Analysis of Aspartame and its Hydrolysis Products by ~ h i n k a ~ chromatography er Alfred R. Conklin Wllmington College, Wllmington, OH 45177 Undergraduate lahoratory exercises involving the separation and identification of amino acids have been developed (1-4). Also experiments that demonstrate the hydrolysis of prvreinsund simple polypeptidesand thesubsequent identification of amino arids have been reported ( 4 . 5). These exercises use a variety of thin-layer add colu~nchromatographic procedures and a variety of solvent systems. For some years I have been developing a lahoratory experiment that would allow students to separate and identify amino acids found in food. This exercise is for u s e in a nonmajors or general chemistry laboratory. Because the background of the students in such courses is limited, the experiment must deal with a simple protein or polypeptide that most students would know about. An additional ohjective would be for this experiment to serve as an introduction to chromatographic techniques. Accomplishing these objectives is complicated because of the variety of amino acids found in common proteins. Of course, small polypeptides, such as oxytocin, bradykinin, or encephalins could he used. However, these are expensive, potentially dangerous, and not commonly known by students. Thus, what I am looking for is a polypeptide that is readily available, safe for students to use, and recognized by them. There is a common dipeptide, actually the methyl ester of a dipeptide, that can be used for such an exercise. This is asnartame. better known bv the student as Nutrasweet. Aspartame and its hydrolysis products can easily be separated and identified usinrr s i m ~ l ethin-laver chromatomaphy (6). Other chromato&ap& separation methodsare equally suitable and can be used to separate aspartame from other constituents in food (7). However, thin-layer is preferred for freshman laboratories because i t is simple and fast. Both aspartame and its hydrolysis products, except for methanol, are easily detected by common amino acid visualizing reagents. Aspartame, aspartylmethylphenylalanine, is readily hydrolvzed under acid conditions to its constituent Darts. . . aspar& acid, phenylalanine, and methanol. This reaction occurs r a ~ i d l va t elevated temDeratures and slowlv hut measurabl; with long storage a t room tempera&e. The hydrolysis can be represented
Commercial preparations of solid Nutrasweet, that is, not
as part of drinks, contains components in addition to aspartame. The common powder contains aspartame, dextrose, silicon dioxide, cellulose, etc. One preparation also contains the amino acid leucine. Although most of the additional components pose no problem in analysis, leucine is difficult to separate from phenylalanine. Fortunately, its occurrence is infrequent enough so as not to pose a serious problem in most cases. Solutions of aspartic acid, leucine, and phenylalanine containing 30 mgl25 mL Hz0 work well as standards. For aspartame a solution containing 30 mgI25 mL H20 of pure aspartame or 90-180 mgI25 mL H20 of commercially available tabs or granular (powder) Nutrasweet is adequate. Using these concentrations, one spot (0.5 r L approximately 1mm in diameter) will contain enough amino acid or aspartame to allow easy detection after chromatography and visualization. Chromatography can be carried out using either cellulose or silica gel thin-layer plates. Separation is faster on the former. Plastic-hacked thin-layer sheets allow students t o cut microscope slide size "plates" for the analysis (see figure). This size plate gives adequate separation while allowing maximum speed and economy. For more complex separations, that is, partially hydrolyzed beverage samples, a longer plate cut to fit into a tall form heaker (58 X 140 mm) works well. Plates can be spotted using either Pasteur pipets pulled out to make a capillary tip, capillary tubes pulled t o a finetip, or a microliter syringe. I t is a good idea to have students practice spotting a previously used or a waste piece of plate before starting to spot a plate for development. Elution is carried out with one of two eluants depending on the type of plate used. A mixture of n-hutanol-acetic acid-water (4:1:5 vlv) is a good eluant system for cellulose. For silica gel a solution of n-butanol-acetic acid-water ( 6 2 2 or 12:2:2 vlv) is a good eluant. Plates are spotted and spots allowed to dry, then the plates are placed in a developing chamber containing an appropriate eluant. Development, which is typically carried out in a slide-staining (Copin) jar, or a beaker as described above, is allowed to continue until the solvent front is within a millimeter of the top of the plate. Plastic wrap makes a suitable cover when a heaker is used as a developing chamber. The dried plates are sprayed with either a0.2% solution of ninhydrin (1,3,3-triketohydrindenehydrate) in acetone or 0.10% isatin in acetone. Sprayed plates are allowed to dry and are heated t o hasten the reaction. An infrared lamp works as well or better than putting the plates in the oven t o heat. Both visualizing reagents work well. Ninhydrin gives a typical purplish color upon reaction with amino acids and with asDartame. Isatin Droduces different colors with differen1 amkoacids. Wr ha;e,on occasion, been unable toohtnin \,isualization with old solutions of ninhydrin. Thus. if no spots are observed after heating, check the activity of your visualizing reagent. Volume 64 Number 12 December 1987
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These s a m e nrocedures c a n be used to look for aspartame or its decomposition ~ r o d u c t s in beverages o r other foods. However, beverages are dilute solutions, s o 12 to 15spots (2+ cL), w i t h d r y i n g b e t w e e n spotting, m u s t b e m a d e in t h e s a m e place. Although drying between spotting i s t i m e consuming, it does n o t t a k e long if t h e spots a r e dried using a hair dryer. Experimental Obtain a cellulose thin-layer sheet and cut out several microscope-sire picrrs ro be used in the analysis. T h r fhin-layer
