hydrophilic interaction chromatography (hilic) for small molecules

© 2003 Waters Corporation.

Hydrophilic Interaction Chromatography Hydrophilic Interaction Chromatography (HILIC) for Small Molecules(HILIC) for Small Molecules

Diane Diehl, Eric Grumbach, Bonnie Alden, Pamela Iraneta, Paul Rainville, Jeff Mazzeo, Uwe Neue

Pittsburgh Conference 2003 #2370-8

©2003 Waters Corporation.

Outline• Introduction to HILIC• Development of AtlantisTM HILIC Silica

• Important considerations for HILIC • Sample diluent selection• Equilibration time• Reproducibility

• Column performance• Complementary selectivity to reversed-phase• Enhanced sensitivity for API-MS• Higher throughput


What is HILIC?• HILIC - Hydrophilic Interaction Chromatography

• HILIC is a variation of normal-phase chromatography

• Stationary phase is a polar material such as silica, cyano, amide, etc.

• A high organic-low aqueous mobile phase• i.e. water is the elution solvent

• Mechanism is a combination of weak cation exchange and partitioning into the adsorbed aqueous layer

• An alternative to traditional reversed-phase chromatography for the retention of very polar analytes

• Polar peptides, underivatized amino acids, active pharmaceutical ingredients and polar metabolites


HILIC Retention Characteristics

Cytosine

Greater retention occurs when using greater than 70% organic for polar bases.

N

NH

O

NH2

-3.00

-2.00

-1.00

0.00

1.00

2.00

3.00

0 10 20 30 40 50 60 70 80 90% MeCN

LN (T

rmin

/cm

)

pH 5

pH 4

pH 3


-2.90-2.80-2.70-2.60-2.50-2.40-2.30-2.20-2.10-2.00

0 10 20 30 40 50 60 70 80 90% MeCN

LN (T

rmin

/cm

) pH 5

pH 4

pH 3

Reversed-Phase Retention Characteristics

Cytosine

Greater retention occurs when using less than 20% organic for polar bases.

N

NH

O

NH2


Benefits of HILIC

•Allows the retention of highly polar analytes that would be un-retained by reversed-phase• Complementary selectivity to reversed-phase

•Enhanced sensitivity in mass spectrometry• Utilizing high organic mobile phases(> 80%)

promotes enhanced API-MS response

•Shortens sample preparation procedure• Elimination of evaporation/reconstitution step


Outline• Introduction to HILIC

• Development of AtlantisTM HILIC Silica• Important considerations for HILIC

• Sample diluent selection• Equilibration time• Reproducibility



Development of the New AtlantisTM HILIC Silica Column

•First HILIC column in a family of columns optimized for the retention of highly polar analytes

•Silica column that has been optimized for HILIC• AtlantisTM HILIC Silica is ideal for the retention of very

polar bases



• Development of AtlantisTM HILIC Silica

• Important considerations for HILIC• Sample diluent selection• Equilibration time• Reproducibility



The Importance of Sample Diluent Selection

• In HILIC, it is important for the sample diluent to be 100% organic – remember, the organic mobile phase is the weak solvent.

• The sample diluent can significantly influence peak shape and peak area of your analytes of interest

• However, polar analytes often have low solubilities in organic solvents

• We ran an extensive series of experiments to determine a generic diluent• 75% acetonitrile: 25% methanol is a useful generic

diluent for most polar analytes• This diluent is a compromise between solubility and

peak shape


Influence of Sample Diluent on Peak Shape

Peak shape improvesas % organic in

the diluent increases.

Peak shape can be further improved by

removing the aqueous portion of the diluent.

Compounds Sample Conc.1. 5-Fluorouracil 25 µg/mL2. Uracil 25 µg/mL3. 5-Fluorocytosine 25 µg/mL4. Cytosine 25 µg/mL

AU

0.00

0.05

0.10

Minutes1.00 2.00 3.00 4.00 5.00

H2O

50:50 ACN:H2O

75:25 ACN:H2O

AU

0.00

0.05

0.10

Minutes1.00 2.00 3.00 4.00 5.00

AU

0.00

0.10

0.20

Minutes1.00 2.00 3.00 4.00 5.00

1

1

1

2

2

2

3

3

3

4

4

4


AU

0.00

0.05

0.10

Minutes1.00 2.00 3.00 4.00 5.00

Influence of Sample Diluent on Peak Shape

MeOH

50:50 ACN:MeOH

75:25 ACN:MeOH

Peak shape improves as % acetonitrile in

the diluent increases.

Peak shape is improved by

favoring the non-polarportion of the diluent.

AU

0.00

0.05

0.10

Minutes1.00 2.00 3.00 4.00 5.00

AU

0.00

0.05

0.10

Minutes1.00 2.00 3.00 4.00 5.00

Compounds Sample Conc.1. 5-Fluorouracil 25 µg/mL2. Uracil 25 µg/mL3. 5-Fluorocytosine 25 µg/mL4. Cytosine 25 µg/mL

1

1

1

2

2

2

3

3

3

4

4

4


Equilibration and Reproducibility of AtlantisTM HILIC Silica

•Fast equilibration times• Columns equilibrated in 20 column volumes• As with any column, insufficient equilibration

can cause drifting retention times of analytes

•Excellent reproducibility• With appropriate column equilibration, HILIC

is as reproducible as reversed-phase


Analytes:S. Solvent Peak1. 5-Fluorouracil2. Uracil3. 5-Fluorocytosine4. Cytosine

Reproducibility of AtlantisTM HILIC Silica

AU

0.00

0.10

0.20

0.30

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.00

0.10

0.20

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Injection 500

Injection 1

1 2 43

S

Continuous injections of polar bases under gradient conditions yield excellent reproducibility.



• Development of AtlantisTM HILIC Silica

• Important considerations for HILIC • Sample diluent selection• Equilibration time• Reproducibility



Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

1.00 2.00 3.00 4.00 5.00

Complementary Selectivity to Reversed-Phase

Vo = 0.5 min

Compounds 1. Morphine 2. Morphine 3-β-D-glucuronide

AtlantisTM HILIC Silica4.6 x 50 mm, 3.0 µm90% to 50% ACN

1 2

AtlantisTM dC184.6 x 50 mm, 3.0 µm2% ACN

2

1Vo = 0.65 min

O

N

OH

OH

CH3

H

Morphine

Morphine 3-ß-D-Glucuronide


Peptides unretained on reversed-phase column can be retained by HILIC.

Complementary Selectivity to Reversed-PhaseSeparation of Cytochrome C Tryptic Digest

Peak AnnotationM/Z (Fragment ID)

0

100

%

204 (T2)261 (T6,11)361 (T18)434 (T21)

678 (T14)

634 (T4)

585 (T8)

779 (T15)

729 (T10)964 (T19)

1005 (T12)

634 (T4)

779 (T15) 678 (T14)434 (T21)

964 (T19)

261 (T6,11)204 (T2)

361 (T18)1005 (T12)

585 (T8)

729 (T10)

0

100

%

0 45

AtlantisTM dC184.6 x 50 mm, 3 µm

AtlantisTM HILIC Silica4.6 x 50 mm, 3 µm


Retention Benefits of HILICA

U

0.00

0.05

0.10

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AtlantisTM dC18 4.6 x 50 mm, 3 µm100% Formate Buffer, pH 1 mL/min

Vo = 0.65 min

NH

NHNH

O

OO

NH2

Allantoin

HILIC offers retention when there is no retention by reversed-phase.

Vo = 1.15 min

AU

0.00

0.05

0.10

0.15

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AtlantisTM HILIC Silica4.6 x 50 mm, 3 µm95:5 ACN:Formate Buffer, pH 31.4 mL/min


Enhanced Sensitivity in Mass Spectrometry

AtlantisTM dC18 Peak Area2.1 x 50 mm, 3 µm1. Albuterol 100 pg/µL 782. Bamethan 20 pg/µL 2

AtlantisTM HILIC Silica Peak Area2.1 x 50 mm, 3 µm2. Bamethan 20 pg/µL 93871. Albuterol 100 pg/µL 13131

HILIC requires high volatility solvents which increase sensitivity compared to high-aqueous mobile phases used in reversed-phase.

7.63e3

0

100

%

1.802.13

ES+

239.8

1.07e5

209.90

100

%

1.80

2.13

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

0

100

%

1.311.63

ES+

239.8

1.07e5

209.9

1

2?

12


Simplified Mixed-Mode SPE Procedure for HILIC

Condition/Equilibrate*200 µL methanol/200 µL water

Load150 µL spiked plasma sample

150 µL internal standardwith 2% ammonium hydroxide

Wash200 µL 5% methanol in water

Elute300 µL 40% acetonitrile/60% isopropanol

with 2% formic acid

*Oasis® HLBµElution Plate

Inject eluent directly onto columnEliminate Evaporation and Reconstitution Step


Mixed-Mode SPE:Direct Injection onto HILIC Column

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time

0

100

%

SIR of 2 Channels ES+TIC239.8209.98.97e4

1.49 1.84

NHOH

OH

CH3

Bamethan

NH

OH

OH

OHCH3 CH3

CH3

Albuterol

AtlantisTM HILIC Silica2.1 x 50 mm, 3 µm1. Bamethan 10 pg/µL 2. Albuterol 50 pg/µL

1 2

SPE eluent injected directly onto AtlantisTM HILIC Silica column


Summary

•AtlantisTM HILIC Silica columns offer:• Complementary selectivity to reversed-phase

chromatography • Retention of highly polar basic analytes not retained

by reversed-phase chromatography• Reproducible chromatography with equilibration

times similar to reversed-phase chromatography• Enhanced sensitivity in ESI-MS due to highly volatile

mobile phases (> 80% organic) • Shorter sample preparation procedures due to the

elimination of the evaporation and reconstitution steps and directly injecting the eluent

hydrophilic interaction chromatography (hilic) for small molecules

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