Chromatography Forum

Hello,

I'm a beginner in the method developing world, and could really use your expertise. Currently I'm trying to develop an HPLC method for Alpha Lipoic Acid in soft gel form, using the following, with reference to the USP method:

Column: Waters C18 3.9 mm x 30 cm, L1 packing
Detector: 220 nm PDA
Flow rate: 1.5 ml/min. Injection size: 20 ul
Mobile phase: 0.025 M phosphoric acid:ACN (62:38)
Sample and standard prep.: as stated in the USP method, no modifying at this time.

I did a single injection, for sample and standard. Ran each injection for 30 min. Pressure stable at around 3500 psi, but NO PEAK ! Only something that looks like base line noise running horizontally across ?

I'm afraid there are many places that I went wrong, just could't pinpoint where ? Please share your thoughts. Thanks so much. Hanh

Inject you sample without the column and measure peak area. Make sure that you see a UV signal. Use lower wavelength and more concentrated sample to make sure that you can elute and observe your compound

Make sure you turn-on your detector UV lamp.

I usually analyze alpha lipoic acid in tablet as USP method. I did not any problems with this method. But you can try to increase the injection volume (I injected 50 microl). Pls check again the system or injection module in your HPLC machine