Application Note

Food/Beverage Testing,
Fermentation Monitoring,
Agrochemicals, Biofuels,
Alternative Energy

Analysis of sugars using an

Agilent InfinityLab Poroshell 120
HILIC-Z column

Authors Abstract
Anne Mack and Ta-Chen Wei
An Agilent InfinityLab Poroshell 120 HILIC-Z column was used to separate 11 sugar
Agilent Technologies, Inc.
compounds by both gradient and isocratic elution. The effect of pH and temperature
on glucose anomer separation was explored. It was discovered that a combination
of high pH and low temperature offered the best solution for both peak shape and
column lifetime. The final separation used an ammonium hydroxide mobile phase at
35 °C.
Introduction The InfinityLab Poroshell 120 HILIC-Z Agilent OpenLAB software was used to
phase uses a novel zwitterionic control the system and process the data.
Superficially porous particle LC stationary phase bonded to a robust Table 1 shows the chromatographic
columns are a popular tool in liquid hybrid particle. It is stable up to 80 °C methods that were used. All compounds
chromatography. These columns and up to pH 12. This phase is capable were injected as individual standards.
generate high efficiency at lower of separating analytes over a wide range Table 2 lists the concentrations and
pressure compared to their totally porous of polarity. These HILIC-Z phase features sample solvents.
particle column counterparts. This is make it well suited for challenging The 11 sugar compounds analyzed were
primarily due to a shorter mass transfer high pH separations, offering a robust bought from Sigma-Aldrich. Ammonium
distance and substantially narrower solution. formate, formic acid, ammonium
particle size distribution of the particles
acetate, and ammonium hydroxide were
in the column. The higher efficiency can Experimental also from Sigma-Aldrich. Acetonitrile
be used to speed up analyses or improve
An Agilent 1260 Infinity binary LC with was bought from Honeywell (Burdick
results by increasing resolution and
an Agilent G4218A ELSD was used for and Jackson). Water was 0.2 µm filtered,
sensitivity.
this work. All connecting capillaries 18 molecular weight, from a Milli-Q
To date, superficially porous system (Millipore).
were short, with a 0.12 mm internal
particles have primarily focused on
diameter to minimize system dispersion.
reversed‑phase separations. With the
maturation of superficially porous
particle technology, applications for
Table 2. Sample preparation and injection volumes.
further chemistries and chromatographic
techniques, such as hydrophilic Sugar Prepared as a saturated solution in: Injection volume (µL)
interaction liquid chromatography Xylose CH3CN/H2O (9:1) 0.1
(HILIC), are becoming available. HILIC Arabinose CH3CN/H2O (9:1) 0.1
is well suited for the analysis of polar Fructose CH3CN/H2O (9:1) 0.1
analytes, which are often difficult to Mannose CH3CN/H2O (9:1) 0.2
retain and separate in reversed‑phase Glucose CH3CN/H2O (9:1) 0.4
mode. This Application Note Galactose CH3CN/H2O (9:1) 0.4
demonstrates the UHPLC performance Sucrose CH3CN/H2O (9:1) 0.5
of an Agilent InfinityLab Poroshell 120 Maltose CH3CN/H2O (9:1) 1.0
HILIC-Z, 2.7 µm column, and its ability Lactose CH3CN/H2O (9:1) 1.5
to resolve 11 sugar compounds by both Maltotriose CH3CN/H2O (9:1) 3.0
gradient and isocratic elution. Raffinose CH3CN/H2O (9:1) 7.0

Table 1. Method parameters.

Mobile phase Flow rate Column ELSD

Method Mobile phase A Mobile phase B composition (mL/min) Column temperature (°C) Settings
Water
100 mM Ammonium formate
in water pH 4.5 Competitive HILIC
95–80 %B in 12 minutes,
Figure 1 100 mM Ammonium acetate Acetonitrile 0.4 column, 35, 40, or 80 60 °C, 3.5 bar, 30 Hz
3 minutes re‑equilibration
in water pH 7.0 2.1 × 100 mm, 2.7 µm

0.6 % Ammonium hydroxide

in water
0.3 % Ammonium hydroxide
35
in water
Figure 2 Acetonitrile 90 % B Isocratic 0.4 60 °C, 3.5 bar, 30 Hz
100 mM Ammonium acetate Agilent InfinityLab
80
in water pH 7.0 Poroshell 120 HILIC-Z,
0.3 % Ammonium hydroxide 0.3 % Ammonium hydroxide 85–60 %B in 6 minutes, 2.1 × 100 mm, 2.7 µm
Figure 3 0.4 35 60 °C, 3.5 bar, 30 Hz
in water in acetonitrile 3 minutes re‑equilibration (p/n 685775-924)

0.3 % Ammonium hydroxide 0.3 % Ammonium hydroxide

Figure 4 80 %B Isocratic 0.4 35 60 °C, 3.5 bar, 30 Hz
in water in acetonitrile

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Results and discussion
Sugars can be difficult to analyze by
HPLC, as many compounds experience
anomer separation. Figure 1 shows
that glucose anomer separation can
be controlled by either using high pH,
high temperature, or some combination
thereof. For silica-based LC columns,
these conditions are harsh, and can
negatively impact column lifetime.

H2O, 35 °C

pH 4.5, 80 °C
pH 4.5, 35 °C
PW = 0.1439
pH

pH 7.0, 40 °C
pH 7.0, 80 °C
PW = 0.2669
PW = 0.1267

Mobile phase A) Varies,

B) CH3CN
Gradient 95–80 %B in 12 minutes
Flow rate 0.4 mL/min
pH ~10.8, 35 °C Temperature Varies
PW = 0.2185 ELSD 60 °C, 3.5 psi
Column 2.1 × 100 mm competitor’s HILIC column

Temperature

Figure 1. Effect of pH and temperature on anomer separation of glucose.

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The Agilent InfinityLab Poroshell 120 Figure 3 shows that 11 sugars were Figure 4 shows that an isocratic
HILIC-Z particles are made with a resolved on an InfinityLab Poroshell 120 separation of sugars is also possible.
proprietary zwitterionic bonding on HILIC-Z column, using gradient elution Figure 3 shows that the isocratic
hybrid particles, which makes them with a high-pH mobile phase. Peak shape separation was accomplished in
more stable in high-pH mobile phases. was excellent for all compounds, and approximately the same amount of time
Figure 2 evaluates the Agilent InfinityLab anomer separation was well controlled as the gradient analysis. However, it
Poroshell 120 HILIC-Z column lifetime using ammonium hydroxide (pH ~10.8) does not offer as much resolution for the
under method conditions suitable for and a 35 °C column temperature. Two early-eluting compounds, and sensitivity
sugars. The combination of high pH critical pairs, xylose/arabinose and is not as great for the later-eluting
and low temperature causes no loss glucose/galactose, were not baseline compounds. Despite these small issues,
of performance over 14,000 column resolved. It is likely that these critical the isocratic solution could work well for
volumes. Elevated temperature pair separations could be improved with sugar analyses where gradient elution is
combined with mid-pH offers narrower a longer column and a longer analysis not possible. Note that RI detector flow
peak widths and lower backpressures, time. cells are not compatible with high‑pH
but these conditions also accelerate mobile phases. If RI detection is desired,
column degradation. For the InfinityLab use the combination of mid-pH and
Poroshell 120 HILIC-Z column, the high high temperature. All other method
pH and low temperature combination parameters should remain constant, and
offers a more robust method for sugar similar selectivity is expected.
analysis.

A High pH (~10.8), low temperature (35 °C) B Mid pH (7.0), high temperature (80 °C)
120 Fructose 120 Fructose
Glucose Glucose
115 Sucrose 115 Sucrose
Maltose Maltose
110 110

105 105
Retention time (%)

Retention time (%)

100 100

95 95

90 90

85 85

80 80
0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000
Column volume Column volume

Mobile Phase A) 0.3 % Ammonium hydroxide Mobile Phase A) 100 mM Ammonium acetate pH 7.0
B) CH3CN B) CH3CN
Gradient 90 %B Isocratic 90 %B
Flow rate 0.4 mL/min Flow rate 0.4 mL/min
Temperature 35 °C Temperature 80 °C
ELSD 60 °C, 3.5 psi ELSD 60 °C, 3.5 psi
Column Agilent InfinityLab Poroshell 120 HILIC-Z, Column Agilent InfinityLab Poroshell 120 HILIC-Z,
2.1 × 100 mm, 2.7 µm 2.1 × 100 mm, 2.7 µm

Figure 2. Lifetime comparison of high pH versus high temperature sugar analysis on a 2.7 µm Agilent InfinityLab Poroshell 120 HILIC-Z LC column.

4
mV 4

180 Mobile Phase A) 0.3 % Ammonium hydroxide in H2O 1. Xylose

B) 0.3 % Ammonium hydroxide in CH3CN 2. Arabinose
Gradient 85–60 %B in 6 minutes
160 Flow rate 0.4 mL/min 3. Fructose
Temperature 35 °C 4. Mannose
ELSD 60 °C, 3.5 psi
140 Column Agilent InfinityLab Poroshell 120 HILIC-Z, 5. Glucose
5
2.1 × 100 mm, 2.7 µm
6. Galactose
120
1 7. Sucrose
7 11
8. Maltose
100
9. Lactose
6
2
8 9 10. Maltotriose
80 10
3 11. Raffinose

60

40

20

0 1 2 3 4 5 min

Figure 3. Gradient separation of 11 sugar compounds on an Agilent InfinityLab Poroshell 120 HILIC-Z LC column.

mV 4
Mobile Phase A) 0.3 % Ammonium hydroxide in H2O 1. Xylose
225 B) 0.3 % Ammonium hydroxide in CH3CN
1 2. Arabinose
Isocratic 80 %B
Flow rate 0.4 mL/min 3. Fructose
200
Temperature 35 °C 4. Mannose
ELSD 60 °C, 3.5 psi
175 Column Agilent InfinityLab Poroshell 120 HILIC-Z, 5. Glucose
5
2.1 × 100 mm, 2.7 µm
6. Galactose
150 7. Sucrose
8. Maltose
125 6
9. Lactose
2 3 10. Maltotriose
100
11. Raffinose
75 7

50
8
9
11
25 10

0 1 2 3 4 5 6 7 min

Figure 4. Isocratic separation of 11 sugar compounds on an Agilent InfinityLab Poroshell 120 HILIC-Z LC column.

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Conclusions
The Agilent InfinityLab Poroshell 120
HILIC-Z column is well suited for the
separation of sugars. This column offers
good resolution and peak shape for all
compounds, as well as excellent lifetime
under high-pH conditions.

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This information is subject to change without notice.

© Agilent Technologies, Inc. 2018, 2019

Printed in the USA, January 16, 2019
5991-8984EN