Page 25 - Application Handbook - Liquid Chromatography

Application No.L488
News

n Repeatability Table 2 Intra-day Repeatability of Retention Time (n=6)
Due to the large number of peaks that must be Avg. R.T. Std. Dev. %RSD
separated when conducting peptide mapping, a Peak (min) (min) (%)
gradient with a long shallow slope is required. In this Peak a 9.929 0.027 0.271
analysis, the mobile phase B percentage changes from Peak b 24.669 0.047 0.192
0 % to 45 % over 90 minutes, resulting in a slope of Peak c 36.299 0.042 0.117
0.5 %/min. The optimized low-pressure gradient valve Peak d 48.815 0.033 0.068
of the Nexera-i and mixer selection for use with TFA will Peak e 59.864 0.032 0.054
provide repeatable delivery even with a shallow Peak f 74.535 0.043 0.057
gradient.
Tables 2 and 3 show the intra-day and inter-day
repeatability of retention time, respectively. Fig. 3 shows Table 3 Inter-day Repeatability of Retention Time (n=6)
the IgG tryptic digest chromatogram intra-day
repeatability. Selecting the principal peaks from the Peak Avg. R.T. Std. Dev. %RSD
(min)
(min)
(%)
chromatogram (peaks labeled a to f), we checked their
repeatability. We calculated the intra-day repeatability Peak a 9.907 0.016 0.159
based on the results of six repeat analyses. The inter- Peak b 24.708 0.033 0.132
day repeatability was calculated based on average of Peak c 36.355 0.034 0.093
three analyses per day over a period of six days. In Peak d 48.877 0.034 0.069
peptide mapping using the Nexera-i, it is clear that Peak e 59.901 0.027 0.046
good intra-day and inter-day repeatability is obtained. Peak f 74.555 0.036 0.049

uV
a
110000

100000

90000 d

80000
b c
e
f
70000
60000

50000

40000

30000

20000

10000

0
0 10 20 30 40 50 60 70 80 min

Fig. 3 Intra-day Repeatability of Chromatogram of IgG Tryptic Digest

First Edition: Apr. 2015

For Research Use Only. Not for use in diagnostic procedures.
The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu.
The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its
accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the
use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject
to change without notice.
www.shimadzu.com/an/ © Shimadzu Corporation, 2015