Dextran Calibration Ladder Standard

[ CARE AND USE MANUAL ]

CONTENTS I. INTRODUCTION

I. INTRODUCTION Hydrophilic Interaction Liquid Chromatography (HILIC) coupled with Fluorescent Detection is widely used for fluorescent (FLR) II. STORAGE & STABILITY labeled glycans. Waters ACQUITY® BEH Glycan column operated III. RECONSTITUTION OF DEXTRAN CALIBRATION LADDER in HILIC mode shows drastic improvement in separation e.g., peak resolution and the ability to separate both neutral and acidic glycans, IV. BENEFITS OF USING A CALIBRATION STANDARD compared to the conventional HPLC methods in HILIC mode (Ref. 1). The ACQUITY BEH Glycan column enables scientists to get robust V. EXAMPLES OF USING THE DEXTRAN CALIBRATION and reproducible glycan separation data with less time spent in LADDER ON THE ACQUITY UPLC method optimization. VI. ORDERING INFORMATION 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.

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.

Dextran Calibration Ladder Standard 1 [ CARE AND USE MANUAL ]

For reconstitution, the sample can be diluted with 100 µl of Milli-Q IV. ORDERING INFORMATION water plus 100 µl of Acetonitrile for a 200 µl total dilution (1µg/µl). Description Part Number IV. BENEFITS OF USING A CALIBRATION STANDARD Dextran Calibration Ladder 186006841

Each individual glycan structure has a GU value which is directly ACQUITY UPLC BEH Glycan 1.7µm 2.1 x 50mm 186004740 related to the number of linkage of its constituent monosaccharides. ACQUITY UPLC BEH Glycan 186004741 The GU value can be used to predict structures because each 1.7µm 2.1 x 100mm monosaccharide in a specific linkage adds a given amount to the ACQUITY UPLC BEH Glycan GU value of a given glycan. 186004742 1.7µm 2.1 x 150mm The elution times of glycans are expressed in glucose units (GU) by ACQUITY UPLC BEH Glycan, Vanguard 3-pack 186004739 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 References curve to the dextran ladder, then using this curve to allocate GU 1. Joomi Ahn, Jonathan Bones, Ying Qing Yu, Pauline M. Rudd, Martin Gilar. values from retention times. The GU values for neutral N-glycans are “Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 µm sorbent”, Journal of Chromatography very reproducible with standard deviations of <0.3 between columns, B, 878 (2010) 403–408. this allows direct comparison with database values collected from a 2. Matthew P. Campbell, Louise Royle, Catherine M. Radcliffe, Raymond A. Dwek and range of instruments over a period of time (Ref. 3). 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. V. EXAMPLES OF USING THE DEXTRAN CALIBRATION LADDER Dwek. “A Rapid High-Resolution High-Performance Liquid Chromatographic Method for Separating Glycan Mixtures and Analyzing Oligosaccharide Profiles” ON THE ACQUITY UPLC Analytical Biochemistry, 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 34.00 GU = Glucose Unit Injection Mode: Partial loop needle overfill 32.00

Column: ACQUITY UPLC BEH Glycan, 2.1 x 150mm 30.00 Eluent A: 100mM Ammonium Formate Buffer pH 4.5 28.00 GU-10 GU-15 Eluent B: Acetonitrile 26.00 Weak Needle Wash: Acetronitrile/HPLC Grade Water, (90:10 v/v) 24.00 Strong Needle Wash: Acetronitrile/HPLC Grade Water, (10:90 v/v) 22.00 GU-20 Seal Wash: Acetronitrile/Water (50:50 v/v) 20.00 GU-2 Temperature: 60°C 18.00 GU-5 Detection: Flourescence: λex= 330 nm, λem= 420 nm EU 16.00 Gradient: Time Flow Rate 14.00 (min) (mL/min) %A %B Curve 12.00 initial 0.50 25 75 6 10.00 46.5 0.50 50 50 6 8.00 48 0.25 100 0 6 6.00 49 0.25 100 0 6 4.00 50 0.50 25 75 6 2.00 63 0.50 25 75 6 0.00

0 5 10 15 20 25 30 35 40 45 min

Dextran Calibration Ladder Standard 2 [ CARE AND USE MANUAL ]

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©2012. Waters, ACQUITY and UPLC are registered marks Milford, MA 01757 U.S.A. of Waters Corporation. The Science of What's Possible is a T: 1 508 478 2000 trademark of Waters Corporation. All other trademarks are F: 1 508 872 1990 the property of their respective owners. www.waters.com September 2012 720004463EN Rev A IH-PDF