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CONTENTS
INTRODUCTION TYPES PRINCIPLE INSTRUMENTATION PARAMETERS APPLICATIONS

INTRODUCTION
HPLC -

Fast growing analytical technique

The word HPLC is coined by late.prof.Csaba harvath in 1970. It is defined as

High

performance/ Pressure/ price Chromato Liquid graphy

HPLC
It is also termed as High speed liquid chromatography

or High efficiency liquid chromatography

Mobile phase is forced through the column at high

pressure (>5000 p.s.i) by using a pumping system.

Its simplicity,sensitivity,specifity,selectivity make it ideal

for analysis of many drugs.

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TYPES OF HPLC
y BASED ON MODES OF CHROMATOGRAPHY

NORMAL PHASE

REVERSE PHASE

BASED ON PRINCIPLE OF SEPARATION

ADSORPTION

PARTITION

ION EXCHANGE

SIZE EXCLUSION

NORMAL PHASE MODE: Stationary phase is polar. Mobile phase is nonpolar. Stationary phases - SiO2, -NH2 ,-CN, -NO2, Al2O3 ,-Diol. Mobile phases - non polar organic solvents hexane, heptane, chloroform.

SILICA GEL Silica gel is saturated with silanol (Si-OH) groups. These silanol groups represent the active sites in the stationary phase. OH OH OH Si-OH = Silanol group

-Si-o-Si-o-Si-

Si-o-Si = Siloxane group

Non polar compounds travels faster, elutes first.

REVERSE PHASE MODE: Stationary phase is nonpolar. Mobile phase is polar. Stationary phases - n-octadecyl (RP-18), n-octyl (RP-8) Hexyl(C6) , Pentyl (C5),Butyl (C4) Mobile phases - methanol, acetonitrile, buffers

OCTYL & OCTADECYL GROUPS

The polar nature of silica is reversed i.e. polar to non - polar by incorporating hydrophobic C8,C18 groups into silanol . Chemically bonded octadecyl silane(ODS) widely used stationary phase.

CH3 Si-o-Si-R CH3 R=Hydrophobic carbon chain

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ADSORPTION (liquid-solid) The interaction between solute and surface of adsorbent. Mechanism: The competition between the solute molecule and mobile phase molecule for active adsorbent site is the driving force for separations Adsorbents: Silica gels are more acidic, good for separation of basic materials. Alumina gels are more basic, good for separation of acidic materials
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Eg: Phenol has both hydroxyl(-OH), aromatic groups. -OH groups have more adsorption capabilities than aromatic groups Since OH groups are more polar and capable of hydrogen bonding.

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PARTITION (liquid-liquid) The solid support is coated with liquid stationary phase Mechanism: The relative distribution of solutes between the two liquid phases determines the separation.

Mobile Phase

Stationary Phase

Solvent

Bonded Phase

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ION EXCHANGE Separation is by reversible exchange of ions between the ions present in the ion exchange resin and those present in the liquid mobile phase. TYPES OF ION EXCHANGE RESINS: Cation exchange resin Anion exchange resin Cation exchange resin: It posses ve charged groups such as sulphonic ,carboxylic groups and those will attract +ve charged molecules. Eg: resin-SO3H +Na+ resin-SO3Na + H+
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Anion exchange resin:

It posses +ve charged groups such as amine, quaternary ammonium groups and those will attract ve charged molecules. _ Eg: resin-N(CH3)3OH +Cl resin-N(CH3)3Cl +OH

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SIZE EXCLUSION The solute molecules are excluded on the basis of their hydrodynamic volumes i.e.their size and shape. Larger molecules pass through intercellular spaces between the particles and elutes first. Smaller particles diffuse through the particles and elutes later. Column packing materials/Gels: Dextran (sephadex) Agarose (sepharose,biogel-A) Poly acrylamide (biogel-P) Polystyrenes (bio beads-S)

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PRINCIPLE The resolving power of a chromatographic column increases with column length and the number of theoretical plates per unit length. Smaller the particle size of stationary phase ,the better the resolution. But smaller the particle size ,the greater the resistance to eluant flow.

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INSTRUMENTATION
COMPONENTS: Solvent reservoir (mobile phase container) Solvent delivery system (pump) Sample injection system Pre column Guard column Column (heart of HPLC) Detector Read out device(recorder)

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BLOCK DIAGRAM OF HPLC

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HPLC SYSTEM

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SOLVENT RESERVOIR
It is made up of glass or stainless steel capable of holding 1 lit of solvent . It should be inert. Solvent degassing: It is the process of removing dissolved gases from mobile phase before and during use. Degassing methods: Sparging Heating Stirring Vacuum Sonication Filtration
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Sparging:
Sparging with N2 or He gases remove background absorbance

on a UV detector and quenching(decrease in fluorescent intensity) due to dissolved oxygen.

Heating :
Low levels of heat raise the partial pressure of the solvent there

by reduce the solubility of gas in solution Disadvantage :

Not suitable for when organic solvents present.
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Vacuum:
Vacuum reduces pressure on surface of solvent The mass of gas in solution is proportional to the partial

pressure of the gas at the surface.

So, as pressure reduced, mass of gas in solution also reduced.

Sonication:
Ultrasonicator converts the ultra high frequency to mechanical

vibrations.
These high energy sound waves cause aggregation of bubbles

which float and dissipate

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Filtration :
Vacuum is used in conjugation with 0.45 sintered glass

membrane filter ,a gallon of solvent filtered and degassed in 8 min.

PUMPS
Pump is a device designed to deliver the mobile phase at a

controlled flow rate

There must be no pulses of flow and the output of atleast

5000 psi.
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Pumps types: Constant pressure pumps Constant displacement pumps Constant pressure pumps: These operate by introduction of high pressure gas into the pump. Eg: pneumatic pumps Constant displacement pumps: A motor driven syringe pump where a fixed volume of solvent is forced from pump to column by a piston driven by a motor to give uniform flow rates. Eg: Reciprocating pumps
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PUMPS:

Pulse dampners: These are used to minimize the pulsating flow which is originates from reciprocating pump. Check valves: The entry of solvent from reservoir to pump & exist of solvent to column is regulated by check valves. These control back pressure & flow rate of solvent.
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Mixing chamber:
It mix the solvents in different proportions by using static

mixer or dynamic mixer Mobile phase:

Depending on type of separation, mobile phase choose.

Elution techniques:
Isocratic elution Gradient elution/ solvent programming
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ISOCRATIC ELUTION - In this mode, the mobile phase, either a pure solvent or a mixture, remains the same throughout the run. - Fixed mobile phase strength is used.

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GRADIENT ELUTION
The mobile phase composition changes during the separation. The elution strength of the mobile phase is increased to elute

the more strongly retained sample components. Types:

High pressure gradient elution Low pressure gradient elution

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HIGH PRESSURE GRADIENT ELUTION The mixer is downstream of the pumps; thus the gradient is created under high pressure.

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LOW PRESSURE GRADIENT ELUTION It is designed to mix multiple streams of solvents under low pressure, ahead of a single pump.

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SAMPLE INJECTION SYSTEM

Injector: Apparatus for accurately injecting predetermined amount of sample with mobile phase stream. It can be either manual, auto sampler Types: Septum injectors: Sample solution is injected through a self sealing rubber or teflon disc using a l syringe. Rheodyne injector(loop valve injector): It has fixed volume loop like 20 l or 50 l It has two modes load position and inject mode
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AUTO SAMPLER

RHEODYNE INJECTOR

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Pre column: A small column placed between injector & guard column It removes particulate matter from mobile phase Guard column: A small column placed between the pre column & analytical column It protects the analytical column from contamination of the sample particulate & from strongly retained species.

Guard column

HPLC column

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Analytical column:
Columns are made up of stainless steel and are manufactured to with stand pressures upto 8000 p.s.i Straight columns of length 20-50 cm, diameter 1-4 mm used It is heart of HPLC where separation takes place

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COLUMN PACKING
1.Microporous support: Microporous ramify through the particles of which are generally 5-10 m in diameter Eg: Silica, alumina used in adsorption chromatography 2.Pellicular (superficially porous)support: Porous particles coated onto an inert solid core Eg: Porous glass beads of about 40 m in diameter used in partition chromatography

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Bonded phases: Stationary phase chemically bonded onto an inert support. Eg: liquid phase covalently bonded to the supporting material silica or silicone polymer High pressure slurring technique Used for column packing A suspension of packing is made in a solvent of equal density to the packing material The slurry is pumped at high presure onto a column with a porous plug at its outlet.

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PACKING MATERIALS
TYPE silica silica silica alumina Bonded phase Pellicular pellicular NAME Porasil Lichrosorb-si zorbax Lichrosorb Alox Bonda pak/c-18 zipax corasil 25-37 37-50 spherical spherical
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(m)

SHAPE spherical irregular spherical irregular

37-75 5,10,20,30 5 5,10,20

DETECTOR
An electronic device that quantitatively discerns the presence of separated components as they elute. It monitors the mobile phase as it merges from the column.

TYPES: Bulk property detectors Solute property detectors Bulk property detectors: These detectors are based on differential measurement of property which is common to both solute and solvent. Eg : Refractive index detector (universal detector) conductivity detector, dielectric constant or density.
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Solute property detectors:

These are based on the physical property of solute which is not

exhibited by the pure solvent Eg : UV-Visible detector, fluorescence detector electro chemical detector, photo diode array detector UV-Visible detector
UV/Visible Detector is a versatile, dual-wavelength

absorbance detector for HPLC.

It is based upon light absorption characteristics to the sample.

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Two types: Single/fixed wavelength detectors Multi/variable wavelength detectors Single/fixed wavelength detectors: The absorbance generally measured at 254 nm Multi/variable wavelength detectors : It operates in the region between 190-600 nm

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Refractive index detector:

Non specific and universal detector It is differential refractometer which respond to change in the bulk property of the refractive index of the solution of the component in the mobile solvent system Low sensitivity and specificity.

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Electro chemical detector:

It is based on standard electro chemical principles involving amperometry ,polarography These detectors are very sensitive for substances that are electro active i.e. those that undergo oxidation or reduction at a suitable potential.

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Fluorescence detector: Their sensitivity depends on the fluorescence properties of the components in the eluate. The disadvantage is that some compounds are not flourescent

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Conductivity detector: It is based upon electrical conductivity the response is recorded It is used when the sample has conducting ions like anions and cations

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Photo diode array detector: These are microprocessor-controlled photo diode array spectrophotometers in which light from an uv source passes through the flow cell into a polychromator which disperses the beam so that the full spectrum falls on the array of diodes. The resulting spectra is a 3-D plot of response vs time vs wavelength

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PARAMETERS
Retention time: tR It is the difference in time between the point of injection appearance of peak maximum. tR = tM + tR tM = time spends in mobile phase tR = time spends in stationary phase Retention volume: VR Volume of mobile phase required to elute one half of the compound as indicated by the peak maximum. VR = tR x f f = flow rate of the mobile phase
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Resolution (Rs): It describes the separation power of the chromatographic system. Rs, of two neighboring peaks is defined as the ratio of the distance between two peak maxima. It is the difference between the retention times of two solutes divided by their average peak width. For baseline separation, the ideal value of Rs is 1.5.

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Rt1 and Rt2 are the retention times of components 1 and 2 and W1 and W2 are peak width of components 1 and 2.
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Capacity Factor (k): It is the ratio of the reduced retention volume to the dead volume. It is a measure of how well the sample molecule is retained by a column during an isocratic separation. The ideal value of k ranges from 2-10. tR = retention volume at the apex of the peak (solute) t0 = void volume of the system.
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Column efficiency: Efficiency of the column is expressed by number of theoretical plates n = 16(tR/w)2 n= number of theoretical plates w= width at the base of the peak n = 5.54(tR/w1/2)2 w1/2 = band width at half height Theoretical plate: Theoretical plate is the hypothetical, functional unit of the column. HETP= height equivalent to a theoretical plate
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 HETP is a section of the column in which mobile phase and stationary phase are in equilibrium. Lower the HETP higher is the efficiency of the column i.e, higher the number of theoretical plates, more efficient the column.

HETP = L/n L= length of column n= number of theoretical plates HETP is given by van deemter equation. HETP = A + B/u + Cu A= eddy diffusion term C= effect of mass transfer B= longitudinal diffusion term u = flow rate of mobile phases
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Asymmetry factor: (Tf )

Asymmetry factor describes the shape of chromatographic

peak. A factor > 1 results in a tailing peak A factor < 1 results in a fronting peak
The peak half width, b, of a peak at 10% of the peak

height, divided by the corresponding front half width, a, gives the asymmetry factor.
For a well packed column, an asymmetry factor between

0.9 to 1.1
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Fronting : The front part of a peak in a chromatogram tapers in advance of remainder of the peak. It is due to saturation of stationary phase and can be avoided by using less quantity of sample
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Tailing: It is due to more active adsorption sites It is a measure of how much a band deviates from being perfectly bell-shaped or symmetrical.

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APPLICATIONS
Bio chemical/clinical field: In analysis of amino acids ,catecholamines, nucleic acid bases, nucleosides,nucleotides,oxytocin,peptides,proteins, purines/pyrimidines Forensic field: In the analysis of benzodiazepines, cannabinoids ,cocaine, opiates Environmental field: In the analysis of aldehydes,explosives,herbicides,ketones, pesticides Neutraceutical: Dietary supplements: plant extracts
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Foods & flavours : In the analysis of flavour compounds, food contaminants, organic acids,sugars Pharmaceutical field: In the estimation of analgesics,anti arrhythmics, antibiotics, anti-asthmatics, anti-depressants,anti-diabetics, antiulceratives, bronchodilators, NSAIDS, sedatives. A combination of HPLC and spectrometric techniques (UV, IR,Mass spectrometry) allows simultaneous quantification & identification of analytes HPLC used in quality control testing of drugs ,stability studies,therapeutic monitoring,drug metabolism studies & pharmacokinetic studies
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Determination of Acetaminophen drug by HPLC

Method Conditions: Column : Cogent Bidentate C-18 4 m, 100 Dimensions : 4.6 x 75 mm Mobile phase : Solvent A : 100% DI water Solvent B : + 0.1% acetic acid + 0.005% TFA 100% acetonitrile+0.1%acetic acid +0.005% TFA

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Flow rate Peaks Injection Volume Sample

: : : :

Detection

1.0 ml/min. 1: Impurity 2: Acetaminophen 20 l 1 mg of the compound dissolved in 1 ml of 50% A/50%B solution. Sample for injection diluted 1:15 with 100% A. UV 254 nm

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Note: Acetaminophen (Nacetyl-p-aminophenol, APAP) is an antiinflammatory drug (non steroidal). The drug is used for the management of pain and fever. The method is used for the quality control of acetaminophen.
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Determination of Analgesic Drugs by HPLC

Method Conditions : Column : Cogent Bidentate C18, 4 m, 100. Dimensions : 4.6 x 75 mm Mobile phase : Solvent A : DI water+ 0.1% formic acid Solvent B : Acetonitrile + 0.1% formic acid Isocratic : 78% A / 22%B composition Flow rate : 1 ml/minute Injection Volume : 20l
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Note: Acetaminophen (paracetamol) is analgesic and antipyretic and with Aspirin (NSAID) they are frequently associated in pharmaceutical formulations against the common cold.
Aspirin is used in

prophylactic aspirin therapy to reduce the risk of stroke or death in patients with myocardial infarction.
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Peaks

Sample

Detection :

1. Acetaminophen (Paracetamol) 2. Caffeine 3. Acetylsalicylic acid (Aspirin) 4. Benzoic acid (internal standard) 5. Salicylic acid (degradation product of Aspirin) 0.02 mg of acetaminophen, caffeine and 0.2 mg of aspirin,benzoic acid and salicylic acid were dissolved in 1 ml of 50%A / 50%B solution. UV 254 nm

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1. Acetaminophen (Paracetamol) 2. Caffeine 3. Acetyl salicylic acid (Aspirin) 4. Benzoic acid 5. Salicylic acid

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CONCLUSION
HPLC has emerged as the most popular, powerful and

versatile technique of chromatography Its application areas include QC, process control, forensic analysis, environmental monitoring & clinical testing. Rapidly growing analytical technique, used to identify & separate compounds based on their polarity

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REFERENCES
1. Gurdeep R.Chatwal, Sham K.Anand. Instrumental Methods of Chemical Analysis., 5th edition; 2.624 2.639. 2. Willard merritt dean settle, Instrumental methods of anlysis, 7th edition; 580 _ 650

A.H.Beckett, J.B. Stenlake, Practical pharmaceutical chemistry, 4th edt., part 2: 157-161 4. P.D.Sethi, HPLC quantitative analysis of pharmaceutical formulations, 1st edition; 42-43, 60-63 5. Application note from www.mtc-usa.com
3.

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