Chromatography Forum

Hello! I am creating a calibration curve for five fatty acid methyl esters (Methyl linoleate, Methyl linolenate, Methyl oleate, Methyl palmitate, Methyl stearate) using HPLC-ELSD. The method follows the IP 590 standard with some modifications. The column is silica, 25 cm, 4.8 mm. The mobile phase is 99.3% hexane in channel A and 0.7% ethyl acetate in channel B. The flow rate is 0.6 mL/min. The ELSD detector has a drift tube at 23 °C and nitrogen gas at 2.2 bar.

I am facing serious reproducibility problems with both the areas and the retention time. The retention time is increasing over time, and the areas are decreasing. Before starting the analyses, I conditioned the system for a long period, and even after several analyses, the retention time keeps increasing.

I would really appreciate some help

Thank you

Not what you asked, but I worked on soaps, fatty acids, and fatty acid methyl esters for over 4 decades. And I always used GC-FID for those. Methyl linoleate, Methyl linolenate, Methyl oleate, Methyl palmitate, and Methyl stearate were easy.

^^ agreed.
GC is usually the way to go for this sort of thing.

That said, your LC attempt could benefit from running premixed MP instead of mixing in a 0.7% B "on the fly" - no matter the style of pump, doing that is asking for baseline noise and other problems.

An ELSD unit requires a LOT of training and experience to use with any HPLC method. They require huge volumes of pure gas (N) to operate and regular drift tube and nebulizer cleaning (as all of the samples you inject bake on to the inside of the detector!). A dirty detector may result in peak area changes, but not retention time drift. We recommend them only for the most experienced chromatographers (> 10 years) with formal training, and only when no other detector is available. *Not designed to be used by new or intermediate users to HPLC. BTW: It is common to experience issues with reproducibility using them as a great deal of work must be spent on optimizing your detector to the proposed method (each detector is different and can not use the same settings from one type to another). Please read this article about how to optimize YOUR ELSD to your analysis method (Note: As you are new user, you did not provide us with any of the needed HPLC info or detector model so please understand that the info in this article can still be universally used to optimize any model of ELSD, but you should get local help from an experienced chromatographer to follow the steps).
"Evaporative Light-Scattering Detector (ELSD) Optimization Procedure. Varex ELSD IIA, Technical Note # 0024."; https://www.researchgate.net/publicatio ... ltext=true

Issues with your column: Your silica column is fouled. Column fouling may cause issues with peak area changes (poor reproducibility) as well as retention time drift (or change in Rt over time). To prevent column fouling, you will need to regularly flush the column with pure IPA, at a low flow rate to clean and recondition it for use. Lipids will not fully dissolve in Hexane (your mobile phase) so will accumulate on the column changing your results. Sample solubility in the injection solution AND in the mobile phase needs to be addressed or you will continue to have problems.Make sure everything is dissolved and filtered.

Here is a link to another paper on, "Common Reasons for HPLC Retention Time Drift, Variation or Change" (http://dx.doi.org/10.13140/RG.2.1.5185.3528) which will provide you with troubleshooting information on common causes of retention time drift (or change). The information applies to all types of HPLC methods.