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Care and use manual – Waters Protein Separation Technology ACQUITY UPLC BEH300, C4, 1.7 µm Columns User Manual

Page 4

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[ Care and Use ManUal ]

Protein Separation Technology ACQUITY UPLC BEH300, C

4

, 1.7

�m

4

f. 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 Mass Prep™ Peptide Standard Mixture

(P/N 186002337)

Reconstitute 1 vial in 100 µL 0.1%TFA:5%

Acetonitrile:94.9% Water

Injection Volume: 3.3 µL
Column:

ACQUITY UPLC BEH300, C

4

,1.7 µm, 2.1 x 50 mm

Flow Rate:

0.2 mL/min

Mobile Phase:

A: 0.1% TFA in Water

B: 0.075% in 71.4% Acetonitrile/28.6% Water

Gradient:

Temperature:

40 ˚C

Detection:

220 nm

Figure 1: Typical Peptide Chromatogram

This chromatogram is typical of the results obtained in Waters
laboratories with the method described above, using a 2.1 x 50 mm
column. The retention times will double and triple for 100 mm and
150 mm columns respectively. The exact results observed in any
laboratory will depend on the instrument in use. Similar results can
only be expected with a Waters ACQUITY UPLC System. 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 (P/N 186004900)

Dissolved in 0.1%TFA:5% Acetonitrile:94.9% Water

Injection Volume: 5.0 µL
Column:

ACQUITY UPLC BEH300, C

4

,1.7 µm, 2.1 x 50 mm

Flow Rate:

0.2 mL/min

Mobile Phase:

A: 0.1% TFA in Water

B: 0.075% in 71.4% Acetonitrile/28.6% Water

Gradient:

Temperature:

40 ˚C

Detection:

220 nm

Time (Column Length)

50 mm

100 mm 150 mm

%A

%B

Curve

I

nitial

Initial

Initial

100

0

*

30

60

90

30

70

6

32

64

96

30

70

1

50

100

150

100

0

1

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

Time (Column Length)

50 mm

100 mm 150 mm

%A

%B

Curve

I

nitial

Initial

Initial

72

28

*

25

50

75

0

100

6

27

54

81

0

100

1

45

90

135

72

28

1