Waters Protein Separation Technology ACQUITY UPLC BEH300, C4, 1.7 µm Columns - Manual (Page 2)

[ Care and Use ManUal ]
Protein Separation Technology ACQUITY UPLC BEH300, C
4
, 1.7 �m
2
The ACQUITY UPLC BEH300, C
4
, 1.7 µm column is based on the
1.7 µm particles that are characteristic of UltraPerformance LC ® . The
small particle size packing reduces dispersion and band broadening
so that improved resolution, sensitivity, and speed are obtained in
protein separations. It is commonly expected that very high system
backpressures will be observed with such small particles. Proteins
are, however, large molecules that diffuse slowly so that the optimum
linear velocity is much lower than for small molecules. Since the flow
rate is low, system pressure is low. Although the column does not
require the high pressure capability of an ACQUITY UPLC system, the
full performance benefits of this column will only be realized with
the low system volume and low detector dispersion of the optimized
ACQUITY UPLC system.
Note: ACQUITY UPLC BEH300 C
4
, 1.7µm columns are optimally
designed for use with the ACQUITY UPLC system. Performance is
sacrificed when these columns are used on conventional
HPLC systems.
II. ConFIGurInG An ACQuItY sYsteM For use In ProteIn
sePArAtIons
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 (P/N 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 (P/N 71500082502).
b. High Sensitivity Peptide Mixer
The Waters ACQUITY UPLC Binary Solvent Manager 425 μL Mixer
(P/N 205000403) should be installed to provide the most stable and
usable baseline with UV detection. It has been has been specifically
designed for long shallow gradients that require more mixing.
Note: The mixer has a pressure rating of 690 bar (10,000 psi).
c. Peptide Needle Kit
The Waters ACQUITY UPLC Peptide Needle Kit (P/N 205000507)
should be installed. This optional part is available for all ACQUITY
UPLC Sample Managers. This kit has proven to offer improved
reproducibility and recovery for some peptide analysis applications.
The kit includes an alternative needle fabricated from unmodified
PEEKsil™. There are also injection protocols for best general perfor-
mance. This kit has been routinely used in Waters laboratories for the
development and testing of the ACQUITY UPLC BEH300, C
4
, 1.7 µm
column.
d. Flow Restrictor
In many protein separation applications, the optimal conditions of
low flow rate, elevated temperature, and short columns yield very
low total system pressures. In Waters laboratories, a flow restrictor is
fabricated from capillary tubing and placed in the flow path between
the mixer and injector. The device is adjusted to a minimum of 2000
psi total backpressure.
III. GettInG stArted
Each Protein Separation Technology ACQUITY UPLC BEH300, C
4
,
1.7 µm column comes with a Certificate of Acceptance and a Perfor-
mance Test Chromatogram. The Certificate of Acceptance is specific to
each batch of packing material and includes the batch number, analysis
of unbonded particles, analysis of bonded particles, and chromato-
graphic results and conditions. The Performance Test Chromatogram is
specific to each individual column and contains the information: batch
number, column serial number, USP plate count, USP tailing factor,
retention factor, and chromatographic conditions. These data 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.