Chromatography Forum

Hi,

I've been testing out some reagents to find a good internal standard for my samples. By "good" internal standard I just want a nice identifiable peak, since I'm also going to connect our LC to a LC-MS it would be relatively straightforward to identify which peak is my internal standard using MS spectra.

One compound that I'm testing didn't show up on the UV Vis spectrum at all, and I'm wondering if that could be related at all to its low molecular weight (~100g/mol, the compounds I target with my LC have a molecular weight of 500~1500g/mol). I'm aware that the retention time of each compound is related to how polar/nonpolar those compounds are, but was thinking if molecular weight can be related to retention time at all? (For instance, if I was using a GC instead of a LC, I think it would be possible for lighter molecules to evaporate faster and elute pretty quickly? Not sure how that would be the case with the LC. Even in this example the retention time would surely depend on more things than just molecular weight, but just curious)

With UV-Vis detection it's not size that matters but absorbance, which usually depends on having double bonds, preferably several, conjugated double bonds. If it hasn't got any, it won't be detectable.
If you're planning to use LC-MS (I assume electrospray) then the target mustn't be too volatile (otherwise it'll just evaporate; spray chambers are in the business of removing volatile solvents, so if your standard is just another volatile solvent, it will get removed) - and it must have some sort of ionisable functional group (anthracene is great for testing LC systems, not so much for MS).

What analyte did you try to use as the Internal Standard?

lmh wrote:
With UV-Vis detection it's not size that matters but absorbance, which usually depends on having double bonds, preferably several, conjugated double bonds. If it hasn't got any, it won't be detectable.

James_Ball wrote:
What analyte did you try to use as the Internal Standard?

Thanks for pointing out what matters with UV-Vis, I will have to learn about how it works with some literature search... I'm using dimethylglyoxime(dmgH2), I was sure I saw a UV/Vis spectrum of it before and now I can't find it and am wondering if I'm just thinking of with a metal complex of dmgH2. It has two double bonds but not conjugated so it sounds like it's not really ideal for UV-Vis detection.