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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.
Column Chemistry
Silica, amino, cyano, PFP, diol, zwitterion, ion-exchange, specialty
Buffer pH
pH 2 7
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
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
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
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
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
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
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
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
0
3.7 4.2 4.7 5.2 5.7 6.2 6.7 7.2 7.7
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
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
Aqueous buffer pH and thus mobile phase pH changes with addition of organic
Analyte pKa and mobile phase pH change with increasing organic solvent
Sorbent surface chemistry significantly affects selectivity and retention thru differences
in H-bonding and ion-exchange
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