Hplc instrumentation
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This document provides an overview of the key components and functioning of high performance liquid chromatography (HPLC). It describes the main units including pumps, mixing chambers, injectors, columns, detectors, and recorders/integrators. It explains how each component works and its role in the HPLC separation process. Some examples of applications of HPLC are also listed such as quantitative and qualitative analysis of compounds, isolation of drugs, stability studies, and more.
Hplc instrumentation
- 1. M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.
- 2. Pumps – solvent delivery system Mixing unit, gradient controller & solvent degassing Injector – manual or auto injectors Guard column Analytical columns Detectors Recorders & integrators
- 3. Solvents or mobile phases must be passed via column @ high pressure (1000 – 3000psi) b/cos the particle size of stationary phase is few µ (5-10 µ) Diff types of pumps available Mechanical pumps operate with constant flow rate & uses sapphire piston This type of pump is used in analytical scale Pneumatic pumps operate with constant pressure & use highly compressed gas The solvents used must be of high purity preferably HPLC grade & filtered through 0.45 µ filter Check valves: to control the flow rate of solvent & back pressure Pulse dampeners: to dampen ( make slightly wet) the pulses
- 4. 2 types of mixing units Low pressure mixing chamber which uses He for degassing solvents High pressure mixing chamber does not require He for degassing solvents Mixing of solvents is done either by Static mixer: packed with beads Dynamic mixer : uses a magnetic stirrer & operates under high pressure
- 5. In an isocratic separation, mobile phase is of same polarity throughout the process In gradient elution tech, the polarity of the solvent is gradually increased & hence the solvent composition has to be changed. Hence a gradient controller is used when two or more solvent pumps are used for such separations
- 6. Several gases are soluble in org solvents When solvents are pumped under high pressure, gas bubbles are formed – interfere the separation process 3 methods 1.Vacuum filtration: which can remove the air bubbles. But is not always reliable & complete 2. He purging: by passing He through the solvent.Very effective but He is expensive 3. Ultrasonication: by using ultrasonicator, which converts ultra high frequency to mechanical vibrations – this causes the removal of air bubbles
- 7. 1. Septum injectors: injecting through a rubber septum. Not common – has to withstand high pressure 2. Stop flow (online): in which the flow of mobile phase is stopped for a while & the sample is injected through a valve device 3. Rheodyne injector ( loop valve type): the most popular injector. This has a fixed volume loop like 20 µl or 50 µl or more Injector has 2 modes 1. load position: when sample is loaded in the loop 2. Inject mode: when the sample is injected
- 8. It has a very small quantity of adsorbent & improves the life of the analytical column Also acts a a prefilter to remove particulate matter, if any & other material GC has the same material as that of analytical column GC does not contribute to any separation
- 9. The most imp part of the HPLC tech which decides the efficiency of separation Column material: Stainless steel ( mostly used), glass, polyethylene and PEEK (poly ethylene ether ketone) (latest) Column length: 5 cm to 30cm Column diameter: 2mm to 50 mm Particle size: 1 µ to 20 µ Particle nature: spherical, uniform , porous materials are used Surface area: I gm of stationary phase provides surface area ranging from 100-860 sq.m ( an aveg of 400sq.m)
- 10. The functional group present in stationary phase depends on the type of chromatographic separation In normal phase mode – contains silanol groups ( hydroxy group) In reverse phase mode – C18 – Octa Decyl Silane (ODS) column C8 – Octyl column C4 – Butyl column CN – Nitrile column NH2 – Amino column
- 11. 1. UV- detector: used based upon the light absorption characteristics of the sample 2 types of this detector are available a) fixed wavelength detector: which operates at 254 nm where most drug compounds absorb b) variable wavelength detector: which can be operated from 190nm to 600nm 2. Refractive index detector: non-specific or universal detector – not much used- low sensitivity and specificity 3. Flourimetric detector: more specificity & sensitivity Demerit: some compounds are not fluorescent 4. Conductivity detector: 5. Amperometric detector: 6. Photodiode array detector( PDA detector)
- 12. Recorders are used to record the responses obtained from detectors after amplification They record the baseline & all the peaks obtained with respect to time (Rt) But the area of the individual peaks cannot be known Integrators: improved version of recorders with data processing capabilities Rt, height and width of peaks, peak area, % area Integrators provide more information on peaks than recorders
- 13. Qualitative analysis Checking the purity of a compound Presence of impurities Quantitative analysis Multicomponent analysis or determination of mixture of drugs Isolation & identification of drugs Isolation and identification of mixture of compounds Biopharmaceutical & pharmacokinetic studies Stability studies Purification of compounds Environmental applications Forensic applications Biochemical separations Biotech, food analysis