Chromatography is a technique used to separate components of a mixture through differential partitioning between a stationary and mobile phase. There are two main forms: planar and column chromatography. High performance liquid chromatography (HPLC) uses column chromatography with a mobile liquid phase. The basis is the distribution coefficient which describes how a solute distributes between two immiscible phases. HPLC is used for analytical separations and employs a pump to deliver the mobile phase through a column containing particles or bonded stationary phase, followed by detection of eluting analytes. Detectors commonly use UV-visible absorption to quantify separated components. Sample preparation and column parameters impact resolution of analyte peaks from complex mixtures.
2. 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. 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 ) .
4. 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.
6. 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. 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. 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. 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. 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. 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 .
12.
13. 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. 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 .
15. 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. 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 .
17.
18. 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 .
19. 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. 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. 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. 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 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. 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. 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. 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.
29. 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. 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 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. 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. 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 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. 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 .
36. 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. 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. 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. 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. 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. 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. 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 .
43. 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. 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 .
45.
46. 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 .
47. 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. 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 .
49. 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. 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. 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. 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. 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. 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. 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.
57.
58. 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. 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 .
60. 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. 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.