hplc analysis finallllll

GARVITA ARORAI M.PHARM

PHARMACEUTICAL CHEMISTRY

High Performance Liquid

Chromatography

INTRODUCTION TO LIQUID CHROMATOGRAPHY

• It is a versatile technique available to an analyst.• It is simple method and has a high capability for high

resolution separations.• Separations are caused by diverse characteristics like

1) Polarity of the solutes

2) Ionic nature

3) Molecular weight

4) Partitioning ability

5) Ability to form affinity complexes


• The word liquid chromatography is used today is the method in which the separation take place in a packed column.

• The packaging material is the stationary phase and a solid with an inert support coated with a liquid phase.

• A liquid mobile phase is used as eluant.


• They were carried out in glass columns– diameters: 1-5 cm– lengths: 50-500 cm

• Size of the solid particle in stationary phase– diameters: 150-200 m

• Flow rate is low ,Separation times long

Diagram of column chromatography

High PerformanceLiquid Chromatography

PressurePriced

High Performance Liquid Chromatography

• The most widely used analytical separations technique.• Utilizes a liquid mobile phase to separate components

of mixture.• Uses high pressure to push solvent through the column.• Popularity: – Sensitivity.– Suitable for separating nonvolatile species .– widespread applicability to substances that are of prime

interest to industry, to many fields of science.

High Performance Liquid Chromatography is

• Ideally suited for separation and identification of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and a variety of other inorganic substances.

Advantages to HPLC

• Higher resolution and high speed of analysis.• HPLC columns can be reused without repacking or

regeneration.• Greater reproducibility due to close control of the

parameters affecting the efficiency of separation.• Easy automation of instrument operation and data

analysis.• Adaptability to large-scale, preparative procedures.

Advantages to HPLC

• Advantages of HPLC are result of 2 major advances:– stationary supports with very small particle

sizes and large surface areas.– appliance of high pressure to solvent flow.

DIFFERENT TYPES OF HPLC

Modes of Chromatography

Principle of Chromatography

Elution techniques

The scale of operation

Type of Analysis

Reverse Phase mode

Types of HPLC Techniques

Normal Phase mode

Modes of Chromatography

Liquid-liquid partition chromatography

• In this relative method stationary phase is liquid coated onto a solid support.

• The relative distribution of solutes in between two phases determines the separation of substance.

• This can be further classified into

1) Normal phase chromatography – Stationary phase is

polar and mobile is non-polar.

2) Reverse phase chromatography – Stationary phase is

non-polar and mobile phase is polar.

DIAGRAM OF NORMAL PHASE CHROMATOGRAPHY

Polar Solutes

Non- Polar Solutes

Direction of Mobile Phase

DIAGRAM OF REVERSE PHASE CHROMATOGRAPHY

Polar Solutes

Non- Polar Solutes


Size exclusion Chromatography

Adsorption Chromatography

Ion exchange Chromatography

Affinity Chromatography

Principle of Chromatography

Types of HPLC Techniques

ADSORPTION CHROMATOGRAPHY

◊ Adsorption is a surface phenomenon where interaction takes place only on the surface of adsorbent.

◊ The interaction is competitive.◊ Solute having high affinity towards stationary

phase will be adsorbed first compared to less affinity molecules.

◊ Adsorbed solute is replaced from stationary phase by mobile phase.

STATIONARY PHASE

SOLUTES

High Affinity

Low Affinity


◊ The stationary phase in adsorption Chromatography is called "Adsorbent"

Types of adsorbents: (Stationary phase) ◊ Usually a solid such as silica gel, alumina

(Al2O3) or porous glass beads are used.

Types of Mobile Phases:◊ The mobile phases such as heptane, octane,

chloroform etc are used in adsorption chromatography.

SIZE EXCLUSION CHROMATOGRAPHY

• Size-exclusion chromatography is a separation mode based solely on the analyte’s molecular size.

• In this a large molecule is excluded from the pores and migrates quickly, whereas a small molecule can penetrate the pores and migrates more slowly down the column.

SIZE EXCLUSION CHROMATOGRAPHY

It is often called Gel permeation chromatography (GPC) or Gel-filtration chromatography (GFC)

Gel permeation chromatography (GPC) - when used to determine the molecular weights of organic polymers.

The column is packed with cross-linked polystyrene beads of controlled pore sizes.

It is eluted with common mobile phases such as toluene and tetrahydrofuran.

Gel-filtration chromatography (GFC) when used in the separation of water-soluble biological materials.

Ion exchange Chromatography

• In ion-exchange chromatography, the separation mode is based on the exchange of ionic analytes with the counter-ions of the ionic groups attached to the solid support.

• These resins are coated on to the porous glass bead. Separation takes place by exchange of ion of resin with counter ion of mobile phase.

• These are of two types.

1) Cation-ion exchange resin.

2) Anion-ion exchange resin.

Ion-exchange chromatography(IEC), showing the exchange of analyte ion p+ with the sodium counter ions of thebonded sulfonate groups

P+

P+

P+

Na+

Na+

• Typical stationary phases are cationic exchange (sulfonate) or anionic exchange (quaternary ammonium) groups bonded to polymeric or silica materials.

• Mobile phases consist of buffers, often with increasing ionic strength, to force the migration of the analytes.

• Common applications are the analysis of ions and biological components such as amino acids, proteins/peptides, and polynucleotides

• Affinity chromatography: Based on a receptor/ligand interaction in which immobilized ligands (enzymes, antigens, or hormones) on solid supports are used to isolate selected components from a mixture. The retained components can later be released in a purified state.

• Hydrophilic interaction chromatography (HILIC): This is somewhat similar to normal phase chromatography using a polar stationary phase such as silica or ion-exchange materials but eluted with polar mobile phases of organic solvents and aqueous buffers. It is most commonly used to separate polar analytes and hydrophilic peptides.

Other Separation Modes

• Chiral chromatography: For the separation of enantiomers using a chiral-specific stationary phase. Both NPC and RPC chiral columns are available.

• Hydrophobic interaction chromatography: Analogous to RPC except that mobile phases of low organic solvent content and high salt concentrations are used for the separation of proteins that are easily denatured by mobile phases with high concentrations of organic solvents used in RPC.


• Electro chromatography: Uses capillary electrophoresis (CE) equipment with a packed capillary HPLC column. The mobile phase is driven by the electromotive force from a high-voltage source as opposed to a mechanical pump. It is capable of very high efficiency.

• Supercritical fluid chromatography (SFC): Uses HPLC packed columns and a mobile phase of pressurized supercritical fluids (i.e., carbon dioxide modified with a polar organic solvent). Useful for non-polar analytes and preparative applications where purified materials can be recovered easily by evaporating the carbon dioxide. HPLC pumps and GC-type detectors are often used.


Types of HPLC Techniques:

BASED ON ELUTION

TECHNIQUES

Isocratic separation

Gradient separation

BASED ON THE SCALE OF

OPERATION

Analytical HPLC

Preparative HPLC

BASED ON TYPE OF ANALYSIS

Qualitative Analysis

Quantitative Analysis

INSTRUMENTATION

MOBILE PHASE

The mobile phase is the solvent that moves the solute (analyte) through the column. In HPLC, the mobile phase interacts with both the solute and the stationary phase and has a powerful influence on solute retention and separation.

• High solubility for the sample components.• Noncorrosive to HPLC system components.• High purity, low cost, UV transparency.• Other desirable characteristics include low

viscosity, low toxicity, and non-flammability.

HPLC mobile phases should have these characteristics:

Buffers Acidic Mobile Phases Ion-Pairing Additives High pH Mobile Phase

DIFFERENT TYPES OF MOBILE PHASES

• The most common type of solvent reservoir is a glass bottle.

• Most of the manufacturers supply these bottles with special caps, Teflon tubing and filters to connect to the pump inlet and to the sparge gas (helium) used to remove dissolved air.

• Filtration is needed to eliminate suspended particles and organic impurities.

MOBILE PHASE RESERVOIRS

• The main culprit is oxygen (from the air) that dissolves in polar solvents, particularly water.

• The dissolved gases in the mobile phase will come out at column exit or in the detector resulting in sharp peaks.

• The microscopic bubbles can change the nature of the flowing stream making it heterogeneous.

• But peaks can also occur when these microscopic bubbles gradually collect in the detector cell.

Degassing may be accomplished by one or a combination of the following methods:

1) Apply a vacuum to the liquid.2) By Heating.3) Sonication. 4) Sparging.

DEGASSING TECHNIQUES

INTRODUCTIONPumps are used to pass mobile phase at high

pressure and at controlled flow rates.The pump must be capable of generating

pressure of up to 5000 psi at flow rates up to 3 ml/min for analytical situations.

Pumps used in preparative scale HPLC may be required to pump at flow rates of up to 20ml/min.

PUMPS

• Non corrosive & compatible with solvent.• Provide high pressure to push the mobile phase.• High pressure generated by pump should not lead to

an explosion.• Provide constant flow rate to mobile phase.• Easy to change from one mobile phase to another.• Should not leak.• Should have reproducible flow rate and independent

of column back pressure.• It should be easy to dismantle or repair.

IDEAL CHARACTERSTIC OF PUMP

1.Constant flow pumpA). Syringe pump.B). Reciprocating pump. -Single piston -Dual piston2). Constant pressure pump. A). Pneumatic pump -Amplifier pump

TYPES OF PUMP USED IN HPLC

A) SYRINGE PUMP

ADVANTAGES

• Flow is pulse free.• Provide high pressure up to 200-475 atm.• Independent of column back pressure and

viscosity of solvent.• Simple operation.

• Limited solvent capacity.• Gradient elution is not easy.

DISADVANTAGES

CONSTANT FLOW PUMP B) Reciprocating pump(single piston)

RECIPROCATING PUMP (Dual piston)

DIAGRAM OF RECIPROCATING PUMP

• Generate high output pressure.• Ready adaptability to gradient elution.• Provide constant flow rate.• Pressure generated is so high that any back pressure

generated in the column due to mobile phase can be easily overcome.

Pulsed flow must be damped as they produce a base line noise on chromatogram.

ADVANTAGES

DISADVANTAGES

PNEUMATIC PUMP

• A TRIPLE HEADED PUMP.

• A TUBE AN AIR SPACE OR A FLEXIBLE BELLOWS OR TUBE.

• A RESTRICTOR.

PULSE DAMPER

Two heads in different stages of filling as the third is pumping.

TRIPLE HEADED PUMP

COLUMN

MOBILE PHASE

RESERVOIR

Flow of mobile phase

PUMPS

1

23

31 2

Where a flexible metal(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 results.

COLUMN

MOBILE PHASE

RESERVOIR

A TUBE AN AIR SPACE OR A FLEXIBLE BELLOWS OR TUBE

In this method, 25 cm length of 4mm bore, stainless steel tubing packed with 20 micro meter glass beads, is placed between the pump and the column.

25 cm

4 mmCOLUMN PUMP

MOBILE PHASE

A RESTRICTOR

• Pre-columns are packed with inexpensive, coarse silica for mobile phase conditioning function.

• Placed in the HPLC system prior to the sample injector. • Could have larger particle size.• Used to saturate the mobile phase with silica so that the

silica or bonded silica of analytical column packing is not dissolved during use.

• Reduces the problems due to change in the pH of the mobile phase and extends the life of the column.

• Used to remove impurities.

PRE COLUMN

• SEPTUM INJECTION.• MICRO VOLUME SAMPLING VALVES/

SAMPLING LOOP.• RHEODYNE INJECTOR/STOP FLOW.

SAMPLE INJECTION SYSTEM

• Syringe is used to inject the sample through an self sealing inert septum directly in to the mobile phase

• DRAWBACK - Leaching effect of the mobile phase with the septum resulting in the formation of ghost peaks.

SEPTUM INJECTION

SYRINGE INJECTOR

RHEODYNE INJECTOR/STOP FLOW

• Uses syringe, the pumping is stopped and sample is introduced from the head of the column and pumping of the solvent is continued.

RHEODYNE INJECTOR/STOP

FLOW

• Highly sophisticated modern HPLC method for sampling makes use of micro volume sampling devices.

• No interruption of the mobile phase flow.• Volume of sample ranges between 2 µl to over

100µl.Operation of sample loop1)Sampling mode2)injection mode

MICRO VOLUME SAMPLING VALVES/ SAMPLING LOOP

Sampling mode and injection mode

• The column is the main part in HPLC.• Column composed of porous graphite or

particles of polymeric materials like styrene divinyl benzene copolymer material are stable over wide range.

• The column containing small particles of stationary phase because surface area of stationary phase is exposed to sample component in HPLC column.

COLUMNS

• Porous plugs of stainless steel or teflon are used in the end of the column to retain the packaging material.

• It is important in some separation like liquid partition and ion exchange that the column temperature is thermostatically controlled during analysis.

PROPERTIES OF PUMP

• It should withstand the operating pressure.• Materials of column should not be corroded.• It should be non- reactive with mobile phase and the

solute. • It should show satisfactory peak shapes (minimal

tailing).• It should show satisfactory peak width (narrower or

better).• It should give a stable detector base line.• It should have good resolving power.

A) GUARD COLUMN

B) MAIN COLUMN

CLASSIFICATION OF COLUMN

GUARD COLUMN

• Guard column are used in HPLC to protect the analytical column from contaminating substances that may be present in sample or in solvent system.

• The guard column are packed with the same material used in analytical column but have larger particle size than analytical column packaging material.

• Guard column connected between injectors and analytical column.

1)Analytical column 2) Preparative column3)Micro bore4)Semi-preparative column5) New column technology • Radial compression column• Axially compressed column

Main column

ANALYTICAL COLUMN The separation takes place in a stainless steel

tube usually 3-25cm in length with an internal diameter 3-4.6 mm . It is packed with stationary phase.

Materials used to pack column1) Superficially porous or pellicular2) Totally porous

Particle size 3-10 µm

Particle naturespherical, uniformed sized porous material

The price of analytical column depend on the particle size that is a 5nm column cost is more than 10 nm column length, diameter and nature of the packaging material(reverse phase packing cost more ).

TYPE OF ANALYTICAL COLUMN1)COMPRESSION2)CALTRIDGE

COMPRESSION FITTING

CALTRIDGE FITTING

Length 3-25cm 7.5 to 10cm

Inner diameter 3-4.6mm 3-4.6 mm

Particle size 3-10 µm 3-10 µm

• These column are larger as compared to analytical column because large amount of sample to be loaded. They have length 10-25cm and particle size 5-20 micrometer.

• The packaging are attached to the internal column by support includes oxides , carbon , polymer & silica is the most common type.

PREPARATIVE COLUMN

• TYPES:-Magnum 9 Magnum 20 Magnum 40

The micro bore HPLC technique first introduced in 1979.Column with internal diameter less than 2 mm are often referred to as micro bore column.

MICRO BORE COLUMN

Length 15-25 cmInternal diameter 1mmParticle size 3-8 µm

Micro bore column is to get high sensitivity.We compare a typical HPLC separation (10 min at 2ml /min)with a microbore separation consumes 20 times less solvent than conventional separation.

1. Highest possible efficiency.2. Decrease in solvent consumption.3. Small sample requirement.4. Can interface with mass spectrometer.5. Lower detection limits.

ADVANTAGES

SEMIPREPARATIVE COLUMNThese are designed to isolate milligrams

to multigram quantities . These are packed with number of packaging materials for adsorption, reverse phase, size exclusion, ion exchange.

Length 10-25 cm

Internal diameter 0.8-1 cm

Particle size 5-20 µm

These column have been incorporated for separating mixture 1-50gm range.

These are:-1) Radial compression column2) Axially compressed columnHave advantages that both column length &

packing type can be readily changed by operator.

NEW COLUMN TECHNOLOGY

CRITERIA TO CHOOSE

PACKING MATERIALS

•Particle Shape

•Surface Area

•Particle Size

•Bonding Type

•Pore size

•Carbon load

Particle Shape Spherical particles offer reduced back

pressures and longer column life.

Particle SizeSmaller particles offer higher efficiency,

but also cause higher backpressure. Choose 3µm particles for resolving complex, multi-component samples. Otherwise, choose 5 or 10µm particle size.

Surface AreaHigh surface area generally provides

greater retention, capacity and resolution for separating complex, multi-component samples.

Pore SizeLarger pores allow larger solute

molecules to be retained longer through maximum exposure to the surface area of the particles. Choose a pore size of 150Å or less for sample MW 2000. Choose a pore size of 300Å or greater for sample MW > 2000.

Bonding TypeMonomeric bonding offers increased mass transfer

rates, higher column efficiency, and faster column equilibration.

Si

R

R

(CH2)17CH3Si

CH3

CH3

(CH2)17CH3XOH +monomeric

bonding

Si

CH3

X

(CH2)17CH3X+polymeric

bonding

OH

OH O

O

Si

CH3

(CH2)17CH3

Polymeric bonding offers increased column stability, particularly when highly aqueous mobile phases are used. Polymeric bonding also enables the column to accept higher sample loading.

Carbon Load Higher carbon loads generally offer greater resolution and longer run times. Low carbon loads shorten run times and many show a different selectivity.

PACKING MATERIALSSilica–based packing material

• Silica can be altered by reaction with organochlorosilanes or organoalkoxysilanes to give Si–O–Si–R linkages with the surface.

• The attachment of hydrocarbon chains to silica produces a non–polar surface suitable for RPC in which mixtures of water and organic solvents are used as eluents.

• The most popular material is octadecylsilica (ODS), which contains C18 chains, but materials with C1, C2, C4, C6, C8 and C22 chains are also available.

• The latest silica–based bonded phase to be introduced is a long C30 phase, which make it one of the most retentive phases available.

Different types of packing materials

Special Silica Miscellaneous Chemical Moieties are Bound To Silica, They are Designed To Purify Specific Compounds.

Zirconia Packing Materials A Wider pH range and Are Especially Useful For Basic Separations At Ph 10 Or Higher But At Higher Ph The Silica Gel Starts To Dissolve

Polymer–based Packing Materials

Packing Materials Based On Organic Polymers Are Available. For Ex. Unmodified Styrene–divinylbenzene Co–polymers Have A Hydrophobic Character And Can Be Used For Rpc.

Different types of packing materials

Special Silica Miscellaneous Chemical Moieties Are Bound To Silica, They Are Designed To Purify Specific Compounds

Zirconia Packing Materials A Wider Ph Range And Are Especially Useful For Basic Separations At Ph 10 Or Higher But At Higher Ph The Silica Gel Starts To Dissolve

Polymer–based Packing Materials

Packing Materials Based On Organic Polymers Are Available. For Ex. Unmodified Styrene–divinylbenzene Co–polymers Have A Hydrophobic Character And Can Be Used For Rpc.

SPECIAL SILICAA vast range of materials have

intermediate surface polarities that arise from the bonding to silica of organic compounds that contain groups such as phenyl, cyano, nitro, amino, fluoro, sulfono and diols. There are also miscellaneous chemical moieties bound to silica, as well as polymeric packings, designed to purify specific compounds.

Zirconia packing materials• Zirconia is a metal oxide that is more chemically and thermally

stable than silica. • It is unaffected by changes in ionic strength or organic content of

the mobile phase.• Zirconia packings have a wider pH range and are especially

useful for basic separations at pH 10 or higher, where silica gel starts to dissolve.

• Zirconia can be used for RPC and is extremely stable and efficient through surface modification with polymer or carbon coatings.

• Other chemical modifications of zirconia produce packing materials suitable for normal–phase or ion–exchange chromatography.

• Packing materials based on organic polymers are available. For ex. unmodified styrene–divinylbenzene co–polymers have a hydrophobic character and can be used for RPC.

• Polymeric materials are best when a mobile phase that can go beyond the upper pH limits of silica gel (usually pH 6.5 to 7), as they are stable over a wide pH range.

• They also provide different selectivity and retention characteristics to silica–based reversed phase packings. They avoid problems associated with residual silanol groups (e.g. peak tailing).

• Ion–exchange materials of the styrene–divinylbenzene type are available in which sulfonic acids, carboxylic acids or quaternary ammonium groups are incorporated in the polymeric matrix.

Polymer–based packing materials

NORMAL PHASE PACKAGING MATERIAL

System consist of:-Stationary phase- Polar Mobile phase - non polar STATIONARY PHASE - it is made up of either

silica, alumina of siloxane with polar functional group (cyno, diol, amino, dimethylamino)

MOBILE PHASE:- n-hexane ,methylene chloride, chloroform.

The unmodified silica most widely used in HPLC. It has high efficiency and high permeability which allow normal operating pressure less than 2000psi to be used .It has also relative high surface area.

HPLC grade silica consist of totally porous micro particles with spherical irregular shape and diameter 3,5,7or 10 micrometer.

REVERSE PHASE CHROMATOGRAPHY

• The opposite arrangement of normal phase chromatography that is stationary phase non polar and mobile phase polar is found in reverse phase.

• Stationary phase:-it is made up of octadecyl(c-18)or octyl(C-8) chain or butyl(C-4) or phenyl groups.

• Mobile phase:- Methanol, acetonitrile, tetrahydrofuran, water.

DETECTORS

Comparison of classical Gas chromatography and HPLC

Parameter Gas HPLC

Stationary phase large small

Particle size 60-200micron 3-20micron

Column size large small

Column material glass mostly metal

Column packing low pressure often gravity high pressure

Analysis time 5-60min 1-5min

Operating pressure low < 20psi 500-6000psi

Flow rates Low medium-high

Cost Economical Not Economical

Summary

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hplc analysis finallllll

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