Separation technology:High Performance Liquid Chromatography (HPLC)

Fast Protein Liquid Chromatography (FPLC)

Gas Chromatography (GC)

Name: Sarla YadavClass: M. Sc. MBT 3rd sem

Roll No.: 1873

Introduction • Chromatography:• involves the separation of mixtures due to differences in the distribution coefficient

(equilibrium distribution) of sample components between two different phases which are immiscible i.e.• Stationary phase:

• It may be solid or liquid absorbs on the solid support.

• Mobile phase:• Move over or through stationary phase and carries sample along with it thus resulting in the separation of

its component. It may be liquid or gas.

Chro

mat

ogra

phy Planar chromatography

Paper chromatography

Thin layer chromatography

Column chromatography

Adsorption chromatography

Size exclusion chromatography

Ion exchange chromatography

Affinity chromatography

Gas chromatography

HPLC

FPLC

Distribution Coefficient (Equilibrium Distribution )

• The distribution of a solute between the mobile and stationary phases in chromatography is described by κ, the partition coefficient, defined by:

κ=Cs Cm• Cs: concentration of solute in the stationary phase • Cm: concentration of the solute in the mobile phase.• The mobile phase serves to carry the sample molecules through the

chromatographic column

Retention time• During the sample molecules transportation through the column, each analyte is retained according to that

compound's characteristic affinity for the stationary phase. • The time that passes between the sample injection and peak maximum is called the retention time.

• The retention time, tr, is given in seconds by: tr=ts+tm• ts: time the analyte spends in the stationary phase • tm: time spent in the mobile phase it is often referred to as the dead, or void time, as all components spend tm

in the mobile phase• The area underneath each peak is proportional to the amount of corresponding analyte in solution.

Introduction of HPLC• Expanded form:• High Pressure Liquid Chromatography• High Performance Liquid Chromatography

• Separation based on the partitioning, adsorption, ion exchange or molecular sieving properties.• Drawbacks of conventional column chromatography:• Time consuming• Poor resolution

• HPLC overcome these two major drawbacks and gives faster and superior resolution with sharp and compact peaks.

Principle of HPLC• High performance liquid chromatography (sometimes referred to as High

pressure liquid chromatography)is use of high pressure to push a mobile phase solution through the column of stationary phase allowing separation of complex mixture with high resolution.• When a mixture of components are 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 slower.

The component which has less affinity towards the stationary phase travels faster.• Since no two components have the same affinity towards the stationary phase

and hence the components are separated

Instrumentation • The experimental set-up of HPLC mainly involves :

• Solvent reservoir

• Pump

• Damping device

• Sampling device

• Column • Detector • Fraction collector

• Recorder

Instrumentation contd…….• Solvent reservoir:

• Equipped with degassifiers (provided with heater, temperature regulated magnetic stirrer or a condenser)

• Can also be degassed by heating, stirrer, subjecting it to vacuum, ultrasonic vibrations or bubbling helium gas)

• solvent must be of high purity ( it is advisable to introduce a 1-5 µm microfilter prior to pump to prevent the entry of the impurities into the column)

• Pumps:• Column is quite narrow and is packed with superfine particles so there is high resistance to flow of

solvent and high pressure is required to achieve satisfactory and constant flow rates.• Can delivers pulse free solvent flow upto 20ml/min at pressure upto 300-400 atm.• Material in the pump should be chemically resistant to all solvents.• Pump system work on two principles:• Constant pressure• Constant displacement

• Sampling device:• Sample can be introduced into the column by two ways:

• By syringe injection through a septum of an injection port into eluent stream• By a sample loop from which it is swept into the column by the eluent

Instrumentation contd…….• Column:

• Stainless steel column of 15-50cm in height and having 1-4mm diameter• Can withstand very high pressure of upto 5.5*10⁷ Pa• Corrosion resistant• At the end of column, homogenously porous plug of stainless steel or Teflon are used to retain the

packing material and to ensure the uniform flow of the solvent through the column.• In case of impure sample such as urine, blood or crude cell extracts a guard column, 1-2 cm and

having diameter equal to the analytical column, is introduced between the injector and the analytical column, packed with the same material as in the case of analytical column. The guard columns can be replaced at regular intervals.

• Detectors:• Monitor changes in the composition of the eluent coming out of the column. Commonly used are:

• Refractive index detector• UV detector• Electrochemical detector• Fluorometric detector

• Matrices and stationary phases:• Stationary phase or support for stationary phase should be pressure stable.• Three form of column packing materials are available based on nature of the rigid solid

structure:• Microporous support:

• Micropores ramify through particles• 5-10 µm in diameter

• Porous layer beads or pellicular supports:• Thin, porous active layer is coated onto a solid core such as impervious glass beads.• Thickness of porous layer is 1-3 µm. • Size of glass beads is between 25-50 µm.

• Bonded pilases:• Stationary phase is chemically bonded to an inert support such as silica.

Chromatographic separation principle

Commercial name Nature of stationary phase Type of support

Partition ULTRA Pak TSK ODS Octadecylsilane Porous

ULTRA Pak TSK NH₂ Alkylamine Porous

Bondapak-C₁₈/Corasil Octadecylsilane Pellicular

µ Bondapak- NH₂ Alkylamine Porous

Adsorption Corasil Silica Pellicular

Partisil C₈ Octylsilane Porous

Pellumina Alumina Pellicular

Micropak Alumina Microporous

Exclusion Superose Agarose Soft gel

Fractogel TSK Polyvinylchloride Semi-rigid gel

Bio-Glas Glass Rigid solid

Styragel Polystyrene divinyl benzene Semi-rigid gel

Ion exchange Perisorb-KAT Strong acid Pellicular

Partisil-SAX Strong base Porous

Micropak-NH₂ Weak base Porous

Particil-SCX Strong acid Porous

Application of HPLC• Can be used to isolated and purify compounds for further use.• Can be used to identify the presence of specific compounds in a sample.• Can be used to determine the concentration of a specific compound in a sample.• Can be used to performance chemical separation

• Enantiomers• Biomolecules

• It has a vast amount of current and future applications.• Some uses include:

• Forensic: • analysis of explosive, drugs, fibers, etc.

• Proteomics: • can be used to separate and purify protein samples• Can separate and purify other biomolecules such as carbohydrates, lipids, nucleic acids, pigments, steroids.

• Study of disease:• can be used to measure the presence and abundance of specific biomolecules correlating to disease manifestation.

• Pharmaceutical Research:• all areas including early identification of clinically relevant molecules to large-scale processing and purification.

FPLC- A modification of HPLC

• FPLC was introduced by PHARMACIA (Sweden) at 1982. (Pharmacia’s smart system).• FPLC = Fast Protein Liquid Chromatography• FPLC is basically a “protein friendly” HPLC system.• Stainless steel components replaced with glass and plastic.• The chance of denaturation is high because of their stainless steel made instruments which

elevates the inner temperature and resulting denaturation of sample (protein) under investigation• Also many ion-exchange separations of proteins involve salt gradients; thought these

conditions could result in attack of stainless steel system.

• FPLC is an intermediate between classical column chromatography and HPLC.

Difference between HPLC and FPLCHPLC FPLC

Column is made up of steel Plastic or glass columns are used

Pressurized pump generates pressure 0-550 bar (14.6-8000 psi) It is 0-40 bar in case of FPLC

Standard analytical column of 4-5 mm and 10-30 cm in length is used

Microbore column of dimension 1-2mm*10-25cm is widely used

Flow rate is in between 0.010-10ml/min Flow rate is in between 1-499 ml/hr

Not suitable for thermolabile compounds or protein separation Very reliable in separating and purifying proteins

Can separate any molecule Used only for proteins

Follow adsorption chromatography Follow ion exchange and gel filtration chromatography

Sample loading capacity is low (0.5 ml) Sample loading capacity is high (upto 50 ml)

Stationary phase is generally made up of silica Stationary phase is generally made up of agarose

Gas Chromatography• The components of a sample are dissolved in a solvent and vaporized in order to

separate the analytes by distributing the sample between two phases i.e. a stationary phase (solid or liquid phase) and a mobile phase (gas). An inert gas such as He, Ne or Ar are used as a mobile phase.• Two major types:• Gas-solid Chromatography

• Active solid such as silica is the stationary phase• Gas-liquid chromatography

• Liquid coated as a thin film on surface of a solid support is the stationary phase

Process• The process involves the injection of tiny amount of the sample into the head of

the column by microsyringe.• The sample get vaporized due to high temperature of column and carried through

the column by the flow of an inert gas (mobile phase).• Components of sample separate as it passes through the column because they

move at different rates.• There are many types detector with different range of selectivity to identify the

separate substances leaving through the column i.e.• A non specific detector:

• respond to all compounds except carrier gas• A selective detector:

• respond to a range of compounds with a common physical or chemical property.• A specific detector:

• respond to a single chemical compound.

• After detection a chromatogram is generated.

Components of GC• Carrier gas

- He, Ne, H₂, N₂, Ar

• Sample injection port- micro syringe

• Columns2-50 m with 2-4mm internal diameter coiled stainless steel/glass/Teflon which is fitted in the oven

• Detectors-Flame ionization (FID)-Thermal conductivity (TCD)-Electron capture (ECD)-Nitrogen-phosphorus-Flame photometric (FPD)-Photo-ionization (PID)

Carrier gas

• The carrier gas must be chemically inert.• Commonly used gases include nitrogen, helium, argon, and carbon dioxide. • The choice of carrier gas is often dependent upon the type of detector which is

used. • The carrier gas system also contains a molecular sieve to remove water and other

impurities.-P inlet 10-50 psi-F=25-150 mL/min packed column-F=1-25 mL/min open tubular column

Column • There are two general types of column:

• packed and capillary (also known as open tubular).

• Packed columns contain a finely divided, inert, solid support material ( diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.• Capillary columns have an internal diameter of a few tenths of a millimeter. They

can be one of two types; • wall-coated open tubular (WCOT) • support-coated open tubular (SCOT).

• Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase.

• In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed.

• SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns.

Wall of capillary is coated with stationary phaseSmall amount of sample can be applied

Wall coated open tubular columns (WCOT)

Support coated open tubular columns (SCOT)

Stationary phase is in the form of a thin layer on surface of solid support which in turn is packed into the capillary column.Large amount of sample can be applied

Detectors • There are many detectors which can be used in gas chromatography.• Different detectors will give different types of selectivity. • Detectors can be grouped into concentration dependent detectors and mass flow dependent detectors. • The signal from a concentration dependent detector is related to the concentration of solute in the

detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response.

• Mass flow dependent detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas

• Properties of an ideal detector:• Sensitive (10-8-10-15 g solute/s)• Operate at high T (0-400 °C)• Stable and reproducible• Linear response• Wide dynamic range• Fast response• Simple (reliable)• Nondestructive• Uniform response to all analytes

Common GC Detectors

Detector Type Support gases Selectivity Detectability Dynamic range

Flame ionization (FID) Mass flow Hydrogen and air Most organic cpds. 100 pg 107

Thermal conductivity (TCD)

Concentration Reference Universal 1 ng 107

Electron capture (ECD) Concentration Make-up

Halides, nitrates, nitriles, peroxides, anhydrides, organometallics

50 fg 105

Nitrogen-phosphorus Mass flow Hydrogen and air Nitrogen, phosphorus 10 pg 106

Flame photometric (FPD)

Mass flowHydrogen and air possibly oxygen

Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium

100 pg 103

Photo-ionization (PID) Concentration Make-up

Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics

2 pg 107

Factors affecting GC

• Temperature: • The higher the temperature, the more of the compound is in the gas phase.

• Carrier gas flow: • If the carrier gas flow is high, the molecules do not have a chance to interact with the

stationary phase.

• Column length:• The longer the column is the better the separation usually is.

• It does interact less with the stationary phase, hence the retention time is shorter, but the quality of separation deteriorates.

• The trade-off is that the retention time increases proportionally to the column length. There is also a significant broadening of peaks observed, because of increased back diffusion inside the column.

• Amount of material injected: • The injection of too much sample causes poor separation.

• Conclusion:• High temperatures and high flow rates decrease the retention time, but also deteriorate the

quality of the separation.

Application of GC• Qualitative Analysis –• by comparing the retention time or volume of the sample to the standard / by collecting

the individual components as they emerge from the chromatograph and subsequently identifying these compounds by other method

• Quantitative Analysis-• area under a single component elution peak is proportional to the quantity of the detected

component/response factor of the detectors.

• Miscellaneous• analysis of foods like carbohydrates, proteins, lipids, vitamins, steroids, drug and pesticides

residues, trace elements• Pollutants like formaldehyde, carbon monoxide, benzen, DDT etc• Dairy product analysis- rancidity• Separation and identification of volatile materials, plastics, natural and synthetic polymers,

paints, and microbiological samples• Inorganic compound analysis

Thank you