Care and use manual – Waters BioSuite Peptide Analysis Columns User Manual

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[ Care and Use ManUal ]

BioSuite Peptide Analysis Columns

2. The data in Table 1 was generated using a BioSuite C

, 3 μm PA-A

4.6 mm x 250 mm and a 2.1 mm x 250 mm Column.

• Proportionately less system backpressure will be generated using

BioSuite C

, 3.5 µm PA-B columns that contain 3.5 μm material

compared to the 3 μm material contained in the BioSuite C

3 μm PA-A column.

• Proportionately less system backpressure will be generated using

BioSuite Peptide Analysis Columns of lengths less than 250 mm.

3. Recommended pH Range: 2-8.

4. Recommended Organic Solvent/Additive Concentration Range:

• BioSuite C

3 μm PA-A: 0-100% acetonitrile (CH

CN)

Up to 0.1% formic acid (FA) or up to 0.025% trifluoroacetic acid

(TFA)

• BioSuite C

3.5 μm PA-B: 0-100% CH

Up to 0.1% TFA

5. Recommended Temperature Range: 20-50 °C

Note: As indicated above, reduce flow rate when operating at ambient tem-
peratures may be required to avoid excessive column pressure on 250 mm
i.d. columns.

6. Recommended Cleaning Solvents/Procedures: A shift in retention or

resolution may indicate contamination of the column. Flushing with
a neat organic solvent (e.g., 100% CH

CN or 80% Isopropanol/20%

CN) is usually sufficient to remove the contaminants.

7. Recommended Storage: For overnight storage, lower column tempera-

ture to ambient (e.g., from 40 °C to 25 °C) and continuously flush
the column with the mobile phase at 10-20% of the maximum recom-
mended flow rate. If the column is not to be used for several days,
lower column temperature to ambient and store the column in 100%
acetonitrile (CH

CN) without and eluent additives such as formic acid

or TFA. Do not store columns in buffered eluents. If you mobile phase
contained a buffer salt, flush the column with 10 column volumes of
HPLC grade waters before replacing the eluent with 100% CH

CN for

storage. Failure to perform this immediate step could result in precipi-
tation of the buffer salt in the column when 100% CH

CN is introduced.

Completely seal the stored column to avoid evaporation and drying out
the bed.

General Considerations to Maximize Performance of your BioSuite C

3 μm

PA-A and C

3.5 μm PA-B Columns.

II. sample preparatIon

1. Sample impurities often contribute to column contamination.

2. It is preferable to prepare the peptide sample in the mobile phase or a

solvent that is weaker (less organic modifier) than the mobile phase.

3. If the peptide 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 membrane to remove particulates. If the

sample is dissolved in a solvent that contains an organic modifier
(e.g., acetonitrile, methanol, etc.) ensure that the membrane material
does not dissolve in the solvent. Contact the membrane manufacturer
with solvent compatibility questions.

III. mobIle phase ConsIderatIons

Prevent air bubbles from entering the column during its installation, use,
and storage since this may cause degradation of column performance through
the formation of channels in the packed bed. Mobile phases must be thor-
oughly degassed before use. This can be accomplished by vacuum filtration,
helium sparging or by in-line vacuum degassing. In addition to degassing
the solvent, vacuum filtration of the solvent will also prevent small particles
from plugging the column frit. You can use 0.20 µm or 0.45 µm membrane
to filter aqueous and aqueous/organic mobile phases.

Note: Consult with filtration membrane manufacturer for details on solvent
compatibility.

Note:Use high quality reagents, HPLC grade water, and HPLC grade solvents
for preparing eluents. The useful column lifetime is a function of numerous
factors including: the cleanliness and composition of the mobile phase and
the sample; the flow rate and pressure used; and the temperature. Refer to
the section below about “Cleaning” for information on extending column life.

Note: Cleaning, however, is not effective when the column is damaged by
irreversible sample adsorption, channeling, or exposure of the packing mate-
rial to excessive heat or shock.