ELSEVIER

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ACIA Aualytica Chimica Acta 289 W94) 163-167

Investigations on bioanalytical chemistry Part V. Adsorption voltammetry of adenine

Xin Zhao, Wenrui Jin *

Chanist~ Lkpartment, Shunabng Unkmsity, Jinan 250100, China

Jiangxiong Chen, Zuquan Gao, Funing Wang Biology Department, Shandong University, Jinan 250100, China

(Received 25th October 1993)

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The adsorptive and voltammetric behaviour of adenine on a hanging mercury drop electrode was investigated in 0.1 mol/l acetic acid-sodium acetate buffer (pH 3.6). The influences of the preconcentration potential and preconcentration time on the peak current as well as the optimum experimental conditions were discussed. The linear relationship between the peak current and the concentration of adenine was tested. To decrease the detection limit, the 1Sth order derivative technique was used and a detection limit for adenine of 1.8 X 10P9 mol/l was reached when the preconcentration time was only 60 s. This method was applied to determine the adenine content in the hydrolytic product of yeast RNA with satisfactory results.

Key words: Voltammetry; Adenine; Adsorption; Bioanalysis; Nucleic acids; Trace analysis

1. Introduction

The determination of purine and pyrimidine bases is very important in nucleic acid research. Most methods of analysis are based on the ab- sorption of UV light at certain wavelengths [l]. The most suitable concentration range for the UV-spectrophotometric determination of the bases is of the order of magnitude of 10m5 mol/l.

* Corresponding author.

At lower concentrations, this method is not suit- able.

The ability of nucleic bases to yield anodic polarographic currents in an alkaline medium was discovered thirty years ago [2-41. In 1962, Smith and Elving [51 reported the first systematic and detailed study of the electrochemical reduc- tion of adenine. They found that adenine gives a single, large, pH-dependent and largely diffusion- controlled polarographic wave. Coulometry re- vealed that six electrons were involved in the complete reduction of adenine, and spectropho- tometric and chemical investigation of the pro- duction solution revealed that ammonia was pres-

0003-2670/94/$07.00 Q 1994 Elsevier Science B.V. All rights resewed SSDI 0003-2670(93)E0659-U

164 XI Zhao et al. /Analytica Chimica Acta 289 (I!%) 163-167

ent. Dryhurst and Elving [6] studied the cyclic voltammetric behaviour of adenine at a hanging mercury drop electrode (HMDE) and the single, pH-dependent cathodic peak of adenine gave no evidence for reversibility of the electrochemical process. A differential pulse polarographic deter- mination of adenine was used by Temerk and Kamal [7] and a detection limit of 2.2 X 10m6 mol/l was found. Cummings et al. [81 used the same method to determine adenine with a detec- tion limit of 5 X lo-’ mol/l.

Adsorption voltammetry is a newly developed technique for determination of trace and ultra- trace metal ions and organic compounds [g-11]. Lower concentrations can be determined by accu- mulation of the compound at an HMDE before applying voltammetric detection.

In this paper, the adsorption voltammetric characteristic of adenine on an HMDE was stud- ied. The optimum detection conditions were se- lected. When 15th-order derivative adsorption voltammetry was used, a detection limit of 1.8 X lo-’ mol/l was reached when the preconcentra- tion time was only 60 s. The method was used to test the content of adenine in the hydrolytic prod- uct of yeast RNA with satisfactory results.

2. Experimental

2.1. Apparatus

A Model 83-2.5 voltammetric analyzer (Ningde Analytical Instrument Factory) coupled with a Model 3b86-11 X-Y recorder (Yokogawa Hokus- kin) was used in connection with a cell, using potentiostatic control of the electrode potential by means of a three-electrode system, consisting of a Model SH-84 HMDE (Department of Chem- istry, Shandong University) as the working elec- trode, a Pt plate as the counter electrode and an SCE as the reference electrode. The SCE was connected to the analyte via a salt bridge filled with buffer to the same level as in the electrolytic cell. During each determination, the solution was stirred with a PTFE-covered stirring bar, rotated .by a Model Lab-Line 1250-2 magnetic stirrer.

2.2. Reagents and solutions

A 1 mg/ml stock solution of adenine was prepared by dissolving an appropriate amount of adenine (> 95%, Shanghai Dongfeng Biochemi- cal Technological Co.) in water and standard solutions were obtained by diluting the stock so- lution. The stock solution was stored in a refrig- erator at 4°C. Sodium acetate (NaOAc) and acetic acid (HOAC) were analytical reagent grade. All solutions were prepared from doubly distilled wa- ter.

2.3. Procedure

The supporting electrolyte consisted of 0.1 mol/l NaOAc-HOAc (pH 3.6). The solution was deaerated for 20 min with pure nitrogen. Mea- surements were made after a preconcentration time, in which the solution was left to rest for a certain time, t,, and a preconcentration potential, E,, was applied. The response curve was recorded by scanning the potential from E, to - 1.80 V, with a scan rate of 120 mV/s. Each measurement was performed with a fresh drop. All potentials were measured against the SCE.

3. Results and discussion

3.1. Aakolption characteistics of adenine

The electrocapillary curve of adenine in 0.1 mol/l HOAc-NaOAc buffer (pH 3.6) is shown in Fig. 1. After adenine is added to the buffer, the surface tension of the dropping mercury elec- trode (DME) decreases resulting in a shorter drop time, because of the adsorption of adenine on the surface of the DME.

In 0.1 mol/l HOAc-NaOAc buffer (pH 3.61, a reduction peak of adenine at - 1.18 V can be observed. The voltammograms of the reduction of adenine at different preconcentration times, t,, are shown in Fig. 2. The peak current in- creases with increasing t,. It shows the adsorp- tion of adenine on the HMDE. The faster the scan rate, the larger the peak current of adenine. The relationship between the peak current of adenine and the square root of scan rate is shown

X. Zhao et aL /Ana&ica Chhica Acra 289 (1994) 163-167 165

1 drop time /s 3.3. Analytical application

.iJ.o -0.2 -0.4 -04 -0.8 4.0 4.2 -1.b E/V vs.SCE

Fig. 1. Electrocapillary curve of adenine in HOAc-NaOAc buffer. (1) 0.1 mol/l HOAc-NaOAc buffer (pH 3.6); (2) 0.1 mol/l HOAc-NaOAc buffer + 4.12 X 10m4 mol/l adenine.

in Fig. 3. The curve has a positive deviation with increasing scan rate, because of the adsorption of adenine on the HMDE.

The relationship between the adsorption peak current of adenine and the preconcentration po- tential, E,, is shown in Fig. 4. The peak currents are different at different E,, which is characteris- tic for organic materials adsorbed on the surface of the HMDE.

3.2. Optimum experimental conditions

The adsorption/ reduction voltammograms of adenine in three different solutions are studied. In 0.1 mol/l NH,-NH&l buffer (pH 8.91, no reduction peak of adenine on the HMDE can be observed. This confirms that the non-protonated form of adenine is electrochemically irreducible, as Ref. 5 has suggested. In both citric acid- Na,HPO, and HOAc-NaOAc buffer, reduction peak of adenine at around - 1.20 V can be ob- served. The peak potentials of adenine tested in these two solutions are summarized in Table 1. The relationship between the peak current of adenine and the pH of HOAc-NaOAc buffer is shown in Fig. 5. When the pH is between 3.2 to 3.6, a higher peak current can be obtained. HOAc-NaOAc buffer of pH 3.6 is selected for subsequent experiments.

A linear relationship between the reduction peak current and the concentration of adenine can be obtained in the concentration range of 4.3 X lo-*-7.6 X 10m7 mol/l for E, = -0.70 V, u = 120 mV/s, and t, = 30 s by adsorption voltammetry on the HMDE. When t, = 60 s, the detection limit is 2.2 x lo-* mol/l. The devia- tion, calculated from 10 successive measurements of 4.34 x 10S7 mol/l adenine, is f2.0%.

It has been proved that g 1Sth (or 2Sth) order derivative technique has the advantage of further

I 0.05pA

,0.8 -1.0 -1.2 -1.4 E/V vs.SCE

Fig. 2. Adsorption voltammograms of adenine at different preconcentration times, t,: (1) 0; (2) 30; (3) 60; (4) 120 5. 0.1 mol/l HOAc-NaOAc buffer (pH 3.61, 4.34X lo-’ mol/l adenine, E. = - 0.70 V, u = 120 mV/s.

i,/pA 0.35.

ip/yA

0.3

Fig. 3. Relationship between the peak current of adenine and square root of scan rate. Conditions as in Fig. 2.

improvement of the resolution and to increase sensitivity in comparison to conventional linear sweep voltammetry for both the solution phase and the amalgam phase [12-141. In our previous studies, the theory of 15th or 2.5th order deriva- tive adsorption voltammetry has been derived [15] and the technique has been applied to adsorptive voltammetric measurements to increase sensitiv- ity [16,17]. In this work, the 1.5th-order derivative technique is used. Voltammograms of the normal curve and the 1.5th-order derivative curve tested at the same concentration of adenine are shown in Fig. 6. The peak-to-peak eLP value, obtained by 1.5th-order derivative adsorption voltammetry

Table 1 Reduction peak currents and peak potentials of adenine in Na,HPO,-citric acid and HOAc-NaOAc buffer (t, = 30 s, other conditions as in Fig. 2)

PH Na,HPO,-citric acid buffer HOAc-NaOAc buffer (0.2 mol/l) (0.1 mol/l)

i, (/LA) Ep 6’) i, (rA) Ep (VI

2.2 0.110 - 1.20 0.235 - 1.05 2.6 0.092 - 1.25 0.253 - 1.09 3.2 0.152 - 1.29 0.322 - 1.12 3.6 _a _a 0.322 - 1.20 3.8 _a _a 0.244 - 1.24 4.0 _a _a 0.193 - 1.25 4.2 _a _a 0.175 - 1.26 4.6 _a _= _a _a

” Ine peak was covered by aischarge peak or butter.

166 X. Ztao et a,! /Analytica Chimica Acta 289 (1994) 163-167

o.zs-

I_& Oil -0.2 -0L -0.6 -08

E/V vs.SCE

Fig. 4. Relationship between the peak current of adenine and pre-concentration potential. t, = 30 s, other conditions as in Fig. 2.

is much higher than the value of the peak cur- rent, i,, by normal adsorption voltammetry. Us- ing this technique, the concentration of adenine is in agreement with eLP in the range of 2.2 x

1O-8-1.7 x 10e7 mol/l for E, = -0.70 V, u = 100 mV/s and t, = 30 s. When t, = 60 s, the limit of detection is 1.8 X 10m9 mol/l. The deviation cal- culated from ten successive measurements of 6.52 x 10v8 mol/l adenine is f5.1%.

Samples of pure yeast RNA (20.0 mg) and 5 ml HCl (1 mol/l) were put in a sealed glass am- poule. After heating in a boiling water bath for 80 min, the solution were transferred into a 25ml

0.30.

0.20.

Fig. 2.

3.2 3.6 LO u PH

;

Fig. 5. Relationship between the peak current of adenine and the pH of buffer’ solution. t, = 30 s, other conditions as in

X. Zhao et al. /Analytica Chimica Acta 289 (1994) 163467 167

i

/‘s

l

!’

-0.6 -0.8 -1.0 -1.2 -1.4 E/V vs.SCE

Fig. 6. Linear sweep adsorption voltammogram of adenine and its 1.5th-order derivative curve. 4.34X10V8 mol/l ade- nine, t, = 30 s, other conditions as in Fig. 2.

flask and diluted to the mark with water. 10 ~1 of the solution was added to 25 ml HOAc-NaOAc buffer (pH 3.6). Cathodic adsorption voltammetry was applied and the standard addition method was used to determine the content of adenine in the hydrolytic product of yeast RNA. A result of 23.8% was obtained which coincides with the standard content of 25.4% reported in the litera- ture.

4. References

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