Qualitativeversus QuantitativeResults An Experimental Introduction to Data Interpretation Erlc R. Johnson' and Paula Aner Ball State University. Muncie, IN 47306 The interpretation of data ohtained through experimentation isacrucial part of thescientificmethod. While experimental techniques and manipulations t o generate data are relatively easy for students t o grasp in the chemistry lahoratory, teaching data interpretation is much more difficult in such a setting. Analyzing the result of a qualitative test nrovides an exnerimental introduction to data interoretation. This paper describes a n experiment in which the student can ascertain the meanine of a neeative result from a qualitative test by performing a more sensitive quantitative test on the same sample.
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The Experiment Urinary glucose can he qualitatively assayed hy one of several colorimetric strip tests.z These tests utilize glucose oxidase to catalyze the oxidation of D-glucose to D-gluconic acid and hydrogen peroxide. Under the catalytic influence of neroxide eenerated in this reaction neroxidase. the hvdroeen . oxidizes a chromogen, producing a color change indicative of the presence of glucose.3In someof these strip tests, comparison of the intensity of this color to a color chart gives a semiquantitative idea of the amount of glucose present in the urine sample. T h e sensitivities of these glucose oxidaseperoxidase assays are adjusted to give a "negative" result with normal urine. This contrived "negative" result leads many students to the erroneous conclusion that there is no glucose present in their urine sample. However, glucose is present in normal freshly voided urine a t a concentration of 10-20 mg/dL.4 T o measure these relatively low glucose concentrations, a more sensitive quantitative glucose assay must be used. One such assav suitable for the measurement of urinary glucose is the o-toluidine tesL5 D-glucose apparentlv condenses with o-toluidine in hot acetic acid solution to produce N-D-glucosyl-o-toluidineas well as the corresoondine Schiff base. Further reaction of these molecules produces a mixture of several green chromogens which can he auantitated hv determination of their absorbance a t 630 -
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Reagents To prepare these reagents, the instructor will need a balance accurate to the nearest milligram, volumetric flasks (1.0 Land 100 mL), graduated cylinders (2000 mL and 100 mL), and the beakers indicated below. Directions for the proper use of the volumetric glassware used in this experiment can be found in a wide variety of analytical chemistry textbooks or Laboratory manual^.^ Benroic acid solution (2.0 glL). In a 2-L beaker, dissolve 2.0 g henzoic acid in 800 mL distilled water, gently warming if necessary. After cooline. transfer this solution to a 1000-mL ... auantitativelv . volumetric flask. Dilut~to the mark wlth distilled water. Caution: Renzoic acid is a mild mirant u,rkm,eyes,and mucuus memtmmes. Avoid contact with skin and wear appropriate protective eyewear. Stock glucose standard (200 mgldL). Transfer 200 mg of dry reagent-grade glucose to a 100-mL volumetric flask. Add 80 mL of the henzoic acid solution. After dissolving the glucose, dilute to the mark with the benzoic acid solution. o-Toluidine H e a ~ m(50mLIL). l To3.0g thiourea in a 3-L Krlenmeyer flask, use graduated cylinders of appropriate volumes ro ndd 1900 ml. glacial acetic acid snd 100 mL +roluidines. Carefully mix
440
Journal of Chemical Education
until the thiourea is dissolved. Store in a brown bottle at room temperature. Caution: Avoid contact with skin and eyes. Do not pipet by mouth. Dispense this reagent from an all-glass automatic dispenser such as a Repipet (Labindustries, Berkeley, CA). In the absence of such a device, careful dispensing from a glass huret will suffice. Procedure Qualitative Test for Urinary Glucose. Caution: Disposable gloves should be worn when handling urine samples. Glassware that contacts urine should be thoroughly rinsed with water and disinfected by steam autoclaving or by soaking for 5 min in a 20%aqueous solution of household bleach. Avoid skin and clothing contact with the bleach solution. Rinse disinfected glassware with distilled water to remove the bleach solution. Collect a urine sample in a clean, dry glass container. Pour about 30 mL of the urine into a clean, dry50-mL beaker. Place the remaining urine in an ice bath for the quantitative o-toluidine assay. Perform a qualitative glucose strip assay on rh? RO.mL urine sample and the 200-myldl. glucose standard. For the ('linirtix qualitative test IAmes i)ivision. Miles 1.aborntorier. Elkhart. IN).the idlowine instructions are provided: 1. Dip the teat areaof thestrip in urine,and remove immediately. Tap the edge of the strip againdt the rim of the braker to remove excess urine. 2. After exactly 10s, match the color of the reagent patch with the color scale printed on the Clinistix vial label to detect the presence of glucose. Ignore color changes which occur after 10 S.
Fallow the manufacturer's instructions on the eontpiner for other glucose strip tests. Based on the results in this test, make a conclusion an the amount of glucose present in your urine sample. To validate your conclusion from the qualitative glucose assay, perform the o-toluidine quantitative glucose assay as indicated below. Quantitatiue (o-Toluidine) Test for Urinary Glucose. Prior to starting this section, students should review the proper use of pine@. Usine" Mohr oioets . . of the annronriate volumes. dilute the stock glucose standard a h t i o n (2&1'myd~) with the benloic acid solution to make gluwse solutions of the fullowing roncrntmtiona: 100mg/dL,75mg dL,5Umg/dL,25mg,dl,, 10mg,dl.,5mg dL,nnd 0 mg/dL (a hlank containing only benloic acid snlution~.IJsing a Mohr pipet of 1.U mL or less or an appropriat~nyrlnge pipet t e g , Eppendorfj, transfrr 02 . mi. of each glucose rdution into separate iarrr test tubes 119 x 1M mm). Pipet 0.2 mL of the reserved cold urge sample into a separate test tube. Using the Repipet jar or the glass huret, add 7.0 mL of o-toluidine reagent to each tube, mix, and
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Author to whom all correspondence should be addressed. Several alucose s t r i ~tests are wldelv available: Clinistix iAmes Division. ~ i l & ~aborator'~es, Elkhart, IN); ies-l ape (Eli illy andcompany. Indianapols, IN; and Chemstrip uG (Bio-Dynamicslomc. Indianapolis, IN). Keston, A. S. In Abstracts of Papers, 129th Meeting of the American Chemical Society; Dallas. Texas. April 1956: p 31c. Smith. E. L.; Hill. R. L.; Lehman. I. R.; Lefkowitz, R. J.; Handler, P.; White, A. Principles of Biochemistry: Mammalian Biochemistry, 7th ed.; McGraw-Hill: New York, 1983; p 203. Yee. H. Y.: Goodwin. J. F. Anal. Chem. 1973.45.2162-2165. F O &le, ~ see ~ k i o gD. , A.: West, D. M. ~ n a l ~ c a l ~ h e m i s t r y . 4th ed.; Saunders: Philadelphia 1986; pp 552-564.
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dace in a hoiline water hath for ten minutes. Cool the samoles in a cold water hath fur several minuter Remix and measure the ahsorhanre st 630 nm va the 0 mg/dL hlsnk The resulting peen color rs atable for about 30 mins. Plat absorbance at 630 nm vs. glucose concentration to obtain a standard curve. Using this standard curve, determinethe amount of glucose present in your urine sample from its 630-nm absorbance. Basedan the results of the o-taluidineassay, modify your conclusion from the qualitative assay. Considering your results in this experiment, what does a "negative" result from a qualitative test really mean?
Results and Dlscusslon Using the color scale included with the Clinistix qualitative glucose strip tests, normal urine will test "negative". From our experience with this experiment, most students will conclude that the "negative" result from the qualitative test means that no glucose is present in their urine sample. While this conclusion is satisfactory for the consumer, it is inappropriate for science students receiving laboratory
training in the generation and interpretation of experimental data. Results of the quantitative o-toluidine test vary, but nearly all of our students have found 5-40 mg1dL glucose in their urine that tested "negative" in the qualitative assay. Finding a nonzero concentration of glucose in their urine with the otoluidine test leads the student t o the conclusion that a "negative" qualitative test result means that the sample contains less than the minimum amount of glucose detectahle in the qualitative test. Most commercially available strip assays will test "positive" a t a urinary glucose concentration of 50-100 mgIdL. For the Clinistix qualitative assay, the ~ a c k a e einsert indicates that the minimum detectable amount ofglucose in urine is about 100 mg/dL. This ex~erimenthas been auite successful in our introductory biochemistry course, especially with medical technology and dietetics students who can see direct application of this concept to their respective fields. We recently added this experiment to our freshman-level organic and biochemistry course for nursing and health science majors. The student response was enthusiastic and very positive.
Volume 66
Number 5
May 1989
441
