CHROMATOGRAPHY Definition: It is a method used by scientists for separating organic and inorganic compounds so that they can be analyzed

and studied. By analyzing a compound, a scientist can figure out what makes up that compound. The word chromatography means "color writing" which is a material in plant life, a Russian botanist invented chromatography in 1903. It is from the Greek words Chroma meaning color and graphein meaning to write. His name was Mikhail
Semenovich Tswett.

Chromatography Terms

The analyte is the substance that is to be separated during chromatography. Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample. A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing. A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture. A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation. The effluent is the mobile phase leaving the column. An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing. The mobile phase is the phase which consists of the sample being separated/analyzed and the solvent that moves the sample through the column. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). Preparative chromatography is used to purify sufficient quantities of a substance for further use, rather than analysis. The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste. The solute refers to the sample components in partition chromatography. The solvent refers to any substance capable of solubilizing other substance, and especially the liquid mobile phase in LC. The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography.

Applications: find out what is in a solid or a liquid determine what unknown substances are the Police, F.B.I., and other detectives use chromatography when trying to solve a crime determine the presence of cocaine in urine, alcohol in blood, and lead in water

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COLUMN Chromatography
It is a method used to purify individual chemical compounds from mixtures of compounds. 1. An impure sample is loaded onto a column of adsorbant, such as silica gel or alumina. An organic

solvent or a mixture of solvents (the eluent) flows down through the column. Components of the sample separate from each other by partitioning between the stationary packing material (silica or alumina) and the mobile eluent. Molecules with different polarity partition to different extents, and therefore move through the column at different rates. The eluent is collected in fractions. Fractions are typically analyzed by thin-layer chromatography to see if separation of the components was successful. 2. Clamp the column to a ring stand and add enough sand to fill the curved portion of the column. 3. Place a pinch clamp on the tubing, then fill the column 1/4 to 1/3 full with the initial eluent. (The composition of eluent is often changed as the separation proceeds.) 4. Prepare a slurry of silica in the intial eluent by pouring dry silica into a beaker of eluent. (Add a volumne of silica gel, such as 20 mL, to approximately double the volume of eluent, 40 mL.) CAUTION: keep the dry silica in your hood and be careful not to inhale the lightweight substance. 5. Quickly but carefully pour the slurry into the column. Stir and pour immediately to maximize the amount of silica that goes into the column instead of remaining behind in the beaker. You may find a clean spatula or glass rod helpful in transferring the silica. 6. Remove the pinch clamp to allow solvent to drip into a clean flask. Tap on the side of the column with a rubber stopper or tubing to help the silica settle uniformly. 7. Use a Pasteur pipet to rinse any silica that is sticking to the sides of the column. Allow the silica to settle while eluent continues to drip into the flask. 8. Once the silica has settled, carefully add sand to the top of the column. Sand is heavier than silica. If the silica has not settled, the sand may sink into the silica instead of forming a layer on top of it. (You may need to rinse down sand that sticks to the side of the column. 9. Drain eluent from the column until no solvent remains above the surface of the sand. 10. Using a long Pasteur pipet, carefully add your sample to the column. 11. Drain eluent from the column until no sample remains above the surface of the sand. 12. Use ~ 1 mL of eluent to rinse your container and pipet. Add this milliliter of sample to the sand. Drain eluent from the column until no liquid remains above the surface of the sand. 13. Repeat step 12 two or three times to completely transfer your sample onto the silica gel. If you do not do and repeat step 12, your sample will remain in the sand instead of on the silica. Sample remaining in the sand will dissolve in the eluent that you add in step 14, ruining the possibility of good separation of components. 14. Once you have rinsed your sample onto the silica, carefully add eluent to the top of the column. To avoid disturbing the top of the column, it's a good idea to carefully pipet an inch or two of solvent onto the column instead of pouring solvent directly onto the sand. 15. Add more eluent as necessary. The eluent collected prior to the elution of sample can be recycled. The composition of the eluent can be changed as the column progresses. If the eluent composition is to be changed, ALWAYS start with least polar solvent/mixture and change to the more polar solvent/mixture. 16. Analyze the fractions by thin-layer chromatography to determine a) if the fraction contains more than one component and b) if fractions can be combined without affecting the purity of those fractions. PAPER Chromatography Definition: It is an analytical technique for separating and identifying mixtures that are or can be colored, especially pigments. It was invented by two British biochemists, Archer John Porter Martin and Richard Laurence Millington Synge.

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Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90 in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids. 1. A small concentrated spot of solution that contains the sample is applied to a strip of chromatography paper about 2 cm away from the base of the plate, usually using a capillary tube for maximum precision. 2. The paper is then dipped in to a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper, also this can be explained as differential absorption of the solute components into the solvent. 3. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. 4. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibers in the paper. 5. In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same values in a particular solvent. In these cases, two-way chromatography is used to separate the multiple-pigment spots. The chromatogram is turned by ninety degrees, and placed in a different solvent in the same way as before; some spots separate in the presence of more than one pigment. THIN-Layer Chromatography Definition: (TLC) It is a chromatography technique used to separate mixtures. Often used for monitoring chemical techniques and for the qualitative analysis of reaction products. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose immobilized onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated is then dissolved in an appropriate solvent and is drawn up the plate via capillary action, separating the experimental solution based on the polarity of the components of the compound in question. 1. Prepare the developing container. o The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top: Pour solvent into the beaker to a depth of just less than 0.5 cm. To aid in the saturation of the TLC chamber with solvent vapors, line part of the inside of the beaker with filter paper. Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you prepare your TLC plate. 2. Prepare the TLC plate. TLC plates used in the organic chem teaching labs are purchased as 5 cm x 20 cm sheets. Each large sheet is cut horizontally into plates which are 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be. Measure 0.5 cm from the bottom of the plate. Take care not to press so hard with the pencil that you disturb the adsorbent. Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin: the line on which you will "spot" the plate.

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3. Spot the TLC plate The sample to be analyzed is added to the plate in a process called "spotting". If the sample is not already in solution, dissolve about 1 mg in a few drops of a volatile solvent such as hexanes, ethyl acetate, or methylene chloride. As a rule of thumb, a concentration of "1%" or "1 gram in 100 mL" usually works well for TLC analysis. If the sample is too concentrated, it will run as a smear or streak; if it is not concentrated enough, you will see nothing on the plate. The "rule of thumb" above is usually a good estimate, however, sometimes only a process trial and error (as in, do it over) will result in well-sized, easy to read spots. 4. Develop the plate. Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass, and leave it undisturbed on your bench top. Run until the solvent is about half a centimeter below the top of the plate 5. Visualize the spots Most samples are not colored and need to be visualized with a UV lamp. Hold a UV lamp over the plate and mark any spots which you see lightly with a pencil. Beware! UV light is damaging both to your eyes and to your skin! Make sure you are wearing your goggles and do not look directly into the lamp. Protect your skin by wearing gloves. If the TLC plate runs samples which are too concentrated, the spots will be streaked and/or run together. If this happens, you will have to start over with a more dilute sample to spot and run on a TLC plate. GAS Chromatography
It is a type of chromatography in which the mobile phase is a carrier gas, or an unreactive gas, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. 1. Add the sample to be injected to the syringe.

2. Inject the sample into the injector port. Push the needle of the syringe through the injection port and immediately press the plunger to inject the sample, then immediately press the start button on the recorder. 3. Push the needle of the filled syringe through the injector (as far as it will go) and quickly push the plunger. 4. Quickly press the start button on the integrating recorder or the start recording button on the computer Observe the recorder. Within several minutes, it should record several peaks.
5. End the GC run and study the observations.

When you have seen all of the peaks which you suspect are in the mixture, or when the recorder has shown a flat baseline for a few minutes or so, press stop on the recorder.

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