Waters XBridge Protein BEH, C4, 300A, 3.5 µm Columns - Manual (Page 3)

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[ CARE AND USE MANUAL ]
XBridge Protein BEH C
4
, 300Å, 3.5, 5, 10 �m
d. Useful Functional Tests for Benchmarking a New Column
The Column Efficiency Test described above is a useful measure
of the physical state of the packed bed as well as an indicator
of the chemical integrity of the bonded phase. It may also be
useful to benchmark the column performance with a sample that is
more representative of the intended application. Two tests can be
suggested as starting points for benchmarking a new column and
for monitoring a column during its use.
Peptide Mixture Performance Test
Sample: Waters MassPREP™ Peptide Standard Mixture
(P/N 186002337)
Reconstitute 1 vial in 100 μL 0.1% TFA:5%
Acetronitrile: 94.9% Water
Injection Volume: 2.1 mm – 3.3 µL
4.6 mm – 16.0 µL
Column: XBridge Protein BEH C
4
, 300Å
3.5 µm, 2.1 x 50 mm
Flow Rate: 2.1 mm – 0.2 mL/min
4.6 mm – 0.96 mL/min
Mobile Phase: A: 0.1% TFA in water
B: 0.075% in 71.4% acetonitrile/
28.6% water
Gradient:
Gradient Time for Different Column Lengths
50 mm 100 mm 150 mm 250 mm %A %B Curve
Initial Initial Initial Initial 100 0 *
30 60 90 150 30 70 6
32 64 96 160 30 70 1
50 100 150 250 100 0 1
Temperature: 40 °C
Detection: 220 nm
Figure 1: Typical Peptide Chromatogram Using MassPREP Peptide 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.
Protein Mixture Performance Test
Sample: MassPREP Protein Standard Mixture (P/N 186004900)
Dissolved in 0.1% TFA:5% acetonitrile:94.9% water
Protein Sigma P/N Conc. mg/mL
Bovine Ribonuclease A R5500 0.04
Horse Cytochrome c C7752 0.06
Bovine Serum Albumin A8022 0.20
Horse Myoglobin M1882 0.13
Yeast Enolase E6126 0.22
Rabbit Phosphorylase b P6635 0.59
Injection Volume: 2.1 mm – 5.0 µL
4.6mm – 25.0 µL
Column: XBridge Protein BEH C
4
, 300Å ,
3.5 µm, 2.1 x 50 mm
Flow Rate: 2.1 mm – 0.2 mL/min
4.6 mm – 0.96 mL/min