Chromatography Forum

Hi everyone,

New here, new to HPLC as well.

Do you recommend using internal standard for HPLC-DAD analysis? No mass spectra analysis will be performed.

We're going to analyse some alkaloids in a plant sample, around 3 to 6 molecules. No extensive sample prep steps (only hot water extraction and dilution in methanol before the analysis).

So, if we manage to get an optimal resolution for all analytes, still having an IS would add benefits for increasing any means of data quality? Does including an IS becoming more common as a standard? I'm just asking so we can think of ordering it together with other reference standard materials.

Thanks a lot in advance!

Hi "GaussianWaves",

my answer comes a little late, but since noone else wanted to respond, I will give my 2 cts...

There are different ways to use internal standards. If you carry them through your whole sample preparation steps, they can correct for errors like varying volumes or losses by adsorption in these steps. But only if the physical properties are (almost) identical to your analytes.

If added to the final extract, they account for variability in instrument response (aka drift). A correction of matrix effects can usually only be reliably achieved by MS-detection and stable isotope labelled standards. You can't be sure that the matrix effect on your int. standard is the same as for your analyte (because of different retention time).

Since the photometric detection (DAD) is very stable, you don't need to correct for instrument drift.

So AFAIK, int. standards added post-extraction are seldom used in HPLC-UV/VIS-detection. But of course if you have to follow some prescribed method or SOP, you have to use them anyhow.

In more detail IS are explained in this article: https://www.chromatographyonline.com/vi ... -be-used-0

Over 40 years with chromatography: we used internal standard assays maybe 5% of the time. Modern HPLC and GC autosamplers have pretty good precision and accuracy.....