Waters XBridge Protein BEH SEC Columns and Standards User Manual

[ CARE AND USE MANUAL ]

XBridge Protein BEH SEC Columns and Standards

CONTENTS

I. INTRODUCTION

II. SYSTEM CONSIDERATIONS FOR
SEC SEPARATIONS

a. Getting Started
b. Column Installation
c. Column Equilibration
d. Useful Functional Tests for Benchmarking LC System
and XBridge Protein BEH SEC Column

III. COLUMN SPECIFICATIONS AND USE

a. SEC Eluent and Needle Wash Preparation
b. Sample Preparation
c. Column Specification

IV. TROUBLESHOOTING

V. COLUMN CLEANING, REGENERATION
AND STORAGE

a. Cleaning and Regeneration
b. Storage

I. INTRODUCTION

Waters family of XBridge® Protein BEH SEC 200Å and 450Å,

3.5 µm Columns was developed to complement the existing line of
UPLC®-based SEC offerings for use where traditional HPLC-based
instrumentation and methods are employed for peptide or protein
size-exclusion chromatography (SEC). These new HPLC-based, SEC
chemistries are based on the same Waters Ethylene Bridged Hybrid
(BEH)-based particle technology and diol-bonded surface coating
as used in our successful line of UPLC-based SEC columns. This
process offers chromatographers the option and ability to easily
transfer methods based on lab instrumentation and component
resolution or sample throughput needs.

All of Waters BEH-based SEC columns are manufactured in a cGMP,
ISO 9001 certified plant using stringent manufacturing protocols
and ultra pure reagents. Each batch of manufactured material
undergoes a series of standard QC measurements (e.g., particle
and pore size distribution) followed by an application specific
test using appropriate peptide and protein test mixture. A packed
column efficiency test is then performed on every batch approved,
packed SEC column to further help ensure reproducible batch to
batch and column to column performance for use in research or in a
demanding validated method.

[ CARE AND USE MANUAL ]

Uracil (112 Da)

Aprotinin (6.5 KDa)

RNAse A (14 KDa)

Myoglobin (17 KDa)

Ovalbumin (44 KDa)

Conalbumin (75 KDa)

Amyloglucosidase (97 KDa)

IgG (150 KDa)

Thyroglobulin (669 KDa)

IgM (900 KDa)

10

100

1,000

10,000

100,000

1,000,000

10,000,000

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Protein Molecular Weight (KDa)

Normalized Retention Volume (Vr/VC)

XBridge Protein BEH SEC 450Å, 3.5 µm, 100K – 1,500K Daltons

XBridge Protein BEH SEC 200Å, 3.5 µm, 10K – 450K Daltons

Figure 1: Calibration Curves on XBridge Protein BEH SEC 200Å and 450Å Columns

II. SYSTEM CONSIDERATIONS FOR
SEC SEPARATION

a. Getting Started

In order to obtain the best performance from your Waters XBridge
Protein BEH SEC Column, it is important that your LC system be
properly configured. It is recommended that only pre-cut tubing is
used, and that the ID of all connecting tubing is 0.005” or less for
optimal chromatographic performance.

Size-exclusion chromatography may require modifications to
an existing LC system. Please refer to “Size-Exclusion and Ion­Exchange Chromatography of Proteins using the ACQUITY UPLC
System” (P/N 715002147A that can be obtained at

www.waters.com/chemcu) for examples of LC System components

that can affect SEC results.

The sample loop used may affect the performance of your
separation. Optimally, select the smallest volume sample loop that
is required for the application. Sample loops larger than 20 µL are
not recommended.

b. Column Installation

1. Prior to placing the column on the system, purge the solvent
delivery system of any organic or water-immiscible mobile
phases. When connecting the column, orient it in the proper
direction as noted by the arrow on the column inlet side which
indicates the correct direction of solvent flow.

2. Flush column with 100% aqueous buffer, by pumping at a flow
rate of 0.2 mL/min.

3. Ensure that the mobile phase is flowing freely from the column
outlet. Attach the column outlet to the detector using .004”
ID tubing (P/N 430001562). Monitor the system pressure to
ensure the column is within its pressure limitations.

4. Gradually increase the flow rate, by not more than 0.1 mL/min
at a time, as described in Step 2.

5. Once the system pressure has stabilized, ensure that there are
no leaks at either the column inlet or outlet.

c. Column Equilibration

XBridge Protein BEH SEC Columns are shipped in 20% methanol
in water. It is important to ensure mobile-phase compatibility
before changing to a different mobile-phase system. Equilibrate
the column with a minimum of 10 column volumes of the buffer to
be used (refer to Table 1 for column volumes).

Column Dimension Approximate Volume

7.8 x 150 mm 7 mL

7.8 x 300 mm 14 m L

Table 1. Empty Column Volumes in mL (multiply by 10 for flush solvent volume)

XBridge Protein BEH SEC Columns and Standards

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