Chromatography Techniques

Advances in HPLC Instrumentation

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HPLC is a popular method of analysis for herbal products because of its accuracy, precision and not limited by the volatility or stability of the sample compounds. HPLC combined with diode array detector (HPLC-DAD), mass spectrometer (HPLC-MS) have been successfully employed in the qualitative and quantitative determination of various types of phytoconstituents like flavonoids, alkaloids, tannins, triterpenes, glycosides, sterols etc. HPLC methods are used in the determination of drug in biological fluids and pharmaceutical dosage forms. HPLC determination with spectroscopic detection is useful for routine quality control of drugs in pharmaceutical dosage forms and stability studies. Several synthetic molecules also are successfully evaluated both qualitatively and quantitatively. Pharmaceutically important antiviral drugs (lamivudine, stavudine, nevirapine, zidovudine), NSAIDs (acetaminophen, meloxicam, paracetamol, phenylbutazone,), antibiotics (amoxicillin, cefepime, rifampicin), CNS stimulants (amphetamine), betablockers (atenolol), preservatives (benzalkoniumchloride), Diuretics (hydrochlorthiazide), antidiabetics (metformin), anticancer (methotrexate), corticosteroids (prednisone, betamethasone, dexamethasone), anti-allergic (cetrizine), antidepressant (Nitroxazepine, milnacipran), veterinary anaesthetic (ketamine) , anti-inflammatory (sodium osagrel), secretolytic (ambroxol) and several other category drugs were successfully evaluated via HPLC.

HPLC columns are usually packed with porous, or pellicular particles. Pellicular particles are made from polymers, or glass. Pellicular particles are surrounded by a thin uniform layer of silica, synthetic resins, alumina, or an of ion-exchange resin. Partition HPLC uses liquid bonded phase columns. The liquid stationary phase is chemically bonded to the packing material. The packing material is usually hydrolysed silica which reacts with the bond-phase coatings like siloxanes. A chromatographic detector is capable of establishing both the identity and concentration of eluting components in the mobile phase stream. A broad range of detectors are available to meet different sample requirements. Detectors respond to a particular compound only and the response is independent of mobile phase composition and the response of bulk property detectors is dependent on collective changes in composition of sample and mobile phase. Specific detectors are UV-VIS, Photo diode array, fluorescence, and mass spectroscopic detectors. Bulk Property detectors include refractive index, electrochemical and light scattering detectors.