Waters XBridge Peptide BEH C18 130A and 300A Columns User Manual

[ CARE AND USE MANUAL ]

XBridge Peptide BEH C18, 130Å and 300Å Columns

CONTENTS
I. GETTING STARTED

a. Column Installation
b. Column Equilibration
c. Initial Column Efficiency Determination

II. COLUMN USE

a. Sample Preparation
b. Operating pH Limits
c. Solvents
d. Pressure
e. Temperature

III. SCALING UP/DOWN ISOCRATIC METHODS

IV. TROUBLESHOOTING

V. COLUMN CLEANING, REGENERATING
AND STORAGE

a. Cleaning and Regeneration
b. Storage

VI. CONNECTING THE COLUMN TO THE HPLC

a. Column Connectors and System Tubing Considerations
b. Measuring System Bandspreading Volume
c. Measuring Gradient Delay Volume (or Dwell Volume)

Thank you for choosing a Waters XBridge® Peptide BEH C18, 130Å
or 300Å Column. The XBridge Peptide BEH C18, 130Å and 300Å
packing materials were designed to provide excellent peak shape,
high efficiency and excellent stability. The XBridge Peptide BEH C18,
130Å and 300Å packing materials are manufactured in a cGMP,
ISO 9002 certified planted using ultra pure reagent. Each batch
of XBridge Peptide BEH C18 Column material has been qualified
with a peptide separation and the results are held to narrow
specification ranges to assure excellent, reproducible performance
for peptide separations. Every column is tested and a Performance
Test Chromatogram along with a Certification of Acceptance are
provided with each column.

VII. ADDITIONAL INFORMATION

a. Use of Narrow-Bore (3.0 mm i.d.)
b. Impact of Bandspreading Volume on 2.1 mm i.d.
Column Performance
c. Non-Optimized vs. Optimized LC-MS/MS System:
System Modification Recommendations

[ CARE AND USE MANUAL ]

I. GETTING STARTED

Each XBridge Peptide BEH C18, 130Å and 300Å Column
comes with a Certificate of Acceptance and a Performance Test
Chromatogram. The Certificate of Acceptance is specific to each
batch of packing material contained in the Peptide Separation
Technology column and includes the batch number, 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
the information: batch number, column serial number, USP plate
count, USP tailing factor, retention factor, and chromatographic
conditions. These data should be stored for future reference.

a. Column Installation

Note: The flow rates given in the procedure below are for a typical

5 µm packing in a 4.6 mm i.d. column. Scale the flow rate up or

down accordingly based upon the column i.d., length, particle size

and backpressure of the Peptide Separation Technology column

being installed. See Scaling Up/Down Isocratic Separations section

for calculating flow rates when changing column i.d and/or length.

See “Connecting the Column to the HPLC” for a more detailed

discussion on HPLC connections.

b. Column Equilibration

XBridge Peptide BEH C18, 130Å and 300Å 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 listing of empty
column volumes).

To avoid precipitating out 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 HPLC system with 60% methanol in water
prior to introducing 60% methanol/40% buffer mobile phase).

c. Initial Column Efficiency Determination

1. Perform an efficiency test on the column before using it
in the desired application. 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.

1. Purge the pumping system of any buffer-containing mobile
phases and connect the inlet end of the column to the injector
outlet. An arrow on the column identification label indicates
the correct direction of solvent flow.

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 1 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.

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.

3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained
on two different HPLC systems due to the quality of the
connections, operating environment, system electronics,
reagent quality, column condition and operator technique.

XBridge Peptide BEH C18, 130Å and 300Å Columns

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[ CARE AND USE MANUAL ]

Table 1: Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)

Column internal diameter (mm)

Column Length (mm) 1.0 2.1 4.6 10 19 30

50 0.04 0.17 0.83 3.9 14 35
100 0.08 0.35 1.7 7.8 28 70
150 0.12 0.52 2.5 12 42 106
250 - 0.87 4.2 20 70 176

II. COLUMN USE

To ensure the continued high performance of XBridge Peptide BEH C18, 130Å and 300Å Columns follow these guidelines:

a. Sample Preparation

1. Sample impurities often contribute to column contamination.
One option to avoid this is to use Waters Oasis® solid-phase
extraction cartridges/columns or Sep-Pak® cartridges of the
appropriate chemistry to clean up the sample before analysis.

2. It is preferable to prepare the sample in the operating mobile
phase or a mobile phase that is weaker (less organic modifier)
than the mobile phase for the best peak shape and sensitivity.

3. If the sample is not dissolved in the mobile phase, ensure that
the sample, solvent and mobile phases are miscible in order to
avoid sample and/or buffer precipitation.

4. Filter sample with 0.2 µm filters to remove particulates. If
the sample is dissolved in a solvent that contains an organic
modifier (e.g., acetonitrile, methanol, etc.) ensure that the
filter material does not dissolve in the solvent. Contact the
filter manufacturer with solvent compatibility questions.
Alternatively, centrifugation for 20 minutes at 8,000 rpm,
followed by the transfer of the supernatant liquid to an
appropriate vial, could be considered.

b. Operating pH Limits

The recommended operating pH range for XBridge Peptide BEH
C18, 130Å and 300Å 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.

XBridge Peptide BEH C18, 130Å and 300Å Columns

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