Waters XBridge Protein BEH, C4, 300A, 3.5 µm Columns - Manual (Page 4)

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[ CARE AND USE MANUAL ]
XBridge Protein BEH C
4
, 300Å, 3.5, 5, 10 �m
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 mm %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
1
2
3
4
5
6
1 - RNase A
2 - Cyt. C
3 - BSA
4 - Myoglobin
5 - Enolase
6 - Phosphorylase b
Figure 2: Typical Protein Test Mixture Chromatogram using MassPREP Protein
Standard Mixture
This chromatogram is typical of the results obtained in Waters
laboratories with the method described above, using a XBridge
Protein BEH C
4
, 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 C
4
,
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 transfer of the
supernatant liquid to an appropriate vial, could be considered.
b. Operating pH Limits
The recommended operating pH range for XBridge Protein BEH
C
4
, 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.