HILIC-sep Sci Webinar 09132013

HILIC Chromatography:
Theory and Method Development Practices

David S. Bell
Supelco/Sigma/Aldrich
Bellefonte, PA
September 18, 2013
sigma-aldrich.com/analytical
2013 Sigma-Aldrich Co. All rights reserved.
Introduction
1.
2.
3.
4.
Describe the HILIC system and prevailing theories on retention mechanisms

Discuss various HILIC stationary phases and the interactions they exhibit
Discuss method development practices/approaches
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
Biphasic Solvent Distribution at Silica Surface
Si-OH
Homogeneous binary
mobile phase high in
organic content
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Biphasic system
aqueous-rich solvent
Si-OH
aqueous-depleted solvent
HILIC conditions
initial conditions
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
If it were only that simple.

Si-OH
Were bringing them close to a

polar surface and therefore can
expect (should expect) other
strong interactions to take place
Strong dipole interactions (Hbonding) and ion-exchange are
an integral and important part of
HILIC chromatography
Si-OSi-OH
Si-OSi-OH
Si-O-
aqueous-rich solvent
Polar analytes are just that

because they are ionic or exhibit
strong dipoles naturally
interactive
Biphasic system
Si-OH
CH3
aqueous-depleted solvent
HILIC conditions
OH
NH2
HO
Normetanephrine
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.
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
HN
10
Retention (min)
HO
OH
Synephrine
10
20
30
40
50
60
70
80
90
100
% Acetonitrile
Synephrine Ascentis Express HILIC
Synephrine Ascentis Express PFP

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
Retention (min)
0
0
10
20
30
40
50
% Acetonitrile
Synephrine Ascentis Express HILIC (2)
60
70
80
90
100
Synephrine Ascentis Express PFP (2)

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
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
Are HILIC Phases the Same?

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

function of buffer concentration was collected and
interpreted.
Several related compounds were also run simultaneously
and retention and selectivity noted. Interpretation of the
observed results in terms of the dominant interactions
prevalent using each phase is provided.
HO
HO
OH
R2
R3
R1
Si
OH
R2
OH5
10
Selected Probes
Structure
H3C
H3C
pKa(MB)
LogD(8.0)
LogP
MW
name
9.38
-0.37
1.08
165.23
pseudoephedrine
9.38
-0.37
1.08
165.23
ephedrine
9.37
-1.35
0.13
167.21
synephrine
NH
OH
H3C
H3C
ACD/Labs, PhysChemProp, v. 12
NH
OH
CH3
HN
HO
OH
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, ionexchange 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.
12
Response of Ephedrine Retention on Buffer

Concentration on Three HILIC Phases
1.20
1.00
y = -0.6525x + 1.2104
R2 = 0.9984
log k'
0.80
0.60
y = -0.8187x + 1.1259
R2 = 1
0.40
0.20
0.00
0.00
y = -0.2199x + 0.2455
R2 = 0.9934
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)
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
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
15
R2
Proposed Model for Different HILIC Stationary Phases
Aqueous-Organic Mobile Phase

Silica
OH5
F5
HO
HO
OH
HO
F
F
Aqueous
Layer
OH
R1
Si
R3
R2
Aqueous Layer
-
R1 Si
R2
R1
Polar Stationary Phase

16
Attributes of Common HILIC Phases
Stationary Phase
Interactions
Partitioning
Polar
Ionic
Bare Silica
moderate
moderate
Zwitterionic
strong
weak
Amide
strong
weak
Diol/Polyol
strong
weak
PFP
weak
strong
Cyano
weak
strong
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)
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
19
Bath Salt Analytes Hydrophilic, Weak Bases

O
NH
CH3
NH
CH3
CH3
CH3
CH3
Butylone
MDPV
Monoisotopic Mass = 221.105193 Da
O
NH
CH3
methylone
O
NH
CH3
O

O
NH
20
CH3
H3C
ethylone
NH
CH3
CH3
4-fluoromethcathinone
H3C
mephedrone
CH3
methedrone
O
O
CH3
CH3
NH
NH
CH3
CH3
CH3
CH3
Buphedrone

20
Ascentis Express F5, 2 mM Ammonium Acetate

in 90% Acetonitrile
2 mM - 3
A1849_029_B031
1: Scan ES+
194
3.01e8
6.01
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
178
3.04e8
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
208
2.82e8
7.00
8.00
9.00
10.00
1: Scan ES+
276
2.04e8
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
222
3.64e8
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
TIC
1.05e9
7.00
8.00
9.00
5.76
A1849_029_B031
1.00
2.00
3.00
4.00
5.00
4.92
A1849_029_B031
1.00
2.00
3.00
4.00
5.00
6.00
6.76
A1849_029_B031
1.00
2.00
3.00
4.00
3.88
5.00
4.95
A1849_029_B031
0
A1849_029_B031
1.00
2.00
3.00
4.00
3.87
5.00
4.94
5.78 6.01
1.30
1.00
2.00
3.00
4.00
5.00
6.00
6.76
Time
10.00
IEX results in some coelution in this case

21
Ascentis Express HILIC, 2 mM Ammonium

Acetate in 90% Acetonitrile
2 mM - 3
A1849_029_B015
1: Scan ES+
194
2.97e8
7.01
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
178
3.47e8
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
208
3.08e8
5.74
A1849_029_B015
1.00
2.00
3.00
4.00
5.00
6.19
A1849_029_B015
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
276
3.32e8
5.00
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
222
3.91e8
6.00
7.00
8.00
9.00
10.00
1: Scan ES+
TIC
8.22e8
8.00
9.00
3.82
A1849_029_B015
1.00
2.00
3.00
4.00
4.11
4.80
A1849_029_B015
0
A1849_029_B015
1.00
2.00
3.00
4.00
3.82
4.11
5.00
4.80
5.74
6.18
7.01
1.27 1.59
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Time
10.00
IEX + partition provides the needed selectivity in this case

22
Optimized Separation of Bath Salts on Ascentis

Analyte
Express HILIC
1.0
2.0
3.0
System:
4.0
Time (min)
5.0
6.0
rt
MDPV
2.0
Buphedrone
2.2
2.7
butylone
2.9
ethylone
3.4
3.7
mephedrone
4.6
methylone
5.2
methedrone
6.0
7.0
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
Temperature:
35 C
System Pressure:
127bar
Injection Vol:
1uL
MS Detection:
ESI+, 100-1000m/z ,
Sample:
200ng/mL Bath Salts in acetonitrile
Craig R. Aurand, Robert Shirey,

Leonard Sidisky Janusz Pawliszyn,
and Yong Chen, Application of BioSPME for the Enrichment of Illicit
Phenethylamine and Cathinone
Compounds from Biological Samples,
Poster, ASMS national Meeting,
Vancouver, BC, 2012
23
23
Nucleoside Structures Polar Neutral (for the

most part)
NH2
OH
O
N
HN
N
N
O
O
HO
HO
HO
OH
OH
OH
HO
Uridine
Cytidine
Inosine
O
N
HN
H2N
HO
HO
CH3
HN
N
HO
HO
CH3
N
N
NH2
HO
HO
Guanosine
OH
HO
OH
Ribothymidine
5-Methyluridine
HO
OH
5-Methylcytidine
24
Structures
NH2
NH2
NH
H3C
N
N
N
S
HO
HO
HO
HO
HO
OH
HO
2-Thiocytidine
2'-O-Methylcytidine
NH
CH3
CH3
HN
N
HN
H2N
H3C
1-Methyladenosine
NH
7-Methylguanosine
OH
HO
HO
OH
HO
HO
H3C
OH
HO
OH
Pseudouridine
HO
OH
3-Methylcytidine metosulfate
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 acetate, pH 5
Good retention and selectivity reasonably little change as

a function of buffer pH
26
Ascentis Express HILIC

5 mM ammonium acetate, pH 6.9
unadjusted
Good retention and selectivity group of more basic analytes

preferentially retained that are sensitive to changes in buffer pH
27
Ascentis Express F5
5 mM ammonium acetate, pH 6.9 unadjusted
28
Optimized Separation of Nucleosides on Ascentis

Express OH5
7.53
12 NUCLEOSIDES_ASCEXPOH5.CDF
55
50
45
8.6
6.5
9.02
30
25
11.11
15
6.08
5.28
4.62
20
10.29
3.07
Response
35
7.79
2.75
40
2.91
10
5
0
0
6
7
Retention Time (min)
10
11
12
13
column: Ascentis Express OH5, 10 cm x 2.1 mm, 2.7 m (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 L
sample:
10 - 100 g/mL
29
Structures of Yohimbine and Ajmalicine Alkaloids
N
N
H
N
H
H
CH3
H
O
H3C
H3C
O
Yohimbine
OH
Ajmalicine
Relatively nonpolar, basic analytes
Separation of Yohimbe Alkaloid Standards on

Ascentis Express F5
YOHIMBE STANDARDS.CDF1
2
4
5
6
1. Ajmalicine
2. Yohimbine
10
Conditions:
Column: Ascentis Express F5, 10 cm x 3.0 mm, 2.7 m (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 : 35C
Detection: UV. 275 nm
Injection: 2 L
HILIC Analysis of Yohimbe Extract on Ascentis

Express F5
YOHIMBE EXTRACT_F5.ESP
1. Yohimbine
4
5
6
10
Conditions:
Column: Ascentis Express F5, 10 cm x 3.0 mm, 2.7 m (53766-U)
Temperature : 35C
Injection: 2 L
HILIC Analysis of Yohimbe Alkaloids on Ascentis

Express HILIC
1
YOHIMBE STANDARDS_HILIC.CDF
2
YOHIMBE EXTRACT HILIC.ESP
4
5
6
10
4
5
6
Conditions:
Column: Ascentis Express HILIC, 10 cm x 3.0 mm, 2.7 m (53970-U)
Temperature : 35C
Injection: 2 L
10
Ephedrine Alkaloids Polar, Basic Compounds

H3C
H3C
NH
OH
H3C
ephedrine
H3C
H3C
CH3
NH2
N
OH
H3C
H3C
OH
norephedrine
methylephedrine
CH3
NH
OH
OH
HN
NH2
HO
CH3
pseudoephedrine
norpseudoephedrine
OH
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.
34
Optimization of Mobile Phase Ion Concentration

Linear Dependence
1.200
1.000
log k
0.800
0.600
norephedrine
synephrine
methylephedrine
ephedrine
pseudoephedrine
4 mM optimum
0.400
0.200
0.000
2
10
ammonium acetate concentration, mM

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:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Sample:
Discovery HS F5 (PFP), 15 cm x 4.6 mm, 5 m

2 or 10 mM ammonium acetate in 90:10, CH3CN:water
1 mL/min
35C
UV at 215 nm
10 L
mix of ephedrine alkaloids + synephrine at 100 g/mL each in 2
mM ammonium acetate mobile phase
36
LC-MS Analysis of Ephedrine Alkaloids

1
ESI+ 152
8
10
Time (min)
12
8
10
Time (min)
12
14
16
18
14
16
18
ESI+ 168
8
10
Time (min)
12
8
10
Time (min)
12
14
16
18
ESI+ 166
norephedrine
norpseudoephedrine
synephrine
methylephedrine
ephedrine
pseudoephedrine
ESI+ 180
1.
2.
3.
4.
5.
6.
14
16
Understanding
dominant
interactions greatly
facilitates method
development
18
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
38
Method Development in HILIC - Tips

Sample consideration
HILIC is generally restricted to highly polar and/or ionic analytes. Low octanolwater 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)
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
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
41
s
pH
w
pH Measured Following Addition of Organic
Effect of Acetonitrile on pH of Ammonium Acetate

12
11
The pH of the aqueous ammonium

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

Square: 75% ACN,
Diamond: 50% ACN,
Circle: 32.5% ACN
4
3
2
2
10
12
pH Measured Prior to Addition of Organic
w
pH
w
42
Determination of pKa Values using NMR

Amitriptyline
% Acetonitrile
s
w
pKa
Correlation
(R2)
25
9.32
0.9997
50
9.02
0.9996
75
90
8.88
8.34
Analyte
Literature pKa
pKa
Correlation
(R2)
Amitriptyline
9.4
8.34
0.9923
Nortriptyline
9.7
8.92
0.9920
Diphenhydramine
9.0
8.33
0.9978
Verapamil
8.9
7.98
0.9976
Alprenolol
9.7
8.73
0.9855
s
w
0.9956
0.9923
pKa values for bases decrease

with increasing acetonitrile
At 90% each analyte exhibited a
pKa value about 1 full pH unit less
than the literature pKa value
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
44
Balancing HILIC and IEX
IEX dominated
Low ionic concentration

Fluorinated/Cyano phases
Partition dominated
High ionic concentration

Bare silica
OH5 phase/Amide
Zwiterionic
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
46
Acknowledgements
Hillel Brandes
Xiaoning Lu
Hugh Cramer
Craig Aurand
Errol Fernandes
Carmen Santasania
Gaurang Parmar
Wayne Way
Separation Science
Q&A
Follow up questions on HILIC? Contact: Dave.Bell@sial.com

Ascentis Express HPLC Columns are available in 2 particle sizes
and multiple phases. For product information, visit:
sigmaaldrich.com/express
2.7 m
9 Phases
5 m
7 Phases
48

HILIC-sep Sci Webinar 09132013

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HILIC-sep Sci Webinar 09132013

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HILIC Chromatography:

Theory and Method Development Practices

2013 Sigma-Aldrich Co. All rights reserved.

Describe the HILIC system and prevailing theories on retention mechanisms

2013 Sigma-Aldrich Co. All rights reserved.

Biphasic Solvent Distribution at Silica Surface

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

2013 Sigma-Aldrich Co. All rights reserved.

If it were only that simple.

Were bringing them close to a

Polar analytes are just that

2013 Sigma-Aldrich Co. All rights reserved.

Interactions in HILIC Chromatography

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

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Retention Profile of Synephrine on Bare Silica and

Synephrine Ascentis Express HILIC

Synephrine Ascentis Express PFP

Retention Profile of Synephrine on Bare Silica and

Synephrine Ascentis Express HILIC (2)

Synephrine Ascentis Express PFP (2)

2013 Sigma-Aldrich Co. All rights reserved.

2013 Sigma-Aldrich Co. All rights reserved.

Are HILIC Phases the Same?

Using ephedrine as a probe molecule, retention as a

2013 Sigma-Aldrich Co. All rights reserved.

Focus on Understanding the IEX Component

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Response of Ephedrine Retention on Buffer

log buffer concentration (mM)

2013 Sigma-Aldrich Co. All rights reserved.

2013 Sigma-Aldrich Co. All rights reserved.

2013 Sigma-Aldrich Co. All rights reserved.

Proposed Model for Different HILIC Stationary Phases

Aqueous-Organic Mobile Phase

Polar Stationary Phase

Attributes of Common HILIC Phases

2013 Sigma-Aldrich Co. All rights reserved.

Matching the Sample Analytes to the Right

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

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Method Development in HILIC

2. Choose appropriate stationary phases based on potential interactions

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Bath Salt Analytes Hydrophilic, Weak Bases

Monoisotopic Mass = 221.105193 Da

Monoisotopic Mass = 275.152144 Da

Monoisotopic Mass = 207.089543 Da

Monoisotopic Mass = 221.105193 Da

Monoisotopic Mass = 181.090292 Da

Monoisotopic Mass = 181.090292 Da

Ascentis Express F5, 2 mM Ammonium Acetate

IEX results in some coelution in this case

Ascentis Express HILIC, 2 mM Ammonium

IEX + partition provides the needed selectivity in this case

Optimized Separation of Bath Salts on Ascentis

Agilent 1200SL Rapid Resolution, 6210 TOF

Ascentis Express HILIC 10cm X 2.1mm, 2.7um (53939-U)

5mM ammonium formate (98:2 acetonitrile:water)

200ng/mL Bath Salts in acetonitrile

Craig R. Aurand, Robert Shirey,

2013 Sigma-Aldrich Co. All rights reserved.

Nucleoside Structures Polar Neutral (for the

2013 Sigma-Aldrich Co. All rights reserved.