Paper chromatography

Paper chromatography

1. Presented to: Mam Seher Fatima Paper Chromatography

2. Introduction History and Background Types of Paper Chromatography Procedure Steps of Paper Chromatography Techniques of Development Detection of Spots Applications of Paper Chromatography Conclusion Contents

3. Tehreem Abbas Introduction

4. Chromatography is a non-destructive physical technique for separating and identifying the components of a mixture through two phases. Or Chromatography may be defined as a method of separating a mixture of components into individual components through equilibrium distribution between two phases a stationary phase and a mobile phase. Chromatography

5. Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle . They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates Explanation

6. It is defined as a technique in which the analysis of an unknown substance is carried out mainly by the flow of solvent (mobile phase) on specially designed fitter paper (stationary phase). Paper chromatography is a technique used for the separation & identification of relatively small chemical substances by a moving solvent on sheets or strips of filter paper. Paper chromatography is one of the types of chromatography procedures which run on a piece of specialized paper. PAPER CHROMATOGRAPHY

7. All chromatographic methods require one static part (the stationary phase) and one moving part (the mobile phase). The techniques rely on one of the f phenomena: adsorption; partition; ion exchange; or molecular exclusion Paper chromatography usually follow liquid-liquid chromatographic methods stationary phase It is the static part on which separation of mixture is occur.eg whattman filter paper mobile phase The moving part of the chromatographic system which fascinate separation of components Separation depends upon the affinity of constituents with stationary phase

8. The movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. Capillary Action

9. In the beginning of 1860 a scientific concept with the work of FRIEDRICH GOPPELSROEDER who was a pioneer of paper chromatography he developed the theory of capillary analysis by using paper strips while examining wine , milk, alkaloids, dyes and oils among other. His work was the improvement of the work of German scientist Christian Friedrich Schonbein that he introduced the concept in 1865 HISTORY….

10. Chromatography was first developed by the Russian botanist Mikhail Tswett in 1903 as he produced a colourful separation of plant pigments through a column of calcium carbonate. HISTORY…..

11. He was the searching for a way to separate the hidden red and yellow pigments from the green leaves. He published report but chemists paid very little attention to it. There were a few reasons for ignoring the work. HISTORY

12. First, the report was written in Russian, which few Western chemists of the time read. Second, the technique may have seemed too simple to chemists who were used to relying on lengthy extraction, crystallization, or distillation processes to separate mixtures. Within a few years, Tsvett's technique was rediscovered. The rediscovery was by the German organic chemist Richard Martin Willstatter (1872-1942), who was also studying chlorophyll. History

13. Tsswet's book, published in 1910, described a technique that is used today in essentially the same form. New forms of chromatography developed in the 1930s and 1940s made the technique useful for a wide range of separation processes and chemical analysis tasks, especially in biochemistry. HISTORY

14. In 1931 chromatography emerged from its relative obscurity when the German chemist Richard Kuhn and his student, the French chemist Edgar Lederer , reported the use of this method in the resolution of a number of biologically important materials. Beginning in the 1940s paper chromatography found wide application in the Analysis of biologically important compounds, such as amino acids, steroids, carbohydrates, and bile pigments. In this field it replaced, to a large extent, the column technique initiated by Tsvet . Years later, two British researchers, Martin and Synge, improved upon Tsvett's procedure and developed a process called paper chromatography. They were able to use this technique to separate the amino acids in a protein, And were awarded the Nobel prize in chemistry in 1952 for their work in paper chromatography. HISTORY

15. Separation of similar substances by repeated extraction by two immiscible liquids. In standard method of analysis where paper is utilized as a support with one solvent as mobile phase and other as stationary phase. Partition chromatography

16. Adsorption chromatography

17. Separation occurs because of the fact that an equilibrium is established between molecules adsorbed on stationary phase and those which are flowing freely in mobile phase.. Principle

18. Types of Adsorption Chromatography

19. In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cation onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Ion Exchange Chromatography

20. Also known as gel permeation or 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. This causes the larger molecules to pass through the column at a faster rate than the smaller ones. Molecular Exclusion Chromatography

21. Materials Coffee filter (use a brand which is fairly thick, such as the Molinex or the Kafilta cone filter), plastic cups, water, rubbing alcohol, one each of several brands of black felt tip pen or marker (use no more than one brand of "permanent" marker, the rest should be the non-permanent type) GENERAL PROCEDURE OF PAPER CHROMATOGRAPHY

22. Put a small amount of water in a plastic cup, so as to barely cover the bottom of the cup. Assign a number to each pen. Cut the coffee filter into long strips (about 3 - 4 cm wide and 10 cm long). Cut it in such a way that the grain of the filter paper runs parallel to the 10 cm dimension of the strip. Fold each strip along the 10 cm dimension so as to make a crease along the middle of the strip. Identify each strip near one end of the filter paper along the 10 cm dimension. For convenience, this end will be called the top of the paper. Use the same numbering system as used with the pens. Take a black pen and a strip. The two should have the same identification number. Make two small marks with the pen about 2 cm from the bottom of the strip, one on each side of the crease. (It is important to keep the size of the marks small). Repeat with another pen and another numbered strip until all the different pens have been used. Procedure

23. Take a marked strip and stand it in the cup so that the bottom of the strip is touching the water. Make sure that the pen mark stays above the water level. Observe the separation of the ink into different colors as water rises up the coffee filter. Remove the paper from the cup when the water (not the colors) has risen to about 2 - 3 cm from the top. Repeat the above with all the marked strips. Observe the color patterns produced with different pens. Repeat the above using rubbing alcohol in the cup instead of water. Compare the color patterns produced in this case with those produced with the corresponding pens using water PROCEDURE

24. The number of the spots or bands tells you how many compounds are in your substance. Their color and the distance they traveled might help you to identify those compounds. You can try to find out which dyes were used in black marker using other markers from the same package as a reference samples. If certain reference color sample will travel the same distance ( rf ) that one of the black marker colors both of them likely to be the same chemical compound. You can't identify the chemical substance by paper chromatography, but you can roughly analyze the mixture with this simple and neat technique . Understanding the results

25. PROCEDURE DIAGRAME

26. Steps In Paper Chromatography

27. The first step involved in paper chromatography is modification of paper in size or adsorbing some sort of material on the paper. This step depends upon the type of paper chromatographic technique that you are going to use. Paper Modification

28. This step refers to the sample required to be separated. Also the material required for performing the paper chromatography. Red and blue inks, water, fine glass capillary, hard glass boiling tube or gas jar and chromatography paper strip . Materials Required

29. Draw a pencil line 2cm from the edge of the chromatography paper Mix equal quantities of inks in a small pot. Put a very small drop of the mixture of inks in the center of the line on the filter paper strip. Allow it to dry. Place a drop of the mixture on the line. Wait for a while before dripping about 2 more times, this is to ensure there is a concentration of the mixture. Application Of Sample

30. Solvent system is added in the developing jar in such a way that the bottom is covered to a height of at least 1mm by solvent system Inner sides of jar is lined with solvent impregnated paper and top is sealed with lid.  The internal environment is allowed to be saturated with vapors of solvent. If it is not done, the developing plate may lack reproducibility Preparation of environment of glass jar

31. This step includes dipping of paper into the water. This step is very important as due to this step the mobile phase can move over the stationary phase. Thus mobile phase moves along the stationary phase through capillary action . The paper is dipped in solvent in such a manner that the spots will not dip completely into the solvent. The solvent rises up and it is allowed to run 2/3 rd. of paper height for better and efficient result. Immersing The Paper

32. After the solvent has moved a certain distance for certain time, the chromatogram is taken out from the glass jar & position of the solvent front is marked with a pencil. Chromatogram is dried by cold or hot air depending on volatility of solvents. Drying

33. In order to obtain a measure of the extent of movement of a component in a paper chromatography experiment, we can calculate an " Rf value" for each separated component in the developed chromatogram. The Rf value is defined as the ratio of the distance moved by the solute (i.e. the dye or pigment under test) and the distance moved by the the solvent where both distances are measured from the common  Origin  or  Application Baseline. RF VALUE

34. Detection If the substances are colored they are visually detected easily. But for colorless substance, physical and chemical methods are used to detect the spot . Detection

35. Ascending Development. Descending Development. Circular/Radial Development. Wedge Strip Development. Linear/Horizontal Development. Spiral Development. Two Dimensional Development. Different types of development techniques are as follows :

36. The solvent flows against gravity. The spots are kept at the bottom portion of the paper. And kept in a chamber with mobile phase solvent at the bottom. Ascending Development(Go Up )

37. This is carried out in a special chamber where the solvent holder is at the top. The spot is kept at the top and the solvent flows down the paper. In this method the solvent moves from top to bottom so it is call Descending Chromatography. Advantage over Ascending type is that development is faster. Descending Development(A downward Slope )

38. A hybrid of above two techniques is called Ascending-Descending chromatography. Only length of separation increased first ascending takes place followed by descending. Ascending-Descending Development

39. Here the solvent travels from center(mid point) towards periphery of Circular chromatography paper. The entire system is kept in a covered petri dish for development of chromatogram. The wick at the center of paper dips into mobile phase in a Petri dish by which the solvent drains on to the paper and moves the sample radially to form the sample spots of different compounds as concentric rings. Radial mode\Circular Development

40. Circular/Radial Development

41. Here the chromatogram development occurs in two directions at right angles. In this mode the samples are spotted to one corner of rectangular paper and allowed for first development. Then the paper is again immersed in mobile phase at right angle to previous development for second chromatogram. Two Dimensional Development

42. Two Dimensional Development

43. It is a hybrid of two techniques. The upper part of ascending chromatography can be folded over a glass rod allowing the descending development to change over into the descending after crossing the glass rod. The chromatogram is repeatedly developed in the same direction and thus the complete resolution of two or more substances which have R f values close together can be obtained. As the mobile phase one can use either the same solvent system or different solvent systems. Spiral/Ascending Descending

44. This technique chromatography closely resemble to those of column and thin layer chromatography . The components are separate horizontally as solvents movement is . Horizontal technique

45. In this method of development a strip of paper is used vertically. It works on basis of adsorption principle. As the paper absorbs the solvent, the soluble components of the mixture are carried away by the moving solvent which acting as mobile phase. Wedge strip method

46. Analysis of Spot Detection

47. Non destructive Destructive Nondestructive spots can be viewed using physical methods which is by UV detection UV light makes them visible and glow Colorless Spots

48. Destructive types of spots can be viewed by chemical methods through locating agents Locating agents are of 2 types (a) Specific locating agent (b) Non specific locating agents

49. Basically chemical methods involve application of locating agents . Reagents give coloured compounds after reacting. Specific locating agents are basically of 2 types Ninhydrin : forms coloured complexes with amino acids

50. 2,4 Di nitro phenol hydrazine : used o locate aldehydes and ketones Reagents are those which react with specific components having specific functional groups Non specific types form colored compounds with various organic compounds

51. Example of non specific L.A are iodine , ammonia , H2SO4 and H2S Locating agents are applied by (a) Dipping method (b) spraying method

52. Paper chromatography is a simple chromatography technique which has many applications. Its main advantage is that it is not very expensive to perform, and provides clear results. Given below are some important uses of paper chromatography . Separating Colored Pigments Qualitative Analysis Pathology and Forensic Science Analyzing Complex Mixtures Applications

53. Paper chromatography is an effective technique for separating colored pigments from a mixture. A few drops of the mixture of colored pigments are placed on the filter paper (stationary phase) and it is then slowly submerged into a jar of solvent (mobile phase). As the solvent rises up the paper, it dissolves the molecules present in the mixture, their solubility depending on their polarity. Because of different polarity, molecules of each pigment leaves the solution at different places, as the solvent continues to rise up the stationary phase. Thus, each pigment rises up to a particular level on the chromatography paper, and gets separated in the process . Separating Colored Pigments

54. Paper chromatography is used to obtain pure compounds from a mixture. This is done by cutting out and redissolving the patterns formed by each constituent. Also, this technique can be effectively used to remove impurities from chemical compounds. Due to the process of paper chromatography, the impurities get separated from the compound and the pure compound can be obtained . Used in the separation of proteins into homogenous groups for use in medicine . Obtaining Pure Compounds

55. Paper chromatography is one of the methods of qualitative analysis, to analyze or separate the different constituents of a mixture. It is a useful tool for separating polar as well as nonpolar solutes. Pharmaceutical companies use this technique to analyze the different compounds in drugs. Used in the testing of antibiotics and determining the pollutants in water. Qualitative Analysis

56. Paper chromatography is useful in the field of forensic science, for investigation of crime. This is because this process can be successfully carried out even with very small quantities of material. Samples from crime scenes are collected to be analyzed and identified, using this technique . Used in DNA and RNA fingerprinting. Pathological laboratories use paper chromatography to detect the presence of alcohol or chemicals in blood. Pathology and Forensic Science

57. Paper chromatography is used to detect the presence of, or identify certain organic compounds such as carbohydrates and amino acids, from a complex mixture of organic compounds. Used in the separation of amino acids and anions. Analyzing Complex Mixtures