Why Aqueous Normal Phase and HILIC are Different

The Essential Feature of the Aqueous Normal Phase Mode is the ... Overlap of reversed phase and normal phase ... Hydrophilic Interaction Chromatography.
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PITTCON 2009 Chicago, IL

March 2009

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

Hydrophilic Interaction Chromatography (HILIC) uses ordinary Silica-Based Column or zwitterionic polymers

• 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

L-Alanine L-Arginine L-Aspartic acid L-Cystine L-Glutamic acid L-Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tyrosine L-Valine

9 8

Retention time (min)

7 6 5 4 3 2

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

7 Glucosamine

Retention Time (min.)

6

5

4

3

2

1

0 40

50

60

70

80

% Acetonitrile in DI Water + 0.5% FA

90

100

x10 2 + EIC(120.00000-120.20000) Scan Mix4_Gradient09A_Temp25_02.d

Leu

1 0.95 0.9 0.85

Gln

Ile

Pro

0.8 0.75 0.7

Phe

0.65 0.6

Retention Time L-Tryptophan 11.07 L-Leucine 11.22 L-Phenylalanine 11.25 L-Isoleucine 11.31 L-Tyrosine 11.33 L-Methionine 11.55 L-Valine 11.61 L-Aspartic acid 11.83 L-Glutamic acid 11.83 L-Alanine 12.05 Amino Acid

Amino Acid L-Threonine L-Glycine L-Serine L-Proline L-Asparagine L-Glutamine L-Arginine L-Histidine L-Lysine

Retention Time 12.29 12.32 12.52 12.94 13.20 13.35 16.63 16.67 17.01

0.5

His

Arg

0.55

0.45

0.35

Val

Trp

0.4

Asp

0.1 0.05 0.5

11

11.5

12

Lys Asn

Ser

Thr

0.15

Ala

Glu

0.2

Gly

0.25

Met

Tyr

0.3

12.5

13

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.

EXTRACTED ION CHROMATOGRAMS OF ORGANIC ACIDS

maleic acid (1) aconitic acid, trans (2) aconitic acid, cis (3) aconitic acid, impurity (4) fumaric acid (5) citric acid (6) oxaloacetic acid (7)

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

Cytidine-R1 Cytidine-R2 Cytidine-R3 Quinolinedione-R1 Tetramic acid Quinolinedione-R2 Benzopyran

Mol Wt. 397 454 425 536 536 520 396

ApKa 12.12 12.07 12.09 8.65, 8.68 7.91, 10.98 8.64 -

BpKa

Log P

3.73 3.73 3.73 5.36 -

1.54 3.66 2.66 3.46 2.96 2.74 2.80

Mobile Phase: 60:40 acetonitile/water

FOR MORE INFORMATION ON ALL TYPE C SILICA HYDRIDE PHASES

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