[ CARE AND USE MANUAL ]
ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns
CONTENTS
I. INTRODUCTION
II. GETTING STARTED
a. Column Connectors
b. Column Installation
c. Column Equilibration
d. eCord Installation
e. Initial Column Efficiency Determination
III. COLUMN USE
a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature
IV. COLUMN CLEANING, REGENERATING
AND STORAGE
a. Cleaning and Regeneration
b. Storage
I. INTRODUCTION
Thank you for choosing an ACQUITY UPLC Peptide BEH C18, 130Å or
300Å Column. The ACQUITY UPLC Peptide BEH C18, 130Å and 300Å
packing materials were designed specifically for use with the Waters
ACQUITY UPLC System and are manufactured in a cGMP, ISO 9002
certified plant using ultra pure reagents. Each batch of ACQUIT Y
UPLC BEH material is tested chromatographically with acidic, basic
and neutral analytes and has been qualitifed for peptide mapping.
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 are provided on the eCord™ intelligent chip. Each batch of
ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns is also QC
tested with the separation of a protein digest.
V. INTRODUCING e CORD INTELLIGENT
CHIP TECHNOLOGY
VI. REPRESENTATIVE TEST CHROMATOGRAPH
AND CONDITIONS FOR SEPARATION OF
PROTEIN DIGEST
[ CARE AND USE MANUAL ]
II. GETTING STARTED
Each Peptide Separation Technology ACQUITY UPLC Peptide BEH
, 130Å and 300Å Columns comes with Certificate of Analysis
C
18
and a Performance Test Chromatogram embedded within the eCord
intelligent chip. The Certificate of Analysis is specific to each batch
of packing material contained in the ACQUITY UPLC Peptide BEH
, 130Å and 300Å Columns and includes the gel batch number,
C
18
analysis of unbonded particles, analysis of bonded particles, and
chromatographic results and conditions. The Performance Test
Chromatogram is specific to each individual column and contains
such information as: gel batch number, column serial number, USP
plate count, USP tailing factor, capacity factor, and chromatographic
conditions. These data should be stored for future reference.
a. Column Connectors
The ACQUITY UPLC System utilizes tubing and gold plated
compression screws which have been designed to meet stringent
tolerance levels and to minimize extra column volumes.
Optimized column inlet tubing (Part Number 430001084) is
supplied with the ACQUITY UPLC System. The inject valve end of
the tubing is clearly marked with a blue shrink tube marker. Insert
the opposite end of the tubing into the ACQUITY UPLC Column and
tighten the compression fitting using two 5/16-inch wrenches.
For information on the correct column outlet tubing, please refer
to the relevant detector section in the ACQUITY UPLC System
Operator’s Guide (Part Number 71500082502).
b. Column Installation
1. Purge the pumping system of any buffer-containing mobile phases
and connect the inlet end of the column to the injector outlet.
Note: If mobile phase additives are present in low concentrations (e.g.,
ion-pairing reagents), 100 to 200 column volumes may be required
for complete equilibration. In addition, mobile phases that contain
formate (e.g., ammonium formate, formic acid, etc.) may also require
longer initial column equilibration times.
c. Column Equilibration
ACQUITY UPLC BEH130 and BEH300 Columns are shipped in 100%
acetonitrile. It is important to ensure mobile phase compatibility
before changing to a different mobile phase system. Equilibrate the
column with a minimum of 10 column volumes of the mobile phase to
be used (refer to Table 1 for a list of column volumes)
Table 1. Empty Column Volumes in mL (Multiply by 10 for
Flush Solvent Volumes)
Column Length (mm) Internal Diameter 2.1 mm
50 0.2 mL
100 0.4 mL
150 0.5 mL
To avoid precipitating mobile phase buffers on your column or
in your system, flush the column with five column volumes of a
water/organic solvent mixture, using the same or lower solvent
content as in the desired buffered mobile phase. (For example,
flush the column and system with 60% methanol in water prior to
introducing 60% methanol/40% buffer mobile phase.)
d. 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.
2. Flush column with 100% organic mobile phase (methanol or
acetonitrile) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 0.5 mL/min over 5 minutes.
3. When the mobile phase is flowing freely from the column
outlet, stop the flow and attach the column outlet to the
detector. This prevents entry of air into the detection system
and gives more rapid baseline equilibration.
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline have been achieved,
proceed to the next section.
e. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it.
Waters recommends using a suitable solute mixture, as found
in the “Performance Test Chromatogram”, to analyze the
column upon receipt.
2. Determine the number of theoretical plates (N) and use this
value for periodic comparisons.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained
on two different UPLC systems due to the quality of the
connections, operating environment, system electronics,
reagent quality, column condition and operator technique.
ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns
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[ CARE AND USE MANUAL ]
III. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC Peptide
, 130Å and 300Å Columns follow these guidelines:
BEH C
18
a. Sample Preparation
1. Sample must be dissolved in a diluent compatiable with initial
strength of mobile phase.
2. Sample must be completely in solution and free of particulates.
3. To remove particulates the sample may be filted with a 0.2 µm
membrane. If the sample is dissolved in a solvent that contained
an organic modifier (e.g., acetronitrile, methanol, etc.) ensure
that the membrane material does not dissolve in the solvent.
Contact the membrane manufacturer with solvent compatibility
Table 2. Buffer Recommendations for Using ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns from pH 1 to 12
questions. Alternatively, centrifugation for 20 minutes at
8,000 rpm, followed by the transfer of the supernatant liquid to
an appropriate vial, could be considere
d.
b. pH Range
The recommended operating pH range for ACQUITY UPLC BEH columns
is 1 to 12. A listing of commonly used buffers and additives is given in
Table 2. Additionally, the column lifetime will vary depending upon the
operating temperature, the type and concentration of buffer used. For
example, the use of phosphate buffer at pH 8 or above in combination
with elevated temperatures will lead to shorter column lifetimes.
Additive/Buffer pKa Buffer range
TFA 0.3 - Volatile Yes
Acetic Acid 4.76 - Volatile Yes
Formic Acid 3.75 - Volatile Ye s
Acetate ( NH
Format e (NH
Pho sphate 1 2.15 1.15 – 3.15 Non-volatile No Traditional lo w pH buffer, good UV t ransparenc y.
Pho sphate 2 7. 2 6.20 – 8.20 Non-volatile No
4-Methylmorpholine ~8.4 7.4 – 9.4 Volatile Yes Generall y used at 10 mM or less.
Ammonia (N H4OH) 9.2 8.2 – 10.2 Volatile Yes
Ammonium Bicarbonate
Ammonium (Acetate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range.
Ammonium (Formate) 9.2 8.2 – 10.2 Volatile Yes Used in th e 1-10 mM range.
CAPSO 9.7 8.7 – 10.7 Non-Volatile No
Glycine 2.4, 9.8 8.8 – 10.8 Non-Volatile No Zwitterionic buffer, ca n give longer lifetime s than borate bu ffer.
1-Methylpiperidine 10.2 9.3 – 11.3 Volatile Yes U sed in the 1-10 mM range.
CAPS 10.4 9.5 – 11.5 Non-Volatile No
Triethylamine 10.7 9.7 – 11.7 Volatile Yes
(as acetate s alt) Pyrr olidine 11. 3 10.3 – 12.3 Volatile Yes
COOH) 4.76 3.76 – 5.76 Volatile Yes
4CH2
COOH) 3.75 2.75 – 4.75 Volatile Yes
4
9.2 (NH
2CO3
-
)
3
+
)
4
)
6.8 – 11.3 Volatile Yes
10. 3 (H CO
6.3 (H
Volatility
(±1 pH unit)
Used for
Mass Spec
Comments
Ion pair ad ditive, can suppress MS signal, u sed in the
0.02-0.1% range.
Maximum buffering obtain ed when used w ith ammonium acetate
salt. Us ed in 0.1-1.0% range.
Maximum buffering obtain ed when used w ith ammonium
formate salt. Use d in 0.1-1.0% range
Used in t he 1-10 mM range. Note tha t sodium or potas sium salts
are not volatil e.
Used in t he 1-10 mM range. Note tha t sodium or potas sium salts
are not volatil e.
Above p H 7, reduce tem perature/concentrat ion and use a guard
column to ma ximize lifetime.
Keep c oncentration below 10 mM and temper atures
below 30 ˚ C.
Used in t he 5-10 mM range (for M S work keep so urce >150 ˚C ).
Adjust pH w ith ammonium hydro xide or aceti c acid. Good b uffering
cap acity at pH 10.
Note: us e ammonium bicarb onate (NH
car bonate (( NH4)2CO3).
Zwitterionic buffer, com patible with ac etonitrile, used in the
1-10 mM range. Lo w odor.
Zwitterionic buffer, com patible with ac etonitrile, used in the
1-10 mM range. Lo w odor.
Used in t he 0.1-1.0% range. Vol atile only when titrated with
acetic acid (not hydrochloric or phosphoric).
Used a s ion-pair for DNA analysi s at pH 7-9 Mild buffer, gives
long lifetime.
HCO3), not ammonium
4
ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns
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[ CARE AND USE MANUAL ]
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior to
use. Pall Gelman Laboratory Acrodisc® filters are recommended.
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 and
poorer performance.
d. Pressure
ACQUITY UPLC BEH130 and BEH300 Columns can tolerate pressures of
up to 15,000 psi (1034 bar or 103 Mpa).
e. Temperature
Temperatures between 20 ˚C – 55 ˚C are recommended for operating
ACQUITY UPLC Peptide BEH C
to enhance selectivity, lower solvent viscosity and increase mass
transfer rates. Operating at the extremes of pH, temperature, and/or
pressure will result in a shortened column lifetime.
, 130Å and 300Å Columns in order
18
IV. COLUMN CLEANING, REGENERATING
AND STORAGE
a. Cleaning and Regeneration
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 a neat 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.
Table 3. Column Cleaning Sequence
Polar
Samples
1. Water Option 1: Inject repeated 100 µL aliquots of
2. Methanol Option 2: gradient of 10% to 90% B where:
3. Isopropanol
Note: To avoid potentially damaging precipitation within your column (e.g., if
your separation eluent contains phosphate buffer), be certain to flush column
with 5–10 column volumes of water BEFORE using suggested organic eluent
column wash procedures.
Proteinaceous Samples
dimethylsulfoxide (DMSO) using a reduced flow
rate delivering 50% Eluent A and 50% Eluent B
A = 0.1% trifluoroacetic acid (TFA) in water
B = 0.1% trifluoroacetic acid (TFA) in
acetonitrile (CH
3
CN)
Option 3: Flush column with 7M guanidine
hydrochloride, or 7M urea
b. Storage
For periods longer than four days at room temperature, store
the column in 100% acetonitrile. For elevated temperature
applications, store immediately after use in 100% acetonitrile
for the best column lifetime. Do not store columns in buffered
eluents. If the mobile phase contained a buffer salt, flush the
column with 10 column volumes of HPLC-grade water (see
Table 1 for common column volumes) and replace with 100%
acetonitrile for storage. Failure to perform this intermediate step
could result in precipitation of the buffer salt in the column when
100% acetonitrile is introduced. Completely seal column to avoid
evaporation and drying out of the bed.
Note: If a column has been run with a mobile phase that contains formate
(e.g., ammonium formate, formic acid, etc.) and is then flushed with 100%
acetonitrile, slightly longer equilibration times may be necessary when the
column is re-installed and run again with a formate-containing mobile phase.
Use the cleaning routine that matches the properties of the
samples and/or what you believe is contaminating the column (see
Table 3 below). Flush columns with 20 column volumes of HPLCgrade solvents. Increasing mobile phase temperature to 35–55 ˚C
increases cleaning efficiency. If the column performance is poor
after regenerating and cleaning, call your local Waters office for
additional support.
V. eCORD
a. Introduction
The eCord intelligent chip is a new technology that 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.
Waters eCord intelligent chip
ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns
4
[ CARE AND USE MANUAL ]
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. The eCord provides a
solution to easily track the history of column usage.
The eCord chip provides the user with QC test conditions and
results on the column run by the manufacturer. The information
includes mobile phases, running conditions and analytes used
to test the columns. In addition the QC results and acceptance is
placed onto the column.
eCord inserted into side of column heater
b. Installation
Install the column into the column heater. 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 in Empower® and MassLynx® software
allowing the user to access column information on their desktop.
c. Manufacturing Information
The eCord chip provides the user with an overview of the bulk
material QC test results.
d. Customer Use Information
The eCord chip will automatically capture column use data. The
top of the screen identifies the column including chemistry type,
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.
ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns
5
[ CARE AND USE MANUAL ]
VI. REPRESENTATIVE TEST CHROMATOGRAPH AND CONDITIONS FOR SEPARATION/
OF PROTEIN DIGEST
T5
T9-T10
T4
T10
T8
T12
T12-T13
T14
T1
T13-T14
T19C
6.00 9.00 12.00 15.00 18.00 21.00 24.00
Peak Identification
T1 N-AcGDVEK T8 TGPNLHGLFGR
T13-T14 KYIPGTK T15 MIFAGIK
T14 YIPGTK T5 CAQCHTVEK (heme attached)
T4 IFVQK T19 EDLIAYLK
T9-T10 KTGQAPGFSYTDANK T12-T13 GITWGEETLMEYLENPKK
T10 TGQAPGFSYTDANK T12 GITWGEETLMEYLENPK
T19C EDLIAY
T15
T19
Minutes
*Chromatographic Conditions:
Column: 2.1 mm x 100 mm,
Sample: Tryptic digest of cytochrome c;
Flow rate: 0.20 mL/min
Mobile phase A: 0.045% TFA in water;
Mobile phase B: 0.045% TFA in acetonitrile;
Gradient: from 0 to 15% B in 6 min, from 15 to
36% B in 20 min;
Temperature: 35 ˚C; Detection: UV 214 nm
©2014 Waters Corporation. Waters, The Science of What’s
Possible, Oasis, Sep-Pak, Empower, MassLynx, and XBridge
are registered trademarks of Waters Corporation. eCord is a
trademarks of Waters Corporation. All other trademarks are
property of their respective owners.
February 2014 715001465 Rev C VW-P DF
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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