HILIC Chromatography:

Theory and Method Development Practices

David S. Bell
Supelco/Sigma/Aldrich
Bellefonte, PA
September 18, 2013

sigma-aldrich.com/analytical 1

© 2013 Sigma-Aldrich Co. All rights reserved.

Introduction

1. Describe the HILIC system and prevailing theories on retention mechanisms

2. Discuss various HILIC stationary phases and the interactions they exhibit
3. Discuss method development practices/approaches
4. Provide some tips and cautions as they related to method development in
HILIC mode

Aim
• Understand retention mechanisms involved in HILIC – the basis for method
development practices
• Provide concepts and models for retention
• Provide some method development hints/pitfalls to watch for
• Illustrate with a few examples

© 2013 Sigma-Aldrich Co. All rights reserved. 2

Biphasic Solvent Distribution at Silica Surface

initial conditions HILIC conditions

Si-OH Homogeneous binary Si-OH Biphasic system

mobile phase high in

aqueous-depleted solvent
aqueous-rich solvent
Si-OH organic content Si-OH

Si-OH Si-OH

Si-OH Si-OH

Si-OH Si-OH

Si-OH Si-OH

Si-OH Si-OH

When a polar phase is utilized with a mobile phase high in organic

concentration, the more polar water will preferentially adsorb on the surface
creating a semi-stagnant, water-rich stationary phase and a water depleted
mobile phase. Polar analytes can then partition in to aqueous-enriched
phase

© 2013 Sigma-Aldrich Co. All rights reserved. 3

 If it were only that simple….
HILIC conditions

Si-OH Biphasic system

aqueous-depleted solvent
aqueous-rich solvent
Si-O-
• Polar analytes are just that
because they are ionic or exhibit Si-OH
strong dipoles – naturally Si-O-
interactive Si-OH
• We’re bringing them close to a Si-O-
polar surface and therefore can Si-OH
expect (should expect) other
strong interactions to take place CH3 OH

• Strong dipole interactions (H- O NH2

bonding) and ion-exchange are

an integral and important part of HO
Normetanephrine
HILIC chromatography

© 2013 Sigma-Aldrich Co. All rights reserved. 4

Interactions in HILIC Chromatography

Partition – phase transfer of analytes to and from a polar stationary solvent

(aqueous) and relatively non-polar organic mobile phase

Polar interactions – Dipole (hydrogen bonding), induced dipole, etc

Ionic – Ion-exchange with bonded phase or ionized surface silanol groups

One major difference between reversed-phase and HILIC is the relative

importance of these mechanisms. In RP partition dominates and
selectivity/retention is generally ‘tweaked’ by supporting secondary
polar or ionic interactions. In HILIC polar and ionic often dominate.

© 2013 Sigma-Aldrich Co. All rights reserved. 5

 Retention Profile of Synephrine on Bare Silica and
Pentafluorophenyl Phases
13 mM ammonium acetate, pH 6.7: acetonitrile – buffer diluted as organic is added
12

CH3

10 HN

8
HO
Retention (min)

OH
6

Synephrine
4

0
0 10 20 30 40 50 60 70 80 90 100

% Acetonitrile
Synephrine Ascentis Express HILIC Synephrine Ascentis Express PFP

© 2013 Sigma-Aldrich Co. All rights reserved. 6

 Retention Profile of Synephrine on Bare Silica and
Pentafluorophenyl Phases –
13 mM ammonium acetate, pH 6.7: 13 mM AA in acetonitrile – buffer held constant
6

4
Retention (min)

0
0 10 20 30 40 50 60 70 80 90 100

% Acetonitrile
Synephrine Ascentis Express HILIC (2) Synephrine Ascentis Express PFP (2)

© 2013 Sigma-Aldrich Co. All rights reserved. 7

So What?

The previous example shows that there may be very different mechanisms
causing retention and selectivity in HILIC systems:

Bare silica phase exhibits partition (not impacted by buffer concentration)

PFP phase exhibits IEX (highly impacted by buffer concentration)

Differing retention mechanisms give rise to alternative selectivity and

retention – same as in reversed-phase

© 2013 Sigma-Aldrich Co. All rights reserved. 8

So What, What?

A fundamental understanding of underlying retention mechanisms is vital

for:
• Choosing the right set of initial conditions for a given separation problem (column
chemistry, mobile phase additives, aqueous/organic ratios)
• Knowing the correct knobs to turn to facilitate method development and
optimization
• Placing the needed controls on the system to establish a rugged and robust
method

© 2013 Sigma-Aldrich Co. All rights reserved. 9

Are HILIC Phases the Same?
Much like in reversed phase, changing stationary phases in
HILIC provides retention and selectivity differences F
R1
F
Si
This study focuses on the interaction differences that might R2
R1
be expected for three different HILIC stationary phases: F F
Ascentis Express F5 (pentafluorophenyl), Ascentis Express F
HILIC (bare silica) and Ascentis Express OH5
(pentahydroxy phase) F5
HO

Using ephedrine as a probe molecule, retention as a HO

function of buffer concentration was collected and HO

interpreted. OH

R1 OH
R2
Several related compounds were also run simultaneously Si
R2
and retention and selectivity noted. Interpretation of the R3

observed results in terms of the dominant interactions

prevalent using each phase is provided.
OH5

© 2013 Sigma-Aldrich Co. All rights reserved. 10

Selected Probes ACD/Labs, PhysChemProp, v. 12

Structure pKa(MB) LogD(8.0) LogP MW name

H3C
NH

OH
H3C
9.38 -0.37 1.08 165.23 pseudoephedrine

H3C
NH
OH
H3C
9.38 -0.37 1.08 165.23 ephedrine

CH3
HN
9.37 -1.35 0.13 167.21 synephrine

HO
OH

© 2013 Sigma-Aldrich Co. All rights reserved. 11

Focus on Understanding the IEX Component

For any analysis where the potential for IEX exists, a good general practice is to
evaluate the IEX contribution to the retention (and selectivity)

For an ion-exchange process involving singly charged analytes, the dependence of

retention on mobile phase counter ion may be expressed as

Log k = -log[C+]m + logbIEX

Where [C+]m represents the concentration of the competing ion in the mobile phase
and bIEX is a constant for a given system which includes the phase ratio, ion-
exchange capacity of the stationary phase and the ion-exchange equilibrium
constant.

A plot of log k vs log [C+]m will thus yield a slope of -1 when ion-exchange is
solely responsible for retention, whereas the plot would yield a slope of 0
where ion-exchange is not present. The closeness of the resultant slopes
to -1 is an indication of the relative dominance of IEX in the overall
retention process.

© 2013 Sigma-Aldrich Co. All rights reserved. 12

Response of Ephedrine Retention on Buffer
Concentration on Three HILIC Phases
1.20

1.00
y = -0.6525x + 1.2104
R2 = 0.9984
0.80
log k'

0.60
y = -0.8187x + 1.1259
R2 = 1
0.40

0.20
y = -0.2199x + 0.2455
R2 = 0.9934
0.00
0.00 0.20 0.40 0.60 0.80 1.00 1.20

log buffer concentration (mM)

OH5 HILIC F5 Linear (HILIC) Linear (F5) Linear (OH5)

© 2013 Sigma-Aldrich Co. All rights reserved. 13

Discussion

Examining observed retention in the present study:

• Keys
– All three analytes have very similar pKa values and thus should, excluding any
outside interference, IEX the same
– Synephrine is more polar than the ephedrine pair
– The ephedrine pair are identical in physical properties except for their
orientation in space
OH5 –
• Little IEX behavior (lack of surface interactions)
• Synephrine elutes after the ephedrine pair (partitioning)
– Conclusion – close to pure HILIC partitioning

© 2013 Sigma-Aldrich Co. All rights reserved. 14

Discussion (continued)

HILIC (bare silica phase)

• Mid range IEX behavior (surface interactions)

• Synephrine elutes after the ephedrine pair (partitioning)
• Greater overall retention (relative to the two other phases) – mechanistic synergy
– Conclusion: synergistic combination of partitioning and ion-exchange

PFP (F5)

• Near total IEX behavior (surface interactions – at least long range)

• Synephrine elutes prior to ephedrine pair (lack of HILIC partitioning)
– Conclusion: Mainly Ion-exchange

© 2013 Sigma-Aldrich Co. All rights reserved. 15

 Proposed Model for Different HILIC Stationary Phases

Aqueous-Organic Mobile Phase

OH5 Silica F5

HO

HO
OH F
F F
Aqueous Layer
HO
OH

F
R1 F

R2 Si R2 Aqueous Layer R1 Si R1

R3 - - - - - - - R2 -
Polar Stationary Phase

© 2013 Sigma-Aldrich Co. All rights reserved. 16

Attributes of Common HILIC Phases

Interactions
Stationary Phase
Partitioning Polar Ionic

Bare Silica moderate moderate

Zwitterionic strong weak

Amide strong weak

Diol/Polyol strong weak

PFP weak strong

Cyano weak strong

© 2013 Sigma-Aldrich Co. All rights reserved. 17

Matching the Sample Analytes to the Right
Retention Mechanisms

There are many different sets of analytes we may wish to apply HILIC to

A good start is to identify the stationary phase with the appropriate

mechanisms to retain and separate them or at least eliminate those
that do not:
• For example – if we have a sample with polar neutral molecules, we know we will
need partition as they will not interact ionically – F5 would be a bad choice, OH5
and bare silica would provide more promise

A key to utilizing any chromatographic mode is to have some idea of where

the greatest differences lie with respect to the analyte physiochemical
attributes and invoke the appropriate retention mechanism(s)

© 2013 Sigma-Aldrich Co. All rights reserved. 18

Method Development in HILIC

1. Understand the analyte properties

Will they partition (polar?), will they ion-exchange (charged?), how do they
differ most?
2. Choose appropriate stationary phases based on potential interactions
(conversely, eliminate those that do not make sense)
3. Screen chosen stationary phases for retention and selectivity –
isocratic
4. Select phase that best fits the needs of the method and optimize

© 2013 Sigma-Aldrich Co. All rights reserved. 19

 Bath Salt Analytes – Hydrophilic, Weak Bases
O
O O
NH
CH3 CH3 NH
O
CH3

CH3
O N CH3
O
O O
Butylone
O
Monoisotopic Mass = 221.105193 Da MDPV
Monoisotopic Mass = 275.152144 Da methylone
O Monoisotopic Mass = 207.089543 Da

NH CH3 O O

NH NH
CH3 CH3 CH3

CH3 H3C CH3

O H3C O
ethylone
mephedrone methedrone
Monoisotopic Mass = 221.105193 Da
Monoisotopic Mass = 177.115364 Da Monoisotopic Mass = 193.110279 Da
O
O O
NH
CH3 F NH NH
CH3 CH3
CH3
F CH3
CH3

3-fluoromethcathinone Buphedrone
20 4-fluoromethcathinone
Monoisotopic Mass = 181.090292 Da Monoisotopic Mass = 181.090292 Da

© 2013 Sigma-Aldrich Co. All rights reserved. 20

Ascentis Express F5, 2 mM Ammonium Acetate
in 90% Acetonitrile

2 mM - 3
A1849_029_B031 1: Scan ES+
6.01 194
3.01e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B031 1: Scan ES+
5.76 178
3.04e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B031 1: Scan ES+
4.92 208
2.82e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B031 1: Scan ES+
6.76 276
2.04e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B031 1: Scan ES+
3.88
4.95 222
3.64e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B031 1: Scan ES+
4.94 TIC
3.87 5.78 6.01 1.05e9
6.76
%

1.30
7 Time
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

IEX results in some coelution in this case

© 2013 Sigma-Aldrich Co. All rights reserved. 21

 Ascentis Express HILIC, 2 mM Ammonium
Acetate in 90% Acetonitrile
2 mM - 3
A1849_029_B015 1: Scan ES+
7.01 194
2.97e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B015 1: Scan ES+
5.74 178
3.47e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B015 1: Scan ES+
6.19 208
3.08e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B015 1: Scan ES+
3.82 276
3.32e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B015 1: Scan ES+
4.11 4.80 222
3.91e8
%

0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
A1849_029_B015 1: Scan ES+
4.11 4.80 5.74 7.01 TIC
3.82 6.18
8.22e8
%

1.27 1.59
8 Time
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

IEX + partition provides the needed selectivity in this case

© 2013 Sigma-Aldrich Co. All rights reserved. 22
Optimized Separation of Bath Salts on Ascentis
Express HILIC Analyte rt
MDPV 2.0
Buphedrone 2.2
3-fluoromethcathinone 2.7
butylone 2.9
ethylone 3.4
3-fluoromethcathinone 3.7
mephedrone 4.6
methylone 5.2
methedrone 6.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0
Time (min)
System: Agilent 1200SL Rapid Resolution, 6210 TOF
Column: Ascentis Express HILIC 10cm X 2.1mm, 2.7um (53939-U)
Mobile Phase: 5mM ammonium formate (98:2 acetonitrile:water)
Flow: 0.6mL/min
Craig R. Aurand, Robert Shirey,
Temperature: 35 ºC Leonard Sidisky Janusz Pawliszyn,
and Yong Chen, Application of Bio-
System Pressure: 127bar SPME for the Enrichment of Illicit
Phenethylamine and Cathinone
Injection Vol: 1uL Compounds from Biological Samples,
Poster, ASMS national Meeting,
MS Detection: ESI+, 100-1000m/z , Vancouver, BC, 2012
23
Sample: 200ng/mL Bath Salts in acetonitrile

© 2013 Sigma-Aldrich Co. All rights reserved. 23

Nucleoside Structures – Polar Neutral (for the
most part)
NH2 O OH

N
N HN N

O N O N N N
O O
O
HO HO
HO

HO OH HO OH
HO OH
Uridine Inosine
Cytidine

O
O
NH2
N CH3
HN CH3
HN
N

H2N N N
O N
O O N
O
O
HO HO
HO
HO OH
HO OH
Ribothymidine HO OH
Guanosine 5-Methyluridine 5-Methylcytidine

© 2013 Sigma-Aldrich Co. All rights reserved. 24

Structures
NH2
NH2 NH
H3C N
N N
N

HO N O
N S N
N
O
O O
HO HO

HO O
HO OH HO OH
H3C
2-Thiocytidine 1-Methyladenosine 2'-O-Methylcytidine

O CH3 NH
O
+ CH3
N
HN N
HN NH H3C O

H2N N N N O O S OH
O
O O
O O
HO HO HO

HO OH HO OH
HO OH
Pseudouridine 3-Methylcytidine metosulfate
7-Methylguanosine

© 2013 Sigma-Aldrich Co. All rights reserved. 25

 Ascentis Express OH5
5 mM ammonium acetate, pH 6.9 unadjusted

0.1% formic acid, pH 1.9

5 mM ammonium formate, pH 3

5 mM ammonium formate, pH 4

5 mM ammonium acetate, pH 5

Good retention and selectivity – reasonably little change as

a function of buffer pH
© 2013 Sigma-Aldrich Co. All rights reserved. 26
 Ascentis Express HILIC
5 mM ammonium acetate, pH 6.9
unadjusted

0.1% formic acid, pH 1.9

5 mM ammonium formate, pH 3

5 mM ammonium formate, pH 4

5 mM ammonium acetate, pH 5

Good retention and selectivity – group of more basic analytes

preferentially retained that are sensitive to changes in buffer pH
© 2013 Sigma-Aldrich Co. All rights reserved. 27
Ascentis Express F5

5 mM ammonium acetate, pH 6.9 unadjusted

0.1% formic acid, pH 1.9

5 mM ammonium formate, pH 3

5 mM ammonium formate, pH 4

5 mM ammonium acetate, pH 5

© 2013 Sigma-Aldrich Co. All rights reserved. 28

Optimized Separation of Nucleosides on Ascentis
Express OH5 12 NUCLEOSIDES_ASCEXPOH5.CDF

7.53
55

50

45

40

2.75

7.79
35

Response

8.6
30

9.02
6.5
25

3.07

10.29
20

4.62

6.08
5.28
15

11.11
10

2.91
5

0 1 2 3 4 5 6 7 8 9 10 11 12 13
Retention Time (min)

column: Ascentis Express OH5, 10 cm x 2.1 mm, 2.7 µm (53757-U)

mobile phase: (A) 5 mM ammonium acetate, pH 5.0 with acetic acid in 95:5,
acetonitrile:water; (B) 5 mM ammonium acetate, pH 5.0 with acetic acid in 80:20,
acetonitrile:water
gradient: 0% B held for 1 min; to 100% B in 10 min; held at 100% B for 1 min
flow rate: 0.3 mL/min
column temp.: 25 °C
detector: UV at 250 nm
injection: 2 µL
sample: 10 - 100 µg/mL
© 2013 Sigma-Aldrich Co. All rights reserved. 29
Structures of Yohimbine and Ajmalicine Alkaloids

N N
N N
H H H H H H
CH3

H H
O O O
H3C H3C

O OH O
Yohimbine Ajmalicine

Relatively nonpolar, basic analytes

© 2013 Sigma-Aldrich Co. All rights reserved.

Separation of Yohimbe Alkaloid Standards on
Ascentis Express F5
YOHIMBE STANDARDS.CDF1

2
1. Ajmalicine
2. Yohimbine

0 1 2 3 4 5 6 7 8 9 10
Retention Time (min)

Conditions:
Column: Ascentis Express F5, 10 cm x 3.0 mm, 2.7 µm (53766-U)
Mobile Phase: 2 mM ammonium acetate in 90% acetonitrile, pH adjusted
to 6.0 with acetic acid (post addition of organic)
Flow Rate: 0.5 mL/min
Temperature : 35°C
Detection: UV. 275 nm
Injection: 2 µL
© 2013 Sigma-Aldrich Co. All rights reserved.
HILIC Analysis of Yohimbe Extract on Ascentis
Express F5

YOHIMBE EXTRACT_F5.ESP

1
1. Yohimbine

0 1 2 3 4 5 6 7 8 9 10
Retention Time (min)

Conditions:
Column: Ascentis Express F5, 10 cm x 3.0 mm, 2.7 µm (53766-U)
Mobile Phase: 2 mM ammonium acetate in 90% acetonitrile, pH adjusted
to 6.0 with acetic acid (post addition of organic)
Flow Rate: 0.5 mL/min
Temperature : 35°C
Detection: UV. 275 nm
Injection: 2 µL
© 2013 Sigma-Aldrich Co. All rights reserved.
 HILIC Analysis of Yohimbe Alkaloids on Ascentis
Express HILIC
1
YOHIMBE STANDARDS_HILIC.CDF

YOHIMBE EXTRACT HILIC.ESP 1

0 1 2 3 4 5 6 7 8 9 10
Retention Time (min)

0 1 2 3 4 5 6 7 8 9 10
Retention Time (min)

Conditions:
Column: Ascentis Express HILIC, 10 cm x 3.0 mm, 2.7 µm (53970-U)
Mobile Phase: 2 mM ammonium acetate in 90% acetonitrile, pH adjusted
to 6.0 with acetic acid (post addition of organic)
Flow Rate: 0.5 mL/min
Temperature : 35°C
Detection: UV. 275 nm
Injection: 2 µL
© 2013 Sigma-Aldrich Co. All rights reserved.
 Ephedrine Alkaloids – Polar, Basic Compounds

CH3
H3C H3C
NH2
NH N
OH
OH OH
H3C H3C
H3C

ephedrine methylephedrine norephedrine

H3C
NH CH3
OH HN
OH
H3C NH2

HO
CH3
OH

pseudoephedrine norpseudoephedrine synephrine

Bell, D. S., H. M. Cramer, et al. (2005). "Rational method development strategies on a fluorinated liquid chromatography stationary phase: Mobile
phase ion concentration and temperature effects on the separation of ephedrine alkaloids." Journal of Chromatography A 1095(1-2): 113-118.

© 2013 Sigma-Aldrich Co. All rights reserved. 34

Optimization of Mobile Phase Ion Concentration –
Linear Dependence

1.200

1.000

0.800
norephedrine
synephrine
log k

0.600 methylephedrine
4 mM optimum ephedrine
0.400
pseudoephedrine

0.200

0.000
2 10
ammonium acetate concentration, mM

© 2013 Sigma-Aldrich Co. All rights reserved. 35

Experimental

Retention data was first acquired at 2 mM and 10 mM ammonium acetate in 90%

acetonitrile
Acquisitions were performed on a Shimadzu 10A VP HPLC system

Column: Discovery HS F5 (PFP), 15 cm x 4.6 mm, 5 µm

Mobile Phase: 2 or 10 mM ammonium acetate in 90:10, CH3CN:water
Flow Rate: 1 mL/min
Temp.: 35°C
Det.: UV at 215 nm
Inj.: 10 µL
Sample: mix of ephedrine alkaloids + synephrine at 100 µg/mL each in 2
mM ammonium acetate mobile phase

© 2013 Sigma-Aldrich Co. All rights reserved. 36

 LC-MS Analysis of Ephedrine Alkaloids

1 2
ESI+ 152
1. norephedrine
2. norpseudoephedrine
0 2 4 6 8 10 12 14 16 18
Time (min) 3. synephrine
3
4. methylephedrine
ESI+ 168 5. ephedrine
6. pseudoephedrine
0 2 4 6 8 10 12 14 16 18
Time (min)
4

ESI+ 180 Understanding

dominant
0 2 4 6 8 10
Time (min)
12 14 16 18
interactions greatly
5 6
facilitates method
ESI+ 166
development
0 2 4 6 8 10 12 14 16 18
Time (min)

© 2013 Sigma-Aldrich Co. All rights reserved. 37

Notes on Method Development

These few applications highlight the need to:

1) choose a column with the interactions needed for retention/selectivity

based on analyte properties/method goals

2) manipulate retention and selectivity using the strongest variables first –

then hone in with remaining variables
if IEX dominant – buffer concentration/pH
if partition – aqueous/organic composition

The result is facile method development of robust and rugged methods

© 2013 Sigma-Aldrich Co. All rights reserved. 38

Method Development in HILIC - Tips

Sample consideration
• HILIC is generally restricted to highly polar and/or ionic analytes. Low octanol-
water coefficients (Log P generally less than 1) and ionizable bases are
considered good candidates for this mode of chromatography.
• Make sure samples are prepared in weak HILIC solvents (high organic, low salt
concentrations)

Mobile phase conditions

• A good general starting mobile phase for HILIC is 2-10 mM ammonium acetate
or formate in 90% acetonitrile leaving pH unadjusted (~pH7 where both strong
bases and surface silanols are ionized)
• If the analytes are nonionic or IEX is not desirable, a starting point could be 10
mM ammonium formate, pH 3.0, in 90-95% acetonitrile.
• Choice of formate or acetate buffer informed primarily by volatility and MS
compatibility, and solubility at high levels of organic
• Run isocratically if possible, if gradients are necessary, change one variable only
(ie organic or buffer, not both)

© 2013 Sigma-Aldrich Co. All rights reserved. 39

Method Development in HILIC - Tips

System Variables:
It may be difficult to predict the operative retention mechanisms in HILIC;
however, it is imperative to gain some understanding of the mechanisms in
order to facilitate the development and ensure rugged and robust methods
result.

A quick buffer concentration study like the one discussed in this seminar will
provide good insight into the relative dominance of IEX

If IEX is present, buffer concentration, pH and temperature must be controlled

variables

Make sure instrument wash solvents are compatible with HILIC mobile phases,
especially if used in certain injection modes

© 2013 Sigma-Aldrich Co. All rights reserved. 40

Determination of Dominant Interactions

pH adjustment
– Be aware that pH can modify the ionization of both the analyte and the
chromatographic surface
– Modern surface silanols exhibit a range of pKa values – finding the right pH
is often a balance between analyte and surface degrees of ionization
– Buffer concentrations need to be as exact as possible
– Best to measure and state the pH after the addition of organic

© 2013 Sigma-Aldrich Co. All rights reserved. 41

 Effect of Acetonitrile on pH of Ammonium Acetate

12
pH Measured Following Addition of Organic

11
The pH of the aqueous ammonium
10 hydroxide solution was adjusted with
acetic acid prior
w
to the addition of
9 acetonitrile ( pH ). Subsequent pH
w
measurement was taken following the
8 addition of acetonitrile ( s pH ).
w
Each measurement utilized a glass
7 electrode filled with saturated KCl
s
pH calibrated using pH 4, pH 7 and pH 10
w NIST standardized aqueous reference.
6
Measurements were taken at 25ºC.
5
Triangle: 90.0% ACN,
4 Square: 75% ACN,
Diamond: 50% ACN,
3 Circle: 32.5% ACN

2
2 4 6 8 10 12

pH Measured Prior to Addition of Organic

w
w
pH
© 2013 Sigma-Aldrich Co. All rights reserved. 42
 Determination of pKa Values using NMR

Amitriptyline
s Analyte Literature pKa s pKa Correlation
% Acetonitrile w pKa Correlation w
(R2)
(R2)

25 9.32 0.9997 Amitriptyline 9.4 8.34 0.9923

50 9.02 0.9996
Nortriptyline 9.7 8.92 0.9920

75 8.88 0.9956
Diphenhydramine 9.0 8.33 0.9978
90 8.34 0.9923

Verapamil 8.9 7.98 0.9976

• pKa values for bases decrease

with increasing acetonitrile Alprenolol 9.7 8.73 0.9855
• At 90% each analyte exhibited a
pKa value about 1 full pH unit less
than the literature pKa value

© 2013 Sigma-Aldrich Co. All rights reserved. 43

Impact of pH and pKa Variation on Ion-Exchange

In order for an ion-exchange interaction to take place, both the analyte and the
stationary phase must posses opposite charges

Taking only the aqueous-based values:

• Analyte pKa = 8.0
• Mobile phase pH = 7

The degree of analyte ionization would be:

• 10 (pKa – pH) /(1 + 10 (pKa-pH) ) = 0.90 or 90% ionized

In 90% acetonitrile, however:

• Analyte pKa ~ 7.0
• Mobile phase pH ~ 8

The degree of analyte ionization would be only about 10% and thus much less apt
to interact via ion-exchange

It is thus extremely important to take the variation of both pH and pKa into account
when developing methods

© 2013 Sigma-Aldrich Co. All rights reserved. 44

Balancing HILIC and IEX

IEX dominated Partition dominated

Low ionic concentration High ionic concentration

Fluorinated/Cyano phases Bare silica OH5 phase/Amide

Zwiterionic

© 2013 Sigma-Aldrich Co. All rights reserved. 45

Summary

Developed some models to describe what is happening in HILIC

• Take home message: if you have basic analytes in your system, expect IEX
mechanisms to be prevalent –
• Employing and controlling IEX is different than partitioning – need to be prepared
to do both

Showed that different phases offer different degrees of partition and IEX
• OH5/Amide/Zwitterionic – mainly partition
• Bare Silica – partition and IEX
• F5/Cyano – IEX

Demonstrated through application how one can choose and omit column
chemistries based on analyte knowledge
• If analytes are neutral (or acidic), partition will need to be invoked
• If all analytes are bases, IEX and/or partition may be most suitable

Finally provided a few tips on method development and system

parameters to consider

© 2013 Sigma-Aldrich Co. All rights reserved. 46

Acknowledgements
Hillel Brandes Carmen Santasania
Xiaoning Lu Gaurang Parmar
Hugh Cramer Wayne Way
Craig Aurand
Errol Fernandes Separation Science

© 2013 Sigma-Aldrich Co. All rights reserved.

Q&A
Follow up questions on HILIC? Contact: [email protected]

Ascentis Express HPLC Columns are available in 2 particle sizes

and multiple phases. For product information, visit:
sigmaaldrich.com/express

2.7 µm 5 µm
9 Phases 7 Phases

© 2013 Sigma-Aldrich Co. All rights reserved. 48