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HPLC

BIOCHEMISTRY

mprasadnaidu
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HPLC

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  1. M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar. HPLC

  2. Instrumental requirements • Pumps – solvent delivery system • Mixing unit, gradient controller & solvent degassing • Injector – manual or auto injectors • Guard column • Analytical columns • Detectors • Recorders & integrators

  3. PUMP – solvent delivery system • 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. Mixing unit, gradient controller and solvent degassing • 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. Gradient controller • 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. Solvent degassing • 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. Injector – manual or auto injectors • 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. Guard column • 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. Analytical columns • 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. Functional group • 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. Detectors • 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 & integrators • 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. Applications of HPLC • 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

  14. THANK YOU

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