CHROMATOGRAPHY

Submitted to:

Sayema Khanam

Senior lecturer

Dept. of Pharmacy

South East

University

Submitted by:Md. Asaduzzaman Id: 2012000300048Batch: 19th

DefinitionChromatography (from Greek chroma "color and  graphein "to write") 

is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases.

mobile phase = solvent or gasstationary phase = column packing material

Examples of Chromatography

Gas ChromatographyUsed to determine the chemical composition of unknown substances, such as the different compounds in gasoline shown by each separate peak in the graph below.

Paper ChromatographyCan be used to separate the components of inks, dyes, plant compounds (chlorophyll), make-up, and many other substances

Liquid ChromatographyUsed to identify unknown plant pigments & other compounds.

Thin-Layer ChromatographyUses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances.

Application of chromatography 

• The chromatographic technique is used for the separation of amino acids, proteins & carbohydrates.

• It is also used for the analysis of drugs, hormones, vitamins & brain amines.

• Helpful for the qualitative & quantitative analysis of complex mixtures.

• The technique is also useful for the determination of molecular weight of proteins.

History• 1903: Michael Tswett (or Tsvet) a Russian botanist separated the pigments in green 

plant leaves using a glass column packed with 2 μm inulin and ligroin as mobile phase. 

• 1938: Izmailov & Schreiber worked out a procedure where the solid stationary chromatographic phase is distributed as a thin film on a glass plate.

• 1941: Archer John Porter Martin & Richard Laurence Millington Synge published a paper on liquid partition chromatography that set the stage  for gas liquid chromatography.

• 1952: Martin and Synge were awarded the Nobel prize in chemistry for the invention of column partition chromatography, and particularly paper chromatography.

• 1956: Martin & James applied gas chromatography to the separation  of acids and amines.

Michael Tswett Archer John Porter Martin Richard Laurence Millington Synge

Methods of chromatography: Two phases chromatography are:

1. Mobile phase2. Stationary phase

Classification of chromatographic methods Chromatography

Mobile Phase Gas Liquid

Stationary phase Liquid Solid Bonded Liq. Liq Solid Bonded Liq. Ion Ex Polymer Solid

Process (part’n) (adsorp’n) (adsorp’n- (part’n) (adsorp’n) (adsorp’n- (ion (part’n part’n) HPLC part’n) exc’ng) sieving) paper HPLC exclusion chromat

Techniques of chromatography • Techniques by Chromatographic bed shape

Column chromatography

-The stationary phase is held in to a tube made of glass or metal (gel-ion exchange-adsorption)

Planar chromatography Paper chromatography

- A specific type of papers is used as stationary phase. Thin layer chromatography

- The stationary phase is spread on glass or plastic or aluminum sheets.

• Techniques by Physical state of mobile phase Gas chromatography

-The mobile phase is an inert gas nitrogen or helium. Again if the stationary phase is solid it is called: Gas-Solid Chromatography(GSC). When stationary phase is liquid it is called: Gas-Liquid Chromatography(GLC).

Liquid chromatography

-The mobile phase is liquid. In case of separation by adsorption the stationary phase is solid so it is called: Liquid-Solid Chromatography(LSC). If separation occurs through partition, the stationary phase is liquid so it is called: Liquid-Liquid Chromatography(LLC).

• Affinity chromatography Supercritical fluid chromatography

Mechanisms of separation in chromatography are:1. ADSORPTION (LIQUID-SOLID) CHROMATOGRAPHY2. PARTITION (LIQUID-LIQUID) CHROMATOGRAPHY3. ION-EXCHANGE CHROMATOGRAPHY4. Molecular Exclusion Chromatography

5. Affinity chromatography

6. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY1. Adsorption chromatography: It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.

2. Partition chromatography: This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.

Ion Exchange chromatography : In this type of chromatography the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. It has 2 prinicipal types of ion- exchanger is cationic and anionic.

Molecular Exclusion Chromatography (gel permeation/gel filtration): This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume.

Affinity chromatography :

This technique separates proteins on the basis of a reversible interaction between a protein or

group of proteins and a specific ligand coupled to a chromatography matrix. The technique

can be used to separate active bio molecules from denatured or functionally different forms,

to isolate pure substances present at low concentration in large volumes of crude sample and

also to remove specific contaminants.

High-performance liquid chromatography (HPLC)

Liquid  chromatography  (LC)  is  a  separation  technique  in which  the mobile 

phase  is a  liquid. Liquid chromatography can be carried out either  in a column or a 

plane.  Present  day  liquid  chromatography  that  generally  utilizes  very  small  packing 

particles  and  a  relatively  high  pressure  is  referred  to  as high  performance  liquid 

chromatography (HPLC).

  

Uses:•It  has  the  ability  to  separate,  identify,  and  quantitate  the  compounds  that  are present in any sample that can be dissolved in a liquid.•  HPLC  can  be,  and  has  been,  applied  to  just  about  any  sample,  such  as pharmaceuticals,  food,  nutraceuticals,  cosmetics,  environmental  matrices,  forensic samples, and industrial chemicals.

Chromatography detectorA chromatography detector is a device used in gas chromatography (GC) or liquid 

chromatography (LC) to detect components of the mixture being eluted off the chromatography column. There are two general types of detectors: destructive and non-destructive. The destructive detectors perform continuous transformation of the column effluent (burning, evaporation or mixing with reagents) with subsequent measurement of some physical property of the resulting material (plasma, aerosol or reaction mixture). The non-destructive detectors are directly measuring some property of the column effluent (for example UV absorption) and thus affords for the further analyte recovery.

Destructive detectors• Flame ionization detector (FID)• Aerosol-based detector (ABD)• Flame photometric detector (FPD)• Atomic-emission detector (AED)• MS detector• Nitrogen Phosphorus Detector (NPD)• Evaporative light scattering detector (ELSD)

Non-destructive detectors• UV detectors : Fixed or variable wavelength, which includes diode array detector (DAD or 

PDA). The UV absorption of the effluent is continuously measured at single or multiple wavelengths. These are by far most popular detectors for LC.

• Thermal conductivity detector (TCD): Measures the thermal conductivity of the effluent. Only used in GC.

• Fluorescence detector : Irradiates the effluent with a light of set wavelength and measure the fluorescence of the effluent at a single or multiple wavelength. Used only in LC

• Electron capture detector (ECD): The most sensitive detector known. Allows for the detection of organic molecules containing halogen, nitro groups etc.

• Conductivity monitor: Continuously measures the conductivity of the effluent. Used only in LC when conductive eluents (water or alcohols) are used.

• Photoionization detector (PID): Measures the increase in conductivity achieved by ionizing the effluent gas with UV radiation.

• Refractive index detector (RI or RID): Continuously measures the refractive index of the effluent. Used only in LC. The lowest sensitivity of all detectors. Useful when nothing else works and at high analyte concentrations.

• Radio flow detector: Measures radioactivity of the effluent. This detector can be destructive if a scintillation cocktail is continuously added to the effluent.

• Chiral detector: Continuously measures the optical angle of rotation of the elutant. Used only in LC when chiral compounds are being analyzed.

Rf Factor

Retention Factor• It is a number that represents how far a compound travels in a particular solvent.

• It is measured by measuring the ratio of the distance traveled by the compound and the distance traveled by the solvent. 

Thank you for your kind attention