autosampler. The column compartment contains two heatexchangers. The right heat exchanger is used to precondition the mobile
phase before it enters the column. The oven is a dead-air oven, which isgood. The left heat exchanger is used to postcolumn condition the mobile
phase temperature to near the detector cell temperature. The detector
is a diode array detector (DAD) with a high-pressure flow cell. After
passing through the DAD flow cell, the mobile phase is returned to the
backpressure regulator in the SFC conversion module where it is allowed
to drop to atmospheric pressure. After the pressure drop, the fluid breaks
down to two phases. The liquid phase is collected in a trap, while thegaseous phase is vented into a fume hood.
The conversion module contains a powerful Peltier-based chiller plate
mounted behind, and chilling the pump head. Unlike other SFC systems,
the vapor phase from the CO 2 cylinder is drawn off, effectively distilling
it just prior to use. This facilitates the use of low-cost industrial grades of
CO 2 such as beverage grade. The chiller is powerful enough to liquefy the
CO 2, and cool it to below –10 °C, even at 5 mL/min. The incoming fluid isprechilled by passing through a tube imbedded in the chiller plate, before
entering the pump head. A sealed plastic cover shields the cold surface ofthe pump and chiller plate from the lab air. A small pump circulates a glycol
solution through a heat exchanger on the hot side of the Peltier device toremove heat, which is dissipated through a radiator with a fan on the rearof the instrument. Thus, the tubing containing the glycol solution is nevercolder than lab air, eliminating condensation problems.
The A-side of the binary pump (not the booster) is used to meter thecompressed CO 2 to the head of the column. The B-side of the binary pumpis used to deliver the modifier. A pressure transducer mounted in the SFC
conversion module is connected to the outlet of the binary pump througha T-piece and a pilot tube. This transducer monitors the outlet pressure of
the binary pump. This signal is used to control the outlet pressure of thebooster pump to just below the outlet pressure of the binary pump.
Neither side of the binary pump is chilled. The compressibility compensationof the A-side of the binary pump in the HPLC instrument, is set to zero.
The B-side is set to Agilent’s recommendation for the specific modifier.
123

Since the A-side does not compress the fluid, it acts solely to accurately
meter the flow. This separation of compression and metering between
two pumps in series dramatically reduces pressure noise. The conversion
module also contains a low-noise, electronic backpressure regulator.

9.2
UV detector optimization
9.2.1
Software settings

It is fairly simple to optimize a DAD for low noise. The signal wavelength
should be set to near an absorbance maximum. Since spectra are notoften used in routine analysis, the bandwidth can be opened up to allow
more light onto the photodiodes. The DAD averages the signals from all
the diodes used, which tends to lower noise noticeably. However, if someof the diodes are at a wavelength of low absorbance, including them in
the averaging is a poor choice. Either the wavelength should be changedor the bandwidth narrowed.
Many detectors also allow the adjustment of the width of the slit entering
the monochromator. The slit is usually set to the same value as the
bandwidth of the signal beam. However, if spectra are required, the slit
should be reset to its lowest value of 1 or 2 nm, to avoid blurring finestructures in the spectra.
A reference wavelength should be used whenever possible. Some noiseand drift are due to fluctuations in the lamps that occur at all wavelengths.
By using the ratio of the signal to the reference wavelength, such noise
can largely be eliminated. The reference wavelength should be set as
close to the signal wavelength as possible, without allowing significant
absorbance by the sample. The reference bandwidth is often set rather
wide at 80 nm but narrower values such as 40 nm or even lower can beused with only minimal increases in noise.
Situations should be avoided where the signal bandwidth involves
sampling light mostly from one lamp, while the reference is sampling lightfrom the other lamp, since this may increase noise. Such a situation ismost likely when the sample absorbs at relatively high UV wavelengths,
and the reference is set to even higher wavelengths.
A wavelength of 205 or 210 nm is regarded as enabling near universal
detection since most organics absorb at least a little light in that region.
CO 2 is completely transparent to below 190 nm. The UV cut-off formethanol is about 205 nm, which facilitates operation below 210 nm withminimal noise. Both ethanol and isopropanol have a slightly higher UV
cut‑off at about 210 nm. Although seldom recommended, acetonitrile has
a UV cut-off of 195 nm, enabling detection below 200 nm.

124Additives are often acids or bases that generally absorb out to about
230 nm, which can limit the ability to detect compounds with weakchromophores. A high-purity modifier should always be used to avoid
absorbance at higher wavelengths.

9.2.2
Data rate

UV noise increases 10-fold from 2.5 to 80-Hz data rate, as shown in
the upper panel of Figure 9.2. Too low data rate decreases noise but
also degrades peak shape and resolution. Too high data rate results inexcessive noise and loss of sensitivity. Clearly, the filter should be set
to just pass the fastest expected peak from any given column. Agilent
chromatography data systems list filter settings as peak width in minutes,
response time in seconds, and frequency in Hertz. The integrator reports
peak widths in minutes. The filter should be set to the next higher speed.
You can find the best filter setting empirically. After collecting achromatogram with an initial filter setting, decrease the filtering by one
setting (less filtering) and repeat the chromatogram. If the reported
peak width does not change, the original setting was probably correct.
If the reported peak width does change, decrease the setting further
and repeat the chromatogram. Keep lowering the filter until the reported
peak width is unchanged.
With older methods, using 4.6 by 250-mm columns with 5-µm particles,
a filter setting of 2.5 Hz was appropriate. With 3 by 100-mm columns with
1.8-µm particles, a filter setting of 40 Hz is usually adequate.
The actual noise measured is variable, depending on the age of the lamp
and the nature of the cell, among other factors discussed in the next
few sections. In the lower panel of Figure 9.2, an older 6-mm, 1.7-µL
cylindrical flow cell is compared to a 3-mm, 2-µL conical flow cell, bothwith a nearly new lamp. Noise was almost a linear function of frequency.
The noise with the 3-mm cell at 80 Hz was nearly an order of magnitude
lower than the Berger Instruments DAD from 1995 with a 2.5-Hz filter.

125

ASTM noise [mAU]
0.12
0.10
0.08
0.06
0.04
0.02
0

0

10
30 %

20

30

40

20 %

10 %

5%

30

40

50

60

70
80
90
Filter frequency [Hz]

50

60

70
80
90
Filter frequency [Hz]ASTM noise [mAU]
0.12
0.10
0.08
0.06
0.04
0.02
0

0

10
30 %, 6 mm
20 %, 6 mm
10 %, 6 mm

20

30 %, 3 mm
20 %, 3 mm
10 %, 3 mm

Figure 9.2 The effect of filter frequency on optimized ASTM noise with 2.5 mL/min.
Data was collected at 5 to 30 % MeOH, 150-bar outlet pressure using; (upper panel) 3-mm,
2-µL flow cell with a conical flow path and with an older lamp, and (lower panel) with boththe 3-mm, 2-µL flow cell, and a 6-mm, 1.7-µL flow cell with cylindrical flow path and with a
new lamp. The noise with the 3-mm cell nearly doubled as the lamp aged.

1269.2.3
Refractive index

UV detectors designed for HPLC have optics with a focal length that
depends in part on the refractive index (RI) of the fluid passing throughthe cell. RI is a function of the density of fluids. The RI of water at 25 °C
is 1.333. The RI of most other common HPLC solvents lies between 1.328
and 1.375. Detector designers assume an RI within this general range.
The density and the RI of such solvents changes little with pressure.
As shown previously, in Figure 2.1 and Figure 2.3, the density of both pure
CO 2 and modified CO 2 are dependent on temperature and pressure114–116.
Similarly, the RI is a strong function of both temperature and pressure asshown for pure CO 2 in Figure 9.3 and Figure 9.4. The minimum RI value
for pure CO 2 is 1.08, which is far from the RI of any of the normal liquidsolvents used in HPLC. The maximum RI calculated for CO 2 was about
1.24. The addition of a modifier to the CO2 is likely to increase the RI of the
mixture toward the values for the pure solvents. For example, the densityof methanol/CO2 mixtures is always higher than the density of either pure
fluid at the same temperature. Thus, the changes in RI caused by changes
in temperature, pressure or composition can cause large baseline shifts
and potentially increase noise due to changes in the focal length of the cell.
Refractive index [RIU]
Slope = 0.0011/bar

1.24
1.20
1.16
1.12

Slope = 0.0049/bar

1.08
1.04

10

20

Outlet pressure

30
300 bar40

50
200 bar

60

90
70
80
Column temperature [°C]

150 bar

100 barFigure 9.3 Effect of temperature on the refractive index of pure CO2 at four differentpressures. The same small changes in temperature at 100 bar result in much larger changes
in RI than at 300 bar.

127

Refractive index [RIU]
1.30
1.25
1.20
1.15
1.10
1.05
1.00

0
40 °C

100

200

50 °C

80 °C

300

400

500
Pressure [bar]Figure 9.4 Refractive index of pure CO 2 as a function of pressure at three temperatures:
40 °C; 50 °C; 80 °C.
The dashed lines indicate the change in slope between 100 and 200 bar. The same smallchanges in pressure at 100 bar result in much larger changes in RI than at 200 bar.

9.2.3.1
UV flow-cell temperatureand noise

SFC is often conducted at temperatures significantly different from room
temperatures. Fluid leaving the column can be many degrees warmer
or cooler than the flow cell in the detector, which is heated by its internal
electronics. When the fluid enters the flow cell the temperature must
equilibrate, potentially causing large changes in RI within the cell.
This causes changes in the focal length of the optics. Light hitting thewall of the cell can be reflected away from the slit, causing a variationin the light entering the monochromator. This effect generates a varying
baseline signal, which is UV noise.
One of the heat exchangers in the column compartment can be used
to condition the postcolumn mobile phase temperature to match theactual cell temperature, eliminating most noise associated with thermal
differences. A schematic is presented in Figure 9.5.

128

Autosampler

L

R

Columncompartment

Detector

Figure 9.5 Schematic diagram showing the plumbing arrangement for the column
compartment that allows the right heat exchanger to precondition the mobile phase
temperature before it enters the column, and use the left heat exchanger to postcolumn
condition the mobile phase temperature to the detector flow cell temperature.

Many modern UV detector cells have a conically shaped aperture for lightto pass through. In HPLC, this shape has been shown to be importantin minimizing noise due to RI effects caused by short-term variations intemperature. In SFC, this may not be as important. In fact, one 10-mm
path length cell with the conical flow path produced excessive noise when
the temperature of the fluid entering the flow cell was more than a few
degrees different from the flow cell temperature, as shown with the upper
curves in Figure 9.6. The curves were obtained at 5, 10, 20 and 30 %methanol in CO 2 at 2.5 mL/min and 150-bar outlet pressure. Both heatexchangers were varied between 20 and 70 °C. However, when the rightheat exchanger was varied between 20 and 70 °C, while the left heatexchanger was maintained (optimized) at the cell temperature, virtually all
the thermally induced noise disappeared as shown by the lower curves in
Figure 9.6.

129

ASTM noise [mAU]
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

20

25

30

35

No insulator –vary left heat exchanger
30 %
20 %
10 %

40

5%

45

50

55

60
65
70
Temperature [°C]

No insulator –vary right heat exchanger
30 %
20 %
10 %

5%

Figure 9.6 ASTM noise measured at four modifier concentrations using an older 10-mm,
13-µL flow cell with conical flow path and without a thermal insulator. The temperature of
the effluent from the left heat exchanger was varied between 20 and 80 °C just before it
entered the flow cell. The mismatch in temperature caused excessive noise as seen in theupper set of curves. The experiment was repeated except the right heat exchanger wasused to vary the column temperature between 20 and 70 °C, while the left heat exchanger
temperature was constant at 41 °C. Virtually all the temperature-induced noise disappeared
as shown with the near horizontal curves at the bottom of the figure.

Surprisingly, a nearly identical flow cell with a conical flow path but witha ceramic insulator between the cell and the optical bench produced
almost no thermally induced noise. The nonoptimized noise from thisinsulated cell is show as the lower, nearly horizontal curves in Figure 9.7.
The nonoptimized noise curves from the uninsulated flow cell (top curve,
Figure 9.6) are included for comparison. In this instance, the insulator
appeared to be more important than the conical shape. Many SFC
systems as little as a few years old use the uninsulated cell as standard.
Immediate improvement in noise could be expected by switching to thenewer insulated cell.

130

ASTM noise [mAU]
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2

Insulator

0.1
0

20

25

30

35

Insulator
30 %

20 %

10 %

40

5%

45

50
55
60
65
70
Left heat exchanger temperature [°C]
No insulator
30 %
20 %

10 %

5%

Figure 9.7 A nearly identical cell as in Figure 9.6 but with a ceramic insulator thermallyisolating the cell from the optical bench produced dramatically less noise, even when theleft heat exchanger temperature was varied between 20 and 70 °C, as can be seen withthe lower, nearly horizontal curves. The upper curves from Figure 9.6 are included forcomparison.

The fact that the conical shape was not the determining factor in the noiselevel may be due to the fact that the RI of CO 2 and presumably mixturesof CO 2 with modifiers, is much lower than the RI of the normal liquids
used in HPLC, and therefore bends light much less. The flow cells wereoriginally designed for HPLC, where the conical shape was apparently
more important.
On the other hand, a 6-mm, 1.7-µL flow cell with a cylindrical flow pathhas been available for many years and shows similar, but not quite
as severe, performance to the uninsulated 10-mm cell. This was true
regardless of the identity of the modifier, as shown in Figure 9.8.
Two chromatograms collected with this flow cell are shown in Figure 9.9.
In the upper chromatogram, the left heat exchanger was set to 30 °C,
while the flow cell temperature was 41 °C. In the lower chromatogram,the left heat exchanger was optimized. Note the small peaks that could
be distinguished in the lower chromatogram, due to the lower noise.

131

Np-p [mAU]
0.50
0.40
0.30
0.20
0.10

0

10

20
Methanol30

40

Isopropanol

50
Acetonitrile

60

70
80
Temperature [°C]
Methanol/H2O

Figure 9.8 Another older flow cell with a 6-mm path length and 1.7-µL volume, but acylindrical flow path, was similarly evaluated using several modifiers. The results are similar
regardless of the modifier used. This cell was only half as noisy as the 10-mm uninsulated
cell, but much noisier than the 10-mm insulated cell. Using the left heat exchanger to
optimize performance produced the optimized results in Figure 9.2.

132

31 °C

Absorbance [mAU]
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5

3

4

5

6

7

8

9

41 °C

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0

3

4

5

6

7

9
8
Retention time [min]Figure 9.9 Chromatograms collected with the 6-mm flow cell used in Figure 9.8. The
upper curve was collected with the left heat exchanger set to 30 °C. The lower curve wascollected with an optimized left heat exchanger of 41 °C, 100-bar outlet pressure.

Confusingly, an uninsulated 3-mm path length cell with a conicalflow path, but without the insulation, showed little susceptibility totemperature induced RI effects, similar to the 10-mm insulated cell.
However, it was still necessary to adjust the left heat exchanger
temperature to the flow cell temperature to achieve minimum noise as
shown at 40 Hz in Figure 9.10. Insulating that cell with a ceramic spacer
did not improve noise further. Surprisingly this 3-mm cell produced only
87 % of the noise of the longer, much larger volume (13 versus 2-µL) cell.

133

ASTM noise [mAU]
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0

20

25

30

35

40

45

50

55

65
60
Temperature [°C]

Figure 9.10 Yet another flow cell, with a 3-mm path length, 2-µL volume, uninsulated, witha conical flow path produced much less noise than the cell in Figure 9.8. Using the left heatexchanger to optimize further, eliminated noise.

9.2.3.2
Column outlet pressureand noise

All electronic backpressure regulators (BPR) are continuously dithering
their control electronics around the set point. This results in small
variations in the outlet pressure. If not tightly controlled, this dithering
can lead to significant noise. The Agilent analytical SFC system contains astate-of-the-art BPR for SFC with a pressure noise of as little as ±0.05 bar
at 150 bar. This is greater than 10-times better than previous generationsof electronic BPRs for SFC.
As mentioned previously, the RI of CO 2 is a strong function of its density,
which in turn depends on its temperature, pressure and composition.
Despite the low-pressure fluctuations obtained with the Agilent
analytical SFC system, some pressure-related noise can be seen undersome conditions. It should be noted that probably 80 % of all reports
on packed column SFC from before 1990 to after 2008 used 40 °C with
100-bar outlet pressure as the primary set points. In Figure 9.4, it was
shown that at 40 °C and 100 bar the RI of CO 2 changes steeply withsmall changes in pressure. This is an inherent characteristic of the fluid.
In past years this was largely ignored, since other noise sources werelarger and tended to obscure the effect of pressure-induced noise.
The slope of RI against pressure decreases fairly rapidly above
approximately 120 bar. A plot of UV detector noise against outlet pressureis shown in Figure 9.11. The outlet pressure was changed from 100 to
200 bar in several stages at 40 °C. At 100 bar, the noise was about
0.11 mAU with a filter setting of 0.05 min (5 Hz). At 120 bar, the noise
dropped by over 50 % to about 0.04 mAU. At 140 bar, the noise fell134

another 50 % to about 0.02 mAU. In the author’s laboratory the default
settings for outlet pressure and temperature have been 150 bar and 50 °C
for the last 4 to 5 years. For the lowest possible noise, an outlet pressureof 200 bar is recommended.
ASTM noise [mAU]
0.12

40 °C

0.10
0.08
0.06
0.04
0.02
0

20

100

120

140

160

180

220
200
Pressure [bar]Figure 9.11 Effect of outlet pressure on Noise. At 40 °C, the refractive index changes much
more with small changes in pressure at low column outlet pressures such as 100 bar, than athigh outlet pressures such as 200 bar. Conditions: 3 mL/min of 4 % MeOH, 40 °C, filter set
at greater than 0.05 minutes.

With modified mobile phases, especially with modifier concentrations
much above 10%, pressure becomes a secondary control variable withlittle impact on either selectivity or retention. Since the viscosity of thefluids is so low, the pressure drops occurring at optimum flow rates, evenusing sub-2-μm particles, are modest, so a set point of 200 bar for theoutlet pressure has only minor consequences. The column head pressure
should seldom exceed 400 bar, although the system is capable of operating
to 600 bar.
There are instances such as with mixtures of similar nonpolar compounds
where it is desirable to operate at pressures even below 100 bar to
maximize resolution between closely related compounds. Increasing thetemperature tends to flatten out the curves of RI against pressure, which
can help. Under such cases, there is a trade-off between sensitivity andresolution since the RI is an inherent property of the fluid.

1359.2.3.3
Low modifier concentrationand noise

In SFC, the modifier concentration seldom exceeds 50 %, except during
rapid screening runs. Since the range of modifier concentration is lower
than in HPLC, concentrations well below 10 % modifier are often used.
However, at low flow rates and low modifier concentration the modifier
pump may not cycle more than once a minute or even slower. At 2 mL/min
and 5 % modifier, the modifier pump is delivering 100 µL/min. The Agilent
analytical SFC system has a variable stroke length. At that flow rate,
the pump stroke is probably 20 µL. Thus, the pump makes five strokes
per minute.
When the fluid is being compressed to working pressure, no flow leaves
the primary piston, while the secondary piston is withdrawing fluid fromdownstream. A downstream check-valve between the B pump andthe mixing point is intended to prevent this backflow from reaching themixing point.
After the compression stroke, the modifier pump injects additional modifier
to make up for the total shortfall. The flow and composition are accurate
but with a periodic perturbation of the baseline. The mixer must attempt
to average out this slow oscillation in modifier flow, although the variation
may occur only once a minute or slower.
The primary mixer was designed for mainstream HPLC, involving atleast 1 mL/min with 5 % modifier or greater. At low flow or modifier
concentration, the standard mixer has difficulty completely blendingout these perturbations. Since the modifier has a different RI comparedto the CO 2, any oscillation in modifier concentration, particularly at low
concentrations, creates another lower frequency form of noise at themodifier pump frequency.
A plot of noise against modifier concentration is presented in Figure 9.12.
At 2 mL/min, 40 °C and 200 bar, the worst noise was found at 1 %methanol, where the pump was operating at only one stroke per minute.
Any need to operate at such low modifier concentrations is the result ofusing a modifier that is too strong for the solutes.

136

ASTM noise [mAU]
0.12
0.10
0.08
0.06
0.04
0.02
0

0

2

4

6

8

10

16
12
14
Methanol content [%]Figure 9.12 Noise induced by modifier pump at low modifier concentrations. The modifier
pump is operating at low frequency and perturbations in composition at that low frequency
are not being adequately averaged by the standard mixer. Conditions: 2 mL/min, 40 °C,
200 bar. Filter greater than 0.1 minutes.

There are newer versions of mixers that provide variable path lengths suchas the Agilent Jet Weaver mixer, but with much lower volume.
Alternately, avoid low modifier concentrations by decreasing the solvent
strength, that is, the polarity, of the modifier. With a weaker modifier, thecompounds are more retained, requiring higher concentrations. Ethanol
decreases solvent strength significantly, compared to methanol, as
pointed out in Chapter 2 “The Mobile Phase”. Solvent strength is further
decreased with isopropanol. Many other organic solvents are also usable
as modifiers that further decrease solvent strength, increase retention, orchange selectivity. However, some solvents such as chlorinated solvents
have negative environmental impact and are avoided.

9.3
The doubling gradient

There are further implications regarding the rule of thumb that doubling themodifier concentration halves retention. The first small additions have adisproportionate effect on retention but at higher modifier concentrations,
small changes have minimal effects. Nevertheless, modifier concentration
is most often programmed linearly. Thus, at the beginning of a gradient,peaks are pushed together and at the end of the gradient they arebroadened. If the modifier concentration is initially changed only gradually,
but the rate of change is progressively increased during the run, the early
eluters will be better resolved while the late eluters will be sped up. Such
gradients were proposed more than 20 years ago but apparently have notbeen widely embraced.
137

The column hold-up time, t0 , (retention time of an unretained peak) is
a convenient time base for translating methods between columns ofdifferent dimensions. If you start at the lowest feasible percent modifier,some compound might elute in the first few column hold-up times after t0 .
With a properly chosen modifier, most solutes in a complex mixture will
be easily retained at the initial conditions. Holding the initial concentrationfor one or more column hold-up times produces the maximum resolution
under the weakest elution conditions. Doubling the concentration
during each subsequent multiple of t0 results in all peaks emerging with
about equal peak widths, which is similar to an isocratic peak withk approximately equal to 1 to 2. If each doubling of the concentration
reduces retention by a factor of 2, starting from 2 % and progressing
to 64 % should change retention by about 64 %. This type of gradientcan be rapid yet degrade resolution no worse than about 40 % from
optimized isocratic separations.

9.4
Autosampler
considerations
9.4.1
Variable-loop versus
fixed-loop

Many current autosampler designs use the variable-loop or flow‑throughdesign concept, but SFC is incompatible with this approach. In avariable‑loop autosampler, a needle is connected to a high-pressure
metering device (such as a syringe) with a piece of flexible stainless-steel
tubing that acts as the sample loop. During sample loading, the mobile
phase is diverted directly from the pump to the column by switching an
injection valve into bypass (load) mode. Sample can then be withdrawn
from a vial at atmospheric pressure into the needle and loop. After
withdrawing the sample, the needle is pushed into a high-pressureneedle seat, and the injection valve is switched (inject), diverting themobile phase from the pump through the metering device, loop, needle
and seat, then through the valve onto the head of the column. The needle
and the sample loop can remain in the inject position to be flushed bymobile phase in order to minimize carryover.
In HPLC, this approach has many beneficial aspects, since the mobile
phase is essentially an incompressible liquid. Switching the valve to thebypass position isolates the metering device, loop, and needle from theflowing stream and connects the needle seat to waste. The fluid in thisisolated section immediately expands minimally and the pressure drops
to atmospheric. The metering device, loop and needle remain filled withthe liquid mobile phase.
In SFC, the mobile phase behaves like a compressed gas. When thevalve is switched to the bypass position, the contents of the metering
device, connecting tubing (loop), needle and needle seat all expand up138to 500-times their compressed volume, and rapidly vent through the waste
port of the valve to ambient. After this expansion, the whole injection
system is filled with a low density gas. Attempting to withdraw the next
sample results in the cavitation of the metering device, which results in it
being unable to withdraw any further samples from the sample containers.
Variable-loop autosamplers need to be modified for use in SFC byconverting them to fixed-loop operation. An external fixed-volume loop
is connected between opposite ports of a 2-position/6-port valve.
The sample is withdrawn from a vial, and then pushed through the loopwith the same high pressure metering device. When the injection valve
is turned, only the loop and rotor groves experience the high pressure of
the system.
When using an injector program, the needle should be left in the needle
seat, and withdrawn only after the valve returns to the bypass position.
If the needle is prematurely withdrawn from the needle seat before thevalve is switched, the contents of the metering device (about 200 µL), theloop and the needle all rapidly vent as a highly compressed gas into theinterior of the sample compartment, contaminating all the surfaces withpotentially harmful mobile phase components such as some additives.
If the needle is left in the needle seat, all this fluid is vented out the waste
line. The waste line should always be connected to a liquid trap andthe vapor phase should be diverted into a fume hood. Before the next
injection can be made, a wash pump mounted in the SFC module washes
the metering pump, loop and needle with fresh solvent.

9.4.2
Carryover

The SFC control module in the Agilent analytical SFC system contains awash pump that thoroughly washes the injection valve, syringe, connecting
tubing, and needle between injections. This usually results in exceptionally
low carryover. An Example is shown in Figure 9.13. With the highly
concentrated sample, the peak with a reported area of greater than 17,500
was off-scale at greater than 2500 mAU due to detector saturation.
A subsequent blank injection produced a peak with area reported as 0.88.
Carryover in terms of area was about 0.005 %.

139

[mAU]
2500

4.545

0.15

Area = 17,545.3

2000

Blank Area
= 0.88

0.10
0.05

1500

0.00
0.05

1000

3

500

5 [min]

4

0
0

2

4

6

8

10

12
Time [min]Figure 9.13 Carryover of caffeine. A highly concentrated sample was injected followed by
a MeOH blank. Between injections the syringe, connector tubing, needle, needle seat, valve
and external loop were washed with approximately 0.5 mL MeOH. Carryover was 0.005 %.
Conditions: 2 mL/min of 5 % MeOH in CO2, 40 °C, 200 bar. Column: 4.6 by 250 mm, 5 µm,
RX-SIL. Filter greater than 0.05 minutes, 15 µL into a 5-µL loop with 1.5-µL air bubbles oneach side of the sample plug.

9.4.3
Reproducibility

In regulated environments, reproducibility of both retention time andpeak area are required to be better than ±1 % RSD, when signal-to-noise
is greater than or equal to 100. These levels of reproducibility can beachieved easily in SFC, provided full-loop injections are made and the loop
is overfilled at least several times. For example, with a 1.25-µL loop, an
injection volume of 5 to 7 µL should be more than adequate. It is possible
to load the sample such that only the middle, undiluted part of the sample
plug is in the external loop.
Retention-time reproducibility is usually better than 1 % in modern SFC.
However, area reproducibility is also dependent on signal-to-noise.
To achieve less than 1 % area reproducibility, the uncertainty in the signal
can be no better than about ±1 %, if the signal-to-noise is 100. However,with low noise such as 0.01 to 0.02 mAU, a signal-to-noise of 100 canbe achieved with a peak of only 1 to 2 mAU high.
Two sets of reproducibility measurements (n = 10) are presented. In thefirst, a high modifier concentration and high pressure were used, which
produces among the best reproducibility. The first example, presented inTable 9.1, shows the retention time and area reproducibility of caffeine,
theophylline and theobromine. Retention-time reproducibility averaged
0.033 % RSD. Area reproducibility, with a signal-to-noise less than 300
to about 1400, was between 0.1 and 0.29 % RSD.

140

Caffeine

Theophylline

Theobromine

RT

Area

RT

Area

RT

Area

1.420

1471.81.895

2305.2

2.198

273.9

1.419

1471.91.895

2309.8

2.197

275.7

1.420

1472.51.898

2307.9

2.198

275.8

1.419

1472.31.895

2308.7

2.197

273.3

1.420

1473.11.895

2308.8

2.197

274.5

1.419

1472.41.895

2308.5

2.198

274.2

1.420

1474.81.895

2307.0

2.198

274.0

1.420

1475.21.895

2312.8

2.198

273.9

1.420

1474.91.895

2309.0

2.195

274.3

1.420

1475.31.895

2312.9

2.195

274.1

Mean

1.41971473.4

1.89512308.82.1985274.37SD

0.00051.40210.00032.32030.001

0.7524

% RSD

0.04

0.10

0.02

0.10

0.05

0.27

Table 9.1 Reproducibility of caffeine, theophylline and theobromine under isocratic
conditions with high flow rate, high % modifier, and high pressure. Conditions: 40 % MeOH,
200 bar, 4 ml/min, 40 °C. Column: 4.6 by 250 mm, 5 µm, RX-SIL.

The second set of reproducibility measurements were collected using a
10 % per minute gradient in the separation of a mixture of profens andxanthenes. Modifier composition was held constant at 2 % for 1 minute,then 10 % per minute to 42 %, at 3 mL/min, 30 °C and 200 bar, using
a 4.6 by 250 mm, 5-µm silica column. The retention-time RSDs averaged
0.11. The area RSDs averaged 0.38, as seen in Table 9.2.

141

Ibuprofen

Caffeine

Theophylline

Ketoprofen

Theobromine

RT

Area

RT

Area

RT

Area

RT

Area

RT

Area

2.850

507.02

3.372

440.324.497

1183.3

4.883

1875.05.777

383.572.849

503.983.372

439.384.493

1173.94.879

1875.1

5.772

385.082.848

502.803.372

438.884.491

1171.94.878

1873.45.789

384.982.850

507.983.373

442.804.489

1174.7

4.874

1875.7

5.785

384.742.851

507.983.375

441.184.487

1171.3

4.872

1873.85.782

384.872.851

508.223.375

442.504.485

1175.7

4.870

1878.35.759

381.782.853

508.483.377

444.01

4.485

1182.24.871

1890.05.758

385.892.854

508.003.378

441.284.483

1170.04.888

1870.05.753

383.182.852

507.813.377

442.824.481

1172.74.888

1877.2

5.752

382.11

2.852

508.423.377

443.084.480

1178.3

4.884

1880.95.749

385.25

Mean

2.851

507.083.3748441.4

4.48711175.24.8723

1878.95.7814384.12SD

0.00182.00020.00242.109

0.00544.42590.00595.44240.0093

1.3978

% RSD

0.07

0.39

0.07

0.48

0.12

0.38

0.13

0.29

0.18

0.38

Table 9.2 Reproducibility with a 10 % per minute gradient: 3 mL/min of 2 % MeOH for 1
minute then 10 % per minute to 42 %, then hold, 40 °C, 200 bar on a 4.6 by 250 mm, 5 µm,
RX-SIL column.

9.4.3.1
Partial-loop injections

With partial-loop injections, retention-time reproducibility was similar to
full-loop injections, but area reproducibility degraded somewhat. Linearity
was excellent. At greater than 1.5 µL on a 5-µL loop, area RSD was about
2 %. At less than 1 µL, the RSDs are less than 10 %.

9.4.4
Injection volume and theeffects of strong sample
solvent

This is a neglected aspect of SFC that needs far more attention. Samples
are often dissolved in a relatively strong solvent such as methanol. Thispotentially causes a problem, which is also present in HPLC. The mobile
phase in SFC can be rather nonpolar, particularly when the modifier
concentration is low. Injecting a sample dissolved in a strong (polar)
solvent into a low concentration of the same solvent, used as modifier,results in a situation where at least the center of the sample plug is much
more polar than the bulk mobile phase. Solute molecules in the center ofthis band experience a stronger solvent than the rest of the mobile phase,and are subsequently less retained than solute molecules on the edgesof the band. This results in peak broadening, tailing, and loss of resolution
between closely retained peaks.

142

Increasing the injection volume often accentuates the effect. An example
using the separation of four corticosteroids, dissolved in methanol, on a
4.6-mm id column using 8 % methanol as modifier, was shown in Figure2.5. Pure methanol as sample solvent is much more polar (a much stronger
solvent) than 8 % methanol in CO2.
You would expect a linear relationship between the injection volume ofa standard and the peak height of the resulting peaks, if there was no
broadening due to the strong sample solvent effect. However, the peakheights of the solutes were found to be a linear function of injection volume
up to only about 6 to 7 µL, as shown in Figure 9.14. At higher injection
volumes, the peak height rolled off significantly. Similarly, a plot of peakwidth at half height also shows a break in the same vicinity of injection
volumes, although the break is less clear-cut, as shown in Figure 9.15.
Finally, a plot of peak area against injection volume was linear, as shownin Figure 9.16, indicating that the appropriate amounts were being injected
but progressively broadened at higher injection volumes.
Peak height [mAU]
700
600
500
400
300
200
100
0

0

5
Testosterone

10
Estradiol

15

20
Hydrocortisone

25

30
35
Injection volume [µL]

Estriol

Figure 9.14 The effect of strong sample solvent. The effect of injection volume on thepeak height of testosterone, estradiol, hydrocortisone and estriol dissolved in MeOH, using
8 % MeOH in CO2 as the mobile phase at 40 °C, 100-bar outlet pressure. Column: 4.6 by
150 mm, 5 µm, bare silica. Height rolls off above about 8 µL.

143

Peak width [min]
0.30
0.25
0.20
0.15
0.10
0.05
0

5

0

Testosterone

10

15

Estradiol

20
Hydrocortisone

25

30
35
Injection volume [µL]

Estriol

Figure 9.15 The effect of injection volume on the peak width of testosterone, estradiol,hydrocortisone and estriol dissolved in MeOH, using 8 % MeOH in CO2 as the mobile phaseat 40 °C, 100-bar outlet pressure. Column: 4.6 by 150 mm, 5 µm, bare silica. Peak width
starts to degrade about 6-µL injection volume.
Area counts
9000
8000
7000
6000
5000
4000
3000
2000
1000
0

0

5
Testosterone

10
Estradiol

15

20
Hydrocortisone

25

30
35
Injection volume [µL]

Estriol

Figure 9.16 The effect of injection volume on the peak area of testosterone, estradiol,hydrocortisone and estriol dissolved in MeOH, using 8 % MeOH in CO2 as the mobile phaseat 40 °C, 100-bar outlet pressure. Column: 4.6 by 150 mm, 5 µm, bare silica. Peak area is alinear function of the amount injected.

Samples should never be dissolved in a solvent more polar than themodifier in use. For example, the sample solvent should never be methanol
when the modifier is ethanol or isopropanol. If the sample is dissolvedin the same solvent as used as the modifier, the user should strongly
consider switching the sample solvent to a much less strong (less polar)
144

solvent, particularly if the initial modifier concentration during injection
is low (less than 10 % modifier). For analytical situations, this switch toa much less polar solvent is unlikely to create any solubility issues of thesample in the solvent. Most users underestimate the solubility of mostsmaller, drug-like molecules or larger natural-product type molecules inthose less polar solvents.
The results in Figure 9.14, Figure 9.15 and Figure 9.16 were obtainedwith a 4.6 by 150 mm column with 5-µm particles. If the maximum
injection volume, without band broadening, were found for the 4.6-mm
id column, transferring the method to a column with smaller diameteror smaller particles, or both, the injection volumes should be decreased
in direct proportion to the cross-sectional area of the column, and to
the particle diameter. If the maximum injection on a 4.6-mm column
with 5-µm particles is 10 µL, injecting onto a 3-mm id column with thesame particles should be no more than about 4.2 µL. Simultaneously
decreasing the particle size to 1.8 µm, while keeping the total efficiency
the same (decreasing column length) requires an injection volume of no
more than 1.5 µL. Obviously, with small inside diameter columns packed
with sub-2-µm particles, a small loop may be required, which may not bereadily available, commercially. A 1.25-µL loop can be made with a 10-cm
length of 125-µm (0.005-inch) tubing. Using Agilent 1/32-inch outside
diameter tubing with 1/16-inch ends makes this simple and easy to install.
An alternative is the use of partial-loop injections, with a larger loop, but
RSDs may not be adequate.
At higher modifier concentrations, this strong sample solvent effect ismuch less obvious. In fact, at 40 % methanol as modifier, injections of up
to 90 µL of a warfarin standard dissolved in methanol could be injected
onto a 4.6 by 250 mm column packed with Regispack stationary phasewith only modest band broadening, for analytical scale semipreparative
separations.
Before an injection, the wash pump in the Agilent analytical SFC system fills
the loop with a wash solvent, chosen by the user. If this wash solvent is
too strong a solvent, it needs to be displaced by several loop volumes of theactual sample dissolved in a weaker solvent (overfilling the fixed loop).
The worst-case scenario is to perform a partial-loop injection by insertinga small sample plug into the fixed loop, filled with a stronger solvent, but
not displacing most of this stronger solvent with sample. A subsequent
injection would then place a large volume of this highly polar (strong)solvent onto the column with the (weaker) sample plug, sweeping it down
the column with most of the plug experiencing minimal retention, but145some of it having the strong solvent diluted down to where parts of thesample are far more retained. Subsequently, and in particular, the trailingedge is significantly broadened with a major tail.
If you are generating your own injection programs, an air bubble oneither end of the sample plug creates segmented flow, which minimizes
mixing between the sample and the wash solvent but can be usedalso to displace excess wash solvent. Large sample loops should be
avoided. For method development, you should start with a small volume
of a concentrated sample to try to minimize peak widths and maximize
resolution, before trading resolution for sensitivity.

9.5
Miscellaneousconsiderations
9.5.1
Leaks

Liquid-leak detectors mandated by regulators for use in modern HPLC
equipment are incapable of detecting or locating leaks in SFC, except, due
to leaks in the degasser, or in the B-side of the binary pump since thosemodules are involved with pumping normal liquids. Elsewhere in the system,leaks will be gaseous with no dripping liquid. These types of leaks cannot
trigger the liquid-leak detectors.
A visible leak, intermittently sputtering particles of dry ice, is a huge leak,generally meaning that the fitting in question is barely finger-tight. With
such a leak, the software will probably shut down the system before theuser has time to remedy the situation.
Much smaller leaks will be invisible to the naked eye, but may periodicallyfreeze shut, then thaw, creating unstable pressure. This can cause either
the booster pump, or the BPR, or both, to attempt to restabilize the pressure.
The system may never get to the ready state, and will eventually shut down.
Such leaks may make the fitting, where the leak is occurring, feel slightly
cooler than the rest, but this not necessarily true. This should still be
regarded as a major leak.
Built-in software-based leak tests, such as available for the Agilent
analytical SFC control module can assist to detect leaks automatically,
but this involves installing certain hardware items and running diagnostic
software, which could take several hours. However, such functionality
cannot detect the actual position of any leak.
There is a much simpler, faster, and more effective way to locate leaks
based on vast experience in gas chromatography. A drop or two of soapy
water applied to each fitting almost instantly indicates when there is aleak in that fitting, by making either a huge bubble, indicating a giant
leak, or a few tiny bubbles, indicating an almost irrelevant tiny leak. Thereare commercial products designed to perform this task. Alternately, one

146can dilute any household dish-washing detergent to perform the sametask. However, some of the commercial products have a convenient, long
1/16- inch applicator tube that allows easy dispensing of a drop or two of
soapy water onto each fitting.
Every time the chromatographic system is modified (for example, a changeof column), every fitting that was loosened and refitted should be tested
using soapy water immediately after pressurization, to determine whether
even tiny leaks exist. Immediately after applying a drop or two of thesolution, minuscule bubbles of less than to much less than 1-mm diameter
many emerge from the fitting. However, if continuing observation of thefitting fails to find further bubbles, the fitting is tight. This should be thegoal of every fitting in the entire system.

9.5.2
FerrulesHPLC fittings were not necessarily designed to handle low viscosity fluids
such as the mobile phases used in SFC. As a result, fittings are often
overtightened to stop leaks. Two-piece, stainless-steel ferrules tend to
deform after a relatively few number of disconnections and reconnections,and become progressively more difficult to seal. Eventually the ferrules
can jam in the fitting. After deformation starts, it is recommended toreplace the tubing.
Single-piece, stainless-steel ferrules tend not to distort on repeatedretightening. However any slight bend in the tubing near the end can jam
the ferrule in the fitting, making it close to impossible to remove the tube.
Polyimide ferrules tend not to distort and are easy to seal leak-tight,
at least on stainless-steel tubes. However, they can jam in the fitting.
Fortunately they can usually be removed easily, since they are relatively
soft. PEEK ferrules are not recommended for use with stainless-steel
tubing since such ferrules may not adequately hold the tube against thehigh pressures encountered and may fail, allowing the tube to be ejectedfrom the fitting and vent the system. With PEEK tubing the author always
uses stainless-steel ferrules, the tips of which can cut into the tubing,making a permanent geometry.
The most common location for repeatedly making, and remaking
connections is in column end-fittings. Getting a fitting stuck in a column,
often means you cannot get the fitting leak-tight, potentially ruining thecolumn for further work. In the past, the author used a 10-cm piece oftubing connected to each end of a new column, with new tubing, nut
and ferrules, and which was never removed from the column. This moved
the vulnerable fitting out of the column and to the other end of the tube.
147

If that fitting fails, the tubing can almost always still be removed from thecolumn and replaced.
Columns from different manufacturers can have different pilot-hole depths(the length of tubing that protrudes past the ferrule). Depths can change from
1.5 to at least 4 mm. This means that when changing columns, the connector
tubing may also need to be changed, to avoid creating a mixing chamber or
diffusion chamber, which would cause excessive band broadening.
Recently, Agilent has introduced several new fittings that largely solvethese problems. In one type, the polyimide ferrule is attached to the nut.
The nut can be screwed into the fitting until finger-tight. The tube is pushed
through the ferrule until it touches the seat of the fitting. A lever can thenbe engaged, and locked in place, which presses the ferrule firmly against
the seat with a spring of the appropriate tension to make a finger‑tight seal.
These seals are ideal for use with columns with different pilot‑hole depths,
since the depth is adjustable. These fittings are guaranteed for more than
200 make-and-break connections, and are recommended particularly foruse in column end-fittings.

9.6
Ultrahigh performancewith SFC

At the time of writing, there are no standard commercial SFC instruments
configured as a true ultrahigh performance supercritical fluid chromatograph.
In this context, ultrahigh performance SFC involves the use of sub-2-µm
particles in columns with less than 4.6-mm id.
The mere use of such particles and columns does not indicate true
performance. At a minimum, the instrument characteristics should be suchthat at least 90 % of the theoretical efficiency of the column should beachieved at k greater than or equal to 2.
SFC usually does not require an ultrahigh pressure instrument to use
sub-2-µm particles, due to the much lower viscosity and resulting
low-pressure drops of CO 2 -based mobile phases. Even at relatively highmodifier concentrations such as 50 to 60 %, the pressure drop across a
100-mm column packed with 1.8-µm particles seldom exceeds 250 bar.
However, the use of much longer columns or above optimum flow rates
with smaller particles, makes a high-pressure system desirable. Althoughultrahigh pump pressures are not necessary, other aspects of instrument
design determine the capability to use sub-2-µm particles effectively

1489.6.1
Flow cell volumes

Most chromatographs are designed with a specific column in mind, which
dictates the size of all the components particularly that of the UV flow cellused. As a rule of thumb, any UV detector cell should have a volume lessthan one-fifth of the volume of mobile phase containing the fastest peakthat the system is expected to produce. Larger cells cause extracolumn
band broadening, and loss of efficiency and resolution between closely
resolved peaks. For a perfectly packed 4.6 by 150 mm column, packed with
5-µm particles, the volume holding a peak with k equal to 1 is roughly (4σ)
150 µL. A flow cell of the order of 30 µL could be used. With a 4.6 by 150
column with 3.5-µm particles, the peak volume (k equal to 1) would be
about 80 µL, requiring a cell volume of less than or equal to 18 µL, to avoid
broadening the early eluting peaks.
The optimum flow rate for 1.8-µm particles in a 4.6-mm id column
approaches 5 mL/min, which is the maximum flow for the Agilent
analytical SFC system. Decreasing the column inside diameter to 3 mmdecreases the optimum flow rate to about 1.7 to 2.0 mL/min, dependingon the modifier concentration. With a smaller inside diameter, the cellvolume must be significantly smaller. With a 3 by 100 mm column packed
with 1.8-µm particles, the volume holding a peak with k equal to 1 would
be about 15.5 µL and the cell volume would need to be less than 3 µL.
With a 2.1 by 100 mm column packed with 1.7-µm particles, the peakvolume with k equal to 1 would be theoretically 7.3 µL, requiring a cellvolume less than 1.5 µL.
Recently, a new 3 mm, 2-µL flow cell has become available that has aconical through-hole, and is much less susceptible to thermally induced
noise. This type of flow cell is highly recommended when using columns
packed with sub-2-µm particles.

9.6.2
Tubing

The tubing connecting the injection valve to the column, and the column
to the UV detector can also contribute significantly to extracolumn bandbroadening. None of the currently available SFC systems are designed tohave tubing diameter or length to make them truly ultrahigh performance
SFC systems.
The variance of a peak caused by the dispersion of the connecting tubing isdirectly proportional to tubing length, and to the fourth power of the radius.
The standard 175-µm id tubing in the Agilent analytical SFC system has
a too large inside diameter and is too long for use with sub-2-µm particle
packed columns. Therefore, instrumentation to be used with sub‑2‑µm
particles needs to be slightly modified in order to achieve optimum
performance.
149With the smaller 3-mm detector cell and most of the tubing replacedwith 125-µm id tubing, the Agilent analytical SFC system can deliver
approximately 90 % of the efficiency of a 3 by 100 mm column packed
with 1.8-µm particles, and with k as low as 2.5. This type of column can
produce about 20 % higher efficiency in less than one-third of the time,compared to a 4.6 by 150 mm column packed with 3.5-µm particles.

9.7
Hybrid systems switching
between SFC and HPLC

150

The Agilent analytical SFC/UHPLC hybrid system can be configured as a
hybrid system117 that can be switched rapidly back and forth between SFC
and either normal phase or reversed phase HPLC operation, even withina sequence. This is surprising since water is miscible with CO2 at no morethan a few tenths of 1 %. However, once a polar modifier is present, it ismuch easier to flush the system between techniques. An additional pump,typically a quaternary pump, is configured along with a 2-position/10‑port
valve. The regular SFC binary pump is only used in SFC mode. The additional
pump is used only to deliver the HPLC solvent mixture, which is most often
aqueous-based. The two systems share the degasser, autosampler, column
compartment and detector. A schematic diagram is presented in Figure 9.17
for the system operating in SFC mode. Figure 9.18 presents the system
operating in HPLC mode. Both a reversed phase and a polar column aremounted in a single oven on a 2-position/6‑port column-selection valve orusing a method development setup that facilitates automated screeningof multiple columns117.

SFC Mode

2

3

1
10

4

9

5
6

7

8

Degasser
HPLC Pump

CO2
Booster

SFC Pump

BPR

Autosampler

Columncompartment
SFC
Waste

Modifier
trap

HPLC
Waste

Detector

Figure 9.17 Schematic of the Agilent analytical SFC/UHPLC hybrid system configured
for SFC operation.

151

UHPLC Mode

2

3

1
10

4

9

5
6

7

8

Degasser
HPLC Pump

CO2
Booster

SFC Pump

BPR

Autosampler

Columncompartment
SFC
Waste

Modifier
trap

HPLC
Waste

Detector

Figure 9.18 Schematic of the Agilent analytical SFC/UHPLC hybrid system configured
for UHPLC operation.

When the valve is switched to change techniques, a small amount of themobile phase from the other technique may enter either column, but isquickly re-equilibrated. Switchover takes no more than a few minutes.
A mix of eight compounds from three different solute families; caffeine,
theophylline, cortisone, prednisone, hydroxycortisone, prednisilone,sulfamerazine, and sulfaquinolaline were separated118 by both SFC andUHPLC on the same equipment. Six SFC runs were followed by two
washing runs, and a blank to clear the system of the SFC mobile phase.
Six UHPLC injections were performed, followed by one SFC wash and ablank. Four more SFC runs were performed, followed by two washes anda blank, and four UHPLC runs, further washes, and finally, two extra SFC
runs. All the runs were controlled as part of a single automated sequence.
All 12 of the SFC runs were overlaid and are presented in the top of
Figure 9.19. The authors reported that the retention-time RSD for isocratic
separations was between 0.04 and 0.08 %, while gradient runs produced
an RSD between 0.02 and 0.04 %.

152

[mAU]
120
SFC
100
12 overlapped
80
injections
60
40
20
0
0
0.5
1.0

7
1

1.5

4
2

5

3

8

6

2.0

2.5

3.0

3, 4, 5, 6

700
600

HPLC

10 overlapped
injections

500
400

7

8

300

1

200

2

100
0

0

1.0

2.0

3.0

4.0

5.0
Time [min]Figure 9.19 Results of a sequence where six SFC runs, followed by six UHPLC runs, four
SFC runs, four UHPLC runs, and finally two SFC runs were made. The system was flushedwith several gradient washing blanks during switchover between methods. All 12 SFC runs
were overlaid, as seen in the upper chromatograms. Similarly, the reversed phase HPLC runs
were all overlaid as shown in the lower chromatograms.

The 10 UHPLC chromatograms were also overlaid (lower panel in
Figure 9.19), showing retention-time reproducibility with RSDs about
0.2 %. Comparing the results from the two methods demonstrates once
again the orthogonality of the two techniques, since the elution orderis significantly different in the two data sets. Such large differences inselectivity should be welcome in situations where two orthogonal methods
are required. For example, to ensure that trace contaminants are not hiding
under a major peak. In this particular comparison, the UHPLC method uses
a 2.7-µm packing compared to the 5-µm packing used for SFC.

153

9.8
Interfacing to mass
spectrometers

Starting more than 25 years ago, almost every kind of mass spectrometerhas been interfaced to SFC systems. As mass spectrometer designs have
improved, the interfaces have become simpler. In fact, it is now feasible
to directly couple the outlet of the BPR to the inlet of the MS source,
simply using a piece of 125 to 250 µm stainless-steel tubing, although
this tends to broaden peaks somewhat, see Figure 9.20. Most of thepressure drop will occur across the BPR but there will be substantial backpressure caused by the tubing. Nevertheless, by the time the fluid reaches
the source, most of the mobile phase will have vaporized, becoming apoor solvent. A make-up pump delivering a small flow of modifier, usually
methanol, is used to ensure none of the solutes stick to the walls of thetransfer line. There are no issues with changing split ratios when thecomposition changes.

Make-up

Binary Pump

Autosampler
Columncompartment

BPR

MS

Booster

Detector

Figure 9.20 Straightforward interfacing of SFC to MS. The total flow passing through the
BPR is fed into the MS source. A make-up pump adds a small flow of a polar modifier andadditive before the BPR.

154

An alternate interface, consisting of several T-pieces and a 1-meter
length of 50-µm id stainless-steel tubing is shown schematically in Figure
9.21. After the UV detector, the flow is split with a T-piece. Typical split
ratios are 5:1, with the lower flow going to the MS, while most of theflow passes through the BPR. It is useful to have a make-up pump for thisconfiguration also. The make-up flow is added either before, or after the
split, through another T-piece. The restrictor is mounted downstream ofthis second T-piece, and directly onto any APCI or ESI source. The make-up
flow insures there is enough liquid entering the MS for proper ionization,and may be used to minimize changes in split ratio caused by changes incomposition and viscosity.
Make-up

Restrictor
5:1 split

Binary Pump
Booster

BPR

Autosampler
Columncompartment
QTOF with ESI

Detector
to BPR

Figure 9.21 Alternate interfacing scheme where two T-pieces after the detector allow
the addition of modifier from a make-up pump, and allow the flow to be split. In this case,
1 meter of 50-µm stainless-steel tubing was used to create a split ratio of about 1:5 withthe lesser amount going to the MS.

The hybrid system is most versatile when used with a mass spectrometeras an additional detector. The ability to switch rapidly back and forth
between SFC/MS and HPLC/MS is a powerful capability, particularly fortrace analysis where orthogonal methods are needed. The combinationof a normal phase SFC with reversed phase HPLC, should be viewed as
greatly superior to two reversed phase HPLC methods,

155

9.9
Other Detectors

The same kind of split-flow interfacing used with mass spectrometers
is appropriate for a number of detectors mostly used for HPLC. These
detectors operate at atmospheric pressure.

9.9.1
Evaporative light scattering
detectors

The most common detector used in HPLC that operates at atmosphericpressure is based on evaporative light scattering (ELS)119–121. Evaporative
light scattering detectors have been interfaced to SFC systems for morethan 25 years. An ELS detector is a nearly universal, so that the samples
do not need to have a UV chromophore. The same split is appropriate as
described in Section 9.8 “Interfacing to mass spectrometers”.

9.9.2
Gas chromatography
detectors

In the past, SFC systems often deployed a gas chromatograph as thecolumn oven. This made it easy to interface most GC detectors. Most
GC detectors have been interfaced to packed column SFC. They include
the flame ionization detector (FID) electron capture detector (ECD),
nitrogen phosphorous detector (NPD), and sulfur chemiluminescence
detector (SCD).
Most GC detectors were used with a large split ratio with only a small
fraction of the total flow entering the detector (depending on the type)while the rest was vented through the BPR. These detectors were usedmostly with 100 % CO 2, which requires a different form of restrictor
compared to those discussed so far. The restrictors consisted of a 25 to
30-cm piece of 50-µm id fused silica tubing with the tip drawn down toa few µm over the last 1 to 2 mm of length. The pressure drop occurredalmost exclusively over this last short section, keeping solutes in solution
to the last moment. Larger versions of such restrictors can be used withmass spectrometers and ELS detectors but have a tendency to plug andare more difficult to work with.

9.9.2.1
Flame ionization detectors

Flame ionization detection was widely used with both capillary and packed
column SFC, primarily with pure CO2 as the mobile phase, although a few
other pure fluids were used. In a few cases, small concentrations of water
were added to the CO 2 to enable the elution of more polar solutes like freefatty acids.
There are several ASTM SFC methods that are still widely used for the
analysis of aromatics in diesel, and olefins in gasoline. Both use pure
CO 2 and an FID. Unlike most of the older methods that use pressure or
density programming at much higher temperature, both these petroleum
applications use a constant outlet pressure and temperatures near room
temperature, which makes them compatible with the Agilent analytical

156

SFC system. Recently, a standalone FID based on Agilent hardware wasintroduced, which can be interfaced to Agilent chromatography datasystems.
The FID can tolerate only relatively low flows of CO 2. The petroleum
methods have traditionally been performed on either a 4.6-mm id column
or on micropacked columns. By far the fastest, highest resolution, most
reproducible results are obtained with the larger columns, not the
micropacked columns. With 4.6-mm (or 3 or even 2.1-mm) columns, arelatively large split ratio is required, partly because the samples are neat.
The design of the restrictor is a key element. There can be no make-up
flow since the FID is a near universal detector and has a large baseline
offset when a modifier is present. The CO 2 gas flow rate (at atmosphericpressure) into the FID is usually around 10 to 40 mL/min. The flow througha 4.6-mm id column is on the order of 1000 mL/min when measured
at atmospheric pressure. Thus, the split ratio needs to be 25:1 to 100:1.
This requires an integral restrictor (a pinhole with a diameter of 1 to
2 µm) or a linear restrictor of less than 25 µm of significant length. This issimilar to the requirements for most other GC detectors such as a nitrogen
phosphorous detector, the electron capture detector, and many others
although many of these can be used with modified fluids.

9.9.3
Chiral detectors

Several chiral detectors, capable to differentiating the right- andleft‑handedness of enantiomers, which have been widely used in HPLC,
have been adapted for SFC. Both circular dichroism detectors and laser
polarimeters have been available. These detectors are capable of handlingthe full flow through the column at high pressure. There is no splitter
required. Such detectors allow you to determine the elution order of theenantiomers. Screening with various columns usually involves racemic
mixtures, which makes it easier to determine which enantiomer elutes
first. Ideally, when doing enantiomeric excess determinations, the much
lower concentration enantiomer should elute first, avoiding any tailingof the higher concentration enantiomer, thereby enabling more accuratequantification. However, without a chiral detector, there is no easy way
to rapidly determine which enantiomer elutes first without collecting
peaks and analyzing offline.

157

9.9.4
Missing detectors

The most obvious missing detector is a fluorescence detector. All theexisting commercial HPLC fluorescence detectors are incompatible withthe high outlet pressures needed. The mechanical layout of existing
detectors makes it unlikely that a high-pressure cell could be fabricated.
One of the most widely used detectors in HPLC is the refractive index (RI)
detector. From previous discussions, it should be obvious that the largechanges in RI occurring during a gradient of CO 2 with any of the common
modifiers rules out its use.

158

REFERENCES
1. Giddings, J.C., Myers, M.N., McLaren, L.M., Keller, R.A., High Pressure
Gas Chromatography of Nonvolatile Species, Science, 1968, 162, 67–73
2. Dorsey, J.G., Solvatochromic Investigations of Chromatographic
Retention Mechanisms, Chromatography, 1987, 2, 37–41
3. Graham, J.A., Rogers, L.B., Effect of Column Length, Particle Size,
Flow Rate and Pressure Programming Rate on Resolution in Pressure
programmed Supercritical Fluid Chromatography, J. Chromatogr. Sci.,
1980, 18, 75–84
4. Novotny, M., Bertsch, W., Zladkis, A., Temperature and Pressure
Effects in Supercritical Fluid Chromatography, J. Chromatogr., 1971,
61, 17–28
5. Schoenmakers, P.J., Open Columns or Packed Columns for Supercritical
Fluid Chromatography: a comparison, in Supercritical Fluid
Chromatography, Smith, R.M., ed., Royal Society of Chemistry, London,
1988, Chapter 4
6. White, C., Integration of supercritical fluid chromatography into drug
discovery as a routine support tool. Part I. Fast chiral screening andpurification, J. Chromatogr. A, 2005, 1074, 163–173
7. Maftouh, M., Granier-Loyaux, C., Chavana, E., Marini, J., Pradines, A.,
Vander Heyden, Y., Picard, C., Screening approach for chiral separationof pharmaceuticals Part III. Supercritical fluid chromatography for
analysis and purification in drug discovery, J. Chromatogr. A, 2005,
1088, 67–81
8. Weast, R.C., ed., Handbook of Chemistry and Physics, 69th Ed,
CDC Press, Boca Raton FL, Section F-48, 1988
9. Kong, C.Y., Fanazukuri, T., Kagei, S., Binary diffusion coefficients andretention factors for polar compounds in supercritical carbon dioxide bychromatographic impulse response method, J. Supercrit. Fluids, 2006,
37, 359

159

10. Pizarro, C., Suárez-Iglesias, O., Medina, I., Bueno, J.L., Binary
diffusion coefficients for 2,3-dimethylaniline, 2,6-dimethylaniline,
2-methylanisole, 4-methylanisole and 3-nitrotoluene in supercriticalcarbon dioxide, J. Supercrit. Fluids, 2009, 48, 1–8
11. Smith, S.A., Shenai, V., Matthews, M.A., Diffusion in Supercritical
Mixtures: CO2 + Cosolvent + Solute, J. Supercrit. Fluids, 1990, 3,
175–179
12. Mantell, C., Rodrıguez, M., Martinez de la Ossa, E., Measurementof the diffusion coefficient of a model food dye (malvidin
3,5-diglucoside) in a high pressure CO2 + methanol system by thechromatographic peak-broadening technique, J Supercrit. Fluids,
2003, 25, 57–68
13. Walsh, J.M., Ikonomou, G.D., Donohue, M.D., Supercritical Phase
Behavior: The entrainer effect, Fluid Phase Equil., 1987, 33, 295–314
14. Gonzales, B., Calvar, N., Gomez, E., Domingues, A., Density, dynamic
viscosity, and derived properties of binary mixtures of methanol or
ethanol with water, ethyl acetate, and methyl acetate at T = (293.15,
298.15, and 303.15) K, J. Chem. Thermodynamics, 2007, 39,
1578–1588
15. Euerby, M., Fever, M., James, M., Pipe, C., Schad, G., Bayly, K.,
Exploration of Chromatograph Selectivity difference between SFC
and reversed phased UHPLC using the Agilent 1260 Infinity Hybrid
SFC/ UHPLC-DAD/MS, poster presented at SFC, 2012 (available
online)16. Berger, T.A., Wilson, W.H., Packed Column SFC with 220,000 Plates,
Anal. Chem., 1993, 65, 1451
17. Kot, A., Sandra, P., Venema, A., Subcritical and supercritical fluid
chromatography on packed-columns- a versatile tool for theenantioselective separation of basic and acidic drugs, J. Chromatogr.
Sci., 1994, 32, 439–448
18. Berger, T.A., Packed Column SFC, Royal Society of Chemistry, London,
1995
19. Taylor, L.T., (review), Packed column supercritical fluid chromatographyof hydrophilic analytes via water-rich modifiers, J. Chromatogr. A,
2012, 1250, 196– 204160

20. Bola, B., Greig, M., Ventura, M., Farrell, W., Aurigemma, C.M., Li, H.,
Quenzer, T,L., Tivel, K., Bylund, J.M.R., Tan, P., Pham, C., Phillipson,D., SFC/MS in drug discovery at Pfizer, La Jolla, Intl. J. Mass
Spectrom., 2004, 238, 85–97
21. Zheng, J., Pinkston, J.D., Zoutendam, P.H., Taylor, L.T., Feasibility ofsupercritical fluid chromatography/mass spectrometry of polypeptides
with up to 40-Mers, Anal Chem., 2006, 78, 1535–45
22. Liu, J., Regalado, E.L., Mergelsberg, I., Welch, C.J., Extending therange of supercritical fluid chromatography by use of water-richmodifiers, Org. Biomol. Chem., 2013, 11, 4925–4929
23. Patel, M.A., Riley, F., Ashraf-Khorassani, M., Taylor, L.T., Supercriticalfluid chromatographic resolution of water soluble isomericcarboxyl/amine terminated peptides facilitated via mobile phasewater and ion pair formation, J. Chromatogr. A, 2012, 1233, 85–90
24. Patel, M.A., Riley, F., Wang, J., Lovdahl, M., Taylor, L.T., Packed
column supercritical fluid chromatography of isomeric polypeptide
pairs, J. Chromatogr. A, 2011, 1218, 2593–2597
25. Mourier, P.A., Eliot, E., Caude, M., Rosset, R., Tambute, A.,
Supercritical and subcritical fluid chromatography on a chiralstationary phase for the resolution of phosphine oxide enantiomers,
Anal. Chem., 1985, 57, 2819–2823
26. Pescher, P., Caude, M., Rosset, R., Tambute, A., Enantiomericseparation of tertiary phosphine oxides on Pirkles’s chiralstationary phase: mobile phase and temperature optimization,
J. Chromatography, 1986, 37, 159–175
27. Steuer, W., Schindler, M., Schill, G., Erni, F., Supercritical fluid
chromatography with ion-pairing modifiers: separation ofenantiomeric 1,2-aminoalcohols as diasteromeric ion pairs,
J. Chromatogr., 1988, 441, 287–296
28. Berger, T.A., Wilson, W.H., Separation of Anilines, Benzamides,
Benzylamines, and Phenylethylamines by Packed Column SFC,
J. Chromatogr. Sci., 1993, 31, 127
29. Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J., Experimental
and computational approaches to estimate solubility and permeability
in drug discovery and development settings, Adv. Drug Deliv. Rev.,
March 2001, 46, 3–26, doi:10.1016/S0169-409X(00)00129-0. PMID
11259830
161

30. Klesper, E., Corwin, A.H., Turner, D.A., High Pressure Gas
Chromatography above Critical Temperatures, J. Org. Chem., 1960,
27, 700
31. Giddings, J.C., Manwaring, W.A., Myers, M.N., Turbulent Gas
Chromatography, Science, 1966, 154, 146–148
32. Myers, M.N., Giddings, J.C., Ultrahigh pressure gas chromatography
in microcolumns to 2000 atmospheres, Sep. Sci., 1966, 1, 761–776
33. Giddings, J.C., Myers, M.N., King, J.W., Dense gas chromatography
at pressures to 2000 atmospheres, J. Chromatogr. Sci., 1969, 7,
276–283
34. Sie, S.T., Rijnders, G.W.A., High pressure gas chromatography
and chromatography with supercritical fluids, II, Permeability andefficiency of packed columns with high pressure fluids as mobile fluids
under conditions of insipient turbulence, Sep Sci., 1967, 2, 729–753
35. Mourier, P.A., Eliot, E., Caude, M.H., Rosset, R.H., Supercritical andsubcritical fluid chromatography on a chiral stationary phase for the
resolution of the phosphine oxide enantiomers, Anal. Chem., 1985, 57,
2819–2823
36. Cui, Y., Olesik, S.V., High-performance liquid chromatography using
mobile phases with enhanced fluidity, Anal. Chem., 1991, 63,
1812–1819
37. Berger, T.A., Packed Column SFC, RSC Chromatography Monographs
Series, Royal Society of Chemistry, Cambridge, UK, 1995
38. Peaden, P.A., Feldstedt, J.C., Lee, M., Springston, R., Novotny, M.,
Instrumental aspects of supercritical fluid chromatography,
Anal. Chem., 1982, 54, 1090–1093
39. Goldfarb, D.L., Ferna´ndez, D.P., Corti, H.R., Dielectric and volumetricproperties of supercritical carbon dioxide-methanol mixtures at
323.15 K, Fluid Phase Equilibria, 1999, 158(1), 1011–1019
40. Berger, T.A., Density of Methanol-Carbon Dioxide Mixtures at Threetemperatures: Comparison vith Vapor Liquid Equilibria Measurements
and Results obtained from Chromatography, J. High Resolut.
Chromatogr., 1991, 14, 312–316
41. Snyder, L.R., Classification of the Solvent Properties of Common
Liquids, J. Chromatogr., 1974, 92, 223–230

162

42. Snyder, L.R., Classification of the Solvent Properties of Common
Liquids, J. Chromatogr. Sci., 1978, 16, 223–234
43. Snyder, L.R., Principles of Adsorption Chromatography, Dekker,
New York, 1976, Chapter 8
44. Kraml, C., private communication, 2005
45. Deye, J.F., Berger, T.A., Anderson, A.G., Nile Red as a Solvatochromic
Dye for Measuring Solvent Strength in Normal Liquids and Mixturesof Normal Liquids with Supercritical and Near Critical Fluids,
Anal. Chem., 1990, 62, 615
46. Berger, T.A., Deye, J.F., Use of Solvatochromic Dyes to Correlate
Mobile Phase Solvent Strength to Chromatographic Retention in SFC,
Supercritical Fluid Technology, Bright, F.V. and McNally, M.E.P, eds.,
ACS Symposium Series 488, American Chemical Society, Washington,
D.C., 1992
47. Smith, S.A., Shenai, V., Matthews, M.A., Diffusion in Supercritical
Mixtures: CO2 + Cosolvent + Solute, J. Supercritical Fluids, 1990, 3,
175–179
48. Strubringer, J.R., Song, H., Parcher, J.F., High pressure phase
distribution isotherms for supercritical fluid chromatographic systems.
2. Binary isotherms of carbon dioxide and methanol, Anal. Chem.,
1991, 63, 104–108
49. Strubringer, J.R., Song, H., Parcher, J.F., High pressure phase
distribution isotherms for supercritical fluid chromatographic systems.
1. Pure carbon dioxide, Anal. Chem., 1991, 63, 98–103
50. Luo, Z., Xiong, Y., Parcher, J.F., Chromatography with DynamicallyCreated Liquid “Stationary” Phases: Methanol and Carbon Dioxide,
Anal. Chem., 2003, 75(14), 3557–3562
51. Ashraf-Korassani, M., Fessahaie, M.G., Taylor, L.T., Berger, T.A., Deye,
J.F., Rapid and Efficient Separation of PTH-Amino Acids Employing
Supercritical CO2 and an Ion Pairing Agent, J. High Resolut.
Chromatogr., 1988, 11, 352
52. Berger, T.A., Deye, J.F., Ashraf-Korassani, M., Taylor, L.T., Gradient
Separation of PTH-Amino Acids employing Supercritical CO2 andModifiers, J. Chromatogr. Sci., 1989, 27, 105–110

163

53. Stringham, R.W., Chiral separation of amines in subcritical fluid
chromatography using polysaccharide stationary phases and acidic
additives, J. Chromatogr. A, 2005, 1070, 163–170
54. Berger, T.A., Deye, J.F., Role of additives in packed column
supercritical fluid chromatography: suppression of solute ionization,
J. Chromatogr., 1991, 547, 317–392
55. Berger, T.A., Deye, J.F., Separation of Phenols by Packed Column
Supercritical Fluid Chromatography, J. Chromatogr. Sci., 1990, 29,
54–59
56. Philibert, G.S., Olesik, S.V., Characterization of enhanced-fluidity
liquid hydrophilic interaction chromatography for the separationof nucleosides and nucleotides, J. Chromatogr. A, 2011, 1218,
8222–8230
57. West, C., Lesellier, E., Orthogonal screening system of columns forsupercritical fluid chromatography, J. Chromatogr. A, 2008, 1203,
105–113
58. Berger, T.A., Demonstration of High Speeds with Low Pressure Drops
Using 1.8 µm Particles in SFC, Chromatographia, 2010, 72, 597–602
59. Halasz, I., Endele, R., Asshauer, J., Ultimate Limits in High-Pressure
Liquid Chromatography, J. Chromatogr., 1975, 112, 37–60
60. Lin, H.-J., Horvath, S., Band spreading in liquid chromatography.
General plate height equation and a method for the evaluation of theindividual plate height contributions, Chem. Eng. Sci., 1981, 36, 47
61. Poppe, H.H., Kraak, J.C., Influence of thermal conditions on theefficiency of high-performance liquid chromatographic columns,
J. Chromatogr., 1983, 282, 399–412
62. de Villiers, A., Lauer, H., Szucs, R., Goodall, S., Sandra, P., Influenceof frictional heating on temperature gradients in ultra-high-pressureliquid chromatography on 2.1mm I.D. columns, J. Chromatogr. A,
2006, 1113, 84–91
63. Kaczmarskia, K., Poe, D.P., Tarafder, A., Guiochon, G., Pressure,temperature and density drops along supercritical fluid
chromatography columns. II. Theoretical simulation for neat carbon
dioxide and columns packed with 3-µm particles, J. Chromatogr. A,
2012, 1250, 115–123

16464. Zauner, J., Lusk, R., Koski, S., Poe, D.P., Effect of the thermal
environment on the efficiency of packed columns in supercritical fluid
chromatography, J. Chromatogr. A, 2012, 1266, 149–157
65. Poe, D.P., Veit, D., Ranger, M., Kaczmarski, K., Tarafder, A.,
Guiochon, G., Pressure and density drops along supercritical fluid
chromatography columns. I. Experimental results for neat carbon
dioxide and columns packed with 3- and 5-micron particles,
J. Chromatogr. A, 2012, 1250, 105–114
66. Berger, T.A., Characterization of a 2.6 µm Kinetex porous shellhydrophilic interaction liquid chromatography column in supercriticalfluid chromatography with a comparison to 3 µm totally porous silica,
J. Chromatogr. A, 2011, 1218, 4559– 4568
67. Lesellier, E., Latos, A., Lopes deOliveira, A., Ultra high efficiency/lowpressure supercritical fluid chromatography with superficially porous
particles for triglyceride separation, J. Chromatogr. A, 2014, 1327,
141–148
68. Lesellier, E., Efficiency in supercritical fluid chromatography withdifferent superficially porous and fully porous particles ODS bonded
phases, J. Chromatogr. A, 2012, 1228, 89– 98
69. Berger, T.A., Deye, J.F., Effect of Column and Mobile Phase Polarity
using Steroids as Probes in Packed Column Supercritical Fluid
Chromatography, J. Chromatogr. Sci., 1991, 29, 280–286
70. Berger, T.A., Wilson, W.H., Separation of Drugs by Packed Column
SFC: 2. Antidepressants, J. Pharm. Sci., 1994, 83, 287–290
71. Berger, T.A., Deye, J.F., Density of Methanol-Carbon Dioxide
Mixtures at Three Temperatures, J. High Resolut. Chromatogr.,
1991, 14, 312–316
72. Berger, T.A., Wilson, W.H., Separation of Drugs by Packed Column
SFC: 1. Phenothiazine Antipsychotics, J. Pharm. Sci., 1994, 83(3),281–28673. Berger, T.A., Wilson, W.H., Separation of Drugs by Packed Column
SFC: 3. Stimulants, J. Pharm. Sci., 1995, 84, 489–492
74. Berger, T.A., Separation of Phenylurea Herbicides by Packed Column
SFC, J. Chromatogr. Sci., 1994, 32, 25

165

75. Lemmon, E.W., Huber, M.L., McLinder, M.O., NIST Reference
Database 23: Reference Fluid Thermodynamic and Transport
Properties-REFPROP, Version 9.1, National Institute of Standards andTechnology, Standard Reference Data Program, Gaithersburg, 201376. Span, R., Wagner, W.J., A New Equation of State for Carbon Dioxide
Covering the Fluid Range from the Triple Point Temperature to 1100 K
at Pressures up to 800 MPA, J.Phys. Chem. Ref. Data, 1966, 25(6),1509–1596
77. de Reuck, K.M., Craven, R.J.B., Methanol International
Thermodynamic Tables of the Fluid State- 12, IUPAC, Blackwell
Scientific Publications, London, 1993
78. Berger, T.A., Berger, B.K., Separation of Natural Food Pigments
in Saponified and un-Saponified Paprika Oleoresin by Ultra
High Performance Supercritical Fluid Chromatography (UPSFC),
Chromatographia, 2013, 76, 591–601
79. Berger, T.A., Berger, B.K., Separation of 9 Sulfonamde Drugs inabout 4 minutes by Ultra High Performance Supercritical Fluid
Chromatography (UPSFC): with a Feasibility Study for Detection in
Milk, Chromatographia, 2013, 76, 1631–1639
80. White, C., Integration of supercritical fluid chromatography into drug
discovery as a routine support tool. Part I. Fast chiral screening andpurification, J. Chromatogr. A, 2005, 1074, 163–173
81. Maftouh, M., Granier-Loyaux, C., Chavana, E., Marini, J.,
Pradines, A., Vander Heyden, Y., Picard, C., Screening approachfor chiral separation of pharmaceuticals Part III. Supercritical fluid
chromatography for analysis and purification in drug discovery,
J. Chromatogr. A, 2005, 1088, 67–81
82. daSilva, J.O., Coes, B., Frey, L., Mergelsberg, I., McClain, R.,
Nogle, L., Welch, C.J., Evaluation on non-conventional polar modifiers
on immobilized chiral stationary phases for improved resolution ofenantiomers by supercritical fluid chromatography, J. Chromatogr.,
2014, 1328, 98–103
83. Franco, P., Zhang, T., Common approaches for efficient method
development with immobilised polysaccharide-derived chiralstationary phases, J. Chromatogr. B, 2008, 875, 48–56

166

84. Miller, L., Evaluation of non-traditional modifiers for analyticaland preparative enantioseparations using supercritical fluid
chromatography, J. Chromatogr. A, 2012, 1256, 261–266
85. Byrne, N., Hayes-Larson, E., Liao, W., Kraml, C.M., Analysis
and purification of alcohol-sensitive chiral compounds using
2,2,2-trifluoroethanol as a modifier in supercritical fluid
chromatography, J. Chromatogr. B, 2008, 875, 237–242
86. Gasparini, F., Misity, D., Villani, C., Direct resolution in sub- andsupercritical fluid chromatography on packed columns containing
trans-1,2-diaminocyclohexane derivatives as selectors, Trends Anal.
Chem., 1993, 12, 137–144
87. Welch, C.J., Biba, M., Gouker, J.R., Kath, G., Augustine, P., Hosek, P.,
Solving multicomponent chiral separation challenges using a new SFC
tandem column screening tool, Chirality, 2007, 19, 184
88. Hamman, C., Wong, M., Hayes, M., Gibbons, P., A high throughput
approach to purifying chiral molecules using 3 µm analyticalchiral stationary phases via supercritical fluid chromatography,
J. Chromatogr. A., 2011, 1218, 3529–3536
89. Medvedovici, A., Sandra, P., Toribio, L., David, F., Chiral packed
column subcritical fluid chromatography on polysaccharide andmacrocyclic antibiotic chiral stationary phases, J. Chromatogr. A,
1977, 785, 159–171
90. Pirzada, Z., Personick, M., Biba, M., Gong, X., Zhou, L., Schafer,
W., Roussel, C., Welch, C.J., Systematic evaluation of new chiralstationary phases for supercritical fluid chromatography using astandard racemate library, J. Chromatogr. A, 2010, 1217, 1134–1138
91. Zhao, Y., Woo, G., Thomas, S., Semin, D., Sandra, P., Rapid method
development for chiral separation in drug discovery using sample
pooling and supercritical fluid chromatography-mass spectrometry,
J. Chromatogr. A, 2003, 1003, 157–166
92. Garzotti, M., Hamdan, M., Supercritical fluid chromatography coupled
to electrospray mass spectrometry: a powerful tool for the analysis ofchiral mixtures, J Chromatogr B, 2002, 770, 53–61

167

93. Garcia, I., Cruz Ortiz, M., Sarabia, L., Vilches, C., Gredilla, E.,
Advances in methodology for the validation of methods according
to the International Organization for Standardization Application
to the determination of benzoic and sorbic acids in soft drinks byhigh-performance liquid chromatography, J. Chromatogr. A, 2003,
992, 11–27
94. Tfouni, S.A.V., Toledo, M.C.F., Determination of benzoic and sorbic
acids in Brazilian food, Food Control, 2002, 13, 117–123
95. Ping, Q., Hong, H., Xiaoyan, L., Donghao, L., Assessment of benzoic
acid levels in milk in China, Food Control, 2009, 20, 414–418
96. Lino, C.M., Pena, A., Occurrence of caffeine, saccharin, benzoic acidand sorbic acid in soft drinks and nectars in Portugal and subsequent
exposure assessment, Food Chemistry, 2010, 121, 503–508
97. Cressey, P., Jones, S., Levels of preservatives (sulfite, sorbate andbenzoate) in New Zealand foods and estimated dietary exposure,
Food Addit Contam Part A Chem Anal Control Expo Risk Assess,
2009, 26, 604–13
98. Zygmunt, L.C., Gas-liquid chromatographic determination of sorbic
acid and sodium benzoate in table syrup, J. Assoc Off. Anal. Chem.,
1979, 62, 939–42
99. Dong, C., Mei, Y., Chen, L., Simultaneous determination of sorbic
and benzoic acids in food dressing by headspace solid-phasemicroextraction and gas chromatography, J. Chromatogr. A, 2006,
1117, 109–14
100. Hui, L.I., Hu, Y-D., Sun, X-Y., Simultaneous determination of sodium
benzoate and potassium sorbate by ultraviolet spectrophotometry
with nonlinear iterative optimization, Chinese Journal of Analysis
Laboratory, 2006, 10
101. Costa, A.C., Perfeito, L.S., Tavares, M.F., Micke, G.A., Determinationof sorbate and benzoate in beverage samples by capillaryelectrophoresis-Optimization of the method with inspection of ionic
mobilities, J. Chromatogr. A, 2008, 1204, 123–7
102. Chen, Q-C., Wang, J., Simultaneous determination of artificial
sweeteners, preservatives, caffeine, theobromine and theophylline
in food and pharmaceutical preparations by ion chromatography, J
Chromatogr. A, 2001, 937, 57–64

168103. Liu, Q-R., Chu, S-M., Yang, W-C., Zhang, L., Peng, Q-L., Eng, H.G-X.,
Shi, Q-Z., High-performance Ion Chromatographic Determination of
Benzoic Acid in Paprika Oleoresin, Food Science, 2009, 12104. Mihyar, G.F., Yousif, A.K., Yamani, M.I., Determination of Benzoic
and Sorbic Acids in Labaneh by High-Performance Liquid
Chromatography, J. Food Composit. Anal., 1999, 12, 53–61
105. Pylypiw, H.M., Jr., Grether, M.T., Rapid high-performance liquid
chromatography method for the analysis of sodium benzoate andpotassium sorbate in foods, J. Chromatogr. A, 2000, 883, 299–304
106. Fernando Mota, J.M., Ferreira, I.M.P.L.V.O., Cunhaa, S.C., Beatriz,
M., Oliveira, P.P, Optimisation of extraction procedures for analysis
of benzoic and sorbic acids in foodstuffs, Food Chemistry, 2003,
82, 469–473
107. G Arli, G., Lafci, Y., A novel RP-HPLC method for simultaneous
determination of potassium sorbate and sodium benzoate in softdrinks using C(18)-bonded monolithic silica column, Can, J. Sep. Sci.,
2011, May 19, doi: 10.1002/jssc.201100172, Epub ahead of print
108. Berger, T.A., Deye, J.F., Separation of Hydroxybenzoic Acids byPacked Column Supercritical Fluid Chromatography using Modified
Fluids with Very Polar Additives, J. Chromatogr. Sci., 1991, 29, 26–30
109. Berger, T.A., Deye, J.F., Separation of Benzene Polycarboxylic
Acids by Packed Column Supercritical Fluid Chromatography using
Methanol-Carbon Dioxide Mixtures with Very Polar Additives,
J. Chromatogr. Sci., 1991, 29, 141–146
110. Kritsunankula, O., Jakmunee, J., Simultaneous determinationof some food additives in soft drinks and other liquid foods byflow injection on-line dialysis coupled to high performance liquid
chromatography, Talanta, 2012, 84, 1342–1349
111. Smyly, D.S., Woodward, B.B., Conrad, E.C., Determination ofsaccharin, sodium benzoate, and caffeine in beverages by reverse
phase high-pressure liquid chromatography, J Assoc. Off Anal
Chem., 1976, 59, 14–9.
112. Yong-gang, L., Zhao-xu, L., Jun-ping, Z., Yan-hui, Z., Feng, G.,
Determination Method of Benzoic Acid, Sorbic Acid and Saccharin
Sodium Content in Milk and Dairy Produce, Food Science, 2007, 06

169

113. Kreuz, D.M., Howard, A.L., Ip, D., Determination of indinavir,
potassium sorbate, methylparaben, and propylparaben in aqueouspediatric suspensions, J Pharm Biomed Anal., 1999, 19, 725–735
114. Sun, Y., Shekunov, B.Y., York, P., Refractive index of supercritical
CO2-ethanol solvents, Chem. Eng. Commun., 2003, 190, 1–14
115. Tang, H., Gulari, E., Rothe, E.W., Large mirage effect in supercritical
CO2, J. Supercrit. Fluids, 2000, 18, 193
116. Jain, J.P., Rothe, E.W., Superior mirage effect in supercritical CO2:
Experiment and model, J. Supercrit. Fluids, 2005, 35, 280
117. Vollmer, M., Becker, M., Agilent 1260 Infinity Hybrid SFC/UHPLC
System, Agilent Technologies Technical Overview, publication
number 5990-9514EN, 2012118. Euerby, M., Fever, M., James, M., Pipe, C., Schad, G., Bayly, K.,
Exploration of Chromatograph Selectivity difference between SFC
and reversed phased UHPLC using the Agilent 1290 Infinity Hybrid
SFC/UHPLC-DAD/MS, poster presented at SFC 2012 (available
online)119. Nizery, D., Thiebaut, D., Caude, M., Rosset, R., Improved Evaporative
Light-Scattering Detection for Supercritical Fluid Chromatography
with Carbon Dioxide-Methanol Mobile Phases, J. Chromatogr.,
1989, 487, 49–80
120. LaFosse, M., Herbreteau, B., Carbohydrate Analysis by LC and SFC
using Evaporative Light Scattering Detection, Carbohydrate Analysis
by Modern Chromatography and Electrophoresis, Ziad El Rassi(Editor), Elsevier Science B.V., Journal of Chromatography Library,
2002, Vol. 88, Chapter 30
121. Rambla-Alegre, M., Dunkle, M.N., David, F., Sandra, P., Vollmer, M.,
Coupling the Agilent 1290 Infinity Analytical SFC System toan Agilent 1290 Infinity Evaporative Light Scattering Detector,
Agilent Technologies Technical Overview, publication number
5991-0988EN, 2012170

www.agilent.com/chem/sfc
This information is subject to change without notice.
© Agilent Technologies, Inc., 2015
Printed in the USA, July 1, 2015
5991-5509EN