Waters BEH130, BEH300 User Manual

[ CARE AND USE MANUAL ]

ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns

CONTENTS

I. INTRODUCTION

II. GETTING STARTED

a. Column Connectors

b. Column Installation
c. Column Equilibration
d. eCord Installation
e. Initial Column Efficiency Determination

III. COLUMN USE

a. Sample Preparation

b. pH Range
c. Solvents
d. Pressure
e. Temperature

IV. COLUMN CLEANING, REGENERATING
AND STORAGE

a. Cleaning and Regeneration

b. Storage

I. INTRODUCTION

Thank you for choosing an ACQUITY UPLC Peptide BEH C18, 130Å or
300Å Column. The ACQUITY UPLC Peptide BEH C18, 130Å and 300Å
packing materials were designed specifically for use with the Waters
ACQUITY UPLC System and are manufactured in a cGMP, ISO 9002
certified plant using ultra pure reagents. Each batch of ACQUIT Y
UPLC BEH material is tested chromatographically with acidic, basic
and neutral analytes and has been qualitifed for peptide mapping.
The results are held to narrow specification ranges to assure
excellent, reproducible performance. Every column is individually
tested and a Performance Chromatogram and Certificate of Batch
Analysis are provided on the eCord™ intelligent chip. Each batch of
ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns is also QC
tested with the separation of a protein digest.

V. INTRODUCING e CORD INTELLIGENT
CHIP TECHNOLOGY

VI. REPRESENTATIVE TEST CHROMATOGRAPH
AND CONDITIONS FOR SEPARATION OF
PROTEIN DIGEST

[ CARE AND USE MANUAL ]

II. GETTING STARTED

Each Peptide Separation Technology ACQUITY UPLC Peptide BEH

, 130Å and 300Å Columns comes with Certificate of Analysis

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and a Performance Test Chromatogram embedded within the eCord
intelligent chip. The Certificate of Analysis is specific to each batch
of packing material contained in the ACQUITY UPLC Peptide BEH

, 130Å and 300Å Columns and includes the gel batch number,

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analysis of unbonded particles, analysis of bonded particles, and
chromatographic results and conditions. The Performance Test
Chromatogram is specific to each individual column and contains
such information as: gel batch number, column serial number, USP
plate count, USP tailing factor, capacity factor, and chromatographic
conditions. These data should be stored for future reference.

a. Column Connectors

The ACQUITY UPLC System utilizes tubing and gold plated
compression screws which have been designed to meet stringent
tolerance levels and to minimize extra column volumes.

Optimized column inlet tubing (Part Number 430001084) is
supplied with the ACQUITY UPLC System. The inject valve end of
the tubing is clearly marked with a blue shrink tube marker. Insert
the opposite end of the tubing into the ACQUITY UPLC Column and
tighten the compression fitting using two 5/16-inch wrenches.

For information on the correct column outlet tubing, please refer
to the relevant detector section in the ACQUITY UPLC System
Operator’s Guide (Part Number 71500082502).

b. Column Installation

1. Purge the pumping system of any buffer-containing mobile phases
and connect the inlet end of the column to the injector outlet.

Note: If mobile phase additives are present in low concentrations (e.g.,

ion-pairing reagents), 100 to 200 column volumes may be required

for complete equilibration. In addition, mobile phases that contain

formate (e.g., ammonium formate, formic acid, etc.) may also require

longer initial column equilibration times.

c. Column Equilibration

ACQUITY UPLC BEH130 and BEH300 Columns are shipped in 100%
acetonitrile. 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 mobile phase to
be used (refer to Table 1 for a list of column volumes)

Table 1. Empty Column Volumes in mL (Multiply by 10 for
Flush Solvent Volumes)

Column Length (mm) Internal Diameter 2.1 mm

50 0.2 mL
100 0.4 mL
150 0.5 mL

To avoid precipitating mobile phase buffers on your column or
in your system, flush the column with five column volumes of a
water/organic solvent mixture, using the same or lower solvent
content as in the desired buffered mobile phase. (For example,
flush the column and system with 60% methanol in water prior to
introducing 60% methanol/40% buffer mobile phase.)

d. eCord Installation

The eCord button should be attached to the side of the column
heater module. The eCord button is magnetized and does not
require specific orientation.

2. Flush column with 100% organic mobile phase (methanol or
acetonitrile) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 0.5 mL/min over 5 minutes.

3. When the mobile phase is flowing freely from the column
outlet, stop the flow and attach the column outlet to the
detector. This prevents entry of air into the detection system
and gives more rapid baseline equilibration.

4. Gradually increase the flow rate as described in step 2.

5. Once a steady backpressure and baseline have been achieved,
proceed to the next section.

e. Initial Column Efficiency Determination

1. Perform an efficiency test on the column before using it.
Waters recommends using a suitable solute mixture, as found
in the “Performance Test Chromatogram”, to analyze the
column upon receipt.

2. Determine the number of theoretical plates (N) and use this
value for periodic comparisons.

3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained
on two different UPLC systems due to the quality of the
connections, operating environment, system electronics,
reagent quality, column condition and operator technique.

ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns

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