the microscale laboratory tube to stand a t room temperature for 15 min. Pour the reaction mixture onto 5 mL of ice-cold 40% NaOH solution. Stir the solution briefly, filter the yellow product using a Hirsch funnel, and wash the solid with 10 mL of cold water. 'hansfer the solid into a medium-size test tube and add to it 2 mL of 95% ethanol. Boil the solution briefly to dissolve the solid. If not all the solid dissolves add more ethanol gradually while boiling until complete dissolution is obtained. (The total volume of ethanol should not exceed 3 mL.) Add water dropwise to the boiling ethanol solution until the solution becomes slightly cloudy, Cool the solution first a t room temperature, then in ice until crystallization is complete. Filter the yellow crystals using a Hirsch funnel, dry in air, and determine the melting point of the product. Students should be able to collect 115 mg (75% yield) of pure 4-methyl-3-nitroaniline 2 that melts a t 78-79 "C (lit. mp 79 "C (6)). 'H NMR spectrum (CDCL + DMSO-d6 ) shows: 62.25 (3H, singlet), 3.95 (2H, singlet), 6.75-7.25 (3H, multiplet). Preparation of the Acyl Derivative of 4Methyl-3-nihoaniline,3
In a small test tube. dissolve 100 m-c (0.66 mmole) of the 4-m~thyl-3-nitroanillneohtamed above In 0.5 mLofglacial aceticacid. Add 0 2 mL(2 1 mmole,ofacet~canhydrideand one boiling stone. Boil the solution gently for lbmin. Pour the reaction mixture into 5 mL of ice water in a small beaker and stir thoroughly for a t least 2 min. Collect the product by filtration and recrystallize i t from aqueous ethanol. Students typically collect 155 mg (80% yield) of pure light yellow crystals of 4-methyl-3-nitroacetanilide3, m.p. 143-145 "C. 'H NMR spectrum (CDClz) shows: 62.2 (3H, singlet), 2.5 (3H, singlet), 7.3 (lH, doublet), 7.5 (lH, doublet), 8.0 (lH, singlet), 8.14 ( l H , broad singlet). Acylation of p-Toluidine
To a solution of 107 mg (1.0 mmole) ofp-toluidine in 0.5 mL of glacial acetic acid in a small test tube, add dropwise 0.2 mL of acetic anhydride. Boil the solution gently for 15 min then pour into a small beaker containing 5 mL of ice water. Stir the mixture thoroughly and filter the solid using a Hirsch funnel. Wash the solid with 10 mL of cold water and recrystallize it from aqueous ethanol. Students should be able to collect 110 mg (74% yield) of white crystals of 4-methylacetanilide 4 that melts a t 147-148 "C (lit. mp 148.5 "C (6)).'H NMR spectrum (CDC13) shows: 62.12 (3H, singlet), 2.3 (3H, singlet), 7.15 (2H, doublet), 7.35 (2H, doublet), 8.14 (lH, broad singlet). Nitration of 4-Methylacetanilide
In a small test tube, dissolve 100 mg (0.67 mmole) of the 4-methylacetanilide obtained above in a solution consisting of 0.3 mL of concentrated HN03 and 0.1 mL of concentrated HzSO4.Heat the reaction mixture a t 3 0 4 0 "C for 15 min. Pour the content of the tube into a small beaker containing 5 mL of ice water. Stir the mixture and collect the yellow solid by filtration using a Hirsch funnel. Washing the product with 10 mL of cold water and recrystallization from aqueous ethanol gave 9 1 mg (70% yield) of pure 4methyl-2-nitroacetanilide5 that melts a t 91-93 "C (lit. mp 93 "C (6)). A10
Journal of Chemical Education
Hydrolysis (Deprotection) of 4-Methyl-2-nitroacetanilide,5
In a small test tube, suspend 80 mg (0.41 mmole) of 4metbyl-2-nitroacetanilide5 into 0.5 mL of concentrated HCI. Add a boiling stone and reflux the mixture gently for 30 min. Cool the tube in ice and carefullv add 0.5 N NaOH solution dropwise until the solution 1s basic to the litmus oaoer. Collect the vellow orecioitate bv filtration and wash it h t h 10 mL of cold waier. ~ecrystailizationof the product from aqueous ethanol gives 30 mg (70% yield) of yellow crystals 6 that melt a t 116-117 "C (lit. mp 117 "C). 'H NMR spectrum (CDCI3+ DMSO-d6) shows: 62.25 (3H, singlet), 6.6 (2H, broad singlet), 6.75-7.25 (3H, multiplet).
-
Acknowledament Support of this work by East Tennessee State University is gratefully acknowledged. Literature Cited 1. Marnor, S. Lubomlory Methods i n Organic Chemistry: Burgera: Minneapolis, MN. 1981;pp 253-258. 2. Brewster, R. Q.; VanderWerf, C. A; McEwen. W. E. U f i l i z dE r p n m ~ r sin Oqonic Chemistry. 4th ed.; D. Van Nostrand: New York, 19'77:p p 3 4 6 3 4 1 . 3. Nimitz, J.S.E~~riment~inOrgonicC~miBry;Prenti~Hal1: Englewoad. NJ, 1991:
NJ, 1992; pp 860-862. 6. Biglow, L. A. J. Am. Chem Soc
1919.41. 1559.
Improving a Microscale Vitamin C Laboratory Terry L. Helser S.U.N.Y. College at Oneonta Oneonta, NY 13820 The determination of vitamin C in foods or urine is a relevant ( I , and interesting general or biochemical laboratory 12-5 I. However, reagent pn?paration can be expensive and timt:-consuming. Wlule microscale techniques reduce these demands dramatically, the assumpti~mthat both the redox dye and vitamin C are SIJ unstable that they have to he remade each day (2. 3, 8) remains an obstacle. Studies reported here show that this reagent instability is probably the result of microhinl contamination. If the dye and are refrigerated, thev remain fully acvitamin C soluti~~ns tive for a t least two weeks. First. the same 1 mM 2.6-dichloroindo~henolsolution (0.326 g DCPL of d i s t i l l e d ' ~ ~(2,0 61, but'0.4 g DCPL is better) without NaHCOi (8) and ascorbic acid solutions (in 1% oxalic acid in distilled H20) were titrated on several days over a two-week period. Vitamin C samples were added to plastic well plates using calibrated Beral pipets and titrated by counting drops of DCP added (2, 5). The results in the figure show that the reagents remain stable relative to each other for a t least two weeks, if refrigerated. When these DCP and ascorbic acid solutions were tested against freshly made preparations a t several times during the two-week period, the refrigerated dye was fully active for more than a week with a maximum possible loss of only &lo% by 14 days. No loss of reduced ascorbic acid was apparent a t any dilution, even after 2 weeks of refrigerated storage. However, when ascorbic acid:l% oxalic acid solutions were stored a t room temperature, they were stable for a t least a week, but then lost 30-40% of their activity by two weeks.
Vitamin C Stability 70 60 w
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40
C @ 5*C + 0 . 5 " @5*C +
30
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20
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mg/mL @ RT + 0.5 " @ R T
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a s redox catalysis by divalent cations. I n contrast to citric acid, which is a nutrient, oxalic acid may be toxic to many microbes, a s i t is to us. Therefore, to ensure stability, t h e ascorbic acid standards should contain 1% oxalic acid, be made with distilled water, be refrigerated and may be filtered into sterile bottles, as is done with cell culture media. Finally, when 1mL of the DCP solution was titrated with the ascorbic acid solutions, the resulting standard plot was not linear. However, the nonlinear titration is a s accurate (f10%) a s the linear titration of the vitamin with DCP. Why reversing the order of addition would cause such a dramatic difference is not apparent. Summary
Vitamin C concentrations in juices and urine can be determined easily and eco0 10 20 nomically by microscale titration with 2,6-dichloroindopbenol. Both the dye and ascorbic acid standard solutions are staI h y $ Stored ble for a t least two weeks, if refrigerated. The ascorbic acid standards are more staGraph of results when 0.5 mLof 1.0,0.5,and 0.25 mg/mLascorbicacid solutions. ail of which ble with 1%oxalic than citric acid, probcontained 1% oxalic acid, were titrated with a t mM DCP solution when made and after ably because oxalic acid is toxic to mistorage in a refrigerator(@ 5 "C) or at room temperature ("@ R r ' ) for3,5.8,12,and Iddays. crobes. If long-term storage is desirable, The DCP solution also was refrigerated between tests. the standards may be filter sterilized. Finally, titrating dye with vitamin C soluThe possibility of using citric acid to stabilize vitamin C tions produces a nonlinear standard curve but is a s accurate a s the usual technique. solutions also was tested. I t is cheaper and less toxic than oxalic acid, and might chelate divalent cations as effecLiterature Cited tively. While its effect is indistinguishable from oxalic acid 1. Pauling, L.Vitamin C and the Common Cold; Fmeeman:San Francisco,1910. when refrigerated, it does not protect vitamin C a t room 2. Thompson,S.Chemlhk:Ailyn & Bacon:Boaton, 1990;p 211. 3. Johnson. E.R.J. C k m . Educ 1988,65.926927. temperature for more than three days. Solutions of ascor4. Kumsr.V;Courie.P,Haley S. J. Chem. Edue 1992.69.A213-A214. bic and citric acids rapidly lost 80-95% of their activity by 5. Tauslsl.J.C. Personal mmmunication. 6. Gyolgy. R.; Pearson, W. N . . Ed.The Wfamins: Vol. 7. 2nd ad.:Aeedemie:New York. 12 days and contained fungal contaminants. Thus, much of 1967:pp 27-51. the "instability" of vitamin C solutions in chemistry labo7. Barakat. M. 2.; El-Wahah, M.R A,:EISsdr, M. M.Anol. Chpm IS=. 27,536. 8. Haddad, P J Chem. Educ 19T7.54.193. ratories is probably due to microbial metabolism, a s well
0
Volume 72 Number 1 January 1995
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