HILIC Method Development in a Few Simple Steps

HILIC Method Development
in a Few Simple Steps
Monica Dolci, Luisa Pereira, Dafydd Milton and Tony Edge;
Thermo Fisher Scientific, Runcorn, Cheshire, UK
Overview
This poster presents a systematic approach to method development in HILIC.
Guidelines are provided for:
- column selection based on analyte(s) properties
- mobile phase selection, including composition, use of buffers and pH
- separation optimization
Introduction
Hydrophilic interaction liquid chromatography (HILIC) is arguably the most successful approach for
the retention and separation of polar compounds. This technique can be described as a variation of
reversed phase chromatography performed using a polar stationary phase. The mobile phase
employed in HILIC is highly organic in nature (60-70% solvent, typically acetonitrile) containing a
small percentage of aqueous solvent/buffer or other polar solvent. The aqueous portion of the
mobile phase acts as the stronger solvent; it forms an aqueous-rich layer adsorbed to the polar
surface of the stationary phase (as illustrated in Figure 1).
Method Par
Column Selection
It is suggested to m
phases. In general t
degree of stationary
relative hydrophilicit
used as a guide in s
FIGURE 3. Relative
FIGURE 1. Schematic representation of the water-rich liquid layer within the stationary phase
In HILIC
Mobile Phase – Or
In HILIC the mobile
has been demonstr
modifier/aqueous ra
percentage of organ
Polar analytes preferentially partition into this aqueous rich layer and evidence [1] suggests that
they are retained through a complex, combination of:

hydrophilic partitioning of the analyte between the aqueous-rich layer and the bulk of the mobile
phase

hydrogen bonding between polar functional groups and the stationary phase

electrostatic interactions of ionized functional groups

van der Waals interactions between the hydrophobic portions of the bonded ligands of the
stationary phase and the non-polar part of the analytes.
In addition to the HILIC mechanism inherent complexity, there is variety of misinformation
regarding the use of this technique.
Although acetonitrile
organic modifiers ca
RPLC.
Mobile Phase – Do
As a general guidel
retention of charged
Due to their good so
salts of acetic and f
with mass spectrom
Generally, stationar
of buffers than neut
1. What column should be used?
2. What are the best mobile phase starting conditions?
3. What are the common issues in HILIC method development that need to be addressed?
These are the types of question that HILIC users face and will be addressed within this poster.
Electrostatic interac
retention in HILIC, s
typically used. The p
(both attractive and
HILIC Method Development
We recommend the following sequential method development steps:
Ammonium formate
of acid and basic m
FIGURE 1. HILIC method development flow chart
However, the aceta
the surface of the ch
retention times than
Determine
analyte log D,
pKa, solubility
2 HILIC Method Development in a Few Simple Steps
Log D> 1
Mobile Phase Buff
HILIC not
applicable
3. What are the common issues in HILIC method development that need to be addressed?
These are the types of question that HILIC users face and will be addressed within this poster.
Electrostatic interac
retention in HILIC, s
typically used. The p
(both attractive and
HILIC Method Development
We recommend the following sequential method development steps:
Mobile Phase Buff
Ammonium formate
of acid and basic m
FIGURE 1. HILIC method development flow chart
However, the acetat
the surface of the ch
retention times than
Determine
analyte log D,
pKa, solubility
HILIC not
applicable
Log D> 1
Preliminary method
development steps
What is the analyte
overall charge
Neutral,
zwitterionic or
mixture of
acids + bases
Column Group 1:
zwitterion, amide, urea
or Group 2: anion
exchanger, silica
Initial mobile phase: Isocratic
ACN/10 mM NH4OAc 80/20
Buffer pH: 4.8
No
Increase ACN
content
Change in 1-5%
steps
Change pH
Use pKas and the
molecular state required
to determine what pH.
(charged or uncharged)
Change buffer
concentration/buffer type
Typical ranges: 5-20 mM for
mid-polar to polar compounds
100-200 mM for very polar
compounds
Change in 2.5 mM steps
Change column type but within
same group
-ve Charged
(acidic)
+ve charged
(basic)
Column Group 1: zwitterion or amide
Column Group 2a: anion exchanger
Initial mobile phase: Isocratic
ACN/10 mM NH4OAc 80/20
Buffer pH: 6.4
Does the analyte have
suitable retention time
Yes
No
Column Group 1: zwitterion, amide, urea
Column Group 2b: silica
Initial mobile phase: Isocratic
ACN/10 mM NH4OAc 80/20
Buffer pH: 3.3
Are analytes
separated
Increase ACN
content
Change in 1-5%
steps
Change pH
Use pKas and the
molecular state required
to determine what pH.
(charged or uncharged)
Change buffer
concentration/buffer type
Typical ranges: 5-20 mM for
mid-polar to polar compounds
100-200 mM for very polar
compounds
Change in 2.5 mM steps
Change column type but within
same group
Yes
Mobile Phase Buff
When electrostatic a
decrease in retentio
phenomenon is illus
exchanger, with the
acetate increases.
Increased salt conc
stationary phases w
and cytidine on an a
bonding interactions
behavior. The hydro
salt ions in the mob
FIGURE 4. The effec
acids on Thermo Sc
90/10 acetonitrile/am
Working Assay
Method
Optimization
Steps
Change column temperature
Further information on method parameters is given in next section.
Thermo Scientific Poster Note • PN20957_HPLC_2014_E_05/14S 3
Method Parameters Considerations
FIGURE 5. The ef
bases on Hypers
acetonitrile/amm
Column Selection
It is suggested to match the analyte log P or log D values to the degree of polarity of the HILIC
phases. In general terms, the more negative the log P or log D value for an analyte, the greater the
degree of stationary phase polarity required to retain it. The following chart, which illustrates the
relative hydrophilicity and ion-exchange properties for Thermo Scientific™ HILIC columns, can be
used as a guide in stationary phase selection at this stage:
ccessful approach for
ribed as a variation of
he mobile phase
nitrile) containing a
us portion of the
orbed to the polar
FIGURE 3. Relative polarity and ion-exchange characteristics for various HILIC phases
the stationary phase
Column Name
Phase type
Syncronis TM HILIC
Column
Group
Zwitterion
1
AccucoreTM 150-Amide-HILIC
AccucoreTM Urea-HILIC
AcclaimTM HILIC-10
Hypersil GOLDTM HILIC
Hypersil GOLDTM Silica
Syncronis TM Silica
AccucoreTM HILIC
Polyacrylamide
1
Urea
1
Proprietary
1
Polyethyleneimine
2a
Unbonded Silica
2b
Unbonded Silica
2b
Unbonded Silica
2b
Mobile Phase Bu
In general, charge
The figure below s
bare silica and zw
FIGURE 6. The e
Mobile phase: 90
was measured b
Mobile Phase – Organic Content
In HILIC the mobile phase is highly organic (generally 60-70%; at least 3% water is required). It
has been demonstrated that besides the selection of a suitable column chemistry, the organic
modifier/aqueous ratio is a major factor controlling the separation selectivity. An increase in the
percentage of organic solvent leads to an increase in retention.
e [1] suggests that
the bulk of the mobile
se
Although acetonitrile is the most popular solvent used in HILIC, several other polar, water-miscible
organic modifiers can be used. The elutropic strength is generally the inverse to what observed in
RPLC.
Mobile Phase – Do I need a Buffer?
ed ligands of the
information
As a general guideline buffers are added to the mobile phase to reduce peak tailing and/or
retention of charged analytes.
Due to their good solubility in organic solvents, the recommended buffers for HILIC are ammonium
salts of acetic and formic acids. These buffers also have the advantage of being volatile for use
with mass spectrometry and charged aerosol detection.
Generally, stationary phases with a net positive or negative charge require higher concentrations
of buffers than neutral or zwitterionic phases.
be addressed?
within this poster.
Electrostatic interactions are secondary forces which can have important contributions to the
retention in HILIC, since some polar compounds can be charged at the mobile phase pH conditions
typically used. The presence of buffers in the mobile phase can reduce electrostatic interactions
(both attractive and repulsive) between charged analytes and the stationary phase.
Mobile Phase Buffer Type
Ammonium formate and ammonium acetate do not provide significant differences in retention times
of acid and basic model compounds on neutral and zwitterionic phases.
However, the acetate ion has a greater neutralising effect of the electrostatic attractions between
the surface of the charged stationary phase and the oppositely charged analyte, providing shorter
retention times than ammonium formate [2].
4 HILIC Method Development in a Few Simple Steps
The mobile phas
the case for bare
At pH>4-5, the s
have an effect on
illustrated in Figu
FIGURE 7. The e
phase. Mobile p
Mobile Phase Buffer Type
Ammonium formate and ammonium acetate do not provide significant differences in retention times
of acid and basic model compounds on neutral and zwitterionic phases.
However, the acetate ion has a greater neutralising effect of the electrostatic attractions between
the surface of the charged stationary phase and the oppositely charged analyte, providing shorter
retention times than ammonium formate [2].
Mobile Phase Buffer Concentration
When electrostatic attractions are prevalent, an increase in the salt concentration leads to a
decrease in retention of charged solutes on the stationary phases of opposite charge. This
phenomenon is illustrated in Figure 4, which show the separation of an acidic mixture on an anion
exchanger, with the retention of the anionic analytes decreasing as the concentration of ammonium
acetate increases.
Increased salt concentrations result in increased retention of positively charged solutes on
stationary phases with same charge, as demonstrated in Figure 5, where the retention of cytosine
and cytidine on an anion exchanger increases with the salt concentration. Enhanced hydrogenbonding interactions (between the analyte and the stationary phase) are responsible for this
behavior. The hydrogen-bonding interactions are facilitated by the increased population of solvated
salt ions in the mobile phase (salting-out effect).
FIGURE 4. The effect of ammonium acetate concentration on the separation of a mixture of
acids on Thermo Scientific™ Hypersil GOLD™ HILIC (anion exchanger). Mobile phase:
90/10 acetonitrile/ammonium acetate. Analytes: 1. Salicylamide; 2. Salicylic acid; 3. Aspirin
Conclusio
These are some k








Use acetonitri
strength is inv
protic solvents
Have a high o
ensure suffic
An increase in
Use buffer sa
to control rete
Buffer salts co
of up to 90%.
impair MS or
When using g
Do not run gra
gradient.
The charge st
depending on
Reference
1. McCalley, D
2. Guo, Y. and
© 2014 Thermo Fisher S
This information is not int
others.
Thermo Scientific Poster Note • PN20957_HPLC_2014_E_05/14S 5
FIGURE 5. The effect of ammonium acetate concentration on the separation of a mixture of
bases on Hypersil GOLD HILIC (anion exchanger). Mobile phase: 90/10
acetonitrile/ammonium acetate. Analytes: 1. Uracil; 2. Uridine; 3. Cytosine; 4. Cytidine
arity of the HILIC
alyte, the greater the
hich illustrates the
IC columns, can be
HILIC phases
Column Name
Phase type
Syncronis TM HILIC
Column
Group
Zwitterion
1
TM
ccucore
150-Amide-HILIC
AccucoreTM Urea-HILIC
AcclaimTM HILIC-10
Hypersil GOLDTM HILIC
Hypersil GOLDTM Silica
Syncronis TM Silica
AccucoreTM HILIC
Polyacrylamide
1
Urea
1
Proprietary
1
Polyethyleneimine
2a
Unbonded Silica
2b
Unbonded Silica
2b
Unbonded Silica
2b
Mobile Phase Buffer pH
In general, charged compounds are more hydrophilic, and therefore are more retained in HILIC.
The figure below shows the retention factor of acetylsalicylic acid increasing with the buffer pH, on
bare silica and zwitterionic phases:
FIGURE 6. The effect of mobile phase buffer pH on the retention of acetylsalicylic acid.
Mobile phase: 90/10 acetonitrile/100 mM ammonium formate. The mobile phase buffer pH
was measured before the addition of acetonitrile
ter is required). It
stry, the organic
n increase in the
polar, water-miscible
o what observed in
ailing and/or
HILIC are ammonium
ng volatile for use
gher concentrations
ibutions to the
phase pH conditions
static interactions
hase.
The mobile phase buffer pH can also affect the stationary phase charge state; this, for example is
the case for bare silica phases, where the silanol ionisation varies with the mobile phase buffer pH.
At pH>4-5, the silanols are deprotonated, making the silica surface negatively charged. This will
have an effect on the retention of positively charged analytes. The increased retention for cytidine,
illustrated in Figure 7 demonstrates this phenomenon.
FIGURE 7. The effect of mobile phase buffer pH on the retention of cytidine on a bare silica
phase. Mobile phase: 90/10 acetonitrile/100 mM ammonium formate
pH 3.3
pH 4.0
pH 6.4
ces in retention times
ttractions between
e, providing shorter
6 HILIC Method Development in a Few Simple Steps
It is very important to fine tune the
mobile phase buffer pH, when
using bare silica phases for the
analysis of basic compounds, in
order to avoid excessively long
retention times and peak
broadening
g and/or
are ammonium
olatile for use
concentrations
ons to the
se pH conditions
c interactions
.
The mobile phase buffer pH can also affect the stationary phase charge state; this, for example is
the case for bare silica phases, where the silanol ionisation varies with the mobile phase buffer pH.
At pH>4-5, the silanols are deprotonated, making the silica surface negatively charged. This will
have an effect on the retention of positively charged analytes. The increased retention for cytidine,
illustrated in Figure 7 demonstrates this phenomenon.
FIGURE 7. The effect of mobile phase buffer pH on the retention of cytidine on a bare silica
phase. Mobile phase: 90/10 acetonitrile/100 mM ammonium formate
pH 3.3
pH 4.0
pH 6.4
n retention times
It is very important to fine tune the
mobile phase buffer pH, when
using bare silica phases for the
analysis of basic compounds, in
order to avoid excessively long
retention times and peak
broadening
tions between
oviding shorter
eads to a
ge. This
ure on an anion
on of ammonium
utes on
tion of cytosine
ed hydrogenle for this
ation of solvated
Conclusions
These are some key tips in method development and optimisation:





f a mixture of
le phase:
cid; 3. Aspirin



Use acetonitrile or other polar, water-miscible organic modifiers. Remember that the elutropic
strength is inverse to what observed in RPLC. Aprotic solvents give longer retention than
protic solvents.
Have a high organic content, between 60 to 97%; a minimum of 3% water is necessary to
ensure sufficient hydration of the stationary phase.
An increase in organic solvent will lead to an increase in retention.
Use buffer salts such as ammonium acetate and ammonium formate to avoid peak tailing and
to control retention times of charged analytes.
Buffer salts concentrations are 2-20 mM, although 20 mM is recommended for organic content
of up to 90%. Higher concentrations would not be soluble in high levels of organic and could
impair MS or CAD signals.
When using gradients, buffer both mobile phases, do not run buffer gradients.
Do not run gradients from 100% organic to 100% aqueous. We suggest a 97-60% organic
gradient.
The charge state of the stationary phase can affect HILIC retention of ionisable compounds,
depending on the mobile phase pH.
References
1. McCalley, D.V., 2010, J. Chromatogr. A, 1217, 3408.
2. Guo, Y. and Gaiki, S. 2005, J. Chromatogr. A, 1074, 71.
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