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Paper No: 2Analytical Chemistry Module: 32 General : Distribution Coefficient and its implications

Development Team

Principal Investigator Prof. R.K. Kohli & Prof. V.K. Garg & Prof. Ashok Dhawan Co- Principal Investigator Central University of Punjab, Bathinda

Dr. J. N. Babu, Paper Coordinator Central University of Punjab, Bathinda Dr. Varinder Kaur Content Writer Department of Chemistry, Panjab University, Chandigarh Content Reviewer Prof. Ashok Kumar Punjabi University, Patiala

Anchor Institute Central University of Punjab

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Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

Description of Module

Subject Name Environmental Sciences

Paper Name Module General Chromatography : Distribution Coefficient and its implications Name/Title Module Id EVS/AC-II/03

Pre-requisites

Objectives Keywords

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Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

Module 3: General Chromatography: Distribution Coefficient and its implications Objectives: To study the basics of general chromatography and know the following self generated questions. 1. What is chromatography? 2. How chromatography was developed? 3. What is the role of chromatography in daily life? 4. What is the basic principle and terms commonly used in chromatography? 5. How is it categorized? 6. What is distribution coefficient? 7. What are the main applications of chromatography? 8. What are the main advantages of using chromatography?

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Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

MODULE 32:GENERAL CHROMATOGRAPHY: Distribution Coefficient and its implications 1. Description and background

A Russian botanist, Mikhail Tswett was working on the separation of plant pigments i.e. chlorophylls and carotenoids. He packed a glass tube with powdered calcium carbonate and poured finely ground plant extracts dissolved in different . After adding mixture of from the upper end of glass tube, he observed separation of different colors in the column with the downward movement of solvents due to gravity. The appearance of different bands was due to separation of colored compounds in column, so, Tswett coined term ‘chromatography (Greek word ‘chroma’ means color and ‘graphy’ means writing). Afterwards, various scientists worked on the chromatographic methods and it established foundation of separation science.

Chromatography is a technique which is used to separate various closely related chemical components of mixture. It is divided into two categories on the basis of their usage; Analytical chromatography and preparative chromatography (given below).

Analytical chromatography Preparative chromatography

It is used for analytical purposes It is used for separation mainly in commercial applications

It uses small sample sizes (ppm or below) It used large sample sizes

It is used mainly for qualitative or It involves separation of various components quantitative analysis like separation of impurities from medicines, chemical compounds etc.

Some common terms used in chromatography

Before understanding the principle of chromatography, it is important to know some basic terms related to chromatography.

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Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

1. Stationary phase: It is the phase which remains fixed at its position. It provides active sites for interacting the components of sample. It is usually a solid, viscous liquid or a thin coating bonded with a surface.

2. Mobile phase: It is the phase which moves through the stationary phase and is responsible for the movement of components of sample through the stationary phase. It may be a liquid or a gas.

3. Analyte: It is the component (or components) of interest which is (are) under observation or to be analyzed. It may be a liquid, , gas or volatile species.

4. Elute: The mobile phase after passing through the stationary phase bringing the components out of the system and is termed as elute. The process of getting components out from the system with the use of appropriate medium is called as .

5. Eluent: The medium which is introduced to the system to bring the components out of the system is called as eluent. It may be same of different as the mobile phase.

Basically in chromatographic techniques, sample having mixture of various components (or analyte) is exposed to the mobile phase running through the stationary phase; where the components interact with the active sites and adhere to the stationary phase. These components get released again in the mobile phase (or eluent) to come out of the system. However, the stay of components on the stationary phase depends upon the physico-chemical properties of the components, therefore, they release/move at different time intervals. This leads to the separation of components.

Example: Suppose a mixture with three colored components A, B, and C is analyzed using a glass tube filled with silica. The diagrammatical procedure for the separation of components is given below.

In this case, glass tube (column) is filled with silica (stationary phase) and the sample mixture (analyte) is poured from the top of the tube. After running solvent through the column, components move down and get separated. With the continuous addition of solvent they get separated into bands. The component B has weak interaction (than A and C), whereas component A adheres to the stationary phase strongly, therefore, their movement follow the order; component B followed by C and then A. In this way, the band due to component B comes out and is collected in a flask. Similarly, components A and C are collected as they elute with the solvent. 5

Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

Fig 1 (I) Addition of sample mixture into glass tube filled with stationary phase (II) Addition of mobile phase and appearance of bands (III) Downward movement of bands with addition of more eluent (IV) Elution of component B and movement of components A and C downward with the addition of eluent.

Exercise: Separate various components present in green leaf.

Overall, the separation of components depends upon the different affinities of components to adhere the stationary phase and solubilization of components in the mobile phase. If the affinity of a component to interact with the stationary phase is more, it will stick for longer period and come out slowly, and vice versa. In contrast, if the of a component is more mobile phase it will come out rapidly and vice versa. Hence, the separation of components can be achieved by choosing a best combination of stationary phase and mobile phase.

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Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

Distribution of components between two phases

As discussed above, the components of a mixture are distributed between stationary phase and mobile phase. This distribution of a particular component in the two phases achieves equilibrium as given below.

Cmobile Cstationary

Where Cmobile is the concentration of a component in the mobile phase Mobile Stationary phase phase and C stationary is the concentration of that component in stationary phase.

The equilibrium constant (KD) is termed as or distribution coefficient, which expresses relative distribution of components in the two phases.

The distribution coefficient (KD) is ratio of of a component in the stationary phase to the molar concentration of that component in mobile phase. The concentration of the component can be expressed in mass per unit volume or mass per unit mass depending upon the technique.

molar concentration of a component in the stationary phase Partition coefficient = molar concentration of that component in mobile phase

Classification of chromatographic techniques

The chromatographic techniques can be categorized on the basis of different parameters. All the types are summarized below.

On the basis of physical state and nature of stationary and mobile phase

Heterogenous Homogenous chromatography chromatography 1 Liquid-liquid Solid-liquid Solid-gas Liquid- chromatography chromatography chromatography

Size exclusion Ion exchange chromatography chromatography

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Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

On the basis of principle of separation

2 Adsorption chromatography Partition chromatography

Liquid-liquid Liquid-gas Solid-liquid Solid-gas chromatography chromatography chromatography chromatography

On the basis of polarity of phases

Normal phase 3 Reverse phase chromatography chromatography

Liquid-liquid Liquid-gas Solid-liquid Solid-gas chromatography chromatography chromatography chromatography

On the basis of shape of stationary phase

4 Columnar chromatography Planar chromatography

Gas Liquid Column Thin layer Paper chromatograph chromatography chromatography chromatography chromatography y

Overall, the chromatographic techniques can be categorized into the following forms as given below.

Type Stationary phase Mobile phase Solid (cellulose) Liquid Thin layer chromatography Solid (silica or alumina) Liquid

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Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

Column chromatography Solid (silica or alumina) Liquid Size exclusion chromatography Solid (microporous silica beads) Liquid Ion exchange chromatography Solid (cationic or anionic) Liquid Solid (immobilized surfaces) Liquid Gas chromatography Solid or liquid lined on a solid Inert gas surface High performance liquid Solid Liquid chromatography

Advantages of chromatography over other separation techniques 1. It can be used to separate multi-components of a mixture. 2. It can be used for separation without knowing about the exact identity of the component. 3. It can be used in every field such as chemical, biological, biochemical, pharmaceutical and industrial applications. 4. It can be used for all the sizes i.e. sub-nanograms to kilograms. Applications of chromatography 1. It is used for the purification and testing of various components of medicines in pharmaceutical industries. 2. It is used for the separation of hydrocarbons in petroleum industry. 3. It is used in the food industry. 4. The environmental testing laboratories mainly employ chromatographic techniques to monitor the amounts of pollutants like pesticides, polyaromatic hydrocarbons, wastes etc in the environment. 5. It is used to test the drinking water. 6. It is used for the separation of insulin purification, plasma fractionation and enzyme purification. 7. It is also used by forensic laboratories, fuel industries and bioinformatic laboratories. Implications of distribution constant As discussed earlier, a component distributes between two immiscible phases and establishes an equilibrium governed by distribution law. The ratio of activities of the component between two phases is constant and the equilibrium constant may be termed as distribution constant. For example; a 9

Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

component A is distributed between mobile and stationary phase and establishes the following equilibrium. A (mobile)  A(stationary) The equilibrium constant or the distribution constant Kc is given by

Kc = (aA)stationary/(aA)mobile=[Cstationary]/[Cmobile]

Where (aA)stationary is the activity of solute A in the stationary phase, (aA)mobile is the activity of A in mobile phase, Cstationary is the molar concentration of A in stationary phase and Cmobile is the molar concentration of A in mobile phase. A distribution constant allows the calculation of concentration of a component remained in the solution after repeated extractions and helps to obtain an efficient method for extractive separations. For example; the concentration of A remained in the solution after I extractions can be given as follows.

푖 Vaq [A]i = ( ) [A]o VorgK + Vaq

where [A]iis the concentration of A remaining in the aqueous solution after extracting Vaq mL of the solution with an original concentration of [A]0 with i portions of the organic solvent, each with a volume of Vorg. Bibliography  D.A. Skoog; F. J. Holler, T.A. Nieman (1998). Principles of Instrumental Analysis, 5th edition. Orlando, FL: Harcourt Brace College Publishers.  J. Tyson, Analysis. What Analytical Chemists Do. London: Royal Society of Chemistry, 1988. A brief book that succinately discusses what analytical chemists do and how they do it.  R.W. Murray, Analytical Chemistry is what analytical chemists do, Editorial, Anal. Chem., 66 (1994) 682A.  D.C. Harris, Quantitative chemical analysis, 6th Ed  Douglas A. Skoog, James Holler, Stanley R. Crounch, “Principles of Instrumental Analysis”  Willard H.W Merritt, L.L Dean J A Settie FA, Instrumental Methods of Analysis  Douglas A Skoog, Donald M, West Holler Thomson, Fundamentals of Analytical Chemistry, 8th Ed  Galen W. Ewing, Instrumental Methods of Chemical Analysis 10

Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications

 D. C. Harris, Exploring Chemical Analysis, 3rd Ed  J. Mendham, R.C. Denney, J.D. Barnes, M.J.K. Thomas, Vogel's Quantitative Chemical Analysis (6th Edition) 6th Edition  Vladimir V. Rachinskii, The General Theory of Sorption Dynamics and Chromatography, translation of Russian book  C. F. Poole, The Essence of Chromatography, Elsevier 2003  Kevin Robards, P. E. Jackson, Paul A. Haddad, Principles and Practice of Modern Chromatographic Methods, 2004  A. Braithwaite, F. J. Smith, Chromatographic Methods, 4th Edition  Inamuddin, Ali Mohammad, Green Chromatographic Techniques, Springer, 2014  Heftmann, Chromatography: Fundamentals and applications of chromatography and related differential migration methods, Elsevier, 2004.  Green Chromatographic Techniques: Separation and Purification of Organic and inorganic analytes, edited by Dr. Inamuddin, Ali Mohammad, Springer.

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Analytical Chemistry Environmental Sciences General Chromatography : Distribution Coefficient and its implications