Usability of Amide and C28 Core Shell and Fully Porous
Column for Separation of Hydrophilic Compounds
Tomoyasu Tsukamoto1), Norikazu Nagae1), Anders Grahn2), Ralf Jutvik2) and Scott Silver3)
1) ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka, Japan2) Biotech AB, Box 133, 439 23, Onsala, Sweden
3) Innovations United, 300 East 57th Street, Suite 11J New York, NY 10022 USA

Abstract

Collapse or Depermeating

Hydrophilic Interaction liquid Chromatography (HILIC) proposed by Alpert in 1990 has been appliedfor 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/MSanalysis of amino acids was achieved using C28 column and a mobile phase added 5 mM
heptafluorobutyric acid under gradient elution conditions.

Amide vs C28
435

SunShell Amide
52431

SunShell RPAQUA
01234

Time (min)Stop flow
Water permeates
into the pore
Permeating
Water expels
from the pore bycapillarity
Depermeating

Pressure

Figure 6. Schematic diagram of C18 particle

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!

Repellency and Hydrophobicity
Repellency

Repellency ∝ Hydrophobicity

θ water

Water-shedding property• 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.

Comparing between amide and C28
column • • •Hydrophobicity• Each elution order of samples
was almost opposite.
• Only elution order of uracil did
not change

Difficult to mixing with water• 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.

material• 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.

• 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 C28
phase.

Table 4. Physical property of each compounds

Amide vs other HILIC phase
2SunShell HILIC-Amide
1352431

Teflon tubing
with water

Figure 7. Schematic of capillarity

Figure 1. Comparison between reversed phase and HILIC

4Glass tubing
with water

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

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.

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

21C18 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.

Column:SunShell HILIC-Amide 2.6 μm, 100 x 4.6 mm
Acentice Express OH5 2.7 μm, 100 x 4.6 mm
Acentice Express HILIC 2.7 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

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

Solubility(㎎/L)

4090

0.006

0.66

8.84×10-10

T. Enami and N. Nagae, BUNSEKI KAGAKU, 53 (2004) 1309.

Stability of C28

5

Acentice Express OH5

Comparing between SunShell HILIC-Amideand other HILIC columns • • •315

Acentice Express HILIC
00.5

11.5

22.5

33.5Time (min)

25 ºC

00.5

11.5

253

12Time (min)

9080
70

50403020100Initial

1234

Measurement number every stop flow

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

40Sunniest RP-AQUA
Other brand aqua type C18020406080100120140100

Test condition
Mobile phase:20 mM phosphate buffer (pH 8.0)
Flow rate: 1.0 mL/min
Temperature: 40 ºC90
80
7060
5040

Sunniest RP-AQUA

30

Other brand aqua type C18

200100200

Time (h)300400Time (h)Figure 9. Stability of C28 under acidic and basic conditions

3Column: 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

1.52Separation of amino acids with C28
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) SIM

2.521

160

50Figure 4. Separation of glycosides

0.57060

Time (min)080

204190

Test condition
Mobile phase: 0.5% TFA
Temperature: 60 ºC30

2.520.5100100Figure 3. Separation of melamine and cyanuric acid04• 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

Time (min)13Column: 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

Relative retention (%)

110column

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
Sample: 1. Cyanuric acid, 2. Melamine

24Figure 2. Comparison of three kinds of HILIC

234• SunShell HILIC-Amide showedstronger retention than others.
• Amide showed different selectivity
comparing with bare silica HILIC
column.

Applications of HILIC

5Relative plate number of thymine (%)

440 ºC2

Relative retention of thymine (%)

21

Change of retention of thymine at 40 oC(measurement every stop flow for 1 hour)

Column: SunShell HILIC-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: Ambient
Detection: UV@215 nm
Sample: 1. Aspartame, 2. Saccharin, 3. Acesulfame K

3

1.52m/z
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile,Leu
LysMetPheProSerThr
Trp
TyrVal

2.5Time (min)Figure 5. Separation of synthetic sweeteners
0102030

Time (min)2010Retention time/min

Figure 10. Separation of amino acids using UV and MS detections

Conclusions• 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

090175133134122147
14876156132147150
166116106120205182118

Applications of C283211410156972810345Time (min)Figure 11. Separation of organic acids

Column: SunShell RP-AQUA 2.6 μm, 150 x 4.6 mm
Mobile phase: 0.025 M KH2PO4, pH2.5
Flow rate: 1.0 mL/min
Column pressure: 32 MPa for 1.5mL/min
Temperature: 40 ºC
Detection: UV@210nm
Injection volume: 2 μLSample: 1 = Oxalic acid (60 ppm), 2 = Tartaric acid (500 ppm), 3 = Formic acid (1000ppm), 4 = Malic acid (1000 ppm), 5 = Lactic acid (1000 ppm), 6 = Acetic acid (1000ppm), 7 = Diglycolic acid (1000 ppm), 8 = Maleic acid (100 ppm), 9 = Citric acid (1000ppm), 10 = Succinic acid (1000 ppm), 11 = Fumaric acid (10 ppm).