I am trying to estimate polyamines from Arabidopsis leaf tissue by RP-HPLC. first I tried to get the correct peak using different polyamines as Putrescine, Spermidine and Spermine along with i used water as blank. I benzoylated the samples using 5% perchloric acid followed by benzoyl chloride method. extracted the samples with diethyl ether and dried the samples completely by speed vac. dried remaining I have dissolve in methanol (200 ul. and ran in RP- HPLC.
used concentration of polyamines were 100mM.
injecting amount 50ul
flow rate 1ml/min
mobile phase acetonitrile 42%
RP-column 4.6*250 mm
one cycle of the run consisted of a total of 60min at a flow rate of 1 ml/min at 30degree detection at 254 nm wavelength. this included 42% acetonitrile for 25min for PA separation, increased upto 100% acetonitrile during 3min, then 100% acetonitrile for 20min for washing, decreased down to 42% acetonitrile during 3min and finally to 42% acetonitrile for 9min.
but I am getting the similar peaks in the standards samples as in the blank instead of correct peak. i am attaching the papers that I used for the estimation and also sharing the peak images.
please send the suggestions for my problem as soon as possible.
It seems as if the derivatization didn't work, likely because you used perchloric acid. The method doesn't say anything about using perchloric acid and it doesn't make sense, either. Acids protonate the amino groups rendering them less reactive towards benzoylization. The method says that sodium hydroxide shall be used which is a base deprotonating the amino groups (and capturing the HCl formed). Maybe try this.
- Badges
- Science topic