Waters XBridge Columns User Manual

[ Care and Use ManUal ]

XbrIdGe Columns

Thank you for choosing a Waters XBridge™ column. The XBridge™ packing
materials were designed to provid e excellent peak shape, high efficiency,
and excellent stability for acidic and basic mobile phases. The XBridge
packing materials are manufactured in a cGMP, ISO 9001:2000 certi-

™

fied plant using ultra pure reagents. Each batch of XBridge
tested chromatographically with acidic, basic and neutral analytes and
the results are held to narrow specification ranges to assure excellent,
reproducible performance. Every column is individually tested and a
Performance Test Chromatogram is provided with each column along
with the Certificate of Acceptance.

material is

Contents

I. GettInG started

™

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

II. Column use

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

III. sCalInG up/down IsoCratIC methods

IV. troubleshootInG

V. Column CleanInG, reGeneratIon
and storaGe

a. Cleaning and Regeneration
b. Storage

XBridge™ Columns 1

VI. ConneCtInG the Column to the hplC

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

VII. addItIonal InformatIon

a. Use of Narrow-Bore (3.0 mm i.d. Columns)
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
d. Waters Small Particle Size (2.5 µm) Columns –
Fast Chromatography
e. Getting Started with XBridge HILIC Columns
f. Getting Started with XBridge Amide Columns

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I. GettInG started

Each XBridge™ column comes with a Certificate of Analysis and a Perfor­mance Test Chromatogram. The Certificate of Analysis, locat ed on the technical
information CD, is specific to each batch of packing material contained in

™

the XBridge
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. This data data should be stored for future
reference.

column and includes the batch number, analysis of unbonded

a. Column Installation

Note: The flow rates given in the procedure below are for a typical 5 µm pack-

ing 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 XBridge

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

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.

b. Column Equilibration

XBridge™ 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).

For XBridge HILIC columns, flush with 50 column volumes of 50:50
acetonitrile:water with 10 mM final buffer concentration. For XBridge

™

HILIC Amide columns, flush with 50 column volumes of 60:40
acetonitrile:aqueous. Prior to the first injection, equilibrate with 20 col­umn volumes of initial mobile phase conditions (refer to Table 1 for a list
of column volumes). See “Getting Started with XBridge HILIC Columns”
or “Getting Started with XBridge HILIC Amide Columns” for additional
information.

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 n umber of theoret ical plates (N ) and use this value for p eriodic
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.

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.

XBridge™ Columns 2

[ 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 7.8 10 19 30 50

20 – 0.07 0.14 0.33 – – – – –

30 – 0.10 0.21 0.50 – 2.4 8.5 – –

50 0.04 0.17 0.35 0.83 2.4 3.9 14 35 98

100 0.08 0.35 0.71 1.7 4.8 7.8 28 70 –

150 0.12 0.52 1.0 2.5 7.2 12 42 106 294

250 – 0.87 1.8 4.2 – 20 70 176 490

II. Column use

To ensure the continued high performance of XBridge™ columns, follow
these guidelines:

a. Guard Columns

Use a Waters g uard column of matc hing ch emistry and p article size bet ween
the injector and main column. It is important to use a high-performance
matching guard column to protect the main column while not compromis­ing or changing the analytical resolution.

Guard columns need to be replaced at regular intervals as determined by
sample contamination. When system backpressure steadily increases above
a set pressure limit, it is usually an indication that the guard column should
be replaced. A sudden appearance of split peaks is also indicative of a
need to replace the guard column.

b. Sample Preparation

1. Sample impurities often contribute to column contamination. One option

®

to avoid this is to use Waters Oasis

®

columns or Sep-Pak
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.

cartridges of the appropriate chemistry to clean up

solid-phase extraction cartridges/

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 t he sol­vent. Contact the filter manufacturer with solvent compatibility qu estions.
Alternatively, centrifugation for 20 minutes at 8,000 rpm, followed by
the transfer of the supernatant liquid to an appropriate vial, could be
considered.

5. For Hydrophilic Interaction Chromatography (HILIC) separations,
the samples must be prepared in 100% organic solvents (e.g.,
acetonitrile). See “Getting Started with XBridge HILIC Columns”
or “Getting Started with XBridge Amide Columns” for additional
information.

c. Operating pH Limits

The recommended operating pH limits for XBridge™ columns are listed in
Table 2. A listing of commonly used buffers and additives is given in Ta ble 3.
Additionally, the column lifetime will vary depending upon the operating
temperature, the type and concentration of buffer used.

XBridge™ Columns 3

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Table 2: Recommended pH and temperature Limits for XBridge™ Columns at Ambient Temperatures

Name of Column Particle Size Pore Diameter Surface Area pH Limits

XBridge C
XBridge C

18

8

2.5, 3.5, 5 µm 130Å 185 m2/g 1-12 80 °C 60 °C 3.1 µmol/m

2.5, 3.5, 5 µm 130Å 185 m2/g 1-12 60 °C 60 °C 3.1 µmol/m
XBridge Phenyl 2.5, 3.5, 5 µm 130Å 185 m2/g 1-12 80 °C 60 °C 3.0 µmol/m
XBridge Shield RP18 2.5, 3.5, 5 µm 130Å 185 m2/g 2-11 50 °C 45 °C 3.2 µmol/m
XBridge HILIC 2.5, 3.5, 5 µm 130Å 185 m
XBridge Amide 3.5 µm 130Å 185 m

2

/g 1-9 45 °C 45 °C - -

2

/g 2-11 90 °C 90 °C 7.5 µmol/m

Temperature Limits

Low pH High pH

Surface Carbon Load

2

2

2

2

2

Table 3: Buffer Recommendations for Using XBridge™ Columns from pH 1 to 12

Additive/Buffer pKa

TFA 0.3 Volatile Yes Ion pair additive, can suppress MS signal, used in the 0.02-0.1% range.
Acetic Acid 4.76 Volatile Yes Maximum buffering obtained when used with ammonium acetate salt. Used in 0.1-1.0% range.
Formic Acid 3.75 Volatile Yes Maximum buffering obtained when used with ammonium formate salt. Used in 0.1-1.0% range.
Acetate (NH
Formate (NH
Phosphate 1 2.15 1.15 – 3.15 Non-volatile No Traditional low pH buffer, good UV transparency.
Phosphate 2 7.2 6.20 – 8.20 Non-volatile No Above pH 7, reduce temperature/concentration and use a guard column to maximize lifetime.
Phosphate 3 12.3 11.3 - 13.3 Non-volatile No Above pH 7, reduce temperature/concentration and use a guard column to maximize lifetime.
4-Methylmorpholine ~8.4 7.4 – 9.4 Volatile Yes Generally used at 10 mM or less.
Ammonia (NH

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 the 1-10 mM range.
Borate 9.2 8.2 – 10.2 Non-volatile No Reduce temperature/concentration and use a guard column to maximize lifetime.
CAPSO 9.7 8.7 – 10.7 Non-volatile No Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.
Glycine 2.4, 9.8 8.8 – 10.8 Non-volatile No Zwitterionic buffer, can give longer lifetimes than borate buffer.
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 Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.
Triethylamine

(as acetate salt)

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

COOH) 4.76 3.76 – 5.76 Volatile Yes Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.

4CH2

COOH) 3.75 2.75 – 4.75 Volatile Yes Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.

4

OH)

4

9.2

-

10.3 (HCO

)

3

+

9.2 (NH

)

4

10.7 9.7 – 11.7 Volatile Yes Used in the 0.1-1.0% range. Volatile only when titrated with acetic acid (not hydrochloric or phosphoric).

Buffer Range
(±1 pH unit)

8.2 – 10.2

8.2 – 11.3

Volatility Used for Mass Spec Comments

Volatile
Volatile

Yes
Yes

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 (NH

Used as ion-pair for DNA analysis at pH 7-9.

HCO3), not ammonium carbonate ((NH4)2CO3)

4

18%
13%
15%
17%

12%

Note: Working at the extremes of pH, temperature and/or pressure will result in shorter column lifetimes.

d. 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

e. Pressure

XBridge™ columns can tolerate pressures of up to 6,000 psi (400 bar or 40
Mpa) although pressures greater than 4,000 – 5,000 psi should be avoided in

order to maximize column and system lifetimes.
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 poor performance.

f. Temperature

Temperatures between 20 ˚C – 80 ˚C (up to 90 ˚C for XBridge Amide columns)

are recommended for operating XBridge columns in order to enhance selectiv­Degas all solvents thoroughly before use to prevent bubble formation in

the pump and detector. The use of an on-line degassing unit is also recom­mended. T his is especially important when running low pressure gradients
since bubble formation can occur as a result of aqueous and organic
solvent mixing during the gradient.

ity, lower solvent viscosity and increase mass transfer rates. However, any

temperature above ambient will have a negative effect on lifetime which will

vary depending on the pH and buffer conditions used. Under HILIC conditions

XBridge Amide columns can be used at high pH and at high temperatures with-

out issues (see recommended conditions in Getting Started wit h XBridge Amide

section). See Table 2 for recommended pH and temperature limits.

XBridge™ Columns 4