KEY CHROMOPHORE Absorption MAX (nm) STRENGTH
acetylide 177 medium
aldehyde (2) 210 strong
anthracene 252 & 375 strong
azido 190 medium
amine 195 weak
benzene 184 & 255 strong
β-carotene 450 medium
disulfide 194 medium
ether 185 weak
ethylene 190 medium
ketone (2) 190 weak
naphthalane 220 & 286 strong
nitrate 270 weak-strong
nitrite 225 weak
nitro 210 strong
oxime 190 medium
thiol 195 weak
thioketone 205 strong
thioether 194 medium
conjugated ring varies strong
Notes:
- Chromophore conjugation is the process that gives rise to multiple spectral peaks (or shoulders) which are very useful in qualitative identification for HPLC (Spectral fingerprinting). For more information on this topic, I recommend a very well written description of UV/VIS spectroscopy fundamentals at this link.
- Other interesting examples: Carbonyl (aldehyde) as found in Acetaldehyde; 293nm. Carbonyl (ketone) such as found in Acetone; 271nm.
Hi Sir,
ReplyDeleteI am working on biogenic amines and will be using o-phthaldialdehye as the derivatization reagent. Will a UV-Vis detector be appropriate for this. Thank you.
NO. OPA is used to create a fluorophore so a Fluorescence detector (FLD) should be used (excitation wavelength ~ 360 nm emission ~ 455 nm). *For best results, a DAD (UV/VIS) detector should be used inline with the FLD.
DeleteMost of our compounds are sugars and the methods we were provided use 205 nm for detection with methanol. Our testing lab in California has been unable to identify the sample compounds using our uplc system. Does this mean we should not use a uplc method or maybe a uplc method with a different detector?
ReplyDeleteMany sugars can be detected at very low wavelength such as 205 nm (5 nm bw), BUT they will be detected only at higher concentration levels as most sugars have little to no chromophores. You also mentioned that your HPLC method uses METHANOL as the RP solvent. Methanol should not be used for this method as its typical low UV limit is around 230 nm rendering it useless for analysis below that range. A solvent such as ACN would be better as, depending on the purity used, should be good to ~ 195-200 nm. Note: Even with ACN in place of methanol, your detection limits will still be very poor, but if the concentration levels found in your samples are high enough, may be fine (run a series of stds to find the limit of detection using your method).
DeleteRegarding your questions about "uplc", please note that "UPLC" is just a trade-name (brand), not a technique. No matter what type of high pressure liquid chromatography column, particle, instrument you use, the technique is always called or correctly known as "HPLC", not UPLC. *Correct terminology is so very important in science. The issues with your proposed (or your client's) method appear to have nothing to do with brand names. The use of methanol as a mobile phase solvent where an analysis of sugars at 205 nm calls into question the training of the person who developed it. No such method should be used without revision to include appropriate UV clear solvents first.