Waters XBridge Protein BEH, C4, 300A, 3.5 µm Columns User Manual

[ CARE AND USE MANUAL ]

XBridge Protein BEH C4, 300Å, 3.5, 5, and 10 µm Columns

CONTENTS

I. INTRODUCTION

II. GETTING STARTED

a. Column Installation
b. Column Equilibration
c. Initial Column Efficiency Determination
d. Useful Functional Tests for Benchmarking
a New Column

III. COLUMN USE

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

IV. SCALING SEPARATIONS

V. TROUBLESHOOTING

VI. COLUMN CLEANING, REGENERATING,
AND STORAGE

a. Cleaning and Regeneration
b. Storage

I. INTRODUCTION

Thank you for choosing a Waters reversed-phase Protein Column.
The XBridge® Protein BEH C4, 300Å packing material was designed
to provide excellent peak shape, high efficiency, and good recovery
for biological macromolecules that are too large or too hydrophobic
for separation on columns with smaller pores or longer chain
bonded phases. The base particle and bonding chemistry are chosen
to provide exceptional stability at both high and low pH as well as
at high temperature. The XBridge Protein BEH C4, 300Å packing
material is manufactured in a cGMP, ISO9002-certified plant
using ultra-pure reagents. Each batch of XBridge Protein BEH C4
reversed-phase column material has been qualified with a protein
test mixture, and the results are held to narrow specification ranges
to ensure reproducible performance. Every column is individually
tested for efficiency, and a Performance Test Chromatogram along
with a Certification of Acceptance is provided with each column.

VII. CONNECTING THE COLUMN TO THE HPLC

a. Column Connectors and System
Tubing Considerations
b. Measuring System Bandspreading Volume

VIII. MEASURING GRADIENT SYSTEM VOLUME
(OR DWELL VOLUME)

[ CARE AND USE MANUAL ]

II. GETTING STARTED

Each XBridge Protein BEH C4 Column has a Certificate of
Acceptance and a Performance Test Chromatogram. The Certificate
of Acceptance is specific to each batch of packing material 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 3.5 μm packing
in a 4.6 mm i.d. column. Scale the flow rate up or down accordingly based upon
the column i.d of the column being installed. See “Scaling” 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.

1. Purge the solvent delivery system of any buffer-containing
or water-immiscible 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 (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 stable backpressure and baseline have been achieved,
proceed to the next section.

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

Column Internal Diameter

Column
Lengt h (mm)

50 0.17 0.83 3.9 14.2 35.3

75 – – – – 53
100 0.35 1.7 7.9 28.4 70.7
150 0.52 2.5 11. 8 42.5 106
250 0.87 4.2 19.6 70.9 176.7

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 HPLC system with 50% acetonitrile in water
prior to introducing 50% acetonitrile/50% buffer mobile phase.

Column equilibration may be judged initially by stable pressure
and by a stable detector baseline. For a specific application, it is,
however, necessary to test the required duration of equilibration.
The criteria for adequate equilibration include reproducibility of
retention time for major and minor peaks, resolution for critical
pairs, and consistent baseline characteristics.

Note: Low concentration mobile phase additives, particularly those with minimal
buffering capacity may require extended equilibration and re-equilibration
between gradient analyses.

2.1 4.6 10 19 30

c. Initial Column Efficiency Determination

1. Perform an efficiency test on the column before using it in
the desired application. Waters recommends using the solute
mixture and conditions described in the Performance Test
Chromatogram to test the column upon receipt.

b. Column Equilibration

Your XBridge Protein BEH C4, 300Å Column is 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 column volumes).

2. Measure retention of the test compounds and the number of
theoretical plates (N).

3.

Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained
on two diff erent H P L C sy stems due to t he quality of th e
connections, operating environment, system electronics, reagent
quality, condition of column, and operator technique.

XBridge Protein BEH C4, 300Å, 3.5, 5, 10 �m

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[ CARE AND USE MANUAL ]

d. Useful Functional Tests for Benchmarking a New Column

The Column Efficiency Test described above is a useful measure
of the physical state of the packed bed as well as an indicator
of the c hemical integrity of the b onded phas e. It may also be
useful to benchmark the column performance with a sample that is
more representative of t he intende d ap plic ation. Two tests c an be
suggested as starting points for benchmarking a new column and
for monitoring a column during its use.

Peptide Mixture Performance Test

Sample: Waters MassPREP™ Peptide Standard Mixture

(P/N 186002337)
Reconstitute 1 vial in 100 μL 0.1% TFA:5%
Acetronitrile: 94.9% Water
Injection Volume: 2.1 mm – 3.3 µL

4.6 mm – 16.0 µL
Column: XBridge Protein BEH C

3.5 µm, 2.1 x 50 mm
Flow Rate: 2.1 mm – 0.2 mL/min

4.6 mm – 0.96 mL/min
Mobile Phase: A: 0.1% TFA in water
B: 0.075% in 71.4% acetonitrile/

28.6% water

Gradient:

, 300Å

4

Gradient Time for Different Column Lengths

50 mm 100 mm 150 mm 250 mm %A %B Curve

Initial Initial Initial Initial 100 0 *

30 60 90 150 30 70 6
32 64 96 160 30 70 1
50 100 150

Temperature: 40 °C
Detection: 220 nm

250 100 0 1

This chromatogram is typical of the results obtained in Waters
laboratories with the method described above, using a XBridge
Protein BEH C4, 300Å, 3.5 µm, 2.1 x 50 mm Column. The
retention times will double, triple, and be five times greater
for the 100 mm, 150 mm and 250 mm columns respectively.
The exact results observed in any laboratory will depend on
the instrument in use. System volume, gradient generation
mechanism, mixing, design of temperature control, detector cell
dimensions, detector optical properties, and detector electronic
properties all have a direct impact on the observed chromatogram.
The pattern should be similar, however, on any well-functioning,
modern HPLC. This test is exceptionally valuable for monitoring
the life of the column and for troubleshooting separation
difficulties that may arise.

Protein Mixture Performance Test

Sample: MassPREP Protein Standard Mixture (P/N 186004900)
Dissolved in 0.1% TFA:5% acetonitrile:94.9% water

Protein Sigma P/N Conc. mg/mL

Bovine Ribonuclease A R5500 0.04
Horse Cytochrome c C7752 0.06
Bovine Serum Albumin A8022 0.20
Horse Myoglobin M1882 0.13
Yeast Enolase E6126 0.22
Rabbit Phosphorylase b P6635 0.59

Injection Volume: 2.1 mm – 5.0 µL

4.6mm – 25.0 µL
Column:

3.5 µm, 2.1 x 50 mm

Flow Rate: 2.1 mm – 0.2 mL/min

4.6 mm – 0.96 mL/min

XBridge Protein BEH C4, 300Å

,

Figure 1: Typical Peptide Chromatogram Using MassPREP Peptide Standard Mixture

XBridge Protein BEH C4, 300Å, 3.5, 5, 10 �m

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[ CARE AND USE MANUAL ]

Mobile Phase: A: 0.1% TFA in water
B: 0.075% in 71.4% acetonitrile/28.6% water
Gradient:

Time (Column Length)

50 mm 100 mm 150 mm 250 m m %A %B Curve

Initial Initial Initial Initial 72 28 *

25 50 75 125 0 100 6
27 54 81 135 0 100 1

45 90 135 225 72 28 1

Temperature: 40 °C
Detection: 220 nm

6

1 - RNase A
2 - Cyt. C
3 - BSA
4 - Myoglobin
5 - Enolase
6 - Phosphorylase b

4

5

2

1

Figure 2: Typical Protein Test Mixture Chromatogram using MassPREP Protein
Standard Mixture

3

This chromatogram is typical of the results obtained in Waters
laboratories with the method described above, using a XBridge
Protein BEH C4, 300Å, 3.5 µm, 2.1 x 50 mm Column. The
retention times will double, triple, and be five times greater
for the 100 mm, 150 mm, and 250 mm columns respectively.
The exact results observed in any laboratory will depend on
the instrument in use. System volume, gradient generation
mechanism, mixing, design of temperature control, detector cell
dimensions, detector optical properties, and detector electronic
properties all have a direct impact on the observed chromatogram.
The pattern should be similar, however, on any well-functioning,
modern HPLC. This test is exceptionally valuable for monitoring
the life of the column and for troubleshooting separation
difficulties that may arise.

III. COLUMN USE

To ensure the continued high performance of XBridge Protein BEH C4,
300Å, 3.5, 5, and 10 µm Columns, follow these guidelines:

a. Sample Preparation

Sample impurities often contribute to column contamination.
Samples should be free of particles before injection into the system.

It is preferable to prepare the sample in gradient solvent A or in a
mobile phase that is weaker (less organic modifier) than the initial
strength mobile phase. This ensures the best peak shape and.

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.

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 12–50,000 g, followed by the t ran sfer of the
supernatant liquid to an appropriate vial, could be considered.

b. Operating pH Limits

The recommended operating pH range for XBridge Protein BEH
C4, 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 as
well as the type and concentration of buffer used.

XBridge Protein BEH C4, 300Å, 3.5, 5, 10 �m

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