Waters Dextran Calibration Ladder Standard User Manual

[ CARE AND USE MANUAL ]

Dextran Calibration Ladder Standard

CONTENTS

I. INTRODUCTION

II. STORAGE & STABILITY

III. RECONSTITUTION OF DEXTRAN CALIBRATION LADDER

IV. BENEFITS OF USING A CALIBRATION STANDARD

V. EXAMPLES OF USING THE DEXTRAN CALIBRATION

LADDER ON THE ACQUITY UPLC

VI. ORDERING INFORMATION

I. INTRODUCTION

Hydrophilic Interaction Liquid Chromatography (HILIC) coupled
with Fluorescent Detection is widely used for fluorescent (FLR)
labeled glycans. Waters ACQUIT Y® BEH Glycan column operated
in HILIC mode shows drastic improvement in separation e.g., peak
resolution and the ability to separate both neutral and acidic glycans,
compared to the conventional HPLC methods in HILIC mode (Ref. 1).
The ACQUITY BEH Glycan column enables scientists to get robust
and reproducible glycan separation data with less time spent in
method optimization.

In order to take full advantage of the BEH glycan column, Waters
created the 2-aminobenzamide labeled Dextran Calibration Ladder
Standard to assist glycan profiling: glycan profile obtained from
UPLC-HILIC/FLR system can be calibrated against the 2AB-labeled
Dextran ladder and assigned with glucose unit (GU) values (Ref.2).
Waters 2AB-labeled Dextran Calibration Ladder is different than
the existing commercial offerings. The average molecular weight of
the glucose homopolymer is higher (~4,500 Dalton), therefore,
the “workable” GU value range is twice as much as the other
Dextran ladder standards; the observed GU goes from 2 to 30.
This feature improves the large glycan retention time assignment.
The purity and structural integrity of the ladder is assessed by
normal phase HPLC and MS.

Dextran Calibration Ladder Standard

II. STORAGE AND STABILITY

The lyophilized powder is shipped at ambient temperatures, but it
is highly recommended upon receipt of the standard to store it
refrigerated (4°C) until the expiration date printed on the label. Once
reconstituted in should be used immediately for best results.

III. RECONSTITUTION OF THE DEXTRAN CALIBRATION LADDER

One vial of the Dextran Calibration Standard contains 200 µg of a
lyophilized, solid powder. It is contained in a Waters Max Recovery Vial
for supreme ease of use so that it can be directly solubilized and put on
the system for analysis and injection.

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EU

For reconstitution, the sample can be diluted with 100 µl of Milli-Q
water plus 100 µl of Acetonitrile for a 200 µl total dilution (1µg/µl).

IV. BENEFITS OF USING A CALIBRATION STANDARD

Each individual glycan structure has a GU value which is directly
related to the number of linkage of its constituent monosaccharides.
The GU value can be used to predict structures because each
monosaccharide in a specific linkage adds a given amount to the
GU value of a given glycan.

The elution times of glycans are expressed in glucose units (GU) by
reference to a dextran ladder. This ladder is used to calibrate the
LC runs against day-to-day or system-to-system changes. The GU
value is calculated by fitting a fifth order polynomial distribution
curve to the dextran ladder, then using this curve to allocate GU
values from retention times. The GU values for neutral N-glycans are
very reproducible with standard deviations of <0.3 between columns,
this allows direct comparison with database values collected from a
range of instruments over a period of time (Ref. 3).

V. EXAMPLES OF USING THE DEXTRAN CALIBRATION LADDER

ON THE ACQUITY UPLC

IV. ORDERING INFORMATION

Description Part Number

Dextran Calibration Ladder

186006841

ACQUITY UPLC BEH Glycan 1.7µm 2.1 x 50mm 186004740

ACQUITY UPLC BEH Glycan

1.7µm 2.1 x 100mm

ACQUITY UPLC BEH Glycan

1.7µm 2.1 x 150mm

186004741

186004742

ACQUITY UPLC BEH Glycan, Vanguard 3-pack 186004739

References

1. Joomi Ahn, Jonathan Bones, Ying Qing Yu, Pauline M. Rudd, Martin Gilar.
“Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction
chromatography columns packed with 1.7 µm sorbent”, Journal of Chromatography
B, 878 (2010) 403–408.

2. Matthew P. Campbell, Louise Royle, C atherine M. Radcliffe, Raymond A. Dwek and
Pauline M. Rudd. “GlycoBase and autoGU:tools for H PLC-based glycan analysis”,
BIOINFORMATICS A PPLICATIONS NOTE, Vol. 24, no. 9 2008, 1214-1216.

3. Geoffrey R. Guile, Pauline M. Rudd, David R. Wing, Sally B. Prime, Raymond A.
Dwek. “A Rapid High-Resolution High-Performance Liquid Chromatographic
Method for Separating Glyc an Mixtures and Analyzing Oligosaccharide Profiles”
Analytic al Bioc hemistry, 240 (1996) 210-226.

Below is a reference chromatogram with conditions to help provide
an example of the Dextran Calibration Ladder. The elution time of
glycans is expressed in glucose units (GU) by reference to a dextran
ladder, refer to Figure 1. If the column size is different, the gradient/
injection volume can be scaled accordingly.

Figure 1: Example Chromatogram of the Dextran Calibration Ladder with glucose units (GU) labeled peaks scaled from 0.8 – 46.5 minutes.

System Conditions:
Injection Volume: 1.5 µl
Injection Mode: Partial loop needle overfill
Column: ACQUIT Y UPLC BEH Glyc an, 2.1 x 150mm
Eluent A: 100mM Ammonium Formate Buffer pH 4.5
Eluent B: Acetonitrile
Weak Needle Wash: Acetronitrile/HPLC Grade Water, (90:10 v/v)
Strong Needle Wash: Acetronitrile/H PLC Grade Water, (10:90 v/v)
Seal Wash: Acetronitrile/Water (50:50 v/v)
Temperature: 60°C
Detection: Flourescence: λex= 330 nm, λem= 420 nm
Gradient: Time Flow Rate
(min) (mL/min) %A %B Curve
initial 0.50 25 75 6

46.5 0.50 50 50 6
48 0.25 100 0 6
49 0.25 100 0 6
50 0.50 25 75 6
63 0.50 25 75 6

34.00

GU = Glucose Unit

32.00

30.00

28.00

26.00

24.00

22.00

20.00

GU-2

18.00

16.00

14.00

12.00

10.00

8.00

6.00

4.00

2.00

0.00

0 10 15 20

GU-5

5 25 30 35 40 45 min

GU-10

GU-15

GU-20

Dextran Calibration Ladder Standard 2