BioHPLC Column Selection Guide Cover - Agilent Technologies

LC/MS analysis of angiotensin
on Extend-C18
Column: ZORBAX Extend-C18
773700-902
2.1 x 150 mm, 5 µm
Mobile Phase: Acidic Conditions:
A: 0.1% TFA in water
B: 0.085% TFA in 80% acetonitrile (ACN)
Basic Conditions:
A: 10 mM NH4OH in water
B: 10 mM NH4OH in 80% ACN
A
Angiotensin I
Max: 10889
Low pH
B
Angiotensin I
Max: 367225
High pH
TIC (200-1500 m/z)
TIC (200-1500 m/z)
5.0E6
4.0E6
3.0E6
2.0E6
1.0E6
0
5.0E7
4.0E7
3.0E7
2.0E7
1.0E7
0
0
0
Flow Rate: 0.2 mL/min
Gradient: 15-50% B in 15 min
Temperature: 35 °C
2.5
2.5
AII + AIII
5.0 7.5 10 12.5
Time (min)
AII
5.499
AI
AIII
8.477
9.621
12.050
13.261
5.0 7.5 10 12.5
Time (min)
AI
100
80
60
40
20
0
0
+3
227.1
250.9
340.3
398.0
433.0
458.0
433.0
Reversed-Phase HPLC
Both small and large peptides demonstrate selectivity changes at high and low pH. At high pH, due to a change in charge, all three Angiotensins
can be resolved. In addition, the spectral clarity of Angiotensin I is dramatically improved at high pH with the ammonium hydroxide mobile phase.
The Extend-C18 column can be used for the analysis of small peptides at high pH as well.
Reference: B.E. Boyes. Separation and Analysis of Peptides at High pH Using RP-HPLC/ESI-MS, 4th WCBP, San Francisco, CA, Jan. 2000.
MS Conditions: Pos. Ion ESI- Vf 70 V, Vcap 4.5 kV,
N2- 35 psi, 12 L/min., 325 °C
Sample: 2.5 µL sample (50 pmol each)
Angiotensin I, II, III
100
80
60
40
20
0
0
500
+3
500
649.5
+2
659.7
720.3
767.3
844.8
649.0
659.8
+2
1025.2
1000
1000
1179.1
1277.9
1326.3
1373.3
1296.6
1468.8
m/z
+1
m/z
LC30003
UHPLC/HPLC
2