Interplay of Chromatographic

Parameters and Analyte

Physical Properties on
Retention and Selectivity in
Hydrophilic Interaction Liquid
Chromatography

A. Carl Sanchez, Monika M. Kansal, Art Dixon and Ismail Rustamov

Phenomenex, Inc., 411 Madrid Ave.,
Torrance, CA 90501 USA
Introduction
The analysis of polar compounds using mass rationalizing the effect of operating parameters

spectrometric compatible conditions is becoming on analyte-sorbent interactions in HILIC mode. In

increasingly important as drug compounds this presentation, changes in analyte and sorbent

continue trending toward more polar molecules. physical properties in the high organic environment

Hydrophilic interaction liquid chromatography of HILIC are used to explain the observed effects

(HILIC) is a unique separation mode that addresses on retention and selectivity with several model

this growing need; however, further research compounds and chromatographic sorbents.

characterizing this separation mode is needed

to reduce it to practice. The chromatographic

conditions employed in HILIC mode separations

have a dramatic effect on both analyte and

chromatographic sorbent physical properties.

To date, little research has been devoted to

Hydrophilic Interaction Chromatography (HILIC)
HILIC retention mechanism
Primarily partitioning of analytes between water enriched layer of solvent near sorbent surface
and the relatively more hydrophobic bulk eluent

Partitioning based on relative solubility in each layer

H-bonding and Ion-exchange with the sorbent also important
Primary HILIC Chromatographic Parameters

Column Chemistry
Silica, amino, cyano, PFP, diol, zwitterion, ion-exchange, specialty

Type and % Organic

Acetonitrile primary weak eluent
Isocratic: 95-70 % Acetonitrile typical
Gradient: 90-50 % Acetonitrile
Modifiers IPA, methanol, ethyl acetate, others

Buffer pH
pH 2 7

Ionic Strength (IS)

5-10 mM buffer concentration
Probe Compound Physical Properties

p-Aminobenzoic Acid (1) Nicotinamide (2) Riboflavin (3) Nicotinic Acid (4) Pyridoxine (5)
pKa 4.7, H+ pKa 2.7 H+ pKa 3.35 pKa 10.2 pKa 4.7, H+ pKa 3.0 H+ pKa 5.6, pKa 8.6
logP 0.83 logP 0.37 logP - 1.46 logP 0.36 logP 0.77
HO
O OH OH
O
OH
O HO

OH HO
OH OH
H2N N N N O
H2N N
NH
N
N
O

Thiamine (6) Ascorbic Acid (7) Vitamin B12 (8) Folic Acid (9)
H+ pKa 5.5 pKa 4.1, 11.2 pKa 1.59 pKa 2.7, 4.1, 8.9
logP 4.6 logP 1.85 logP 0.90 logP - 0.02
O NH 2
O NH2
NH 2 HO O
HO
-
Cl O H2N
NH2
N
O N
N N+ O N N OH N NH 2
HO Co
O H O O N
S OH N N
N H2N NH2 NH N

O NH
O
HO OH N
HO
NH
N
HO H H
O
O
O
O P
O H
O H
OH
Weak Acid pKa vs. % Organic

pKa of weak acids increase with increasing organic solvent concentration

Sorbent silanols expected to follow similar trend

Acetic acid pKa vs. v/v % Acetonitrile2

7.0

6.5
extrapolated
60-90 % Acetonitrile
s/w pKa

6.0

5.5

5.0

4.5
0 10 20 30 40 50 60 70 80 90 100

v/v % Acetonitrile
Weak Base pKa vs. % Organic

pKas of weak bases decrease with increasing organic solvent concentration

Smaller changes than acids

Ammonia pKa vs % Acetonitrile1

9.5

9.3

9.1

8.9
s/w pKa

8.7
extrapolated
8.5 60-90 % Acetonitrile1

8.3

8.1

7.9

7.7

7.5
0 10 20 30 40 50 60 70 80 90 100

v/v % Acetonitrile
 Basic Analyte % Ionization
As in RPLC, ionization of analytes greatly effects retention in HILIC
Retention in HILIC generally follows ionization profile
In HILIC retention increases with increased ionization

Pyridoxine ionization profile vs. pH in water

100
90
80
OH
% ionization

70
60
50 HO
40 OH
30
20
10
N
0
1.7 2.7 3.7 4.7 5.7 6.7 7.7

pH
 Zwitterionic Analyte % Ionization
For zwitterionic analytes, all ionizable groups must be considered to understand
retention changes with pH
p-amino benzoic acid (PABA) micro species vs. pH in water
4 species: neutral, ionized acid, ionized base, zwitterion

PABA micro species vs. pH in water

100

90

80
O
70
% ionization

60

50
OH

40
H2 N
30

20

10

0
1.7 2.7 3.7 4.7 5.7 6.7 7.7

pH
% ionized base % zwitterion % neutral % ionized acid
 High Organic Concentration Effect on Analyte pka and
% Ionization
High organic concentration shifts pKas and changes ionization profile
Decreased ionization due to organic solvent gives decreased retention

Ionization profile pyridoxine in 90 v/v % acetonitrile and 100 % water

100

90
OH
80

70
HO
% ionization

60
OH
50

40
N
30

20

10

0
1.7 2.7 3.7 4.7 5.7 6.7 7.7

pH
ionzation profile 90 % Acetonitrile % ionization profile water
 Shifting pka and % Ionization PABA
With high organic solvent concentration the difference between acidic and basic
pKas increase which reduces the number of species present in solution

Ionization profile PABA in 90 v/v % acetonitrile and 100 % water

100

90 O

80

70
OH
% ionization

60
H 2N
50

40
ionization ionization
profile water profile 90 %
30
Acetonitrile
20

10

0
3.7 4.2 4.7 5.2 5.7 6.2 6.7 7.2 7.7

pH
ionization profile 90 % Acetonitrile ionization profile water
Analyte Retention vs. % Ionization in 90% Acetonitrile
In HILIC, retention increases with increasing polarity
Increasing ionization increases polarity and thus increases retention
For pyridoxine, ionization changes only slightly at the two mobile phase pHs used
Retention is not expected to be significantly affected

100

90 OH
80

70 HO
% ionization

low pH mobile OH high pH mobile

60
phase 90 v/v % phase 90 v/v %
50
Acetonitrile N Acetonitrile
40

30

20
~5% change in ionization
with pH change
10

0
3.7 4.2 4.7 5.2 5.7 6.2 6.7 7.2 7.7

pH
% ionized base 90 % Acetonitrile % neutral 90 % Acetonitrile
Zwitterionic Analyte Retention vs. % Ionization in 90 %
Acetonitrile
Due to pKa shifts, only the acidic group of PABA ionizes
Basic pKa decreased while acidic pKa increased
For PABA, ionization changes significantly at the two mobile phase pHs used
Retention is expected to change significantly
120
O
100

OH
% ionization

80

low pH mobile high pH mobile

60
phase 90 v/v % H2N phase 90 v/v %
Acetonitrile Acetonitrile
40
90 % change in ionization
20
with pH change

0
3.7 4.2 4.7 5.2 5.7 6.2 6.7 7.2 7.7

% zwitterion 90 % Acetonitrile pH % neutral 90 % Acetonitrile

% ionized base 90 % Acetonitrile % ionized acid 90 % Acetonitrile
Pyridoxine & PABA Retention Aqueous pH 3.2 & 5.8
Significant retention and selectivity changes with mobile phase pH change
5 m LUNA HILIC
For pyridoxine, minor change in ionization between pHs, no change in retention
For PABA, major change in ionization which results in major change in retention

Conditions Gradient Program:

Column: 150 x 4.6 mm Time % Acetonitrile H 2O Buffer
Flow Rate: 2 mL/min 0 90 5 5
Temperature: Ambient 2.5 90 5 5
10.0 50 45 5
12.5 50 45 5

OH

H 2N

OH

HO
OH

N
Effect of Column Chemistry H-bonding & Ion-Exchange
Significant retention and selectivity differences with changes in column chemistry
Likely due to differences in H-bonding and ion-exchange

Conditions Gradient Program:

Column: 150 x 4.6 mm Time % Acetonitrile H 2O Buffer
Flow Rate: 2 mL/min 0 90 5 5
Temperature: Ambient 2.5 90 5 5
10.0 50 45 5
12.5 50 45 5
pH Effect on Column Properties
Thiamine (quaternary amine) adsorbed on silica at pH 5.8, elutes at pH 3.2
Surface silanols ionized at pH 5.8

Conditions Gradient Program:

Column: 150 x 4.6 mm Time % Acetonitrile H2O Buffer
Flow Rate: 2 mL/min 0 90 5 5
Temperature: Ambient 2.5 90 5 5
10.0 50 45 5
12.5 50 45 5

NH2
Cl-
+
N N
S OH
N
Conclusions
Retention and selectivity changes in HILIC can be understood by the effect of chromatographic
conditions, such as pH and percent organic, on analyte and sorbent physical properties

The high organic concentration used in HILIC significantly alters analyte, sorbent and buffer pKas

pKas of acids increase with increasing organic concentration

pKas of bases decrease with increasing organic concentration

Aqueous buffer pH and thus mobile phase pH changes with addition of organic

Analyte pKa and mobile phase pH change with increasing organic solvent

Both of these affect ionization and thus retention

In HILIC analyte retention increases with increased ionization

Sorbent surface chemistry significantly affects selectivity and retention thru differences
in H-bonding and ion-exchange

H-bonding and ion-exchange character of analytes and sorbents affected by changes

in ionization
 References
1. Espinosa S., et al Anal. Chem 2002, 74, 3809-3818
2. Roses R., et al J of Chromatogr. A 2002, 982, 1-30
3. Alpert, A.J. J. Chromatogr. 1991, 177-196
4. Naidong, W. J. Chromatogr. B 2003, 796, 177-196
5. Hemstrom, P.; Irgum, K. J. Sep. Sci. 2006, 29, 1784-1821
6. Olsen, B.A. J. Chromatogr. A 2001, 913, 113-122
7. Guo, Y.; Gaika S. J. Chromatogr. A 2005, 1074, 71-80
8. Strege, M.A. Anal. Chem. 1998, 70, 2439-2445
9. Yoshida, T. J. Biochem. Biophys. Methods 2004, 60, 265-280

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