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HPLC

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Title: 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 .

3
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 ) .

4
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

5
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6
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 .

7
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 .

8
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

9
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 .

10
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 .

11
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 .

12
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13
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 .

14
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 .

15
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

16
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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18
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 .

19
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 .

20
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 .

21
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 .

22
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 .

23
  • (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.

24
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 .

25
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 .

26
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 .

27
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
    .

28
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29
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 .

30
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 .

31
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 .

32
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 .

33
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

34
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.

35
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 .

36
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 .

37
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.

38
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 .

39
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 .

40
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 .

41
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 .

42
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 .

43
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 .

44
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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46
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 .

48
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 .

49
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 ) .

50
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 .

51
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 .

52
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.

53
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 .

54
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 .

55
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 .

56
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57
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58
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.

59
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 .

60
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

61
contd
  • For diagnosis of different porphyrias .
  • Thyroxine , uric acid .
  • Nucleic acid analysis, oliginucleotides ,
    steroids , amino acids , serotonin , measurement
    of isoenzymes .

62
  • (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.

63
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