3. High Pressure Liquid Chromatography
or
High Performance Liquid Chromatography
What is it?
Separation technique based on solid stationary phase +
liquid mobile phase
How can achieve separation?
By partition
adsorption
ion exchange
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4. CLASSIFICATION OF HPLC:-
1. NORMAL PHASE: Stationary phase: Polar
Mobile phase: Non polar
Eg. Assay of Pilocarpine, Tacopherol, Piperazine
2. REVERSE PHASE: Stationary phase: Non polar
Mobile phase: Polar
Eg. Assay of Nifedipine, Sulphamethoxazole
Partition chromatography is used for hydrocarbon soluble
compound having molecular weight of lesser than
1000gm/mole.
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5. 3. ION-EXCHANGE CHROMATOGRAPHY (IEX)
Based on the different affinities of the ions for the
oppositely charged ions in the resin or adsorbed
counterions in the hydrophobic stationary phase.
Consider the exchange of two ions A and B between the
solution and exchange resin E :
A·E + B B·E + A
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6. 4. SIZE-EXCLUSION CHROMATOGRAPHY (SEC)
SEC is the method for dynamic separation of molecules
according to their size.
The separation is based on the exclusion of the molecules
from the porous space of packing material due to their
steric hindrance.
Hydrodynamic radius of the molecule is the main factor
determining its retention.
In general, the higher the hydrodynamic radius, the
shorter the retention.
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7. HPLC INTRUMENTATION CONSIST OF
Degasser
Solvent Reservoir( HPLC solvent reservoir systems)
Pumps
Pre Guard Column
Sample injection system
Columns
Detector
Recorder and integrators
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9. DEGASSER
Degassing of mobile phase is required because bubble has
property to expand or compress.
Degasser is needed to remove dissolved air
1) By Subjecting the mobile phase under vacuum.
2) By Purging with fine spray of an inert gas at lower
solubility such as Argon and Helium.
3) By heating and ultrasonic stirring.
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11. HPLC SOLVENT RESERVOIR SYSTEMS
These are the glass bottles use to store the mobile phase.
The mobile phase is pumped under pressure from one or several
reservoirs and flows through the column at a constant rate.
Desirable feature in the solvent delivery system is the capability
for generating a solvent gradient.
Filtration is needed to eliminate suspended particles and organic
impurities.
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12. PUMPS
Pass mobile phase through column at high pressure
and at controlled flow rate.
Performance of pump directly affects the Rt,
reproducibility, detector sensitivity.
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13. IDEAL CHARCETRISTIC OF A PUMP
Non corrosive and compatible with solvent.
Provide High pressure to push mobile phase
Provide constant flow rate to mobile phase.
Easy to change for one mobile phase to another.
Should have reproducible flow rate and independent of column
back pressure.
Should not leak & should be easy to dismantle and repair.
High pressure generated by pump 5 October 2012 should not lead to explosio1n3.
14. TYPE OF PUMP USED IN HPLC
1) Reciprocating pump
2) Displacement pump
3) Pneumatic pump
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16. WORKING
Contains reciprocating piston that moves back and forth in
hydraulic chamber.
By the movement of piston solvent flow into the column under
high pressure.
When piston moves backward inlet valve open while exit valve
closes. This result in mobile phase being drawn into the main
chamber (cylinder).
The reduction in volume in main chamber due to forward motion
of piston result in mobile phase moving out of the exit valve
u5n Odcteobre rh 2i0g12h pressure. 16
17. DISADVENTAGE
Pulsed flow which must be damped as they produce a base line
noise on the chromatogram
ADVANTAGES
Generate high output pressure (upto10000 poise).
Ready adaptability to gradient elusion.
Provide constant flow rate.
Pressure generated is so high that any back pressure generated in
the column due to higher viscosity of stationary phase can be
easily overcome.
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19. WORKING
Works on the principle of positive solvent pressure.
Consist of screw or plunger which revolves continuously driven
by motor.
Rotatory motion provides continuous movement of the mobile
phase which is propelled by the revolving screw at greater speed
and pushes solvent through small needle like outlet.
Consist of large syringe like chamber of capacity 250 – 500 ml.
Double syringe pumps have also been developed in which one
piston is delivering the solvent to the column while other one is
refilled from the reservoir.
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20. ADVANTAGES
Flow is pulse free.
Provide high pressure upto 200 – 475 atm.
Independent of column back pressure and viscosity of solvent.
Simple operation.
DISADVENTAGE
Limited solvent capacity
Gradient elution is not easy.
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22. WWOORRKKIINNGG
The driving air is applied, piston moves, inlet closes & outlet
open pushing mobile phase to the column.
Pressure on solvent is proportional to the ratio of piston usually
50: 1.
A lower pressure gas source of 1- 10 atm can be used to generate
high liquid pressure .( 1 – 400 atm )
About 70 ml of the mobile phase is pumped from every stroke.
ADVENTAGES: Pulse free flow & Generates high pressure.
DISADVANTAGES: 1) Limited volume capacity (70 ml )
2) Pressure output and flow rate depends on the viscosity and
column back pressure.
3) Gradient elusion is not possible. 22
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23. SAMPLE INJECTION SYSTEM
Septum injectors
Stop flow Septumless injection.
Rheodyne injector / loop valve type.
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24. SEPTUM INJECTION PORT.
Syringe is used to inject the sample through an inert septum
directly into the mobile phase.
Drawback: - leaching effect of the mobile phase in contact with
septum, which may give rise to ghost peaks.
STOP FLOW SEPTUMLESS INJECTION.
Flow of mobile phase through the column is stopped while
Syringe is used to inject the sample.
Drawback: formation of ghost peak.
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25. RHEODYNE INJECTOR / LOOP VALVE TYPE.
Sample is introduced in the column without causing interruption
to mobile phase flow.
Volume of sample ranges between 2 μl to over 100 μl.
Operation of sample loop.
Sampling mode
Injection mode.
Sample is loaded at atmospheric pressure
into an external loop in the micro volume
sampling valve, & subsequently injected
into mobile phase by 5 October 2012 suitable rotation of the valve. 25
26. COLUMN
Made up of stainless steel or heavy glass to withstand the
pressure.
The columns are usually long (10 – 30 cm) narrow tubes.
Contains stationary phase at particle diameters of 25 μm or less.
The interior of column should be smooth and uniform.
Column end fitting are designed to have a zero void volume.
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27. CLASSIFACTION OF CLOUMN
column
Main column Guard column
Analytical column Preparative column
Standard column
Narrow bore
Short fast column
Micro preparative
Preparative column
Macro preparative
27
A) BASED ON APPLICATION
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28. B) BASES OF COMPONENTS
Bonded phase column
Column where liquid is inpermagneted on solid inert
support
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29. ANALYTICAL COLUMN
STANDARD COLUMN
• Internal diameter 4 – 5 mm and length 10 – 30 cm.
• Size of stationary phase is 3 – 5 μm in diameter.
• Used for the estimation of drugs, metabolites, pharmaceutical
preparation and body fluids like plasma.
NARROW BORE COLUMN
Internal diameter is 2 – 4 mm.
Require high pressure to propel mobile phase.
Used for the high resolution analytical work of compounds with
very high Rt.
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30. SHORT FAST COLUMN
Length of column is 3 – 6 cm.
Used for the substances which have good affinity towards the
stationery phase.
Analysis time is also less (1- 4 min for gradient elusion & 15 –
120 sec for isocratic elusion).
PREPARATIVE COLUMN
Used for analytical separation i.e. to isolate or purify sample in
the range of 10-100 mg form complex mixture.
Length – 25- 100 cm
Internal diameter – 6 mm or more.
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31. TYPES OF PREPARATIVE COLUMN
Micro preparative or semi preparative column
Modified version of analytical column
Uses same packaging and meant for purifying sample less
then 100 mg.
Preparative column
Inner diameter – 25 mm .
Stationary phase diameter – 15- 100 μm
Macro Preparative Column
Column length – 20 – 30 cm
Inner diameter – 600 mm
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32. GUARD COLUMN
They are placed anterior to the separating column.
Serve as a protective factor that prolongs the life and
usefulness of the column.
They are dependable column designed to filter or remove
Particles that clog the separation column.
Compounds and ions that could ultimately cause baseline
drift, decrease resolution , decrease sensitivity and create
false peaks.
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33. BONDED PHASE COLUMN
Here the molecules, comprising the stationary phase i.e. the
surface of the silica particles, are covalently bonded to a silica
based support particles.
The most popular bonded phase, siloxanes, are formed by
heating the silica particles in dilute acid for the day so as to
generate the reactive Silonal group.
- OH OH OH
ו ו ו
- Si – O – Si - O - Si -
5 ו O c t o b e r 2 ו 0 1 33 ו ו 2
34. Silonal group is the treated with organochlorosilane.
These bonded phases are stable between the pH range 2 – 9 and
upto temperature of 80º C.
Bonded phase is made with a linear C 18 hydrocarbon, also
know as ODS (octadecyl silane) bonded phase. Used in
pharmaceutical analysis or separation of less polar components.
An alkyl nitrile column or cyano column which has 12 carbon
atoms with the last atom appearing as a nitrile group (CN),
moderately polar column.
Amino alkyl bonded phase column which is normally C 8, last C
atom bearing NH2 group, Polar column. Use full in separation of
CHO, peptides, amino acids.
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35. Advantages
Can withstand high pressure exerted by mobile phase.
Life of column is more.
No bleeding effect
Disadvantages
Very expensive
Manually can not be fabricated
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36. COLUMN WHERE LIQUID IS INPERMAGNETED
ON SOLID INERT SUPPORT.
These are not use widely now days.
Stationary phase dose not have the strength to stay in the
column on account of the physical forces exerted by the
mobile phase at very high pressure.
Amount of loading on inner support is minimum
Stationary phase starts bleeding out of the column and can
cause resistance to mass transfer.
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37. METHOD OF PACKING
Depends on the mechanical strength & Particle size of the
stationary phase.
Particle size greater then 20 μm – dry packing
Particle size lesser then 20 μm – slurry packing / wet packing.
WET / SLURRY PACKING
Particle size with diameter less then 20 μm can only be placed
wet as a suspension.
Suspension should be stable, it should not sediment, and
agglomentation should be avoided.
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38. DRY PACKING
Particle size greater then 20 μm filled into vertical clamped
column in small quantity.
Deposition is done by tapping or vibrating the column.
Column is unclamped and the tapped on the firm surface to
obtain dense and reproducible packing.
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40. DETECTORS
Based on the application, the detectors can be classified into
Bulk property detectors
Solute property detectors.
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41. BULK PROPERTY DETECTORS
Compare an over all change in physical property of mobile phase
with or without an eluting solute.
These types of detectors tend to be relatively low sensitive and
require temperature control.
e.g. Refractive index detector.
SOLUTE PROPERTY DETECTORS
They respond to a physical property of the solute that is not
exhibited by the pure mobile phase.
These detectors are more sensitive, detect the sample in
nanograms quantity.
e.g. Uv visible detector , Electrochemical detector, Fluorescence
d5e Otcetocbetro 2r0.12 41
42. ULTRAVIOLET VISIBLE DETECTOR
They measure the ability of a sample to absorb light. This can be
accomplished at one or several wavelengths.
A light source deliver a monochromatic parallel light beam
which passes through a cell swept by the column effluent, and
falls on photocell.
Selective in nature, detect only those solutes that absorb Uv/
visible radiation
E.g. alkenes, aromatic compounds and compound having
multiple bonds between C and O, N or S.
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43. BASICALLY THREE TYPES OF ABSORBANCE DETECTORS
ARE AVAILABLE
Fixed Wavelength Detector
Variable Wavelength Detector
Diode Array Detector
FIXED WAVELENGTH DETECTOR
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44. Detectors which do not allow changing the wavelength of the
radiation called fixed-wavelength detectors.
In this, most of the light may be emitted at a one wavelength,
with most single wavelength UV lamps.
Low-pressure mercury lamp emits very intense light at 254 nm.
By filtering out all other emitted wavelengths, utilize only 254
nm line to provide stable, highly sensitive detectors capable of
measuring subnanogram quantities of any components which
contains aromatic ring
The 254 nm was chosen since the most intense line of mercury
lamp is 254 nm, and most of UV absorbing compounds have
some absorbance at 254 nm.
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46. Variable wavelength detector employs a lamp that emits light
over a wide range of wavelengths and by using a
monochromator, light of a particular wavelength can be
selected for detection purposes.
Depending on the sophistication of the detector, wavelength
change is done manually or programmed on a time basis into
the memory of the system.
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47. DIODE ARRAY DETECTOR
It is also a multiwavelength UV detector, but functions on an
entirely different principle.
The UV photo diode-array detector.
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48. FLUORESCENCE DETECTORS
Very sensitive, but very selective.
By definition, it will detect only those materials that will
fluoresce or, by appropriate derivatization can be made to
fluoresce.
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49. Fluorescence occurs when compounds having specific functional
groups are excited by shorter wavelength energy and emit higher
wavelength radiation.
Fluorescence is often collected at right angle to excitation beam.
With all sample cells, scattered radiation from the excitation
source is selectively removed with cut off or band pass filters
placed before photomultiplier tube.
Most important detectors for use in trace analysis both in
environmental and forensic analysis.
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50. REFRACTIVE INDEX DETECTOR OR
DIFFERENTIAL REFRACTOMETER
The detection principle involves measuring of the change in
refractive index of the column effluent passing through the flow-cell.
It responds to any solute whose refractive index is significantly
different from that of the mobile phase.
Principle: it is based on two principles.
Deflection ( deflection type refractometer)
Reflection (reflection type refractometer)
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51. DEFLECTION TYPE REFRACTOMETER.
Measure the deflection of a beam of a monochromatic light by double
prism.
Eluent passes through one half of prism & pure mobile phase to other
half known as reference compartment.
Reference and sample compartment are separated by diagonal glass
divider.
Auto zero is used to set, out put signal to zero when mobile phase is in
both the compartments.
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52. Tungsten lamp provides beam of light collimated through lens
and passes through Eluent and reference compartment.
Reflected by the mirror through the same compartment again
The beam of light is focused on a beam splitter before passing
into the photo detector.
Refractive index of the mobile phase is changed due to the
presence of solute, the beam from the sample compartment is
deflected which produces the change signal that is proportional
to the concentration of solute.
Advantages
Wide range of linearity.
Covers entire refractive index range.
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53. REFLECTION TYPE REFRACTOMETER
Measure change in % of reflected light at glass liquid interface as
the reflective index of liquid changes.
Based on the Fresnel's law of reflection which states
“The amount of liquid reflected at a glass- liquid interface varies
with the angle of incidence and the refractive index of the liquid”
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54. working:
Two collimated beams from the projector (light source & lens)
illuminate the reference and sample cell.
Cells are formed of Teflon gasket, which is clamped between the
cell prism and a stainless steel reflecting back plate.
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55. As the light of beam is transmitted through the cell interfaces, it
passes through the liquid film and imposes on the surface of the
reflecting back plate.
Diffused, reflected light appears as two spots and passes through
the lens and detected by photo detector.
The ratio of the reflected light to transmitted light is function of
refractive index of the two liquid, the illumination of the cell
back plate is direct measure of the refractive index of the liquid
in each chamber
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56. ELECTROCHEMICAL DETECTOR OR
AMPEROMETRIC DETECTOR
It is based on the measurements of the current resulting from an
oxidation/reduction reaction of the analyte at a suitable electrode.
The level of the current is directly proportional to the analyte
concentration
Also called coulometric detector.
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57. RECORDER AND INTEGRATORS
Recorders are used to record the response obtained from the
detector after amplification. They record the baseline and all the
peaks obtained, with respect to time. Retention time for all the
peaks can be calculated.
Integrators are improved versions of recorder with data
processing capabilities. They can record the individual peaks
with retention time, height and width of peak, peak area, etc.
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58. DERIVATIZATION
The most commonly used detector in HPLC is 254 nm UV
detector, many methods have been developed to introduce or
enhance chromophores that will absorb light at this wavelength.
Also, reactions have been developed to produced a fluorophore
for the purposes of fluorimetric detection.
While it is common to derivatize analytes in order to improve
chromatographic properties, the emphasis in this section will be
on derivatization for the benefit of detectability.
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59. Derivatization may be either pre-column or post-column.
PRE-COLUMN: Derivatization-Injection-Separation-Detection.
Ex. Treatment of ketosteroids with 2, 4, DNP,
Benzoylation of hydroxysteroids,
Esterification of fatty acids.
POST-COLUMN: Injection-Separation-Derivatization-Detection.
Ex. Reaction of amino acid with ninhydrin,
Reaction of fatty acids with o-nitrophenol,
Thermal or acid/phenol treatment of carbohydrates.
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60. IDEAL CHARACTERISTICS
The ideal derivatization reaction is rapid, goes to completion,
produces a stable product.
Product has suitable chromatographic & spectral properties.
The unreacted derivatizing reagent should not interfere with the
chromatographic separation.
The derivatization reactions are characteristics of
functional group, their description will be classified according to
functional group.
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61. 1. CARBOXYLIC ACIDS
Ex.
PDBI (O-p-nitrobenzyl-N,N’-diisopropylisourea) &
1-(p-Nitro)benzyl-3-p-tolytriazine also reacts with carboxylic
acids to produce esters.
4-Bromomethyl-7-methoxycoumarin (BMC) reacts with
carboxylic acids to form a fluorigenic product.
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62. 2. ALCOHOL
Activated carboxylic acid derivatives such as acyl chlorides are
the most common reagents.
This reaction gives a product that has a molar absorptivity at 254
nm too low to be analytically useful.
P-nitrobenzoyl chloride, 3,5-dinitrobenzoyl chloride & anisyl
chloride form esters that have much higher molar absorptivity.
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63. 3. AMINES
The same acylating reagents used for alcohol can also be used
for amines.
R-NH2 + R’COCl R’CONHR + HCl
This reaction has been used for the analysis of tobramycin
in serum.
10 & 20 amines react with 7-chloro-4-nitrobenzyl-2-oxa-1,3-
diazole(NBD chloride) to produce a fluorescent derivative by
displacement of 7-chloro group.
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64. 4. ALDEHYDES & KETONES
Nucleophilic addition to a carbon-heteroatom double bond are
most frequently employed for derivatization of carbonyl
compounds.
A prototype reaction is the condensation of a ketone with
2,4-dinitrophenylhydrazine(2,4-DNPH) to form the hydrazone.
5 October 2012 64
65. REFERENCES
B.K.Sharma, Instrumental method of chemical
analysis, GOLE Publishing House, Page no 292-304.
Ashutosh kar, Pharmaceutical drug analysis -2nd
edition, page no 453 456,459,466
Dr.A.V.kasture, Pharmaceutical analysis vol-2, page
no 52, 53.
Elena katz, Roy Eksteen, Peter Schoenmarkers, Neil
Miller, Handbook of HPLC, volume 78, Special
Indian Edition, page no. 536-550.
Munson, Pharmaceutical Analysis, Page no. 76-80
http//hplc.chem.shu.edu/new/hplcbook/detector
5 October 2012 65
