Waters ACQUITY UPLC BEH Glycan, 1.7 µm Columns, Glycan Performance Test Standard - Manual (Page 2)

[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH Glycan, 1.7 �m Columns 2
Note: ACQUITY UPLC BEH Glycan, 1.7 µm columns are optimally
designed for use with the ACQUITY UPLC, ACQUITY UPLC H-Class,
and ACQUITY UPLC H-Class Bio systems. The expected separations
will not be obtained on a conventional HPLC system because of
excessive dispersion and pressure limitations.
II. GETTING STARTED
Each ACQUITY UPLC BEH Glycan column has a Certificate of Analysis
and a Performance Test Chromatogram. The Certificate of Analysis is
specific to each batch of packing material and includes the batch number
and analyses of the physical and chemical properties of the particle.
Particle size and pore structure are analyzed prior to bonding. The
carbon and nitrogen content of the bonding are measured to insure
consistent coverage. The selectivity of each batch is also assessed with
the chromatographic separation of 2-AB labeled N-linked glycans from
human IgG. The complex mixture of IgG glycans includes high mannose
structures as well as neutral and acidic complex structures. The
retention times and retention time differences of selected components
are used as the quality control test for each batch of packing material.
The Performance Test Chromatogram is specific to each individual
column and contains the following information: batch number, column
serial number, backpressure, USP plate count, reduced plate
height (RPH), USP tailing factor, retention factor (k’), peak width,
and chromatographic conditions. These data can be found on the eCord
supplied with each column and should be stored for future reference.
a. 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.
b. Column Connectors
The ACQUITY UPLC, ACQUITY UPLC H-Class, and ACQUITY UPLC
H-Class Bio systems utilize tubing and gold plated compression
screws which have been designed to meet stringent tolerance levels
and to minimize system dispersion.
For the ACQUITY UPLC system, columns should be attached to the
injector with a column stabilizer, of which there are 4 types:
 205000291 50 or 100 mm column
 205000365 150 mm column
 205000489 HTCH 50 or 100 mm
 205000494 HTCH 150 mm
The first two parts are for the original heater and differ in the tubing
arrangement to allow 150 mm columns to be used with a VanGuard
™
pre-column or in-line filter while stabilizing the solvent temperature.
The second two parts are for the newer high temperature column heater
(HTCH). Optimized column inlet tubing 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 (or finger tighten the knurled nut).
If this column will be used on an ACQUITY UPLC H-Class or
ACQUITY UPLC H-Class Bio system, simply connect the column to
the active preheater supplied on the system using the gold fingertight
fittings. There is only one configuration of column stabilizer on the
ACQUITY UPLC H-Class and ACQUITY UPLC H-Class Bio systems.
For more information on fittings and connective tubing, please refer to
the relevant sections of the operator’s guides for the ACQUITY UPLC,
ACQUITY UPLC H-Class, and ACQUITY UPLC H-Class Bio systems.
c. Column Installation
Note: The flow rates given in the procedure below are for a typical
1.7 µm packing in a 2.1 mm i.d. column.
1. Purge the solvent delivery system of any buffer-containing
or water-immiscible mobile phases and connect the inlet end
of the column to the injector outlet. An arrow on the column
identification label indicates the correct direction of solvent flow.
2. Flush the column with 100% organic mobile phase (acetonitrile)
by setting the pump flow to 0.1 mL/min and increase the flow
to 0.25 mL/min over 3 minutes. Increase the aqueous phase
to 90 % over 10 minutes. Note the backpressure. Decrease
aqueous phase to starting conditions (22% aqueous in the test
chromatogram).
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.