problem regarding poor resolution of glycine peak in HPLC

[avinash lomash]

hi there.

i am a beginner and operating agilent HPLC-1200 series analysing amino acids with column, 5 um, 2.1x200 mm. i am facing a serious problem when peaks of histidine and glycine merges, sometimes in standard also,but what to do in samples.. i am using TEA & THF IN BUFFER BUT STILL GETTING POOR RESSOLUTION .

how can i quantitates both the peaks, how authentic is to quantitate manually using peak split tool.

thanks
AVINASH

[upamaniah]

hi Avinash,
what is the column chemistry you are using, detector and what are other amino acids present in sample.
Vijay

[avinash lomash]

hello vijay,
conditions are as below......

Column= amino acid column 5 um, 2.1x200 mm
Derivatizing agents= OPA & FMOC
Detector= FLD (fluorescence detector)
Plasma , urine & CSF samples are diluted with N/10 HCL in ratio 1:3, but not standard
Mobile Phase= mobile phase A(sodium acetate trihydrate+7.2+methanol+water)
mobile phase B(sodium acrtate trihydtate+water+TEA+ph=7.2+THF)
Run time= 25 min. with mobile phase A+ 5 min post run with mobile phase B

Pure amino acids standard of 25 pico mol contains 17 amino acids =
aspartic acid
glutamic acid
serine
histidine
glycine
threonine
alanine
arginine
tyrosine
cysteine
valine
metheonine
isoleusine
leusine
lysine
proline

of which histidine and glycine are merged.

[upamaniah]

Dear Avinash,
thanks for sharing more info, check with shodex.com for more info, one link given below.
http://www.shodex.com/english/dc010914.html
if you need the column, please write to me vlvijayaragavan@inkarp.co.in
V.L.Vijayaragavan
Inkarp Ins Pvt Ltd.
9500015541
Chennai, India