3
Figure 3 shows that the ProPac SCX-10 column can
provide high resolution comparable to the reversed-phase
separations that use formic acid as a mobile phase
modifier. This is in contrast to many ion-exchange
columns that yield incomplete resolution and poor peak
shape in peptide mapping. Figure 4 shows that a reversed-
phase separation with trifluoroacetic acid (TFA) can be
better than the cation-exchange separation.
Figure 3. Chromatograms of a myoglobin tryptic digest separated by 1) a
ProPac SCX-10 column, 2) a Thermo Scientific Acclaim PolarAdvantage II (PA2)
column, and 3) an Acclaim 300 C18 column with formic acid as the mobile phase
modifier of reversed-phase separation.
100
mAU
-10
50
mAU
-10
100
mAU
-10
0
5
10
15
20
25
30
Minutes
(1)
(2)
(3)
Columns:
1
. ProPac SCX-10, 10 µm (4 × 250 mm, P/N 075725)
2
. Acclaim
™
PA2, 3 µm (3.0 × 150 mm, P/N 063705)
3
. Acclaim 300, C18, 3 µm (3.0 × 150 mm, P/N 063684)
Mobile Phase:
1
. A: 20 mM TEAP, pH 2.0, 50% acetonitrile
B: 100 mM sodium perchlorate in A
2
. A: 98% water, 2% acetonitrile, 0.1% formic acid
B: 98% acetonitrile, 2%water, 0.08% formic acid
3
. Same as 2
Gradient:
1
. 0–20 min, 0–100% B, at 1.4 mL/min
2
. 0–5 min, 0% B; 5–28 min, 0–100% B, at 0.6 mL/min
3
. 0–5 min, 0% B; 5–28 min, 0–100% B, at 0.5 mL/min
Inj. Volume:
20 µL
Temperature:
30 °C
Detection:
UV, 214 nm
Sample:
Myoglobin (from equine heart) tryptic digest
Sample Preparation: Reduce, alkylate, dialyze, and digest with trypsin overnight
Figure 4. Chromatograms of a myoglobin tryptic digest separated by 1) a
ProPac SCX-10 column, 2) an Acclaim PA2 column, and 3) an Acclaim C18
column with TFA as the mobile phase modifier of reversed-phase separation.
100
mAU
-10
50
mAU
-10
100
mAU
-10
0
5
10
15
20
25
30
Minutes
(1)
(2)
(3)
Columns:
1
. ProPac SCX-10, 10 µm (4 × 250 mm, P/N 075725)
2
. Acclaim PA2, 3 µm (3.0 × 150 mm, P/N 063705)
3
. Acclaim 300, C18, 3 µm (3.0 × 150 mm, P/N 063684)
Mobile Phase:
1
. A: 20 mM TEAP, pH 2.0, 50% acetonitrile
B: 100 mM sodium perchlorate in A
2
. A: 98% water, 2% acetonitrile, 0.04% TFA
B: 98% acetonitrile, 2%water, 0.03% TFA
3
. Same as 2
Gradient:
1
. 0–20 min, 0–100% B, at 1.4 mL/min
2
. 0–5 min, 0% B; 5–28 min, 0–100% B, at 0.6 mL/min
3
. 0–5 min, 0% B; 5–28 min, 0–100% B, at 0.5 mL/min
Inj. Volume:
20 µL
Temperature:
30 °C
Detection:
UV, 214 nm
Sample:
Myoglobin (from equine heart) tryptic digest
Sample Preparation: Reduce, alkylate, dialyze, and digest with trypsin overnight
ProPac SCX-10
(10 μm)
Acclaim PA2
(3 μm)
Acclaim 300 C18
(3 μm)
Formic acid as the mobile phase modifier in a reversed-phase separation
73
79
140
TFA as the mobile phase modifier in a reversed-phase separation
73
136
158
Table 1. Peak capacity comparison of three columns.
Table 1 summarizes peak capacity of an ion-exchange
column, the Dionex ProPac SCX-10, and two reversed-
phase columns, the Acclaim PA2 and Acclaim 300. Peak
capacity(n
C
) was calculated by the equation:
n
C
= 1 +
t
G
W
where
t
G
is the gradient time and W the peak width
measured at 4
σ
(13.4% of the peak height). For detailed
information, refer to Dionex (now part of Thermo
Scientific) Technical Note (TN) 74.
4
