Waters ACQUITY UPC2 Torus Columns User Manual
[ CARE AND USE MANUAL ]
ACQUITY UPC2 Torus 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. Additives
d. Pressure
e. Useable Flow-Rate Ranges and Expected ACQUITY UPC2
System Pressures
f. Band Spreading Minimization
IV. TROUBLESHOOTING
V. COLUMN CLEANING, ADDITIVES,
AND STORAGE
a. Cleaning
b. Effects of Additives
c. Aging
d. Storage
I. INTRODUCTION
2
Thank you for choosing an ACQUITY UPC
Torus Columns are specifically designed to use the complete
range of capabilities of the ACQUITY UPC2 System to achieve fast,
robust achiral separations. ACQUIT Y UPC2 Torus Columns simplify
the method development process with four completely new and
innovative 1.7 µm chemistries for UltraPerformance Convergence
Chromatography.™ These columns are designed for excellent
peak shape that eliminate or reduce need for additives, coupled
with added selectivity for a wide range of compounds and
improved robustness.
These columns are based on new bonding technology used to
create an adaptive selectivity platform. Starting with the ethylene
bridged hybrid (BEH) particle, the patent pending bonding consists
of a two stage functionalization of chromatographic particles.
The first stage controls retention characteristics and is applied
to each sorbent. The individual second stage selectors impart
the observed selectivity and control peak shape.
®
Torus™ Column.
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 ]
Achiral
Chemistry
Torus 2- PIC 2-Picolylamine Spherical 1.7 130 0.7 185 Proprietary
Torus DEA Diethylamine Spherical 1.7 130 0.7 185 Proprietary
Torus Diol High-density Diol Spherical 1.7 130 0.7 185 Proprietary
Torus 1-AA 1-Aminoanthracene Spherical 1.7 130 0.7 185 Proprietary
Table 1. Physical Characteristics.
The ACQUITY UPC2 packing materials are designed for use
with the ACQUITY UPC2 Systems and are manufactured in an
ISO-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.
Stationary Phase
Particle
Shape
Particle
Size (µm)
Pore Size (Å)
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.
Pore Volume
(cc/g)
Surface Area
(m2/g)
Endcapped
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 can be found on
www.waters.com/chemcu).
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.
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
SFC columns, while in use, are under pressure with CO2 and
possible modifiers as a supercritical fluid. A major safety concern
is frostbite caused by adiabatic cooling when CO2 decompresses
1. Make sure your co-solvent pump is primed and has an adequate
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.
2. Connect both the inlet and outlet of the column to the
SFC system.
3. If the column is still filled with a solvent, use a low flow rate
and back pressure setting (100 bar) to start pumping CO2
and modifier through the column.
4. If you see frosting on the column at the inlet or outlet, tighten
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.
ACQUITY UPC2 Torus Columns Care & Use
2
[ CARE AND USE MANUAL ]
ACQUITY UP
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.)
Thermal and chemical equilibration are equally important
chromatography. Ensure that the mobile phase is preheated before
entering the column to prevent a thermal miss-match. This can be
2
done by enabling active preheating on a UPC
®
instrument.
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.) It is recommended to use Waters Quality
Control Reference Material (QCRM) (p/n 186007950)
which contains a mixture of standards specifically chosen
for the ACQUITY UPC2 Trefoil and Torus Columns. For more
information, please review the ACQUIT Y UPC2 Columns
Brochure (literature code 720004275EN).
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 1. 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.
C2 Column
VanGuard Pre-Column
2. Determine the number of theoretical plates (N) and use this
3. Repeat the test at predetermined intervals to track column
ACQUITY UPC2 Quality Control Reference Materials (QCRM)
Intended Use Contents
Provides
convergence
chromatographic
performance
information for
both chiral and
achiral modes
value as a benchmark for periodic comparisons.
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.)
1. 0.50 mg/mL (+/-) trans-Stilbene oxide
2. 0.50 mg/mL Thymine
3. 0.50 mg/mL Sulfamethoxazole
4. 0.50 mg/mL Sulfamethizole
In a 1 mL solution of 75:25 ACN:MeOH
Place wrench here
Ferrule
Collet
Place wrench here
Flow
Figure 1. Connecting a VanGuard Pre-column to an ACQUITY UPC2 Column.
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).
ACQUITY UPC2 Torus Columns Care & Use
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