Solvents and of Countercurrent Distribution. the Second Illustrates

Home , Paper chromatography

DOCUMENT RESUME ED 028 945 SE 006 618 By-Canis. Frank M. How to Use Chromatography as a Science Teaching Aid. National Science Teachers Association, Washington, D.C. Pub Date 69 Note-12p. Available from-National Science Teachers Association, 1201 Sixteenth Street, N.W., Washington, D.C. 20036 ($0.35 Stock No. 471-14578) EDRS Price MF-$0.25 HC Not Available from EDRS. Descriptorri-Biology, Chemistry, *Chromatography, College Science, *Instruction, *Laboratory Techniques, Resource Materials, *Science Activities, Secondary School Science Identifiers-National Science Teachers Association Presented arefiveprocedureswhichpermittheeffectiveteaching of chromatography with equipment which is readily available, economical, and simple in design. The first procedure involves a study of solute partition in two immiscible solvents and of countercurrent distribution. The second illustratesthe use of unidimensional ascending paper chromatography in separating the same mixture of colored components used in the first procedure. The third procedure illustrates the separationofthreecommon sugarsbyunidimensional ascendingpaper chromatography. The fourth procedure demonstrates the separation of amino acids by ascending two-dimensional paper chromatography. The fifth _procedure involves the separation of a dye mixture using thin-layer chromatography. (DS) U.S. DEPARTMENT OF HEALTH, EDUCATION & WELFARE

OFFICE Of EDUCATION

THIS DOCUMENT HAS BEEN REPRODUCED EXACTLY AS RECEIVED f yPERSON 02 ORGANIMION ORIGINATING IIPOINTS Of VIEW OR SIAM 00 NOT NECESSARILY REPRESENT OFFICIAL MICE Of EDUCA

E POSITION OR POLICY. as a science teaching aid

Frank M. Ganis Chairman, Department of Biochemistry 1 Schoolof Dentistry University of Maryland, Baltimore

WHY TEACH CHROMATOGRAPHY? Instruction of the subject matter follows a logical sequence,witheachprocedurestressing a Chromatography is an extremely efficient tech- particular aspect ofchromatography.The nique for the separation of mixtures of chemical interrelationships become apparent to the student substances.It was named and popularizedas a asheprogressesfromthesimpletothe practical method of chemical analysis by Mikhail comparatively more complex techniques. More Tswett, a Russian botanist who, in 1903, described sophisticated chromatographic procedures [ 1] such the successful separation of a mixture of colored as column chromatography,,electrochromatography, plant pigments using a simple glass column filled ion exchange, and gas chromatography become with calcium carbonated 11 Since thattime, the more understandable and meaningful to the student procedure has been modified, improved, andex- upon completion of these basic procedures. tended so that today practically all disciplines of chemistry utilize some form of chromatography for The first procedure, in twoparts, invulves a study both colored and non-colored materials. of solute partition in two immisciblesolvents and This technique is widely used for the microanalysis of countercurrentdistribution.Itemploysa of fission products of atomic piles, of foods for the separatoryfunnel ortest tube, to effect the detection of additives, and of drugs, dyes, pesti- separation of a mixture of coloredcomponents. cides, perfumes, vitamins, antibiotics, and hor- A second procedure illustratesthe use of uni- mones. In medicine it plays a most important dimensionalascendingpaperchromatography role in the detection of normal and abnormalcon- for the separation of thesame mixture. The latter stituents of blood, urine, saliva, and other body method employs an ordinarytest tube and cork for tissues. It has helped scientists to find metabolic the chromatography "chamber." A thirdprocedure pathways and has helped to clarify various genetic illustrates the separation of threecommon sugars defects. byunidimensionalascendingpaperchroma- In view of its general applicability and versatility, it tography. The fourth procedure demonstratesthe would be desirable to include in science programs separationofaminoacids,accomplishedby some training in the essentials of chromatography. ascending two-dimensionalpaper chromatography, One could seriously question the practicality ofa and a fifth involves the separation ofa dye mixture teaching program in chromatography, however, if using thin-layer chromatography. he considers the cost of commercially available chromatography equipment, the space required for such equipment, and the time available for student PROCEDURE 1: ILLUSTRATION OF participation in chromatography exercises. These SOLVENT PARTITION OF SOLUTES realistic limitations were considered in the design The student must first understand the concept of of this series of five procedures in chromatography solute separation in a two-phase solvent system to which permit the effective teaching of the subject appreciate the effectiveness of chromatographic withequipmentwhichisreadilyavailable, techniques. This can best be demonstrated by using economical, and simple in design and use. All of a separatory funnel, a mixture of solutes, and two the procedures may be carried out during ordinary immiscible solvents in which the solutes are soluble classroom or laboratory periods by the students to a differential degree. The degree of solubility of themselves or as demonstrations by the teachers. each component is based upon its chemical nature "PERMISSION TO REPRODUCE THIS COPYRIGHTED MATERIAL BY MICROFICHE ONLY HAS BEEN GRANTED By Nat. SCi.Teach. Assn. or affinity for a particular sol- layer, and in contrast, the congo TO ERIC AND ORGANIZATIONS OPERATING UNDER AGREEMENTS WITH THE U. S. OFFICE OF EDUCATION. vent phase. If two solutes, A and red and bromcresol green will re- FURTHER REPRODUCTION OUTSIDE THE ERIC SYSTEM B, with widely differing solubility main in the water phase. If the REQUIRES PERMISSION OF THE COPYRIGHT OWNER." characteristics are used, such that lower chloroform phaseisre- one solute is 'Aighly soluble in one movedand additionalchloro- solvent phase and the other is form extractions of the water highly soluble in the other phase, phase are made,additional the solutes will become quickly chloroform-soluble dye will be distributed, or partitioned, into extractedeachtime,butin the appropriate solvent phase. In diminishing quantities. chromatography, suchdistribu- tions are expressed by the term Materials partitioncoefficientand desig- Separatory funnel2 nated by the symbola which is 3 test tubes to collect extracts defined as the ratio of the solu- Chloroform Indicator dye mixture3 Copyright 1969 by the bilitiesof each soluteinthe National Science Teachers respective solvent phases at equil- Add200milligrams(mg) Association librium. Mathematically, sudan IV, 100 mg bromcresol A department of the concentration of solute A green, and 200 mg congo red a in upper (less dense) phase National Education Association A = to 50 milliliters (m1) ethanol. 1201 16th St., N.W. concentration of solute A Shake, then dispense in a bot- Washington, D.C. 20036 in lower (heavier) phase tlewith a dropper. Do not concentration of solute B filter the mixture. a in upper solvent phase B = Method Stock No. 471-14578 concentration of solute B Price 35 cents in lower solvent phase Add 10 ml of chloroform and 10 Orders of $2 or less A consideration of a will indicate ml of water to a clean separatory must be prepaid the degree of solubility or separa- funnel. The two liquids form two tion of the solutes. For example, immisciblephases.Shakethe if the solutes have a values of 0.2 indicator dye mixture and add ADVISORY COMMITTEE and 0.3 in a given two-phase sol- three drops of it to the funnel. vent system, this would indicate Shake the funnel gently once or T. R. Porter, Chairman a similarity in solubility of each Sebastopol, California twice and observe the partition solute in each of the two immis- ofthesolutesintheliquid Kathora Remy cible solvent phases.These phases. After settling, collect the San Antonio Independent solutes would bedifficultto lower phase into a test tube or separate by solvent partition. If, School District beaker and set it aside. To the San Antonio, Texas however, the a values were 0.2 separatory funnel, add another and 8.0, the solutes would be dif- 10mlof chloroform, make Thomas J. Ritzinger ferentially soluble in each phase another separation, and collect Stout State University and consequently easilysepar- the lower phase into another test Barron County Campus able. tube. Repeat this step once again Rice Lake, Wisconsin and compare the collected lower Part A: Solute Partition phases from the three consec- by Separatory Funnel L. Jean York utive ex tractions. The University of Texas If a solute mixture of a dye, at Austin sudan IV, and two indicators, 1See Source List. Small quantities of the bromcresol green and congo red, materials required to carry out each of the NSTA HEADQUARTERS are added to a separatory funnel procedures described in this pamphlet may containing the immiscible two- be secured at nominal cost from Russler Glassblowing Services, Post Office Box 3577, Mary E. Hawkins phase solvent system chloroform Baltimore, Maryland 21214. Associate Executive and water, the solutes become partitionedaccordingtotheir 21f separatory funnelsare not available, Secretary and the procedure may be carried out in a test Director of Publications solubility in the respective sol- tube. In this case, the layers arc carefully vents after mixing and settling of separated by use of an ordinary glass pipet Eleanor Snyder thesolvents. Sudan IV, being or medicine dropper. Editorial Assistant morechloroform-soluble,will 3Thesame indicator-dye mixture is used in predominate in the chloroform Procedures I and 2. 2 The students will note that practically all of the red It will be easy for the students to comprehend that color of the sudan IV is obtained in the first extrac- for complex mixtures or for mixtures containing tion and very little in the second and third extrac- very closely related solutes, a large number of test tions. This reflects the highly soluble nature of this tubes might be required to effect a reasonable dye in chloroform. In contrast, the indicator dyes separation. Some countercurrent distribution sys- bromcresol green and congo red, beingmore water tems, for example, are composed of several hun- soluble, are left behind in the water phase. Because dred test tubes. of widely differing partition coefficients, therefore, this mixture is easily resolvable in only a few These procedures serve to illustrate effectively that ex trac tions. it is possible to separate a mixture of solutes by taking advantage of the solubility characteristics of Part B: Separation of Solutes the components in two immiscible solvents. The by Countercurrent Distribution second procedure (Part B) allows a more con- venient and more efficient separation than does the When solutes are not clearly differently soluble in first procedure (Part A). two immiscible solvents (for example, when they have very similar partition coefficients or when PROCEDURE2: SEPARATION OF SOLUTES complex mixtures of solutes are to be separated), a BY PAPER PARTITION CHROMATOGRAPHY greater number of separatory funnels is required to A refined application of the separatory funnels or effect a separation. Because separatory funnels are testtube procedures can be shown by paper expensive and tend to become cumbersome when chromatography. Paper chromatography employs used in larger quantities, it becomes more practical filter paper as a solid supporting medium, but to use a series of test tubes. Such a series makes utilizes the same principles as those involved in more separatory steps possible; hence, a more solute separation in a separatory funnel or test efficient separation of components. This technique tubes. The paper, because of the porous nature of is known as countercurrent distribution. the cellulose matrix, can be considered equivalent to a series of test tubes involving an infinitely large Materials number of separatory steps. In a closed vessel con- 5 test tubes and rack taining a solvent of chloroform and water, an Pipet and rubber bulb equilibrium is reached when the chloroform be- Chloroform comes saturated with water and the water becomes Indicator dye mixture3 saturated with chloroform. This occurs in both the liquid and the vapor phases. If a solute mixture is Method placed as a spot on a strip of paper which is dipped into the solvent, a solvent front appears to move To the first of the 5 test tubes placed in a rack, add across the spot to bring about a separation of the 2 ml of chloroform and 2 ml of water. To each of components as the solvent advances. Since the the remaining 4 tubes add only 2 ml of chloroform cellulose matrix of the filter paper has such a high (This volume can be approximate). To the first affinity for water, it binds it firmly. Thus, the tube, add 1 drop of the indicator dye mixture and water phase appears to remain stationary and con- mix by gentle shaking. Allow to settle, then trans- stitutes the non-mobile stationary phase of the fer the upper water layer to the second tube with a chromatographic system; whereas the chloroform, pipet and rubber bulb and again mix. Continue this being immiscible with water, appears to move procedure by successively mixing each tube and freely as the mobile phase of the system and the transferring the upper water layer to each of the immiscibility of the two solvents is enhanced. Such remaining tubes. When the water layer reaches the a two-phase system can be efficiently utilized for last tube, add 2 ml of water to tubes 1 through 4 the separation of the components according to only. Mix the layers in each tube, allow to settle, their partition coefficients. then compare the colors of both phases of all tubes Adsorption of the solutes on the cellulose support against a background of white paper. may also occur. Separation is possible in this case The students will observe that this procedure has not only because of solubility of solutes in two resultedina good separation of the chloro- immiscible solvents, but also because of the struc- form-soluble sudan IV, which appears to remain in tural features of the solute molecules which allow thefirsttube in contrast to the water-soluble for differential attraction to the stationary sup- bromcresol green and congo red dyes which appear port. to move into the last tube. The intermediate tubes 2, 3, and 4 contain very little of each of the com- In paper chromatography, an important term used ponents in the respective phases. to denote the distance of movement of solutes is

3 designated as the Rf value. Rf is an expression of a cedure 1, the chloroform-soluble sudan IV begins ratio equal to the distance a substance moves to separate almost immediately, contrasted with with reference (R) to the front (f). The front desig- the water-soluble components which appear to nates the actual solvent line formed as the solvent remain at the starting line. The chromatogram advances up or down the paper by capillary action. should be removed when the solvent front has If the solvent moves upward, the technique is re- approached the uppermost third of the strip. ferred to as ascending chromatography; if it moves downward, descending chromatographyis ob- Remove the filter paper strip, mark the position of served. To understand the Rf value one can con- the solvent front, and dry the paper strip for three sider the situation in which a solute moves 6 centi- or four minutes. Thestudents should be able to ex- meters (cm) on paper when the solvent front has plain this separation on the basis of partitioning moved 10 cm. The ratio is then 0.6; if the solvent and solubility. front advances only 9 cm and the solute moves 6 cm, then the ratio becomes 6/9 or0.666. The Rf value makes possible the qualitative description of Part B: Separation Based Upon Adsorption thecomparative movement of compounds in Using a stationary support such as filter paper to chromatography.. effect resolution, solute adsorption may be in- Materials volved in the chromatographic process. In Part A of this procedure, separation occurred primarily by Indicator dye mixture3 a partitioning of the solutes in the twoimmiscible Test Tube4 200 x 25 millimeters (mm) solvents. The filter paper provided the stationary Filter paper strip (Whatman No. 4, support for the separation. It is also possible to 2 x 24 centimeters (cm) separate the dyes which remained at the starting Cork stopperNo. 10 line in Part A, by taking advantage of an adsorptive Wood applicator sticks affinity of the indicator dyes for the cellulose base Chloroform of the paper. In this case, the dyes are solubilized in one solvent, water, and separation occurs pri- Part A: Separation Based on Solvent Partition marily by a differential adsorption of the various components on the filter paper. Method On the strip of filter paper, draw a pencil line 3 cm Method from one end and place at the midpoint of this line a very small drop of theindicator dye mixture After drying the chromatogram used in Part A, (shake the mixture before use) with an applicator place the filter paper strip in 2 ml water in another stick. The diameter of the spot should be about 0.5 clean, dry test tube "chamber." Separation begins cm or less. After spotting the paper, preparethe as soon as the water flows over thedye mixture. testtube "chamber" for chromatography by Allow the separation to proceed until the fastest- adding 2 ml of chloroform to a completely dry5 moving component approaches a point midway test tube6. Gently lower the filter paper into the between the slowest-moving component and the test tube, and with the cork stopper, loosely placed previouslyseparatedchloroform-solublecom- in the mouth of the test tube, adjust the position ponent. of the strip so that the spotted end of the paper is just immersed in the chloroform. The applied spot The components have separated by selective ad- should not be touching the upper surface of the sorption of the indicator dyes on the filter paper. chloroform. Carefully seat the cork stopper into One of the components is more soluble in water the test tube without buckling the paper. than the other; hence, it moves farther. As the organic solvent passes over the applied spot, a separation of the components occurs.As in Pro- PROCEDURE 3: SEPARATION OF SUGARS BY UNIDIMENSIONAL ASCENDING 4Recommended size. Other sizes may be used. In this case, cut PAPER CHROMATOGRAPHY filter paper accordingly. Do not allow paper to touch sides of test tube. The final strip should not buckle and should be long enough to protrude above the cork stopper. In the previous procedures, the indicator dyes were 5Water is not added to the system since the cellulose in the filter easily separable because they had widely differing papcA attains enough water vapor from air to insure theadequacy partition coefficients, and were directly observable of the water phase in this system. 6This procedure involving the use of a test tube and cork is because they were colored. This procedure extends similar to one described by Rockland and Dunn 121. the earlier observations by demonstrating that

4 paper chromatography can aso be utilized for the Solvent: more difficult separation of closely related, non- ethyl acetate .. 12 parts colored compounds which can be visualized by pyridine 5 parts appropriate dipping techniques. In this case, three water 4 parts sugars are separated in ordinary canning jars by unidimensional ascending paper chromatography. Dip or sprayreagentsforsugarlocalization: (1) silver nitrate Materials (saturated solution in water) 0 1 volume (2) acetone 20 volumes 2-quartwide-mouth Mason (canning) jar with (3) 0.5 percent sodium hydroxide (Na0H) cover (or other appropriate screw-cap wide-mouth in ethanol jar) (4) 2N ammonium hydroxide (NH4OH) Aluminum foil (12 or 13 cm square) if required Filter paperWhatman No. 4 (20 cm square) Wood applicator sticks Stapler mounted on bench with 2 "C" clamps Method (see Figure 1) Prepare the wide-mouth jar for use as a chamber by Aqueous sugar solutions: adding to the jar 60 ml of a mixture of the solvent (1)1 percent glucose ethyl acetate-pyridine-water. To prevent possible (2) 2 percent ribose solvent attack upon the cover, place the piece of (3) 3 percent lactose aluminum foil over the top of the jar, and replace (4) a mixture containing the cover of the jar securely. Allow the solvents in 1 percent glucose the chamber to reach equilibrium. (Avoid un- 1 percent ribose necessary exposure to the solvent mixtures since 3 percent lactose pyridine has an unpleasant odor.)

Figure 1. Device for forming paper cylinders. Nail a block of wood perpendicularly to the tapered end of a wooden support. Mount a stapler on theblock of wood, and clamp the support to a bench. To use the stapler, one person holds the paper and another staples. The edges of the paper should not quite touch.

41=1111.

.d1111.

5 After the student's name is written on the filter these sugars are very water-soluble, the adsorption paper, draw astarting line in pencil across one was more significant. This canbe explained on the dimension of the paper, 3 cm from the edge. Place basis of the molecular structure of the sugars. four pencil dots along the line at 3-cm intervals. Ribose is a 5-carbon pentose and moved fastest, glu- Label 3 dots with the names of the sugars and the cose is a 6-carbon hexose andmoved to an inter- fourth dot "mixture." The sugar samples are to be mediate position, and lactose is a 12-carbon di- placed upon the dots in such a manner as to pre- saccharide and moved slowest in this system. Each vent their spread beyond a 0.5-cm diameter circle. carbon atom of the sugar has a hydroxyl group Large spots will interfere with separation. attached. By hydrogen bonding these hydroxyl goups have a high affinity forthe many hydroxyl Immerse a separate applicator stick in each of the groups contained in thecellulose of the filter paper sugar solutions to be analyzed,and touch each matrix.Ingeneral, the more solute hydroxyl stick perpendicularly to the appropriate starting groups there are on an atom, the more attractionit line dot. (If desired, a sugar "unknown" may also has for the filter paper support, and the slower be applied to a starting line dot.) After the spots moving the solute in this chromatographic system. are dry, staple the paperwithout overlapping, along the length of the paper at both ends and in PROCEDURE 4: TWO-DIMENSIONAL PAPER the center as shown in Figure 1. CHROMATOGRAPHY OF AMINO ACIDS The separation of the sugars in a single ascending Place the paper cylinder carefully into the jar, dimension was possible because the partition co- replace the jar cover, and "develop" the chroma- efficients were differentiated enough in solubility togram for 11/2 hours or longer, if time permits. and adsorptive characteristics. In contrast, when However, the chromatogram should be removed the components of a complex mixture have very before the solvent front reaches the upper edge of similar partition coefficients, as in the case of the cylinder so that Rf values can be calculated. amino acids, some overlapping of the positions taken by the amino acids would occur in the unidimensional paper chromatogram and differences After development, remove the chromatogram would not be distinguishable. It would be possible, from the chamber by catching a small hook (a bent however,to obtain good separationif,after paper clip will do) on the top staple andmark the separating the components in one dimension, one position of the solvent front in pencil. Dry it in a dried the paper, turned it 90 degrees, and intro- hood or other well-ventilated area. duced it into a different solvent so that the solvent frontmoves ata right angle to the original Remove the staples from the cylinder and dip the direction. The second solvent is such that the parti- chromatogram rapidly through a saturated solution tioncoefficientsof those components which of the silver nitrate in acetone, to localize the overlapped are now sufficiently different to permit sugars on the dried chromatogram (3). The pre- betterseparation. This processiscalled twu- cipitate formed in the reagent does not interfere dimensional paper chromatography. with the procedure. Avoid actual contact with the Materials silver nitratereagent to prevent discoloration of hands. Allow the excess acetone on the paper to Two 2-quart wide-mouth Mason (canning) jars drain, then evaporate. When it is dry, dip the paper with covers (or other appropriate screw-cap, through the sodium hydroxide in another tray and wide-mouth jars) again allow it to drain and dry. Excessive back- Aluminum foil (12 or 13 cm square) ground discoloration may be neutralized by passing Filter paperWhatman No. 4 (20 cm square) the chromatogram through another solution of 2N Wood applicator sticks ammonium hydroxide. The spots appear imme- Stapler mounted on bench with 2 "C" clamps diately. (see Figure 1) Solvents: Prepared by volume First dimension (Solvent 1) To calculate the Rf value of each sugar, record the distance from the center of each spot to the Chloroform 40 parts starting line and the distance which the solvent Methanol 40 parts front traveled. A comparison of the Rf values of Ammonium hydroxide20 parts the "known" and "unknown," if any, should en- (concentrated) able the students to identify the sugars. Second dimension (solvent 2) Butanol 60 parts Both solvent partition and adsorption on the filter Glacial acetic acid 20 parts paper played a role inthis separation. Since all of Water 20 parts

6 Amino acid mixture sion (approximately 2 to 21/2 hours).Remove the arginine paper and dry in a hood or other well-ventilated lysine area. glutamic acid glycine Remove the staples from the dried paper, hold it alanine diagonally corner to corner and pass it rapidly, face valine up, through a solution of ninhydrin reagent which leucine has been added to a tray just before use. Allow the phenylalanine paper to drain, then transfer the paper to an oven tyrosine (75 degrees C) for three minutes, or allow it to dry histidine in the air for slower color development. The spots Themixtureiscomposed of a0.1percent should be outlined with pencil and the color of aqueous solution of each amino acid in 10 percent each noted immediately since the colors will fade isopropanol (noncolored alcohol) and 2 percent with time. hydrochloric acid (6M). Compare the separated amino acid positions with Reagent for localization those given in the standard map (Figure 2). Note 0.25 percent ninhydrin in acetone (do not place also the variety of colors given by the amino acids. in tray until ready for use). The similar structural characteristics of the amino METHOD acids require a two-dimensional chromatographic systemtoassure adequate separation. A dis- Add 50 ml of SolventIto one jar and 50 ml of advantage of this method is the longer develop- Soh znt 2 to the second jar, then cover the tops ment times required in the two systems. Faster with aluminum foil, and cap the jars. Next, drawa systems have been reported, but these are asso- 0.5-cm circle at a point 2 cm from each edge of the ciatedwithpoorerresolution.However,the lower right hand corner of the filter paper. Within method is valuable in that a wide variety of food this circle, apply some of the amino acid mixture proteins may be analyzed after hydrolysis into with a wooden applicator stick so that the solution their constituent amino acids. By using a variety of fills the circle. Immediately after application allow solvent systems, one can determine the amino the spot to air dry. acid content of a large number of proteins. [3] Roll the filter paper into a cylinder and carefully staple the edges at the top, middle, and bottom PROCEDURE 5: SEPARATION OF DYES with a stapler, as shown in Figure 1. The edges BY THIN-LAYER CHROMATOGRAPHY should approximate each other, but should not be touching. (Avoid handling the paper excessivelyso An elegant refinement of chromatography, intro- that the free amino acids found in skin secretions duced within thelast few years, involves the do not cause extraneous spots to appearon the separationof substances on athinlayer of finished chromatogram.) adsorbent materials; hence thename thin-layer chromatography. The procedure is similar to that Remove the cover of the jar containing Solvent 1, of paper chromatography in that principles of sol- and lower the paper cylinder into the jar7 so that vent equilibrium, solvent partition of solutes, and the amino acid spot is near, but not touching, the adsorption are involved.Itdiffers from paper solvent surface. Replace the aluminum foil and the chromatography in that the solid adsorbentper- lid.Without disturbing the jar, allow approxi- mits a greater quantity of material to be applied mately 40 to 50 minutes for the solvent to ascend and the solid supporting medium, usually glassor the paper until the front is approximately 2cm film,is not involved in the separatory process from the top. Remove the paper witha bent which includes principles of elution analysis and paper clip and dry in a hood. adsorption isotherms. [4]

After dryingthepaper, carefully remove the In thin-layer chromatography,an adsorbent med- staples and form a cylinder in the second dimen- ium, commonly silica gel and a binder, is used to sion. Staple the paper, place it in the jar7 containing coat a solid support such as glass or plastic. The solvent 2, and allow for development in this dimen- material to be analyzed is spotted upon the adsorbent material, and the latter is developed in a solvent system in a closed atmosphere to allow separa- 7Lach dimension of thepaper should be developed in the direction tion of the components. The greatest advantages of indicated on the aminoacid map shown in Figure 2. thin-layer chromatography are its speed, greater

7 sensitivity, and resolving power. Consequently, the are available from Matheson Coleman & Bell, Post technique is much more efficient than is paper Office Box 85,East Rutherford, New Jersey chromatography. All of the separations described 07073.

in Procedures 1 through 4 can be accomplished with thin-layer chromatography techniques, but in Procedures II, III, IV: some cases, different solvent systems would need to be employed. The necessary modifications are Filter paper (Whatman No. 4) is available from described in detail in the literature. [4,5] H. Reeve Angel and Co., Inc., Clifton, New Jersey 07014. To demonstrate the effectiveness of the thin-layer Wood applicator sticks are available from Peer- technique, a mixture of three dyes is separated in a less Wood Applicators, Diamond Match Company, very simple system using a thin layer of silica gel B.F.D. Divisions, 733 Third Ave., New York, New on film. York 10001. Materials Procedure IV: Test tube - 25 x 200 mm Cork stopper-No. 10 The amino acid mixture is included in Kit No. Thin-layer chromatography strips cut to fittest 20 available from Nutritional Biochemicals, 21010 tube Miles Avenue, Cleveland, Ohio 44128. Dye mixture forthin-layer ehromatography8 The amino acid reagent, 1X45 Ninhydrin, is Wood applicator sticks available from Matheson Coleman & Bell, Post Chloroform Office Box 85,East Rutherford, New Jersey Method 07073. The reagent can also be purchased as an aerosol To the dry test tube, add 1 ml of chloroform and spray called Spray-Tec No. 101 Ninhydrin in Ace- cover with the cork stopper to assure equilibrium. tone, available from Mann Research Laboratories, 136 Liberty Street, New York, New York 10006. With an applicator stick, apply a very small spot of the dye mixture 1 cm from one end of the thin Procedure V: layer strip. Remove the cork from the test tube and introduce the strip carefully into the test tube, The thin-layer sheets are Eastman Chromagram which is held at an angle. Return the tube toa Sheets, Type K 301R2, and are available from Dis- vertical position andrecap with the stoppei. Ob- tillation Products Industries, Rochester, New York serve the very rapid separation of the components 14603. of the dye mixture which should separate in ten to The dye mixture is Eastman Test Dye Mixture fifteen minutes. Remove the strip after thesepara- for TLC No. 6077, available from Distillation Prod- tion is complete. Calculate the Rf values. ucts Industries, Rochester. New York 14603.

Upon completing this procedure, the students will REFERENCES be impressed with the simplicity and tremendous separatory power of this technique. The speed of I.Heftmann, E. Chromatography. Reinhold Pub- separation and a comparison of the results of thin- lishing Corp., New York, 1961. layer and paper chromatography will make clear the advantages of the former over the latter tech- 2.Rockland,L. B., and Dunn, M.S."Capillary- nique. Ascent Test Tube Method for Separating Amino AcidsbyFilterPaperChromatography." SOURCE LIST Science 109: 539-40; May 27, 1949. Small quantities of materials used in the pro- 3.Smith, I., Editor. Chromatographic Techniques. cedures may be procured from the dealer listed in William Heinemann, Ltd., London, England. footnote I. 1958. Procedures I and II: 4.Stahl,Egon,Editor.Thin-LayerChroma- Indicator dye mixture: SX1115 Sudan IV, tography: A Laboratory Handbook. Academic BX1160 Bromcresol green, and CX1905 Congo red Press, Inc., New York. 1965. 5. 8,not use the indicator-dye mixture used in Procedures 1 and 2. Randerath, Kurt (translated by D.D. Libman). See footnote 1 or Source List for procuring the dye mixture used in Thin-Layer Chromatography. Academic Press, Procedure 5. Inc., New York, 1963.

8 '0

Solvent System#2

Figure 2.

Map of amino acid mixture separated by two-dimensional paper chromatography. Origin Is at intersectionof ordinate and abscissa.

1. Glutamic acid 6. Alanine 2. A rginine 7.Tyrosine 3. Histidine 8.Valine 4. Lysine 9. Phenyklanine 5. Glycine 10. Leucine

9 NOTES

10 NOTES

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