hi evrybody,

I am using the internal standard technique for building a calibration curve for PHB((Polyhydroxybutyrate).My internal standard is benzoic acid.Before GC, I used 2ml 2 mg /mL PHB（in chloroform), 2 ml of methanol containing3% (v/v) concentrated H2SO4 (with 200mg/L internal standard in it). I heated it at 105 degree for4 hours, then added 1 mL water. I took the bottom phase (chloroform part) and dried in in sodium sufate before for GC analysis.

And for GC i used column HP-INNOWAX from agilent. The GC procedure was: inlet200degree,FID detector 250 degree,oven staring with 80 ,holding for 1.5min and then increase to 140degree at speed of 30/min,and then inceasing at 40/min till 240 degree,holding for 5min.

for the first time,i got two peaks for blank and 3 peaks for standards. but after that ,every time i ran GC i got 3 peaks in blank and samples with same time.

i feel so confused what's the problems? and i also ran PHB Without internal standard, there are also three peaks. but for the first time ,there are indeed only two peaks for the blank.and i repeated running the sample that i made the first time, it still only had 2 peaks.

hope someone can help me to analysis what's wrong with it.thanks!