The ACE HILIC range consists of three complementary phases … · 2017-07-07 · ¾ A systematic...
Transcript of The ACE HILIC range consists of three complementary phases … · 2017-07-07 · ¾ A systematic...
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.
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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
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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
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Isocratic10 mM ammonium formate pH 3.0 in
MeCN/H2O (94:6 v/v)
8. Caffeine and Related Substances: Optimisation
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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.
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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
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20 100
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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
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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.
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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
[email protected] www.ace-hplc.com