Any chemical compound could interact with the electromagnetic field. Beam of the electromagnetic radiation passed through the detector flow-cell will experience some change in its intensity due to this interaction. Measurement of this changes is the basis of the most optical HPLC detectors.
Radiation absorbance depends on the radiation wavelength and the functional groups of the chemical compound. Electromagnetic field depending on its energy (frequency) can interact with electrons causing their excitation and transfer onto the higher energetical level, or it can excite molecular bonds causing their vibration or rotation of the functional group. The intensity of the beam which energy corresponds to the possible transitions will decrease while it is passing through the flow-cell. According to the Lambert-Bear law absorbance of the radiation is proportional to the compound concentration in the cell and the length of the cell.
The electromagnetic spectrum is traditionally divided into several regions:
|infrared (IR)||2,500 - 50,000 nm|
|near infrared||800 - 2,500 nm|
|visible||400 - 800 nm|
|ultraviolet (UV)||190 - 400 nm|
Three major regions (IR, visible, and UV) are used in the spectroscopy. In liquid chromatography, IR spectrophotometers have found only limited use. There are few transparent polar liquids which can be used as the mobile phase. On the other hand, spectrophotometers working in the range (200 - 600 nm) are used widely as LC detectors.
UV and visible region of the electromagnetic radiation corresponds to the excitation of the relatively low energy electrons such as pi-electrons, or non-paired electrons of some functional groups. For example, n-alkanes could absorb in the UV region below 180 nm. s-electrons require high energy radiation to get excited and to show absorption of the radiation. But any compounds which have benzene ring will show absorbance at 205-225 and 245-265 nm. The last corresponds to the excitation of conjugated p-electrons of the benzene ring.
The majority of organic compounds can be analyzed by UV/VIS detectors. Almost 70% of
published HPLC analyses were performed with UV/VIS detectors. This fact, plus the relative
ease of its operation, makes the UV detector the most useful and the most widely used LC