Evaluation of Core Shell Type HILIC
Amide Column as First Choice
Tomoyasu Tsukamoto and Norikazu Nagae1) ChromaNik Technologies Inc., Namiyoke, Minato-ku, Osaka, 552-0001 Japan

Abstract
Hydrophilic Interaction liquid Chromatography (HILIC) proposed by Alpert in 1990 has been applied for analysis of many 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 mobile phase for HILIC. It is said that HILIC separation is achieved by partition between a mobile phase and a water rich layer on thestationary phase. Therefore, it is important for HILIC that a stable water rich layer can be formed on the stationary phase. We believe that a HILIC column as a first choice must have the following four characteristics; (1)the base material must be highly hydrophilic, (2) the functional group must have high water retention capacity and be densely bonded on the base material, resin or silica, (3) the functional group and base material musthave a little electrostatic interaction, and (4) the spacer chain between the functional group and the base material must have a reduced hydrophobicity. In this study, we selected amide groups for a HILIC stationary
phase due to non-electrostatics and high hydrophilicity. The amide type core shell particle was evaluated whether it satisfied the characteristics of the above requirements. The characteristics of SunShell HILIC-Amide
used as a core shell type amide column was investigated using the evaluation method for HILIC reported by Kawachi and coworkers [1]. The bonding density of amide groups of SunShell HILIC-Amide was about 3.3μmol/m2, and the efficiency was achieved up to 220,000/m. This HILIC column showed not only hydrophilicity as high as PolyHYDROXYETHYL and Amide-80 column using the uridine test method but also very lowanion and cation exchange interactions. In case of comparison of core shell type HILIC columns on separation of nucleobases, SunShell HILIC-Amide indicated the longest retention time. Finally analyses of glycosides,
melamine and synthetic sweeteners were achieved.
[1]. Y. Kawachi, et al., J. Chromatogr., A 1218 (2011) 5903-5919.

HILIC Mechanism
H2OOOOOOSiSiOH

O

O

1Si

H2O

O

Mobile Phase

H2OSi
O

Si

O

 Character of HILIC
OH

O

O

O

O

Si

OSi

O

SiSi-H2O

O

H2O

OO

Si

OH

O
OSi

O

O

OSi
O

O
OO

Si
O

OOO

O

Si

OOH

O

H2O O

Si

H2O Si

O

HOO

O

H2O

1-

OH

H2O
Si

O

ODS silica
(RP)

OH
OH

O

O

2H2O
Si

H2O Si

H2O O

SiAnalyte

O

OHO
O
O

H2O

HO

O

Si

O

Si

H2O

O

Si

O

O-Si

O

O

Hydrophilic
Partitioning

SiSiOO

SiO

O

Si

O

OAnalyte

H2O

H2O O

Si

Si

OHSiOOOOO

2Silica gel(HILIC)OHOO

Stationary Phase for HILIC

O-H2O

Polar organic solvent suchas methanol can be used.
HILIC separation bases onpartition of sample
between mobile phase andwater layer formed on
stationary phase.
The higher polarity of acompound, the longer theirretention time.

Figure 1. Image of HILIC separation mechanism

Hydrophilic interaction
Ion exchangeinteraction

Electrostaticinteraction
OO-SAnion
exchange
group

Zwitter iongroup
ON

Hydrogen BondingCation
 Comparing with RP column
exchange Main interaction is hydrophilicgroup
Diol group
Opartition but secondary
AmidegroupOHinteraction also works
O S+ONNOHO+CH3

CH3

relatively strong.
 All columns aren’t end-capped.
 Electrostatic functional groups
are often used for HILICOHFigure 2. Structure of functional groups for HILIC columns
O

Work of the Secondary Interaction

What is Needed as First Choice for HILIC

Main interaction

 The secondary interaction
was used to change theselectivity of the columns.
Mix mode type
 The secondary interaction
also was reduced by an end
capping for C18.
 It is very difficult to lose thesecondary interaction onadsorbents completely.

Secondaryinteraction

Controlled secondary
Interaction type

Existing adsorbent

Residual silanolgroup

A polar groupembedded C18

Table 1. Comparison of hydrophilicity
ColumnZIC-HILIC (5 mm)
ZIC-HILIC (3.5 mm)
Nucleodur HILIC (3 mm)
Amide-80 (5 mm)
XBridge Amide (3.5 mm)
PolySULFOETHYL (3 mm)
PolyHYDROXYETHYL (3 mm)
CYCLOBOND I (5 mm)
LiChrospher Diol (5 mm)
Chromolith Si
HALO HILIC (2.7 mm)
COSMOSIL HILIC (5 mm)
Sugar-D (5 mm)
NH2 -MS (5 mm)
SunShell HILIC-Amide (2.6 mm)

Uridine（U） Vidaravine（V） Adenosine （A）

Conditions：Mobile phase: Acetonitrile/ammonium acetate buffer(20 mM, pH = 4.76) = 90:10 [v/v]Linear velocity; 1.0 mm/s,UV detection wave length; 254 nm, Column oven temperature; 30 ℃

Columns were evaluated theirhydrophilicity using uridine
The longer retention time of uridine, thehigher hydrophilicity
HILIC columns have differenthydrophilicity
It is necessary to choose a HILIC
column properly as well as a RPcolumn.

SunSell HILIC-Amide showed
the third highest hydrophilicityin these columns.

USPTSa(SPTS/U)U

TMPACk (U)20 mMk (TMPAC)

SunShell HILIC-Amide
452431k (U)20 mM

k (SPTS)ZIC-HILIC (5 μm)

2.11

0.69

0.33

2.11

3.32

1.57

ZIC-HILIC (3.5 μm)

2.10

0.56

0.27

2.10

3.45

1.64

Nucleodur HILIC (3 μm)

2.20

1.13

0.51

2.20

3.14

1.43

Amide-80 (5 μm)

3.30

0.89

0.27

3.30

4.57

1.38

XBridge Amide (3.5 μm)

2.55

0.74

0.29

2.55

1.890.74

PolySULFOETHYL (3 μm)

1.58

0.25

0.161.58

1.380.87

PolyHYDROXYETHYL (3 μm)

3.92

0.87

0.22

3.92

3.340.85

CYCLOBOND I (5 μm)

0.70

3.32

4.73

0.70

0.450.63

LiChrospher Diol (5 μm)

1.50

0.95

0.63

1.50

1.73

1.16

Chromolith Si

0.31

0.06

0.19

0.31

5.25

16.94

HALO HILIC (2.7 μm)

0.64

0.20

0.31

0.64

9.03

14.11

COSMOSIL HILIC (5 μm)

1.60

1.28

0.80

1.60

0.78

0.49

Sugar-D (5 μm)

1.58

3.00

1.90

1.58

0.39

0.25

NH2-MS (5 μm)

2.442.010.82

2.440.690.28

SunShell HILIC-Amide (2.6 μm)

2.93

1.29

0.48

2.93

1.18

0.44

Column:SunShell HILIC-Amide 2.6 μm, 100 x 4.6 mm
Ascentis Express OH5 2.7 μm, 100 x 4.6 mm
Ascentis Express HILIC 2.7 μm, 100 x 4.6 mm
Mobile 1.0 mL/min
Temperature: 40 oC
Detection: UV@250 nm,
Sample : 1.Tymine, 2. Uracil, 3. Uridine, 4. Cytosine, 5. Cytidine

Ascentis Express OH5

5Ascentis Express HILIC
00.5

11.5

2Time (min)2.5

3Figure 3. Comparison of three kinds of HILIC
column3.5Trimethylphenylammonium
chloride (TMPAC)

Ion exchange interaction was evaluated using theabove ion compounds and uridine.
The smaller the separation factor of uridine and ion
compounds, the lower ion exchange interaction.

The separation factors of both on SunShell
HILIC-Amide were small. Consequently,SunSehll HILIC-Amide had low ion exchangeinteraction.

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

2100.5

11.522.5Time (min)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

24Sodium p-toluenesulfonate (SPTS)

Applications of HILIC

Comparing between SunShell HILIC-Amide andother HILIC columns • • •21Amide groupFigure 4. Separation of melamine and cyanuric acid53selecteda(TMPAC/U)

Column23HN

Evaluation of Ion Exchange

Amide vs other HILIC phase
1O

Table 2. Comparison of ion exchange ability
AVk (A) k (V)1.55 2.32
1.51 2.28
2.33 3.40
3.80 4.90
2.81 3.64
1.15 1.39
3.75 4.93
1.36 1.68
2.50 3.30
0.73 0.85
1.59 1.87
2.20 3.00
1.88 2.72
2.13 2.90
3.55 4.84

2.11
2.10
2.20
3.30
2.55
1.58
3.92
0.70
1.50
0.31
0.64
1.60
1.58
2.44
2.93

RMix mode column

Evaluation of Hydrophilicity
Uk (U)

1. The base material must be highly hydrophilic.
2. The functional group must have high water
retention capacity and be densely bonded on
the base material, resin or silica.
3. The functional group and base material musthave a little electrostatic interaction.
4. The spacer chain between the functionalgroup and the base material should be as
short as possible.

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

400.5

11.5

22.5

Time (min)Figure 5. Separation of glycosides
2Column: 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 KConclusions
 Sunsehll HILIC-Amide had high hydrophilicity and low ion exchange

comparing with other HILIC columns.
 SunShell HILIC-Amide showed the longest retention time in all core shell
type HILIC columns.
 Characteristics of a HILIC column depends on a functional group whichshows different selectivity individually as well as a RP columns.

10

0.5

13

1.5

2

2.5

Time (min)Figure 6. Separation of synthetic sweeteners