The last term in Equation 16 is negligible compared with the preceding one. These relationships lead to the equivalence point values
= 2.j
2
x
10-3
=
1 188
[CN-1
+
6 08 X lo6 [CN-I2
[CN-] = 4 . 3 X B = 7 . 6 X lO-’O;
c
concentration a t 4.25 X 10-14J1. Substitution of this concentration ion into Equation 14 leads to
x
10-8
The silver ion concentration is thus negligible and that of the silver sulfite complex is less than 0.3% of the argentocyanide concentration. Hence the limiting current a t the equivalence point should be very small. I n the titration of 2 X 10F3Xcyanide in a solution that is also 0.1J1 and 2 X 1 0 - 3 ~ 1in sulfite and iodide, respectively, added silver ion passes quantitatively into argentocyanide until precipitation of silver iodide fixes the silver ion
This gives [CN-] = 1.82 X 10-5 and indicates that the end point is about 1% early. The negative error should increase by a factor of 10 for each 100-fold decrease in the initial cyanide concentration. The trend of the results in Table TI1 is thus in the right direction. LITERATURE CITED
(1) Johnston, H. L., Cuta, F., Garrett, A. B . , J . Am. Chem. SOC.55,2311 (1933). (2) Kolthoff, I. M., Sandell, E. B.., “Textbook of Ouantitative Inoreanic Analysis,” p. $74, Macmillan, -Xew York, 1943.
(3) Kolthoff, I. )I., Stock, J. T., Analyst 80, 860 (1955). (4) Kolthoff, I. M., Stock, J. T., J . Am. Chem. SOC.78, 2081 (1956). (5) Kolthoff, I. M., Tanaka, K., AKAL. CHEX 26, 632 (1954).
(6) Laitinen, H. A., Jennings, W. P., Parks, T. D., IXD.ENG.CHEM.,ANAL. ED.18, 574 (1946). (7)Laitinen, H. A., Kolthoff, I. RI., J . Phys. Chem. 46, 1Oi9 (1941). (8)Latimer, IT. M., “Oxidation Potentials,” pp. 74j 137, 191. Prentice-Hall, New Jersey, 1956. (9) Ricci, J. E., J . Phya. Colloid Chern. 51, 1375 (1947). (10) Stock, J. T., Turner, K. R., Chem. Ind. (London)1961, 1710.
RECEIVED for review November 22, 1901. Bccepted April 18, 1962. Division of Analytical Chemistry, 141st Meeting, SCS, Washington, D. C.. Uarch 1962. Taken mainly from the Ph.D. thesis of Fujiko Shinozuka, University of Connecticut, 1962. Work supported in part by the U. S. Atomic Energy Commission
I
.
I
Analytical Utilization of the Polarographic and Voltammetric Behavior of Purines and Pyrimidines DAVID L. SMITH’ and PHILIP J. ELVING Department o f Chemistry, University of Michigan, Ann Arbor, Mich.
b The polarographic and voltammetric behavior at the dropping mercury and stationary graphite electrodes of purine, pyrimidine, and several of their derivatives, including those of biological and medicinal importance, i s discussed from the viewpoint of its analytical significance and applicability. The conditions are outlined under which one purine or pyrimidine can be determined in the presence of related compounds. The possible analysis of mixtures i s illustrated by a procedure for the determination of the three most important nucleic acid components-adenine, cytosine, and guanine-in the presence of each other, The qualitative potentialities of the electroactivity of the purines and pyrimidines are indicated, e.g., identification of the three hydroxypurines-hypoxanthine, xanthine, and uric acid-in the presence of each other.
C
ERTAIN purines and pyrimidines are
of special interest because of their occurrence in cells as catalysts of biochemical reactions or as important constituents of nucleic acids; others, being antimetabolites, are of clinical interest in cancer chemotherapy. Concurrent 1 Present address, Chemical Research Division, The Upjohn Co., Kalamazoo, Mich.
930
ANALYTICAL CHEMISTRY
with the increasing study of these classes of compounds has been a n increasing demand for their analytical determination. This paper summarizes past work of analytical significance based on the reduction of purines and pyrimidines at the dropping mercury electrode (D. M.E.), e.g., (6)and new data b y the present authors involving reduction a t the D.hI.E. and oxidation at the stationary graphite electrode (S.G.E.). The polarographic and voltammetric procedures described can be employed to followthe course of chemicalreactions, t o test the effectiveness of fractionation or isolation procedures, and to identify and estimate quantitatively x i t h speed and precision several purines and pyrimidines; in a few cases, the analysis of mixtures, difficult to accomplish b y other methods, is possible. The fundamental structures of purine (A) and pyrimidine (B) are as follows (these may obviously be altered by tautomeric shifts in the compounds or their derivatives; the numbering shown is that used in Chemical Abstracts) :
3 A
9
1
B
EXPERIMENTAL
Chemicals. Purines and pyrimidines from t h e follon-ing sources were used: adenine, guanine, cytosine, and thymine from Sutritional Biochemicals Corp ; purine, isoguanine, hypoxanthine, pyrimidine, and isocytosine from RIann Research Laboratories, Inc. ; 2-hydrouypyrimidine from K & K Laboratories; 2-amino-4methylpyrimidine from Dougherty Chemicals ; 4-amino-2, 6-dimethylpyrimidine from Fluka AG; uracil, xanthine, uric acid, and 2-aminopyrimidine (practical grade) from Eastman Organic Chemicals (the latter compound was recrystallized once from m t e r and twice from hot benzene). Elemental analysis, polarography, spectrophotometry, and chromatography showed the compounds to be of sufficient purity for polarographic study. Buffer solutions (Table I) were prepared from analytical reagent grade chemicals. Nitrogen used for deoxygenating was purified and equilibrated by bubbling i t successively through alkaline pyrogallol solution, sulfuric acid, and distilled water. Apparatus. Polarograms and voltammograms were automatically recorded with a Leeds & S o r t h r u p T y p e E Electro-Chemogrnph, employing a mater jacketed H-cell (IO), maintained a t 25.0” & 0.1’ C. and containing a saturated calomel reference electrode (S.C.E.). The wax-impregnated graphite electrode was prepared and conditioned as previously described ( 5 ) . Damping of the polarographic
instrument, which affects greatly the peak current obtained at, solid elect'rodes, was equivalent to galvanometer performance. Capillary m values in milligrams per second, determined in distilled xvat'er (open circuit) a t 25' C., were as follom: ( A ) 2.69 a t 30 em. of mercury; ( B ) 1.85 (40 em.); (C) 3.07 (60 em.); ( D ) 2.64 (40 cni.); ( E ) 2.15 (40 cm.) and 3.23 (60 em.); ( F ) 1.85 (40 cm.) and 2.82 (60 em.). Drop-times, measured a t the potentials of interest, n-ere generally betn-een 3 and 4 seconds. The cell resistance, measured with a General Radio Type 650A\ impedance bridge, \\-as a l m j - s helow 300 ohms (usually below 150) ; consyuent'ly, potentials were not correcte-i for iR drop. General Polzrsgraphic Procedure. Test solutions \?-ere prepared by diluting a k n o n n volume of stock solution to 50.0 nil. in a volumetric flask with t h e desired buffer; t h r pEf of this solution was measured. About 10 ml. of t,est solution was transferred t o t h e H-c-ell, purged n i t h nitrogen for about 10 minutes, and then polarographed. Triton X-100 (concentration 0.00270) was used in a feiv cases to suppress masiina. .I portion of the buffer solution was treat'ed in ident'ical fashion to obtain the background curve. The residual current x t s subtracted arithmetically from the total current ; Elfaand id n-ere determined graphically, ut,ilizing the average of the recorder trace. General Voltammetric Procedure. Test solutions were usually prepared :is in the General Polarographic Procedure; in a fe\\- case': t h e compound \vas weighed out and diluted with huffer directly. T h e graphite electrode 11-as resurfaced before each run. 'To ensure rapid and complete wetting of the electrode by t h e test solution, the electrode n-as immersed in a 0.0037, Triton X-100 solution for 1 minute, follon-ed by rinsing v i t h a portion of the t'est, solution. The graphite electrode was positioned in the cell, electrical contact being niade via a n alligator elip, and \vas equilibrated a t the initial potential (0.1 volt) for ca. 1 min. before polarization at' 200 mv. per minute toward more positive potential. The background electrolyte solutions were treated identically to obtain the residual current, 15-hich was subtracted froni the total current to obtain i,. Precautions were taken to aroid stirring or vibration of the test solut'ion, e.g., the circulating pump of the constant beniperatiire system was always turned off prior to polarization. Simultaneous Determina?ion of Adenine, Cytosine, a n d Guanine. A simple calibration procedure is as follon-s: Polarograph a standard 0.5mJf lidenine solution in 0.5X KCl with HC1 added to give pH 2.0, employing 0.00270 Triton X-100 as a maximum suppressor, and measure the adenine id,/Ca(pH 2 ) ratio for the particular capillary used; polarograph a st'andard 0.5mM cytosine solution in 0.25M acetate buffer of p H 4.7; record the potential at, xhich id is measured and the id,'C, ratio;
a t the same potential a t which the cytosine wave was measured, measure the adenine wave at p H 4.7, and record the id/Ca (pH 4.7) ratio. Unknown adenine concentrations can be determined with satisfactory accuracy directly from i d / C a (pH 2); in the absence of adenine, cytosine can be determined directly from i d C,. T o determine cytosine in the presence of adenine, the following procedure is employed. By aliquoting a stock solution of the mixture, prepared two solution samples of identical concentration, adding KCI, HCl, and Triton X-100 to one aliquot to give 0.5X KC1, p H 2.0, and 0.002% Triton X-100 a t final volume; to the other, add SaOAc and H o l e to make the solution 0.25-11 in SaOAc and p H 4.7 a t final iolume. Polarograph the p H 2 solution from - 1.0 to - 1.6 r olts and deteimine the adenine concentration from i , C, (pH 2 ) . llultiply the adenine cuiient obtained a t pH 2.0 by the ratio i d i C a (pH 2) id C, (pH 4.7) to gire f h e current expected for this concentration of adenine a t p H 4.7 in the absence of . acetate solution cytosine [ i d ( A ) ] The is then polarographed froni -1.0 to -1.6 Tolts and i d @is) subtracted from the total current, id(A C), to give the current due to cytosine, nhich is then used to calculate the concentration of cytosine from &ICc. To obtain satisfactory accuracy, the adenine and cytosine waves a t p H 4.7 must be measured a t the same potential; superior accuracy could undoubtedly be achieved by preparing a standard calibration curve for a range of concentrations. Guanine is determined independently in 2 X H2S04 solution by the voltammetric procedure; adenine and cytosine do not interfere. Uracil and thymine do not interfere in a n y of the procedures described.
+
DETERMINATION OF PURINES AT D.M.E.
Purine, adenine (6-aminopurine) , hypoxanthine (6-hydroxypurine) , and 2,6diaminopurine are reduced in aqueous solution, whereas guanine (2-amino-6hydrosypurine), xanthine (2,6-dehydroxypurine) , and uric acid (2,6,8trihydroxypurine) are not reduced within the potential range normally available. Purine, adenine, and hypoxanthine are reduced only in acidic solution; 2,6-diaminopurine, which has been investigated ( I S , 14) only a t low pH, is also likely nonreducible under alkaline conditions. The order of ease of reduction is purine > adenine, 2.6diaminopurine > hypoxanthine. The mechanisms of electrochemical reduction of purine, adenine, and hypoxanthine have been investigated (16); purine is reduced in two diffusion-controlled 2e stages, adenine undergoes a single 6e reduction, and hypoxanthine apparently undergoes only a 2e reduction. The adenine and the second purine reduction waves are complicated b y the catalytic effect of the prod-
Table 1. Buffer and Background Electrolyte Solutions
Buffer So.
1 2
3 4 5 6
Composition pH 0 4- 2 9 KCl +HC1 2 . 2 - 8 0 SaoHPOI 7HD, citric acid monohydrate KC1 3 7- 5 7 KaOhc H0.4~ 6.1- 7 0 KC1 8 5- 9 2 KH,Cl SH3 11.0-13 2 KC1 KaOH
+
+ + +
ucts produced on hydrogen evolution and by the preqence of a chemical reaction during the adenine reduction. Purine. Typical polarographic data for purine het'ieen pH 1 and 11 are given in Table 11. T 1 1 ~average diffusion current constant, I = i d Cm213t1~6, betn-een p H 2 and 6 is 2 5 0.4 and 6.3 i 0 8 for naves I and 11, respectively; the corresponding values a t p H 4.7 are 3.59 i 0.12 and 6.09 ik 0.65. The concomitant reduction of hydrogen ion during the wave I1 process accounts for its I value being larger than that expected for a 2e reduction (16). The heights of both naves increase slightly with decreasing pH. Both waves shift linearly to more negative potential n i t h increasing pH; betn-een pH 1 and 6, E1 2 equals -0.697 - 0.083 p H for wave I, and -0.902 0.080 pH for n ave 11,Le., the separation of the wives remains nearly constant. A maximum appears on n a v e I1 a t concentrations greater than ca. O.lmJI in buffer 1; addition of the nonionic surfactant, Triton X-100. eliminates this maximum and shifts both purine n aves to more negative potential by about 20 my. per 0.00270 Triton added. The height of wave I is not appreciably affected b y increasing Triton X-100 concentration, but wave I1 decreases until it approximately equals nave I. K a v e I1 is less well defined a t higher purine concentration ; this phenomenon is related to the catalytic rffect of the reduction product 011 the evolution of hydrogen, e.g.. hydrogen evalution in pH 4.7 acetate buffer, determined polarographically, is ea. 0.2 volt more positive in the presence of 1.25niX purine wave I1 product, produced by controlled-potential electrolysir, than in the background electrolyte alone. The greater the concentration of this product a t the mercury drop, the more positive is the potential of hydrogen e\ olution. Either or both of the purine waves can be used for its determination; nhen a choice exists, wave I is recommended since the precision nith n hich the m v e I1 current can be measured is strongly affected by the factors described. K h e n wave I is employed, purine can be determined in the presence of all of the other purines examined.
*
VOL. 34, NO. 8, JULY 1962
931
Table 11.
Effect of pH, Concentration, Drop-Time (Head of Mercury), and Temperature on the E,!* and id of Purine"
Concn.,
Buffer No.
mu
15
PH 1.78
0.16
16,c
1.78
0 Iti
1
L85
0.52
1b,c
1.85
0.52
3
3.71
0.39
3c
3.71
0.39
3
3.71
0.52
3c
3.71
0.52
3
4.71
0.16
3
4.71
0.39
3c
4.71
0.39
3
4.71
1.oo
3d
4.71
1 .oo
3
5.72
0.39
3c
5.72
0.39
3
5.72
0.52
3c
5.72
0 52
4 5 6
6.2 9.2 11.3
0.39 0.39 0.39
El/2,
pa. I 1.29 I1 2.15 I 1.56 I1 2.50 I 4 49 I1 Maximum I 5.56 I1 Maximum I 2.55 I1 4.37 I 3.18 I1 5.44 I 3.57 I1 6.08 I 4.41 I1 6.97 I 1.17 I1 2.16 I 2.87 I1 4.93 I 3.54 I1 6.06 I 7.77 I1 11.6 I 9.35 I1 15.8 I 2.86 I1 4.77 I 3.35 I1 5.47 I 4.10 I1 6 . 5 5 I 4.98 I1 7.64 KO wave observed No wave observed ;Yo wave observed
84 27 80 20 09
-Volt 0.836 1.104 0.838 1.105 0.850
4 17
0,852
3 15 5 48 3 23 5 60 3 30 5 65 3 34 5 32 3 51 6 57 3 53 5 86 3 59
1.001 1.193 1,008 1.202 1.002 1.197 1,005 1.201 1.078 1.274 1.088 I . 287 1.093 1.296 1.097 1.298 1.105 1.310 1,172 1.361 1,188 1.377 1,178 1.365 1.182 1.372
I
id,
3 6 3 6 4
6 22 3 76
5 4 7 3 5 3 5 3
6 3 5
69 52 75 55 98 43 66 79 14 80 93
Three commercial (Mann Research Laboratories, Inc.) lots of purine were used in obtaining this table of data. Ionic strength was 0.25M for all runs. All runs were made with capillary E. * Maximum appeared on second wave. At concentrations greater than ea. 0.20mM, it was impossible to measure i d and E,/%accurately. c Mercury height was 60 cm. in these runs, but 40 em. in all others. d Runs were made at 40' C.; all others were at 25' C.
Table 111. Variation of Adenine Current with Concentration in 0.5M Acetate Buffer, pH 5.50
Concn., mN
id,
E1 12,
- Volt I 1.438 3.83 10.1 0.200 9.33 1.440 0.300 5.23 9.77 1.438 0.400 7.38 7.34 9.22 1.441 0.424 9.60 1.441 8.94 0.500 9.13 1.440 8.64 0.504 9.09 1.439 10.46 0.607 8.78 1.442 14.34 0.874 8.54 1.442 1.044 16.65 7.83 1.443 22.65 1.540 7.27 1.444 2.120 29.00 a All runs made a t 25' C. a t mercury height of 30 cm. using capillary B. Each horizontal line represents average of two or more runs. 932
w.
ANALYTICAL CHEMISTRY
Adenine. Reduction of adenine a t the D.M.E., first reported by Heath (9), has been confirmed b y others (8, 13-15), and has been succesPfully applied to its determination in biological samples (6, 7 , 11, I%?), although prior separations are usually required. I n the present investigation, adenine was studied between p H 1 and 13 and in 0.05M BuoNBr solution. It exhibits a single wave only in acidic solution, Le., near p H 5 the wave height begins to decrease until a t p H 6.5 no wave appears. The average Z value in chloride and acetate buffers between pH 1.2 and 5.5 is 10.2 & 0.7 a t the 0.2mit-1adenine concentration level. A report (14) that adenine is not reduced in acetate buffer is not substantiated. Since the adenine wave occurs, espe-
1.0 1.2 POTENTIAL, VOLTS V S S.C.E. Figure 1 . Voltammograms of guanine in 2M HzS04 Millimolar concentrations of guanine indicated on each curve
cially a t higher pH, close to the background electrolyte decomposition potential, its limiting current is difficult to measure with precision. This proximity is related to the lowering of the overpotential for hydrogen ion reduction by adenine and its reduction product. Even in alkaline solution, where adenine reduction does not occur, the background discharge occurs at a more positive potential than in the absence of adenine. El,* in constant ionic strength McIlvaine buffer (4) varies linearly with p H (El/* = -0.975 - 0.090 PH) and agrees closely with those found in chloride and acetate buffers. I n pH 1.2 to 2.9 chloride buffer, El,z becomes more negative with increasing adenine concentration, verifying Heath's observation (9); El,*, however, is independent of adenine concentration in acetate buffer. Experimental factors such as buffer system used, p H selected, and use of a maximum suppressor may markedly affect the diffusion current and are consequently important in the use of the adenine wave for analysis. The adenine diffusion current constant in hIcIlvaine buffer is relatively constant (5.6 =t0.3) between p H 2 and 5 with a slight decrease between pH 3 and 4 where a small postwave, superimposed on the background discharge, appears; this Z is ca. 40y0 lower than in chloride and acetate buffers. I n pH 5.5 acetate buffer the I value decreases approximately linearly with increasing adenine concentration (Table 111); this may be related to the effect of adenine and its reduction product in
Ion ering the overpotential for hydrogen evolution, Le., as the adenine concentration is increased, hydrogen ion is more readily reduced, resulting in a lessening of the demarcation between the adenine and discharge waves. At constant adenine concentration, I seems to decrease slightly with increasing p H in chloride buffer; measurements are difficult, how-ever, because a t pH 1.2 to 2.9 a maximum appears; the latter can be suppressed with 0.0027, Triton X-100 with only a small decrease in the adenine wave height. The wave height continues to decrease markedly, however, with increasing Triton X-100 concentration; above 0.00670 Triton X-100, the wave is practically obliterated. Other maximum suppressors would probably have a similar effect. A 0.0027, Triton X-100 concentration is sufficient to eliminate the postivave observed in p H 2.0 to 2.9 chloride solution and p H 2.7 to 4.0 1IcIlvaine buffer. I n the general polarographic procedure given under Experimental, 0.5X chloride solution of p H 2 containing 0.00270 Triton X-100 is recommended as the background medium. Hypoxanthine. Hypoxanthine was investigated betn-een pH 2 and 13: no evidence of reduction is observed in alkaline solution. Since polarograms of hypoxanthine are actually ill defined inflections on the background discharged, Le., the T a r e follows the background discharge n-ith increasing p H , accurate measurement of its ElirpH relation was not attempted. Acetate buffer of p H 5.7 yields the most well defined wave (I = 2.76, El,* = -1.61) ; point-by-point subtraction of the residual current is required t o give a limiting current suitable for quantitative estimation. Hamer, Kaldron, and Woodhouse (8) found a fairly well defined limiting current a t pH 1.1 ( I = 2.6, calculated from their data). Because of the difficulty in measuring the hypoxanthine reduction wave a t the D.M.E., its anodic wave a t the S.G.E. (vide infra) is recommended for quantitative analysis. DETERMINATION OF PURINES AT S.G.E.
Each of the purines investigated with the exception of purine itself gives an anodic wave a t the graphite electrode ('Table IV) ; these waves will be designated as oxidation waves, although the actual oxidation of the compounds vias not proved by macroscale electrolysis : consequently, it is impossible with the data currently available t o speculate as to the origins of the faradaic oxidation currents. With the exception of the wave exhibited by xanthine a t pH 3.7, all of the oxidizable purines give an anodic wave of the shape shown by guanine (Figure 1); xanthine a t pH 3.7 shows two consecutive current peaks separated
by ca. 0.06 volt, the first being sharper Table IV. Anodic Voltammetry a t and larger in magnitude than the second. Graphite Electrode" The compounds in order of ease of oxidation (increasinglymore positive half-peak Adenine potentials a t constant pH) are uric acid > xanthine > guanine > isoZoncn., mM guanine > adenine > hypoxanthine > purine. 1 000 2M HZSO, 36.0 72.0 1.34 35.8 7 1 . 6 1.23 The half-peak potentials, E,,z, be0.500 2 . 3 35.1 70.2 1.08 0,500 3 . 7 come more positive (oxidation becomes 71.7 71.7 1.06 1.000 4 . 7 more difficult) with decreasing pH for 50.0 71.4 1.04 0,700 4 . 7 all of the compounds, and are also 37.4 74.8 1.02 0.500 4 . 7 affected by the nature of the background 22.6 7 6 . 3 1 . 0 1 0.300 4 . 7 15.8 79.0 1.00 0.200 4 . 7 electrolyte. Because of poor solubility, 3 7 . 1 74.2 1.01 0.500 5.7 saturated solutions of some purines were 24.7 49.4 1.11 0,500 6 . 3 sometimes employed to determine E,n 23.1 46.2 0.74 0.500 9.2" variation with buffer component and pH. Hypoxanthine Since deviations from linearity between No wave peak current, i,, and concentration 1.020 2;M H&OI - . observed usually occur at concentrations below 0.510 2 . 3 3 2 . 8 64.4 1.26 ca. 0.2mM1 the use of calibration curves 61.7 1.14 0 510 60.6 1.14 is recommended. 0 408 63.8 1.13 0 204 Adenine. The previou.1:- reported 61.9 1.08 0 510 i,,'C value for adenine a t the S.G.E. 1 4 . 5 60.6 1.04 0 510 5 . 7 was obtained TT ith the polarograph 25.2 50.0 1.16 0.510 6 . 3 highly damped ( 5 ); the data in Table Xanthine IV were obtained with galvanometer 2-If H,SO, 24.6 49.2 1.01 0.50 equivalent damping. Between p H 2.3 3.7d . . . . . . 0.84 and 5.7, i,/C is 72.7 f 1.9 ga./mAMa t . . . 0.78 4.7d the 0.5mM concentration level. I n pH . . . 0.71 5.7d 4.7 acetate buffer i,/C increases from Uric Scid 71.7 to 79.0 pa./mJI between 1.0 and 0.62 2M HzSOld . . . ... 0.2mM adenine; consequently, for best 0.45 3.7 4 . 6 31 0.15 accuracy a calibration curve is required. 0.33 5.7 5.0 34 0.15 I n ammonia buffer, adenine gives an Guanine inflection close t o the background discharge ; point-by-point subtraction of 48.2 1.02 1 o.i n~ . 48.9 1.02 0.505 the residual current yields a well de52.0 1.02 0.2026 fined wave. 55 1.02 0,101 Guanine and Isoguanine. These Isoguanine difficultly-soluble compounds were investigated only in 2M H,SOd, where 2M HnSOI 57.0 57.0 1.06 1 .oo 29.2 5 8 . 4 1.05 0.50 both give rvell defined anodic waves. 12.7 63.5 1 0 5 0.20 Between 1.0 and 0.20mM guanine, i,/C is 49.7 i. 1.9 ga./mJI. -4s in the Rate of polarization: 200 mv./min.: damping of polarographic instrument was case of most compounds investigated a t equivalent to galvanometer performance. the S.G.E., i,/C increases with de* Background electrolytes: pH 2.3: creasing concentration; it is 57.5 0.7 0.251M SazSOa HZSOd; pH 3.7 to 5.7: ga./mJI between 1.0 and 0.5mM iso0.23M NaOrlc HOAc; pH 6.3: 0.25iM XaZSOd; pH 9.2: 0.25M SHICl NHs. guanine; a significant positive deviac Point-by-point subtraction of the tion occurs at the 0.2mZI level. I n background was necessary to shorn a well 2M H2S04,either guanine or isoguanine defined wave. can be determined in the presence of d Saturated solutions were employed. e Sample also contained 1.00m-W adenine, but not in the presence of each adenine, 0.5mM cytosine, 0.5mJf thymine, other, Le., the adenine wave occurs a t and 0.5mM uracil. 1.34 volts compared to 1.02 and 1.05 volts for guanine and isoguanine, respectively. I n 231 H2S04, xanthine is oxidized at about the same potential as guanine and isoguanine, and would uric acid were not extensively studied interfere ; hypoxanthine, however, does because of poor solubility. The E,/z not show an anodic wave in this medium. values of the three compounds differ Hydroxypurines. HYPOXASTHIKE,sufficiently to allow detection of one in XA4XTHINE, .4ND V R I C A C I D . .kt the presence of the others. Uric acid, constant pH, t h e E,,,* values of the already in a well oxidized state, not hydroxypurines become l e v poqitire only gives an anodic wave but is, in and the iJC values decrease n i t h the fact, the most easily oxidized of the increasing number of hydroxy subcompounds tested. stituents (Table IV). Between pH Simultaneous quantitative determination of the three hydroxypurines is not 2.3 and 5.7, i,/C for hypoxanthine practical, since simple current-concena t the 0.5mM concentration level is tration relationships do not exist for 61.8 f 1.6 pa./mJf. Xanthine and
*
++
VOL 34, NO. 8, JULY 1962
+
933
consecutive peak currents; it may be possible, however, t o develop empirical methods. The anodic behavior of the hydroxypurines may be most useful as a means of identification or as a rapid and simple method for checking for the presence of these compounds. DETERMINATION OF PYRIMIDINES
Reduction at the D.M.E. Cavalieri and Lowy (5)first reported the reduction in acidic solution of pyrimidine and the following derivatives : 2-amino-, 4 amino-, 4-hydroxy-. 2-amino-4methoxy-, 2,4-diamino-, 4,6-diamino-, 4,5,6triamino-, 4-amino-6-hydroxy-, and 1,40-dimethylthymine; they reported several tri- and tetrasubstituted pyrimidines to be nonreducible. Hamer et al. (8) confirmed the reduction of 4-aminoand 2-pyrimidine a t pH 1.1 (assuming
diffusion-control, I values, calculated from their data, are 7.7 and 2.4, respectively) ;they obtained awave for cytosine (2-hydroxy-4aminopyrimidine)in p H 5 acetate buffer ( I = 6.4, calculated from their data). Uracil (2,Pdihydroxypyrimidine), isocytosine (2-amino-4-hydroxypyrimidine), and thymine (2,4dihydroxy-5-methylpyrimidine)did not give reduction waves (experimental conditions not specified) (8). 2.5 - Dimethyl - 4 - aminopyrimidine gives a single wave; I is 4.8 betxeen p H 3 to 6, and decreases to zero a t ea. pH 9 ; Eltz varies linearly with p H (El,*= -1.06 - 0.076 p H us. X.C.E.) between pHland8(1). 2-Amino- and 2-amino-6-methylpyrimidine each give one wave in the pH region of 7.4 to 8.9, \%-hereas2-amino-4chloro- and 2-amino-4-chloro-6-methylpyrimidine each yield two waves ( I S ) .
Table V.
Effect of pH, Concentration, and Drop-Time (Head of:Mercury)-.on t h e Eli2and id of Pyrimidine. Buffer Concn., -Eli?, niJl I i d , pa. Volt KO, PH 1 0.5 2 500 11.15 2.10 0.632 1
0 5
4 060 0 440 0 440 0 440 0 440 0 273 0 273
18.90 1.86 2.35 1.81 2.26 1.28 2 2.9 I 1.19 I1 1 . 4 0 3 3.7 0 440 I 1.60 I1 2 . 0 4 3b 3.7 0 440 I 2,l4 I1 2 . 4 0 2 4.0 0 273 I 1.29 I1 1 . 3 6 3 4.7" 0 440 I 1.91 I1 2 . 1 3 3b 4.7 0 440 I 2.33 I1 2 . 5 9 2 4.8 0 273 I 1.34 I1 1 . 2 5 0 273 2.58 0 273 2.55 0 440 3.89 0 440 4.72 2 850 27.25 3 5.7 4 970 43.30 0 273 2.47 0 273 2.40 0 273 2 46 0 273 2.42 0 273 111 2 34 IS' 2 . 3 2 8.0 0 273 111 2 . 2 3 IS- 2 . 4 6 5 9.2d 0 440 7.45 5 9 .2 0 440 i.39 5b 9.2 0 440 8.49 5 0.2 1 370 23.30 5 9.2 1 470 25.40 5 9.2 1 470 2 5 . io 6 13.1 0 440 5.04 6 13.1 0 440 5.09 a Al! funs made at 25' C.; ionic strength was 0.45.1l for runs for the others. Capillary F was used for runs xith buffer 2,
2.18 1.99 2.06 1.94 2.00 2.43 2.26 2.70 1.72 2.33 1.91 2.17 2.49 2.64 2.08 2.33 2.08 2.35 2.58 2.41 5.00 4.94 4.29 4.28 4.68 4.69 4.79 4.66 4.81 4.73 4.58 4.6 4.38 4.97 8.51 8.44 7.97 8.62 8.78 9.03 6.01 6.07
0.632 0.6i4 0.673 0.751 0.752 0.772 0.841 1,174 0,967 1.226 0.972 1.223 0.968 1.184 1.076 1.237 1.078 1.240 1.067 1.198 1,140 1.147 1.217 1.220 1.243 1.263 1.193 1,224 1.258 1.300 1.351 1.637 1.400 1.640 1.535 1.535 1,539 1.543 1.536 1.538 1.847 1.843 n-ith buffer 2 and 0.25.11 and capillary E for t h e
others. Mercury height was 60 cm in these runs, but 40 cm. in all others. The two waves are almost merged at pH 4.7; consequently, it was difficult t o measure the individual currents. The sum of the two currents, however, gives a true measure of total current at plateau of second wave. Waves at pH 5.0 and 9.2 have inflections near E l ( ? . e This polarogram has an inflection close t o the background discharge (Eli? 1.64). N
934
ANALYTICAL CHEMISTRY
The first chloro compound wave was attributed t o reductive dehalogenation with replacement b y hydrogen; the second coincided with the single wave of the parent 2-aminopyrimidines, and was consequently ascribed to reduction of the pyrimidine nucleus. Of the pyrimidines examined over the whole p H range in the present study, pyrimidine, 2-aniinopyrimidine, 2amino-4-methylpyrimidine, 2-hydroxypyrimidine, 4-amino-2,6-dimethylpyrimidine, and cytosine are reduced in aqueous solution, while isocytosine, thymine, and uracil are not reduced. The electrochemical reduction mechanisms for several pyrimidines have been investigated (3, 1 7 ) . Compounds substituted with a tautomeric group in the 4-position generally give higher currents and are more difficult to reduce by a t least 0.2 or 0.3 volt than those unsubstituted a t this position. Significantly. all of the compounds nonreducible n-ithin the available euperimental potential range are substituted a t the 4-position. Pyrimidine. Pyrimidine (Table V) gives a single polarographic wave in highly acidic solution (nave I ) ; a t ca. p H 3 m v e I1 appears. nhich is masked by the background discharge below thiq p H . Kaves I and I1 are approuimately equal in height. TTave I is pH-dependent (E12 = -0.576 0.105 pH); since wave I1 is essentially independent of pH, the t n o naves merge near p H 5 to form pH-dependent wave 111, whose diffusion current constant is about equal to the sum of waves I and 11. S e a r p H 7.2 n-ave I T appears; this wave is about equal in height to wave 111 and is essentially independent of pH. Since Lvave I11 is pH-dependent, it ultimately merges with m v e IT' near pH 9.2 t o form pH-dependent wave V. ~5 hose height is about tn ice the height of wave 111. Wave I11 is thus a combination of waves I and II. and nave T' is a combination of waves 111 and IS'. The coulometric n values (I?') are 1, 1, 2, 2, and 4 for waves I through V, coIisecut ively . Pyrimidine is the only compound investigated IT hich yields an additional reduction wave in alkaline solution; the appearance of a new wave a t about pH 7.2 positively identifies pyrimidine even in the presence of the 2-aminopyrimidines and of 2-hydroxypyrimidine, which are also reduced in alkaline solution. The heights of all the pyrimidine waves are directly proportional to concentration over a 1%-ideconcentration range nhen conditions of pH, buffer component, and ionic strength are controlled. 2-Amin9- and 2-Amino-4-methylpyrimidine. The three males obw v e d for thme compounds (Tables YI and YII) correspond in general
behavior t o those observed for pyriniidine in acidic solution, Le., wave I is p H d e p e n d e n t , wave I1 is essentially p H - independent, and they merge t o form pH-dependent wave
111. The 2-amino compounds can easily be distinguished from pyrimidine by their polarographic behavior, Le., they do not exhibit naves in basic solution corresponding t o vvaves IT' and V of pyriniidine, and the merger of waves I and I1 occurs a t a higher p H than in the case of pyrimidine. 2-Amino- and 2-amino-4methylpyrimidine can be distinguished from each other by E1 2 differences, especially at higher pH, Le., in p H 9.2 ammonia buffer their wave I11 values are - 1.44 and - 1.60 volts, respectively. The diffusion current constants of the three waves observed in acidic solution for pyrimidine and2-amino-and 2-amino4-methylpyrimidine are very similar. 2-Hydroxypyrimidine. This pyrimidine gives a single diffusion-controlled l e wave over the entire p H range, nhose Eli? equals -0.530 Table VI. Effect of pH, Concentration, Drop-Time (Head of Mercury), and Temperature on the Ell2 and id of 2Aminopyrimidinea
PH 2.1 2.lb 2.1c 2.9 4.0
id,
pa.
I 2.21 2.07 2.62 2.17 2.13 2.37 2.25 2.22 2.25 2.18 2.19 2.07 2.17 2.00 2.23 2.13 2.33 2.05 2.39 1.90 4.32 4.16 '4.27
-Eiiz, Volt 0.786 0.788 0.784 0.827 0.913 1.375 1,006 1.369 1.030 1.372 1.055 1.369 1.060 1.378 1,134 1.373
2.14 2.50 2.53 2.10 I 2.06 I1 2.20 4.8 I 2.16 I1 2.06 5.0 I 2.16 I1 2.02 5.2 I 2.08 I1 1.92 5.2b I 2.58 I1 2.32 5.9 I 2.12 I1 1 . 9 8 I 2.22 6.3 1,188 I1 1.90 1.386 6.8 I 2.26 1.240 TI 1.76 1.389 7.2d 4.02 1.338 7.6 1.371 3.86 8.06 1.408 2.38 8.0 4.15 1.417 3.82 8.0" 4.62 5.02 1.428 8.0, 1.418 7.32 3.94 8.O b J 1.420 8.76 3.81 9.2 4.00 4.31 1.440 11.8b' 4.2 4.1 1.89 Buffer 2 (0.45M) was employed for all runs except the last two, which nere made in 0.2534 buffer 5 and 0.45M buffer 6, respectively. The mercury height was 60 cm. in these runs, but 40 cm. in all of the others. Temperature was 40" C. for these runs; it was 25' C. for the others. An inflection was apparent near the Ei12. e
0 3OmJd 2-aminopyrimidine. Concentration in these runs was 1.00mM 2-aminopyrimidine; it was 0.50mM in all others, exrept for one run. 0 This wave is actually an inflection vcry close to the background discharge. f
0.078 p H ; the general behavior of this wave corresponds t o that of pyrimidine wave I in acidic solution. Except for a significant decrease in p H 3.7 acetate buffer, I is remarkably constant between 0.03 a t the 0.5mM p H 2 and 8 (2.06 concentration level). I is 2.12 i 0.05 between 0.15 and 1.67mM in p H 2.9 l\lcIlvaine buffer. Cytosine. Cytosine gives a fairly ne11 defined wave, which lies close t o the background discharge, i n p H 3.7 to 5.7 acetnte buffer (Eli2= -1.125 - 0.073 p H ) ; in 0.554 acetat
