Evaluation of amide and C28 phases for separation ofhydrophilic compounds
1)
1)
2)
2)
1)Tomoyasu Tsukamoto , Norikazu Nagae , Anders Grahn , Ralf Jutvik and Vinay D. Gaitonde

1) ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka, Japan2) Biotech AB, Box 133, 439 23, Onsala, Sweden

Abstract

Collapse or Depermeating

Hydrophilic Interaction liquid Chromatography (HILIC) proposed by Alpert in 1990 has been applied
for analysis of hydrophilic compounds. Amide, diol, polyol, bare silica, ion exchange and zwitter ion
phases have been used as a hydrophilic stationary phase along with an organic solvent rich mobilephase. A polar group embedded C18 or a long alkyl chain phase such as C30 or C28 also have beenused to separate hydrophilic compounds without change in retention using an aqueous mobile phaseon a reversed-phase mode. The reason why these reversed-phases can showed no change in
retention under an aqueous condition is that a low contact angle of water on the surface of the poreof these reversed-phase packing materials makes an aqueous mobile phase keep in the pore
because pressure yielded by capillarity is less atmospheric pressure, so that retention doesn’tchange. Both HILIC stationary phases and reversed-phases have completely opposite
characteristics each other. Therefore both HILIC and reversed-phase modes are useful for
separation of hydrophilic compounds. It is important to understand separation behavior of eachmode. In this study, an amide column and a C28 column were compared and evaluated to separatehydrophilic compounds. SunShell HILIC-Amide and Sunniest RP-AQUA (C28) and SunShell RPAQUA (C28) were used to separate nucleobases, amino acids and hydrophilic vitamins. Whennucleobases were separated on HILIC and reversed-phase modes using an amide column and aC28 column, each elution order of samples is said to be opposite. Only uracil, however, showed aspecific elution. It was considered that the polarity of uracil under an organic solvent rich condition
was different from that on water rich condition to be due to keto-enol tautomerization. LC/MS
analysis of amino acids was achieved using C28 column and a mobile phase added 5 mM
heptafluorobutyric acid under gradient elution conditions.

C18 phases exhibit decreased and poorly reproducible
retention under more than 98% aqueous conditions.
This problem traditionally has been explained as being
the result of ligand collapse. Nagae1-3 ascertained,
however, that the mobile phase was being expelledfrom the pores of the packing material.
When the surface of packing materials isn’t wet bywater, water used as a mobile phase expels from thepore of the packing material by capillarity. This is areason why reproducibility in retention is low under
100% aqueous conditions. Reversely pressure aroundthe packing material makes water permeate into thepore of the packing material to overcome a force
worked by capillarity.

4SunShell Amide
523
1

SunShell RPAQUA
23

4Time (min)Figure. 1 Chromatograms of nucleobases• Repellency is expressed as a contact angle of water
on a material.
• The larger a contact angle, the stronger repellency, if
the contact angle is more than 90 degree.

4524• Each elution order of samples
was almost opposite.
• Only elution order of uracil did
not change

Trifluoromethane

Octadecane(C18)

Octane(C8)

Octacosane(C28)

Contact angle(θ)

120°

126°

140°

108°

Partition coefficient (LogP)

0.64

9.18

5.18

14.09

Solibility(㎎/L)

4090

0.006

0.66

8.84×10-10T. Enami and N. Nagae, BUNSEKI KAGAKU, 53 (2004) 1309.

Stability of C28

Comparing between SunShell HILIC-Amideand other HILIC columns • • •40 ºC

12Change of retention of thymine at 40 oC(measurement every stop flow for 1 hour)531• SunShell HILIC-Amide showedstronger retention than others.
• Amide showed different selectivity
comparing with bare silica HILICcolumn.

5Ascentice Express HILIC00.5

11.522.5

33.5Time (min)25 ºC

12Time (min)34Column: SunShell RP-Aqua 2.6 μm, 75 x 4.6 mm
Mobile phase: 10mM Phosphate buffer pH7.0
Flow rate: 1.0 mL/min
Temperature: 40 ºC and 25 ºC
Sample: 1. Cytosine, 2. Uracil, 3. Thymidine,
4. Uridine, 5. Thymine

Applications of HILIC
Column: SunShell HILIC-Amide 2.6 μm, 100 x 4.6 mm
Mobile phase: acetonitrile: 5 mM phosphate Buffer (pH6.9) = 75:25
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: UV@220 nm
NH2OSample: 1. Cyanuric acid, NH NH
2. Melamine N N

1009080706050403020100Initial

1234Measurement number every stop flow

SunShell RP-Aqua showed more than 97%of reproducibility for retention using 100%
aqueous buffer as a mobile phase.

1.52O

2.5

NH

NH2 NO

10090

Relative retention (%)

1NH2Time (min)Figure. 3 Chromatogram of melamine and cyanuric acid

Test condition
Mobile phase: 0.5% TFA
Temperature: 60 ºC

8070605040Sunniest RP-AQUA30

Other brand aqua type C1820020406080

100120140100Test condition
Mobile phase:
20 mM phosphate buffer (pH 8.0)
Flow rate: 1.0 mL/min
Temperature: 40 ºC

90807060
5040Sunniest RP-AQUA

30

Other brand aqua type C18200100

200

Time (h)13Column: SunShell HILIC-Amide 2.6 μm, 100 x 4.6 mm
Mobile phase: acetonitrile: 25 mM phosphate Ammonium (pH4.9) = 8:2
Flow rate: 1.0 mL/min
Temperature: Ambient
Detection: UV@215 nm
Sample: 1. Helicin, 2. Salicin,
3. Arbutin,
4. Rutin
OH2400.5

11.52OHOH H OHHHHHOOOHOHOH H OH
HH
HHOO
OH

2.5OHOH H OH
HH
HHOOOHOHFigure. 4 Chromatogram of glycosides
200.51OHOH

22.5

O
NHH CH3ONH2HTime (min)O400

Figure. 9 Stability of C28 under acidic and basic conditions

Separation of amino acids with C28OH

Column: Sunniest RP-AQUA 5 μm, 150 x 2.0 mm
Mobile phase:A) 5mM HFBA (Heptafluorobutyric acid)B) 5mM HFBA in Acetonitorile/water(9/1)%B 0% to 20% in 20 min
Flow rate: 0.2mL/min
Temperature: 40 oC
Detection: Quattro Micro API (ESI positive) SIMColumn: SunShell Amide 2.6 μm, 100 x 4.6 mm
Mobile phase: acetonitrile: 25 mM phosphate buffer (pH2.5) =8:2
Flow rate: 1.0 mL/min
Temperature: AmbientDetection: UV@215 nmO
Sample: 1. Aspartame,
2. Saccharin,
3. Acesulfame K

3

1.5

O

O

300

Time (h)O
CH3HOH O O
HOH
HOO
H
HHOH
H
OH
H
OH
OHHOHHTime (min)1• C28 column can be used
under the 100% aqueous
mobile phase condition.
• C28 column is usefulcolumn for separation ofhydrophilic compounds.
• C28 column has betterstability to acidic and basic
conditions than a polargroup embedded C18column.

Figure. 8 Separation of nucleobases and retention time stability

21530with HILIC columns

0.5

14Figure. 2 Different separation of nucleobases

02

Relative retention of thymine (%)

44• C28 has the smallest contact
degree comparing with C18and C8.
• In C28, the pressure for amobile phase to go out frompore is smaller thanatmospheric pressure.
• Aqueous mobile phase isn’t
expelled from the pore of C28phase.

Table. 1 Physical property of each compounds

Ascentice Express OH5

3• Repellency and hydrophobicity areindependent each other.
• Those two parameters are out of proportional.
• When hydrophobicity is high, it doesn’t meanthat repellency is always high.
• Capillarity depends on a contact degree.

• Hydrophobicity is expressed as the ratio of concentrationsof a compound between water and n-octanol using a
mixture of both solvents.
• This value is well known as LogPow.

52Repellency ∝ Hydrophobicity

material

Relative plate number of thtmine (%)

3Teflon tubing
with water

Difficult to mixing with water

Column:SunShell HILIC-Amide 2.6 μm, 100 x 4.6 mm
Ascentice Express OH5 2.6 μm, 100 x 4.6 mm
Ascentice Express HILIC 2.6 μm, 100 x 4.6 mm
Mobile phase: acetonitrile:
20 mM ammonium acetate(pH4.7) =8:2
Flow rate: 1.0 mL/min ,Temperature: 40 oC
Detection: UV@250 nm,Sample : 1.Tymine, 2. Uracil, 3. Uridine, 4. Cytosine, 5. Cytidine

SunShell HILIC-Amide

1Glass tubing
with water

Figure. 7 Schematic of capillarity

θ water

Water-shedding property

23Formula of Capillarity:
h=2g cosq /(rrg)g : Surface tension
r: Density of liquid

Hydrophobicity

Amide vs other HILIC phase
1Figure. 6 Schematic diagram of C18 particle

Repellency

Comparing between amide and C28
column • • •41Pressure

What does “Dewetting” mean?
A surface state changes from wetting to un-wetting? The surface of C18
is always un-wetting even if water exists in the pore, so that expressionof “dewetting” is not scientific. Depermeating is a scientific expression!

Column:SunShell Amide 2.6 μm, 100 x 4.6 mm
SunShell RPAqua 2.6 μm, 100 x 4.6 mm
Mobile phase:
Amide: Acetonitrile: 20 mM ammonium acetate(pH4.7) =8:2
RPAqua: 20 mM phosphate buffer(pH7.0)
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: UV@250 nm
Sample: 1. Tymine, 2. Uracil, 3. Uridine, 4. Cytosine, 5. Cytidine

50

Water expels
from the pore bycapillarity
Depermeating

Repellency and Hydrophobicity

23

Water permeates
into the pore
Permeating

1) N. Nagae, T. Enami and S. Doshi, LC/GC North America October 2002.
2) T. Enami and N. Nagae, American Laboratory October 2004.
3) T. Enami and N. Nagae, BUNSEKI KAGAKU, 53 (2004) 1309.

Amide vs C28
1Stop flowm/zAlaArgAsnAspCysGlnGlu
GlyHisIle,LeuLysMetPheProSerThr
Trp
TyrVal-(I)-(I)OO
2+(IV)
SH2
NH

O-(I) -(I)O O
2+(IV)
K
SH2NO
O

CH3

OH

Figure. 5 Chromatogram of synthetic sweeteners
0Conclusions• Nucleobases were separated using an amide column and a C28 column, eachelution order of samples is said to be opposite.
• Only uracil showed a specific elution. It was considered that the polarity of uracil
under an organic solvent rich condition was different from that on water richcondition to be due to keto-enol tautomerization.
• LC/MS analysis of amino acids was achieved using C28 column and a mobile
phase added 5 mM heptafluorobutyric acid under gradient elution conditions.
• Both amide and C28 column were useful for analysis of hydrophilic compounds

1020Time (min)30020

10Time (min)Figure. 10 Chromatogram of amino acids and MS detection

HPLC Column

90
175
133
134
122
147
148
76
156
132
147
150
166
116
106
120
205
182118