Chromatography Forum

I´ve been used the same methodology to analyze two flavonoids (rutin and quercetin) since last year. First analyzes were correct, however with time, excessive peak tailing arose. Now both blocked frit and split peak are presents in my analyzes including in my standards samples . I changed to another column and the problem persisted. My samples are eluted with methanol and 20ul is injected in HPLC with a flow rate 0.8mL/min. I use C-18 Shimpack VP-ODS 4.6 mm X 25 cm and 5 um of diameter.

Gradient Elution
Water with 0,1% TFA (A) and methanol with 0,1% TFA (B)
0-10 min 50-70%B
10-13 min 70%B
13-14 min 70-100%B
14-16 min 100%B
16-17 min 100-50%B
17-25 min 50%B

Thank you for help me!

Try using and adapting the method below. Always use a guard column (it's cheaper than replacing an analytical column). Even though it's for plants I think you can use it for a dietary supplement (which has a lot of other crap called excipients).

http://www.scielo.br/scielo.php?script= ... 3000100008

The method in that article was the first that I had tried to use, because my research involves de same plant specie. Even adjusting, this methodology was not good for my analyzes. Thanks!

Peak splitting usually occurs when your compound can take two possible paths, then it arrives at the detector at two different times. This can be due to an inefficient column, or a loose fitting in the sample path somewhere. Have you noticed lesser pressure than normal?