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A SEMINAR ON LIQUID CHROMATOGRAPHY Presented By: MR.Laxman.S.Vijapur . Dept. of Pharmaceutics


CONTENTS History Introduction Parameters used in HPLC Theory of HPLC Types of HPLC technique Instrumentation Applications


HISTORY OF HPLC The origins of Liquid Chromatography began in the early 1900’s with the work of the Russian botanist, Mikhail S. Tswett. His famous studies focused on separating compounds (leaf pigments), which were extracted from plants using a solvent. During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flow through time, thus reducing purification times of compounds being isolated by column chromatography By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above the previous techniques. Computers and automation added to the convenience of HPLC. Improvements in type of columns and thus reproducibility were made as such terms as micro-column, affinity columns, and Fast HPLC began to immerge.


INTRODUCTION Liquid chromatography is the method, in which a dilute solution of sample is passed through a column packed with solid particles. Thus, liquid is passed through vertical columns under gravitational flow. This is passed with slow rate especially if the packing granules are small enough to give efficient separation, then the delivery under gravity decreases even up to a few drops per minute Thus to increase flow rate and get efficient separation is to force the liquid by a positive displacement pump or by gas pressure. This versatility can be achieved by making certain modifications in columns & by using smaller diameter & smaller surface area of columns with suitable modification leading to development of so called HPLC


PRINCIPLE OF SEPARATION IN HPLC When a mixture of components is introduced into a HPLC column, they travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent travels slowest the component which has less affinity towards the stationary phase travels faster. Since no two components have the same affinity towards the stationary phase, the components are separated.




ADVANTAGES OF HPLC Separation is fast and efficient. Separation & analysis of very complex mixture. Accurate quantitative measurement. Both aqueous & non-aqueous sample can be analyzed. Separated components can be easily collected. Determination of multiple components in a single analysis.


DISADVANTAGES OF HPLC HPLC is for non-volatile substances not for volatile substance Substances GC is an alternative. It is for high polarity or ionic samples. It is for high mol. Weight substances.


PARAMETERS used in HPLC Retention time ( Rt ) : It is the difference in time between the point of injection and appearance of peak maxima. It is the time required for 50% of a component to be eluted from the column. Measured in min or sec


RETENTION VOLUME It is the volume f mobile phase required to elute 50% of the component from the column Retention volume= retention time x flow rate


SEPARATION FACTOR (S): It is the ratio of the partition coefficient of the two substances concerned . S=Kb/Ka= t b -t o / t a -t o Where, t o = retention time of unretained substance K b ,K a = partition coefficients of b & a t b , t a = retention time of substance b & a S= depends on liquid phase ,column temp


THEORIES OF HPLC PLATE THEORY: According to this theory, the chromatographic column consists of continuous, narrow, horizontal plates of equal units called as “theoretical plates”. Though these units are hypothetical, they give rise to a very useful method for the practical measurement of column efficiency. 3 basic assumptions are made, namely: All the solute particles are placed on the first theoretical plate. Equilibrium of the solute between the two phases is instantaneous & complete within each theoretical plate. The partition coefficient of all components remains constant

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The theory measures the column efficiency & the factors which influence the efficiency are: Number of theoretical plates (N) Height equivalent to theoretical plates (HETP) Resolution

Number of theoretical plates (N)::

Number of theoretical plates (N): Efficiency of a column is expressed by the number of theoretical plates. It can be determined by N= Number of theoretical plates r =Retention time (min or sec) w=peak width at base (mm or cm) If the number of theoretical plates is high, then the column is said to be highly efficient If the number of theoretical plates is low, then the column is said to be less efficient For gas chromatographic columns, a value of 600/metre is sufficient. But in HPLC, high values like 40,000 to 70,000/metre are recommended.

Height equivalent to theoretical plates (HETP)::

Height equivalent to theoretical plates (HETP): It is the length of the column neccesary to obtain the equilibrium of the solute particles between the two phases & is given by HETP = length of the column / no. of theoretical plates Where L= length of the chromatographic column N= no. of theoretical plates

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HETP is given by Van Deemeter equation: HETP=A+B/u + Cu Where, A=eddy diffusion term or multiple path diffusion which arises due to packing of the Column. This is unaffected by the flow rate. This can be minimized by uniformity in packing B= longitudinal diffusion term or molecular diffusion which depends on flow rate C= effect of mass transfer which depends on flow rate. u= flow rate or velocity of the mobile phase


EDDY’S DIFFUSION In packed columns the solute and mobile phase molecules travel along many paths of different length, thus solute molecules have different residence time. This results into peak broadening. This broadening depends upon the size of packing particles, the shape and the manner of packing and also on the column diameter. Eddy diffusion can be decreased by using smaller particle size but it is easier to obtain regular packing with larger rather than smaller particles hence particle size should be optimum. Thus the Eddy diffusion can be minimized by using small particles of uniform size and smaller diameter columns. Generally the particle size upto 100-120 mesh range and the columns with 1/8 inch inner diameter are used for good resolution


LONGITUDINAL DIFFUSION or MOLECULAR DIFFUSION Molecules move from solution of higher concentration to lower concentration by diffusion i.e. The concentration of the solute particles is lower at the edges than in the center of the band and hence concentration gradient exists which results in band broadening. This is called Longitudinal Diffusion. High solute diffusivity leads to band broadening with consequent loss of efficiency To reduce the longitudinal diffusion, the diffusivity of the solute in the mobile phase is decreased by increasing the pressure. Also by increasing the viscosity of the mobile phase (optimum flow rate).


MASS TRANSFER RESISTANCE This term describes the effect of the amount and viscosity of liquid on the solid support. A low viscosity, low vapour pressure solvent with good, absolute and differential solubility for the sample should be used. Also low liquid loadings (1 – 10 %) have the advantage of fast analysis Low liquid loadings, however, reduce the sample capacity and may require highly inactive solid supports.

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Resolution (R): It is defined as the distance between two adjacent peak maxima divided by their average peak width. R = 2(Rt 1 -Rt 2 )/W 1 +W 2


RATE THEORY, Plate theory failed to explain the ways to improve the performance of the column, which the rate theory did. this theory explained the fact that the mobile phase flows continuously & that the solute particles are constantly being transported & partitioned in the column. It can be explained by Van Deemeter equation: Where,h=HETP λ =measure of packing irregularities of stationary phase d p = particle diameter of the stationary phase γ =tortuosity D g =diffusivity of the solute in mobile phase d f =film thickness

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D L =diffusivity of the solute in the liquid phase µ =average linear velocity of the mobile phase This equation can be written as ; A= Eddy’s diffusion B= Longitudinal diffusion C= Mass transfer resistance A plot of h versus u as shown in the figure gives a hyperbola. To obtain a minimum h (maximum effiency),the constants A, B, C must be minimised.


COLUMN EFFICENCY The efficiency of column entirely depends upon the amount of the spreading of solute molecules. The efficiency or the performance of the column can be measured by the equation N=16(VR/WB) 2 VR= Retention volume of the solute WB= Distance along the base line between the point of injection & a perpendicular dropped from the maximum of peak of interest


TYPES OF HPLC TECHNIQUES A. Based on modes of chromatography 1.Normal phase mode 2.Reverse phase mode B.Based on principle of chromatography 1.Adsorption chromatography 2.Ion exchange chromatography 3.Size exclusion/gel permeation chromatography 4.Affinity chromatography 5.Chiral chromatography C.Based on elution techniques 1.Isocratic separation 2.Gradient separation D.Based on the scale of operation 1.Analytical HPLC 2.Preparative HPLC E.Based on the type of Analysis 1.Qualitative analysis 2.Quantitative analysis

F.Based on the internal diameter of the column employed for HPLC:

F.Based on the internal diameter of the column employed for HPLC



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METHOD/DESCRIPTION Reversed Phase HPLC columns Ion Pair HPLC Normal Phase HPLC WHEN IS THE METHOD PREFERED First choice for most samples, especially neutral or non ionised compounds that dissolve in water, organic mixtures Acceptable choice for Ionic or Ionisable compounds especially, bases or cations. Good second choice when reversed phase or ion pair HPLC is ineffective: First choice for lipophilic samples that do not dissolve well in water : organic mixtures.

1.Normal phase chromatography :

1.Normal phase chromatography Mechanism: Retention by interaction of the stationary phase’s     polar surface with polar parts of the sample molecules. Stationary phase: Si0 2 , Al 2 0 3 , -NH 2 , -CN, -Diol, —NO 2 Mobile phase : Heptane, hexane, cylohexane, CHCI 3 , dioxane, methanol. Application : Separation of non-ionic, non-polar to medium    polar substances


2.REVERSE PHASE MODE CHROMATOGRAPHY Mechanism: Retention by the interaction of the stationary phase’s non-polar hydrocarbon chain with non-polar parts of the sample molecules . Stationary phase : n-octadecyl(PR-18), n-octyl (RP-8), ethyl(RP-2), phenyl ( CH 2 ) n -CN , (CH 2 ) n -diol Mobile phase: Methanol or Aceronitrile / water or buffer Application: Separation of non-ionic and ion-forming non-polar to medium polar substances ( carboxylic adds—>hydrocarbons). If the ion forming substances (as carboxylic adds ). Have to be separated; a pH control by buffers is necessary.


1. ION-EXCHANGE CHROMATOGRAPHY Mechanism Retention by reversible ionic bonds to charged groups on the stationary phase. Stationary phase Strong weak Cation exchange S0 3 COO- Anion exchange NR 3 NHR 2 Mobile phase: Aqueous buffer system Application: separation of substances which form ions : a. Ion-organic ions. b . Organic acids, organic bases c Proteins d. Nucleic acids

Size exclusion chromatography :

Size exclusion chromatography Here a mixture of components with different molecular size are separated by using gels. The gel used acts as molecular sieve and hence a mixture of substances with different molecular sizes is separated Soft gels like dextran, agarose or polyacrylarmide are used Semi rigid gels like polystyrene, alkyl dextran in non aqueous medium are also used The mechanism of separation is by steric and diffusion effects.


AFFINITY CHROMATOGRAPHY uses the affinity of the sample with specific stationary phases. This technique is mostly used in the field of biotechnology, microbiology , biochemistry etc


CHIRAL PHASE CHROMATOGRAPHY This method is used for the separation of the optical isomers by using Chiral stationary phases. The stationary phases used for this type of chromatography are mostly chemically bonded silica gel Different principles operate for different types of stationary phases for different samples.


BASED ON THE SCALE OF OPERATION Analytical HPLC: used only for the analytical purpose and the recovery of the sample is not done since the quantity of sample used is very small usually in micrograms. Preparative HPLC: here the individual fractions of pure compound can be collected using fraction collector and the collected samples can be reused Eg.separations of few grams of a mixture.


BASED ON THE TYPE OF ANALYSIS Quantitave analysis: is used to identify the compound, detect impurities and to find the number of components. It is done by the retention time values. Qualitative analysis : is done to detect the amount of individual or several components in a mixture. It is done by comparing the peak area of standard and sample


STATIONARY PHASES Stationary phases are particles which are usually about 1 to 20 μm average diameter (often irregularly shaped). Polar (“Normal” Phase): Silica, alumina Cyano, amino or diol terminations on the bonded phase Non-Polar (“Reversed Phase”) C18 to about C8 terminations on the bonded phase Phenyl and cyano terminations on the bonded phase


IMPORTANT NOTE Either silica or bonded silica materials should not be used above pH 8 because silica itself begins to dissolve at around pH 8. Chemically bonded materials also get cleaved off at pH 2.0 Adsorbed compounds can usually be removed from silica columns by flushing with methanol or water and from bonded phase columns by flushing with methanol or dichloromethane In Adsorption chromatography, there is no additional phase on the stationary phase particles (Silica, alumina) In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin

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The Mobile Phase in HPLC:

The Mobile Phase in HPLC solvate the analyte molecules and the solvent they are in Be suitable for the analyte to transfer “back and forth” between during the separation process Must be compatible with the instrument (pumps, seals, mixing, detector, etc) compatible with the stationary phase readily available (often use liters/day) Of adequate purity Spectroscopic and trace-composition usually Not too compressible (causes pump/flow problems

Mobile Phases in HPLC::

Mobile Phases in HPLC: The polarity index is a measure of the relative polarity of a solvent It is used for identifying suitable mobile phase solvents The more polar your solvent is, the higher the index You want to try to choose a polarity index for your solvent (or solvent mixture) that optimize separation of analytes Usually the index is a starting point The polarity of any mixture of solvents to make a mobile phase can. be modeled to give a theoretical chromatogram Usually, optimization of solvent composition is experimental A similar number is the Eluent Strength Increasing eluent strength or polarity index values mean increasing solvent polarity Remember; the analyte(s) and samples must be mobile phase and stationary phase compatible

Eluotropic series for some common solvents:

Eluotropic series for some common solvents

Effect of Solvent:

Effect of Solvent


INSTUMENTATION OF HPLC Degassing system Pump solvent delivery system Check valves Pulse damper Pre-columns Guard column Flow splitter Auto sampler Sample injection port Column


DEGASSING SYSTEM Sparging/Bubbling Vacuum filtration Ultra sonication


PUMP-SOLVENT DELIVERY SYSTEM The pumps are used to pass mobile phase through the column at high pressure because the particles that are used pack HPLC columns are small enough ie: <50μm and also particle size of packing material is 5-10 μm To prevent solvent flow from gravity pumps that develop pressure up to 5000psi are needed to force the mobile phase through column so that solvent stream enters the instrument at constant flow rate/pressure. In addition to this the pumps used in HPLC should have the following features


PUMPS Generation of pressure upto 5000psi Flow rate ranging from 0.1 to 100ml/min Flow control & flow reproducibility of plus or minus 0.5% It should be composition resistant and give a pulse free out put Pumps are thus categorized into 1.Mechanical pumps Displacement pumps Reciprocating pumps 2.Pneumatic pumps



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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.

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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.


2. RECIPROCATING PUMPS WORKING Pressure from a gas cylinder delivered through a large piston drivers the mobile phase. Pressure on the solvent is proportional to the ratio of piston usually 50: 1.

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3. CHECK VALVES They are used to control the flow of solvent & back pressure 4. PULSE DAMPER Pulse are used to dampen or reduce the pulses observed from the wavy    baseline caused by pumps


PULSE DAMPER Important damping methods include A triple headed pump : - two heads in different stages of filling as the third is pumping. A tube with an air space or a flexible bellows or tube : Here a gas (air space) or a flexible metal vessel takes up some of the solution energy. When pump refills, this energy is released and a smooth pressure pulsation result. A restrictor : - In this method, a 25 cm length of 4 mm bore .stainless steel tubing. Packed with 20µm glass beds, is placed between the pump and the column.


5. PRE-COLUMNS A pre-column is packed with 37-53μm silica particles (saturator column). It is being fitted between pump & the injector valve ensures that the mobile phase is fully saturated with the silicate ions prior to the sample injection. Thus its use reduces the adverse effects of low or high pH mobile phases which in turn substantially extend the life of the column. Their use is recommended in ion-exchange chromatography using buffered aqueous phase.


6. GUARD COLUMN A short column placed between the sample injector and the inlet of the main ("analytical") column The guard column is packed    with the same kind of packing as the main analytical column, and is    intended to absorb or pick up impurities in the sample or mobile phase that    might damage the main column & increase the life time of main analytical   column.


7. FLOW SPLITTER When a differential type of detector is used the flow of solvent is split just before it enters the sample injection port so that one portion directly goes to the reference side of the detector & a portion to the analytical column housed in a constant temperature chamber


8. AUTOSAMPLER Here in this type of instruments their will be a piston metering syringe type pump to suck the prestabilised sample volume into a line & than transfer it to the relatively large loop (approx 100ml) In a standard six port valve. The simplest Autosampler utilizes the special vial & displace the sample through the needle in to the valve loop. Most of the Autosamplers are microprocessor controlled & can serve as a master collector for the whole instrument


9 . SAMPLE INJECTOR PORT Septum injector Stop flow septum less injection Rheodyne injectors

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SEPTUM INJECTION PORT . Syringe is used to inject the sample through a self sealing inert septum directly into the mobile phase. Drawback: - leaching effect of the mobile phase with the septum resulting in the formation of ghost peaks. STOP FLOW SEPTUMLESS INJECTION. Flow of mobile phase through the column is stopped for a while. Syringe is used to inject the sample. Drawback: formation of ghost peak.


RHEODYNE INJECTORS Operation of sample loop. sampling mode Injection mode. Sample is loaded at atmospheric pressure into an external loop in the micro volume sampling valve, and subsequently injected into the mobile phase by suitable rotation of the valve. Micro volume sampling valve operation of a Sampling loop.

10. Analytical Column:

10. Analytical Column Analytical column is most important part of the instrument dimensions of column are Column length: 5cm to 30cm Column diameter: 2mm to 50mm Particle size: 1μ to 20 μ Particle nature: Spherical, uniform sized, porous materials are used




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. ( signal is increased 4 times ) Require high pressure to propel mobile phase. Used for the high resolution analytical work of compounds with very high Rt

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SHORT FAST COLUMN Length of column is 3 – 6 cm. Used for the substances which have good affinity towards the stationary 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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Preparative column are of three type : 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


PACKING OF COLUMN The most widely used method of packing column is by high pressure slurring technique. A suspension of packing is made in a solvent of equal density to the packing material. The slurry is then rapidly pumped at high pressure onto a column with a porous plug at its outlet. The resulting bed of packed material with in the column can then be prepared for use by running the developing solvent through the column, hence equilibrating the packing with the developing solvent. When hard gels are packed, it is necessary for them to be allowed to swell first in the solvent to be used in the chromatographic process before packing under pressure. Soft gels cannot be packed under pressure and have to be allowed to pack from a slurry in the column under gravitational sedimentation only, in a similar way of packing conventional column


METHOD OF PACKING Depends on the mechanical strength of stationary phase. Particle size of the stationary phase. Particles of greater then 20 µm – dry packing Particles of lesser then 20 µm – slurry packing / wet packing. 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


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.


DETECTORS Criteria Selectivity Sensitivity and detection limit Stability Reproducibility Economically affordable It should only record the component of interest

UV absorption detectors:

UV absorption detectors Fixed wavelength detectors Multi wavelength detectors Photo diode array detectors

Fixed wavelength detectors:

Fixed wavelength detectors

Diode array detector:

Diode array detector Here broad emission source like deuterium lamp is collimated by an achromatic lens Sample is subjected to all wavelength generated by the lamp Dispersed light from gratings allowed to fall on to diode array Array contain hundreds of diodes, output from each diode is sampled by a computer


FLUORESCENCE DETECTOR The single wavelength excitation fluorescence detector is probably the most sensitive LC detector that is available, but is achieved by forfeiting versatility. A diagram of a simple form of the fluorescence detector is shown in figure. The excitation light is normally provided by a low pressure mercury lamp which is comparatively inexpensive and provides relatively high intensity UV light at 253.7 nm. Many substances that fluoresce will be excited by light of this wavelength The excitation light is focused by a quartz lens through the cell. A second lens, set normal to the incident light, focuses the fluorescent light onto a photo cell. A fixed wavelength fluorescence detector will have a sensitivity (minimum detectable concentration at an excitation wavelength of 254 nm) of about 1 x 10-9 g/ml and a linear dynamic range of about 500 with a response index of 0.96 < r <1.04.


ELECTROCHEMICAL DETECTORS This detector is based on the measurements of the current resulting from oxidation/reduction reaction of the analyte at a suitable electrode. Since the level of the current is directly proportional to the analyte concentration, this detector could be used for quantification

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The eluent should contain electrolyte and be electrically conductive. Most of the analytes to be successfully detected require the pH adjustments. The areas of application of electrochemical detection are not large, but the compounds for which it does apply, represent some of the most important drug such as phenol, catecholamines, nitrosamines, and organic acids are in the picomole (nanogram) range The specificity, and sensitivity make it very useful for monitoring these compounds in complex matrices such as body fluids and natural products .

Deflection detectors :

Deflection detectors The optical schematic of the deflection detector is shown in below. This detector based on the deflection principle of refractometry where the deflection of a light beam is changed when the composition in the sample flow-cell changes in relation to the reference side (as eluting sample moves through the system). When no sample is present in the cell, the light passing through both sides is focused on the photodetector (usually photoresistor). As sample elutes through one side, the changing angle of refraction moves the beam. This results in a change in the photon current falling on the detector which unbalances it. The extent of unbalance (which can be related to the sample concentration) is recorded on a strip chart recorder.

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The advantages of this type of detector are: (1) Universal response; (2) Low sensitivity to dirt and air bubbles in the cells; and (3) The ability to cover the entire refractive index range from 1.000 to 1.750 RI      with a single, easily balanced cell.

Refractive index detectors:

Refractive index detectors It is very sensitive to changes in ambient temp,pressure,flow rate It can not be used for the gradient elution technique Extremely used for the comp that not adsorb in uv region and not fluorescence


WORKING It passes the visible light through two compartments The differential refractometer monitors the deflection of a light beam caused by the diff in refractive index between the contents of the sample and the reference cell The beam of the light from lamp passes through an optical mask That confines the beam to the region of the cell The lens collimates the light beam which passes through both the cells to a mirror The mirror reflects the beam back to a lens which focus it on to a photocell


APPLICATIONS It is widely used in the separation and analysis of the polymers Used in case of those polymers that contains more than six monomers RI is directly proportional to the concentration of the polymer and is practically independent of mol. weight


DERIVATIZATION This a technique of treatment of the sample to improve the process of separation by column or detection by the detectors. The main purpose of derivatisation in HPLC is to improve detection specifically when determining traces - of solutes in complex matrices. The derivatisation is of two types: i. Pre-column off-line derivatisation ii. Post-column on-line derivatisation


PRE-COLUMN OFF-LINE DERIVATISATION This done to improve some properties of the sample for separation by column. By this technique the components are converted to more volatile thermostable derivatives resulting in improved separation and less tailing. It is done when the components to be analysed are less volatile and thermolabile i.e., heat sensitive. E.g., carboxylic acids, sugars, phenols, alcohols, etc They can be converted to less polar compounds by using reagents like BSA (Bis tnmethyl silyl acetatnide) reagent They can also be converted to acetyl derivative or criflouro derivative

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MERITS It does not require any modification to the instrument (a plus point when compared to the post.-column methods) DEMERITS a) The formation. of a stable and well defined product is an absolute necessity b) Presence of excess reagent or by products may invariably interfere with separation c) Sometimes the derivatisation changes the entire chromatographic properties of the sample which is not desirable.


POST-COLUMN ON-LINE DERIVATISATION This done to improve the response shown by detector The components may not be detected by detector unless derivatisation is done. The components can be converted in such a way that their ionization or affinity towards electrons is increased. This method is essentially a on-line detection technique where the flow rate is neither stopped or altered. The examples of post column derivatization reactions for use with UV detectors include: 1. Reaction of amino acids with ninhydrin 2. Reaction of fatty acids with o-nitrophenol 3. Reaction of ketones with 2,4-DNP - 4. Thermal or acid Phenol treatment of carbohydrates


REAGENTS FOR DERIVATISATION The reagent are employed for the derivatisation of compounds either for enhancing UV/ visible radiation (called chromatags) or for the reaction of non flourescent reagent molecules (called fluorotags) with solutes to yield fluorescent derivatives


DERIVATIZATION FOR UV DETECTORS: Ninhydrin, a chromotag, is commonly employed to yield corresponding derivatives of. amino acids that show absorption specifically at about 570nm as shown in the following reaction


DERIVATIZATION FOR FLUORESCENCE DETECTORS Dansyl chloride (a fluorotag) is invariably used to obtain fluorescent derivatives of proteins, amines and phenolic compounds, the excitation and emission wavelengths being 335nm,


COMPARISON OF HPLC & GC HPLC GC 1. Here vast majority of solutes can be used for separation 2. Efficiency in HPLC is 5000 plates/m 3. Possible to vary pH, concentration, components of the mobile phases, flow rate, pressure for better separation 4. Sample is not destroyed & fraction can be collected 5. Less speed compared to GC 6. The mobile phase competes actively with the standard phase for solute molecules 1. In GC solute must be volatile 2. Efficiency in GC is 2000 plates/m 3. In this only flow rate, temperature can be varied 4. Sample are destroyed 5. High speed of resolution 6. Gases are inert & have no affinity for solute

HPLC Vs Classical column:

HPLC Vs Classical column Parameters C.C HPLC Stationary Phase- Particle Size Column Size Length x int.diameter Column material Column packing pressure Large 60-200 μ Large 0.5-5m x 0.5-5cm i.d. Glass Slurry packed at low pressure Small 3-20 μ Small 5-50cm x 1-10mm i.d . Mostly metal Slurry packed at high presure

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Parameters C.C HPLC Operating pressure Flow rates Sample load Column efficiency i.e. Cost Low(<20psi) Low to very low Low to medium (g/mg) (Low) < 500 theoretical plates/m Low- Few hundreds High(500-3000psi) Medium-High (often>3ml min Low to very low ( μ g) (High) often > 1,00,000 plates/m High- Few lakhs

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Parameters C.C HPLC Detector flow cell volume Types of stationary phases available Scale of operation Large – 300 to 1000 μ l Limited range Preparative Scale Low 2 to 10 μ l Wide range Analytical and Preparative Scale


APPLICATIONS OF HPLC Quality control testing of drugs In Qualitative & Quantitave analysis Therapeutic monitoring of drug metabolism studies Separation & control of impurities In analysis of biological fluids Stability studies Study of metabolic pathways in basic biochemical pathways Separation of positional isomers, enantiomers, Optical isomers Industrial applications a. Determination of synthetic intermediates ex: atenolol b. In determining traces of impurity ex:Tolnafate c. Stability studies ex Acyclovir


REFRENCES Fundamentals of Analytical chemistry by Skoog, West, Holler, Crouch. Pharmaceutical analysis by James. W. Munson. Pharmaceutical Analysis - Dr. A. V. Kasture. Instrumental Analysis by G.R.Chatwal Internet source

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