Introduction chromatography. Types of chromatographic techniques

Definition of terms used in chromatography
Types

1. Separation techniques Distillation Sublimation Precipitation Extraction Crystallisation Floatation Dialysis El e ct r o d eposi t ion Chromatography

2. Mixtures & Compounds Mixture – Two or more substances that are mixed together, but not chemically combined . Examples of mixtures ... Air – mixture of gases Bowl of cereal – mixture of cereal and milk Soda pop – mixture of soda syrup, water, and CO 2 gas Fog –water suspended in air Kool-Aid – mixture of water, sugar, and flavor crystals Examples of compounds ... Salt –Sodium and chlorine combined chemically Water –Hydrogen and oxygen combined chemically Carbon Dioxide – Carbon and oxygen combined chemically Compounds – Two or more elements that are chemically combined .

3. Solutions Solutions are mixtures in which one substance is dissolved in another. Solutions have two parts: solute and solvent The solute is the substance that is dissolved. The solvent is the substance that does the dissolving Solubility - A measure of how much of a given substance will dissolve in a liquid. A substance that does not dissolve in water is called insoluble. A substance that does dissolve in water is called soluble.

4. ADVANTAGES OF CHROMATOGRAPHY OVER OTHER METHODS Se p arati o n o f m i x t u res o f che m icals hav i ng c l o s er physical or chemical properties. Gentle method. Separation in even micrograms is possible. Simple, rapid and efficient technique.

5. History The subject of Chromatography was introduced into scientific world in a very modest way by M. Tswett in 1906. He employed a technique to separate various pigments such as chlorophylls and xanthophylls by passing the solution of these compounds into the glass column which was packed with finely divided calcium carbonate. After the later, Thompson and Way had realized the Ion Exchange properties of soils. after three decades, i n 1935 Ada m s Almost observed the Ion Exchange characteristics and H o lm e s i n crushed phonograph. This observation opened the field for preparation of Ion Exchanged resins. T h e concept of G a s - Liquid C h r oma t ography w a s f i rst introduced by Martin and Synge in 1941 .

6. They were also responsible for the development in Liquid- Liquid chromatography. In 1944 , from Martin laboratory, the separation of amino acid by paper chromatography was reported. In 1959 , a technique known as Gel Filtration chromatography was observed which is used to separate low molecular weight substances from high molecular substances. In 1960 , further improvement in liquid chromatography led to the development of High Performance Liquid Chromatography . In 1970 improvement in the field of adsorption chromatography in the form of Affinity chromatography which was mainly based on biological interactions.

7. Supercritical fluid chromatography is a hybrid of gas and liquid chromatography and combine advantageous feature of the both gas and liquid chromatography. T he entire twentieth century can be named as the century of chromatography .

8. Ch rom ato gra phy Is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster.

9. What attracts the scientists to Chromatography? Chromatography is a way to separate two components based on a specific characteristics. What makes chromatography so useful... The results are reproducible with better accuracy than the other techniques. Chromatography can separate more complex mixtures than the other techniques. Chromatography is less time consuming and cheaper.

10. Gas Chromatography Used to determine the chemical composition of unknown substances, such as the different c o m po u nds i n g a s o l i ne s h o wn b y e a c h separate peak in the graph below. Paper C h r oma t ography Can be used to separate the components of inks, dyes, plant compounds (chlorophyll), make- up, and many other substances Liquid C h r oma t ography Used to identify unknown plant pigments & other compounds. Thin-Layer C h r omatography Uses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances. Examples of Chromatography

11. What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separate Analyze Identify Purify Qu a nti f y Components M i xtu r e

12. Illustration of Chromatography Components Affinity to Stationary Phase Affinity to Mobile Phase Blue ---------------- Insoluble in Mobile Phase Black         Red        Yellow           M i xtu r e Components Separat i on Stationary Phase Mobile Phase

13. Uses for Chromatography Chromatography is used by scientists to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample

14. Uses for Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect blood or alcohol levels in a patient’s blood stream Law Enforcement – to compare a sample found at a crime scene to samples from suspects Environmental Agency – determine the level of pollutants in the water supply Manufacturing Plant – to purify a chemical needed to make a product

15. CHROMATOGRAPHIC MECHANISMS Adsorption Partition Ion exchange Size-exclusion

16. ADSORPTION Based on relative polarities . Compounds having high affinity towards the stationary phase travel slower Compounds having lesser affinity towards the stationary phase travel faster No two components have same affinity for a combination of stationary phase, mobile phase and other conditions.

17. PARTITION Based on relative solubility. Solutes will be distributed according to their partition coefficients. Components which are more soluble in the stationary phase – Travel slower Components which are less soluble in the stationary phase –Travel faster No two components have same partition coefficient for a particular combination of stationary phase, mobile phase and other conditions.

18. ION EXCHANGE Stationary phase contains fixed charged groups and mobile counter ions Counter ion exchange with ions of solute Reversible exchange of ions takes place between similar charged ions of solute in the mobile phase and that of an ion exchange resin .

19. SIZE EXCLUSION Retention depends on the extend to which the solute molecules are trapped in the pores of inert stationary phase. This depends on size of molecules. Smaller molecules diffuses into the pores of the stationary phase – Travel slower. Larger molecules do not enter the pores of stationary phase – Travel faster.

20. Classification Of Chromatography On the basis of interaction of solute to the stationary ph a s e On the basis of ch roma t o g ra p h i c bed shape On the basis of physical state of mobile ph a s e Adsorption C hr oma t og r a ph y Partition Chroma t o gr a p h y Io n E x ch a n g e C hr oma t og r a ph y Size Exclusion Chroma t o gr a p hy Two Di men s i onal Three Di men s i onal Thin Layer Chroma t o gr a p hy Paper Chroma t o gr a p hy Column C hr oma t og r a ph y Liquid Chroma t o gr a p hy Gas Chroma t o gr a p hy Super Cr itical Fluid Chromatograph y

21. Types of Chromatography

22. Terminology I nflu e nt – The liquid e n terin g th e colum n . E f flu e nt – The liquid living th e colum n . Elution – The process by which the adsorbed ions are removed from the column. El u e n t – The s o lution used fo r e l ution. El u ate – The s o lution obtained a s resul t o f e lution.

23. HOW TO CHOOSE A METHOD Polarity of the sample. Solubility and volatility of sample. Resolution required. Concentration of analyte. Detection limit. Physical and chemical properties of sample.