Hydrophilic Interaction Chromatography (HILIC) for Small Molecules
Transcript of 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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
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-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
©2003 Waters Corporation.
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
©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
©2003 Waters Corporation.
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
©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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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.
©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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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
©2003 Waters Corporation.
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