CHROMATOGRAPHY UV and Fluorescence Detectors 1 UV detector

CHROMATOGRAPHY

UV and Fluorescence Detectors 1. UV detector: using a flow cell, most common type 2. Fluorescence detector: Fluorescence is measured after excitation of the eluate with a laser. Employ deuterium, tungsten or xenon lamps with monochromators to choose the optimum λ. A special type of UV detectors: the photodiode array detectors (PDA) which records the spectrum of each solute as it is eluted. The linearity range for this type of detectors is in a 5 orders of magnitude. Adv. : very sensitive Disadv. : response to few analytes. Derivatization of originally nonfluorescing analytes by covalent attachment of fluorophors to analytes either prior to chromatographic separation, or by adding derivatization reagents to the eluate between column and detector. The flow cell 2

Refractive Index Detector A differential refractometer (DRI), or refractive index detector (RI or RID) is a detector that measures the refractive index of an analyte relative to the solvent. Used when detecting substances with limited or no UV absorption. These chemical components included alcohols, sugars, fatty acids, polymers and carbohydrates 3

2. 4. 4 Evaporative light scattering detector (ELSD) commonly used for analysis of compounds where UV detection might be a restriction and therefore used where compounds do not efficiently absorb UV radiation, such as sugars, antivirals, antibiotics, fatty acids, lipids, oils, phospholipids, polymers, surfactants, terpenoids and triglycerides. ELSDs is related to the charged aerosol detector (CAD)

Principles of operation ELSDs analyze solvent after elution from HPLC. As the eluent passes from an HPLC, it is mixed with an inert carrier gas and forced through a nebulizer, which separates the liquid into minute aerosolized droplets. These droplets then pass into a heated drift tube, where the mobile phase solvent is evaporated off. As the mobile phase evaporates, the droplets become smaller and smaller until all that is left is minute particles of dried analyte. These particles are pushed through the tube by the carrier gas to the detection region. In this region, a beam of light crosses the column of analyte and the scattering of light is measured by a photodiode or photomultiplier tub.

Advantages of HPLC - HPLCis more sensetive than normal liquid chromatography. -It requires a smaller amount of the mixture. - It is fast, it takes few minutes to several hours for the separation. - It has a variety of SPand MP. - Apparatus is simpler and easier to use than gas chromatography. - It is applicable to macromolecules and to small molecular weight compounds. Disadvantages - Like GC, it requires special complex and expensive equipment.