Waters XBridge Peptide BEH C18 Care And Use 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 SYSTEM

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

VIII. ADDITIONAL INFORMATION

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

IX. REPRESENTATIVE TEST CHROMATOGRAPH
AND CONDITIONS FOR SEPARATION OF
PROTEIN DIGEST

X. CAUTIONARY NOTE

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 plant 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. eCORD INTELLIGENT CHIP TECHNOLOGY

(applies only to XBridge XP 2.5 m,
≤4.6 mm I.D. Columns )

1

[ CARE AND USE MANUAL ]

. 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 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 column’s: 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 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 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 System” for a more detailed
discussion on HPLC connections.

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.

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.

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.

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.

2XBridge Peptide BEH C18, 130Å and 300Å Columns

[ 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 3.0 4.6 10 19 30

50 0.04 0.17 0.35 0.83 3.9 14 35
100 0.08 0.35 0.71 1.7 7.8 28 70
150 0.12 0.52 1.06 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 m 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 8000 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.

3XBridge Peptide BEH C18, 130Å and 300Å Columns

[ CARE AND USE MANUAL ]

Table 2. Buffer Recommendations for Using XBridge Peptide BEH C18, 130Å and 300Å Columns from pH 1 to 12

Additive/Buffer pK

a

Buffer
range

TFA 0.3 – Volatile Yes

Acetic acid 4.76 – Volatile Yes

Formic acid 3.75 – Volatile Yes

Acetate
(NH4CH2COOH)

4.76 3.76–5.76 Volatile Yes

Formate (NH4COOH) 3.75 2.75–4.75 Volatile Yes

Phosphate 1 2.15 1.15–3.15 Non-volatile No

Phosphate 2 7.2 6.20–8.20 Non-volatile No

Phosphate 3 12.3 11.3–13.3 Non-volatile No

4-Methylmorpholine ~8.4 7.4–9.4 Volatile Yes Generally used at 10 mM or less.

Ammonia (NH4OH) 9.2 8.2–10.2 Volatile Yes

Ammonium
bicarbonate

10.3 (HCO

9.2 (NH

-

)

3

+

)

4

8.2–11.3 Volatile Yes

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 the 1–10 mM range.

Borate 9.2 8.2–10.2 Non-volatile No

CAPSO 9.7 8.7–10.7 Non-volatile No

Glycine 2.4, 9.8 8.8–10.8 Non-volatile No

1-Methylpiperidine 10.2 9.3–11.3 Volatile Yes Used in the 1–10 mM range.

CAPS 10.4 9.5–11.5 Non-volatile No

Triethylamine
(as acetate salt)

10.7 9.7–11.7 Volatile Yes

Pyrrolidine 11.3 10.3–12.3 Volatile Yes Mild buffer, gives long lifetime.

Volatility

(±1 pH unit)

Used for

mass spec

Comments

Ion pair additive, can suppress MS
signal, used in the 0.02–0.1% range.

Maximum buffering obtained when
used with ammonium acetate salt.
Used in 0.1–1.0% range.

Maximum buffering obtained when
used with ammonium formate salt.
Used in 0.1–1.0% range.

Used in the 1–10 mM range.

Note: Sodium or potassium salts are not volatile.

Used in the 1–10 mM range.

Note: Sodium or potassium salts are not volatile.

Traditional low pH buffer, good UV
transparency.

Above pH 7, reduce temperature/
concentration and use a guard column
to maximize lifetime.

Above pH 7, reduce temperature/
concentration and use a guard column
to maximize lifetime.

Keep concentration below 10 mM and
temperatures below 30 °C.

Used in the 5–10 mM range (for MS work
keep source >150 °C ). Adjust pH with
ammonium hydroxide or acetic acid.
Good buffering capacity at pH 10.

Note: Use ammonium bicarbonate (NH4HCO3),
not ammonium carbonate ([NH4]2CO3).

Reduce temperature/concentration and
use a guard column to maximize lifetime.

Zwitterionic buffer, compatible with
acetonitrile, used in the 1–10 mM range.
Low odor.

Zwitterionic buffer, can give longer
lifetimes than borate buffer.

Zwitterionic buffer, compatible with
acetonitrile, used in the 1–10 mM range.
Low odor.

Used in the 0.1–1.0% range. Volatile
only when titrated with acetic acid (not
hydrochloric or phosphoric). Used as
ion-pair for DNA analysis at pH 7–9.

4XBridge Peptide BEH C18, 130Å and 300Å Columns