Ella D. Coppola and J. Gordon Hanna The Conneticut Agricultural Experiment Station 123 Huntinqton Street New ~ a c e n06504 ,
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Simple Fluorimetric Analysis of Glycine in Dietetic Beverages A student experiment
The objective of this paper is to present a simple fluorimetric experiment for the determination of as little as 0.02% glycine in commercially available dietetic beverages. T h e experiment requires no sample preparation or extraction and can b e performed easily within the one three-hour laboratory period by undergraduate students enrolled in a n instrumental analvsis course. Since the bannine of cvclamates, glycine has been used increasingly as a masking aeent to curtail the after-taste of saccharin in some dietetic beverages. Recently, the m o u n t of glycine in manufactured beverages has been limited by federal regulation to a maximum of 0.20% in the finished product. The experiment can be used to determine glycine in dietetic samples in which i t is a declared ingredient or to detect a minimum of 0.020% of glycine in samples in which it is not declared. T h e p'H dependence of the glycine fluorophor also can be studied as part of the overall experiment.
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Theory This experiment is based on the work of Udenfriend e t al.' who recently introduced a new reagent, 4-phenylspiro[furan-2(3H), 1'-phthalanl-3,3'-dione, commonly called fluorescamine. for the determination of ~ r i m a r vamines and primary amino acids a t picomole levels. Fluorescamine reacts instantaneously and specifically a t room temperature in alkaline media, with primary amines such as elvcine to form a stable fluoro~horand non-fluorescent ~ y d r o l y s i sproducts according to the reaction shown in Fieure 1. This principle was adapted for the determination of glycine in a variety of commercial dietetic beverage sample^.^ T h e reaction is;nstantaneous and the fluoresc&ce maxima for the glycine fluorophors are unchanged for a t least 30 min after the fluorescamine addition.
Fluorescarnine
GLycine
FLuorophor
Figure 1. The reaction of fluarescaminewith glycins to yield a tluwophor. The reaction is carried out in alkaline medium at mom temperature.
solution (15-30 mg of fluoreseamine, Fluramm,Roche Diagnostics, Nutley, N.J. 07110, dissolved in 100 ml acetone) to obtain a final volume of 5.0 ml. To prepare a blank, pipet 2.0 ml of water into a test tube and add 2 ml of borate buffer and 1.0 ml of the fluoreseamine solution. Make the measurements of the standards and blank at 366 nm exeitation wavelength and at 480 nm fluorescence wavelength with the sensitivity control of the spectrofluorimeter at 6 and the selector control at X-1. Measure the relative fluorescence intensity by adjusting the highest standard (3.2 ppm glycine) to 100%and the blank to 0%.Plot the values of the remaining standards as shown in Figure 2. Fluorescence intensity is directly praportional to glycine concentration with an experimental slope of about 31% relative fluorescence per 1.0 ppm glycine at an effective pH of 9.40.
Fluorescence Spectra Fill one cell with the glycine-fluorescamine solution containing 3.2 ppm glycine and a second one with the blank solution and place them in the spectrofluorimeter. To register the complete excitation soectrum. first set the emission waveleneth scale at 480 nm. Then senn thb exeitat~onmanually from 350 h -470 n m and record the intensity values at 2-nm imervslr near the peaks at 366. 405, and 4% nm. Ten-nanometer intervals can be used for the other points of the curve. To obtain the complete emission speetrum, set the excitation wavelength at 366 nm, manually increase
Equipment A Perkin-Elmer Model 203 spectrofluorimeter equipped with a manual scan was used in this experiment; however, another eauivalent t w e of fluorimeter can be used. If a complete s k d y of the incitation and emission fluorescence spectra is not considered necessary, a ratio fluorimeter equipped with a 366-nm excitation wavelength filter and a 480-nm emission wavelength filter may be substituted for the spectrofluorimeter. A mixer, vortex type, was used to rapidly agitate the buffered solution in each test tube prior to the fluorescamine addition. Experlmental
Standard Curve Preparation To prepare a standard curve, pipet 2.0, 1.5, 1.0, and 0.5 ml of 8.00 ppm glycine (aminoacetieacid) into (19 X 150 mm) test tubes. Add 0, 0.5, 1.0, and 1.5 ml of distilled water, respectively, so that the final volume in eaeh test tube is 2.0 ml. Pipet 2.0 ml of a borate buffer (0:20 M boric acid solution adjusted to pH 8.70 with sodium hydroxide) into eaeh test tube. As each test tube is rapidly shaken on a vortea-type mixer, add from a pipet 1.0 ml of fluorescamine
' Udenfriend. S.. Stein. S.. Boblen. P.. Dairman.. W... Leimerub-
er, W., and Weigel;, M., science, 178; 871 (1972). Coppola, E. D., and Hanna, J. G., J. Ass. Offic. Anal. Chem.,
57,1265 (1974). 322 / Journal of Chemical Education
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2 3 Glycine, ppm
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Flgure 2. Plot of relative fluorescence intensity versus glycine. Glycine solution is at LH 9.40.
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Wavelength, nm Figure 3. Excitation specbum 01 a 3.2 ppm glycine solution observed at 480 nm.
the emission wavelenkh valuer from 430 to -610 nm, and record the intensity readings for each IO-nm interval. Typical exvilntion and emission spectra are shown in Figures 3 and 4.
Fioure 4. Emission spectrum of a 3.2ppm glycine solution with excitation at 366 nm.
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pH Dependence The intensity of the fluorophor resulting from the reaction of a 3.2 ppm glycine solution with fluoreseamine is significantly pH dependent. The student can ascertain this dependence easily by changing the pH of the 0.20 M buffer solution with boric acid or with sodium hydroxide to achieve a range of pH values for the buffer solution from pH 1.M)-10.00. The apparent final pH of the solutions will range from approximately 7.M0.6 because of the presence of acetone. A typical curve showing the pH dependence of the fluorophor is shown in Figure 5. Maxima are obtained a t final apparent pH values of 9.2-9.4. Procedure for Dietetic Beverages with Declared Glycine Pipet a 5-ml sample of any commercial dietetic beverage listing glycine as an ingredient into a 500-ml volumetric flask. Dilute to volume with distilled water. Pipet 12.5 ml of this prepared sample into a 100-ml volumetric flask. The dilution factor is then 800. Take a 2.0-ml aliquot and proceed as for the standard glycine samples. Read the glycine concentration in ppm from the standard curve. Convert into percent glycine with the equation (ppm glycine) X dilution factor % Glycine = 104 Run triplicate measurements to check precision. Screening Procedure for Dietetic Beverages with Undeclared Glycine Pipet a 5.0-ml sample of any commercial dietetic beverage not listing glycine as an ingredient into a 500-ml volumetric flask. Dilute to volume with water. Pipet 2.0 ml of this diluted sample into a test tube and proceed as for the standard glyeine samples. This dilution factor is 100. A reading of 12% relative fluorescence or less. corresoondine to 0.0040% elvcine or less. is considered neelieih l e . ' ~ h i shlank tkurescenee i;.probahly due to rhc presen;eUof trace amounts of primary amines ur ui nnthally orcurrmp. flurreqcenee suhvtanr~sin the carbonated aarer or in the arrif~cialcoloring and flavoring ingredients
Figure 5. Plat of relative fluwoscence intensity versus pH. Sollrtion: 3.2ppm glycine. Results and Discussion As we reported e l ~ e w h e r e t, ~ h e average recovery of glycine was 100.3% with a standard deviation of f3.76% for nine determinations of glycine added t o commercial dietetic beverages. T h e fluorescence intensity of t h e glycine-fluorescarnine fluorophor indicates a potential detection limit of 1.000 X M glycine. However, i n diet drinks, t h e blank fluorescence eauivalent t o a ~ o r o x i m a t e,l v0.0040% glycine iimirs rhe minimum detectable amount t o 0.0200%. T h e method is not affected h\. colored solutions or hv the presence of saccharin, benzoate and citrate additives a n d does not require prior separation or extraction. Therefore, i t is a simple a n d fast way t o determine glycine i n dietetic beverages.
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Volume 53. Number 5. May 1976 / 323
