[ 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
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[ 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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[ CARE AND USE MANUAL ]
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.
dissolved in a solvent, ensure that the membrane material
does not dissolve in the solvent. Contact the membrane
manufacturer with solvent compatibility questions. Acrodisc®
filters are recommended (refer to the Waters Quality
Parts,® Chromatography Columns and Supplies Catalog for
additional information). Please consider that some analytes
can be retained on certain membrane materials and result
in lower recovery (or lower detector signal) than expected.
Alternatively, centrifugation for 20 minutes at 8000 rpm,
followed by the transfer of the supernatant liquid to an
appropriate vial could be considered.
b. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Solvents containing suspended
particulate materials will generally clog the outside surface
of the inlet distribution frit of the column. This will result in
higher operating pressure, reduced column lifetime, and
compromised performance.
III. COLUMN USE
To ensure the continued high performance of ACQUITY UPC2
Columns, follow these guidelines:
a. Sample Preparation
1. Sample impurities often contribute to column contamination.
Waters offers both solid-phase extraction (SPE) and
supercritical fluid extraction (SFE) options. For SPE, use
Oasis® Solid-Phase Extraction Cartridges/Columns or
Sep-Pak® Cartridges of the appropriate chemistry to cleanup
the sample before analysis. For more information, visit to
www.waters.com/sampleprep. Alternatively, Waters offers
the MV-10 ASFE Supercritical Fluid Extraction System for high
throughput extractions from a wide variety of sample matrices.
2. Consider preparing the sample in a weak solvent (such as
heptane) for the best peak shape and sensitivity. Using weak
sample diluents may avoid peak distortion due to “strong
solvent effects”. In particular, stronger solvents can impact
peak shapes of low retaining analytes.
c. Additives
ACQUITY UPC2 Torus Columns can safely be used with commonly
used acidic and basic additives used in supercritical fluid
chromatography (SFC) such as trifluoroacetic acid (TFA), formic
acid, ammonium acetate, ammonium formate, ammonium
hydroxide, and organic amines (such as diethylamine and
triethylamine) and ammoniated methanol. Typical concentrations
of 20 mM or 0.2% can be used safely. Consider the volatility,
solubility and detector compatibility when choosing an
appropriate additive. Additives tend to improve peak shape and
control the retention characteristics of analytes, but can also
impart different selectivities. It is recommended to flush and
remove additives and salts from the column before placing the
column into storage.
d. Pressure
ACQUITY UPC2 Column hardware and packing materials are
packed and tested to pressures of up to 6,000 psi (414 bar or
41 Mpa).
3. If the sample is not dissolved in the mobile-phase modifier,
ensure that the sample, solvent and mobile phases are miscible
in order to avoid sample precipitation. Filter sample with
0.2 μm membranes to remove particulates. If the sample is
ACQUITY UPC2 Torus Columns Care & Use
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[ CARE AND USE MANUAL ]
Diluted/Distorted Sample Band
Dimension
100% CO
Predicted Max Flow Rate
(mL/min)
2
Predicted Max Pressure
(psi)
Predicted Max Flow Rate
(mL/min)
60/40 CO2/MeOH
Predicted Max Pressure
2.1 x 50 mm 3.45 5581 2.00 6 011
2.1 x 75 mm 2.65 5970 1.35 5938
2.1 x 100 mm 2.05 5969 1.05 6060
2.1 x 150 mm 1.4 0 5951 0.70 6002
3.0 x 50 mm 4.00 4179 3.60 5946
3.0 x 75 mm 3.75 4897 2.60 5959
3.0 x 100 mm 3.45 5499 2.00 5919
3.0 x 150 mm 2.70 5977 1.40 6023
Table 2. Useable Flow Rate Ranges and Expected ACQUITY UPC2 System Pressures.
e. Useable Flow-Rate Ranges and Expected ACQUITY UPC2
System Pressures
Table 2 estimates maximum flow rate and system pressure which
can be achieved without over-pressuring the ACQUITY UPC
System. The ACQUITY UPC2 System has a maximum flow rate of
4 mL/minute and maximum pressure of 6000 psi (~400 Bar).
2
IV. TROUBLESHOOTING
1. One of the most common problems with regards to columns
is incorrect or insufficient priming of the co-solvent/modifier
pump. If no peaks are observed after an injection or unusually
long retention times are observed, check the priming of the
co-solvent/modifier pump first.
(psi)
The predicted flow rates and expected backpressure information
is based on the ACQUITY UPC2 System BPR (Backpressure
Regulator) set at 1800 psi (~120 Bar).
f. Band Spreading Minimization
The ACQUITY UPC2 System has been designed to minimize band
spreading. Deviation from Waters specified tubing could result
in deterioration of chromatographic performance due to band
spreading induced by inappropriate tubing i.d. Figure 2 shows the
influence of tubing internal diameter on system band spreading
and peak shape. As can be seen, the larger tubing diameter causes
excessive peak broadening and lower sensitivity.
0.005 inches
0.020 inches
0.040 inches
2. If you see frosting on the column at the inlet or outlet, tighten
the compression screw on that side. If tightening doesn’t work,
depressurize the system and the column, and then replace the
fitting that is not sealing correctly.
3. If you continue to see frosting on the column, turn off the CO2
and vent the system. Allow the column to depressurize fully
before disconnecting the inlet or outlet. Please contact your
Waters representative for additional support.
V. COLUMN CLEANING, EFFECTS OF ADDITIVES,
AGING, AND STORAGE
Changes in peak shape, peak splitting, shoulders on the
peak, shifts in retention, change in resolution or increasing
backpressure may indicate contamination of the column.
Flushing with high concentrations of organic solvent, taking care
not to precipitate buffers, is usually sufficient to remove the
contaminant. If the flushing procedure does not solve the
problem, purge the column using the following cleaning and
regeneration procedures.
Figure 2: Effect of connecting tubing on system.
ACQUITY UPC2 Torus Columns Care & Use
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[ CARE AND USE MANUAL ]
a. Cleaning
Use the cleaning routine that matches the properties of the
samples and/or what you believe is contaminating the column
(see Table 4). Placing the column on an ACQUITY UPLC® System
(or HPLC system) will enable the use of a wide range of solvents
and water to improve cleaning. Flush columns with 20 column
volumes of solvent. Increasing column temperature increases
cleaning efficiency. If the column performance is poor after
cleaning and regenerating, call your local Waters office for
additional support.
Note: Using an ACQUITY UPC2 System is not recommended for
the cleaning and regeneration procedures because recommended
solvents and high concentrations of water are immiscible with
supercritical CO2. Rather, the use of an ACQUITY UPLC System
is recommended for cleaning and regeneration.
Polar Samples Non-Polar Samples
1. Water (could also be
a mixture of acetonitrile
and water)
2. Methanol 2. Tetrahydrofuran
3. Tetrahydrofuran 3. Dichloromethane
4. Methanol 4. Hexane
5. Water
6. Mobile Phase 6. Mobile Phase
Table 3: Column Cleaning Sequence.
1. Isopropanol (or an
appropriate water/
Isopropanol mixture)
5. Isopropanol (followed
by an appropriate water/
isopropanol mixture)
b. Effects of Additives
Use of co-solvent additives, such as ammonia or organic
amines, can be used successfully with all Torus columns.
Consider the volatility and detector compatibility when choosing
an appropriate additive. Additives tend to improve peak shape
and control the retention characteristics of analytes, but can
also impart different selectivities. An ammonia additive works
particularly well on Torus DIOL and Torus 1-AA to improve peak
shapes and reduce retention variability. Ion pairing additives
should be used with caution on Torus phases, particularly
those with exposed amines (e.g. Torus 2-PIC and Torus DEA).
Counter ions, such as trifluoroacetate, can bind very strongly or
irreversibly with the stationary phase. It is recommended that
dedicated columns be used with particular ion pairing additives.
c. Aging
Research at the Waters Corporation confirms that there may be
changes in retention with SFC columns due to packing material
surface changes. This is commonly referred to by SFC industry
experts as “Aging or retention drift.” Mobile-phase additives
and methanol can cause changes to the silica or hybrid surface
resulting in retention shifts. When working with a new column,
the column can be flushed with CO2/methanol to eliminate
or reduce further shifts in retention. Storage in 100% CO2 is
recommended to ensure the column retention and selectivity
will be the same from the end of one test to the beginning of
another. The 100% CO2 will halt the aging process and results
in no additional retention shifts.
Retest the column after using the cleaning procedure to
determine if the specific problem has been fixed. If so, continue
using the column, avoiding samples and solvents that may clog
the column inlet.
Torus columns were designed and developed to not show
the characteristic retention and selectivity changes related
to aging. When exposed to CO2/Methanol mobile phases,
these columns show little or no shifts in retention over extended
periods of time.
d. Storage
Purging out any additives with a CO2/Methanol mobile phase
is recommended and storing columns in pure CO2.
ACQUITY UPC2 Torus Columns Care & Use
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[ CARE AND USE MANUAL ]
VI. INT RODUCING ECORD INTELLIGENT
CHIP TECHNOLOGY
a. Introduction
The eCord Intelligent Chip will provide the history of a column’s
performance throughout its lifetime. The eCord is permanently
attached to the column to assure that the column’s performance
history is maintained in the event that the column is moved from
one instrument to another.
Figure 3. eCord Intelligent Chip.
At the time of manufacture, tracking and quality control
information will be downloaded to the eCord. Storing this
information on the chip will eliminate the need for a paper
Certificate of Analysis.
c. Manufacturing Information
Once the user installs the column, the software will automatically
download key parameters into a column history file stored on
the chip. In this manual, we explain how the eCord will provide
a solution for easily tracking the history of the columns, reduce
the frustration of paperwork trails, and give customers the
reassurance that a well-performing column is installed onto
their instruments.
b. Installation
Install the column into the ACQUIT Y® Column Manager. Plug
the eCord into the side of the column heater. Once the eCord is
inserted into the column heater the identification and overall
column usage information will be available allowing the user to
access column information on their desktop.
eCord Fob
Figure 5: Manufacturing results stored on an eCord.
Figure 4. Installing the eCord Intelligent Chip.
ACQUITY UPC2 Torus Columns Care & Use
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[ CARE AND USE MANUAL ]
d. Column Use Information
The eCord Chip provides the customer with column use data,
column dimensions and serial number. The overall column
usage information includes the total number of samples, total
number of injections, total sample sets, date of first injection,
date of last injection, maximum pressure and temperature.
The information also details the column history by sample set
including date started, sample set name, user name, system
name, number of injections in the sample set, number of
samples in the sample set, maximum pressure and temperature
in the sample set and if the column met basic system suitability
requirements. Up to 50 sample sets can be stored on the eCord
Chip. In addition, the eCord provides two-way communications
between the eCord Chip and Empower® Software.
VII. ACQUITY UPC2 COLUMN FAMILY
The ACQUITY UPC2 Torus Columns Care & Use Manual is one of
three manuals for the ACQUIT Y UPC2 Column family. The other
two Care & Use Manual are available at www.waters.com/chemcu
ACQUITY UPC2 Trefoil™ Columns Care & Use Manual
- 720004828EN
ACQUITY UPC2 BEH, CSH, and HSS Columns Care & Use Manual
- 720004349EN
Figure 6. Column use information.
Waters, The Science of W hat’s Possible, ACQUITY U PC,2 UPC,2 Sep-Pak, Oasis, Waters Quality Parts, ACQUITY, and Empower
are registered trademarks of Waters Corporation. Trefoil, eCord, UltraPerformance Convergence Chromatograp hy, VanGuard,
and Torus are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2014 Waters Corporation. Produced in the U.S.A. October 2014 720005203EN TC-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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