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[ CARE AND USE MANUAL ]
ACQUITY UPC2 Trefoil 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
III. COLUMN USE
a. Sample Preparation
b. Solvents
c. Additives
d. Pressure
e. Band Spreading Minimization
IV. TROUBLESHOOTING
V. COLUMN CLEANING, ADDITIVES,
AND STORAGE
a. Cleaning
b. Effects of Additives
c. Storage
I. INTRODUCTION
2
Thank you for choosing an ACQUITY UPC
ACQUITY UPC2 Trefoil Columns are specifically designed to use
the complete range of capabilities of the ACQUIT Y UPC2 System
to achieve fast, robust chiral separations. ACQUITY UPC2 Trefoil
Columns are uniquely designed for the ACQUITY UPC2 System
to enable both selectivity and speed in chiral separations and to
reduce method development time. ACQUIT Y UPC2 Trefoil Columns
consist of modified polysaccharide coated stationary phases for
broad-spectrum chiral selectivity. T he higher order structure of
the polysaccharides have specific spatial orientations that impart
chiral selectivity through well-chosen modifications or selectors.
ACQUITY UPC2 Trefoil AMY1, CEL1, and CEL2 column
chemistries are complementary to each other and independently
offer different retention characteristics for separating chiral
compounds. Selectivity can be further enhanced by using
co-solvents and additive blends that most favorably modulate
chiral recognition. These columns are designed to separate
enantiomers, stereoisomers, metabolites, degradants, and
impurities with greater resolution and speed.
®
Trefoil™ Column.
VI. INT RODUCING ECORD INTELLIGENT
CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing Information
d. Column Use Information
VII. ACQUITY UPC2 COLUMN FAMILY
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[ CARE AND USE MANUAL ]
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
min
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
min
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
min
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
N
N
ACQUITY UPC2 Trefoil Columns for Chiral Separations
ACQUITY UPC2 Trefoil AMY1
2.5 µm Columns
Amylose tris-(3,5-dimethylphenylcarbamate)
min
O
O
Flavanone
ACQUITY UPC2 Tref oil C EL1
2.5 µm Columns
Cellulose tris-(3,5-dimethylphenylcarbamate)
ACQUITY UPC2 Trefoil CEL2
2.5 µm Columns
Cellulose tris-(3-chloro-4-methylphenylcarbamate)
Chiral separations were all run using the 2-minute screening method.
Trefoil Column: 2.1 x 50 mm
Co-solvent: 1:1 MeOH:IPA w/20 mM NH4OH
Gradient: 3–60% in 1.5 min, 60% for 0.5 min
Flow rate: 1.2 mL/min
Temperature: 40 °C
ABPR: 3,200 psi
UV Detection: 220 nm
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.
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
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.
min
min
OH
O
O
Oxprenolol
Tetramisole
H
N
S
ACQUITY UPC2 Trefoil Columns Care & Use
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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 volume. 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.
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
(see section IIIb).
2. Connect both the inlet and outlet of the column to the
UPC2 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.
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.
e. Initial Column Efficiency Determination
1. Before use, perform an efficiency test on the column,
using suitable analytes dissolved in a weaker injection solvent
to avoid peak distortion from strong injection solvent effects
(see section IIIb). 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).
ACQUITY UPC2 Quality Control Reference Materials (QCRM)
Intended Use Contents
Provides
convergence
chromatographic
performance
information for
both chiral and
achiral modes
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
150 mm 0.5 1.0
Table 1. Empty Column Volumes in mL (multiply by 10 for flush
solvent volumes).
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
Column Internal Diameter (mm)
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.
ACQUITY UPC2 Trefoil Columns Care & Use
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[ CARE AND USE MANUAL ]
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.
ACQUITY UPC2 Trefoil Columns are designed for supercritical
(compressed CO2) applications. ACQUITY UPC2 Trefoil Columns
may be used with compressed CO2 and the following co-solvents
(individually and in miscible combinations): alcohols (typically
methanol, ethanol, isopropanol), acetonitrile and alkanes
(typically heptane and hexane). Under SFC conditions, the
co-solvent elution strength is in the following order: methanol >
ethanol > isopropanol > acetonitrile > alkanes. When dissolving
samples for analysis, use an injection solvent that is weaker
to avoid peak distortion. Employing blends of methanol,
isopropanol, acetontrile and/or heptane commonly maintains
analyte solubility while avoiding strong injection solvent
peak distortion.
Water tends to have a irreversible effect on selectivity. While not
damaging the polysaccharide, the selectivity will be permanently
effected upon exposure.
ACQUITY UPC2 Trefoil Columns contain a modified polysaccharide
coating which can be damaged when exposed to stronger solvents.
Solvents that must be avoided include:
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
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.
Ketones such as acetone, methyl ethyl ketone (MEK)
Halogenated solvents such as chloroform, dichloromethane,
methylene chloride
Ethyl acetate
Tetrahydrofuran (THF)
Methyl tert-butyl ether (MTBE)
Toluene
Dimethyl formamide
Dimethyl sulfoxide
N-Methylformamide
c. Additives
ACQUITY UPC2 Trefoil 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
ACQUITY UPC2 Trefoil Columns Care & Use
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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).
e. 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 1 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
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.
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, ADDITIVES 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 1: Effect of connecting tubing on system.
Diluted/Distorted Sample Band
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
to improve cleaning.1 Flush columns with 10 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.
Polar Samples Non-Polar Samples
1. M ethanol 1. Isopropanol
2. Isopropanol 2. Heptane
3. Methanol 3. Isopropanol
4. Mobile Phase 4. Mobile Phase
Table 2: Column Cleaning Sequence.
ACQUITY UPC2 Trefoil Columns Care & Use
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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.
b. Effects of Additives
Use of co-solvent additives (e.g. ammonia, organic amines, formic
acid, trifluoroacetic acid) can be used successfully with all Trefoil
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.
c. Storage
If additives were used, flush the column using a minimum of
5 column volumes 50% MeOH/CO2. Store the columns in
100% CO2.
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.
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.
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 3. Installing the eCord Intelligent Chip.
Figure 2. eCord Intelligent Chip.
ACQUITY UPC2 Trefoil Columns Care & Use
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[ CARE AND USE MANUAL ]
c. Manufacturing Information
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.
Figure 5. Column use information.
VII. ACQUITY UPC2 COLUMN FAMILY
The ACQUITY UPC2 Trefoil 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
Figure 4: Manufacturing results stored on an eCord.
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, 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 720004828EN TC-PDF
ACQUITY UPC2 Torus™ Columns Care & Use Manual
- 720005203EN
ACQUITY UPC2 BEH, CSH, and HSS Columns Care & Use Manual
- 720004349EN
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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