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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.
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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
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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+
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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
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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
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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 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.
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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.
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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 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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