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High Pressure/Performance Liquid

Chromatography
Introduction
Principle/Theory
Instrumentation
Application

-Developed in last 1960s & early 1970s.
-Most widely used of all analytical separation techniques.
-It is used for purifying ,identifying & quantifying the individual components of the mixture.
-Based on conventional liquid column chromatography.

1)Liquid-solid. 2)Liquid-liquid

3)Ion exchange. 4)Size exclusion.

Working principle/Theory

i)Adsorption. ii)Partition( normal/reversed phase).
iii)Ion exchange. iv)Size exclusion.
-Facts that brought about the difference:
i)Column packing material & particle size(3-5m) in contrast to earlier(30-70m) .
ii)Design of chromatographic equipments.
-Close packing of small particle in column reduces flow rate.
-To maintain suitable flow , high pressure should be applied.
-Better and faster separation is result.
-Hence the name given High performance or High pressure.

Matches with gas chromatography in the following respects:
i)High resolving power.
ii)Speed of separation.
iii)Continuous monitoring of the column efficiency.
iv)Accurate/precise quantitative measurement.
v)Reproducible analysis using the same column.
vi)Automation.
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Versatility over gas chromatography:

i. Not limited to volatile & thermally stable sample.
ii)Wider choice of mobile & stationery phases.
Instrumentation:
Mode of operations
i)Isocratic ii)Gradient

i)Isocratic:
One particular mobile phase composition is used through out the analysis
ii)Gradient:
Mobile phase composition is altered gradually to give gradient elution.

1)Solvent reservoir
2)Pumps
3)Sample injection system.
4)The column.
5)The detector.
6)Recorder
7)Data handling device & microprocessor control.

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1)Solvent reservoir:
-One or more glass reservoirs( 500ml or more
capacity).

-Provisions are made to remove dissolved gases & dust from the liquids.
-Dissolve gas can lead irreproducible flow rates & band spreading.
-Both dust ,bubbles interfere performance of column detector.
2)Pumps:
-Required to deliver a constant/pulsate free flow of mobile phase.
-Should pressurise the mobile phase from 0.1- 55MPa (14.6-8000psi).
-400-1500 psi for analytical work.
-Flow rate 0.01-10ml min
-1
.
-Two types of pumps are in use:
a)Screw driven syringe type.
b)Reciprocating type.
b)Reciprocating type.
-It is commonly used.
(dual & triple head reciprocating type ~ pulse-free flow)
-Pulse causes excessive noise at high levels of sensitivity & low pressure may cripple the detection small
quantity of sample.


3)Sample injection system:
a)Sample is directly injected into column (through a self-sealing septum). (old technique)
b)Sample is deposited in a loop(10-20l)then swept by a valving action into column by the mobile phase.
& transferred immediately before the column inlet.
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4)Column
-High quality stainless steel polished internally to a mirror finish.
-Standard analytical column:
Int.Dia: 4-5mm, L=10-30cm

-Shorter column:
Int.Dia.:4-5mm, L= 3-6cm

-Packing material size:3-5m
Partition HPLC:
a)Normal phase: Mobile phase is less polar , hence the least polar solute is eluted first.

-Nowadays polar phases are chemically bonded to silica.
Si-(CH
2
)
3
-O-CH
2
-CH-CH
2 ,
Licrosorb Diol.

OH OH
Si-(CH
2
)
3
-CN e.g, Bondpak CN

Si-(CH
2
)
3
-NH
2 ,
e.g., Polygosil NH
2


b)Reversed-phase chromatography:
(More polar mobile phase), hence the most polar solute is eluted first
-Silica is chemically bonded to less polar functional group through (Si-O-Si-C).
-Surface silanol gr. of silica reacts with an organochlorosilane reagent.

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-Where, R= C
6
H
13
(hexyl), C
8
H
17
(Octyl), C
18
H
37
(Octadecyl).
-The most frequenty used bonded phase is C-18 in pharmaceutical analysis.
Retention or elution of the solute is controlled by:
(in reversed phase partition chromatography)

1)Composition of mobile phase& flow rate.
2)pH of mobile phase(ion suppression).
3)Ion pairing agent.
Ion exchange HPLC
-Packing materials are based on the cross-linked polystyrene-divinylbenzene resins
or
ion exchange residues chemically bonded to silica.
Size exclusion HPLC(Gel permeation)

-Material used are crossed-linked polystyrene divinylbenzene resins
or silica microspheres(5-15m diameter).
-Solutes are separated according to their molecular size & shape
-Larger molecules are eluted first.
5)Detectors:
-are to monitor the mobile phase as it emerges from the column.
-able to give a linear response over a wide range concentration range.
-Detection of solute depends upon :
i)Bulk property ii)solute property
Refractive index UV/absorption,
Fluorescence,
Electrochemical
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Types of detectors:
i)UV detector(Variable wavelength/ photo diode array)
-Most widely used in HPLC.
-Normally operates in UV region.
-Comprises

Light source,

A dispersing device to select radiation of
appropriate wavelength, flow cell to measure
absorbance of eluate,

Photomultiplier or photo diode array (PDA)
detector for the measurement of intensity
of transmitted light.
-Both single & double beam instruments are available.
-In variable wavelength detector any wavelength along 190-360nm can be selected with the help of grating
monochromator.
-Photodiode array(multichannel) detector is of the very advanced type.
-In this, polychromatic light from UV source is passed through the flow cell.

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-The emergent radiation is diffracted by a grating & then falls on an array of photodiodes(1042 diodes).
-Each diode receives narrow wavelength band.
-A microprocessor scans the array of diodes many times a second.
-The spectrum so obtained may be displayed on a screen.
ii)Fluorescence detectors:
-Sensitivity depends upon the fluorescence property of eluate in cell.
iii)Refractive index detector(Differential)
-It is the closest approach to the universal detector.
-It responds to the bulk property i.e. refractive index of the mobile phase as it leaves the column.
-The presence of solute changes the refractive index.
-It is not applicable in gradient elution type HPLC.
-It is suitable for detection of carbohydrates, alcohols other which do not show other specific property
for their specific detection.
iv)Electrochemical detector:

-Based on standard electrochemical principles, i.e amperometry, voltametry & polarography.
-Very sensitive for oxidising & reducing substances at the suitable potential.
-Useful in the assay of low level of endogenous catecholamine in biological tissues, pesticides,
tryptophan derivative & many drugs.
6)Recorder & 7)Data handling device & microprocessor control
-Peak area, height vs. retention time displayed automatically as a chromatogram
-Peak area Vs time required for evaluation.
-The chromatogram can be stored in computer or be printed as appropriate.
Application:
-It is impossible to review briefly the range of application of HPLC, however-

i) Qualitative

ii) Quantitative
i)Qualitative;

Identification:
a)Comparing the retention time of Sp. & standard reference substance for identification.
b)If diode array detector is available then it is possible to obtain a spectrum with Abs.Vs wavelength. This
further consolidate the proof gathered in (a).
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Detection of the impurities & related substances:
-4-epianhydrotetracycling, anhydrotetracycline in tetracycline.
-Additional substances in ciprofloxacin & in glimepiride.
ii)Quantitative

-Chromatography is mainly a resolving technique,
-However it is useful in quantitative analysis.
Peak area/height concentration.
-Peak area is preferred over peak height.
-Peak area is relatively independent of mobile phase composition.

Techniques of concentration calculation:
a.Direct comparison(single point standardisation.
-Chromatograms of sample & standard solution are developed one by one.
-Sample concentration is calculated comparing the peak area of the standard solution.
b.Calibration by external standards.
c.Calibration by internal standards.
Quantitative pharmaceutical analysis:

a)Quality control testing of drugs & medicines for compliance with specifications.
e.g.
Omeprazol(RP-18/Phosphate Buffer ) at 302nm ,
Paracetamol(RP-18/Sodium butane sulphonate in water : methanol: Formic acid) at 243nm

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b)Stability studies.

c)Therapeutical monitoring, drug metabolism, and pharmacokinetic studies.
d)Increasingly is being used for the assay of medicines in natural products, e.g. antibiotics &
hormones.

-It reduces dependence on biological assays.

e)It is also effective in the separation of geometrical
isomer & racemates, e.g

-E-isomer in Tamoxifen citrate.

-In the limitation of Diasteroisomers in Fenoterol HBr