Care and use manual – Waters ACQUITY UPLC CSH130 C18 1.7 μm and XP 2.5 μm Columns User Manual

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[ CARE AND USE MANUAL ]

ACQUITY UPLC CSH130 C

Columns

I. GETTING STARTED

Each Peptide Separation Technology ACQUITY UPLC CSH130 C

column

comes with Certificate of Analysis 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
CSH130 C

column and includes the gel batch number, 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 chromato-
graphic 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.

Note: Waters recommends using finger tight fittings when connecting Waters
CSH130 C

, 2.5 µm XP column to an HPLC. To ensure a proper connection to

your 2.5 µm XP column, Waters recommends p/n

to connect to the outlet of your column. This

will ensure correct seating of the HPLC System’s connection tubing ferrules to
your 2.5 µm XP column and minimize undesired band broadening.

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, with HPLC-grade water, of any

buffer-containing mobile phases and connect the inlet end of the
column to the injector outlet.

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.

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 CSH130 C

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)

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.)

Column Length (mm)

Internal Diameter 2.1 mm

0.2 mL

100

0.4 mL

150

0.5 mL