Evaluation of the Temperature Influence on
Retention for HILIC of Glucose Oligomers
Labeled with 2-Aminobenzamide: Retention
Thermodynamics and Practical Influence on
Separation of Glycans
DaMauro De Pra and Frank Steiner, Thermo Fisher Scientific, Germering, Germany
Overview
Purpose: Improve the understanding of retention
mechanisms of glycans labeled with 2 aminobenzamide
(2-AB) in amide columns.
Methods: 2-AB-glucose oligomers were separated in
isocratic and gradient modes at various temperatures.
Results: Selectivity and peak broadening improve at high
temperature.
Introduction
Separation of glycans with amide columns is widely used
in glycomics and in biopharmaceutical analysis. Better
understanding of the retention mechanism is beneficial for
the development of future stationary phases and for
optimum method development.
In this work we carried out an initial evaluation of the
retention characteristic of linear glucose oligomers labeled
with 2-AB. Additionally, based on the collected information,
we provide some indication on how to improve the method
resolving power without compromising on analysis time.
Methods
Liquid Chromatography
System: Thermo Scientific™ Dionex™ UltiMate™ 3000
BioRS UHPLC system equipped with low pressure
gradient pump LGP-3400RS, fluorescence detector FLD3400RS, and pre-column heater.
Mobile Phases: A- ammonium formate 0.1M pH 4.5. Bacetonitrile
T (°C) %B
20
80
-8.6E+
30
80
-9.1E+
40
80
-9.2E+
50
80
-9.8E+
20
76
-1.8E+
30
76
-1.9E+
40
76
-2.1E+
50
76
-2.2E+
20
66
-9.7E+
30
66
-1.1E+
40
66
-1.1E+
50
66
-1.1E+
According to the
selectivity betwe
monomer unit, is
observation app
content (80 %B
content (66 %B)
The increased se
observed in the V
retention factor d
oligomers are mo
FIGURE 2. Van’t
oligomers obtain
0.4 mL/min.
Detector Setting: Ex. 320 nm, Em 420 nm, sensitivity 8,
Filter wheel 370 nm, data collection rate 2Hz, response
time 4 sec
Column: Thermo Scientific™ Accucore™ 150-Amide-HILIC
2.6 m, 2.1 × 250 mm
Sample Preparation
Dextran ladder standard was labelled in house with 2-AB
according to standard procedure. Final concentration was
0.1 μg/μL in acetonitrile/ammonium formate buffer = 9/1
v/v
4
3.5
3
2.5
2
ln k'
1.5
1
0.5
0
0.01
2.6
Chromatography Data System
Thermo Scientific™ Dionex™ Chromeleon™
Chromatography Data System Software Version 7.2
Results
Contribution of Additional Glucose Units to Retention
2.4
2.2
ln k'
2
1.8
1.6
2 Evaluation of the Temperature Influence on Retention for HILIC of Glucose Oligomers Labeled with 2-Aminobenzamide: Retention Thermodynamics and Practical
1.4
Influence on Separation of Glycans
The plots logarithm of retention factor (ln k’) vs Glucose
ΔG0
(J/mo
0.01
0.
2.4
Thermo Scientific™ Dionex™ Chromeleon™
Chromatography Data System Software Version 7.2
2.2
Results
ln k'
2
1.8
Contribution of Additional Glucose Units to Retention
The plots logarithm of retention factor (ln k’) vs Glucose
Units (GU) at a given temperature and mobile phase
composition can be interpolated by linear equation. This
behavior is typically found for homologous series. Therefore,
the relative importance of the 2-AB group appears negligible
relative to the carbohydrate chain.
1.6
1.4
0.01
0.02
The Van’t Hoff plots of F
observation indicates a
thermodynamics with te
maxima are observed.
Further studies are requ
mechanism of glucose o
to explain behavior such
FIGURE 1. Example of ln(k’) vs GU plot. Isocratic elution
at 66% B and 20 °C; flow rate 0.4 mL/min.
The retention of linear g
mode is in agreement w
isocratic elution: selectiv
Figure 3 shows that sma
are less retained at high
oligomers are more reta
3
2.5
Gradient Separation of
Different Temperature:
y = 0.3985x + 0.4705
R² = 0.9979
2
ln k' 1.5
1
FIGURE 3. Retention t
different temperatures
in 40 minutes. Flow ra
0.5
0
GU (i)
GU(i+1)
GU(i+2)
GU(i+3)
GU(i+4)
35
The slope of plots such as the one in Figure 1 represents
the natural logarithm of the selectivity between the
oligomer GU(i+1) and oligomer GU(i).
Based on the assumption that the 2-AB-glucose oligomers
behave as series of homologous (i.e. 2-AB does not
influence retention), the variation of Gibbs Free Energy
related to the transfer from mobile to stationary phase,
when one glucose monomer is added to the oligomer, can
be calculated by the following formula:
Retention Time (min)
30
25
20
15
10
5
0
10
20
Thermo Scientific Poster Note • PN71311 ISC2014-EN 0814S 3
30
adening improve at high
columns is widely used
tical analysis. Better
chanism is beneficial for
ry phases and for
al evaluation of the
ucose oligomers labeled
he collected information,
w to improve the method
ising on analysis time.
x™ UltiMate™ 3000
with low pressure
escence detector FLD-
mate 0.1M pH 4.5. B-
20
80
-8.6E+03
30
80
-9.1E+03
40
80
-9.2E+03
50
80
-9.8E+03
20
76
-1.8E+03
30
76
-1.9E+03
40
76
-2.1E+03
50
76
-2.2E+03
20
66
-9.7E+02
30
66
-1.1E+03
40
66
-1.1E+03
50
66
-1.1E+03
Table 1. Estimated contribution
to Gibbs Free Energy involved
in the transition of one
monomer (glucose) unit from
mobile to stationary phase, at
different temperatures and
isocratic conditions. Flow rate
0.4 mL/min.
The increased selectivity at high temperature can also be
observed in the Van’t Hoff plots of Figure 2, where the
retention factor differences between 2-AB- labeled glucose
oligomers are more pronounced at high temperatures
FIGURE 2. Van’t Hoff plots for 2-AB-glucose linear
oligomers obtained at 76% and 66 % organic. Flow rate
0.4 mL/min.
4
76%B
3.5
3
core™ 150-Amide-HILIC
2.5
GU(j+3)
2
GU(j+2)
1.5
GU(j+1)
ln k'
1
led in house with 2-AB
Final concentration was
m formate buffer = 9/1
GU(j)
10
8
6
4
2
10
Gradient separat
different tempera
peak capacity
It was observed th
linear oligomers c
increasing temper
sharper when incr
effects contribute
shows that peak c
20 °C to 50 °C. Th
changing the anal
0.01
0.02
0.03
0.04
0.05
0.06
1/T (°C-1)
66%B
2.4
FIGURE 5. Peak
separation of GU
Gradient 80 to 5
rate: 0.4 mL/min
145
2.2
ln k'
2
140
GU(i+2)
GU(i+1)
GU(i)
1.6
135
130
125
4 Evaluation of the Temperature Influence on Retention for HILIC of Glucose Oligomers Labeled with 2-Aminobenzamide: Retention Thermodynamics and Practical
on Separation of Glycans
ctor (ln k’)Influence
vs Glucose
20
0
1.8
ose Units to Retention
12
0.5
2.6
meleon™
ftware Version 7.2
Besides influencin
temperature affec
phase and station
mass transfer is lo
consequently pea
common in any liq
observed for all ol
FIGURE 4. Peak
oligomers at diff
50% acetonitrile
According to the Gibbs free energy values of Table 1, the
selectivity between glucose oligomers differing by one
monomer unit, is more favorable at high temperature. The
observation applies to mobile phase with high organic
content (80 %B and 76 %B), whereas at lower organic
content (66 %B) the same behavior is not clearly visible.
420 nm, sensitivity 8,
n rate 2Hz, response
Gradient Separat
Different Temper
PWHH (sec)
s were separated in
arious temperatures.
ΔG0
(J/mol)
Peak Capacity
ing of retention
th 2 aminobenzamide
T (°C) %B
1.4
0.01
0.02
0.03
0.04
0.05
0.06
120
UltiMate™ 3000
ow pressure
nce detector FLD-
0.1M pH 4.5. B-
oligomers are more pronounced at high temperatures
FIGURE 2. Van’t Hoff plots for 2-AB-glucose linear
oligomers obtained at 76% and 66 % organic. Flow rate
0.4 mL/min.
nm, sensitivity 8,
e 2Hz, response
4
76%B
3.5
3
150-Amide-HILIC
2.5
GU(j+3)
2
GU(j+2)
1.5
GU(j+1)
ln k'
1
GU(j)
0.01
0.02
0.03
0.04
0.05
0.06
FIGURE 5. Peak capa
separation of GU2 to
Gradient 80 to 50% a
rate: 0.4 mL/min.
1/T (°C-1)
66%B
2.4
on™
e Version 7.2
145
2.2
ln k'
2
Units to Retention
140
GU(i+2)
GU(i+1)
1.8
GU(i)
1.6
1.4
0.01
0.02
0.03
0.04
0.05
0.06
1/T (°C-1)
FIGURE 3. Retention time of linear oligomers at
different temperatures. Gradient 80 to 50% acetonitrile
in 40 minutes. Flow rate: 0.4 mL/min.
30
125
120
15
Gradient Separation of Linear Glucose Oligomers at
Different Temperature: Effects on Retention
35
130
110
The Van’t Hoff plots of Figure 2 are not linear. This
observation indicates a change of the retention
thermodynamics with temperature. Negative slopes and
maxima are observed.
The retention of linear glucose oligomers with gradient
mode is in agreement with the behavior observed for
isocratic elution: selectivity increases at high temperature.
Figure 3 shows that smaller oligomers (up to GU6-GU7)
are less retained at high temperature, whereas longer
oligomers are more retained at high temperature.
GU(i+4)
135
115
Further studies are required to elucidate the retention
mechanism of glucose oligomers with amide phases, and
to explain behavior such as those of Figure 2.
GU(i+3)
It was observed that th
linear oligomers can be
increasing temperature
sharper when increasin
effects contribute to inc
shows that peak capac
20 °C to 50 °C. The inc
changing the analysis
0
2.6
plot. Isocratic elution
/min.
3
0.5
n house with 2-AB
concentration was
mate buffer = 9/1
ln k’) vs Glucose
mobile phase
ar equation. This
ous series. Therefore,
up appears negligible
20
Gradient separation o
different temperature
peak capacity
Peak Capacity
™
10
GU12
20
This work was perform
however it is expected
reached for branched
are required to confirm
Conclusion
 Isocratic retention
labeled linear gluc
to the behavior of
 Gibbs Free Energ
indicate that isocr
temperature
 Selectivity for grad
temperature
 Peaks of labeled l
at high temperatu
Thermo Scientific Poster Note • PN71311 ISC2014-EN 0814S 5
GU11
25
 The peak capacity
FIGURE 3. Retention time of linear oligomers at
different temperatures. Gradient 80 to 50% acetonitrile
in 40 minutes. Flow rate: 0.4 mL/min.
n Retention for HILIC of Glucose
)
de: Retention Thermodynamics and
one in Figure 1 represents
ans
ectivity between the
(i+2)
GU(i+3)
GU(i+4
35
GU12
GU11
30
he 2-AB-glucose oligomers
us (i.e. 2-AB does not
on of Gibbs Free Energy
bile to stationary phase,
added to the oligomer, can
ormula:
. Estimated contribution
s Free Energy involved
ransition of one
er (glucose) unit from
to stationary phase, at
nt temperatures and
c conditions. Flow rate
min.
Retention Time (min)
GU(i).
GU10
25
GU9
20
GU8
GU7
15
GU6
10
GU5
5
GU4
 Gibbs Free
indicate tha
temperature
 Selectivity fo
temperature
 Peaks of lab
at high temp
 The peak ca
increases w
© 2014 Thermo Fisher Scientific
Inc. and its subsidiaries. This inf
infringe the intellectual property
GU3
0
10
20
30
40
50
GU2
60
T (°C)
Gradient Separation of Linear Glucose Oligomers at
Different Temperature: Effects on Peak Broadening
Besides influencing the retention thermodynamics,
temperature affects the mass transfer rate between mobile
phase and stationary phase. Peak dispersion generate by
mass transfer is lower at higher temperature, and
consequently peaks are narrower. This behavior, extremely
common in any liquid chromatography technique, is
observed for all oligomers between GU2 and GU12.
FIGURE 4. Peak Width at Half Height (PWHH) of linear
oligomers at different temperatures. Gradient 80 to
50% acetonitrile in 40 minutes. Flow rate 0.4 mL/min.
temperature can also be
of Figure 2, where the
een 2-AB- labeled glucose
at high temperatures
GU12
12
GU11
10
PWHH (sec)
rgy values of Table 1, the
omers differing by one
at high temperature. The
ase with high organic
ereas at lower organic
vior is not clearly visible.
GU10
GU9
8
GU8
GU7
6
GU6
GU5
4
GU4
GU3
2
10
20
30
40
50
60
GU2
T (°C)
-AB-glucose linear
66 % organic. Flow rate
6
Gradient separation of linear glucose oligomers at
different temperatures. Practical consequences on
peak capacity
It was observed that the retention window of 2-AB glucose
linear oligomers can be enlarged significantly (Figure 3) by
increasing temperature. Additionally all peaks become
Evaluation of76%B
the Temperature Influence onsharper
Retention for
HILIC ofincreasing
Glucose Oligomers
Labeled with 2-Aminobenzamide:
Retention Thermodynamics and Practical
when
temperature
(Figure 4). Both
Influence on Separation of Glycans
effects contribute to increase of peak capacity. Figure 5
10
30
40
50
60
GU2
Gradient separation of linear glucose oligomers at
different temperatures. Practical consequences on
peak capacity
It was observed that the retention window of 2-AB glucose
linear oligomers can be enlarged significantly (Figure 3) by
increasing temperature. Additionally all peaks become
sharper when increasing temperature (Figure 4). Both
effects contribute to increase of peak capacity. Figure 5
shows that peak capacity is increases of about 16% from
20 °C to 50 °C. The increase of capacity occurred without
changing the analysis time.
76%B
GU(j+3)
GU(j+2)
GU(j+1)
GU(j)
0.06
FIGURE 5. Peak capacity at different temperature for
separation of GU2 to GU12 labeled linear oligomers.
Gradient 80 to 50% acetonitrile in 40 minutes. Flow
rate: 0.4 mL/min.
66%B
145
140
GU(i+2)
GU(i+1)
Peak Capacity
05
20
T (°C)
ucose linear
organic. Flow rate
5
GU3
2
temperatures
GU(i)
0.06
135
130
125
120
115
110
inear. This
etention
ative slopes and
15
20
25
30
35
40
45
50
55
T (°C)
e the retention
mide phases, and
ure 2.
se Oligomers at
tention
s with gradient
observed for
high temperature.
up to GU6-GU7)
hereas longer
mperature.
igomers at
o 50% acetonitrile
GU12
GU11
This work was performed with linear glucose oligomers;
however it is expected that similar conclusions can be
reached for branched 2-AB-glycans. Further experiments
are required to confirm this hypothesis.
Conclusion
 Isocratic retention at a given temperature of 2-ABlabeled linear glucose oligomers can be approximated
to the behavior of homologous series
 Gibbs Free Energy measurements and Van’t Hoff plots
indicate that isocratic selectivity improves at high
temperature
 Selectivity for gradient separations is improved at high
temperature
 Peaks of labeled linear glucose oligomers are narrower
at high temperature
Thermo Scientific Poster Note
•
PN71311 ISC2014-EN 0814S 7
are required to confirm this hypothesis.
se Oligomers at
tention
rs with gradient
r observed for
t high temperature.
(up to GU6-GU7)
whereas longer
mperature.
ligomers at
o 50% acetonitrile
.
0
GU12
GU11
GU10
GU9
Conclusion
 Isocratic retention at a given temperature of 2-ABlabeled linear glucose oligomers can be approximated
to the behavior of homologous series
 Gibbs Free Energy measurements and Van’t Hoff plots
indicate that isocratic selectivity improves at high
temperature
 Selectivity for gradient separations is improved at high
temperature
 Peaks of labeled linear glucose oligomers are narrower
at high temperature
 The peak capacity of 2-AB-glycan separations
increases with temperature
GU8
GU7
GU6
© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific
Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might
infringe the intellectual property rights of others.
PO71311-EN 0814S
GU5
GU4
GU3
60
GU2
www.thermofisher.com
©2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its
subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not
intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local
sales representative for details.
Africa +43 1 333 50 34 0
Australia +61 3 9757 4300
Austria +43 810 282 206
Belgium +32 53 73 42 41
Brazil +55 11 3731 5140
Canada +1 800 530 8447
China 800 810 5118 (free call domestic)
400 650 5118
Denmark +45 70 23 62 60
Europe-Other +43 1 333 50 34 0
Finland +358 9 3291 0200
France +33 1 60 92 48 00
Germany +49 6103 408 1014
India +91 22 6742 9494
Italy +39 02 950 591
Japan +81 6 6885 1213
Korea +82 2 3420 8600
Latin America +1 561 688 8700
Middle East +43 1 333 50 34 0
Netherlands +31 76 579 55 55
New Zealand +64 9 980 6700
Norway +46 8 556 468 00
Russia/CIS +43 1 333 50 34 0
Singapore +65 6289 1190
Sweden +46 8 556 468 00
Switzerland +41 61 716 77 00
Taiwan +886 2 8751 6655
UK/Ireland +44 1442 233555
USA +1 800 532 4752
PN71311-EN 0716S