I have been trying to carry out the Stewart assay on lipid extraction to detect the phospholipid content. I have used the Bligh/Dyer technique to isolate lipids. I tend to do this in triplicates. The problem I am finding is that the first reading is always higher than the second reading, and the second reading is always higher than the third reading. I am using a quartz cuvette to take the readings as I am working with chloroform, I wash the cuvette with acetone. I am trying to create a standard curve, but the readings are not very linear. So, I am wondering if time is an important factor in this assay?
Dear Yanis Pantoja I found a technical note (see enclosed file) in the following discussion: https://www.researchgate.net/post/Can_you_send_Stewart_assay_protocol_for_liposomal_suspensions
it states that the sample needs at least 15-20 minutes. Hope this solves your problem.
Best regards.
PS. I also enclosed the original paper (see enclosed file).
Dear Rob Keller thank you very much for your help, I really appreciate it. I was not waiting 15-20 minutes, and that might explain why the standard curve does not look great.
The chloroform can affect the readings if you are using any plastic based materials in your assay... do you have access to TLC-FID of GC-MS, they will give better results for your phospholipids. If yes let me know and I can give you a method for analysis
If you take reading by spectrophotometry with chloroform or DCM, you should use glass quartz cuvette and take the reding at 15 or 20 degree Celsius due to volatility and follow the same for standard.