The Essential Feature of the Aqueous Normal Phase Mode is the ... Overlap of reversed phase and normal phase ... Hydrophilic Interaction Chromatography.
Why Aqueous Normal Phase and HILIC are Different Joseph J. Pesek Department of Chemistry San Jose State University San Jose, CA, 95192 Maria T. Matyska Microsolv Technology Corporation Eatontown, NJ 07724
The Essential Feature of the Aqueous Normal Phase Mode is the Presence of the Hydride Surface
ORDINARY SILICA
O O Si O O Si O O Si O
HYDRIDE SILICA
O OH OH OH
O Si O O Si O O Si O
O O O O
Si O Si O Si O
H H H
WHAT IS HILIC RETENTION? HILIC Retention 14
Retention Time (min)
12 10
Hydrophilic Compound Hydrophobic Compound
8 6 4 2 0 0
20
40
60
% Organic in Mobile Phase
80
100
WHAT IS AQUEOUS NORMAL PHASE RETENTION? A continuum of retention that provides a transition from the reversed-phase to the normal phase modes with water as a constituent in the mobile phase Three distinct retention patterns are possible: 1. No overlap of reversed phase and normal phase retention for two or more compounds. 2. Overlap of reversed phase and normal phase retention for two or more compounds. 3. Individual compounds that can be retained by both reversed phase and normal phase modes.
No overlap of reversed phase and normal phase retention for two or more compounds. ANP 1 16 14
ANP Compound Reversed-Phase Compound
Retention Time (min)
12 10 8 6 4 2 0 0
20
40
60
% Organic in Mobile Phase
80
100
Overlap of reversed phase and normal phase retention for two or more compounds.
ANP 2 45 ANP Compound Reversed-Phase Compound
40
Retention Time (min)
35 30 25 20 15 10 5 0 0
20
40
60
80
100
% OPrganic in Mobile Phase
Aqueous normal phase (1) and reversed-phase (2) compounds at three mobile phase compositions: A, 50:50 acetonitrile, DI water; B, 80:20 acetonitrile, DI water; and C, 85:15 acetonitrile, DI water.
Individual compounds that can be retained by both reversed phase and normal phase modes ANP 3 20 18
Retention Time (min)
16 14 12 10 8 6 4 2 0 0
10
20
30
40
50
60
% Organic in Mobile Phase
70
80
90
100
DIFFERENCES BETWEEN AQUEOUS NORMAL PHASE AND HILIC Aqueous Normal Phase Silica Hydride-Based Column
• Retains nonpolar compounds by reversed phase mechanism • Retains polar compounds by normal phase mechanism • Both reversed phase and normal phase mechanisms can operate simultaneously • Can separate samples with both polar and nonpolar compounds
• Retains polar compounds by a normal phase mechanism • Does not retain nonpolar compounds • Cannot usually separate samples having both polar and nonpolar compounds
USE OF VARIOUS MOBILE PHASE SOLVENTS IN ANP
Acetonitrile is the most common organic mobile phase component for ANP but others are possible
Methanol is usually only possible with strongly basic compounds
Acetone is possible when using MS detection. ANP behavior similar to acetonitrile
Retention time vs % acetone in the mobile phase
1 0 40
50
60
70
80
% Acetone
Retention Time With Acetone
90
100
EFFECT OF TEMPERATURE IN ANP OPPOSITE TO REVERSED-PHASE FOR MANY SOLUTES
EFFECT OF STATIONARY PHASE ON ANP RETENTION Hydride Based Cholesterol: Choline/Acetylcholine 30 Acetylcholine Choline
20
15
10
5
0 40
50
60
70
80
90
100
% Acetonitrile in DI w ater + 0.5% FA
Hydride Based BD C18: Choline/Acetocholine 25
20 Choline Retention time (min.)
Retention time (min.)
25
Acetylcholine 15
10
5
0 40
50
60
70
% Acetonitrile in DI w ater + 0.5% FA
80
90
100
GRADIENT REPRODUCIBILITY
The table shows retention time reproducibility for nine amino acids at two temperatures. Four replicates were performed at each temperature. The reproducibility was 0.28% or better for the amino acids. This is a significant improvement over what is usually observed for most HILIC analyses, especially considering this is gradient data with only a 5 minute re-equilibration time between runs.
ANP RESULTS IN IMPROVED SENSITITY WHEN USING MS FOR DETECTION
SIGNAL INTENSITY AS A FUNCTION OF MOBILE PHASE COMPOSITION GLUCOSE SIGNAL INCREASES AS % ACETONITRILE INCREASES UP TO 95% IN THE MOBILE PHASE
COLUMN EFFICIENCY OF ANP STATIONARY PHASES Plots of efficiency (HETP) vs. flow rate
(A) glucose and sorbitol on DH column (2.1 x 150 mm, particle size 4 µm) in a 80:20 ACN/DI water + 0.1% formic acid mobile phase.
(B) Comparison of commercial HILIC (4.6 x 150 mm, particle size 3.5 µm) and DH (4.6 x 150 mm, particle size 4.0 µm) columns for ANP retention of uracil.
EXAMPLES OF HYDROPHILIC COMPOUNDS RETAINED BY ANP Glucosamine on Bidentate C18 8
13.5 14 14.5 Counts (%) vs. Acquisition Time (min)
15
15.5
16
16.5
17
Extracted Ion Chromatogram Of Nineteen Amino Acid Separation All of the critical amino acid pairs (those that are isobaric or have masses within one mass unit ) are separated under these conditions except for the Leucine / Isoleucine pair.
NUCLEOTIDES ON UNDECENOIC ACID COLUMN 1 = adenosine-3’,5’-cyclic monophosphate; 2 = adenosine-5’-monophosphate; 3 = adenosine-5’-triphosphate; 4 = thymidine-5’-triphosphate; 5 = uridine-5’- triphosphate; 6 = cytosine-5’-triphosphate; 7 = guanosine-5’-triphosphate. 85:15 90% acetonitrile/10% DI water + 0.1% ammonium formate/ DI water + 0.1% ammonium formate.
Highly polar metabolites: Uridine 5′-diphosphate (UDP) and phosphorylated sugars analyzed using Diamond Hydride column.
1 - the monitored MRM transitions were m/z 535 to m/z 323 2 - the monitored MRM transitions were m/z 564 to m/z 322 3 - UDP hexanolamine (internal standard) - the monitored MRM transitions were m/z 502 to m/z 258
ANP RETENTION CAN BE OBSERVED FOR HYDROPHILIC PEPTIDES
GENERAL PEPTIDE STRUCTURE: Ac-AXEXAHKAY-NH2
SEPARATION OF A MIXTURE OF POLAR AND NONPOLAR COMPUNDS Compound No. Compound Type 1 2 3 4 5 6 7
Discussion of Chromatograms. Chromatograms A & B. Peak 1 (Metformin) eluted with longer retention time as the concentration of Acetonitrile was increased.
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