Peptide synthesis can result in numerous peptide products because of deletions, truncations, and side reactions. The quality of every peptide synthesized by UWBC is therefore analyzed by high pressure liquid chromatography (HPLC) and mass spectrometry.
The HPLCs at UWBC have photodiode array detectors (PDA) which allow us to monitor several UV wavelengths and infer the identity of chromaphores on the peptide. For example, removal of FMOC can be problematic for some peptides, so we frequently monitor at 260 nm. The following table consists of wavelengths we use to infer the presence of different chromaphores on the peptide.
Amide bond
| 215 nm
|
Fluorenylmethyoxycarbonyl (FMOC)
| 260 nm
|
| Fluorescein | 445 nm
|
| Rhodamine | 555 nm
|
| Coumarin | 370 nm
|
| Dabsyl | 510 nm
|
| Benzophenone | 265 nm
|
| Dinitrophenol | 360 nm
|
para-nitro phenylalanine
| 355 nm
|
4,4-dimethyl-2,6-dioxocyclohex-1-ylidene (Dde)
| 297 nm
|
| tetramethylrhodamine | 558 nm
|
Our mass spectroscopy is done at UWBC Mass Spec facility. Check out their website for instrumentation and rate information. Delta mass is a useful website which provides clues to the cause of loss and gain of mass in a peptide. The site includes common side chain protecting groups and amino acids.