Using Core Shell Columns
for Improved Separation of Pharmaceutical Compounds
by Supercritical Fluid Chromatography
William P. Farrell¹, Nicole C. Aurigemma¹, Cristine M. Aurigemma¹,Toshiyuki Ono²
Ono Norikazu Nagae
Nagae³,¹Pfizer, Inc., La Jolla Laboratories, San Diego, United States.
²Nacalai USA, Inc., San Diego, United States.
³ChromaNik Technologies, Inc., Osaka, Japan.

Abstract

Superficially porous (core shell) particles consist of a non-porous silica core surrounded by a thin shell of porous silica, have gained in popularity as a solid
support for chromatographic phases over the last decade. Core shell particles offer reduced resistance to solute mass transfer into the porous structure due to thesmall diffusion distances which leads to very fast and efficient separations. Since the overall particle size is larger, core shell particles operate with lower pressure
than sub-2 micron particles, and do not require specialized instrumentation to deal with the increased pressure. Similar benefits of using superficially porous
particles have been demonstrated for supercritical fluid chromatography (SFC)(SFC), but the selection of available phases is limited to those for reversed phase,
phase or baresilica. In this study, we evaluate a 2.6-micron superficially porous 2-ethylpyridine stationary phase made specifically for SFC applications. We compare its
performance to several available superficially porous HILIC (hydrophilic interaction liquid chromatography) or unbonded silica phases, as well as fully porous,
sub-2 micron silica and 2-ethylpyridine columns. Using a mixture of 17 small drug-like compounds under similar SFC conditions, we determined thatsuperficially porous 2-ethylpyridine columns outperformed comparable silica phases for overall selectivity.
Introduction

Results and Discussion

SFC analysis has increased in prevalence in the last ten years for the analysis of pharmaceuticalcompounds, as it benefits from high peak capacities and efficiencies.[1-3] Run times on SFC systems are
typically 3-5 times faster and are cost-efficient for long term use. The most common mobile phase solventsfor SFC are compressed CO2 mixed with some organic modifier, typically an alcohol. The lower viscosityand higher diffusivity of this fluid offer less pressure drop across the column relative to aqueous-based
solvents under similar flow and temperature conditions.[4]Recent studies have found that the utilization of particles <3 μm in diameter for SFC provides anadditional benefit to separation efficiency.[5-7] Compared to conventional fully porous particles (FPP),
solid core particles (SCP) consist of solid core silica microspheres surrounded by a thin, porous outershell. The advantages of using SCPs include faster solute mass transfer due to decreased diffusion
distances, where molecules no longer have to traverse the longer distances previously found on the totallyporous FPP.[8] Several groups suggest that SCPs are similar to sub-2 micron particles but without thepressure limitations of the micronized particles. For instance, Berger [9] concluded that SCP particles
offered a 10-fold decrease in analysis time, with better resolution, at lower pressures than columns withfully porous, 3μm particles, under conventional pressure limitations (e.g. < 400 bar).
To date, evaluation of SCP columns by SFC has been limited to those used primarily for reversed phase orbbaresilica.
iliI thisIn
hi study,
d we createdd andd evaluatedld a 2.6-µm
26core shellh ll 2-ethylpyridine
2 h l idi stationaryiphasehmade specifically for SFC applications. We compare its performance to that of two superficially porous
bare silica phases, a fully porous sub-2μm silica phase, and a fully porous 2-ethylpyridine phase.

Comparison of HILIC and 2-Ethylpyridine Columns
Three different silica columns and two 2-ethylpyridine columns were evaluated in the selectivity differences
as shown in Figure 2. The 2-ethylpyridine columns demonstrated enhanced selectivity over the bare silica
columns, as expected. The addition of the basic aryl functional group near the surface silanols enhance theselectivity of a wide variety of compounds and provide retention inversion relative to elution on HILIC oralkyl-bonded phases, which resulted in increased popularity of the 2-EP phase.
7164

142

5 109 13 15811 12 14 1694599(1) Flurbiprofen(2) Naproxen(3) Ketoprofen(4) Caffeine(5) Warfarin(6) Thymine(7) Etophylline(8) Uracil

Viridis BEH 2-EP

Viridis BEH
1617

Ascentis HILIC
1715710

Experimental
Chemicals and ReagentsThe compounds used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) and prepared
to a concentration of ~1mg/mL in methanol. The structure of the analytes are presented in Figure 1.

Kinetex HILIC

134

SunShell 2-EP350

137

3

1. Caffeine
2. Etophylline
3. Thymine
4. Uracil
5. Flurbiprofen
6. Naproxen
7. Ketoprofen
8. Cortisone
9. Warfarin
10. Hydrocortisone
11. Prednisolone
12. Acetamidophenol
13 Sulfamethazine
14. Sulfamethoxazole
15. Sulfadimethoxine
16. Sulfaquinoxazline
17. Sulfamethizole

141516300

125250(10) Cortisone(11) Sulfamethoxazole(12) Sulfadimethoxine

1712002116150

9100

10(13) Hydrocortisone(14) Sulfamethazine(15) Prednisolone(16) Sulfaquinoxaline

Figure 2: Chromatogram of the 17component mix run. Gradient elution of
5-20% methanol over 4 minutes at
2.5mL/min flow rate; outlet pressure 150
bar and column temperature 55ºC.
The retention order of sulfamethazine(14) on the 2-EP columns is inverted
compared to that of sulfamethoxazole(11) and sulfadimethoxine (12) on thesilica columns, in addition to thesignificant increase in selectivity.
On the 2-EP columns
columns, caffeine (4) andthymine (6) elute before the acidic
compounds flurbiprofen (1), naproxen (2)and ketoprofen (3)The SunShell 2-EP column demonstrated
the best overall baseline separation withrespect to the columns tested.

An optimized gradient separations of the 17-component mix for the SunShell 2-EP is presented in Figure 3,
where all 17 components are separated in less than 2 minutes. A similar separation was achieved using theAcquity Viridis BEH 2-EP (Figure 4) with the exception of co-eluting sulfamethazine (13) andsulfamethoxazole (14) peaks.
mAU(9) Acetamidophenol17

12 14 161161715
13

1082

16

1715

12 14

117

5

16

SunShell 2-EP

11 1213 15
9 10

13

10

8

6

4

2

7

53
15

1114 1237 8123114

2

63

3

864

50

8

Figure 3: Chromatogram of the separation for
the 17-component mix using the Sun Shell 2-EPcolumn. A methanol gradient of < 2 minutes
was used on the Agilent 1260 Infinity SFC
system. SFC conditions: flow rate: 4.0mL/min;outlet pressure 160 bar; column temperature
55⁰C. Gradient program: 5.0-7.5% in 0.20 min,then 7.5-20% in 1.3 min and held at 20% for 0.2
min.

00.25

(17) Sulfamethizole

0.5

0.75

11.25

1.5

1.75

min

13,14mAU

Figure 4:Chromatogram of the separation fortthee 17-component
7 co po e t mix using
us g thet e Acquity
cqu ty U
UPCC2Viridis 2-EP column. 16 of the 17 componentswere resolved. A methanol gradient of < 2minutes was used on the Agilent 1260 Infinity
SFC system. SFC conditions: flow rate 3.5mL/min; outlet pressure 160 bar; and column
temperature 70⁰C. Gradient program: 5.0-12.5%
in 1.0 min, 12.5% for 0.25 min, then 12.5-20%
in 0.75 min.
.

700

Acquity Viridis 2-EP

600

500

Figure 1: Seventeen drug-like compounds utilized to evaluate the columns in this study. They are
numerically ordered based on elution from the Acquity UPC2 Viridis BEH (silica) column using theconditions listed in the Analysis Conditions section.
Analysis Conditions
Analysis was performed using the columns listed below and under the following conditions unlessspecifically noted. The mobile phase consisted of carbon dioxide (A) and methanol (B) with a gradientprofile from 5-20% B at a flow rate of 2.5 mL/min and outlet pressure at 150 bar. The columns weremaintained at a temperature of 55˚C and sample injection volume was 1µL.
Fully Porous Particle (FPP) columns
The fully porous particle columns used were the Waters Acquity UPC2 Viridis BEH (silica) and the Waters
Acquity UPC2 Viridis BEH 2-ethylpyridine (2-EP), with 1.7 µm diameter particles and column dimensionsof 3.0 mm i.d. x 100 mm length.
Solid Core Particle (SCP) columns
The solid core particle columns used were the Ascentis Express HILIC (Supelco, Bellefonte, PA, USA)with 2.7 µm diameter particles; the SunShell 2-EP (Nacalai USA, San Diego, CA, USA) with 2.6 µm
diameter particles; and the Phenomenex Kinetex HILIC (Torrance, CA, USA) with 2.6 µm diameterparticles; The dimension of each column was 3.0 mm i.d. x 150 mm in length.
When using small particle columns, one must expect an increase in pressure relative to 3 or 5 µm particle
columns. In order to avoid excessive column pressure for the 1.7 µm particles, the 100 mm length BEH
columns were used.

154

16

3400

1300

127

5

172
11620098100

10000.2

0.4

0.6

0.8

11.2

1.4

1.6

1.8minConclusions
In this study, we have demonstrated the advantages of using the superficially porous particle-type 2ethylpyridine column on retention, selectivity, and peak shape in comparison to comparable sub-2µcolumns.
lFurther,F h theh superficiallyfi i ll porous particles
i l exhibit
hibi lowerlcolumnlpressure drops
dandd havehtheh
potential to produce highly efficient separations without specialized high pressure pumping systems(> 400 bar). The superficially porous particle-type 2-ethylpyridine phase has demonstrated significant
advantages over bare silica phases for fast, efficient SFC analysis.

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