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  1. 1. Chromatography -- what does it mean? It means ―to write with colors‖ -- literally translated from its Greek roots chroma and graphein. Chroma means color and graphein means to write. --Definition: ―A physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it.‖ (McGraw-Hill Science & Technology Encyclopedia) ―A technique for separating and analysing the components of a mixture of liquids or gases. Chromatography depends on the selective absorption of the different components in a column of powder (column chromatography) or on a strip of paper (paper chromatography). Chromatography is one of the techniques used to identify specific drugs in a urine sample.‖ (Oxford Dictionary of Sports Science & Medicine) ―Any of various techniques for the separation of complex mixtures that rely on the differential affinities of substances for a gas or liquid mobile medium and for a stationary adsorbing medium through which they pass, such as paper, gelatin, or magnesia.‖ (American Heritage Dictionary) Chromatography can be defined as: that technique for the separation of mixture of solutes in separation is brought about by the differential movement of the individual solutes through a porous medium under the influence of a moving solvent. OR Chromatography is a separation technique which involves the differential migration of a multi- component sample through a packed bed. The degree of retardation of one component relative to another is an indication of this differential. This differential is a result of a number of interactions that may occur between the components and the system. These interactions include----- Partition, Adsorption, Ion exchange and Gel filtration. In biological, chemical, phytochemical and pharmaceutical sciences it is frequently necessary to separate, isolate, purify and identify the components of complex mixtures. These complex mixtures are not easily resolved by simple physical and chemical means such as distillation, fractional distillation, crystallization and fractional crystallization etc. It is possible, however, to achieve such a separation rapidly by the process of CHROMATOGRAPHY.
  2. 2. Why use chromatography? The key here is separation. But what is the importance of separation in the lab? Separation of chemical components is vital in any type of chemical analysis. When trying to identify an unknown substance, the sample must first be simplified as much as possible into its constituent compounds. The unknown can then be characterized by individual identification of its parts. This does not imply that the separated chemical components are recovered after the separation and analyzed. Usually, the analytes are irretrievable. Separated compounds are compared to known standards. As with most chemical exploration, it is important to have an idea of what compounds are being searched for in the first place. History: The history of chromatography begins during the mid-19th century. Chromatography, literally "color writing", was used—and named— in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll. Chromatography was first developed by the Russian botanist Mikhail Tswett 1872-1919. In 1903 as he produced a colorful separation of plant pigments through a column of calcium carbonate. He called the new technique chromatography because the result of analysis was written in colors along the length of adsorbent column. New types of chromatography developed during the 1930s and 1940s made the technique useful for many types of separation process. Chromatography became developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several types of chromatography method: paper chromatography, gas chromatography, and what would become known as high performance liquid chromatography. Since then, the technology has advanced rapidly. Researchers found that the main principles of Tswett's chromatography could be applied in many different ways, resulting in the different varieties of chromatography. Simultaneously, advances continually improved the technical performance of chromatography, allowing the separation of increasingly similar molecules. Chromatography has since developed into an invaluable laboratory tool for the separation and identification of compounds. Although color usually no longer plays a role in the process, the same principles of chromatography still apply. The principle behind Chromatography is: The rate of migration of the solute depends upon the rate of interaction of the solute with the two phases, one being the mobile phase and the other stationary phase as the compounds travel through
  3. 3. the supporting medium. Chromatography can separate a mixture into its components with great precision. In fact, it can be used to distinguish between two very similar components, such as proteins that may be different only by a single amino acid. The conditions under which the separation process takes place are also not severe, allowing the use of chromatography on delicate products. With the right materials and operating conditions, chromatography is capable of purifying any soluble or volatile substance. Uses of Chromatography: Chromatography is extensively used in the semiconductor industry, especially in the identification of contaminants that cause yield, quality, and reliability problems. Chromatography is used to separate particles and contaminates in chemical plants. For example, in the chemical industries, pesticides and insecticides like DDT in the groundwater and PCBs (Polychlorinated biphenyls) are removed by the process of chromatography. As a major testing tool, chromatography is used by government agencies to separate toxic materials from the drinking water and also to monitor air quality. One of the significant chromatography uses is made in pharmaceutical companies, who specialize in making medicines. Chromatography is used by pharmaceutical companies to prepare large amounts of pure materials that are further required in making medicines. Also, it is used to check the presence of any contamination in the manufactured compounds. In the field of organic chemistry and pharmacy, chiral compounds are very close to each other in terms of atomic or molecular weight, element composition, and the physical properties. However, they exist in two different forms, called as the enantiomers and optical isomers. Both these compounds though may appear to be same, have very different chemical properties. So, in pharmacy, chromatography becomes crucial to analyze the exact chiral compound so that correct medicines can be manufactured. For instance, a compound called as thalidomide has two optical isomers and one of the isomers can cause birth defect if a pregnant woman consumes it in early stages of pregnancy. So, it is important to carefully separate the isomers. Other important chromatography uses are in the food industry where proper food maintenance is necessary to ensure quality. Chromatography is used as a technique to separate the additives, vitamins, preservatives, proteins and amino acids. Some other chromatography uses are in the detection of drugs or medications in the urine and the separation of traces of chemicals in the case of fire in houses or buildings. It is also very popular in forensic science for investigative purposes. Chromatography technology has gained immense industrial popularity in the past few decades as it can separate chemicals that just differ even in their atomic orientations in space. These were some of the chromatography uses that are used in various technological pursuits in chemical industries.
  4. 4. THIN LAYER CHROMATOGRAPHY (TLC) TLC is universal analytical technique in chemical analysis for organic and inorganic matter. HISTORY- In 1938 Izmailov and Shraiber describe basic principle used it for separation of plant extract. In 1958 Stahl mainly created with bringing out the work on preparing plates and separation of wide variety of compound. TLC is simple and rapid method carrying out using thin layer of adsorbent on plates. ADVANTAGES:  Low cost  Simple and rapid  Short analysis time  All spots can be visualized  Adaptable to most pharmaceuticals  Uses small quantities of solvents  Requires minimal training  Reliable and quick  Minimal amount of equipment is needed  Densitometers can be used to increase accuracy of spot concentration PRINCIPLE:  TLC is included under both adsorption and partition chromatographs.  Separation of component may result due to adsorption or partition or both phenomenons depend upon nature of adsorbent used on plate and solvent system used for development. TLC SUPERIOR OVER OTHER METHOD:  It requires little equipment  Require little time for separation  It is more sensitive  Very small quantity of sample require for analysis  The method use for adsorption, partition, ion exchange chromatography  Components which are separated can be recovered easily.  Quantitative separation of spot and zone are possible.  For identification is permitted spraying of corrosive agent Supporting Surfaces:
  5. 5. In this type a thin layer of a solid coating material is spread on a suitable supporting surface. Types of Supporting Surfaces: 1. Glass Plates 2. Plastic sheets 3. Aluminum sheets. Stationary phase (coating material): A plate of TLC is coated by a solid matter as a stationary phase. The coated material has 0.1- 0.3mm in thickness. TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent with a small amount of inert binder. This mixture is spread as thick slurry on an unreactive carrier sheet. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. Stationary phase has two components: 1. Additive 2. Adsorbent 1. ADDITIVES: These include binders and indicators. i. Binder: These are materials used to hold the thin layer of the coating material into the surface of the supporting plates. Types of binders: a. CaSO4 (Plaster of Paris) or Gypsum (10-15%) b. Silicon dioxide c. Starch (1-3 %) d. Organic polymers e.g. polyvinyl alcohol. ii. Indicator: These are materials mixed with the coating material and binder to help locating the spots on the TLC. Fluorescent indicator will make it florescence during the UV light exposure. The most common used indicator is the fluorescent materials (silica gel 60 F254). 2. ADSORBENTS:
  6. 6. Adsorbent used such as silica gel, alumina, kieselguhr. The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC.  Silica gel: Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups. So, at the surface of the silica gel you have Si-O-H bonds instead of Si-O-Si bonds. The diagram shows a small part of the silica surface. The surface of the silica gel is very polar and, because of the -OH groups, can form hydrogen bonds with suitable compounds around it as well as van der Waals dispersion forces and dipole-dipole attractions. Some modified silica is also used in certain purposes. Silica gel G Silica gel with average particle size 15µm containing 13% calcium sulfate binding agent. Used in wide range pharmacopoeial test Silica gel G254, i.e., Silica gel G with fluorescence added has same application with Silica gel G where visualization is to be carried out under UV light.  Alumina: The other commonly used stationary phase is alumina - aluminium oxide. The aluminium atoms on the surface of this also have -OH groups attached. Anything we say about silica gel therefore applies equally to alumina.
  7. 7.  Kieselguhr:  Cellulose Cellulose powder of less than 30µm particle size. Factors consider for adsorbent: 1. Characteristic of compound to be separated 2. Solubility of compound 3. Nature of substance to be separated 4. To see whether compound is liable to react chemically with adsorbent. 5. Adsorbent particle size 6. Adsorbent do not adhere to glass plate. i. Inorganic adsorbents:  Silica  Silica gel  Alumina  Calcium phosphate  Glass powder  Kieselguhr  Magnesium silicate  Calcium silicate  Phosphate  Ferric & Chromic oxides  Zinc carbonate & zinc Ferro cyanides  Bentonites ii. Organic adsorbents: Normal cellulose powder Charcoal & activated carbon Starch Sucrose Mannitol Dextrin gel
  8. 8. Mobile phase (solvent system): The ability of mobile phase to move up is depend on the polarity itself Volatile organic solvents are preferably used as mobile phase. Choice of mobile phase depends on nature of substance to be separated. And also depend on adsorbent material to be used. Polarity of solvent and substance to be separated plays important role in selection. Purity of solvent also important. Factor affecting mobile phase-  Nature of the substance to be separated.  Nature of the stationary phase used.  Mode of chromatography.  Nature of separation.  Suitable eluents are usually selected by trial and error method, literature review  The solvent used should be of high purity.  Other factor which are taken into consideration while selecting solvents include polarity, solubility etc.  Combination of two solvents gives better separation than with a single solvent Solvent used – 1. Petroleum ether 2. Benzene 3. Carbon tetrachloride 4. Chloroform 5. Diethyl ether 6. Ethanol 7. Methanol 8. Acetone 9. Dichloromethane 10. Diethyl form amide Preparation of chromatographic plate: 1. Size: Heksana 0 Butanol 3.9 Chloroform 4.1 Methanol 5.1 Ethanol 5.1 Acetonitrile 5.8 Air 9.0
  9. 9.  Glass plate or plastic plate used to sprayed adsorbent.  Standard size of plate is 20 X 5cm, 20 X 10 cm, 20 X 20cm.  Plate surface is flat and regular.  Standard film thickness is 250 um.  Thicker layer 0.5to 2 mm used for preparative separation. 2. Method for application of adsorbent on the plate- 1. Pouring- adsorbent of homogeneous particle size made in slurry and pour on plate. 2. Dipping- it used for small plate by dipping two plate back to back in slurry of adsorbent in chloroform or other volatile solvent. 3. Spraying- simply by spraying slurry on plate 4. Spreading- slurry spread by using spatula or glass rod 3. Activation of plate:  After spreading plate allowed to dry and activated by heating about 1000 cfor 30 min.  Plate made with volatile organic solvent may not require further drying  For activation plate is placed in hot oven at temperature 120-150 for one hour. This will eliminate the extra water which occupy in silica gel. Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups. 4. Sample application (spotting):  Given sample should dissolve in any volatile substance and polarity of that solvent should low.  The apparatus used is capillary tube, micropipette or calibrated glass syringes for application of sample on TLC. PRECAUTION: NOT TOUCH THE SURFACE OF STATIONARY PHASE. This causes the distraction of stationary phase and retard the movement of moving phase on stationary phase. How does thin layer chromatography work? The stationary phase - silica gel
  10. 10.  The area of application is kept as small as possible for sharper and greater resolution of sample.  For preparative work sample applied in narrow band  The pipette, loop or syringe use for applying sample.  The spot should be within 2-5 mm diameter.  For preparative work sample up to 4 mg is applied on starting line.  The spots must be about 1-1.5cm away from the bottom of the plate and 0.5 cm away from the plate sides and 0.5 cm away from each other. 5. Development:  Chromatographic Jars (Tanks) made of Glass with air-tight lids of different sizes containing the mobile phase are used for developments. The solvent must be left in the Jars enough time before developing the plates for saturation.  TLC plate placed vertically in rectangular chromatography tank or chamber.  Glass and stainless steel is suitable chamber.  If tank is not saturated, solvent will evaporate and affect the RF value.  Development should be carried out at room temp. by covering chamber with glass plate. Producing the chromatogram We'll start with a very simple case - just trying to show that a particular dye is in fact a mixture of simpler dyes.
  11. 11. A pencil line is drawn near the bottom of the plate and a small drop of a solution of the dye mixture is placed on it. Any labeling on the plate to show the original position of the drop must also be in pencil. If any of this was done in ink, dyes from the ink would also move as the chromatogram developed. When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a covered beaker. It is important that the solvent level is below the line with the spot on it. The reason for covering the beaker is to make sure that the atmosphere in the beaker is saturated with solvent vapour. To help this, the beaker is often lined with some filter paper soaked in solvent. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the plate. As the solvent slowly travels up the plate, the different components of the dye mixture travel at different rates and the mixture is separated into different coloured spots. The diagram shows the plate after the solvent has moved about half way up it. The solvent is allowed to rise until it almost reaches the top of the plate. That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase. Measuring Rf values If all you wanted to know is how many different dyes made up the mixture, you could just stop there. However, measurements are often taken from the plate in order to help identify the compounds present. These measurements are the distance travelled by the solvent, and the distance travelled by individual spots. When the solvent front gets close to the top of the plate, the plate is removed from the beaker and the position of the solvent is marked with another line before it has a chance to evaporate. These measurements are then taken:
  12. 12. 6. Development of chromatogram: a) Ascending development- Plate after spotting placed in chamber and flow of solvent from bottom to top. b) Descending – In this flow of solvent from reservoir to plate is by means of filter paper strip. Solvent moved from top to bottom. The Rf value for each dye is then worked out using the formula: For example, if the red component travelled 1.7 cm from the base line while the solvent had travelled 5.0 cm, then the Rf value for the red dye is: If you could repeat this experiment under exactly the same conditions, then the Rf values for each dye would always be the same. For example, the Rf value for the red dye would always be 0.34. However, if anything changes (the temperature, the exact composition of the solvent, and so on), that is no longer true. You have to bear this in mind if you want to use this technique to identify a particular dye.
  13. 13. APPLICATION: 1- Qualitative:  Identification through comparison of the Rf value with that of Reference material.  Determination of Complexity of mixtures. That will be indicated from number of spots.  Determination the purity of materials.  Monitoring the progress of Chemical reactions.  Monitoring of column chromatography.  Development of finger print TLC for extracts volatile oils or pharmaceutical preparation for future identification and comparison. In this application plates 5×5, 5×10 cm with thin film of coating material are usually used. 2- Quantitative: In this case an accurate volume of samples are applied using syringes. The dimensions of plates range from 5x10 to 20x20 according to the number pf spots used. The plates are developed as usual in the chromatographic tanks. After development the concentration of material can be determined by:  Spot area measurement: Which is directly proportional to the conc. of materials?  Photodensitometry: Measure transmittance, reflection or fluorescence of spots.  Radioactivity: For radioactive material. These measurements are done using TLC Scanner connected to computer that performs all calculations. 3- Preparative TLC: In preparative application 20×20 plates with thick layer of adsorbent 0,25m are used. The mixture is applied as bands and a pilot or guide spots may be used in one side of the plate to enable the detection of the spots location. Problems commonly occur in TLC and how to solve a. The spot shape Is too broad
  14. 14. Diameter is supposed to be < 1-2mm b. The movement of solvent Should be straight up Unproportionality in stationary phase surface will inhibit the movement of solvent c. streaking formation - caused by too concentrated sample What if the substances you are interested in are colourless? There are two simple ways of getting around this problem. Using fluorescence You may remember that I mentioned that the stationary phase on a thin layer plate often has a substance added to it which will fluoresce when exposed to UV light. That means that if you shine UV light on it, it will glow. That glow is masked at the position where the spots are on the final chromatogram - even if those spots are invisible to the eye. That means that if you shine UV light on the plate, it will all glow apart from where the spots are. The spots show up as darker patches. While the UV is still shining on the plate, you obviously have to mark the positions of the spots by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will disappear again. Showing the spots up chemically In some cases, it may be possible to make the spots visible by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures. The chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with
  15. 15. amino acids to give coloured compounds, mainly brown or purple. In another method, the chromatogram is again allowed to dry and then placed in an enclosed container (such as another beaker covered with a watch glass) along with a few iodine crystals. The iodine vapour in the container may either react with the spots on the chromatogram, or simply stick more to the spots than to the rest of the plate. Either way, the substances you are interested in may show up as brownish spots. Using thin layer chromatography to identify compounds Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids. A small drop of the mixture is placed on the base line of the thin layer plate, and similar small spots of the known amino acids are placed alongside it. The plate is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5. The left-hand diagram shows the plate after the solvent front has almost reached the top. The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin. There is no need to measure the Rf values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colours. In this example, the mixture contains the amino acids labelled as 1, 4 and 5.
  16. 16. What separates the compounds as a chromatogram develops? As the solvent begins to soak up the plate, it first dissolves the compounds in the spot that you have put on the base line. The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards. How fast the compounds get carried up the plate depends on two things: How soluble the compound is in the solvent. This will depend on how much attraction there is between the molecules of the compound and those of the solvent. How much the compound sticks to the stationary phase - the silica get, for example. This will depend on how much attraction there is between the molecules of the compound and the silica gel. Suppose the original spot contained two compounds - one of which can form hydrogen bonds, and one of which can only take part in weaker van der Waals interactions. The one which can hydrogen bond will stick to the surface of the silica gel more firmly than the other one. We say that one is adsorbed more strongly than the other. Adsorption is the name given to one substance forming some sort of bonds to the surface of another one. Adsorption isn't permanent - there is a constant movement of a molecule between being adsorbed onto the silica gel surface and going back into solution in the solvent. Obviously the compound can only travel up the plate during the time that it is dissolved in the solvent. While it is adsorbed on the silica gel, it is temporarily stopped - the solvent is moving on without it. That means that the more strongly a compound is adsorbed, the less distance it can travel up the plate. In the example we started with, the compound which can hydrogen bond will adsorb more strongly than the one dependent on van der Waals interactions, and so won't travel so far up the plate. What if both components of the mixture can hydrogen bond? It is very unlikely that both will hydrogen bond to exactly the same extent, and be soluble in the solvent to exactly the same extent. It isn't just the attraction of the compound for the silica gel which matters. Attractions between the compound and the solvent are also important - they will affect how easily the compound is pulled back into solution away from the surface of the silica And what if the mixture contained amino acids other than the ones we have used for comparison? There would be spots in the mixture which didn't match those from the known amino acids. You would have to re- run the experiment using other amino acids for comparison.
  17. 17. However, it may be that the compounds don't separate out very well when you make the chromatogram. In that case, changing the solvent may well help - including perhaps changing the pH of the solvent. This is to some extent just a matter of trial and error - if one solvent or solvent mixture doesn't work very well, you try another one. (Or, more likely, given the level you are probably working at, someone else has already done all the hard work for you, and you just use the solvent mixture you are given and everything will work perfectly!)