Using Hydrophilic Interaction Chromatography
(HILIC) for the Retention of Highly Polar Analytes
Eric S. Grumbach, Diane M. Diehl and Jeffrey R. Mazzeo
©2003 Waters Corp.
Outline
ƒ Introduction
ƒ HILIC – Definition and Benefits
ƒ Important considerations for HILIC
– Mobile phase considerations
– Solvent selectivity
ƒ Column performance
–
–
–
–
–
Column reproducibility
Complementary selectivity to reversed-phase
Enhanced ionization in mass spectrometry
Low limits of detection
Direct injection of SPE eluent
ƒ Summary
© 2003 Waters Corporation
Introduction
ƒTraditional options for retaining polar analytes
– Ion pairing or ion exchange
ƒ Only works if analytes ionize
ƒ Often not compatible with mass spectrometry (MS)
– Manipulate pH to make compound neutral
– Reversed-phase columns designed for the retention of
polar analytes (such as the Atlantis™ dC18)
ƒ Used under highly aqueous conditions, often resulting in poor
MS sensitivity
What happens when you’ve tried all of these options
and you STILL have inadequate retention???
Let’s take a look at HILIC…
© 2003 Waters Corporation
What is HILIC?
ƒ HILIC - Hydrophilic Interaction Chromatography
– Term coined in 1990 to distinguish from normal-phase*
ƒ HILIC is a variation of normal-phase chromatography
without the disadvantages of using solvents that are not
miscible in water
– “Reverse reversed-phase” or “aqueous normal-phase”
chromatography
ƒ Stationary phase is a POLAR material
– Silica, cyano, amino, diol
ƒ The mobile phase is highly organic (> 80%) with a smaller
amount of aqueous mobile phase
– Water (or the polar solvent(s)) is the strong, eluting solvent
*Alpert, A. J. J.Chromatogr. 499 (1990) 177-196.
© 2003 Waters Corporation
HILIC Retention on Silica
• Polar analyte partitions into and out of adsorbed water layer
• Charged polar analyte can undergo cation exchange with charged silanol groups
• Combination of these mechanisms results in enhanced polar retention
© 2003 Waters Corporation
HILIC vs. Reversed-Phase
Retention Characteristics
H
N
O
N
Retention
NH+
3
Cytosine
HILIC
RP
0
10
20
30
40
50
60
% MeCN
70
80
90
HILIC offers dramatically more retention than
reversed-phase for very polar bases.
© 2003 Waters Corporation
Alden, Iraneta
Benefits of HILIC
ƒRetention of highly polar analytes not retained by
reversed-phase
ƒComplementary selectivity to reversed-phase
ƒEnhanced sensitivity in mass spectrometry
–High organic mobile phases (> 80%) promotes
enhanced ESI-MS response
ƒShortens sample preparation procedure
–Elimination of evaporation/reconstitution step by
directly injecting the organic phase
© 2003 Waters Corporation
Outline
ƒ Introduction
ƒ HILIC – Definition and Benefits
ƒ Important considerations for HILIC
– Mobile phase considerations
– Solvent selectivity
ƒ Column performance
–
–
–
–
–
Column reproducibility
Complementary selectivity to reversed-phase
Enhanced ionization in mass spectrometry
Low limits of detection
Direct injection of SPE eluent
ƒ Summary
© 2003 Waters Corporation
Mobile Phase Considerations:
Before You Start
ƒ Wash solvents
– Seal wash: 10% acetonitrile in water
– Needle wash: 1:1 acetonitrile:water
– Purge Solvent: initial mobile phase conditions (without
salt, additive or buffer)
ƒ Initial conditions: 95% to 70% acetonitrile
– Remember, acetonitrile is the weaker solvent
– At least 5% should be a polar solvent (i.e., water or methanol)
ƒ Where do I start?
– Initial scouting gradient from 95 to 50% acetonitrile
– If analytes are not retained, try 95% acetonitrile isocratic
© 2003 Waters Corporation
Mobile Phase Considerations:
Before You Start
ƒ Buffers/additives
– Phosphate salt buffers are not recommended due to precipitation in the highly
organic mobile phase (phosphoric acid is OK)
– Ammonium formate, pH 3*; ammonium acetate, pH 5*; 0.2% formic acid, pH
2.5*, 0.2% phosphoric acid, pH 1.8*
For optimum performance and reproducibility it is
recommended that a concentration of 10 mM buffer or
0.2% of an additive be introduced ON COLUMN
ƒ To increase retention of analytes, replace some of the water with
another polar solvent (i.e., methanol, isopropanol)
– In HILIC, these polar solvents are weaker eluters than water
– Peak shapes and reproducibility may be compromised by completely removing
the polar solvent (e.g., water)
Let’s take a closer look…
*The actual pH of the mobile phase may be 1 pH unit higher due to the highly organic mobile phase
Canals, I.; Oumada, F. Z.; Roses, M.; Bosch, E. J. Chromatogr. A. 911 (2001) 191-202.
© 2003 Waters Corporation
Solvent Strength: HILIC
Solvent
Water
Methanol
Ethanol
Isopropanol
Acetonitrile
Acetone
Tetrahydrofuran
© 2003 Waters Corporation
Strongest
In HILIC, utilizing
a less polar
solvent can
increase the
retention
of polar analytes.
Weakest
Increased Retention with
a Less Polar Solvent
2
Compounds
1. 5-Fluorouracil
2. Uracil
3. 5-Fluorocytosine
4. Cytosine
0.50
4
Initial 90 ACN: 5 H2O: 5 buffer
Final 50 ACN: 45 H2O: 5 buffer
1
3
1.00
1.50
2.00
2.50
Minutes
2
1
3.00
4
3
0.50
1.00
1
2
1.50
2.00
2.50
Minutes
3.00
1.00
1.50
2.00
2.50
Minutes
4.00
4.50
5.00
Initial 90 ACN: 5 MeOH: 5 buffer
Final 50 ACN: 45 MeOH: 5 buffer
3.50
4.00
4.50
5.00
Replace MeOH
with IPA
Initial 90 ACN: 5 IPA: 5 buffer
Final 50 ACN: 45 IPA: 5 buffer
3
0.50
3.50
Replace H2O
with MeOH
4
3.00
3.50
4.00
4.50
5.00
Retention increases by using a less polar solvent.
© 2003 Waters Corporation
Grumbach
Outline
ƒ Introduction
ƒ HILIC – Definition and Benefits
ƒ Important considerations for HILIC
– Mobile phase considerations
– Solvent selectivity
ƒ Column performance
–
–
–
–
–
Column reproducibility
Complementary selectivity to reversed-phase
Enhanced ionization in mass spectrometry
Low limits of detection
Direct injection of SPE eluent
ƒ Summary
© 2003 Waters Corporation
Reproducibility of
AtlantisTM HILIC Silica
1
3
2
4
S
Injection 1
Injection 100
Injection 200
Injection 300
Analytes:
S.
Solvent Peak
1.
5-Fluorouracil
2.
Uracil
3.
5-Fluorocytosine
4.
Cytosine
Injection 400
Injection 500
Injection 600
Injection 700
Injection 800
0.50
1.00
1.50
2.00
2.50
Minutes
3.00
3.50
4.00
4.50
5.00
Continuous injections of polar bases under
gradient conditions yield excellent reproducibility.
© 2003 Waters Corporation
Grumbach
Retention Benefits of HILIC
AtlantisTM dC18
4.6 x 50 mm, 3 µm
100% Formate Buffer, pH 3
1.0 mL/min
k=0
0.10
AU
Vo = 0.65 min
0.05
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
k = 1.09 AtlantisTM HILIC Silica
0.15
H
N
5.00
Minutes
O
O
O
N
H
Allantoin
4.6 x 50 mm, 3 µm
95:5 ACN:Formate Buffer, pH 3
1.0 mL/min
Vo = 1.15 min
AU
0.10
NH
H2N
0.00
0.05
0.00
0.50
1.00
1.50
2.00
2.50
Minutes
3.00
3.50
4.00
4.50
5.00
HILIC offers retention when there is no retention by
reversed-phase.
© 2003 Waters Corporation
Grumbach
Complementary Selectivity
to Reversed-Phase
HO
1
2
AtlantisTM HILIC Silica
4.6 x 50 mm, 3 µm
90% to 50% ACN
Vo = 0.5 min
1.00
2.00
2
3.00
4.00
O
H
N
HO
CH3
1. Morphine
5.00
Minutes
AtlantisTM dC18
4.6 x 50 mm, 3 µm
2% ACN
1
Vo = 0.65 min
2. Morphine 3-ß-D-Glucuronide
0.50
1.00
© 2003 Waters Corporation
1.50
2.00
2.50
Minutes
3.00
3.50
4.00
4.50
5.00
Grumbach
Reversed-Phase Conditions:
Separation of Cytochrome C Tryptic Digest
100
Peak Annotation
M/Z (Fragment ID)
204 (T2)
261 (T6,11)
361 (T18)
434 (T21)
AtlantisTM dC18
779 (T15)
%
4.6 x 50 mm, 3 µm
678 (T14)
634 (T4)
729 (T10)
964 (T19)
1005 (T12)
585 (T8)
0
ƒ Extremely polar peptides are not retained under
reversed-phase conditions even with the aid of
Atlantis™ dC18, a column specifically designed for the
retention of polar analytes
ƒ Atlantis™ HILIC Silica can aid in the separation and
identification of these very polar peptides
© 2003 Waters Corporation
Rainville
Complementary Selectivity to Reversed-Phase:
Separation of Cytochrome C Tryptic Digest
100
Peak Annotation
M/Z (Fragment ID)
204 (T2)
261 (T6,11)
361 (T18)
434 (T21)
AtlantisTM dC18
779 (T15)
%
4.6 x 50 mm, 3 µm
678 (T14)
634 (T4)
729 (T10)
964 (T19)
1005 (T12)
585 (T8)
0
634 (T4)
AtlantisTM HILIC Silica
100
4.6 x 50 mm, 3 µm
779 (T15)
%
678 (T14)
434 (T21)
585 (T8)
261 (T6,11)
204 (T2)
729 (T10)
964 (T19)
1005 (T12)
361 (T18)
00
45
Polar peptides not retained by reversed-phase can be retained by HILIC.
© 2003 Waters Corporation
Rainville
Enhanced Sensitivity
in Mass Spectrometry
LC/MS conditions were optimized individually for both HILIC and RP.
100
1.84
1
100
1.75 e3
2
2.18
ES+
%
%
239.9
Reversed
Phase
0
1.00
2.00
3.00
4.00
5.00
Time
1
1.84
2
209.9
2.8e5
0-50% ACN
2.18
AtlantisTM dC18
Peak Area
2.1 x 50 mm, 3 µm
10.0 µL injection volume
1. Albuterol 100 pg/µL
2. Bamethan 50 pg/µL
21 (ND)
14 (ND)
0
1.53
100
ES+
2
HILIC
239.9
%
209.9
1
2.8e5
1.92
95-50% ACN
0
0.50
1.00
1.50
2.00
2.50
Time
3.00
3.50
4.00
4.50
AtlantisTM HILIC Silica Peak Area
2.1 x 50 mm, 3 µm
10.0 µL injection volume
2. Bamethan 50 pg/µL
1. Albuterol 100 pg/µL
110085
19567
5.00
HILIC requires high volatility solvents which increase sensitivity
compared to high-aqueous mobile phases used in reversed-phase.*
© 2003 Waters Corporation
*Naidong, W.; Shou, W.; Chen, Y-L.; Jiang, X. J. Chromatogr. B. 754 (2001) 387-399.
Grumbach
Mass Spectrometer
Detection Limits
Approximate Limit of Detection
(Analyte Dependant)
Single Quadrupole
MS
(SIR) mode ES+
Triple Quadrupole
MS
(MRM) mode ES+
Reversed-phase
HILIC
10 ng/mL
1 ng/mL
1 ng/mL
0.1 ng/mL
[100 fg/µL]
HILIC-ESI-MS will achieve at least 10x higher sensitivity with high organic
mobile phases than typical RP-ESI-MS with high aqueous mobile phases.
© 2003 Waters Corporation
Extremely Low Detection Limits on a
Single Quadrupole Mass Spectrometer
ACh
100
HC
3
CH
3
CH
3
+
O
N
HC
3
ES+
146.4
3.19e5
O
%
100
HC
3
Ch
HC
3
%
OH
+
N
ES+
104.2
1.78e5
CH
3
AtlantisTM HILIC Silica
2.1 x 50 mm, 3 µm
20.0 µL injection volume
Waters® ZQ™ single quadrupole MS
SIR m/z: 104.2, 118.3, 146.4
Analyte
Concentration
1. Acetylcholine (Ach) 100 fg/µL
2. Choline (Ch)
100 fg/µL
3. Betaine (I.S.)
5000 fg/µL
0
100
CH
H C
3
N
O
Bet (I.S.)
3
+
%
O
-
86% ACN
H C
3
0
1.00
2.00
3.00
4.00
5.00
Time
© 2003 Waters Corporation
ES+
118.3
1.32e6
6.00
7.00
8.00
9.00
Detect 100 fg/µL on a
single quadrupole MS!
Traditional SPE Methods for
Reversed-Phase Chromatography
ƒ Traditional SPE methods often contain an elution step
that consists of high organic content
ƒ To make this extracted sample compatible with your
mobile phase, you must first evaporate the high organic
eluent then reconstitute in some portion of aqueous
ƒ Evaporation and reconstitution are often the most lengthy
steps in an SPE procedure*
ƒ In HILIC, the high organic eluent can be directly injected
on the column, thus eliminating the need for evaporation
and increasing your throughput
© 2003 Waters Corporation
*Jemal, M., Teitz, D., Ouyang, Z., J.Chromatogr. B, 732 (1999) 501.
Generic Oasis® HLB SPE
Procedure for Polar Bases Using HILIC
Condition/Equilibrate*
200 µL methanol/200 µL water
Load
75 µL spiked plasma sample
75 µL internal standard
with 2% ammonium hydroxide
Wash
200 µL 5% methanol in water
*Oasis® HLB
µElution Plate
Elute
75 µL 40% acetonitrile/60% isopropanol
with 2% formic acid
Inject eluent directly onto column
Eliminate Evaporation and Reconstitution Step
© 2003 Waters Corporation
Generic Oasis® HLB SPE:
Direct Injection onto HILIC Column
HO
H3C
H3C
HO
NH
OH
OH
NH
CH3
CH3
Albuterol
Bamethan
1.49
100
1
OH
AtlantisTM HILIC Silica
2.1 x 50 mm, 3 µm
1. Bamethan 10 pg/µL
2. Albuterol 50 pg/µL
1.84
SIR of 2 Channels ES+
239.8
209.9
8.97e4
2
Polar species from matrix
elute after analytes
(normally seen in solvent
front using RP)
%
0
0.00
0.50
1.00
1.50
2.00
2.50
Time
3.00
3.50
4.00
4.50
5.00
SPE eluent injected directly onto AtlantisTM HILIC Silica column
© 2003 Waters Corporation
Grumbach
Summary:
When to Use HILIC
AtlantisTM dC18
or XTerra®
Yes
Start Here
1
2
Can it be
retained by
RP?
Yes
Is the polar
compound
a base?
Yes
3
No
Sufficient
Mass Spec
sensitivity?
2
No
AtlantisTM
HILIC Silica
Note:
dC18 = High aqueous, Low pH
®
XTerra = High aqueous, High pH
AtlantisTM HILIC Silica = Low aqueous, Low pH
AtlantisTM
© 2003 Waters Corporation
No
Can it be
retained by
RP?
Yes
No
Alternate separation technique
such as ion exchange,
ion-pair, etc.
AtlantisTM
dC18
Summary
ƒAtlantisTM HILIC Silica columns offer:
–Retention of highly polar basic analytes
–Complementary selectivity to reversed-phase
–Excellent reproducibility
–Enhanced sensitivity in ESI-MS
–Shorter sample preparation procedures
ƒElimination of the evaporation and
reconstitution steps by directly injecting the
final organic phase of the sample prep
procedure
© 2003 Waters Corporation
References
1. Alpert, A. J. J. Chromatogr. 499 (1990) 177-196.
2. Canals, I.; Oumada, F. Z.; Roses, M.; Bosch, E. J.
Chromatogr. A. 911 (2001) 191-202.
3. Naidong, W.; Shou, W.; Chen, Y-L.; Jiang, X. J.
Chromatogr. B. 754 (2001) 387-399.
4. Naidong, W.; Rapid Commun. Mass. Spectrom. 16
(2002), 1613-1621.
© 2003 Waters Corporation
Acknowledgements
Bonnie Alden
Pamela Iraneta
Paul Rainville
Uwe Neue
Doug McCabe
Tom Walter
Michael Savaria
Susan Karn
© 2003 Waters Corporation