Waters Dextran Calibration Ladder Standard - Manual (Page 2)

[ CARE AND USE MANUAL ]
Dextran Calibration Ladder Standard 2
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
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.
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
186004741
ACQUITY UPLC BEH Glycan
1.7µm 2.1 x 150mm
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, Catherine M. Radcliffe, Raymond A. Dwek and
Pauline M. Rudd. “GlycoBase and autoGU:tools for HPLC-based glycan analysis”,
BIOINFORMATICS APPLICATIONS 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 Glycan Mixtures and Analyzing Oligosaccharide Profiles”
Analytical Biochemistry, 240 (1996) 210-226.
System Conditions:
Injection Volume:
1.5 µl
Injection Mode: Partial loop needle overfill
Column: ACQUITY UPLC BEH Glycan, 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/HPLC 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
EU
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
5 25 30 35 40 45 min
GU-2
GU-5
GU-10 GU-15
GU-20
GU = Glucose Unit
0 10 15 20
Figure 1: Example Chromatogram of the Dextran Calibration Ladder with glucose units (GU) labeled peaks scaled from 0.8 – 46.5 minutes.