A systematic and rationally designed method development strategy can aid in

streamlining the method development process.

In HILIC, column stationary phase and mobile phase pH are the most critical

parameters affecting selectivity.

The step-by-step method development strategy proposed in this poster therefore

provides a powerful means by which to probe selectivity of a new application.

The ACE HILIC-A, HILIC-B and HILIC-N phases have been shown to provide

complimentary selectivity, ideal for method development.

Screening an analyte mixture on these three phases has been demonstrated as

an effective method development strategy for selecting an appropriate stationary

phase/mobile phase combination.

Optimisation can be achieved by altering parameters such as ionic strength, %

organic and temperature.

12. Summary

ACE® is a registered trademark of Advanced Chromatography Technologies Limited. ACE UltraCore™ and EBT™ are trademark of Advanced Chromatography Technologies Limited.

mV

0

250

mV

0

250

min0 2 4 6 8 10 12 14 16 18

mV

0

250

1 2

12

1

2

HILIC-N: pH 3.0

(Neutral phase)

HILIC-A: pH 3.0

(Acidic phase)

HILIC-B: pH 3.0

(Basic phase)

11. Example 2: MMA and Succinic Acid

Very strong retention was observed at pH 6.0 on all phases (data not shown).

At pH 3.0, the analytes proved difficult to separate on the ACE HILIC-N and ACE

HILIC-A phases due to similar analyte properties.

The ACE HILIC-B provided additional retention and selectivity for the two analytes.

Use of the screening protocol provided a separation of these challenging analytes

on the ACE HILIC-B phase with no further development required.

Columns: ACE 5 HILIC-N 150 x 4.6 mm

ACE 5 HILIC-A 150 x 4.6 mm

ACE 5 HILIC-B 150 x 4.6 mm

Mobile phase: 10 mM ammonium formate pH 3.0 in

MeCN/H2O (90:10 v/v)

Flow Rate: 1.5 mL/min.

Temperature: 25 C

Detection: ELSD, Evaporator: 30 C, Nebuliser: 30 C,

Gas speed: 1 SLM

2. MMA1. Succinic acid

10. Example 2: MMA and Succinic Acid

Elevated levels of methylmalonic acid (MMA) in blood/urine is routinely tested for

as an indicator of vitamin B12 deficiency.

The isobaric species succinic acid may be present in biomatrices at concentrations

up to 50x that of MMA.

Chromatographic separation of these two components with good retention to

eliminate matrix interference is therefore essential for accurate determination.

Physico-chemical properties are similar and suitable for HILIC mode

The two compounds were screened on the three ACE HILIC phases.

The two acids were predicted to possess a single negative charge at pH 3.0 and a

double negative charge at pH 6.0, therefore these two mobile phase pH’s were

selected for screening

MMA

pH logD

3.0 -0.43

6.0 -3.21

Succinic acid

pH logD

3.0 -0.40

6.0 -3.30

min0 1 2 3 4 5 6

0

50

1

2

3

4

5

9. Caffeine and Related Substances: Final Method

Column: ACE 5 HILIC-N 150 x 4.6 mm

Mobile phase: A: 10 mM ammonium formate pH 3.0 in MeCN/H2O (96:4 v/v),

B: 10 mM ammonium formate pH 3.0 in MeCN/H2O (50:50 v/v)

Gradient: 0-100%B in 15 minutes

Flow Rate: 1.5 mL/min.

Temperature: 15 C

Detection: UV, 275 nm

Sample: 1. Caffeine, 2. Theophylline, 3. Theobromine, 4. Xanthine,

5. Hypoxanthine

Reduced temperature was utilised to achieve separation of the mixture on the ACE

5 HILIC-N at pH 3.0.

A small increase in acetonitrile in the gradient starting conditions was also found to

be beneficial

First published in Chromatography Today, Volume 8, Issue 4, Nov/Dec 2015

Gradient Screen

0

50

1

2 3

45

min0 1 2 3 4 5 6

1

min0 2 4 6 8 10 12

0

50

23

45

Isocratic10 mM ammonium formate pH 3.0 in

MeCN/H2O (94:6 v/v)

8. Caffeine and Related Substances: Optimisation

1

25 C

0

50

2 3

45

min0 1 2 3 4 5 6 min0 1 2 3 4 5 6

0

501

2 3

45

15 C

Isocratic or gradient mode

Isocratic conditions were assessed and found to increase the retention of later

eluting compounds but failed to improve resolution between the critical pair.

Effect of Temperature

Reducing the temperature was found to improve the resolution of both impurity peak

pairs.

0

50

1

2 3

4 5

0

50

1

23

45

0

50

min0 1 2 3 4 5 6

1

45

23

mAU

0

50 HILIC-A: pH 3.0(Acidic phase)

1

2 3

45

HILIC-N: pH 3.0(Neutral phase)

0

50

1

2 3

45

0

50 HILIC-B: pH 3.0(Basic phase)

1

45

2,3

min0 1 2 3 4 5 6

HILIC-A: pH 4.7(Acidic phase)

HILIC-N: pH 4.7(Neutral phase)

HILIC-B: pH 4.7(Basic phase)

7. Caffeine and Related Substances: Gradient Screen

Gradient screen of caffeine and related compounds at pH 3.0 and 4.7. Conditions as per table 1. Sample: 1. Caffeine, 2. Theophylline, 3. Theobromine, 4. Xanthine, 5. Hypoxanthine

The HILIC-N at pH 3.0 provided the most potential for a full separation.

First published in Chromatography Today, Volume 8, Issue 4, Nov/Dec 2015

The screening protocol was used to develop a HILIC method for caffeine

and related substances.

All analytes are polar neutral with negative logP values.

The mixture was screened on the ACE HILIC-A, HILIC-B and HILIC-N

phases at pH 3.0 and 4.7.

1. CaffeinelogP -0.13

3. TheobrominelogP -2.08pKa = 9.2

2. TheophyllinelogP -0.17pKa = 7.8

4. XanthinelogP -1.99pKa = 7.95

5. HypoxanthinelogP -1.74pKa = 2.3, 9.4

6. Example 1: Caffeine and Related Substances5. Screening Conditions

Table 1

Column ACE HILIC-A, ACE HILIC-B and ACE HILIC-N,

150 x 4.6 mm

Isocratic screening 10 mM ammonium formate in MeCN/H2O (90:10 v/v)

Ammonium formate at pH 3.0, 4.7 or 6.0.

Gradient screening Line A: 10 mM ammonium formate in MeCN/H2O (94:6 v/v)

Line B: 10 mM ammonium formate in MeCN/H2O (50:50 v/v)

Ammonium formate at pH 3.0, 4.7 or 6.0.

Gradient:

Flow rate 1.5 mL/min

Temperature 25 C

Detection Dependent on sample

Time (mins.) %B

0 0

15 100

20 100

21 0

41 0

Isocratic and gradient screening is performed as follows:

4. ACE MD Protocol

Assess method robustness

Return to isocratic mode

Return to gradient mode

Yes No

Run either the gradient or isocratic screening protocol using the appropriate pH on the three ACE

HILIC phases (Table 1).

Is the retention window narrow?

Assess if the separation can be achieved using

isocratic conditions

Optimise separation by investigating other

parameters i.e. temperature, ionic strength

Did isocratic analysis improve the

separation?

Yes

No

Yes

No

Select 2 pH values from pH 3.0, 4.7 and 6.0

based on logD/pKa dataUse pH 3.0, 4.7 and 6.0

Start

Method Developed

Is method robust?

Yes

No

Are analyte properties

known?

Is the retention window wide?

Assess if separation can be achieved using

gradient conditions

Did gradient analysis improve the

separation?

Yes

Yes

For Gradient:

For Isocratic:

No

Select the most promising column/mobile phase combination

from screening data

No

3. Effect of stationary phase and pH

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

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30

40

50

60

70

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

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20

30

40

50

60

70

min0 2 4 6 8 10 12 14 16 18

mAU

0

10

20

30

40

50

21/3

4

5/6

7 8

9

1/4

2

3

5

6/87

9

1 23

4

5

67

89

ACE HILIC-A

ACE HILIC-N

ACE HILIC-B

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

20

40

60

80

100

120

1

2

34

5/6

789

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

10

20

30

40

50

60

70 1/4

2

3

5

6/87

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min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

10

20

30

40

50

60

70

12

3

4

5

7 6/8

9

ACE HILIC-N

pH 4.7

ACE HILIC-N

pH 3.0

ACE HILIC-N

pH 6.0

Stationary Phase Mobile Phase pH

Columns: 150 x 4.6 mm, 10 mM ammonium formate pH 4.7 in

MeCN/H2O (90:10 v/v), flow rate: 1.5 mL/min, temperature: 25 °C,

detection: UV, 254 nm. Sample: 1. p-aminobenzoic acid, 2. 4-

hydroxybenzoic acid, 3. nicotinamide, 4. acebutolol, 5. adenine, 6.

mandelic acid, 7. tyramine, 8. atenolol, 9. 2’-deoxyguanosine

Columns: 150 x 4.6 mm, 10 mM ammonium formate in MeCN/H2O

(90:10 v/v), flow rate: 1.5 mL/min, temperature: 25 °C, detection: UV,

254 nm. Sample: 1. p-aminobenzoic acid, 2. 4-hydroxybenzoic acid,

3. nicotinamide, 4. acebutolol, 5. adenine, 6. mandelic acid, 7.

tyramine, 8. atenolol, 9. 2’-deoxyguanosine

2. Selectivity in HILIC

In HILIC, the column stationary phase has a significant effect on chromatographic

selectivity.

The ACE HILIC range consists of three complementary phases specifically

designed to offer maximum selectivity differences – ideal for method

development:

ACE HILIC-A

ACE HILIC-B

ACE HILIC-N

Mobile phase pH is also a powerful parameter and can affect ionisation of

analytes and the stationary phase itself.

Method development strategies based on screening different stationary phases

and mobile phase pH are therefore the optimum choice.

Buffer concentration and temperature are less influential, however can be used to

fine-tune methods.

ACE HILIC-B

ACE HILIC-NS=74ACE HILIC-A

S = 0, Identical selectivity

S = 100, Completely orthogonal

*Generated from comparison of retention data for 54 compounds at pH 3.0

1. Introduction

Exploring chromatographic selectivity is a powerful approach for LC method

development.

1.00 1.05 1.10 1.15 1.20 1.250.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5000 10000 15000 20000 25000

0 5 10 15 20 25

N

k

a

a

N

k

Re

so

luti

on

(R

s)

Efficient method development requires a logical

exploration of the key chromatographic parameters

affecting selectivity.

Rationally designed method development strategies

assess key parameters and allow well informed

decision making leading to robust stationary phase /

mobile phase selection.

Method development strategies based on column /

mobile phase screening and optimisation are

commonly utilised for reversed phase.

For HILIC, such strategies are less common / less well

defined.

Zhao, J.H. and P.W. Carr. Analytical Chemistry,

(1999) 71, 2623-2632

This poster demonstrates a simple, step-by-step approach to HILIC method

development, based on the concept of exploring column selectivity.

A Practical, Selectivity Based Hydrophilic

Interaction Liquid Chromatography (HILIC)

Method Development Protocol

Alan P McKeown

Advanced Chromatography Technologies Ltd, 1 Berry Street, Aberdeen, Scotland, AB25 1HF UK

UHPLC and HPLC Columns

info@ace-hplc.com www.ace-hplc.com