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440 — Voltammetric on-line analysis of molecules of biological importance
459
440 A VOLT .BIOLOGICAL
OIkLlkE A1c;IMETRTC IMPORTANCE
Department of Chemistry.
ANALYSIS
OF MOLE-
OF
l
Uniuersi& College Cork. Cork (Repubiic of Ireland)
. . A. lVA6KA Department of Analytical Chemistzy, Abe Akademi
SF-20500
&o
(Finland)
JS. BURMICZ EC & G Brookdeal, Bmcknell. Berks. (Englan+) LE. DAVIDSON John
Wyeth
& Brother.
Huntercombe
Lane South,
Taplow.
Maidenhead,
Be&s_
(England)
Y. VANEESORN Chinng Mai University.
Chiang bfai (Thailand)
(Manuscript received December 9th 1980)
SuBfMARY The on-line voltammetric behaviour of several groups of biologically important molecules, (a) 1,4-benzodkzepines, (b) 2-thiobarbituric acids and (c) thioamides, is investigated. A flow through voltammetric cell based on the wall jet principle is described and the d-c. and puke working modes are studied in on-line situ~tioas. Direct oxidation at the glassy carbon electrode, direct oxidation at a krcury coated glassy carbon electrode and cathodic stripping on this mercury coated glassy carbon electrode have been evaluated with respect to analytical optimkation, i.e., in terms of linear range, limit of detection etc.
INTRODUCXTON The need to develop rapid methods of anaIysis’for compounds of ck.ical and pharmaceutical importance has resulted in on-line.~polarographic and voltammetic methods for-their analysis. In these methods either the stream containing
* Invited lecture at the IIIrd Brno Symposium on Molecular Biophysics: Ekctroanalysis Biopolymers, August 3lst-September 5th. 1980, Kupakwice (CSS_RR.). 0302-4598/81/000~~00/~02.50
0 1981 Ekvier
Sequoia SAA.
of
Reference
Pt, c C d.m.e. d,m.e.
benzodiazopince phenols &cted drugs benzodlazopines pro&e
16 20 30 120
d,m.e.
d,m,e.
,velecteddrug8
60
Working electrode
Compounde etudled
Number of eamplee per hour
TABLE1 Summaryof Bornevoltammetric on.llne onnlyeoeof orgnnicmoleculeo
0.4-10 mg/cm3 16-26 ng/cm3 4 x lo-66 Xlo+ g/cm3 10’3-10’6 M 0.1 mg/cm3 6-GOlrglcrn’
d,c. ; o,c,
d,c. d,c. DPP
i$P
sampled
Cone, range atudled
Mode
0.3 1
1.6
1.4
Relative standard deviation
461 the ekctrgactiyg gderiaI is continuously monitored by an electrochemical detitor [l.Z] or a-large number of samples are analysed by repetitively passing them pa&the detector and recording the current.produced by each sample 13-7 1. Some of these results are collected in Table 1. The great selection of electrochemical detectors which have been developed for high-performance liquid chromatography [9-151 can also be used where chromatographic separation is not required. Electrochemical detectors can also be used in flow injection analysis where a smell volume of the analyte is aspirated into a flowing background electrolyte. This method has been successfully applied to the analysis of some organic compounds [12,1S,L7]. This paper is ccncerned with the voltammetric on-line analysis of several groups of compounds which are of clinical and pharmaceutical importance and which have heen subjected to detailed polarographic end voltaxnmetric investigation in quiescent solutions as the literature will bear out: (a) 1,4berzz+eeepines [l&22,23] chlordiazepoxide (I), medazepani (II), n.itmz.e~ @I), amino-nitrazepam (IV), lomzepam (V) and flurazepam (VI); (b) 2-tiuM@bituric acids [19,21,23) 5ethyl-5’-(l-methylbuty!)-2-thiobarbituric acid (VII), ~-methyl-5&hyl-5’-(1-methylpropyl)-2-thiobarbituric acid (VIII), 1,3-dimethy15;ethy1-5’-(p~hloropheny1)-2-thiobarbituric acid (DC) and 2-methylthiobarbituric acid (X); (c) ~triuumides [20,21] 5,6,7,8-tetrahydro-3-methylquinoline-S-thiocarboxamide hydrochloride (XI) and 5,6,7,8-tetrahydro-3-methylquinolineS-(Nmethyl)-thiocarboxamide hydrochloride (XII) and 5,6,7,8-tetrahydro-3-methylquinoline-&(N-ethyl)-thiocarboxamide (XIII). This on-line voltammetric behaviour has been studied at a glassy carbon indicator electrode as part of a cell based on the wall jet principle. Such a solid electrode is essentially easier to handle in on-line situations and has a constant surface area in comparison to dropping mercury and hanging mercury drop elecbodes. High ffow rates can also be used thus increasing the sensitivity of the resulting analytical method due to increase in mass transport to the electrode surface. Direct oxidation at this electrode, direct oxidation at the electrode coated with a thin mercury film and cathodic stripping on this mercury coated glassy carbon electrode have all been evaluated with respect to optimi&ion of the voltemmetric signal for analytical purposes. EXPERIMENTAL
Appam
fus
The electrochemical flow through cell used in this work is of the wall jet type and its performance and use_fu.lnesshave been evaluated in a separate work 1231. The incoming’sohrtion impinges on the surface of the working electrode which is a glassy carbon electrode of 3 mm diemeter. A platinum auxil&y electide is placed near the working eleCf30de and when the solution leaves the cell it passes
462
the tip of the saturated calomel reference electrode_ No contamination of the working electmde is thereby caused by the reference electrode, a factor which is particularly important when a mercury coated electrode is used at positive potentials, with Hg,Cl* being a possible surface contaminant. Ail potentials in this work are measured against the saturated calomel electrode. The ceil volume is adjustable and a volume of about 80 mm3 (fi) has been used throughout in this study. A P4R 174A polarograph has been used in the voltammetric-measurements and the solutions are either pumped to the cell with a three-bar peristaltic pump with variable rotation speed or the flow is effected hydrostatically from elevated electrolyte and sample containers. This has resulted in flow-rates from 2-5 cm3 min-’ being used. The currents have been recorded with a strip chart recorder and the measurements performed at 22°C. Techniques
In the direct oxidation studies, the supporting electrolyte was first pumped through the electrochemical cell. After a steady background current had been obtained, the analyte solution was pumped and the current change recorded. When high current ranges were used in the d-c. working mode, a steady background current was obtained immediately whereas for low ranges (e-g. 0.02-0.2 ti) a period of 2-15 min was required to achieve steady values. The pulse mode generally required more time for attainment of a steady background current; at tile lowest current range used (1 @) the time required was 5-10 min. The mercurycoated glassy carbon electrode in the d-c_ mode behaved sk.niIarlyin this respect to the glassy carbon electrode. When a new mercury surface was plated it took 5-15 min before a steady background current was achieved_ When the current range was kept constant for a series of different concentrations of the same electroactive compound, the background current reached its steady-state value in the d-c. mode about 20 s after the changeover from analyte to supporting ekxtsol~ when the glassy carbon electrode was used with OF without the mercury f&n; l-2 min was needed for the pulse mode. The mercury film on the glassy carbon electrode was plated by electrolytic deposition at -1.0 V for 4 min from a flow of lo’* M mercury (II) nitrate solution in 0.01 M nitric acid pumped at a rate of ca. 2 cm3 mi.n-‘. After the plating, the electrode was held at to.1 V for 2 min, to remove any co-plated metallic impurities_ The film was mechanically removed daily and replated as above on the following morning. Occasional anomalous results were traced to defects in the film; these disappeared when a new film was plated. In the cathodic stripping method the supporting electrolyte solution was first pumped through the celI with an applied potential of -1.0 V for 1 min and then the pump was stopped and the applied potential switched off. The anaIyte was then pumped through the cell while the applied potential was stiIl at the off position. After 1 m.in the chosen plating potential (+0_15 + to.2 V) was applied to the working electrode for 4 min. During the pIating period the flow-rate was kept constant. At the end of the plating time, the applied potential was switched off and the pump was sttpped. The soluti&n of the sup,porting elec-
463
trolyte was then pumped through the cell while the applied potential to the electrode was still at the off position. It was found that no potential should be applied to the electrode, not even the plating potential, when the flow was changed from the analyte to the buffer; otherwise no stripping current was observed. After passing the buffer through the cell for 1 min the pump was stopped. The stripping process was then initiated by scanning the potential from the plating potential to -0.8 V at a scan rate of 10 mV S-I during which the stripping voltammogram was recorded. RESULTS
AND DISCUSSION
(a) 1.4~Benzodiazepines The oxidative voltammetric behaviour of the benzodiazepines, chlordiaxepoxide (I), medazepam (II), nitmzepam (III), amino-nikaxepam (IV), lorazepam (V) and fluraxepam (VI) has been investigated at the glassy carbon electrode in quiescent solution and the following results were obtained (Table 2) CM]. Although the >C=N’ group, involving the 4-N atom, is the functional group which is reduced in these compounds at both mercury 1243 and carbon 1251 indicator electrodes, it would appear that this group is not involved in benzodiaxepine electro-oxidation at the glassy carbon electrode. Preliminary results, to be confirmed by a more in-depth forthcoming publication 1261, indicate that the 1-N atom is involved in elecko-oxidation reactions initially forming a nitrogen radical cation and leading to tail-to-tail coupling, in a similar manner to the corresponding reactions of secondary and tertiary aromatic amines. This would appear to be the case for medaxepam (II), amino-nitrazepam (IV) and lorazepam (V). Where the 1-N substituent is b-ulkierthan a methyl group, and contains nitrogen atom(s) in the side chain, then electio-oxidation is likely to take place on these nitrogen atom(s). Two cases in point would appear to be chlordiazepoxide (I) and flurazepam (VI). If the substituent in position 7 is strongly electron withdrawing, as is the case with nitraxepam (III), then the decreased electron density on the 1-N atom, primarily due to resonance delocalis&ion of the lone pair on the 1-N atom, precludes electro-oxidation at this site TABLE
2
Voltammekic behaviour of some 1.4-benzodiazepines cent solution [ 181
at a glassy carbon electrode in quies-
Benzodiazepine
UP (V vs. .s.c.e.) in pH 4 BR buffer
Chlordiiepoxide(1)
+1.04 t0.78, t1.0 no wave cl.23 +1.15 +1.22
Medazepam(II)
Nitrazepam(III) Amino-nitrazepam(IV) Lorazepam( V) PIurazepam(vI)
464
in the available potential range. This investigation (Table 2) was followed up by a study of the on-line voltammetric behaviour of these skl,4benzodiazepines at a glassy carbon electrode using a detector potential set at t1.2 3 and a ‘supporting electrolyte of pH 4 Britton-Robinson buffer which contained lG% methanol to minimise adsorption problems. The differential pulse mode was used to increase the sensitivity of the method. All compounds,. except nitrazepam (RI) gave satisfactory signals for on-line analysis. Results on the analysis of tablet formulations using this method in conjunction with a Carlo-Erba Automatic Analyser have been presented 118-J_ (b)
Z-Thiobarbituric
acids and (c)
thioamides
(i) Direct electro-oxidation on a thin mercury firm using d-c. voltammefry. The results of these studies are summarized in Table 3 where also the experimental
conditions are given together with ranges where a Linear reIationship was obtained between the faradaic current and concentration of the different depolarisers. The detection limits for the studied compounds are also given i;l Table 3. Due to the poor responses of compounds (X) and (XIII) they were no& further studied using this on-tie system. Compound (X) was found not to give any current by the hydrodynamic d-c. voltammekic technique at any concentration levels and using the pH range B-11 with Britton-Robinson buffers. This is contrary to the investigation of this compound using polarography in quiescent solutions [ 191. The reason may be that the compound may not be able to give any significant electrochemical reaction at the thin mercury film under fIowing stream conditions. The structure of compound (X) is different from compounds (VII), (VIII) and (IX) in that the sulphur atom at the 2-position has a substituent methyl group thus preventing its reaction with mercury to form a mercury salt with adsorption properties on the mercury surface as for compounds (VII), (VIII) and (IX). If
TABLE
3
Direct electrooxidation Compound
2-Thiobarbituric acids VII IX X Thioamides XI
on a thin mercury fii
using d.c. voltammetry.
u applied WI
PH
Linear =ge WI
Detection limit WI
+0.1 co.1 +0.1 +0.1
8 8 8 11
1 x 10*-l x 104 5 x 10-6-l x 104 5 x lOd-5 x 10‘5 no reaction
5x 10-7 1x10” 2x10* -
to.25 eo.2 +0.2
4 4 4
5 x 10-8-l x 10-5 1 x 10-6-l x 104 no reaction
5 x 10-B 5 x 10” -
465
compound (X) gives an &nodic reaction as suggested Cl9 ] it will therefore react differently. from compounds (VII), (VIII) and .(IX) with reactant and/or product absorption being’significantly dimimshed. Either this and/or the kinetics of the electrochemical reaction will influence this compound’s amenabiliQ7 to on-line analysis. The upper limits of the linear ranges of the 2-thiobarbituric acid derivatives are determined by their solubility in aqueous supporting electrolytes. Thioamides, existing as hydrochlorides, are rather soluble in these electrolytes but at high concentrations the electrode surface was found to be fouled resulting in a decreased signal and hence not useable for analytical purposes. The fouling can be due to the adsorption of the thioamides on the surface which partly can be a result of their chemical reaction with metallic mercury. AIternatively, the product of the electEochemiw.l reaction can adsorb on the surface and hence prevent thioamide molecules re&ching it. The upper limits of the linear ranges of the thioamides are concentrations where a constant signal is observed. The voltammetric flow-through cell can also be applied to cathodic stripping [ 231. In this study the compounds (VII), (VIII), (IX), (XI) and (XII) are investigated by an on-line cathodic stripping method, For the 2-thiobarbituric acid derivatives +0.15 V was taken as the plating potential and for the thioamides CO.2 V. In cathodic stripping the cell volume was increased to approximately 100 mm3 (fl). In this way the flow velocity near the surface was reduced allowing the products of electrolysis to be adsorbed on the mercury film. In the d-c. oxidation method it was more advantageous to have small cell volumes where the high flow velocity near the surface removed some of the electrochemical reaction products thus mechanically keeping the electrode surface clean. Results kom the cathodic stripping analysis are given in Table 4. As can be seen in Table 4 the thioamides do not plate on the thin mercury film in the flowing stream. This is obviously attributable to the inabiliw of the plated compound to adhere satisfactorily to the mercury film. It is postulated that although some electrolysis will take place the plated compound is swept away kom the surface by the stream. Plating was slightly improved by increas(ii) Cathodic stripping on a thin mercury film
TABLE Cathodic
4
strippingon the thin Hg-film
Compound
pH
Plating potential 09
stripping potential w
Linear =wze WI
M vm Ix XI XII
8 8 8 4-3 4-6
+0.15 +0.15 +0_15 +0.2 eo.2
-0.10 -0.65 4.63 -
5 x 106-S 1 x 10-7-1 5 x 10-7-2 no plating no plating
Detection limit WI x 10-s x lo* x 10-5
5x106 1 x 10-6 1 x 10-7 -
TABLE
5
Linear concentration ange for the determination of some sulphurcontaining organic compounds by static and on-line voltammetrir methods (oxidation processes used except wil* otherwise stated) Compound
Linear concentration range DSP. (static)*
C.S.V. (static)
dx. (on-line)
C.S.V. (on-line)
5,5’-Disubstituted 2thiobarbiturate(VII)
2 x lo’lxlo+M=
lXlO”1x10-M
1 x lo+lXlO-+hf
5 x 10-6: 5XlOSM
1,5,5’-Tkisubstituted 2thiobarbitwate(VIlI)
5 x lo*4 x 16+-Ma
2 x lO4lXl05M
5 x lo+lX104M
1 x lo-‘lxlO*M
1,3,5,5’-Tetrasubstituted I-thiobarbitwate(M j
5 x 10-64x10*hf=
1 x lo+6x10dM
5 x lO”5 x 10-5 M
5 x lo-‘2 x 10-s M
Primary thioamide
5 x lo-‘5xlO”M
5 x 10-aIO+ M
5 x lo-81~10-~M
-
a Reduction processes used.
ing the cell volume but even with large cell volumes reproducible results were not obtained. The 2-thiobarbituric acid derivatives did plate well on the thin mercury film. Compound (VIII) was found to plate most satisfactorily giving the smallest’ relative standard deviation and lowest detection Emit of the compounds studied. From the stripping potentials in Table 4 it can be seen that the products of electrolysis of compounds (VIII) and (IX) appear the same and from this potential value, HgS would appear to be the most probable product [27]. Compound (VII) behaves differently forming presumably a thiobarbiturate mercury salt.as . observed in quiescent solutions [28] _ Log I vs. log c plots of compounds (VII), (VIII) and (IX) gave slopes of 1.0,0.7 and 0.7 showing that plating of compounds (VIII) and (IX) is reproducible but not quantitative 1293. CONCLUSIONS
Hydrodynamic d-c. voltammetry has been successfully applied to the analysis of some 1,4benzodiiepines, 2-thiobarbituric acid derivatives and thioamides. An increase in sensitivity in most cases has been achieved mainIy due to the hydrodynamic nature of the fIowing stream as compared to vokunmetry in quiescent solution (Table 5). Several factors do influence the improved sensitivity. The diffusion layer adjacent to the electrode is reduced to a very narrow region allowing fast diffusion of the electroactive molecules to the elecfzode surface and hence an improved mass transport from the solution to the electrode is obtained, The greater surface area of a solid electrode when compared with a dropping mercury electrode leads to an additional increase in sensitivity. With application of a constant potential no current is needed to charge the double
467
layer decreas ing the background current and hence improving the sensitivity.
& demo-ted in this work such sulphur~ontaining compounds which form a mercury salt can be aualysed by on-line methods either by direct oxidation on the mercury film or by.cathodic stripping. Although a compound may give a well defined response when analysed in quiescent solution its analysis by on-line methods may fail as demon&at& with compounds (X) and (XIII) in the d-c. oxidation mode, Compounds (v]tr), (VIII) and (IX) could be analysed by on-line ca!hodic stripping whereas compounds (XI) and (XII) gave poor results although they have been analysecl successfully by cathodic stripping in rather complex matrices in quiescent soIutions 1271. All the studied compounds in general exhibit larger linear concentration ranges and lower detection limits with the proposed on-line methods than by analysis in quiescent solutions. The detection limit of compound (XI) with the on-line d-c. oxidation method is in fact sim&r to that of the cathodic stripping method in quiescent solution 127 1. It is believed that such on-line voltammetric studies will be of particular value in studying the interaction of drugs with biopolymers and their components by analysis of these complex matrices using the combination of high-performance liquid chromatography and electrochemical detectors.
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Zs. Feher. G. Nagy. EC.Toth and E. Pungor. Analyst
440 A VOLT .BIOLOGICAL
OIkLlkE A1c;IMETRTC IMPORTANCE
Department of Chemistry.
ANALYSIS
OF MOLE-
OF
l
Uniuersi& College Cork. Cork (Repubiic of Ireland)
. . A. lVA6KA Department of Analytical Chemistzy, Abe Akademi
SF-20500
&o
(Finland)
JS. BURMICZ EC & G Brookdeal, Bmcknell. Berks. (Englan+) LE. DAVIDSON John
Wyeth
& Brother.
Huntercombe
Lane South,
Taplow.
Maidenhead,
Be&s_
(England)
Y. VANEESORN Chinng Mai University.
Chiang bfai (Thailand)
(Manuscript received December 9th 1980)
SuBfMARY The on-line voltammetric behaviour of several groups of biologically important molecules, (a) 1,4-benzodkzepines, (b) 2-thiobarbituric acids and (c) thioamides, is investigated. A flow through voltammetric cell based on the wall jet principle is described and the d-c. and puke working modes are studied in on-line situ~tioas. Direct oxidation at the glassy carbon electrode, direct oxidation at a krcury coated glassy carbon electrode and cathodic stripping on this mercury coated glassy carbon electrode have been evaluated with respect to analytical optimkation, i.e., in terms of linear range, limit of detection etc.
INTRODUCXTON The need to develop rapid methods of anaIysis’for compounds of ck.ical and pharmaceutical importance has resulted in on-line.~polarographic and voltammetic methods for-their analysis. In these methods either the stream containing
* Invited lecture at the IIIrd Brno Symposium on Molecular Biophysics: Ekctroanalysis Biopolymers, August 3lst-September 5th. 1980, Kupakwice (CSS_RR.). 0302-4598/81/000~~00/~02.50
0 1981 Ekvier
Sequoia SAA.
of
Reference
Pt, c C d.m.e. d,m.e.
benzodiazopince phenols &cted drugs benzodlazopines pro&e
16 20 30 120
d,m.e.
d,m,e.
,velecteddrug8
60
Working electrode
Compounde etudled
Number of eamplee per hour
TABLE1 Summaryof Bornevoltammetric on.llne onnlyeoeof orgnnicmoleculeo
0.4-10 mg/cm3 16-26 ng/cm3 4 x lo-66 Xlo+ g/cm3 10’3-10’6 M 0.1 mg/cm3 6-GOlrglcrn’
d,c. ; o,c,
d,c. d,c. DPP
i$P
sampled
Cone, range atudled
Mode
0.3 1
1.6
1.4
Relative standard deviation
461 the ekctrgactiyg gderiaI is continuously monitored by an electrochemical detitor [l.Z] or a-large number of samples are analysed by repetitively passing them pa&the detector and recording the current.produced by each sample 13-7 1. Some of these results are collected in Table 1. The great selection of electrochemical detectors which have been developed for high-performance liquid chromatography [9-151 can also be used where chromatographic separation is not required. Electrochemical detectors can also be used in flow injection analysis where a smell volume of the analyte is aspirated into a flowing background electrolyte. This method has been successfully applied to the analysis of some organic compounds [12,1S,L7]. This paper is ccncerned with the voltammetric on-line analysis of several groups of compounds which are of clinical and pharmaceutical importance and which have heen subjected to detailed polarographic end voltaxnmetric investigation in quiescent solutions as the literature will bear out: (a) 1,4berzz+eeepines [l&22,23] chlordiazepoxide (I), medazepani (II), n.itmz.e~ @I), amino-nitrazepam (IV), lomzepam (V) and flurazepam (VI); (b) 2-tiuM@bituric acids [19,21,23) 5ethyl-5’-(l-methylbuty!)-2-thiobarbituric acid (VII), ~-methyl-5&hyl-5’-(1-methylpropyl)-2-thiobarbituric acid (VIII), 1,3-dimethy15;ethy1-5’-(p~hloropheny1)-2-thiobarbituric acid (DC) and 2-methylthiobarbituric acid (X); (c) ~triuumides [20,21] 5,6,7,8-tetrahydro-3-methylquinoline-S-thiocarboxamide hydrochloride (XI) and 5,6,7,8-tetrahydro-3-methylquinolineS-(Nmethyl)-thiocarboxamide hydrochloride (XII) and 5,6,7,8-tetrahydro-3-methylquinoline-&(N-ethyl)-thiocarboxamide (XIII). This on-line voltammetric behaviour has been studied at a glassy carbon indicator electrode as part of a cell based on the wall jet principle. Such a solid electrode is essentially easier to handle in on-line situations and has a constant surface area in comparison to dropping mercury and hanging mercury drop elecbodes. High ffow rates can also be used thus increasing the sensitivity of the resulting analytical method due to increase in mass transport to the electrode surface. Direct oxidation at this electrode, direct oxidation at the electrode coated with a thin mercury film and cathodic stripping on this mercury coated glassy carbon electrode have all been evaluated with respect to optimi&ion of the voltemmetric signal for analytical purposes. EXPERIMENTAL
Appam
fus
The electrochemical flow through cell used in this work is of the wall jet type and its performance and use_fu.lnesshave been evaluated in a separate work 1231. The incoming’sohrtion impinges on the surface of the working electrode which is a glassy carbon electrode of 3 mm diemeter. A platinum auxil&y electide is placed near the working eleCf30de and when the solution leaves the cell it passes
462
the tip of the saturated calomel reference electrode_ No contamination of the working electmde is thereby caused by the reference electrode, a factor which is particularly important when a mercury coated electrode is used at positive potentials, with Hg,Cl* being a possible surface contaminant. Ail potentials in this work are measured against the saturated calomel electrode. The ceil volume is adjustable and a volume of about 80 mm3 (fi) has been used throughout in this study. A P4R 174A polarograph has been used in the voltammetric-measurements and the solutions are either pumped to the cell with a three-bar peristaltic pump with variable rotation speed or the flow is effected hydrostatically from elevated electrolyte and sample containers. This has resulted in flow-rates from 2-5 cm3 min-’ being used. The currents have been recorded with a strip chart recorder and the measurements performed at 22°C. Techniques
In the direct oxidation studies, the supporting electrolyte was first pumped through the electrochemical cell. After a steady background current had been obtained, the analyte solution was pumped and the current change recorded. When high current ranges were used in the d-c. working mode, a steady background current was obtained immediately whereas for low ranges (e-g. 0.02-0.2 ti) a period of 2-15 min was required to achieve steady values. The pulse mode generally required more time for attainment of a steady background current; at tile lowest current range used (1 @) the time required was 5-10 min. The mercurycoated glassy carbon electrode in the d-c_ mode behaved sk.niIarlyin this respect to the glassy carbon electrode. When a new mercury surface was plated it took 5-15 min before a steady background current was achieved_ When the current range was kept constant for a series of different concentrations of the same electroactive compound, the background current reached its steady-state value in the d-c. mode about 20 s after the changeover from analyte to supporting ekxtsol~ when the glassy carbon electrode was used with OF without the mercury f&n; l-2 min was needed for the pulse mode. The mercury film on the glassy carbon electrode was plated by electrolytic deposition at -1.0 V for 4 min from a flow of lo’* M mercury (II) nitrate solution in 0.01 M nitric acid pumped at a rate of ca. 2 cm3 mi.n-‘. After the plating, the electrode was held at to.1 V for 2 min, to remove any co-plated metallic impurities_ The film was mechanically removed daily and replated as above on the following morning. Occasional anomalous results were traced to defects in the film; these disappeared when a new film was plated. In the cathodic stripping method the supporting electrolyte solution was first pumped through the celI with an applied potential of -1.0 V for 1 min and then the pump was stopped and the applied potential switched off. The anaIyte was then pumped through the cell while the applied potential was stiIl at the off position. After 1 m.in the chosen plating potential (+0_15 + to.2 V) was applied to the working electrode for 4 min. During the pIating period the flow-rate was kept constant. At the end of the plating time, the applied potential was switched off and the pump was sttpped. The soluti&n of the sup,porting elec-
463
trolyte was then pumped through the cell while the applied potential to the electrode was still at the off position. It was found that no potential should be applied to the electrode, not even the plating potential, when the flow was changed from the analyte to the buffer; otherwise no stripping current was observed. After passing the buffer through the cell for 1 min the pump was stopped. The stripping process was then initiated by scanning the potential from the plating potential to -0.8 V at a scan rate of 10 mV S-I during which the stripping voltammogram was recorded. RESULTS
AND DISCUSSION
(a) 1.4~Benzodiazepines The oxidative voltammetric behaviour of the benzodiazepines, chlordiaxepoxide (I), medazepam (II), nitmzepam (III), amino-nikaxepam (IV), lorazepam (V) and fluraxepam (VI) has been investigated at the glassy carbon electrode in quiescent solution and the following results were obtained (Table 2) CM]. Although the >C=N’ group, involving the 4-N atom, is the functional group which is reduced in these compounds at both mercury 1243 and carbon 1251 indicator electrodes, it would appear that this group is not involved in benzodiaxepine electro-oxidation at the glassy carbon electrode. Preliminary results, to be confirmed by a more in-depth forthcoming publication 1261, indicate that the 1-N atom is involved in elecko-oxidation reactions initially forming a nitrogen radical cation and leading to tail-to-tail coupling, in a similar manner to the corresponding reactions of secondary and tertiary aromatic amines. This would appear to be the case for medaxepam (II), amino-nitrazepam (IV) and lorazepam (V). Where the 1-N substituent is b-ulkierthan a methyl group, and contains nitrogen atom(s) in the side chain, then electio-oxidation is likely to take place on these nitrogen atom(s). Two cases in point would appear to be chlordiazepoxide (I) and flurazepam (VI). If the substituent in position 7 is strongly electron withdrawing, as is the case with nitraxepam (III), then the decreased electron density on the 1-N atom, primarily due to resonance delocalis&ion of the lone pair on the 1-N atom, precludes electro-oxidation at this site TABLE
2
Voltammekic behaviour of some 1.4-benzodiazepines cent solution [ 181
at a glassy carbon electrode in quies-
Benzodiazepine
UP (V vs. .s.c.e.) in pH 4 BR buffer
Chlordiiepoxide(1)
+1.04 t0.78, t1.0 no wave cl.23 +1.15 +1.22
Medazepam(II)
Nitrazepam(III) Amino-nitrazepam(IV) Lorazepam( V) PIurazepam(vI)
464
in the available potential range. This investigation (Table 2) was followed up by a study of the on-line voltammetric behaviour of these skl,4benzodiazepines at a glassy carbon electrode using a detector potential set at t1.2 3 and a ‘supporting electrolyte of pH 4 Britton-Robinson buffer which contained lG% methanol to minimise adsorption problems. The differential pulse mode was used to increase the sensitivity of the method. All compounds,. except nitrazepam (RI) gave satisfactory signals for on-line analysis. Results on the analysis of tablet formulations using this method in conjunction with a Carlo-Erba Automatic Analyser have been presented 118-J_ (b)
Z-Thiobarbituric
acids and (c)
thioamides
(i) Direct electro-oxidation on a thin mercury firm using d-c. voltammefry. The results of these studies are summarized in Table 3 where also the experimental
conditions are given together with ranges where a Linear reIationship was obtained between the faradaic current and concentration of the different depolarisers. The detection limits for the studied compounds are also given i;l Table 3. Due to the poor responses of compounds (X) and (XIII) they were no& further studied using this on-tie system. Compound (X) was found not to give any current by the hydrodynamic d-c. voltammekic technique at any concentration levels and using the pH range B-11 with Britton-Robinson buffers. This is contrary to the investigation of this compound using polarography in quiescent solutions [ 191. The reason may be that the compound may not be able to give any significant electrochemical reaction at the thin mercury film under fIowing stream conditions. The structure of compound (X) is different from compounds (VII), (VIII) and (IX) in that the sulphur atom at the 2-position has a substituent methyl group thus preventing its reaction with mercury to form a mercury salt with adsorption properties on the mercury surface as for compounds (VII), (VIII) and (IX). If
TABLE
3
Direct electrooxidation Compound
2-Thiobarbituric acids VII IX X Thioamides XI
on a thin mercury fii
using d.c. voltammetry.
u applied WI
PH
Linear =ge WI
Detection limit WI
+0.1 co.1 +0.1 +0.1
8 8 8 11
1 x 10*-l x 104 5 x 10-6-l x 104 5 x lOd-5 x 10‘5 no reaction
5x 10-7 1x10” 2x10* -
to.25 eo.2 +0.2
4 4 4
5 x 10-8-l x 10-5 1 x 10-6-l x 104 no reaction
5 x 10-B 5 x 10” -
465
compound (X) gives an &nodic reaction as suggested Cl9 ] it will therefore react differently. from compounds (VII), (VIII) and .(IX) with reactant and/or product absorption being’significantly dimimshed. Either this and/or the kinetics of the electrochemical reaction will influence this compound’s amenabiliQ7 to on-line analysis. The upper limits of the linear ranges of the 2-thiobarbituric acid derivatives are determined by their solubility in aqueous supporting electrolytes. Thioamides, existing as hydrochlorides, are rather soluble in these electrolytes but at high concentrations the electrode surface was found to be fouled resulting in a decreased signal and hence not useable for analytical purposes. The fouling can be due to the adsorption of the thioamides on the surface which partly can be a result of their chemical reaction with metallic mercury. AIternatively, the product of the electEochemiw.l reaction can adsorb on the surface and hence prevent thioamide molecules re&ching it. The upper limits of the linear ranges of the thioamides are concentrations where a constant signal is observed. The voltammetric flow-through cell can also be applied to cathodic stripping [ 231. In this study the compounds (VII), (VIII), (IX), (XI) and (XII) are investigated by an on-line cathodic stripping method, For the 2-thiobarbituric acid derivatives +0.15 V was taken as the plating potential and for the thioamides CO.2 V. In cathodic stripping the cell volume was increased to approximately 100 mm3 (fl). In this way the flow velocity near the surface was reduced allowing the products of electrolysis to be adsorbed on the mercury film. In the d-c. oxidation method it was more advantageous to have small cell volumes where the high flow velocity near the surface removed some of the electrochemical reaction products thus mechanically keeping the electrode surface clean. Results kom the cathodic stripping analysis are given in Table 4. As can be seen in Table 4 the thioamides do not plate on the thin mercury film in the flowing stream. This is obviously attributable to the inabiliw of the plated compound to adhere satisfactorily to the mercury film. It is postulated that although some electrolysis will take place the plated compound is swept away kom the surface by the stream. Plating was slightly improved by increas(ii) Cathodic stripping on a thin mercury film
TABLE Cathodic
4
strippingon the thin Hg-film
Compound
pH
Plating potential 09
stripping potential w
Linear =wze WI
M vm Ix XI XII
8 8 8 4-3 4-6
+0.15 +0.15 +0_15 +0.2 eo.2
-0.10 -0.65 4.63 -
5 x 106-S 1 x 10-7-1 5 x 10-7-2 no plating no plating
Detection limit WI x 10-s x lo* x 10-5
5x106 1 x 10-6 1 x 10-7 -
TABLE
5
Linear concentration ange for the determination of some sulphurcontaining organic compounds by static and on-line voltammetrir methods (oxidation processes used except wil* otherwise stated) Compound
Linear concentration range DSP. (static)*
C.S.V. (static)
dx. (on-line)
C.S.V. (on-line)
5,5’-Disubstituted 2thiobarbiturate(VII)
2 x lo’lxlo+M=
lXlO”1x10-M
1 x lo+lXlO-+hf
5 x 10-6: 5XlOSM
1,5,5’-Tkisubstituted 2thiobarbitwate(VIlI)
5 x lo*4 x 16+-Ma
2 x lO4lXl05M
5 x lo+lX104M
1 x lo-‘lxlO*M
1,3,5,5’-Tetrasubstituted I-thiobarbitwate(M j
5 x 10-64x10*hf=
1 x lo+6x10dM
5 x lO”5 x 10-5 M
5 x lo-‘2 x 10-s M
Primary thioamide
5 x lo-‘5xlO”M
5 x 10-aIO+ M
5 x lo-81~10-~M
-
a Reduction processes used.
ing the cell volume but even with large cell volumes reproducible results were not obtained. The 2-thiobarbituric acid derivatives did plate well on the thin mercury film. Compound (VIII) was found to plate most satisfactorily giving the smallest’ relative standard deviation and lowest detection Emit of the compounds studied. From the stripping potentials in Table 4 it can be seen that the products of electrolysis of compounds (VIII) and (IX) appear the same and from this potential value, HgS would appear to be the most probable product [27]. Compound (VII) behaves differently forming presumably a thiobarbiturate mercury salt.as . observed in quiescent solutions [28] _ Log I vs. log c plots of compounds (VII), (VIII) and (IX) gave slopes of 1.0,0.7 and 0.7 showing that plating of compounds (VIII) and (IX) is reproducible but not quantitative 1293. CONCLUSIONS
Hydrodynamic d-c. voltammetry has been successfully applied to the analysis of some 1,4benzodiiepines, 2-thiobarbituric acid derivatives and thioamides. An increase in sensitivity in most cases has been achieved mainIy due to the hydrodynamic nature of the fIowing stream as compared to vokunmetry in quiescent solution (Table 5). Several factors do influence the improved sensitivity. The diffusion layer adjacent to the electrode is reduced to a very narrow region allowing fast diffusion of the electroactive molecules to the elecfzode surface and hence an improved mass transport from the solution to the electrode is obtained, The greater surface area of a solid electrode when compared with a dropping mercury electrode leads to an additional increase in sensitivity. With application of a constant potential no current is needed to charge the double
467
layer decreas ing the background current and hence improving the sensitivity.
& demo-ted in this work such sulphur~ontaining compounds which form a mercury salt can be aualysed by on-line methods either by direct oxidation on the mercury film or by.cathodic stripping. Although a compound may give a well defined response when analysed in quiescent solution its analysis by on-line methods may fail as demon&at& with compounds (X) and (XIII) in the d-c. oxidation mode, Compounds (v]tr), (VIII) and (IX) could be analysed by on-line ca!hodic stripping whereas compounds (XI) and (XII) gave poor results although they have been analysecl successfully by cathodic stripping in rather complex matrices in quiescent soIutions 1271. All the studied compounds in general exhibit larger linear concentration ranges and lower detection limits with the proposed on-line methods than by analysis in quiescent solutions. The detection limit of compound (XI) with the on-line d-c. oxidation method is in fact sim&r to that of the cathodic stripping method in quiescent solution 127 1. It is believed that such on-line voltammetric studies will be of particular value in studying the interaction of drugs with biopolymers and their components by analysis of these complex matrices using the combination of high-performance liquid chromatography and electrochemical detectors.
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Zs. Feher. G. Nagy. EC.Toth and E. Pungor. Analyst