HPLC.

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HPLC

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

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Instrumental requirements

• Pumps solvent delivery system
• Mixing unit, gradient controller solvent
  degassing
• Injector manual or auto injectors
• Guard column
• Analytical columns
• Detectors
• Recorders integrators

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PUMP solvent delivery system

• Solvents or mobile phases must be passed via
  column _at_ 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

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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

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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

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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

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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

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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

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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)

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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

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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)

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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

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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

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