Seminar 3 Hplc

8/3/2019 Seminar 3 Hplc

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CONTENTS

INTRODUCTION

TYPES

PRINCIPLE

INSTRUMENTATION

PARAMETERS

APPLICATIONS

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INTRODUCTION

HPLC - Fast growing analytical technique

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

It is defined as

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performance/Pressure/

priceChromato

graphy

High

Liquid



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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.

It¶s simplicity,sensitivity,specifity,selectivity make it ideal

for analysis of many drugs.



TYPES OF HPLC

y BASED ON MODES OF CHROMATOGRAPHY

y BASED ON PRINCIPLE OF SEPARATION

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REVERSE PH

ASE NORMAL PHASE

ADSORPTION PARTITIONION

EXCHANGESIZE

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.

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SILICA GEL Silica gel is saturated with ³silanol´ (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.

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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

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OCTYL & OCTADECYL GROUPS

The polar nature of silica is reversed i.e. polar to non - polar byincorporating hydrophobic C8,C18 groups into silanol .

Chemically bonded octadecyl silane(ODS) widely usedstationary phase.

CH3

Si-o-Si-R

CH3

R =Hydrophobic carbon chain

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ADSOR PTION (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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Stationary PhaseMobile Phase

Bonded PhaseSolvent

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.



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ION EXCHANGE

Separation is by reversible exchange of ions between theions present in the ion exchange resin and those present inthe 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 ,carboxylicgroups and those will attract +ve charged molecules.Eg:

resin-SO3H + Na+ resin-SO3 Na + H+



Anion exchange resin:

It posses +ve charged groups such as amine, quaternaryammonium 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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PR INCIPLE

The resolving power of a chromatographic column increaseswith 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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INSTRUMENTATIONCOMPONENTS:

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 solutionDisadvantage :

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 dissipate22



Filtration : Vacuum is used in conjugation with 0.45 µ 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 togive 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 solventto 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 iscreated 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 µl syringe.

Rheodyne injector (loop valve injector):

It has fixed volume loop like 20 µl or 50 µl

It has two modes load position and inject mode

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AUTO SAMPLER R HEODYNE INJECTOR

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Pre column:

A small column placed between

injector & guard column

It removes particulate matter from

mobile phaseGuard column:

A small column placed between

the pre column & analytical column

It protects the analytical columnfrom contamination of the sample

particulate & from strongly retained species.

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Guard column

HPLC column



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 PACK ING

1.Microporous support:

Microporous ramify through the particles of which aregenerally 5-10 µm 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 µm 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 silicaor silicone polymer

High pressure slurring technique Used for column packing

A suspension of packing is made in a solvent of equal densityto the packing material

The slurry is pumped at high presure onto a column with a porous plug at it¶s outlet.

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PACK ING MATER IALS

TYPE NAME DIAMETER (µm)

SHAPE

silica Porasil 37-75 spherical

silica Lichrosorb-si 5,10,20,30 irregular

silica zorbax 5 sphericalalumina Lichrosorb Alox 5,10,20 irregular

Bonded

phase

Bonda pak/c-18

Pellicular zipax 25-37 spherical

pellicular corasil 37-50 spherical

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DETECTOR

An electronic device that quantitatively discerns the presenceof 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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R efractive 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 thecomponent in the mobile solvent system

Low sensitivity and specificity.

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Electro chemical detector:

It is based on standard electro chemical principles involvingamperometry ,polarography

These detectors are very sensitive for substances that areelectro 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 thecomponents 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 arrayspectrophotometers in which light from an uv source passesthrough 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 vswavelength

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PARAMETERS

Retention time: tR

It is the difference in time between the point of injectionappearance of peak maximum.

tR = tM + t¶R

tM = time spends in mobile phaset¶R = time spends in stationary phase

Retention volume: VR

Volume of mobile phase required to elute one half of thecompound as indicated by the peak maximum.

VR = tR x f

f = flow rate of the mobile phase

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Resolution (R s

):

It describes the separation power of the chromatographicsystem.

R s, of two neighboring peaks is defined as the ratio of thedistance between two peak maxima.

It is the difference between the retention times of two solutesdivided by their average peak width.

For baseline separation, the ideal value of R s is 1.5.

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Rt1 and Rt2 are the retention timesof components 1 and 2 andW1 and W2 are peak width of components 1 and 2.



Capacity Factor (k¶):

It is the ratio of the reducedretention 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.

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tR = retention

volume at the apexof the peak (solute)t0 = void volume of the system.



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 thecolumn.

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 thecolumn.

HETP = L/nL= length of column

n= number of theoretical plates

HETP is given by van deemter equation.

HETP = A + B/u + CuA= eddy diffusion term B= longitudinal diffusion term

C= effect of mass transfer 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

4m, 100Å

Dimensions : 4.6 x 75 mmMobile phase :

Solvent A : 100% DI water

+ 0.1% acetic acid + 0.005% TFA

Solvent B : 100% acetonitrile+0.1%acetic acid+0.005% TFA

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Flow rate : 1.0 ml/min.

Peaks : 1: Impurity2: Acetaminophen

Injection Volume : 20 l

Sample : 1 mg of the compound dissolved

in 1 ml of 50% A/50%B solution.

Sample for injection diluted 1:15

with 100% A.

Detection : UV 254 nm

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Note:

Acetaminophen (N-acetyl-p-aminophenol,

APAP) is an anti-

inflammatory drug (nonsteroidal).

The drug is used for themanagement of pain andfever.

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, 4m, 100Å.

Dimensions : 4. 6x75mm

Mobile phase :

Solvent A : DI water+ 0.1% formic acid

Solvent B : Acetonitrile + 0.1% formic acid

Isocratic

composition : 78% A / 22%B

Flow rate : 1 ml/minute

Injection Volume : 20µl

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Note:

Acetaminophen

(paracetamol) is analgesicand antipyretic and withAspirin ( NSAID) they arefrequently associated in

pharmaceuticalformulations against the

common cold.

Aspirin is used in prophylactic ³aspirintherapy´ to reduce

the risk of stroke or deathin patients withmyocardialinfarction.

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Peaks : 1. Acetaminophen (Paracetamol)

2. Caffeine3. Acetylsalicylic acid (Aspirin)

4. Benzoic acid (internal standard)

5. Salicylic acid (degradation product

of Aspirin)Sample : 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.

Detection : 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

It¶s application areas include QC, process control, forensicanalysis, 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 Methodsof Chemical Analysis., 5th edition; 2.624 ± 2.639.

2. Willard merritt dean settle, Instrumental methods of anlysis,

7th edition; 580 _ 6503. 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, 60-635. Application note from www.mtc-usa.com

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Seminar 3 Hplc