Waters ACQUITY UPC2 BEH, HSS, and CSH Columns User Manual
[ CARE AND USE MANUAL ]
ACQUITY UPC2 BEH, CSH, and HSS Columns
CONTENTS
I. INTRODUCTION
II. GETTING STARTED
a. Safety Considerations
b. Column Connectors and Installation
c. eCord Installation
d. Column Equilibration
e. Initial Column Efficiency Determination
f. VanGuard Pre-Columns
III. COLUMN USE
a. Sample Preparation
b. Solvents
c. Pressure
d. Useable Flow-Rate Ranges and Expected ACQUITY UPC2
System Pressures
e. Band Spreading Minimization
IV. TROUBLESHOOTING
V. COLUMN CLEANING, AGING, REGENERATION,
AND STORAGE
a. Cleaning
b. Aging
c. Regeneration
d. Storage
I. INTRODUCTION
2
Thank you for choosing an ACQUITY UPC
UPC2 Columns feature Ethylene-Bridged Hybrid (BEH), Charged-
Surface Hybrid (CSH™) and High-Strength Silica (HSS) particle
technologies to provide a wide range of selectivities, excellent peak
shape, and high efficiency. The ACQUITY UPC2 packing materials
are designed for use with the ACQUITY UPC2 Systems and are
manufactured in an ISO 9000 certified plant using ultra-pure
reagents. Each batch of ACQUITY UPC2 material is tested and 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 is
provided on the eCord™ Intelligent Chip.
ACQUITY UPC2 Columns are designed and tested specifically for
use on ACQUITY UPC2 Systems. ACQUITY UPC2 Columns will
exhibit maximum chromatographic performance and benefit
ONLY when used on holistically-designed ACQUITY UPC2 Systems
since these systems and columns were created and designed to
operate together. For these reasons, Waters cannot support the
use of ACQUITY UPC2 Columns on any system other than an
ACQUITY UPC2 System.
®
Column. The ACQUITY
VI. INT RODUCING ECORD INTELLIGENT
CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing Information
d. Column Use Information
VII. ACQUITY UPC2 COLUMN FAMILY
[ CARE AND USE MANUAL ]
Chemistry
BE H 2-Ethylpyridine (2-EP) Spherical 1.7, 3. 5 130 185 0.7 9 No BEH
BEH (Unbonded) Spherical 1.7, 3. 5 130 185 0.7 N/A N/A BEH
CSH Fluoro-Phenyl Spherical 1.7, 3.5 130 185 0.7 10 No CSH
HSS C18 SB Spherical 1.8, 3.5 100 230 0.7 8 No HSS
Table 1. Physical Characteristics.
Particle
Shape
II. GETTING STARTED
Each ACQUITY UPC2 Column comes with a Certificate of Analysis
and a Performance Test Chromatogram. The Certificate of Analysis
is specific to each batch of packing material and includes the gel
batch number, physical characterization, analysis of unbonded
particle, analysis of bonded particles, and a SFC chromatographic
batch test. The Performance Test Chromatogram is specific to
each individual column and contains the following information:
gel batch number, column serial number, USP plate count, USP
tailing factor, capacity factor, and chromatographic conditions
under normal-phase LC conditions. These data should be stored for
future reference.
a. Safety Considerations
An SFC column, while in use, is under pressure with CO2 and
possible modifiers as a supercritical fluid. A major safety concern
is frostbite caused by adiabatic cooling when CO2 decompresses
from a fluid to a gas at atmospheric pressure. Pay attention to any
frosting on the column or system connections. This indicates a
leak, usually with temperatures far below 0 °C.
Any small leak could produce a situation where the LEL (lower
exposure limit) is reached. Laboratories should be equipped with
CO2 and/or O2 sensors when carbon dioxide (CO2) is in use.
b. Column Connectors and Installation
ACQUITY UPC2 Systems utilize tubing and connectors which have
been designed to meet stringent tolerance levels and to minimize
extra column volumes. For information on system tubing and
connectors, please refer to the ACQUITY UPC2 System Operator’s
Guide (Part Number 720004226EN).
Note: Scale the flow rate up or down accordingly based upon
the column i.d., length, particle size, and backpressure of the
ACQUITY UPC2 Column being installed.
Particle
Size (µm)
Pore
Size (Å)
Surface Area
(m2/g)
1. Make sure your co-solvent pump is primed and has an adequate
2. Connect both the inlet and outlet of the column to the
3. If the column is still filled with a solvent, use a low flow
4. If you see frosting on the column at the inlet or outlet, tighten
Pore Volume
(cc/g)
solvent/modifier supply before performing injections. It is
recommended to use CO2 with purity level of 99.97%
(food grade) and use high-quality chromatography grade
solvents (typically methanol) as organic modifier.
SFC system.
rate and back pressure setting (100 bar) to start pumping
CO2 and modifier through the column.
the finger-tight fitting or compression screw on that side. If you
continue to see frosting, turn off the CO2 and vent the system.
Allow the column to depressurize fully before disconnecting
the inlet or outlet to troubleshoot the leaking issue.
Carbon
Load (%)
Endcapped
Technology
c. 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.
d. Column Equilibration
ACQUITY UPC2 Columns are shipped dry. Equilibrate the column
with a minimum of 10-column volumes of the mobile phase prior
to use. (Refer to Table 2 for a listing of empty column volumes.)
e. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it.
Waters recommends using a suitable solute mixture to analyze
the column upon receipt. We recommend that weaker solvents
are selected as injection solvents. Peak distortion can occur
due to strong solvent effects, resulting in lower efficiency
values. (Methanol can be considered a strong solvent under
SFC conditions.)
Particle
ACQUITY UPC2 Torus Columns Care & Use
2
[ CARE AND USE MANUAL ]
ACQUITY UP
2. Determine the number of theoretical plates (N) and use this
value as a benchmark for periodic comparisons.
3. Repeat the test at predetermined intervals to track
column performance over time. (Slight variations may
occur if performance tests are performed on two different
ACQUITY UPC2 Systems due to the quality of the connections,
operating environment, system electronics, reagent quality
and column condition.)
Empty Column
Volumes (m L)
Column Length 2.1 mm 3.0 mm
50 mm 0.2 0.4
75 mm 0.3 0.6
100 mm 0.4 0.8
150 mm 0.5 1.0
Table 2. Empty Column Volumes in mL (multiply by 10 for f lush
solvent volumes).
Column Internal Diameter (mm)
f. VanGuard Pre-Columns
VanGuard™ Pre-columns are 2.1 mm i.d. x 5 mm length guard
column devices designed specifically for use on the ACQUIT Y
UPC2 Systems. VanGuard Pre-columns are packed with the same
chemistries and frits as our 2.1 mm i.d. ACQUITY UPC2 Columns.
VanGuard Pre-columns are designed to be attached directly to the
inlet side of an ACQUITY UPC2 Column.
Note: In order to ensure void-free and leak-free connections, the
VanGuard Pre-column is shipped with the collet and ferrule NOT
permanently attached. Care must be taken when removing the
O-ring that holds these two pieces on the pre-column tubing.
Installation Instructions
1. Remove VanGuard Pre-column from box and shipping tube and
remove plastic plug.
2. Orient pre-column so that male end is facing up and
carefully remove rubber O-ring that holds collet and ferrule
in place during shipping (collet and ferrule are not yet
permanently attached).
3. Orient ACQUITY UPC2 Column perpendicular to work surface
so that column inlet is on the bottom (facing down), with
column outlet on top (facing up).
4. From below, insert VanGuard Pre-column into ACQUITY UPC2
Column inlet and hand-tighten (collet and ferrule are not yet
permanently attached).
5. While pushing the VanGuard Pre-column into the column inlet,
turn assembled column and pre-column 180˚ so that pre-
column is now on top.
6. Tighten with two 5/16” wrenches placed onto ACQUITY UPC2
Column flats and VanGuard Pre-column hex nut (male end) as
shown above.
7. Tighten 1/4 turn to set collet and ferrule.
8. Check that ferrule is set by loosening connection and
inspecting the ferrule depth. A properly set ferrule depth will
resemble other connections in the ACQUITY UPC2 System.
9. Reattach pre-column, connect VanGuard Pre-column and
analytical column combination to the ACQUITY UPC2 System,
apply mobile-phase flow and inspect for leaks.
Figure 1. Connecting a VanGuard Pre-column to an ACQUITY UPC2 Column.
C2 Column
Place wrench here
Ferrule
Flow
VanGuard Pre-Column
Collet
Place wrench here
ACQUITY UPC2 Torus Columns Care & Use
3
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