Page 105 - Application Handbook - Liquid Chromatography

Application No.L457
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n Linearity of Calibration Curves
Four standard mixtures in the following concentration
ranges were prepared: acetate and formate ions 2.5 to
20 mg/L, chloride ion 0.5 to 4 mg/L, and TFA ion 5 to
40 mg/L. Then measurements were done using an
electroconductivity detector to create calibration curves,
which are shown in Fig. 2.

a) b) c) d)
Area Area Area Area
4000 15000
Acetate 25000 Formate 20000 Chloride TFA
2
2
2
R =0.998 R =0.998 R =0.998 R =0.998
2
3000 20000
15000
10000
15000
2000 10000
10000
5000
1000 5000
5000
0 mg/L 0 mg/L 0 mg/L 0 mg/L
0.0 5.0 10.0 15.0 20.0 0.0 5.0 10.0 15.0 20.0 0.0 1.0 2.0 3.0 4.0 0 10 20 30 40
Fig. 2 Linearity of Calibration Curves
a) Acetate, b) Formate, c) Chloride, d) TFA
n Analysis of Counterions
In the example shown in Fig. 3, a standard solution of The mole ratio between the principal component and
hydroxocobalamin containing acetate (70 mg/L : counterions was angiotensin Ⅰ : TFA ion = 1:6.2.
0.05 mmol/L) is analyzed. The quantitative value Furthermore, repeated analysis of each of the standard
obtained for the counterions (acetate ions) was solutions resulted in an area repeatability (n = 6) that
3.5 mg/L (0.06 mmol/L). The mole ratio between the was favorable, with acetate at 0.52 % i n
principal component and counterions was hydroxocobalamin and TFA at 1.15 % in angiotensin Ⅰ.
hydroxocobalamin : acetate ion = 1:1.2. The conditions of the analysis are shown in Table 2.
In the example shown in Fig. 4, a standard solution of
angiotensin Ⅰ containing TFA (65 mg/L) is analyzed. The
quantitative value obtained for (TFA ions) was 23 mg/L
(0.2 mmol/L).

uV(×1,000) uV(×1,000)
4.5
˙Peak ˙Peak
1.2
1. Acetate 4.0 1. TFA
3.5
1.0 1
%RSD = 0.52 % 3.0 %RSD = 1.15 %
1
0.8 2.5
2.0
0.6
1.5
0.4 1.0
0.5
0.2
0.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0min 0.0 2.0 4.0 6.0 8.0 10.0 min
Fig. 3 Chromatogram of Hydroxocobalamin Acetate Fig. 4 Chromatogram of Angiotensin Ⅰ TFA Salt

First Edition: May. 2014

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