Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, April 2, 2016

Chromophores, UV Absorbing for HPLC Analysis

A compound's absorption coefficient relates to its "strength". I find it useful to know which compounds can (and cannot) be easily detected by UV/VIS and a quick analysis of their chemical groups can provide an answer. Please note that the actual measured absorbance maximums will vary depending on the solution that the compound is dissolved in. Beta-Carotene is included as a very interesting structural example because it is composed of long chains of conjugated double bonds (isoprene units) which are cyclised at each end. Here are some other popular examples:

KEY CHROMOPHORE        Absorption MAX (nm)  STRENGTH

acetylide                                    177                           medium
aldehyde                                    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                                        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

Note: 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.

Data supplied from "Instrumental Methods of Analysis"; Willard, Merritt & Dean; D. Van Nostrand Co. Inc., (1965).