HPLC

1
High Performance Liquid Chromatography

• M.Prasad Naidu
• MSc Medical Biochemistry,
• Ph.D.Research Scholar

2
Introduction

• Chromatography is a physical process whereby
  components ( solutes ) of a sample mixture are
  separated by their differential distribution
  between stationary mobile phases .
• Planar column are two basic forms of
  chromatography .
• High performance liquid chromatography is a form
  of column chromatography .

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contd

• During column chromatography process mobile phase
  carries the sample through the column containing
  stationary phase .
• As the mobile phase flows through the stationary
  phase the solutes may
• Reside only on stationary phase ( no migration )
  ,
• Reside only in the mobile phase ( migration with
  mobile phase ) ,
• Distribute between two phases ( differential
  migration ) .

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contd

• The basis of all forms of chromatography is
  partition or distribution coefficient ( Kd ) .
• Kd describes the way the solute distribute it
  self between two immiscible phases .
• Distribution coefficient is a constant at a
  given temperature for two immiscible phases A B
  .
• concentration in phase A
• Kd
• concentration in phase B

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Column Chromatography

• In column chromatography , the stationary phase
  may be pure silica or polymer , or it may be
  coated onto , or chemically bonded to, support
  particles .
• The stationary phase may be coated into a tube ,
  or it is coated on inner surface of the tube .
• When the mobile phase is liquid it is called
  liquid chromatography ( LC ) .
• When the stationary phase in LC consists of
  smaller diameter particles the technique is high
  performance liquid chromatography .

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principle

• In analytical liquid chromatography the mobile
  phase or eluent , exits from the column passes
  through a detector or a series of detectors that
  produce a series of electronic signals that are
  plotted as a function of time distance or volume
  , the resulting graph is a chromatogram .
• The retention time ( tR ) is the time taken for
  each analyte peak to emerge from the column .

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contd

• Under defined chromatographic conditions tR is
  a charcteristic of the analyte .
• The volume of the mobile phase required to elute
  the analyte under defined chromatographic
  conditions is referred to as retention ( or )
  elution volume ( VR ) .
• VR tR Fc

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Contd

• Eluting solutes are displayed graphically as a
  series of peaks , they are frequently referred to
  as chromatographic peaks .
• These peaks are described in terms of peak width
  , peak height peak area .
• The data represented by the chromatogram are used
  to help identify quantify the solutes .

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contd

• Most important parameter in column chromatography
  is the partition ratio ( or )
  capacity ratio K .
• Capacity ratio has no units it is a measure of
  the additional time the analyte takes to elute
  from the column relative to an unretained or
  excluded analyte that does not partition into
  stationary phase .

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contd

• K tR tM VR VM
• tM VM
• Capacity ratios characterize the column
  performance .
• The success of any chromatographic procedure is
  measured by its ability to separate completely (
  resolve ) one analyte from a mixture of similar
  compounds .
• Peak resolution ( Rs )is related the properties
  of the peaks .

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contd

• Rs 2 ( tRB tRA )
• WA WB
• tRA tRB are the retention times of compounds A
  B respectively , WA WB are base widths of
  peaks for A B , respectively .
• When Rs 1.5 the separation of the two peaks is
  99.7 complete .
• In most practical cases Rs value of 1.0
  corresponds to 98 of separation , are adequate
  for quantitative analysis .

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contd

• Peak asymmetry has many causes ,
• Application of too much analyte to the column ,
• Poor packing of the column ,
• Poor application of the sample to the column or
  solute support interactions .

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contd

• Chromatography columns consists of number of
  adjacent zones each zone is called theoretical
  plate its length in the column is called plate
  height .
• The more efficient the column the greater the
  number of theoretical plates are involved .
• N 16 ( tR/W )2

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contd

• The plate number can be increased by increasing
  the column length, but there is a limit to this
  because the retention time peak width increases
  proportionally L , where as the peak height
  decreases as the square root of N .

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contd

• Good resolution is determined by the following 3
  functions
• Selectivity ,
• Efficiency ,
• Capacity .
• Selectivity is a measure of inherent ability of
  the system to discriminate between structurally
  related compounds .
• Two structurally related compounds differ in Kd
  or K .
• Ratio of partition coefficient of two compounds
  gives relative retention ratio ,a .

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contd

• Efficiency is the measure of diffusion effects
  that occur in the column to cause peak broadening
  over lap .
• Capacity is a measure of the amount of material
  that can be resolved without causing peaks to
  overlap irrespective of actions like gradient
  elution .

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Principle of HPLC

• The limit to the length of the column is due the
  problem of peak broadening .
• The number of theoretical plates is related to
  the surface area of the stationary phase
  therefore smaller the particle size of the
  stationary phase , the better is the resolution.
• The Smaller the paritcle size , the greater is
  the resistance to flow of the mobile phase .

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contd

• The resistance in flow causes back pressure in
  the column that is sufficient to damage the
  matrix structure of the stationary phase .
• The new smaller particle size stationary phases
  that can withstand high pressures caused dramatic
  development in the column chromatography .

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Instrumentation

• The increased resolution achieved in HPLC
  compared to classical chromatography is primarily
  the result of adsorbents of very small particle
  size ( less then 20µm ) large surface areas .
• The smallest gel beads used in gel exclusion
  chromatography are superfine grade with diameters
  of 20-50µm .
• A combination of high pressure adsorbents of
  smaller size leads to high resolution power
  short analysis time in HPLC .

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• (1) Solvent reservoirs, (2) Solvent degasser, (3)
  Gradient valve, (4) Mixing vessel for delivery of
  the mobile phase, (5) High-pressure pump, (6)
  Switching valve in "inject position", (6')
  Switching valve in "load position", (7) Sample
  injection loop, (8) Pre-column (guard column),
  (9) Analytical column, (10) Detector (i.e. IR,
  UV), (11) Data acquisition, (12) Waste or
  fraction collector.

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Solvent reservoir

• Solvent reservoir should have a capacity of at
  least 500 ml for analytical applications , but
  larger reservoirs are required for preparative
  work .
• In order to avoid the bubbles in the column
  detector the solvent must be degassed .
• Several methods are there for degassing
• 1) By warming the solvent ,
• 2) By vigorous stirring with magnetic stirrer ,
• 3) By ultrasonication ,
• 4) By subjecting solvent to vacuum or by bubbling
  helium gas through the solvent reservoir .

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Pump

• Typical requirements for a pump are
• 1 ) it must be capable of pressure outputs of at
  least 500 psi preferably up to 5000 psi .
• The main feature of good pumping system is that
  it can capable of outputs of at least 5x107
  pascals ( 7200 psi ) .
• 2) Pump should have a controled , reproducible
  flow delivery of about 1ml/min for anlytical
  applications up to 100ml/min for preparative
  applications .
• 3 ) it should yield pulse free solvent flow
• 4) It should have a small hold up volume .

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Injection port

• The correct application of the sample on to the
  HPLC column is particularly important factor in
  achieving successful separations .
• Two injection methods are existing
• First method makes use of a microsyringe to
  inject the sample either directly on to the
  column packing or onto a small plug of inert
  material immediately above the column packing .
• The second method of sample injection retains the
  column pressure by use of a loop injector .

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contd

• Metal loop has as fixed small volume that can be
  filled with sample .
• By means of an appropriate valve switching system
  , the eluent from the pump is channelled through
  the loop , the outlet of the loop leads directly
  onto the column .
• Therefore sample is flushed on to the column by
  eluent without interruption of flow to the column
  .

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contd

• Repeated application of highly impure samples
  such as sera , urine , plasma or whole blood are
  preferably deproteinated because they decrease
  the resolving power of the column .
• To prevent the above problem a guard column is
  frequently installed between the injector the
  analytical column .

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Contd

• Guard column is a short column of the same
  internal diameter packed with material similar
  to analytical column .
• The packing in the guard column retains
  contaminating material can be replaced at
  regular intervals .

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Sample preparation

• Sample preparation is essential preliminary
  action in HPLC , particularly for the test
  compounds in a complex matrix such as plasma ,
  urine , cell homogenate .
• For analysis of drugs in biological fluids sample
  preparation is relatively much simpler.
• Sample preparation is done by clean up techniques
  they are
• Solvent extraction ,
• Solid phase extraction ,
• Column switching newer supercritical fluid
  extraction ( under research )
• Derivatization .

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Sample Derivatization

• For HPLC analysis many analytes are chemically
  derivatized before or after chromatographic
  separation to increase their ability to be
  detected .
• Eluted amino acids are reacted with ninhydrin in
  post column reactor , the resulting chromogenic
  species are detected by photometer .

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contd

• Aliphatic amino acids , carbohydrates , lipids
  other substances do not absorb UV can be detected
  by chemical derivatization with UV absorbing
  functional groups .
• Precolumn derivitization for amino acids
  peptides is by phenyl isothiocyanate , dansyl
  chloride for UV column detection .
• Precolumn derivatization for fatty acids ,
  phospholipids is by phenacyl bromide for UV
  column detection .
• Post column derivatization for carbohydrates is
  by orsinol sulphuric acid for UV column
  detection

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Column

• Column is made up of stainless steel .
• Column has to withstand pressures of up to 5.5 X
  107 pascal .
• Straight columns of 15 50 cm length 1
  4mm diameter has flow rate of 2
  cm3/ min.
• Preparative columns have an internal diameter of
  25 mm has flow rate of 100 cm3 / min.

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Matrices Stationary phases

• Three form of column packing matrices are
  available they are
• Microporous supports ( micropores ramify
  through the particles which are generally 5
  10 µm in diameter ),
• Pellicular ( superficially porous ) supports in
  which porous particles are coated on to an inert
  solid core such as a glass bead of 40 µm in
  diameter ,
• Bonded phases in which stationary phase is
  chemically bonded to an inert support such as
  silica .

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contd

• For adsorption chromatography , adsorbents such
  as silica alumina are available as microporous
  or pellicular forms which are suitable for HPLC .
• Pellicular forms have high efficiency but low
  sample capacity therefore microporous supports
  are preferred .
• For partition chromatography bonded phases are
  used .

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contd

• In normal phase liquid chromatography the
  stationary phase is a polar compound such as
  alkylnitrile or alkylamine the mobile phase is
  a nonpolar solvent such as hexane .
• For reversed phase liquid chromatography
  stationary phase is a nonpolar compond such as
  octasilane (OS) or octadecylsilane (ODS), the
  mobile phase is a polar solvent such a water /
  acetonitrile or water / methanol.

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contd

• Cross linked microporous polystyrene resins are
  widely used suitable ion exchange resins for HPLC
  .
• Stationary phase for exclusion separations are
  porous silica , glass , polystyrene or
  polyvinylacetate beads are available in a range
  of pore size .

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contd

• The support for affinity separation are similar
  to those for exclusion separations .
• The spacer arm ligand are attached to the
  supports by chemical bonding .
• Chiral stationary phases contain proteins that
  are composed of amino acids each of which has a
  stereocenter ( except glycine ) commonly used
  are alfa 1 acid glycoproteins ( AGP ) ,human
  serum albumin ( HAS ) .
• Semirigid as well as nonrigid gels have limitted
  role in HPLC stationary phase .

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Column packing

• The major priority in packing of a column is to
  obtain a uniform bed of material with no cracks
  or channels .
• Rigid solids as well as hard gels should be
  packed as densely as possible but without
  fracturing the packing process .
• Most widely used technique for column packing is
  the high pressure slurrying technique .

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Mobile phase

• The choice of mobile phase to be used in any
  separation will depend on the type of separation
  to be achieved .
• Eluting power of the solvent is related to its
  polarity.
• The components of the applied sample are
  separated by the continuous passage of the mobile
  phase through the column , this is known as
  elution development .

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contd

• Column development is of 2 types
• 1)Isocratic elution ,
• 2)Gradient elution .
• Column development using a single liquid as the
  mobile phase is known as an isocratic elution .
• In order to increase the resolving power of the
  mobile phase , it is necessary continuously to
  change its pH , ionic concentration or polarity
  this is known as gradient elution .

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contd

• In order to produce a suitable gradient , two
  eluents have to be mixed in the correct
  proportions prior to their entering the column.
• Gradient elution uses separate pumps to deliver
  two solvents in proportions predetermined by a
  gradient programmer .
• All solvents for use in HPLC systems must be
  specially purified because traces of impurities
  can affect the column interfere the detection
  system especially when measuring absorbance below
  200nm .

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contd

• Purified solvents are available commercially ,
  but even with these solvents 1 5 µm microfilter
  is generally introduced into the system prior to
  the pump .
• All solvents are degassed before use .
• Gassing can alter column resolution interfere
  with continuous monitoring of the effluent .

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Pumping system

• The purpose of the pump is to provide a constant
  , reproducible flow of solvent through the column
  .
• Two types of pumps are available
• Constant pressure pump ,
• Constant volume pump .

47
contd

• Constant pressure pumps produce a pulseless flow
  through the column , but any decrease in the
  permeability of the column will result in lower
  flow rates for which the pumps will not
  compensate .
• Constant pressure pumps are seldom used in
  contemporary liquid chromatography .
• Constant displacement pumps maintain a constant
  flow rate through the column irrespective of
  changes within the column .

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contd

• Two types of constant displacement pumps are
  available
• Motor driven syringe type pump ,
• Reciprocating pump ( most commonly used form of
  constant displacement pump ) .
• All constant displacement pumps have in built
  safety cut off mechanisms , so that if the
  pressure within the chromatographic systems
  changes from preset limits the pump is
  inactivated automatically .

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Detector

• The sensitivity of the detector system must be
  high stable to respond to the low
  concentrations of each analyte in the effluent.
• Most commonly the detector is a variable wave
  length detector based upon UV visible
  spectrophotometry since few compounds are colored
  visible detectors are of limited value .
• Detector is capable of measuring absorbance units
  down to 190 nm wave length has sensitivities as
  low as 0.001 absorbance units for full scale
  deflection ( AUFS ) .

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contd

• Variable wave length detector operates at a wave
  length selected from a given wave length range .
• Thus the detector is tuned to operate at the
  absorbance maximum for a given analyte or set of
  analytes which enhances greatly the applicability
  selectivity of the detector.
• Acetonitrile methanol two widely used solvents
  in reversed phase chromatography have minimum UV
  absorption at 200nm .

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contd

• Most biomolecules like proteins , nucleic acids,
  vitamins , steroids , pigments aromatic amino
  acids absorb strongly in 220 365 nm range .
• Aliphatic amino acids , carbohydrates , lipids
  other substances do not absorb UV can be detected
  by chemical derivatization with UV absorbing
  functional groups .

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contd

• UV detectors have many positive characteristics
  highly sensitive ,
• small sample volumes
  ,
• linearity over wide range
  concentrations ,
• non destuctiveness to sample
  
• suitability for gradient elution.

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contd

• Fluorescence detectors are extremely valuable for
  HPLC because of their sensitivity but the
  technique is limited by the fact that relatively
  few compounds fluoresce .
• Electrochemical detectors are extremely sensitive
  for electro active species .
• The sensitivity of UV absorption , fluorescence
  electrochemical detection can be increased
  significantly by the process of derivatisation ,
  where by the analyte is converted pre or post
  column to a chemical derivative .

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contd

• Diode arrays are used as HPLC detectors because
  they rapidly yeild spectral data over the entire
  wave length range of 190 600 nm in about 10
  milliseconds .
• Incorporation of computer technology into HPLC
  has resulted in cost effective , easy to operate
  automated systems with improved analytical
  performance .

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Contd

• The area or height of each chromatographic peak
  is determined from the stored data in computer
  used to compute the analyte concentration
  represented by each peak .
• Fast protein liquid chromatography this provides
  a link between classical column chromatography ,
  HPLC .
• FPLC uses experimental conditions intermediate
  those of column chromatography HPLC .

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HPLC with Mass spectrometer

• Narrow-bore columns (1-2 mm) are used for in this
  application .
• Liquid chromatography-mass spectrometry (LC-MS,
  or alternatively HPLC-MS) is an analytical
  chemistry technique that combines the physical
  separation capabilities of liquid chromatography
  (or HPLC) with the mass analysis capabilities of
  mass spectrometry.

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Application of HPLC

• HPLC has had big impact on separation of
  oligopeptides proteins .
• FPLC a modified version useful in separation of
  proteins .
• HPLC coupled with electrochemical detector is
  useful in assay of catecholamines ,vitamins (ADE
  ,niacin , thiamine) antioxidants .
• HPLC has role in quantification of various
  hemoglobins in hemoglobinopathies .
• HPLC coupled with MS is useful in measuring
  cortisol in blood saliva .

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contd

• HPLC is useful in cytokine measurement .
• Useful in assay of HbA1c .
• Useful in assay of fructosamine .
• 5 hydroxy idole acetic acid serotonin can be
  assayed.
• The pharmaceutical industry regularly employs
  Reverse Phase HPLC to qualify drugs before their
  release.
• Assay of plasma urinary catecholamines , plasma
  urinary metanephrines

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contd

• For diagnosis of different porphyrias .
• Thyroxine , uric acid .
• Nucleic acid analysis, oliginucleotides ,
  steroids , amino acids , serotonin , measurement
  of isoenzymes .

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• (1) Solvent reservoirs, (2) Solvent degasser, (3)
  Gradient valve, (4) Mixing vessel for delivery of
  the mobile phase, (5) High-pressure pump, (6)
  Switching valve in "inject position", (6')
  Switching valve in "load position", (7) Sample
  injection loop, (8) Pre-column (guard column),
  (9) Analytical column, (10) Detector (i.e. IR,
  UV), (11) Data acquisition, (12) Waste or
  fraction collector.

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