Question about Piracetam and its unstable peak.

[ultima_86]

It is a really big trouble. I have prepared every as the following:
Buffer: Na3PO4 0.02M adjust pH 7.0 with H3PO4.
Mobile phase: Buffer : CH3OH = 95:5
I run the HPLC with following conditions:
Flow rate: 0.5ml/min;
Wavelength: 220nm
C8 column 250x4.0
Run time: 20 minute
I use Buffer as Blank and to dissolve my sample.
I weight 250mg Piracetam to 50ml flask, dissolve with Buffer and sonicate in 1 minute then make up to volume with Buffer. After that , I draw 5ml and dilute to 50ml flask with the same solvent then filter it.
And here is my problem:
I often get 2 peaks: one is Piracetam (RT = 9.0) and another is Unknown Impurity (RT = 5.0). Peak due to Piracetam is very stable but the Unknown Impurity isn't even I filter 3 vials from only 1 flask, I always get the disparity in area (1.5 times or even double, triple). And the peak area even is unstable day by day. Sometimes is vey high (30000 - 35000) but sometimes is very low (4000 - 10000). I am sure that I did 100% correctly in procedure. I am looking forward your opinion and any idea helping me stabilize the Unk. Impurity peak area will be appreciated. Thanks for reading and helping!!!
I hope to get solution from you

[Gerhard Kratz]

My first question is:
what column temperature do you have? At pH7 it is critical to have a stable column temperature.
Please check.
Good luck.

[ultima_86]

Sorry for not giving you enough information. I don't use column temperature. My room temperature is about 24 or 25 Celsius.
If my problem is column temperature. So, how many Celsius should I increase for my column temperature?
And one more problem, the buffer seem to be unstable after a few hours at room temperature. In the morning, I use buffer I have prepared to make my sample and get the Impurity's peak area is about 9000 - 14000. But in the evening, with the same buffer the peak area is always higher (20000-45000 sometimes even 80000). Today I tried to use the buffer after preparing as soon as possible and I will tell you the result tomorrow. Thanks for your help!