[ 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
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[ 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
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[ CARE AND USE MANUAL ]
Diluted/Distorted Sample Band
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.
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.
c. Pressure
ACQUITY UPC2 Column hardware and packing materials can
tolerate pressures of up to 18,000 psi (1241 bar or 124 Mpa).
Pressures greater than 5000 – 6000 psi should be avoided in
order to maximize column and ACQUITY UPC2 System lifetimes.
d. Useable Flow-Rate Ranges and Expected ACQUITY UPC2
System Pressures
Table 3 estimates maximum flow rate and system pressure which
can be achieved without over-pressuring the ACQUITY UPC
2
System. The ACQUITY UPC2 System has a maximum flow rate of
4 mL/minute and maximum pressure of 6000 psi (~400 Bar).
The predicted flow rates and expected backpressure information
is based on the ACQUITY UPC2 System BPR (Backpressure
Regulator) set at 1800 psi (~120 Bar).
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 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
Figure 2: Effect of connecting tubing on system.
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.
ACQUITY UPC2 Torus Columns Care & Use
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[ CARE AND USE MANUAL ]
ACQUITY UPC2 Columns, 1.7 µm
100% CO
Dimension
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
Predicted Max Flow Rate
(mL/min)
2
Predicted Max Pressure
(psi)
Predicted Max Flow Rate
(mL/min)
60/40 CO2/MeOH
Predicted Max Pressure
ACQUITY UPC2 Columns, 3.5 µm
100% CO
Dimension
2.1 x 50 mm 4.00 3549 4.00 4963
2.1 x 75 mm 4.00 4096 3.75 5973
2.1 x 100 mm 3.85 4530 3.00 5979
2.1 x 150 mm 3.50 5386 2.10 5946
3.0 x 50 mm 4.00 3042 4.00 3703
3.0 x 75 mm 4.00 3275 4.00 4283
3.0 x 100 mm 4.00 3528 4.00 4906
3.0 x 150 mm 4.00 4063 3.80 5965
Predicted Max Flow Rate
(mL/min)
2
Predicted Max Pressure
(psi)
Predicted Max Flow Rate
(mL/min)
60/40 CO2/MeOH
Predicted Max Pressure
(psi)
(psi)
Table 3. Useable Flow Rate Ranges and Expected ACQUITY UPC2 System Pressures.
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, AGING, REGENERATING,
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.
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
ACQUITY UPC2 Torus Columns Care & Use
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[ CARE AND USE MANUAL ]
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 4: Column Cleaning Sequence.
1. Isopropanol (or an
appropriate water/
Isopropanol mixture)
5. Isopropanol (followed
by an appropriate water/
isopropanol mixture)
c. Regeneration
If you have noticed a significant change in retention or selectivity,
an additional regeneration process may restore your column’s
performance. There are two main sources of retention or
selectivity change for ACQUITY UPC2 Columns, assuming no
damage has been done to the column bed. Mobile-phase additives
and methanol can cause changes to the silica or hybrid surface.
The effects can be gradual or expeditious and may be permanent.
Columns showing peak shape, retention or selectivity changes
are best cleaned and regenerated on an ACQUIT Y UPLC System.
Before placing the column to be regenerated on a LC system,
it should be free of mobile-phase co-solvents and additives.
To do this, flow 100% CO2 through the column (3.0 x 100 mm) at
1 mL/min for five minutes (40 °C and 2000 psi at the ABPR).
For other column dimensions, the flow rate should be recalculated
appropriately to avoid overpressuring the system.
Based on the stationary phase to be regenerated, different rinsing
agents should be used as to not damage the packing material.
High-pH rinsing agents (pH > 8) should be avoided for cleaning
silica-based materials. Low-pH rinsing agents (pH < 1) should be
avoided for cleaning silica-based materials with ligand bondings.
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. 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.” 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.
Step 1. Unbonded and bonded silica-based materials
should be rinsed with 200+ column volumes of pure water.
All hybrid-based materials (e.g., BEH and CSH) can be
rinsed with either 200+ column volumes of pure water or
30+ column volumes of 0.1% N,N-Dimethylbutylamine
(or similar tertiary amine) in water.
Step 2. All columns should be rinsed with an additional
10-column volumes of 90/10 IPA/water to remove the mobile-
phase additives.
Step 3. All columns should be rinsed with 10-column
volumes of isopropanol to remove any residual water before
returning the column to an ACQUIT Y UPC2 System.
After regeneration, please reinstall and equilibrate the ACQUIT Y
UPC2 Column on the ACQUIT Y UPC2 System. Retest the column
after using the cleaning procedure to determine if the specific
problem has been fixed.
ACQUITY UPC2 Torus Columns Care & Use
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[ CARE AND USE MANUAL ]
d. Storage
Purging and storing a column in pure CO2 is recommended.
This will eliminate or minimize retention changes of the
column during storage. Storing the column in methanol is
not recommended.
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.
eCord Fob
Figure 4. Installing the eCord Intelligent Chip.
c. Manufacturing Information
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.
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.
Figure 5: Manufacturing results stored on an eCord.
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
UPC2 BEH, CSH, and HSS 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/CU
ACQUITY UPC2 Trefoil™ Columns Care & Use Manual
- 720004828EN
ACQUITY UPC2 Torus™ Columns Care & Use Manual
- 720005203EN
Figure 6. Column use information.
Waters, The Science of W hat’s Possible, ACQUITY U PC,2 Sep-Pak, Oasis, Waters Quality Parts, ACQUITY,
ACQUITY U PLC, and Empower are registered trademarks of Waters Corporation. Trefoil, eCord, 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 720004349EN 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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