Waters XSelect CSH130 C18 3.5 μm and 5 μm Columns User Manual

[ CARE AND USE MANUAL ]

XSELECT CSH130 C18, 3.5 µm AND 5 µm COLUMNS

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, REGENERATING AND STORAGE

a. Cleaning and Regeneration
b. Storage

Thank you for choosing a Waters XSelect™ CSH™ HPLC column. XSelect
CSH130 C18, 3.5 µm and 5 µm Columns feature Waters Charged Surface
Hybrid (CSH) Technology which provides excellent peak shape, high
efficiency and loading capacity for basic compounds when using acidic,
low ionic strength mobile phases. This same particle technology is used
in the ACQUITY UPLC CSH130 C18, 1.7 µm and 2.5 µm XP family of
HPLC columns, thus enabling seamless transferability between HPLC and
UPLC® system platforms. The XSelect CSH130 C18 packing materials
were designed and are manufactured in a cGMP, ISO 9001 certified
manufacturing facility using ultra pure reagents. Each batch of XSelect
CSH130 C18 material is tested chromatographically with acidic, basic
and neutral analytes as part of qualification for use in peptide mapping.
The results are held to narrow specification ranges to assure excellent,
reproducible performance. XSelect CSH130 C18 batches are also QC tested
with a gradient separation of a tryptic digest of cytochrome c using 0.1%
formic acid containing eluents. Finally, every shipped column is individually
tested for packed bed efficiency and a Performance Chromatogram and
Certificate of Batch Analysis is available upon request.

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)

VII. ADDITIONAL INFORMATION

a. Use of Narrow-Bore (2.1 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

XSelect CSH130 C18 Columns 1

[ CARE AND USE MANUAL ]

I. GETTING STARTED

Each XSelect CSH130 C18 Columns 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.

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.

b. Column Equilibration

Peptide Separation Technology 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.

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.

XSelect CSH130 C18 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 4.6 10 19 30 50

50 0.04 0.17 0.83 3.9 14 35 98

100 0.08 0.35 1.7 7.8 28 70 196

150 0.12 0.52 2.5 12 42 106 294

II. COLUMN USE

To ensure the continued high performance of XSelect CSH130 C18 Columns,

follow these guidelines:

a. Guard Columns

Use a Waters guard column of matching chemistry and particle size between

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® solid-phase

extraction cartridges/columns or Sep-Pak® cartridges of the

appropriate chemistry to clean up the sample before analysis.

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.

c. Operating pH Limits

The recommended operating pH range for XSelect CSH130 C18, 3.5 µm

and 5 µm columns are 1 to 11. 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.

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.

XSelect CSH130 C18 Columns 3

[ CARE AND USE MANUAL ]

Table 2: Buffer Recommendations for Using XSelect CSH130 C18 Columns from pH 1 to 11

Additive/Buffer pKa Buffer range Volatility

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 (NH4CH2COOH) 4.76 3.76 – 5.76 Volatile Yes Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.

Formate (NH4COOH) 3.75 2.75 – 4.75 Volatile Yes Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.

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 (NH4OH) 9.2 8.2 – 10.2 Volatile Yes Keep concentration below 10 mM and temperatures below 30 ˚C.

Ammonium Bicarbonate 10.3 (HCO

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)

-

)

8.2 – 11.3 Volatile Yes Used in the 5-10 mM range (for MS work keep source >150 ˚C ). Adjust pH with

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).

(±1 pH unit)

Used for

Mass Spec

Comments

ammonium hydroxide or acetic acid. Good buffering capacity at pH 10

Note: use ammonium bicarbonate (NH4HCO3), not ammonium carbonate ((NH4)2CO3).

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

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 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.

e. Pressure

XSelect CSH130 C18 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.

f. Temperature

Temperatures up to 80 ˚C are recommended for operating XSelect CSH130

C18 columns in order to enhance selectivity, 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.

XSelect CSH130 C18 Columns 4