HPLC instrument

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

 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 .

 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
1) Reside only on stationary phase ( no migration ) ,
2) Reside only in the mobile phase ( migration with
mobile phase ) ,
3) Distribute between two phases ( differential
migration ) .

 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

 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 .

 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 .

 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

 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 .

 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 .

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

 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 .

 Peak asymmetry has many causes ,
1) Application of too much analyte to the column ,
2) Poor packing of the column ,
3) Poor application of the sample to the column or
solute support interactions .

 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

 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 .

 Good resolution is determined by the following 3
functions :
1) Selectivity ,
2) Efficiency ,
3) 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 ,α .

 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 .

 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 .

 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 .

 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 .

 (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.

 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 .

 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 .

 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 .

 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 .

 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 .

 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 .

 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 .

 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 .

 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

 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.

 Three form of column packing matrices are
available they are :
1) Microporous supports : ( micropores ramify
through the particles which are generally 5 –
10 µm in diameter ),
2) 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 ,
3) Bonded phases : in which stationary phase is
chemically bonded to an inert support such as
silica .

 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 .

 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.

 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 .

 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 .

 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 .

 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 .

 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 it’s pH ,
ionic concentration or polarity this is known as
gradient elution .

 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 .

 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 .

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

 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 .

 Two types of constant displacement pumps are
available :
1) Motor driven syringe type pump ,
2) 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 .

 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 ) .

 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 .

 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 .

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

 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 .

 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 .

 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 .

 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.

 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 (AD&E ,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 .

 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

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

HPLC instrument

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