Chromatography Forum

Recently I was troubleshooting peak splitting in UPLC H-Class using an Acquity® UPLC HSS T3 1.8 μm, 2.1 x 150 mm PDA e-lambda detector. MP A is a Buffer, MP B is 15% Buffer: 85% ACN. Gradient start at 97% A 3% B, Weak 90% H2O:ACN, Strong 50% ACN. I clean the instrument/column as Manufacturer recommend. After several trials troubleshooting the peak splitting with no results, instrument detector disconnects from Empower. I turned off the detector, then back on and now peak splitting it's not observed. For me makes no sense but I will like to know if someone experiment this and if it is something with detector reset.

The fact that the instrument looses connection to the detector during use must be examined ON-SITE. Please contact someone knowledgeable in your local area to have the instrument configuration looked over (as there are hundreds of reasons why this may happen).

Peak splitting, if that is what you have observed (you did not include any chromatogram and it appears you are new to HPLC so we can not be sure), can be caused by many things. The most obvious causes are due to NOT dissolving the sample into mobile phase OR column fouling, BUT here is a link to an authoritative article on the topic of why it happens:

*"HPLC Peak Splitting. Common Reasons For It " ; https://hplctips.blogspot.com/2016/09/h ... s-for.html

Operating an HPLC system takes many years just to learn the basics (~ 5-years for basic level). A lot of training is required over many years so please consider getting some expert help so you can learn more about this technique and improve your analysis method.

I would compare size of result file to be sure that data acquisition rate is the same.
Regards,
PS

2 things -

Starting a gradient at 97%/3% is just asking for trouble right out of the gate. Do the math and get your A phase made to your starting conditions so you can go 100% A at the start.

Peak splitting also happens a lot when the pKa of the thing getting split is too close to the pH of the MP. A 2 pH unit difference is usually enough to remedy this.