Chromatography Forum

Hi

One of my research work required quantification of Cyromazine and melamine in several aqueous samples. Therefore I used the method in following publication (https://doi.org/10.1093/chromsci/bmt089) and used a 85:15 ratio methanol: water system as a mobile phase and C18 column as non polar stationary phase to perform reverse phased liquid chromatography. HPLC machine used was a shimadzu 20A series. Detector was a UV-Vis with a deuterium lamp. I used the same flowrate of 0.7 milliliters per minute for mobile phase as mentioned in the publication and used hydrochloric acid to balance pH. However results were quite absurd and I have attached a file of the results along with this. There was a broad elute dragging along the whole chromatogram. I am quite new to chromatography. Please help me out.
https://ibb.co/h11mTPN

1. In order to confirm the paper's chromatographic conditions try injecting 10-20 uL of 100 ppm solution of CYR and MEL (do not SPE this solution). Be sure you have a flat baseline before injection!

2. Then run the same chromatogram but use the solution above but after SPE.

3. Since CYR and MEL like water, you may have to use 50/50 MeOH/water as your mobile phase, buffered with pH 3 phosphate buffer (or 0.1% Formic acid since HCl is too strong)!

There is just a drifting baseline on the picture.
MeOH concentration is 15% in the article.

It looks like nothing was injected, or theres no flow through the flow cell.

The mobile phase was made incorrectly: prepare .1% TFA first, mix it with methanol 85:15 v/v. TFA appears to be an IP reagent, so it cannot be replaced with HCl.