BioHPLC Column Selection Guide Cover - Agilent Technologies

Reversed-Phase HPLC
Peptides: Effect of TFA concentration
Column: ZORBAX 300SB-C8
883995-906
4.6 x 150 mm, 5 µm
Mobile Phase: A: Water and TFA
B: ACN and TFA
Flow Rate: 1.0 mL/min
Gradient: 0 min 0% B
30 min 30% B
Temperature: 40 °C
Detector: UV 254 nm
Sample: Peptide Standards S1-S5, decapeptides
differing slightly in hydrophobicity, 6 µL
Peptides/proteins:
Effect of elevated temperature
Column: ZORBAX 300SB-C3
883995-909
4.6 x 150 mm, 5 µm
Mobile Phase: A: 5:95
ACN:Water with 0.10% TFA (v/v%)
B: 95:5
ACN:Water with 0.085% TFA (v/v%)
Flow Rate: 1.0 mL/min
Gradient: 15-53% in 20 min, posttime 12 min
Temperature: Ambient – 60 °C
Detector: UV 215 nm
Sample: Polypeptides
Tips & Tools
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Ambient
Peptide Sequence
0.05% TFA
1 2
3
4
0 2 4 6 8 10 12 14 16 18 20
Time (min)
4
8 10 12 14 16 18 20 22 24
Retention Time (min)
5
5
6
7 8
45 °C
35 °C 60 °C
S1 S2 S3 S4 S5
S1 S2 S3
S3
S2 S1
4, 5
0 2 4 6 8 10 12 14 16 18 20
Time (min)
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6
0.25% TFA
6
1.0% TFA
6
8 10 12 14 16 18 20 22 24
5
4
S4 S5
8 10 12 14 16 18 20 22 24
S4
LCBP026
S5
LCBP021
1. Leucine Enkephalin
2. Angiotensin II
3. RNase A
4. Insulin (BOV)
5. Cytochrome c
6. Lysozyme
7. Myoglobin
8. Carbonic anhydrase