ISSN:0003-2654    

DOI:10.1039/AN97499FX021

出版商:RSC    

年代:1974

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Volume 99, No. 1179, Pages 313-384 June, 1974THE ANALYSTTHE JOURNAL OF THE SOCIETY FOR ANALYTICAL CHEMISTRYCONTENTSPageORIGINAL PAPERSA Direct Gas-chromatographic Method for the Determination o f BasicNitrogenous Drugs in Pharmaceutical Preparations-N. D. Greenwoodand I. W. Guppy . . . . . . . . . . . . . . . .A Method for the Determination o f Volatile Fatty Acids in the BloodPlasma o f Ruminant Animals-J. W. Gardner and G. E. Thompson . .Quantitative Determination o f the Enantiomeric Purity of SyntheticPyrethroids. Part II. S-Bioallethrin-F, E. Rickett and P. B. Henry . .The Detection and Determination o f Polynuclear Aromatic Hydrocarbonsby Luminescence Spectrometry Utilising the Shpol'skii Effect at 77 K-G. F. Kirkbright and C. G. de Lima .. . . .. . . . . . .Application o f the Spectrophotometric Determination of Nickel andCobalt in Mixtures With Bipyridylglyoxal Dithiosamicarbazone t o theAnalysis o f Catalysts-J. L. Baharnonde, D. PBrez Bendito and F. Pino . .Ionic Polymerisation as a Means o f End-point Indication in Non-aqueousThermometric Titrimetry. Part VI. The Determination o f Thiols-E. J. Greenhow and Miss L. H. Loo . . a . . . . . . . . .Determination o f Ammonia Levels in Water and Wastewater With anAmmonia Probe-W. H. Evans and 6. F. Partridge . . .. . .The Coulometric Determination o f Trace Levels of Sulphur in GalliumPhosphide-K. Gijsbers, L. Bastings and R. van de LeestBook Reviews . . .. . . . . .. . . . . . . . .Erratum . . . .. . . .- . . . . . . . . . . . . . . .. .353326330338356360367376381384Summaries o f Papers in this Issue ' . . . vi, vii, xii, xivPrinted for the Society for Analytical Chemistry by Heffers Printers Ltd., Cambridge, EnglandAll communicationsto beaddressedtothe Managing Editor,TheAnalyst, 9/10Savile Row, London, W l X l AFEnquiries about advertisements should bs addressed to J. Arthur Cook, 9 Lloyd Square, London, WC1X 9BAEntered as Second Class at New York, U.S.A., Post Offic

ISSN:0003-2654    

DOI:10.1039/AN97499BX023

出版商:RSC    

年代:1974

数据来源: RSC

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vi SUMMARIES OF PAPERS IN THIS ISSUE [June, 1974Summaries of Papers in this IssueA Direct Gas -chromatographic Method for the Determination of BasicNitrogenous Drugs in Pharmaceutical PreparationsA gas-chromatographic method has been developed for the direct assayof basic nitrogenous drugs, present as salts, in a range of pharmaceuticalpreparations. A glass column was packed with 3 per cent. silicone OV-17 onGas-Chrom Q, 80 to 100 mesh, and maintained isothermally a t temperatureswithin the range 200 to 270 "C, as appropriate for the drug to be determined.Calibration data are given for eighteen drugs, and the gas-chromatographicprocedures have been subjected to a statistical evaluation. Five determina-tions performed on each of eight standard pharmaceutical preparations gavecoefficients of variation of less than 2.2 per cent.The subsequent applica-tion of the method, based upon duplicate determinations, to a larger series ofstandard pharmaceutical preparations gave recoveries within the range 96 to104 per cent. of the known concentration of the test drug. The procedurecan now be used as part of a routine quality control specification in place ofa variety of non-specific classical methods.N. D. GREENWOOD and I. W. GUPPYRegional Quality Control Laboratory, Pharmacy Department, Leeds GeneralInfirmary, Great George Street, Leeds, LSl 3EX.Analyst, 1974, 99, 313-325.A Method for the Determination of Volatile Fatty Acids in the BloodPlasma of Ruminant AnimalsAn improved method for the determination of volatile fatty acids in theblood plasma of ruminant animals is described.The acids are extracted astheir sodium salts with isopropyl alcohol and then dissolved in 9 + 1 diethylether - formic acid for determination by gas - liquid chromatography.J. W. GARDNER and G. E. THOMPSONDepartment of Physiology, The Hannah Research Institute, Ayr, KA6 5HL,Scotland.Analyst, 1974, 99, 326-329.Quantitative Determination of the Enantiomeric Purity ofSynthetic PyrethroidsPart 11. S-BioallethrinS-Bioallethrin consists primarily of ( + )-allethronyl-( + )-trans-chrysanthe-mate, but technical samples contain small amounts of other allethrin isomers.The ratio of diastereoisomers can be measured directly from the nuclearmagnetic resonance spectrum after using a europium shift reagent, whenmany of the resonances split into two distinct diastereoisomer signals.If theenantiomeric purity of the chrysanthemate moiety is determined independently,then the absolute enantiomeric purity of the allethrin sample can be calcu-lated. Standard deviations for the measurement of laboratory or technicalsamples were between 0.3 and 0.7 per cent.The accuracy of the method was verified by independently determiningthe enantiomer ratios of various allethrolone samples by gas chromato-graphy of their diastereoisomeric ( - )-a-methoxy-a-trifluoromethylphenyl-acetic esters, esterifying with natural ( + ) -trans-chrysanthemic acid and re-determining the enantiomeric purity by nuclear magnetic resonance.Goodagreement was achieved between the two determinations.The nuclear magnetic resonance method should also be suitable formeasuring the diastereoisomer ratio of cis-allethrins.F. E. RICKETT and P. B. HENRYWellcome Research Laboratories (Berkhamsted), Berkhamsted Hill, Berkhamsted,Hertfordshire.Analyst, 1974, 99, 330-337June, 19741 SUMMARIES OF PAPERS I N THIS ISSUEThe Detection and Determination of Polynuclear AromaticHydrocarbons by Luminescence Spectrometry Utilising the Shpol’skiiEffect at 77 KThe luminescence emission spectra of twenty-three polynuclear aromatichydrocarbons (PAH) have been examined in n-alkane solvents a t 77 K. TheShpol’skii effect, in which narrow-band (quasi-linear) emission spectra areobtained under these conditions when a monochromator of adequate resolvingpower is used, is shown to be readily observed for twelve of the compoundsexamined in these solvents.Quasi-linear emission spectra have also beenobtained in tetrahydrofuran for some of the PAH compounds examined.These emission spectra provide for unambiguous qualitative identification ofPAH compounds a t trace concentrations in solution; this effect is demon-strated by identification of the compounds present in an eight-componentmixture of PAH compounds.Measurement of the low-temperature quasi-linear luminescence intensitycan be applied quantitatively to the determination of these compoundsprovided that a standard additions procedure is employed in conjunctionwith the use of an internal standard to ensure sufficient accuracy and precision.G.F. KIRKBRIGHT and C. G. de LIMAChemistry Department, Imperial College, London, S.W.7.Analyst, 1974, 99, 338-354.viiApplication of the Spectrophotometric Determination of Nickel andCobalt in Mixtures With Bipyridylglyoxal Dithiosemicarbazone tothe Analysis of CatalystsBipyridylglyoxal dithiosemicarbazone forms a complex with nickel( 11)a t pH 5.2, which can be extracted into chloroform (Amax. = 410 nm). Asimilar complex is obtained with cobalt(II), but is not extractable in thissolvent, thus allowing nickel and cobalt to be determined in mixtures. Twoprocedures are proposed for the accurate analysis of such mixtures in which1 p.p.m. of one of the ions can be determined accurately in the presenceof as much as 6 p.p.m. of the other. One of the procedures has been appliedto the determination of nickel and cobalt in industrial catalysts and theresults obtained have been compared with those obtained by atomic-absorp-tion spectrophotometry. Satisfactory results were obtained.J. L. BAHAMONDE, D. PEREZ BENDITO and F. PIN0Department of Analytical Chemistry, University of Seville, Seville, Spain.Analyst, 1974, 99, 355-359

ISSN:0003-2654    

DOI:10.1039/AN97499FP061

出版商:RSC    

年代:1974

数据来源: RSC

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xii SUMMARIES OF PAPERS I N THIS ISSUE [June, 1974Ionic Polymerisation as a Means of End-point Indication inNon- aqueous Thermometric TitrimetryPart VI. The Determination of ThiolsAlkyl and aryl thiols have been determined in the presence of carboxylicacids and phenols by means of acid - base catalytic thermometric titrimetry.Two titrations are carried out, with acrylonitrile and acetone as the end-point indicators. With the former indicator, thiol groups are not deter-mined, so that the difference between the titration values obtained by usingthe two methods of end-point indication is a measure of the thiol content.The thiol content of 2-mercaptothiazoline, 4,6-dihydroxypyrimidine-2-thiol (2-thiobarbituric acid), purine-6-thiol and 2-mercaptobenzimidazole canbe determined by the same procedure.In the titration of 2-thiohydantoin,4-hydroxypyrimidine-2-thiol (2-thiouracil), 2-mercaptobenzoxazole and2-mercaptobenzothiazole, however, both end-point methods give the sametitration value. These apparently anomalous results can be explained if itis accepted that the last four heterocyclic thiols exist in the thione tautomericform in dimethylformamide solution. Some thioamides also titrate as acids,and differences between titration values obtained by using the two methodsof end-point indication can again be attributed to thione - thiol tautomerism.Thiols can be determined conveniently in amounts down to 0.01 mequiv,ie., about 2 mg of dodecane-1-thiol, with 0.1 M titrants. In instances whenthe acrylonitrile method can be used for the direct determination of the thiolfunction, 0.001 M titrant can be used and the lower level of determination isthen about 0.0001 mequiv.E.J. GREENHOW and Miss L. H. LOODepartment of Chemistry, Chelsea College, University of London, Manresa Road,London, S.W.3.Analyst, 1974, 99, 360-366.Determination of Ammonia Levels in Water and Wastewater Withan Ammonia ProbeThe application of an ammonia probe has been investigated for discretelaboratory measurement of ammonia levels in a variety of waters. The probedisplays a Nernstian response for the range 0.2 to 40 mg 1-1 of ammoniacalnitrogen in a stirred 0.1 M sodium hydroxide solution containing 0.01 Methylenediaminetetraacetic acid. Recoveries of added ammonia from awide range of water samples are satisfactory.Both these recoveries of addedammonia and repeated calibrations of the probe suggest a precision of 4 percent. for ammoniacal nitrogen concentrations greater than 0.4 mg 1-1 and0-015 mg 1-1 for concentrations less than 0-4 mg 1-l; the statistical limit ofdetection is 0-03 mg 1-l. Good agreement is obtained with existing methodsbased on distillation and spectrophotometric measurement for a furtherrange of samples, but the limit of detection and the precision a t low levelssuggest that accurate determination in potable waters would be difficult.The probe can also be used to determine albuminoid nitrogen by takingthe difference between the ammoniacal nitrogen and the total free plusalbuminoid nitrogen obtained by distillation.Values obtained in this wayagree with those obtained by existing methods subject to the precision of theprobe being acceptable.W. H. EVANS and B. F. PARTRIDGEDepartment of Trade and Industry, Laboratory of the Government Chemist,Cornwall House, Stamford Street, London, SEl 9NQ.Analyst, 1974, 99, 367-375XiV SUMMARIES OF PAPERS I N THIS ISSUE [June, 1974The Coulometric Determination of Trace Levels of Sulphur in GalliumPhosphideSulphur present in gallium phosphide has been determined. At 950 "Cgallium phosphide reacts with platinum and hydrogen to form Ga,Pt, andplatinum phosphide (PtP,) , and sulphur is catalytically converted intohydrogen sulphide. The latter is absorbed in an alkaline medium and deter-mined by controlled-potential coulometry at a silver-gauze electrode. Thedetermination of 0.05 pg of sulphur is shown to be possible, the blank valueby this method amounting t o 0.01 pg of sulphur.K. GIJSBERS, L. BASTINGS and R. van de LEESTPhilips Research Laboratories, Eindhoven, The Netherlands.Analyst, 1974, 99, 376-380

ISSN:0003-2654    

DOI:10.1039/AN97499BP067

出版商:RSC    

年代:1974

数据来源: RSC

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JUNE, 1974 THE ANALYST Vol. 99, No. 1179 A Direct Gas-chromatographic Method for the Determination of Basic Nitrogenous Drugs in Pharmaceutical Preparations BY N. D. GREENWOOD AND I. W. GUPPY (Regional Quality Control Laboratory, Pharmacy De$artment, Leeds General Infirmary, A gas-chromatographic method has been developed for the direct assay of basic nitrogenous drugs, present as salts, in a range of pharmaceutical preparations. A glass column was packed with 3 per cent. silicone OV-17 on Gas-Chrom Q, 80 to 100 mesh, and maintained isothermally at temperatures within the range 200 to 270 “C, as appropriate for the drug to be determined. Calibration data are given for eighteen drugs, and the gas-chromatographic procedures have been subjected to a statistical evaluation. Five determina- tions performed on each of eight standard pharmaceutical preparations gave coefficients of variation of less than 2.2 per cent.The subsequent applica- tion of the method, based upon duplicate determinations, to a larger series of standard pharmaceutical preparations gave recoveries within the range 96 to 104 per cent. of the known concentration of the test drug. The procedure can now be used as part of a routine quality control specification in place of a variety of non-specific classical methods. Great George Street, Leeds, LS1 3EX) THE assay of basic nitrogenous drugs confronts the pharmaceutical analyst with a wide range of chemical and physico-chemical methods. Such methods include non-aqueous titrati~n,l-~ total nitrogen determination2p6 and ultraviolet spectroph~tometry,~,~ all of which are open to the criticism of being non-specific towards a given drug, although they are included in [several current pharmac~poeias.~s&~~ The tetraphenylboron precipitation method of Johnson and King14 and Bonnard,15 has been applied to the assay of alkaloids in eye drops16J7 and is currently specified in the B.P.C.18 Other non-specific titrimetric m e t h o d ~ ~ s ~ ~ - ~ ~ can also be used in the assay of basic nitrogenous drugs, and again several such methods are specified in the various pharma~opoeias.~J2 Gas - liquid chromatography permits a standard procedure to be applied to the majority of preparations, which is far more specific than alternative “classical” methods.The examination by gas - liquid chromatography of many basic nitrogenous drug substances (in an organic solvent) has been reported in the literat~re,~~-~’ and it has become a standard method for their determination in biological r n a t e r i a l ~ .~ ~ - ~ ~ These drugs are conventionally chromatographed as the free bases after extraction (if necessary) from alkaline solution into a suitable organic solvent .23~~69~9J~,46--50 Gas chromatography is by now a well established technique in pharmaceutical anaIysi~~~s~6,~~--6* and its applications within a hospital pharmaceutical laboratory have been d i s c u s ~ e d . ~ ~ - ~ ~ A direct approach will often be indicated for the examination of basic nitro- genous drugs as they are normally present in pharmaceutical preparations as the stable salt of a mineral acid in aqueous solution.The direct injection of the salts of basic nitrogenous drugs has been r e p ~ r t e d ~ ~ ~ ~ ~ , ~ ~ (see also Greenwood, N. D., unpublished work) and would be expected to result in the on- column liberation of the corresponding free bases, either by thermal di~sociation,3~~~6,68 or on account of the natural basicity of the support.23 Many reports regarding injection as the salts are of a qualitative nature, although some quantitative results have been reported, notably by Koehler and Hefferren,23 who advocated a direct method for the determination of a range of local anaesthetics in pharmaceutical preparations, and Rader and Aranda,34 who examined a wide range of drugs. Umbreit, Nygren and Testa71 evaluated methods for the direct determination of trace amounts of the salts of several aliphatic amines in water samples.313 @ SAC and the authors.314 GREENWOOD AND GUPPY: DIRECT GAS CHROMATOGRAPHY OF BASIC [Analyst, Vol. 99 The typical adsorption problems that are associated with basic nitrogenous compounds in genera129171-80 may be encountered with these drugs, leading to the phenomena of g h ~ s t i n g ~ ~ p ~ ~ , ~ ~ and t a i l i ~ g . ~ ~ - ~ ~ These effects can be counteracted by various means, including silanisation of the s u p p ~ r t , ~ ~ ~ ~ modification of the support with alkali,26,29s47s66s65166171--74184188189 the use of an alkaline pre-co1umn,65J1~90-92 the coating of a low loading of stationary phase on to glass or the preparation of volatile derivati~es.~~1~~J’6 This last approach was rejected, because it could involve complex and time-consuming procedures.The use of a very inert support such as Gas-Chrom Q (Applied Science Laboratories) would be expected to minimise the adsorption effects, and Alber67 has advocated the use of 3.0 per cent. silicone OV-17 on this support as an “all purpose gas - liquid chromatographic column for pharmaceuticals.” In a series of chromatograms, the resolution of a comprehensive range of drugs was illustrated, and the examples included a number of basic nitrogenous drugs, some of which were injected directly as a salt. Good peak symmetry was maintained even for those drugs with comparatively long retention times, but no quantitative results were reported, nor was the significance of this phenomenon discussed.A variety of stationary phases can be used in the analysis by gas - liquid chromato- graphy of basic nitrogenous ~ 0 m p 0 ~ n d ~ , ~ ~ ~ ~ ~ ~ ~ 8 ~ ~ 5 the most commonly reported being sili- cones.23-29~33s35s54157s76191 The increased thermal stability of the OV silicones over the estab- lished silicones such as SE-30 is obviously advantageous when dealing with comparatively involatile drugs; this stability is of particular significance when drugs such as codeine, morphine, papaverine and quinidine, which require high column temperatures in order to obtain symmetrical peaks within a reasonable retention time, are to be chromatographed on the same column as less complex drugs, such as lignocaine, pethidine and procaine. Even so, problems may be encountered with those drugs, such as ephedrine and fenfluramine, which require comparatively low column temperatures.Thus, columns such as that described by Alber67 and others97198 would appear to present an ideal choice, as they combine an OV silicone with a very inert support. That they are suitable was confirmed by a preliminary evaluation, in which almost perfect peak symmetry was observed from a wide range of drugs. A typical chromatogram is shown in Fig. 1, which illustrates the resolution of several local anaesthetics covering a range of structures. A Fig. 1. Mixture of local anaesthetics injected as the salts; A, %methocaine hydrochloride ; B, butacaine sulphate ; C, cinchocaine hydrochloride ; L, lignocaine hydrochloride monohydrate ; and P, procaine hydrochloride.Column temperature, 235 “C GLOSSARY OF TERMS- The following terminology is used throughout this paper. Standard solution-A solution containing a known concentration of the drug under test in aqueous solution (normally 1.0 per cent. mlV).June, 19741 NITROGENOUS DRUGS I N PHARMACEUTICAL PREPARATIONS 315 Internal marker solution-A solution containing a known concentration of the compound to be used as the internal marker in a given assay (normally 1.0 per cent. m/V). Reference solution-A solution containing an aliquot of the standard solution together with the fixed amount of the internal marker and diluted with water to the required volume (normally 25.0 ml). Dilution-The preliminary dilution of the sample (if necessary) prior to the preparation of the test solution.Test solution-A solution containing an aliquot of the sample under test, together with the fixed amount of the internal marker for a given assay, and diluted to the required volume with water (normally 25-0 ml). Standard Pharmaceutical preparation-A preparation prepared according to the formula usually used within this department, or as specified in a pharmacopoeia, so as to contain a known concentration of a test drug. EXPERIMENTAL MATERIALS- All of the drugs were to B.P. or B.P.C. specification as appropriate, with the exceptions of procaine hydrochloride and papaverine hydrochloride, which were of laboratory-reagent grade. The other constituents, including antibacterials, antioxidants and stabilisers, were of grades suitable for incorporation into pharmaceutical preparations.REAGENTS- For calibration purposes a standard 1.0 per cent. m/V aqueous solution of each drug was prepared and stored in a sealed amber-glass bottle for subsequent stability studies. The formulated products were prepared in accordance with the specification used in the manufacturing units within this hospital, normally B.P. or B.P.C., as set down in the relevant tables. GAS CHROMATOGRAPHY- A Pye Series 104 gas chromatograph, fitted with dual flame-ionisation detectors, was used in conjunction with a 10 mV full-scale deflection potentiometric recorder and an electronic integrator. The chromatograph included a wide-range amplifier module, thus enabling the integrator to be used to full advantage. A glass column, 1 m long by 4 mm internal diameter, was packed with 3 per cent.silicone OV-17 on Gas-Chrom Q, 80 to 100 mesh6' (Phase Separations Ltd.), which had been conditioned at 270 "C overnight. Argon was used as carrier gas at a flow-rate of 50 ml min-1. The detectors were maintained at the same temperature as the column, and the flame gases were hydrogen at 50 ml min-l and air at 600 ml min-l. Aliquots of each solution (of about 1 p1) were injected into a heated zone above the column packing, at a temperature about 50 "C higher than that of the column (corresponding to setting number 2 on the heater control). The column temperatures appropriate to each drug investigated in this study are listed in Table I. Only one column position was used at any given time; no balancing of column bleed rates was attempted, as a steady base-line was obtained at the attenuation settings that were employed during this work.CALIBRATION PROCEDURES- Aliquots (1, 2, 3, 5 , 7 and 10 ml) of the standard solution of the drug under test were pipetted into a series of 25-ml calibrated flasks and either 3.0 or 5.0 ml of the internal marker solution (equivalent to 30 or 50 mg) were added, the amount depending upon the detector response to the two compounds involved. It was necessary to substitute a 15-ml aliquot of the standard quinidine sulphate solution in place of the 1-ml aliquot. Details of the internal marker applicable to a given drug are listed in Table I. Similar calibrations were prepared for certain drugs which may also be present at concentrations below those covered by these graphs, namely atropine sulphate and hyoscine hydrobromide, by using solutions of the test drug and internal marker at concentrations of 0.1 per cent.316 GREENWOOD AND GUPPY: DIRECT GAS CHROMATOGRAPHY OF BASIC [Analyst, Vol.99 TABLE I SUMMARY OF INTERNAL MARKERS, GAS - LIQUID CHROMATOGRAPHIC CONDITIONS AND CALIBRATION DATA Volume of test solution = 25 ml Test drug Amethocaine hydrochloride . . .. Atropine sulphate . . .. .. Butacaine sulphate . . .. .. Cinchocaine hydrochloride . . .. Cocaine hydrochloride . . .. .. Codeine phosphate . . .. .. Eserine sulphate .. .. .. Hornatropine hydrobromide . . .. Hyoscine hydrobromide .. .. Morphine sulphate . . .. .. Oxybuprocaine hydrochloride . . .. Papaverine hydrochloride . . .. Papaverine sulphate .. .. .. Pethidine hydrochloride .. .. Pilocarpine hydrochloride . . .. Procaine hydrochloride . . .. .. Quinidine sulphate . . .. .. Lignocaine hydrochloride monohydrate Internal marker* r I d e n t i t y g B 50 B 50 Ci 50 B 30 D 30 Ci 50 Cm 30 D 30 Ci 50 Cm 50 Ci 50 P 30 Ci 50 Ci 50 Cm 50 B 50 D 30 Ci 50 Gas - liquid chromatographic column temperaturel'c 240 245 255 255 235 255 220 235 255 205 255 235 270 270 210 245 215 270 Linear range of the calibration 20 to 100 20 to 100 10 to 100 20 to 100 10 to 100 10 to 100 10 to 50 10 to 100 20 to 100 10 to 100 20 to 100 10 to 100 20 to 100 20 to 100 10 to 100 10 to 100 20 to 100 20 to 150 graph/mg * Added as a 1.0 per cent. m/V solution; 1 ml = 10 mg. Internal markers: B, butacaine sulphate; Ci, cinchocaine hydrochloride ; Cm, chlorpheniramine maleate ; D, diphenhydramine hydrochloride ; and P, procaine hydrochloride. The peak area ratio of the test drug to internal marker was then calculated. The method was calibrated in duplicate for each drug by plotting a graph of the peak area ratio versus its concentration, thus establishing the linear range applicable to a given drug (see Table I).Subsequently, only two reference solutions were injected alongside each test solution in order to define the slope of the line.99 Certain drugs, including atropine sulphate, eserine sulphate and hyoscine hydrobromide, decomposed o n - c o l u ~ n n , ~ ~ ~ ~ ~ thus giving rise to two or more major peaks, as illustrated in Fig. 2. Nevertheless, accurate calibration data could be obtained by using the summed areas of the peaks, although this on-column decomposition detracts from the ability of the Fig.2. Typical chromato- grams from drugs that exhibit on-column decomposition. A, atropine sulphate (column temperature, 245 "C) ; and B, eserine sulphate (column temperature, 220 "C)June, 19741 NITROGENOUS DRUGS I N PHARMACEUTICAL PREPARATIONS 317 method to detect breakdown products within the actual preparations. This phenomenon became exaggerated with atropine methonitrate, which gave several peaks, thus preventing accurate measurement of the peak areas. The analysis by gas - liquid chromatography of tropane alkaloids in general, has been reviewed by Achari and Newcornbeloo and the determination of atropine and hyoscine in pharmaceutical preparations is well documented.lol Ephedrine hydrochloride could not be satisfactorily chromatographed on this column because at lower temperatures excessive tailing was observed and, if the temperature was elevated, it was eluted on the tail of the solvent peak.Data relating to the gas-liquid chromatography of this drug are, however, particularly well documented in the litera- STATISTICAL EVALUATIONS- A series of reference solutions (normally ten) containing the drug under test (50 mg), together with the appropriate internal marker, was prepared for each of the three drugs cinchocaine hydrochloride, lignocaine hydrochloride monohydrate and procaine hydro- chloride. Fifteen aliquots (each about 1 pl) of one solution in each series, together with certain other test solutions, were injected in order to ascertain the precision that can be expected from the gas-liquid chromatographic system.The peak area ratio arising from each injection, and hence the over-all standard deviation (o), was calculated, a being defined thus: ture. 34,39,46,102-104 where X represents the sum of N individual peak area ratios. Two aliquots (of about 1 pl) of each solution were then injected and the average peak area ratio was calculated for each solution. Again, the standard deviation was calculated for the series of solutions in order to ascertain the over-all precision of the dilution and the gas - liquid chromatographic procedures. The calculations were performed on an Olivetti Programma 101 electronic desk computer. GENERAL PROCEDURE- An amount of the sample within the linear range of the calibration graph was added to a 25-ml calibrated flask.The internal marker was then added as a 1.0 per cent. m/V solution and the contents of the flask were diluted to the mark. Two aliquots of a standard solution of the drug under test were also diluted in a similar manner, so as to contain concentrations close to that of the sample; one of these aliquots would normally correspond to the concentration expected from the sample. Full details of the sampling procedure are given in Table 11. TABLE I1 SAMPLING INFORMATION REGARDING TYPICAL PHARMACEUTICAL PREPARATIONS Test drug Amethocaine hydrochloride . Atropine sulphate . . Butacaine sulphate . . Cinchocaine hydrochloride . Cocaine hydrochloride (plus adrenaline, 1 + 999) Preparation and standard* Eye drops, B.P.C.Pastilles, L.G.I. Topical solution, L.G.I. Eye drops, B.P.C. Injection, B.P. Eye drops, L.G.I. Jelly, L.G.I. Eye drops, B.P.C. Eye drops, L.G.I. Aliquot to be taken for the Concentration assaylml of the test drug, per cent. vn/V Sample Reference A -I 1.00 5.0 7.0, 5.0 6-00? t 7.0, 5.0 2.00 3.0 7.0, 5.0 1.00 5.0 7.0, 5-0 0.50 10.0 7.0, 5.0 0.125 5.05 7.0, 5-04 0.06 lO*O$ 7.0, 5-05 0.04 15.05 7.0, 5-09 0.50 10.0 7.0, 5.0 2.00.t 'IT 7-0, 5-0 4.00 10.0 to 50.0 8.0, 5.0 take 10.0 2-00 3.0 7.0, 5.0 5.00 5-0 to 50.0 7.0, 5.0 take 10.0318 GREENWOOD AND GUPPY: DIRECT GAS CHROMATOGRAPHY OF BASIC [Analyst, Vol. 99 TABLE II-continued Test drug Codeine phosphate . . .. Homatropine hydrobromide . . Hyoscine hydrobromide .. Lignocaine hydrochloride monohydrate .. .. Morphine sulphate . . .. Oxvbumocaine hvdrochloride ($hi fluoresce& sodium) Papaverine hydrochloride Papaverine sulphate . . Pethidine hydrochloride Pilocarpine hvdrochloride Procaine hydrochloride (plus adrenaline, 1 + 49999) . . .. Quinidine sulphate . . Comfiound 9vepaYations- .. .. .. .. .. .. .. .. Aliquot to be taken for the Concentration assaylml Preparation and of the test drug, A * standard* Injection, L.G.I. Eye drops, B.P.C. Eye drops, L.G.I. Eye drops, B.P.C. Injection, B.P. Topical solution, L.G.I. Injection, B.P. Eye drops, L.G.I. Solution, L.G.I. Isoprenaline spray co., B.P.C. Injection, B.P. Eye drops, B.P.C. Injection, L.G.I. Injection, L.G.I. Injection, B.P. Injection, L.G.I. homatropine hydrobromide Eye drops' B'P-C' Eye drops, B.P.C.Cocaine hydrochloride plus Cocaine hydrochloride plus homatropine hydrobromide per cent. m/V Sample 6.00 2.00 1-00 1.00 0.50 5.00 2-00 1.50 1.00 0-50 0.125 4.00 1-50 1.00 0.40 0.30 2-50 2-50 6.00 1.00 4.00 3.00 2-00 1-00 2.00 2.00 1.00 0.50 0.125 2.00 6.00 4.00 2.00 2.00 2.00 5.0 to 50.0 take 10.0 3.0 6.0 5.0 10.0 5.0 to 50.0 take 10.0 3.0 5.0 5.0 10.0 15.0 5.0 to 50.0 take 15.0 3.0 5.0 10.0 15.0 10.0 to 50.0 take 10.0 10.0 to 50.0 take 10.0 10.0 to 50.0 take 5.0 5.0 5.0 to 50.0 take 15.0 10.0 to 50-0 take 10.0 3.0 5.0 3.0 3.0 5-0 10.0 20.0 3-0 10.0 to 50.0 take 10.0 10.0 to 50.0 take 10.0 3.0 Reference 7.0, 5.0 7.0, 5.0 7.0, 5.0 7.0, 5.0 7.0. 5.0 7.0, 5.0 7-0, 5.0 8.0, 5.0 7-0, 5.0 7.0, 5.0 3.0, 2.0 7.0, 5.0 7.0, 5.0 7.0, 5.0 5-0, 3.0 5-0, 3.0 7.0, 5.0 7.0, 5-0 7.0, 5.0 7.0, 5-0 7.0, 5.0 7.0, 5.0 7-0, 5.0 7.0, 5.0 7-0, 5-0 7.0, 5.0 7-0, 5.0 7.0, 5-0 3.0, 2.0 7.0, 5.0 15.0, 10-0 * Where a preparation is not the subject of a monograph in the 1968 editions of the British Pharma- copoeia (B.P.) or British Pharmaceutical Codex (B.P.C.), the formula prepared within this hospital (L.G.I.) is given.t Solid and semi-solid preparations are expressed as a percentage m/m. The preliminary dilution for amethocaine hydrochloride pastilles is prepared by dissolving about 5 g in warm distilled water (about 40 ml) in a 100-ml calibrated flask. The solution is then cooled and diluted to the mark, and 15.0 ml are taken for the assay. 5 The concentrations of the internal marker and standard reference solutions are 0.10 per cent.m/V. 7 The test solution for cinchocaine hydrochloride jelly is prepared by transferring about 2-5 g of the sample to a 25-ml calibrated flask; the internal marker is then added, followed by distilled water to a volume of about 20 ml. The jelly is then dispersed throughout the solution, which is diluted to the mark, after the frothing has been allowed to subside. ** The reference solutions for the simultaneous determination of two drugs will contain both of the test drugs plus the internal marker. For example: cocaine hydrochloride . . . . 80mg homatropine hydrobromide , . 50 mg diphenhydramine hydrochloride . . 30 mg distilled water . . .. .. to 25mlJune, 19741 NITROGENOUS DRUGS I N PHARMACEUTICAL PREPARATIONS 319 Two aliquots (of about 1 p1) of each of the three solutions were then injected in a sequence that would rninirnise the effects of any short-term instability in the chromatographic system.The results were calculated from the following equationg9 to the full capacity of the laboratory calculator (floating decimal point) and subsequently rounded to the required number of decimal places (two or three). where C, is the concentration of the test drug in the sample, Rs, the peak area ratio resulting from the test solution, C, and C2 are the concentrations of the test drug in the two reference solutions, giving peak area ratios R, and R,, respectively, V is the volume of the test solution (normally 25.0 ml), A , the volume of the aliquot of the sample taken for the assay and D, the dilution of the sample (if any).Cs, C, and C2 can be in per cent., m/V or m/m as appropriate. A , V and D are all normally expressed in millilitres, except for solid or semi- solid preparations, when A is expressed in grams. EVALUATION OF THE METHOD- A series of eight typical pharmaceutical preparations, containing known concentrations of amethocaine hydrochloride, lignocaine hydrochloride monohydrate, oxybuprocaine hydro- chloride or procaine hydrochloride, was prepared, and five replicate determinations were performed on each preparation. The results from each analysis were then subjected to a statistical evaluation in order to ascertain the reliability of the method as a whole. The evaluation of the method was extended according to a procedure described previ- ously.99 Duplicate assays were performed on a number of typical pharmaceutical preparations containing known concentrations of the test drug.They were not sterilised, as this procedure could cause some decomposition. All of the assays were performed at room temperature with the exception of the eye drops containing oxybuprocaine hydrochloride (0.3 per cent. m/V) $,!us fluorescein sodium (0.125 per cent. m/V), which were sampled at 37 "C in order to avoid precipitation.105J06 Certain more complex formulations, notably oral medications, cannot be assayed by this direct method because they give rise to a large, tailing solvent peak. This effect is typified by Codeine Linctus B.P.C.,lo7 which contains 0.3 per cent. m/V of codeine phosphate. This drug is eluted on the solvent peak but accurate quantitative results could not be obtained by the measurement of peak areas or. by triangulation of the peak height measurements.A typical chromatogram is illustrated in Fig. 3. No results relating to such preparations are reported in this paper, but methods based on an extraction of the free base are documented in the l i t e r a t ~ r e . ~ ~ ~ 29933&34 SIMULTANEOUS DETERMINATION OF COMPOUND PREPARATIONS- Certain preparations include two (or more) basic nitrogenous drugs, and typical formula- tions were evaluated according to the standard procedure, by using reference solutions con- taining both compounds in admixture. As part of the calibration procedure, it was estab- lished that no interaction occurred between the two drugs by the examination of two addi- tional series of solutions containing various amounts of one drug in the presence of a fixed amount of the other.The application of the method to compound preparations is dependent upon two principal factors, namely the concentration of the two drugs, and their resolution by the gas - liquid chromatographic system. The selection of a compound that is eluted between the two test drugs represents the optimum choice as the internal marker. Although cocaine hydro- chloride and homatropine hydrochloride, which may be present together in certain eye drop formulations,lO* can be resolved by the gas - liquid chromatographic system at a temperature of 235 "C (Fig. 4), any significant reduction in the temperature results in excessive tailing of both peaks, thus precluding the use of an internal marker that is eluted with an intermediate retention time.Three compounds were evaluated in a preliminary series of experiments, but diphenhydramine, which is eluted before both drugs, was selected as the internal marker for their simultaneous assay as it gave the most reproducible results. The simultaneous determination of these two drugs as the free bases has been suggested previously.109320 GREENWOOD AND GUPPY: DIRECT GAS CHROMATOGRAPHY OF BASIC [Analyst, Vol. 99 Fig. 3. Typical c h r o m a t o g r a m following the direct injection of Codeine Linctus B.P.C. (di- luted 1 in 5 with water). Column temperature, 255 "C STABILITY OF THE REFERENCE SOLUTIONS- Fig. 4. Chromatogram obtained following the injec- tion of cocaine hydrochloride (C) plus homatropine hydro- bromide (H).Column temperature, 235 "C The use of freshly prepared standard solutions on each occasion is time consuming, especially in a laboratory that handles a large number of samples. In order to ascertain the feasibility of a monthly preparation regimen, a series of standard 1.0 per cent. m/V aqueous solutions of certain drugs (see Table VI) were stored in sealed, amber-glass bottles at room temperature for a period of at least 4 weeks. No antibacterial agents or chemical preserva- tives were added. Each stored solution was then examined by gas - liquid chromatography alongside a freshly prepared standard solution of the same drug. A 5.0-ml aliquot of each solution was diluted to 25.0 ml, together with the appropriate internal marker (see Table I), and two aliquots (of about 1 p1) of each dilution were injected into the chromatograph. The average peak area ratio was calculated for each solution, and the recovery of the test drug in the stored solution was calculated from the equation- Mean peak area ratio for stored solution Mean peak area ratio for fresh solution Recovery, per cent.= x 100 RESULTS AND DISCUSSION The chromatographic results confirm that excellent peak symmetry and good resolution of closely related structures can be expected, even from those drugs with comparatively long retention times (see Fig. 1). In general, peak tailing was absent, but certain drugs did exhibit a minimal degree of tailing, which did not adversely affect the accuracy or precision.Although the gas - liquid chromatographic column can be maintained at elevated tempera- tures (about 250 "C) for prolonged periods of time (1 to 2 months) it was found to be necessary to repack the initial portion of the column every 2 to 3 months. The entire column was repacked after 4 to 6 months, when indicated by a deterioration in peak symmetry and in the reproducibility of peak area ratios between duplicate injections of the same solution. Deterioration may be partly due to the effects of the acids that are liberated by the dissociation of the salts, although no adverse effects upon the flame-ionisation detector arising from this source110 have been observed.June, 19741 NITROGENOUS DRUGS I N PHARMACEUTICAL PREPARATIONS 32 1 The calibration data are summarised in Table I, and the linear working range covers test solutions at least containing between 20 and 100 mg of the test drug, with the exception of quinidine sulphate.The calibration graphs prepared for atropine sulphate and hyoscine hydrobromide, based upon the 0.1 per cent. m/V solutions, both gave a linear range from 1 to 10mg. Concentration Fig. 5 . involatile drugs Calibration graph typically obtained with Most of the drugs investigated in this study gave rise to the typical calibration graph of involatile compound~~~~99+1-~~~ shown in Fig. 5, in which an initial non-linear portion gives way to a wider linear working range, which, if produced, intersects the y-axis at some point other than the origin. The significance of this type of calibration graph has been discussed previouslygg and gives rise to the necessity to prepare two reference solutions.Once the linear working range for a given drug has been established, it is not necessary to construct a full graph during the routine application of the method, but merely to define the slope of the line. TABLE I11 STATISTICAL EVALUATIONS OF THE GAS-CHROMATOGRAPHIC PROCEDURES (a) Replicate injections of one solution- Coefficient of Number of Mean peak Standard variation, Test drug* injections area ratio deviation per cent. Atropine sulphate . . .. 10 1.078 0.054 5.01 Cinchocaine hydrochloride . . 15 1,741 0.029 1.67 Cocaine hydrochloridef . . .. 10 1.166 0.037 3-17 Homatropine hydrobromidef . . 10 0.984 0.018 1-83 Lignocaine hydrochloride monohydrate .. .. .. 15 1.172 0.009 0.77 Procaine hydrochloride . . .. 15 1.086 0.038 3.50 (b) Dzlplicate injections of several solutions- Coefficient of Number of Mean peak Standard variation, Test drug* injections area ratio$ deviations per cent. Cinchocaine hydrochloride . . 8 1.745 0.01 3 0.74 Lignocaine hydrochloride monohydrate . . .. .. 10 1.180 0.022 1.86 Procaine hydrochloride . . .. 10 1.089 0.020 1.84 * Internal markers as specified in Table I. t Determined simultaneously. 1 Mean of the individual mean peak area ratios from each solution. 3 Applied to the mean peak area ratios from each solution.322 GREENWOOD AND GUPPY: DIRECT GAS CHROMATOGRAPHY OF BASIC [Analyst, Vol. 99 The statistical evaluations of the gas - liquid chromatographic system and the sampling techniques are summarised in Table 111.As might be expected, atropine sulphate, which decomposed on-column, gave a higher coefficient of variation than the other drugs not demonstrating this effect. The data resulting from the application of the full method to representative series of determinations, which are reported in Table IV, indicate that a high degree of accuracy and precision can be expected. The results from the two concentrations of amethocaine hydrochloride in eye drops confirm that the procedure will readily differentiate between preparations containing similar concentrations of the same drug. Coefficients of variation of about 2 per cent. or less were obtained from all these preparations. TABLE IV STATISTICAL EVALUATION OF THE METHOD AS APPLIED TO TYPICAL PHARMACEUTICAL PREPARATIONS Concentration of the test drug, per cent.m/V & Test drug and preparation Added Found* Coefficient of variation, per cent. Standard error of the meant Standard deviationt Amethocaine hydrochloride eye drops, B.P.C. 1968 .. .. .. .. .. .. Amethocaine hydrochloride eye drops, B.P.C. 1968 .. .. .. . . .. .. Lignocaine hydrochloride monohydrate injec- tion, B.P. 1968 .. .. .. .. Lignocaine hydrochloride monohydrate inj ec- tion, B.P. 1968 .. .. Lignocaine hydrochloride monohydrate injec- tion, B.P. 1968 (plus adrenaline, 1 + 99 999) Oxybuprocaine hydrochloride eye drops . . Oxybuprocaine hydrochloride eye drops (plus fluorescein sodium, 0.125 per cent. %/V) . . Procaine hydrochloride injection, U.S.P. (1970) .. .. 0.96 0.96 0.0 12 0*005 1.26 1-10 1.09 0.024 0.011 2.20 0.60 0.60 0.009 0.004 1-80 6.10 5-17 0.053 0.024 1-03 2.00 1.96 0.41 0.40 0.026 0.005 0.012 0.002 1.33 1-26 0.30 0.30 1.05 1.05 0.006 0.008 0.003 0.004 2-00 0.76 * Mean of five determinations.t Defined in the text. $ Standard error of the mean is defined thus: S.E.M. = 5. d N A detailed comparison of the gas - liquid chromatographic procedure with alternative methods was not undertaken as part of the investigation, because chromatography is validated by the statistical data and the results obtained from its subsequent application to typical analyses, which are summarised in Table V. Recoveries within the range from 96 to 104 per cent. of the amount of test drug known to be present were obtained during the application of the method to a wide range of drugs, including the simultaneous assay of cocaine hydrochloride and homatropine hydrobromide.These recoveries are similar to those reported previously by Rader and Aranda.34 Eye drops containing eserine sulphate, which decomposes on-column, gave variable recoveries, but a gas-chromatographic method of assay for this drug as the trimethylsilyl derivative has been described previou~ly.1~~ The preparations containing atropine and hyoscine yielded good quantitative results. Certain drugs among those which required a comparatively high column temperature, such as morphine sulphate and papaverine hydro- chloride, did not yield reproducible recoveries within the set limits and consequently no results for them are reported in Table V.Similarly, papaverine and quinidine sulphates gave variable results, but the effect was not so marked. No explanation for this phenomenon is evident, but repeated assays on the standard preparations gave varied recoveries over an unacceptably wide range. The salts of papaverine gave over-all borderline results, and the method cannot be regarded as being satisfactory for this drug. The stability studies on the standard reference solutions of certain drugs, which are reported in Table VI, confirm that, in general, the replacement of these solutions at monthly intervals is justified. It is axiomatic that the raw materials to be used in the preparation of these reference solutions should be subjected to assay by an appropriate method, in order to ascertain accurately their purity.June, 19741 NITROGENOUS DRUGS IN PHARMACEUTICAL PREPARATIONS TABLE V SUMMARY OF RESULTS FROM THE APPLICATION OF THE GAS - LIQUID CHROMATOGRAPHIC METHOD TO A SERIES OF TYPICAL PHARMACEUTICAL PREPARATIONS 323 Results by gas - liquid chromatography: Concentration , L 3 Test drug Amethocaine hydrochloride Atropine sulphate .. Butacaine sulphate . . Cinchocaine hydrochloride Cocaine hydrochloride . . (plus adrenaline, 1 + 999) Codeine phosphate . . Homatropine hydrobromide Hyoscine hydrobromide .. .. .. .. .. .. . . .. .. .. .. .. .. ,. .. .. .. .. Lignocaine hydrochloride monohydrate Papaverine sulphate . . * . .. Pethidine hydrochloride .. .. Pilocarpine hydrochloride . . .. Procaine hydrochloride . . .. .. Qumidine sulphate . . .. .. Cocaine hydrochloride .. .. .. Cocaine hydrochloride . . .. .. (pZus adrenaline, 1 + 49 999) .. Contpound pyepavations- (plus hornatropine hydrobromide) . . (plus hornatropine hydrobromide) . . Preparation and standard* Pastilles, 1 g, Topical solution, Eye drops, B.P.C. Eye drops, B.P.C. Eye drops, B.P.C. Injection, B.P. Eye drops, L.G.I. Jelly, L.G.I. Eye drops, B.P.C. Eye drops, L.G.I. Injection, L.G.I. Eye drops, B.P.C. Eye drops, B.P.C. Eye drops, L.G.I. Injection, B.P. Topical solution, L.G.I. Isoprenaline spray co., B.P.C. Injection. B.P. Eye drops, B.P.C. Eye drops, B.P.C. Eye drops, B.P.C. Injection, L.G.I. Iniection, L.G.I. L.G.I. L.G.I. Injection; L.G.I. Iniection. B.P. Injection; L.G.I. Eye drops, B.P.C. Eye drops, B.P.C. of the test drug, per cent. Concentration, w v per cent.m/V 6.877 2.00 1.00 0.60 0-126 0.06 0.60 4.05 5.00 6.00 2-10 1-05 0.60 1.60 4.00 2-60 5-00 4.00 3.00 1.00 2-00 0.60 0.125 2.00 6.07 2.00t 4-00 1.96 2.00 2.06 6.78, 6-90 1.98, 1.98 1.02, 1.04 0.52, 0-62 0.121, 0.128 0.059, 0.064 0.51, 0.51 2.04, 2.06 4.12, 4-06 4.89, 4.99 6.08, 6-00 2.06, 2.09 1.04, 1.04 0.50, 0.61 1.54, 1.63 4.04. 4-06 2-66, 2.68 6.14, 6-05 3-98, 3.86 2-96, 2-86 0.99, 1.02 1.96, 1.97 0.48, 0.48 0.124, 0.123 1.96 6.24, 6.84 3-93, 3.82 1.93, 1-87 1.96, 1.96 2.12. 2-08 Average recovery, per cent. 99.6 99.0 103-0 104.0 99.6 102.6 102.0 102.6 101.0 98.8 100.7 98.8 99.1 101.0 102.3 101.3 102.6 101.9 98.0 97.0 100.6 98.3 96.0 98.8 97-6 99-6 96.9 97.4 98.0 102.4 *, t Defined in the footnotes to Table 11. $ In general, the assays were performed in duplicate, the recoveries being based upon the average of the two determinations.TABLE VI SUMMARY OF RESULTS FROM THE STABILITY STUDIES ON THE STANDARD DRUG SOLUTIONS Length of time under Test drug storagelweeks Atropine sulphate . . . . Cinchocaine hydrochloride , . Cocaine hydrochloride . . .. Codeine phosphate . . .. Homatropine hydrobromide . . Hyoscine hydrobromide .. Morphine sulphate . . ,. Papaverine hydrochloride . . Pilocarpine hydrochloride . . Quinidine sulphate . . .. 8 6 8 7 9 6 7 7 9 8 Recovery, as compared with a freshly prepared solution, per cent. 99.4 100-4 99-6 99.8 101.9 98.9 101.3 98.4 100.9 103.0324 GREENWOOD AND GUPPY: DIRECT GAS CHROMATOGRAPHY OF BASIC [Anahst, VOl. 99 CONCLUSIONS A statistical evaluation of the gas-chromatographic method has shown that satisfactory results can be obtained.This was confirmed during the subsequent application of the method to a series of typical pharmaceutical preparations that contain a number of basic nitrogenous drugs as salts. The method has now been in routine use for over 18 months and has proved to be reliable and convenient. The authors are grateful to all their colleagues who have assisted in various aspects of the study, particularly Mr. C. 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Abstr., 1969, 70, Calo, A., Cardini, C., and Quercia, V., Boll. Chim.-Farm., 1969, 108, 175. Cardini, C., Bucci, B. T., and Calo, A., Ibid., 1969, 108, 180. Drug Houses Ltd., Poole, Dorset, 1961. 1964, p. 700. Part I, The Athlone Press, London, 1968, p. 146. -, oP. cit., p. 792. Part 11, The Athlone Press, London, 1970, p. 192. Part I, The Athlone Press, London, 1968, p. 225. Amer.Chem. SOC., 1960, 82, 3791. 71111.June, 19741 NITROGENOUS DRUGS I N PHARMACEUTICAL PREPARATIONS 325 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. Zimmerer, R. O., and Grady, L. T., J . Pharm. Sci., 1970, 59, 87. Brochmann-Hanssen, E., Ibid., 1962, 51, 1017. Hishta, C., and Lauback, R. G., Ibid., 1969, 58, 745. Solomon, M. J., Crane, F. A., Wu-Chu, B. L., and Mika, E. S., Ibid., 1969, 58, 264. Houtman, R. L., Ibid., 1968, 57, 1975. Molina, J., and Poe, R. D., J . Pharm. Pharmac., 1968, 20, 481. Boon, P. F. G., and Sudds, W., Ibid., 1967, 19, 88s.Cometti, A., Bagnasco, G., and Maggi, N., J . Pharm. Sci., 1971, 60, 1074. Quercia, V., Merli, F., and Boniforti, L., Boll. Laboratori Chim. Prov., 1968, 19, 457. Brochmann-Hanssen, E., and Svendsen, A. B., Pharmazie, 1965, 20, 591. Paris, M., Produits Probl. Pharm., 1964, 19, 281. Kuleshova, M. I., Rudenko, B. A., and Ryabtseva, I. M., Farmatsiya, 1969, 18, 80. Patel, J. A., Amer. J . Hosp. Pharm., 1970, 27, 411. Greenwood, N. D., “Gas Chromatography in the Analysis of Pharmaceutical Preparations,” presented at the Chemical Society/Society for Analytical Chemistry Symposium, “Analysis 72,” Imperial College, London, September 1972. -, Scan, 1973, 3, 5. Thompson, G. F., and Smith, K., Analyt. Chem., 1965, 37, 1591. Fontan, C. R., Smith, W. C., and Kirk, P.L., Ibid., 1963, 35, 591. Alber, L. L., J . Ass. Off. Analyt. Chem., 1969, 52, 1295. Vessman, J., Acta Pharm. Suec., 1964, 1, 183. MacDonald, A., jun., and Pflaum, R. T., J . Pharm. Sci., 1964, 53, 887. “Perkin-Elmer Gas Chromatography Application,” No. GC-DS-020, Perkin-Elmer Corporation, Umbreit, G. R., Nygren, R. E., and Testa, A. J., J . Chromat., 1969, 43, 25. Cincotta, J. J., and Feinland, R., Analyt. Chem., 1962, 34, 774. Smith, E. D., and Radford, R. D., Ibid., 1961, 33, 1160. Metcalfe, L. D., and Schmitz, A. A., J . Gas Chromat., 1964, 2, 15. Hantzsch, S., Ibid., 1968, 6, 228. Vanden-Heuvel, W. J. A., Gardiner, W. L., and Horning, E. C., Analyt. Chem., 1964, 36, 1550. Di Lorenzo, A., and Russo, G., J . Gas Chromat., 1968, 6, 509. Ives, N. F., and Giuffrida, L., J .Ass. 08. Analyt. Chem., 1970, 53, 973. Palframan, J. F., and Walker, E. A., Analyst, 1967, 92, 71. Ottenstein, D. M., J . Gas Chromat., 1963, 1, 11. Simonaitis, R. A., and Guvernator, G. C., 111, Ibid., 1967, 5, 49. O’Donnell, J . F., and Mann, C. K., Analyt. Chem., 1964, 36, 2097. Hantzsch, S., Talanta, 1966, 13, 1297. Knight, H. S., Analyt. Chem., 1958, 30, 2030. Horning, E. C., Moscatelli, E. A., and Sweely, L. L., Chem. & Ind., 1959, 751. Reiser, R. W., Analyt. Chem., 1964, 36, 96. Bohemen, J., Langer, S. H., Perrett, R. H., and Purnell, J. H., J . Chem. Soc., 1960, 2444. Nelson, J., and Milun, A., Chem. 6 Ind., 1960, 663. James, A. T., Analyt. Chem., 1956, 28, 1564. Peterson, P. E., and Tao, E. V. P., J . Org. Chem., 1964, 29, 2322. James, A. T., Martin, A. J. P., and Smith, G. H., Biochem. J., 1952, 52, 238. Hishta, C., and Bomstein, J., Analyt. Chem., 1963, 35, 924. Hishta, C., Messerly, J. P., Reschke, R. F., Fredericks, D. H., and Cooke, W. D., Ibid., 1960, Filbert, A. M., and Hair, M. L., J . Gas Chromat., 1968, 6, 150. Vessman, J., and Schill, G., Svensk. Farm. Tidskr., 1962, 25, 601. Anthony, G. M., Brooks, C. J. W., and Middleditch, B. S., J . Pharm. Pharmac., 1970, 22, 205. Stromberg, L. E., J . Chromat., 1971, 63, 391. Gas Chromat. Newsl., 1973, 14, 1. Greenwood, N. D., J . Hosp. Pharm., 1972, 30, 196. Achari, R., and Newcombe, F., Planta Med., 1971, 19, 241. Nieminen, E., Farm. Aikak., 1971, 80, 263. Nanikawa, R., Kotokus, S., Sasaki, H., and Sakaguchi, S., Nippon Hoigaku Zasshi, 1968, 22, 16; Elefant, M., Chafetz, L., and Talmage, J. M., J . Pharm. Sci., 1967, 56, 1181. Wesselman, H. J., and Koch, W. L., Ibid., 1968, 57, 845. Fenton, P. J., BY. J . Ophthal., 1965, 49, 205. -, Ibid., 1965, 49, 504. “British Pharmaceutical Codex 1973,” The Pharmaceutical Press, London, 1973, p. 722. “British Pharmaceutical Codex 1973,” The Pharmaceutical Press, London, 1973, p. 690. Greenwood, N. D., J . Hosp. Pharm., 1971, 29, 240. Gough, T. A., and Walker, E. A., Analyst, 1970, 95, 1. Vollmer, K. O., and Zinnermann, F., Arzneimittel-Forsch., 1970, 20, 995. Gudzinowicz, B. J., and Clark, S. J., J . Gas Chromat., 1965, 3, 147. Greenwood, N. D., Pharm. J., 1973, 210, 431. Teare, F. W., and Borst, S. I., J . Pharm. Pharmac., 1969, 21, 277. Nonvalk, Connecticut, U.S.A., December 1964. 32, 880. Chem. Abstr., 1969, 70, 90785. Received Sefitember 14th, 1973 Accepted December 17th, 1973

ISSN:0003-2654    

DOI:10.1039/AN9749900313

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

326 Analyst, June, 1974, Vol. 99, @. 326-329 A Method for the Determination of Volatile Fatty Acids in the Blood Plasma of Ruminant Animals BY J. W. GARDNER AND G. E. THOMPSON (Department of Physiology, The Hannah Research Institute, Ayr, KA 6 5HL, Scotland) An improved method for the determination of volatile fatty acids in the blood plasma of ruminant animals is described. The acids are extracted as their sodium salts with isopropyl alcohol and then dissolved in 9 + 1 diethyl ether - formic acid for determination by gas - liquid chromatography. THE accurate determination of volatile fatty acids in human or animal blood is difficult to achieve and has been attempted by a variety of methods.1-9 Many of these methods have involved the use of steam distillation, but it has been shown that steam distillation of the small amounts involved here is an irreproducible process and that considerable losses may occur.10 In the present work it has been found possible to extract the volatile fatty acids from plasma, as their sodium salts, with isopropyl alcohol, thereby eliminating the need for steam distillation.Previous techniques for the determination of the acids after extraction have used either paper or gas - liquid chromatography. The paper-chromatographic methods have depended on the production of a coloured derivative of the acids, which can be determined by densito- metry. Tranger3 prepared the hydroxamates from volatile fatty acid methyl esters and determined them by measuring the intensity of the colour formed with iron(II1) chloride. Considerable losses were reported when using this method.The gas - liquid chromatographic analyses of volatile fatty acids have generally been carried out in dilute solutions of mineral acid, especially metaphosphoric acid, and it is very difficult to obtain good results with such methods.ll Ghosting1, and anomalous peak broadening13 have been observed, and poor peak shapes are frequently obtained.14 In order to overcome these difficulties, volatile fatty acid derivatives have been pro- duced and determined by gas - liquid chromatographic methods.l5J6 Preliminary efforts, made in this laboratory, to apply these techniques to the volatile fatty acids in blood have been unsuccessful, probably because the minute amounts of acids present make it difficult to convert all of the acid into the derivative.A simple way of overcoming the problem of the gas - liquid chromatographic analysis of solutions of volatile fatty acids is to inject them on to the column in an organic acid. Zerilli, Brambilla and Rimorinis have suggested 9 + 1 acetone - formic acid as a suitable solvent system. In the present work 9 + 1 diethyl ether - formic acid has been found to give sharper peaks and less solvent tailing. EXPERIMENTAL REAGENTS- Formic acid (Analar)-This acid was fractionally distilled from a 500-ml flask by using a 0-5 m x 20 mm lagged glass column, packed with Fenske helices and fitted with a partial take-off head. The first 40 ml of distillate collected were redistilled in semi-micro apparatus and the first 10 ml of distillate collected were again fractionally distilled in the same apparatus.The first 2 ml of this distillate were found to be pure on gas - liquid chromatographic analysis and contained no traces of acetic, propionic and butyric acid, which had been present in the original volume of formic acid. Isopropyl alcohol-Used after fractional distillation with a 1-m Vigreux column. Diethyl ether (May and Baker Ltd., Proaalys)-Used without further purification. Samples of acetic, propionic, isobutyric, butyric, isovaleric (3-methylbutanoic) and valeric acids were supplied by BDH Chemicals Ltd. and were of the highest purity available. n-Hexanoic acid (Sigma Chemical Co. Lid.)-This reagent was better than 99 per cent. pure, and gas - liquid chromatographic analysis indicated that it contained none of the C, to C, volatile fatty acids.As preliminary investigation had indicated that n-hexanoic acid was 0 SAC and the authors.GARDNER AND THOMPSON 327 not present in appreciable amounts in sheep or bovine plasma it was chosen as an internal standard for the method. Aqueous solutions of the acid were made up to contain lob2 mg ml-l and were used as described in the following section. METHOD SAMPLE EXTRACTION AND PREPARATION FOR GAS - LIQUID CHROMATOGRAPHY- Take a 10-ml sample of blood and centrifuge it at 3500 r.p.m. for 15 minutes. Transfer, by pipette, 6.0 ml of the supernatant plasma into another centrifuge tube and add 1.0 ml of the n-hexanoic acid standard solution. The volumes of sample and internal standard must be accurately measured.Adjust the mixture to pH 9 to 10 by adding 0.05 M sodium hydroxide solution dropwise, then add 40 ml of redistilled isopropyl alcohol and remove the protein precipitate by centrifugation at 3500 r.p.m. for 30 minutes. Decant the supernatant liquid and evaporate it to dryness on a rotary film evaporator, the final 1 ml of solution being taken to dryness in a small (10 ml) B14 test-tube. Seal the top of this tube with a layer of Para- film (Gallenkamp Co. Ltd.) and inject 200 p1 of freshly mixed 9 + 1 ether - formic acid (triple distilled) through the film with a syringe. Seal the resultant hole immediately with another layer of Parafilm. Agitate the contents of the tube vigorously on a Whirlimixer and allow them to stand for 15 minutes. Finally, withdraw 10 pl of the solution from the tube and inject it on to the gas - liquid chromatographic column.GAS CHROMATOGRAPHY- A Pye Unicam, Model 104, dual-column gas chromatograph, fitted with flame-ionisat ion detectors, was used, and separations were carried out by using glass columns that were 214 cm long by 6 mm o.d., packed with 17 per cent. neopentyl glycol adipate (Phase Separations Ltd.) in 3 per cent. orthophosphoric acid supported on Embacel (May and Baker Ltd.) of 60 to 100 mesh (R. S. Reid, personal communication). Condition the packed column at 150 "C over- night. For volatile fatty acid analysis use the following conditions : column temperature, 105 "C; detector temperature, 150 "C; and nitrogen flow-rate, 60 ml min-l. (Optimise the hydrogen and air flow-rates for maximum response.) Inject the mixture of acids directly on to the column packing.Under these conditions typical retention times in minutes of the acids are (retention times relative to n-hexanoic acid in parentheses) : acetic, 3.13 (0.095) ; propionic, 5.5 (0.167) ; isobutyric, 7.0 (0.212) ; butyric, 9.38 (0.284) ; isovaleric, 12.5 (0.379) ; valeric, 17.5 (0.53) ; and n-hexanoic, 33.0 (1.0). PREPARATION OF CALIBRATION GRAPH- Aqueous solutions of each of the C, to C, volatile fatty acids were prepared in a range of concentrations in the expected physiological ranges (Le., acetic acid from 1 to 4 mg per 100 ml and the other acids from 0.01 to 0-04 mg per 100 ml). These solutions were made up in cali- brated glassware and standard mixtures of the acids were prepared from them in order to calibrate the method.Known amounts of each mixture were then taken through the analytical procedure exactly as a plasma sample would have been. The peak areas given by the individual acids were measured relative to that of the internal standard. The use of peak height x width at half-height as an estimate of peak area was found to give the closest results to cutting and weighing the chart paper and the former method was used. RESULTS AND DISCUSSION- Fig. 1 shows the calibration graph obtained for acetic acid and Fig. 2 those for propionic, isobutyric, butyric, isovaleric and valeric acids. Each graph approximates to a straight line and it was possible to determine a calibration factor for each acid that was the slope of the line in the calibration graph.It was then possible to calculate the original concentration of each acid in every blood sample, knowing the area of each peak and the amount of internal standard that had been added, by applying the following equation: (1) Peak area of acid x mass of n-hexanoic acid Peak area of n-hexanoic acid x calibration factor Mass of acid = * * ' * A typical chromatogram of a sample of sheep plasma is shown in Fig. 3. Four experi- ments were carried out in order to test the completeness of recovery of known amounts of328 GARDNER AND THOMPSON : DETERMINATION OF VOLATILE FATTY ACIDS [Afialyst, Vol. 99 0 -0 0 m u .- .- 16 m 0 f 12 m Y m a --. 9 v 8 0 .w u .- m 4 L m Y m p. 0 0 10 20 30 40 Mass of acetic acidbmass (1 0-2 mg) of n-hexanoic acid j I I I I Fig.1. Calibration graph for determina- tion of acetic acid. Slope = 0.44 added volatile fatty acids from plasma. Four separate plasma samples were each divided into two equal portions and one portion of each was used to determine the amounts of the individual volatile fatty acids present initially. A measured amount of a standard aqueous mixture of the acids was added to the remaining portion of each plasma sample and the resulting solutions were analysed. It was then possible to calculate the recovery of each acid, knowing the initial and final amounts. Results are shown in Table I as the individual acids present before and after addition of the standard mixtures. I I I I 1 I I I 0 5 10 15 20 I ' 35 Ti me/ m i nutes Fig. 3. A typical chromatogram of a sample of sheep hepatic portal vein plasma showing: 1, acetic; 2, propionic; 3, isobutyric; 4, butyric; 5, isovaleric; 6, valeric; and 7, n-hexanoic acid peaks. Attenuation factors: 1, x 2000; 2, x 500; 3, x 100; 4, x200; 5, x 100; 6, x 50; and 7, x200 A blank performed on 10 ml of distilled water contained no detectable amounts of any of the C, to C, acids and the results can therefore be taken as a measure of the accuracy of the method.The mean recoveries all lie in the range 92 to 106 per cent. There are evidently no substantial losses, except of propionic acid, but in this instance the mean is probablyJune, 19741 I N THE BLOOD PLASMA OF RUMINANT ANIMALS TABLE I RECOVERY OF KNOWN AMOUNTS OF VOLATILE FATTY ACIDS ADDED TO PLASMA 329 Original amount . . Amount added .. Total amount recovered Recovery, per cent. . . Original amount . . Amount added . . Total amount recovered Recovery, per cent. . . Original amount . . Amount added . . Total amount recovered Recovery, per cent. . . Original amount . . Amount added . . Total amount recovered Recovery, per cent. . . Mean recovery, per cent. Standard deviation . , Acetic acid/mg 0-182 0.210 0.380 96.9 0.146 0.210 0.362 101.7 0.168 0.420 0.575 97.8 0.185 0.420 0.580 95.9 98-1 2.5 Propionic acid/mg 0.0037 0.0020 0.0058 101.7 0.00 17 0.0020 0.0035 94.6 0.0045 0.0041 0.0082 95.3 0.0049 0.0041 0.0070 77.8 92.3 10.3 Isobutyric acid/mg 0.0003 0.0020 0.0025 108.7 0~0002 0.0020 0.0024 109.0 0.0006 0.0039 0.004 1 91.1 0.0015 0.0039 0.0057 105.5 103.6 8.5 Butyric acid/mg 0-0027 0.0019 0.005 1 110.8 0.0012 0.0019 0.0029 0.0025 0.0039 0.0065 93.5 103.1 0.0017 0-0039 0*0060 107.2 103.7 7.5 Isovaleric acid/mg 0.0006 0.0019 0.0027 108.0 0*0003 0.0019 0.0024 109.0 0.0008 0.0037 0.0049 108.9 0.0008 0.0037 0-0044 97.8 105.9 5.4 Valeric acid/mg 0*0008 0.00 13 0.0018 85.7 0.0003 0-0013 0.0015 93.8 0.0006 0.0026 0.0035 109.4 0.0004 0.0026 0.0034 112.3 100.6 13.0 artificially low, owing to the final recovery figure of 77.8 per cent.The over-all accuracy of the method could possibly be increased by the use of an integrator for the measurement of peak area. CONCLUSION The method described in this paper for the determination of volatile fatty acids in plasma has been shown to be quantitative and reproducible for the peripheral blood of ruminants. It has the distinct advantage, over published methods involving steam distillation, of relative speed and good recoveries.The gas-chromatographic technique is accurate and reliable and, after several months of continuous use, there is no evidence of column deterioration or the build-up of unstable products. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. REFERENCES Annison, E. F., Biochem. J., 1954, 58, 671. Erwin, E. S., Marcos, G. J., and Emery, E. M., J . Dairy Sci., 1961, 44, 1768. Tranger, M., Vet. Archiv., 1963, 33, 248. Storry, J. E., and Millard, D., J . Sci. Fd Agric., 1965, 16, 417. Tanaka, K., Budd, M. A., Effron, M. L., and Isselbacker, K. I., Proc. Natn. A cad. Sci. U.S.A ., 1966, Kurtz, D. J., and Levy, M. L., Clinica Chim. Acta., 1971, 34, 463. Ross, J. P., and Kitts, W. D., J . Dairy Sci., 1971, 54, 1824. Mahadevan, V., and Zieve, L., J . Lipid Res., 1969, 10, 338. Zerilli, L. F., Brambilla, E., and Rimorini, N., Atti. Soc. ITtaZ. Sci. Vet., 1971, 25, 173. Edwards, G. B., McManus, W. R., and Bigham, M. C., J. Chromat., 1971, 63, 397. Holman, R. T., Editor, “Progress in the Chemistry of Fats and Other Lipids,” Volume 12, Pergamon Geddes, D. A. M., and Gilmour, M. N., J. Chromat. Sci., 1970, 8, 394. Ackman, R. G., Ibid., 1972, 10, 506. OttensJein, D. M., and Bartley, D. A., Ibid., 1971, 9, 673. Downing, D. T., and Greene, R. S., Analyt. Chem., 1968, 40, 827. Umeh, E. O., J. Chronmt., 1970, 51, 147. 56, 236. Press, Oxford, 1972, p. 258. Received October Sth, 1973 Accepted December 31st, 1973

ISSN:0003-2654    

DOI:10.1039/AN9749900326

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

330 Analyst, June, 1974, Vol. 99, PP. 330-337 Quantitative Determination of the Enantiomeric Purity of Synthetic Pyrethroids Part II.* S-Bioallethrin BY F. E. RICKETT AND P. B. HENRY (Wellcome Research Laboratories (Berkharnsted), Berkhamsted Hill, Berkhamsted, Hertfordshire) S-Bioallethrin consists primarily of ( + )-allethronyl-( + )-trans-chrysanthe- mate, but technical samples contain small amounts of other allethrin isomers. The ratio of diastereoisomers can be measured directly from the nuclear magnetic resonance spectrum after using a europium shift reagent, when many of the resonances split into two distinct diastereoisomer signals. If the enantiomeric purity of the chrysanthemate moiety is determined indepen- dently, then the absolute enantiomeric purity of the allethrin sample can be calculated.Standard deviations for the measurement of laboratory or technical samples were between 0.3 and 0.7 per cent. The accuracy of the method was verified by independently determining the enantiomer ratios of various allethrolone samples by gas chromato- graphy of their diastereoisomeric ( - )-a-methoxy-a-trifluoromethylphenyl- acetic esters, esterifying with natural ( + )-trans-chrysanthemic acid and re- determining the enantiomeric purity by nuclear magnetic resonance. Good agreement was achieved between the two determinations. The nuclear magnetic resonance method should also be suitable for measuring the diastereoisomer ratio of cis-allethrins. THE chrysanthemate allethrin, which is an insecticide, can occur in eight isomeric forms; the chrysanthemic acid moiety exhibits both optical and geometrical isomerism and the allethrolone moiety is optically active.This insecticide was introduced in 1949l as the racemic mixture of cis- and tyans- chrysanthemates, in the approximate ratio 25 : 75. Greater insecticidal activity has since been obtained by resolving the acid into the (+)-tram form (bi0allethrint)~-4 and the com- pound is now becoming commercially available with the allethrolone also resolved into the (+)- or S-enantiomer, under the name S-bioallethrin or Esbiol: [( +)-allethronyl-( +)-trans- chry sant hemat el. Polarimetry is a useful method for checking the composition of commercial S-bioallethrin samples as the isomer with the highest insecticidal activity also exhibits the highest negative rotation5 (the values originally published by LaForge, Green and Schechters9' have proved to be inaccurate).However, the method can give only an indication of the minimum amount of (+)-allethronyl-( +)-trans-chrysanthemate present in the sample without defining the other constituents. A more specific method of determining the enantiomeric purity is therefore desirable. An earlier paper8 described a method for determining the enantiomeric purity of cis or trans forms of the chrysanthemic acid moiety by hydrolysis of the allethrin and analysis by gas chromatography of the diastereoisomers formed by reaction of the (+)- and (-)-acids with (+)-or-methylbenzylamine. Analogous methods cannot be used for the allethrolone moiety because it is not possible to recover the free alcohol by hydrolysis of allethrin unless the semicarbazone is first ~repared.~ The method described here provides a means of directly measuring the ratio of diastereoisomers in laboratory and technical S-bioallethrin samples, by use of nuclear magnetic resonance spectroscopy, from which the enantiomeric purity of the allethrolone can readily be calculated.The nuclear magnetic resonance spectra of some natural cyclopropanes with lanthanide shift reagents has been described by Crombie, Findley and Whiting.lo * For details of Part I of this series, see reference list, p. 337. $ Registered trade name, Roussel Uclaf S.A., Romainville, France. @ SAC and the authors. Proposed B.S.I. approved name.RICKETT AND HENRY EXPERIMENTAL REAGENTS- 331 Bioallethrin- ( -J- ) -Allet hron yl- ( + ) -trans-chrysanthemat e, technical grade.S-Bioallethrin- ( + ) -Allet hronyl- ( + ) -trans-chrysant hemat e. Both labor at ory prepared and technical samples were used, the latter supplied by Roussel Uclaf S.A. under the trade name Esbiol. a-( &)-trans-Allethrin-A crystalline racemate of (-)-allethronyl-( +)-trans-chrysanthe- mate and (+)-allethronyl-( -)-trans-chrysanthemate, melting-point 50 to 51 "C, prepared by the method of Schechter, LaForge, Zimmerli and Thomas.11 S-Allethrolone-One sample was kindly supplied by Dr. M. Elliott of the Rothamsted Experimental Station, Harpenden, Herts. Other samples were prepared by hydrolysis of S-bioallethrin semicarbazones by using established procedure~l~s~~ and were blended with (&)-allethrolone (Benzol Products Inc., Edison, New Jersey).(+)-Pyrethrolone-This was supplied by Dr. M. Elliott of Rothamsted Experimental Stat ion. (-)-trans-Chrysanthemoyl chloride-Boiling-point 82 "C (0.5 mm Hg) ; [a]i2 - 24.6" (14.5 per cent. in 2,2,4-trimethylpentane). The chloride was prepared from (+)-trans- chrysanthemic acid that had been obtained by hydrolysis of pyrethrum extract.14 Shift reagent, Eu(fod-d,) ,-Tris- (1 ,l ,l ,2,2,3,3-heptafluoro-7,7-dimethy1-d6-octane-4,6- dioned,) europium(II1) (from Nuclear Magnetic Resonance Ltd., High Wycombe, Bucks). Carbon tetrachloride (nuclear magnetic resonance grade)-Uvasol (E. Merck), dried over type 3A molecular sieve. (-)-cc-Methoxy-cc-trij7aoromethyl~henylacetyl chloride [( -)-MTPA ~hloride]-[a]~~ - 129" (5.0 per cent.in carbon tetrachloride) ; boiling-point 99 "C (12 mm Hg) ; prepared from (-)-MTPA, [a]:' - 72" (neat) (Ralph N. Emanuel Ltd., Wembley, Middlesex) using published methods.15 (-)-Menthol. Nuclear magnetic resonance spectra were recorded on a Vanan T-60 spectrometer by use of high-resolution tubes with tetramethylsilane as internal standard. NUCLEAR MAGNETIC RESONANCE MEASUREMENT OF THE DIASTEREOISOMER RATIO OF The sample (50 mg, 96 to 100 per cent. in total esters) and Eu(fod-d,), (200 mg) were dissolved in the carbon tetrachloride (0.4 d) and filtered into a nuclear magnetic resonance spectrometer tube. Signals for the a- and /3-diastereoisomers of the cyclopropane "CH, (see below) were found at 3.57 and 3.10 p.p.m. downfield of tetramethylsilane, respectively.Peak areas were recorded on a sweep width of 250 Hz and the average of five integrams (each of 50 s sweep time) was taken. S-BIOALLETHRIN- GAS - LIQUID CHROMATOGRAPHIC DETERMINATION OF ALLETHROLONE ENANTIOMERS- (-)-MTPA chloride (22 mg) in 2 ml of dry benzene was added slowly (over 5 minutes) at room temperature to a stirred solution of allethrolone (10 mg) and dry pyridine (5 pl), also in 2 ml of dry benzene. The mixture was stirred at room temperature for 1 hour, then refluxed for 2 hours and evaporated in vacuo at 30 "C to a volume of approximately 1 ml. This product was purified by chromatography on 1 g of Woelm neutral aluminium oxide (Brockmann activity 111) contained in a Pasteur pipette and eluted with 15 ml of benzene. The solvent was evaporated in vacuo and the residue made up to 10 ml with ethyl acetate in preparation for gas - liquid chromatographic analysis, which was performed on a Pye, Series 104, gas chromatograph fitted with a flame-ionisation detector and using a 20 foot x Q inch glass column packed with 6 per cent.LSX-3 on 100 to 120 mesh Gas-Chrom Q. The column oven temperature was 210 "C and the carrier gas (nitrogen) flow-rate 8.5 ml min-l. Retention times were: for (-)-allethronyl-( -)-MTPA, 70.5 minutes; and for (+)- allethronyl-( -)-MTPA, 74.1 minutes. ENANTIOMERIC PURITY OF (-)-MTPA- A solution of 8 mg of (-)-menthol, 10 mg of (-)-MTPA chloride and 50 pl of dry pyridine in 3 ml of dry benzene was refluxed for 2 hours, evaporated in vacuo and made up332 RICKETT AND HENRY: QUANTITATIVE DETERMINATION OF THE [Analyst, Vol.99 to 5 ml with ethyl acetate. The menthyl esters were analysed on a 50 m x 0.2 mm glass capillary column coated with free fatty acid phase (FFAP), operated under the following conditions : column temperature, 163 "C ; injector temperature, 209 "C; carrier gas, nitrogen at 14 p.s.i. inlet pressure. The solution (1 pl) was injected, using an inlet split ratio of approximately 100: 1. The preparation of the column and the equipment used have been described previously.8 Retention times were : (-)-menthyl-( -)-MTPA, 36.4 minutes; and for (-)-menthyl-( +)- MTPA, 37.1 minutes. RECONSTITUTION OF S-BIOALLETHRINS- (-)-trans-Chrysanthemoyl chloride (230 mg), dissolved in 5 ml of dry benzene, was added dropwise over 15 minutes to the S-allethrolone (150 mg) and 0.1 ml of dry pyridine in 5 ml of dry benzene, stirred at 0 "C.The mixture was stirred at room temperature over- night, diluted with 20 ml of diethyl ether, washed with 10-ml portions of 2 M hydrochloric acid, water, saturated sodium hydrogen carbonate solution, then water again and dried over anhydrous calcium sulphate. The solvent was evaporated in vacuo to approximately 1 ml and the product purified by chromatography on Woelm neutral aluminium oxide (Brockmann activity 111), being eluted with 25 per cent. ether in n-hexane. RESULTS AND DISCUSSION Diastereoisomers often exhibit chemical shift differences in their nuclear magnetic resonance spectra, particularly where the protons are close to the asymmetric centres.16117 It has previously been observed18 that synthetic pyrethroids prepared from racemic alcohols show diastereoisomeric non-equivalence for one of the cyclopropane methyl groups.The spectrum of bioallethrin recorded at 60 MHz in solution in carbon tetrachloride shows evidence of diastereoisomeric non-equivalence for three signals [Fig. 1 (a) and 1 ( b ) ] , at 1.25 p.p.m. (cyclopropane "CH,), 1.98 p.p.m. (cyclopentenolone jCH,) and 2.25 p.p.m. (cyclopentenolone 'H) but the separations are not sufficient to allow quantitative determina- tion of the diastereoisomer ratio. With the addition of a europium shift reagent the diastereo- isomers are differentially shifted to lower field, causing many of the resonances to split into two separate diastereoisomer signals [Fig. 2 (a)].The nuclear magnetic resonance spectrum of allethrin, obtained by using a shift reagent, has recently been published by Sugiyama et aZ.l9 The optimum conditions for the quantitative measurement of the diastereoisomer ratio are obtained by using 50 mg of allethrin sample and 200 mg of Eu(fod-d,), dissolved in 0.4 ml of carbon tetrachloride, thus giving a mole ratio of pyrethroid to shift reagent of 0.88: 1. With these concentrations the cyclopropane "CH, gives two signals, at 3.57 p.p.m. and 3.10 p.p.m. for the a- and /3-diastereoisomers, respectively. The #I-diastereoisomer peak consists of the two enantiomers, (+)-allethronyl-( +)-trans-chrysanthemate and (-)- allethronyl-( -)-trans-chrysanthemate, while the a-diastereoisomer comprises the (+) (-) and (-) (+) forms [cf., a-(&)-trans-allethrin].The "CH, peaks are ideal for quantitative measurement, being intense singlets well separated from interfering resonances. The assignments were confirmed by comparing the spectra of bioallethrin [Fig. 2 (a)], S-bio- allethrin [Fig. 2 (b)] and a-(-J-)-trans-allethrin [Fig. 2 (c)]. Measurement of the diastereoisomer ratio does not, of course, give a direct result for the enantiomeric purity of the sample. The (+) to (-) ratio of the chrysanthemic acid moiety can be determined independentlys and, with the nuclear magnetic resonance measurements described above. the enantiomeric Duritv of the allethrolone can be calculated by applying L d the following equation: S-allethrolone, per cent. = and hence S-bioallethrin, per cent.100 (P + x - 100) 2x - 100 x (P + x - 100) - 2x - 100June, 19741 ENANTIOMERIC PURITY OF SYNTHETIC PYRETHROIDS. PART I 333 where P is the observed percentage of the P-diastereoisomer peak and x is the percentage of (+)-enantiomer in the trans-chrysanthemic acid. * In practice this calculation may not be necessary, for if the chrysanthemic acid is of high enantiomeric purity and the allethrolone is predominantly in the (+) form, then the amount of (-)-allethronyl- (-)-trans-chrysanthemate present must be low and the P-diastereo- isomer peak will give an approximate measure of the S-bioallethrin content. d m ? n n H h i 8-0 7 .O 6.0 5.0 4.0 3-0 24 b a TR c 14 0 p.p.m. Fig. 1. Nuclear magnetic resonance spectra of (a) bioallethrin, and (b) S-bioallethrin recorded in solution in carbon tetrachloride without shift reagent.Assignments according to Bramwell et aL1* An optically pure sample of S-bioallethrin was not available for use as a standard to check the accuracy of the nuclear magnetic resonance method. Instead, samples of allethrolone were independently analysed for (+) to (-) ratio, esterified with natural (+)- trans-chrysanthemic acid and used as secondary nuclear magnetic resonance standards. (-)--a-Methoxy-a-trifluoromethylphenylacetic acid [( -)-MTPA] is an established reagent for determining the enantiomeric purity of alcohols by nuclear magnetic resonance spectros~opy.~5~~~ The (-)-MTPA esters of ( j-)-allethrolone give two signals, separated by 8.5 Hz, for the diastereoisomeric cyclopentenolone JCH, [Fig.3 ( a ) ] ; no shift reagents were required in this instance. The lower field resonance at 2-03 p.p.m. was assigned to the (+)-allethronyl ester by comparison with the (-)-MTPA ester of natural (+)-pyrethrolone [Fig. 3 ( b ) ] ; the configurations of the groups around the cyclopentenolone ring are identical in the two compounds,21 the only difference being in the lengths of the side chains. * In the derivation of this equation it is assumed that the enantiomers of acid and alcohol are ran- domly distributed, i.e., that no kinetic resolution has taken place during the esterification stage.334 RICKETT AND HENRY: QUANTITATIVE DETERMINATION OF THE [Analyst, VOl. 99 Although the nuclear magnetic resonance signals were sufficiently well separated for quantitative measurement, an alternative and preferred method for measuring the diastereoisomer ratio was by gas chromatography of the (-)-MTPA esters on a 20-foot glass column packed with 6 per cent. LSX-3 on Gas-Chrom Q support.The resolution obtained 1.. s 8 .O 7.0 6.0 5 .O 4.0 3.0 2.0 1.0 0 p.p.m. Fig. 2. Nuclear magnetic resonance spectra of (u) bioallethrin, (b) S- bioallethrin, and (c) E-( j-)-trans-allethrin with shift reagentJune, 19741 ENANTIOMERIC PURITY OF SYNTHETIC PYRETHROIDS. PART I 335 between the diastereoisomers was 1-45, only marginally above the base-line. Duplicate samples of allethrolone containing 50 to 91 per cent. of (+)-enantiomer were esterified with (-)-MTPA chloride and each was analysed twice by this procedure. Peak areas were measured by use of a disc integrator and the standard deviations calculated from the four analyses of each sample were 0.3 per cent. 0 i" J ~ ~ ~ ~ ~ I ~ ~ ~ ~ ~ 1 1 1 1 ~ ~ 1 ~ 1 1 ~ 1 1 I 1 ~ l l I I 8 .O 7 -0 6.0 5.0 4.0 3.0 2 .o 1 .o 0 p.p.m.Fig. 3. Nuclear magnetic resonance spectra of the ( -)-a-methoxy-a-trifluoro- methylphenylacetic esters of (a) ( f )-allethrolone and ( b ) (+)-pyrethrolone As the (-)-MTPA is a synthetic rather than naturally derived chemical it was considered pertinent to check the enantiomeric purity of the batch used for these determinations. This was accomplished by analysing the diastereoisomeric (-)-menthy1 esters on a 50 m x 0.25 mm glass capillary column coated with FFAP. A base-line separation between the diastereo- isomers was obtained and from four determinations the ratio of (-)-MTPA to (+)-MTPA was found to be 99.3: 0.7 with a standard deviation of 0.2.The enantiomer ratios of the allethrolone samples were corrected for the small amount of (+)-MTPA present in the acid forming the derivatives by using equation (1) where, in this instance, P was the observed percentage of the (+)-allethronyl-( -)-MTPA gas - liquid chromatographic peak and x the percentage of (-)-MTPA in the acid. The corrected values are given in Table I. S-Bioallethrin samples were reconstituted from the allethrolones by use of natural (+)-trans-chrysanthemic acid and the (+) to (-) ratios determined by the nuclear magnetic336 RICKETT AND HENRY: QUANTITATIVE DETERMINATION OF THE [Ana&Sf, VOl. 99 TABLE I PERCENTAGE OF ( +)-ENANTIOMER IN ALLETHROLONE SAMPLES DETERMINED BY GAS - LIQUID CHROMATOGRAPHY AND NUCLEAR MAGNETIC RESONANCE METHODS (+)-Allethrolone by esters, per cent.50.1 76.5 87.7 91-5 91.9 g.1.C. of (-)-MTPA (+)-Allethrolone by n.m.r. of (+)-trans- chrysanthemates, per cent. 50.0 77.0 87-5 91-4 92.3 resonance method. (As, in this instance, the acid is enantiomerically pure, the diastereo- isomer ratio is equal to the enantiomer ratio of the alcohol.) The results given in Table I are the average of five integrations; standard deviations were between 0-3 and 0.7 per cent. The enantiomer ratios of the allethrolone samples determined by the nuclear magnetic resonance method thus compared well with the gas - liquid chromatographic values, all of the differences being less than the combined standard deviations.n H aa I aG I w L Fig. 4. Nuclear magnetic resonance spectra of cis-allethrins with shift reagent. (a). ( j-)-Allethronyl-( +)-cis-chrysanthemate and (b), (+)- allethronyl- ( + ) -cis-chrysanthemateJune, 19741 ENANTIOMERIC PURITY OF SYNTHETIC PYRETHROIDS. PART I 337 Other constituents of technical S-bioallethrin are esters of cis-chrysanthemic acid and free chrysanthemic acid (each comprising generally less than 2 per cent. of the total). When using equivalent amounts of shift reagent, neither compound gave peaks lying within the area from 3.0 to 3.6 p.p.m. that was used for these measurements. No increases in the standard deviations were observed for measurements of technical S-bioallethrin samples ; multiple scan accumulation did not significantly improve precision.The method would also appear to be suitable for determining the diastereoisomer ratio of cis-allethrins. Fig. 4 (a) and (b) shows the nuclear magnetic resonance spectra, after treatment with the shift reagent, of (&)-allethronyl-( +)-cis-chrysanthemate and (+)- allethronyl-( +)-cis-chrysanthemate, respectively (the latter being contaminated with approximately 13 per cent. of the (-)-allethronyl isomer). The cyclopropane aCH3 peaks at 2.88 p.p.m. and 2.27 p.p.m. cannot be used in this instance because the latter overlaps with the methyl peaks of the isobutenyl groups. However, signals from both 'CH, and bCH3 are fully resolved and either should be suitable for quantitative measurement. gift of 1. 2. 3. 4. 5.6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. The authors are grateful to Dr. M. Elliott of Rothamsted Experimental Station for the valuable materials. REFERENCES Stoddard, R. B., and Dove, W. E., Soap Sanit. Chem., 1949, 25, 118. Fales, J. H., Bodenstein, 0. F., Waters, R. M., and Fields, E. S., Soap Chem. Spec., 1970, 46 (12), Chadwick,'P. R.,' Pest. Scz., 1971, 2, 161. Procida S.A., Puteaux, France, technical information. LaForge, F. B., Green, N., and Schechter, M. S., J . Org. Chern., 1954, 19, 457. --- , Ibid., 1956, 21, 455. Rickktt, F.' E., Analyst, 1973, 98, 687. LaForge, F. B., Green, N., and Schechter, M. S., J . Amer. Chem. SOC., 1952, 74, 5392. Crombie, L., Findley, D. A. R., and Whiting, D. A., Tetrahedron Lett., 1972, 39, 4027. Schechter, M. S., LaForge, F. B., Zimmerli, A., and Thomas, J. M., J . Amer. Chem. Soc., 1961, Maciver, D. R., Pyrethrum Post, 1968, 9, 41. Elliott, M., J . Chem. SOC., 1964, 5225. Sawicki, R. M., Elliott, M., Gower, J. C., Snarey, M., and Thain, E. M., J . Sci. Fd Agric., 1962, Dale, J. A., Dull, D. L., and Mosher, H. S., J . Org. Chem., 1969, 34, 2543. Mislow, K., and Raban, M., Topics Stereochem., 1966, 1, 22. Raban, M., and Mislow, K., Ibid., 1967, 2, 199. Bramwell, A. F., Crombie, L., Memesby, P., Pattenden, G., Elliott, M., and Janes, N. F., Telra- Sugiyama, T., Kobayashi, A., and Yamashita, K., Agric. Biol. Chem., 1973, 37, 1497. Koreeda, M., Weiss, G., and Nakanishi, K., J . Amer. Chem. SOC., 1973, 93, 239. Begley, M. S., Crombie, L., Simmons, D. J., and Whiting, D. A., Chem. Commun., 1972, 1276. NOTE-Reference 8 is to Part I of this series. 78. , Ibid., 1971, 47 (l), 64. 73, 3541. 13, 172. hedron, 1969, 25, 1727. Received October 24th, 1973 Accepted November 28th, 1973

ISSN:0003-2654    

DOI:10.1039/AN9749900330

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

338 Analyst, June, 1974, Vol. 99, pp. 338-354 The Detection and Determination of Polynuclear Aromatic Hydrocarbons by Luminescence Spectrometry Utilising the Shpol’skii Effect at 77 K BY G. F. KIRKBRIGHT AND C. G. DE LIMA (Chemistry Department, Imperial College, London, S . W.7) The luminescence emission spectra of twenty-three polynuclear aromatic hydrocarbons (PAH) have been examined in n-alkane solvents at 77 K. The Shpol’skii effect, in which narrow-band (quasi-linear) emission spectra are obtained under these conditions when a monochromator of adequate resolving power is used, is shown to be readily observed for twelve of the compounds examined in these solvents. Quasi-linear emission spectra have also been obtained in tetrahydrofuran for some of the PAH compounds examined.These emission spectra provide for unambiguous qualitative identification of PAH compounds a t trace concentrations in solution; this effect is demon- strated by identification of the compounds present in an eight-component mixture of PAH compounds. Measurement of the low-temperature quasi-linear luminescence intensity can be applied quantitatively to the determination of these compounds provided that a standard additions procedure is employed in conjunction with the use of an internal standard to ensure sufficient accuracy and precision. THE detection and determination of trace concentrations of polynuclear aromatic hydro- carbons (PAH) is of extreme importance as most of these compounds are toxic and many are carcin0genic.l The development of methods for their unambiguous identification and accurate determination in samples of air, water, foods and petroleum products and effluents is therefore necessary.Methods based on solution spectrofluorimetry and spectrophosphorimetry have been widely employed for these purposes.2-6 One of the difficulties with these techniques arises from the relatively broad-band excitation and emission spectra observed for PAH compounds in many solvents at room temperature and even at low temperature in those solvents which form optically transparent glasses that are suitable for luminescence spectro- metry with right -angle illumination. As a consequence, methods for the determination of particular PAH compounds by luminescence spectrometry under these conditions may suffer interference caused by the overlapping excitation or emission spectra of other similar compounds present in the sample.It is usually necessary, therefore, to resort to preliminary separation of the compound by chromatography or extraction before determining it by fluorimetry or phosphorimetry. In complex PAH mixtures, such separations are frequently necessary before even qualitative identification of individual compounds present can be made by these techniques. A further problem arises from the fact that most commercially available fluorescence spectrometers utilise relatively low-resolution, high-aperture monochromators to permit detection of the weak luminescence emission obtained with trace concentrations of the species to be determined. Thus, even if structured luminescence emission is present, it may be difficult to observe with the spectral resolution attainable with this type of monochromator.In 1952, Shpol’skii, Il’ina and Klimova’ reported that some aromatic compounds, when included in the crystalline matrix formed at 77 K or below by use of selected n-alkane solvents, exhibited extremely well resolved fine structure in their luminescence emission spectra. This phenomenon was confirmed by Bowen and Brocklehurst .8 The observation under these conditions of line-like structure, in which individual lines may be less than 0.1 nm in half- width, can be explained by the postulate that the solute analyte molecules become embedded in the crystalline solvent lattice formed on cooling. The solute molecules are thus held in strictly oriented positions and at low concentrations are separated by large distances so that Q SAC and the authors.KIRKBRIGHT AND DE LIMA 339 they do not interact.In contrast to the case that applies in solvents which form transparent glasses at low temperature, where the glass does not show a short-range order and the elec- tronic transitions are very sensitive to variation in the molecular field, in the crystalline solid solutions produced for PAH compounds in n-alkane solvents the solute molecules experience a well defined molecular field that gives rise to sharp-line (quasi-linear) electronic spectra.9 The spectra exhibit the vibrational frequencies of the centres in the ground state; several workers have shown that good correlation can be achieved with results obtained independently from infrared or Raman spectra.1°-12 The quasi-linear spectra of more than 100 organic compounds have been recorded and the application of the technique has been reviewed.13-17 It has been observed that the molecular dimensions of the n-alkane solvent used must be matched to those of the solute molecule in order to obtain well defined quasi-linear spectra.16 The early studies of the Shpol’skii effect indicated that measurement of the quasi-linear spectra obtained at low temperature for PAH and other compounds should provide a powerful tool for fundamental investigation of molecular structure and for the sensitive, and extremely selective, detection and determination of these compounds.Despite the predictions of advantages to be gained by the application of the Shpol’skii effect, which were recorded in the early litera- ture, Winefordner and Lucasiewiczl8 have commented adversely on its potential and outlined possible difficulties associated with its application to quantitative analysis.These workers have also stated that few papers have presented a detailed description of the use of the technique in a specific analytical method and which reported the usual data pertaining to reproducibility, accuracy and precision, and that the availability of suitable commercial luminescence instrumentation severely limits its applications. While those papers which have been concerned with the Shpol’skii effect may in many instances lack adequate reproducibility, accuracy and precision data, a considerable number of publications have described the application of the quasi-linear luminescence measurements to both qualitative and quantitative examination of real samples.Thus Il’ina and Personovlg have applied the technique to the detection of 1,12-benzoperylene in Jurassic and Cretaceous sedimentary rocks and perylene in tertiary sediments. Dikun20 identified pyrene, 1 ,Z-benzoyyrene, 3,4-benzopyrene, perylene, 1,lZ-benzo- perylene and 1,2,7,8-dibenzanthracene in smoked fish and polluted air by using their quasi- linear spectra in n-hexane solvent at 77 K ; with the same technique, he also detected o- phenylenepyrene, 1,2,4,5-dibenzopyrene and 3,4,9,10-dibenzopyrene in polluted air. Gurov and Novikov21 identified anthracene, pyrene, 3,4-benzopyrene, 1,12-benzoperylene, perylene and coronene in soil and snow samples using the quasi-linear luminescence spectra obtained at 77 K in n-hexane solvent. Parker and Hatchard22 have applied the technique using n- octane - cyclohexane solvent to an investigation of an unusual photo-reaction of 3,4-benzo- pyrene in solutions containing polymer.Eichhoff and Kohler23 determined 3,4-benzopyrene in the atmosphere by a method based on direct measurement of the absolute intensity at 403.0 nm of its quasi-linear emission in n-heptane at 79 K. P e r ~ o n o v ~ ~ determined 3,4-benzopyrene by using the quasi-linear emission of coronene for internal standardisation in order to avoid random errors caused by variations in experimental conditions, and D i k ~ n ~ ~ devised a similar method for determining 3,4-benzopyrene by using 1,12-benzoperylene as internal standard.Muel and Lacroix26 and Jager27 utilised a standard additions procedure for the determination of 3,4-benzopyrene in cigarette smoke, alcoholic- drinks, water26 and exhaust fumes;27 these workers employed n-octane solvent at 83 or 77 K and measurement of the luminescence intensity at 403.0 nm with standard additions of 3,4-benzopyrene. Personov and Teplitskaya2* and Florovskaya, Teplitskaya and Personov29 have used the standard additions method for the determination via their quasi-linear luminescence emission of 3,4-benzopyrene, 1,12-benzoperylene and perylene in rocks and minerals of different origin. Khesina and c o - w o r k e r ~ , ~ ~ , ~ ~ in methods that involve the use of both standard additions and internal standardisation, have described the determination of 9,lO-dimethyl-1 ,Z-benzan- thracene, 1,2-benzanthracene, 1,2,5,6-dibenzanthracene, pyrene, 3,4-benzopyrene, perylene and 1,12-benzoperylene.Dikun, Krasnistkaya, Gorelova and Kalinina32 have compared the standard additions, internal standard and combined methods for the determination of 3,4- benzopyrene, using its quasi-linear luminescence emission at low temperature. In the course of the investigations of the determination of various PAH compounds described above,340 KIRKBRIGHT AND DE LIMA: DETECTION AND DETERMINATION OF [Analyst, Vol. 99 qualitative and quantitative methods for their determination in smoked f i ~ h , ~ 2 diesel engine exhausts33 and industrial Several possible difficulties that might hinder utilisation of the Shpol’skii effect for trace analysis have been reported, the first of which is the necessity to choose a suitable alkane solvent in order to observe the effect.Owing to the limited number of such solvents available it may not be possible to stimulate quasi-linear luminescence emission for some PAH compounds; conversely, this would possibly be advantageous if other compounds are to be determined in the presence of these non-emitting species. Shpol’skii, Klimova, Nersesova and Glyadkov~kii~j and Bolotnikova and N a ~ m o v a ~ ~ have investigated the influence of mole- cular aggregation and energy transfer on the intensity of quasi-linear luminescence emission. At low concentrations the spectra may be obscured owing to band emission from molecules aggregated inhomogeneously in the sample and not present in the crystalline matrix, while at high concentrations molecular aggregates excluded from the solvent lattice and which exhibit strong absorption but only weak emission may be formed.These effects might give rise to a restricted concentration range over which the luminescence emission exhibits a linear dependence on solute concentration, and to non-reproducible intensity from sample to sample, i.e., poor precision. In addition, Dokunikhin, Kizel, Sapozhekov and S01oda~~ have observed that both the intensity and width of quasi-linear emission lines is dependent on the rate of freezing of sample solutions. In view of these considerations, it is surprising that the relatively large number of reports of the successful application of measurement of quasi-linear lumines- cence emission to real samples outlined above have appeared.For this reason, and in order to evaluate the potential of an apparently powerful selective technique of analysis, we have studied the Shpol’skii effect for a series of twenty-three PAH compounds in several n-alkane solvents. The technique is similar in its operation to conventional luminescence spectrometry, except for the requirement of a monochromator with moderately high resolution, and direct experiments can be undertaken in order to investigate its potential for qualitative and quantitative analysis. have been established. EXPERIMENTAL APPARATUS- A double monochromator spectrofluorimeter (American Instrument Co., Maryland) fitted with a potted RCA 1P 28 photomultiplier tube, xenon arc lamp continuum source and low-temperature sample cell accessory was employed so as to obtain preliminary spectral data at low resolution, thus facilitating the choice of excitation wavelength for higher- resolution studies in which a mercury-vapour discharge lamp source was used.Spectra were displayed on a Bryans X-Y recorder (Model 21000). The quasi-linear luminescence emission of the compounds studied was recorded using the apparatus of higher resolution. Radiation from a medium pressure mercury-vapour discharge lamp (Wotan Hg/3) was focused into a light-tight sample cell compartment by using two silica lenses of 45 mm diameter and 75 and 50-mm focal length. Interference filters of narrow band width (50 x 50 mm, with a half-band width of 14 nm at 250 nm and 30 nm at 300 nm) were inserted between the source and sample-cell compartment for selection of the excitation wavelength.The sample tubes employed were constructed from silica tubing (Spectrosil) and were 200 mm in length, of 3 mm i.d. and 1-mm wall thickness and were sealed at one end. These tubes were used with the silica Dewar flask from the low-temperature accessory of the spectrofluorimeter used for the low-resolution studies. The liquid samples were plunged into liquid nitrogen contained in the Dewar flask so as to achieve rapid freezing, and the flask was then placed in the sample cell compartment so that the incident radiation was slightly de- focused at the surface of the frozen sample.This defocusing of the incident radiation was found to give rise to more reproducible signal intensities than when the source radiation was brought to a focus at the sample surface. A coil of Nichrome wire was positioned within the sample-cell compartment so as to be adjacent to the outer wall of the Dewar flask when the latter was placed in position. This wire was heated by passing a low a.c. current through it in order to minimise frosting of that part of the Dewar flask surface which is irradiated by the source and viewed by the detection system. A scanning grating monochromator (Optica, Model CF4) with a reciprocal linearJune, 19741 POLYNUCLEAR AROMATICS BY LUMINESCENCE SPECTROMETRY 341 dispersion at the exit slit of 1.6 nm mm-1 was positioned so that its optical axis was at 90" to that of the source and sample cell.Luminescence from the sample cell was focused on to the entrance slit of the mono- chromator by using a composite biconvex silica lens (40 mm in diameter and of 35-mm focal length). An end-window photomultiplier tube (EM1 9601B) was attached at the exit slit of the monochromator and operated at 1200 V by using a Brandenburg EHT supply. The luminescence signal was recorded directly at a potentiometric chart recorder (Servoscribe, Model RE 511.20) although for some experiments a microammeter (RCA, Model WV-84C) was employed for signal registration. REAGENTS- The n-alkane solvents used were n-pentane, n-hexane, n-octane and n-decane. These solvents were of laboratory-reagent grade and were used without further purification.Cyclo- hexane (laboratory-reagent grade) was purified by percolation through silica gel (60 to 120 mesh), which had been activated overnight at 120 to 130 "C. EPA solvent [diethyl ether - iso- pentane - ethanol (5 + 5 + a)] was prepared with ether that had been dried with sodium wire, isopentane dried with sodium wire and percolated through silica gel and ethanol treated with potassium hydroxide and redistilled. Tetrahydrofuran was treated with potassium hydroxide and distilled. Other solvents for study of the matrix were used without pre-treatment. MATERIALS- Samples of pure polynuclear aromatic hydrocarbon compounds were kindly donated by Tobacco Research Council Laboratories, Harrogate, British American Tobacco Co., South- ampton, and Shell Research Ltd., Thornton Research Centre, Chester.4,9-Di-t-butylpyrene and 3,5,8,10-tetraisopropylpyrene were kindly provided by Professor Arne Berg, University of Aarhus, Denmark. PROCEDURE - Low-resolution excitation and emission spectra were recorded in EPA solvent at 77 K with the Aminco spectrofluorimeter with the low-temperature cell attachment or at room temperature with a silica sample cell (10 x 10 x 30 mm). For examination of the quasi- linear luminescence spectra stock solutions of the PAH compounds were prepared in cyclo- hexane; these solutions were diluted to 20 or 2 pg ml-l concentration with the appropriate n-alkane and cyclohexane so that the final solutions examined contained 10 per cent. V/V of cyclohexane. These solutions were transferred into the silica sample tubes, which were then introduced directly into the Dewar flask cell that contained liquid nitrogen. Rapid freezing of the sample solutions was thus obtained.After the initial vigorous boiling action of the liquid nitrogen had subsided, the luminescence emission spectrum was scanned (using a pre-selected excitation wavelength) at 6 nm min-1. RESULTS SPECTRAL CHARACTERISTICS- Cyclohexane is a generally suitable solvent for PAH compounds and its presence in concentrations up to 10 per cent. V/V in the n-alkane solvents used for low-temperature luminescence work has been demonstrated not to disturb appreciably the spectra ~ b t a i n e d . ~ ' . ~ ~ . ~ ~ As only small amounts of some of the PAH compounds examined were available, it was therefore decided to use cyclohexane for their dissolution and n-alkane - cyclohexane mixtures (90+10) for dilution of the stock PAH solutions for the examination of their luminescence spectra.For some PAH compounds quasi-linear luminescence emission is observed only at 77 K when an alkane solvent of matching molecular dimensions is employed. Thus, in a survey of the occurrence of the Shpol'skii effect for a range of twenty-two PAH compounds, it has been necessary to employ several n-alkane solvents in order to choose that which was the most suitable for each compound. An examination of earlier work26,30,31J34 revealed that n-octane had proved to be suitable as a solvent for study of the Shpol'skii effect for some PAH compounds. Initially, therefore, the low-temperature luminescence emission spectra of all twenty-two compounds were342 KIRKBRIGHT AND DE LIMA: DETECTION AND DETERMINATION OF [Analyst, VOl.99 recorded in n-octane - cyclohexane (90+ 10). Excitation wavelengths were selected from the corresponding low-temperature excitation spectra that were obtained by using the low- resolution double-monochromator spectrofluorimeter. The wavelength was chosen so as to correspond to that of the most intense low-wavelength excitation peak in order to minimise observed scattered light during the study of the emission spectrum. Of the compounds examined in n-octane - cyclohexane solvent at 77 K, triphenylene, chrysene, perylene, pyrene, 3,5,8,10- t e t raisoprop ylp yrene, 3,4-benzopyrene, 1,2- benzop yrene, 1,2,3,4-dibenzopyrene, 1,2,4,5-dibenzopyrene, 3,4 , 9,10-dibenzopyrene, 1 2-benzan t hracene and 1,2,5,6-dibenzan- thracene exhibit quasi-linear luminescence in which emission peak half-widths of 0.5 nm or less were recorded.* J U' 458.0 v) 358 0 m L 405 Wavelengthhm Fig. 1. Emission spectra at 77 K of (a) phenanthrene in n-hexane - cyclohexane; (b) triphenylene in n-octane - cyclohexane; (c) chrysene in n-octane - cyclohexane; (d) perylene in n-octane - cyclo- hexane; and (e) coronene in n-hexane The spectra observed for 2 or 20 pg ml-l solutions of these compounds in n-octane - cyclohexane at 77 K are shown in Figs. 1 to 5. The compounds 3,4,8,9-dibenzopyrene (Fig. 6), indeno[l,2,3-cd]pyrene, benzo[a]naphth0[8,1,2-~de]naphthacene (Fig. 7), S-methylchol- anthrene, 7,12-dimethyl-1,2-benzanthracene (Fig.3), 3-methylpyrene, 4,9-di-t-butylpyrene *The nomenclature used follows that of reference 1.Luminescence intensity - 392.25 408.5 41 4.0 423.75 -426.5 432.75 453.5344 KIRKBRIGHT AND DE LIMA: DETECTION AND DETERMINATION OF [Ana&Si!, VOl. 99 (Fig. 5) , phenanthrene (Fig. 1) and 9,lO-dimethylanthracene (Fig. 3) exhibited intense but broad-band luminescence emission in n-octane - cyclohexane solvent. In these spectra, typical peak half-widths greater than 1.0 nm were observed. Anthracene exhibited a very broad spectrum (Fig. 6). Some of these compounds (phenanthrene, anthracene and 9,lO- dimethylanthracene) were examined in n-hexane - cyclohexane solvent and gave somewhat narrower half-band widths in their luminescence spectra (Figs.1, 3 and 6). 3,4,8,9-Diben- zopyrene was examined also in n-decane - cyclohexane (90 + 10) and gave a more well defined spectrum than that obtained in n-octane - cyclohexane (Fig. 6). A schematic presentation of the luminescence characteristics of fifteen of these compounds appears in Fig. 8. Fig. 3. Emission spectra a t 77 K of (a) 9,lO-dimethylanthracene in n-hexane - cyclohexane; and ( b ) 1,2-benzanthracene, (c) 7,12-dimethyl-1,2-benzanthracene, (d) 3-methylcholanthrene and (e) 1,2,5,6- dibenzanthracene in n-octane - cyclohexane Most early observations of the Shpol’skii effect for PAH compounds were made utilising n-alkane solvents and it has generally been accepted that matrices formed from the straight- chain hydrocarbon solvents of suitable molecular dimensions are required in order to obtain quasi-linear emission spectra.A striking demonstration of the need to select the correct n-alkane solvent can be made by comparison of the spectrum obtained for anthracene in n-hexane - cyclohexane (Fig. 6), in which quasi-linear emission is obtained, with the spectrum for anthracene in n-octane - cyclohexane (Fig. 6), in which only a very broad emission is observed. The restriction of the effect to relatively non-polar solvents, however, would result in limited analytical utility and restrict the study of the technique to those compounds which are soluble in these solvents.June, 19741 m m m m ? ? -.- aJ t 5) E C .- -I 345 Wavelengthhm Fig. 4. Emission spectra a t 77 K of some dibenzopyrenes in n-octane - cyclo- hexane: (u) 3,4,9,10- dibenzopyrene; (b) 1,2,4,5-dibenzopyrene; and (c) 1,2,3,4-di- benzop yrene A preliminary qualitative study of the use of other solvents was therefore undertaken.The model PAH compound chosen for study was coronene; this hydrocarbon exhibits a simple and well defined quasi-linear emission spectrum in n-hexane (Fig. 1). The emission spectra of solutions cor,t aining 20 pg ml-l of coronene in dioxan, pentanol, carbon tetrachloride, chloro-Luminescence intensity Luminescence intensity 382.25 384.25 t 3 9 4 - 2 5 398.0 = 378.0 399.0 Hg \ e 460.0 476.0 479.0 483.5 b n n 450.0 n 3 381.0 386.0 391.5 393.0 396-0 402.0 411.0 41 4.0 416.0 375.0 "5- 375.0 381.25 382.75 384.0 386.25 387.75 391.75 393.0 395-0 396.0 1 Hg j R 379.2 383.75,m $ (bi 520 500 440 Wavelengthhm Emission spectra at 77 K in n-octane - cyclohexane of (a) indeno[l,2,3-cd]pyrene; and (b) benzo[u]naphtho[8,1,2-cde]naphthacene 34 Wavelengthhm348 KIRKBRIGHT AND DE LIMA: DETECTION AND DETERMINATION OF [Analyst, Vol. 99 form, bromoform, diethyl ether, dimethylformamide, 1,1,2,2-tetrachloroethane and tetra- hydrofuran were examined at 77 K.Only with tetrahydrofuran was a well defined quasi- linear emission spectrum observed for coronene [Fig. 9 (a)]. The corresponding phosphores- cence emission spectrum for coronene is shown in Fig. 10. Of twenty-three PAH compounds studied, quasi-linear emission was also observed for 4,9-di-t-butylpyrene, 1,2-benzopyrene and 1,2,5,6-dibenzanthracene in tetrahydrofuran. The spectra observed for these compounds are shown in Figs.9 and 11 and the data recorded are listed in Table I. Although spectral emission band widths that were greater than expected for the Shpol’skii effect were observed for 3-methylcholanthrene, perylene and 3,4,9,10-dibenzopyrene in tetrahydrofuran as shown in Figs. 9 and 11, sharp and useful spectra were obtained for these compounds. : LDO 445 507 370 ‘I 420 Wavelengthh Fig. 9. Emission spectra at 77 K of some PAH compounds in tetrahydrofuran: (a) coronene; (b) perylene ; (c) 1,2-benzopyrene (phosphorescence spectrum) ; and (d) 1,2-benzopyrene (fluorescence spectrum) Good agreement is observed in the wavelength assignments made for the principal quasi-linear luminescence emission maxima in this work with those recorded earlier for several of the compounds studied by other ~ o r k e r s .~ ~ , ~ * - ~ ~ The wavelength reproducibility of these emission maxima and the relative freedom from overlap of the narrow-band “quasi-line” spectra compared with that obtained in solution at room temperature or in glass-forming organic solvents such as EPA at 77 K suggest that the quasi-linear spectra may be extremely useful for qualitative identification of PAH compounds. Fig. 2 shows the luminescence emission spectrum at 77 K for pyrene in EPA glass and the corresponding quasi-linear emission spectrum at 77 K in n-octane - cyclohexane. Both spectra were recorded with the high- resolution instrumentation. The gain in structure obtained by utilising the Shpol’skii effect and the possibility of less ambiguous identification of this compound in the presence of others is clearly seen.In order to demonstrate the “fingerprinting” ability of the technique, a synthetic mixture of eight PAH compounds was prepared and its low-temperature luminescence emission spectrum was recorded at 77 K in n-octane - cyclohexane solvent; Fig. 12 shows theJune, 19741 POLYNUCLEAR AROMATICS BY LUMINESCENCE SPECTROMETRY 349 570 Wavelengthhm Fig. 10. Phosphorescence emission spectra of coronene in n-hexane (broken line) and in tetra- hydrofuran (solid line) 430 435 390 450 Ln ld) Wavelengthhm Fig. 11. Emission spectra a t 77 K of some PAH compounds in tetrahydrofuran: (a) 4,9-di-t-butyl- pyrene ; (b) 1,2,5,6-dibenzanthracene ; (c) 3-methylcholanthrene ; and (d) 3,4,9,10-dibenzopyrene350 KIRKBRIGHT AND D E LIMA: DETECTION AND DETERMINATION OF [AfldySt, VOl.99 emission spectrum obtained. Each of the eight hydrocarbons present in the mixture is readily identified from the principal luminescence emission maxima observed. Even when some overlap occurs for certain principal peaks, there is sufficient information present in the minor features of the spectrum of each compound to permit its detection by using alternative peak wavelengths. TABLE I EMISSION AT 77K OF SOME PAH COMPOUNDS IN TETRAHYDROFURAN Excitation wavelength 300 nm Compound .. Wavelengths of principal emission maxima Coronene . . 424.5 (s), 425-0 (m), 431.25 (w), 443.5 (vs), 444.0 (s), 450.75 (m), 451.5 (m), 452.0 (m), 453-0 (m), 472.0 (m), 482.0 (w) p : 515.0 (m), 525.0 (m), 528-0 (w), 546.0 (w), 547.0 (m), 549.25 (m), 554.5 (m), 555.75 (m), 562.0 (vs) .. 448.0 (m), 453.25 (vs), 457.25 (m), 461.0 (m), 465.0 (w), 473.75 (s), 475.0 (s), 480.0 (m), 481.25 (m), 483.0 (m), 488.0 (w) . , 374.75 (m), 376.50 (m), 378.25 (w), 381.0 (w), 382.5 (m), 386.0 (vs), 388-5 (m), 390.75 (w), 394-5 (m), 396.0 (m), 397.0 (m), 399.0 (m), 406.25 (m), 407.5 (m), 409.0 (m) p : 533.25 (s), 542-75 (m), 544.25 (m), 546.0 (w) 3,4,9,10-Dibenzopyrene 433.5 (vs), 438.75 (w), 448.5 (w), 461-25 (m), 465.5 (d,w) 3-Methylcholanthrene . . 393.25 (vs), 397.5 (w), 414.25 (m), 415.5 (m), 417.5 (m), 420.5 (m) 1,2,5,6-Dibenzanthracene 392-25 (m), 394-25 (vs), 395.0 (m), 397.5 (w), 405.75 (w), 414.5 (vw), 415-5 (w), 416.25 (m), 418-0 (m). 4,9-Di-t-butylpyrenc .. 374.75 (m), 375.5 ( s ) , 376.0 (vs), 383.25 (m), 384.0 (m), 384.5 (m), 385.25 (m), 387.0 (m), 388-5 (m), 389.25 (w), 392-5 (m), 394.25 (m), 395.5 (m), 396-0 (m), 397.0 (m), 397.5 (m) Perylene . . 1,2-Benzopyrene .. Concentration of all compounds in tetrahydrofuran : 20 pg ml-1. vs, very strong emission: s, strong; w, weak; m, medium; d, diffuse; p, phosphorescence emission. Wavelengths in italics indicate the most intense peaks. The detection limits given for the compounds studied can be defined as that concentration of hydrocarbon in the solvent employed which gives a signal to noise ratio of 2 at the wave- length of the principal quasi-linear emission. The wavelengths (nm) used and the values (pg ml-l) obtained for these compounds were: phenanthrene (345-5,O.l) ; triphenylene (462.25, 0.1) ; chrysene- (360.5, 0.05) ; perylene (451.0, 0.05) ; pyrene (371.75, 0.05) ; 3.4-benzopyrene (403.0, 0.005) ; 1,2-benzopyrene (387.75, 0.07) ; 1,2,3,4-dibenzopyrene (395.25, 0.08) ; 1,2,4,5- dibenzopyrene (395.5, 0-08) ; 3,4,8,9-dibenzopyrene (449.25, 0.04) ; 3,4,9,10-dibenzopyrene (431.5, 0.1) ; anthracene (377.0, 0.05) ; 1,2-benzanthracene (383.75, 0.1) ; 7,12-dimethyl-1,2- benzanthracene (397.75, 0.2) ; 3-methylcholanthrene (392.5, 0.2) ; 1,2,5,6-dibenzanthracene (394.25, 0.1) ; 9,lO-dimethylanthracene (405.75, 0.05) ; 3-methylpyrene (375.0, 0.2) ; 4,9-di-t- butylpyrene (375.0, 0-2) ; 3,5,8,10-tetraisopropylpyrene (379.25, 0.1) ; indeno[l,2,3-cd]pyrene (462.5, 0.3) ; benzo[rn]naphtho[8,1,2-~de]naphthacene (444.75, 0.3) ; and coronene (445.0, 0.1).These limits are estimates only and would be subject to considerable improvement if a more efficient optical arrangement for collection of the emitted radiation and more sophisti- cated detector electronics were used. QUANTITATIVE STUDIES- The variation in intensity of luminescence emission at 77 K with concentration was examined for the four dibenzopyrene compounds available to us in n-octane - cyclohexane solvent, and the intensity of the quasi-linear emission for 3,4,9,10-dibenzopyrene, 1,2,4,5- dibenzopyrene and 1,2,3,4-dibenzopyrene was measured at 43160, 395.50 and 395.25 nm, respectively. This variation was also examined for 1,2,3,4-dibenzopyrene at its broad emission peak at 470-25 nm. Measurement at this wavelength, rather than at 395.25 nm, would be advantageous in the determination of this compound in the presence of 1,2,4,5-dibenzopyrene as it avoids interference from the emission of the latter compound at 395-5 nm.The lumi- nescence growth curves obtained are plotted logarithmically in Fig. 13. For the compounds examined, the quasi-linear emission intensity is linear over a con- centration range of two orders of magnitude. Fig. 13 also shows that similar intensity, slope and range of linearity are obtained at both 395.25 and 470.25 nm for the luminescence growthJune, 19741 POLYNUCLEAR AROMATICS BY LUMINESCENCE SPECTROMETRY 351 I I I II I I I I ’ 500 g & h Wavelengthhm Emission spectra of a synthetic mixture in n-octane - cyclohexane a t 77 K: Fig. 12. (u) pyrene; (b) 1,2-benzanthracene; (6) 3-methylcholanthrene; (d) 1,2,5,6-dibenzanthracene ; (e) 1,2,4,5-dibenzopyrene; (f) 3,4-benzopyrene; (g) 3,4,9,10-dibenzopyrene ; and (h) 3,4,8,9-dibenzopyrene curve of 1,2,3,4-dibenzopyrene.Although 3,4,8,9-dibenzopyrene does not show quasi-linear luminescence emission in n-octane - cyclohexane solvent at 77 K it can be seen in Fig. 13 that a similar intensity and range of linearity is observed for the luminescence of this compound when measured at the wavelength of its more intense emission at 449.25 nm. Although it appears that direct measurement of the intensity of quasi-linear luminescence emission is capable of permitting the direct quantitative determination of compounds such as the dibenzopyrenes, in the examination of real samples several difficulties exist that lead to the requirement for the use of a standard additions procedure and the use of an internal standard.Thus when other compounds are present with the PAH compound to be deter- mined, their absorption spectra may overlap that of the analyte molecule and give rise to an “inner filter” effect and low fluorescence intensities ; energy transfer between the analyte molecule and others present in the sample may also lead to inaccurate values of intensity of quasi-linear emission compared with those expected in the absence of other compounds; the352 KIRKBRIGHT AND DE LIMA: DETECTION AND DETERMINATION OF [Analyst, Vol. 99 use of the standard additions method of analysis minimises these inaccuracies. Variation of experimental measurement conditions, such as rate of freezing or the reproducibility with which the sample cell can be placed in the optical path, can lead to poor precision in measure- ment of quasi-linear luminescence intensity.These effects can be minimised by the use of an internal standard in quantitative work. A combined standard addition - internal standard procedure was adopted for quanti- tative determination of the dibenzopyrene compounds in n-octane - cyclohexane solvent. The internal standard employed was selected so that the wavelength of its quasi-linear lumi- nescence emission did not overlap that of the quasi-linear emission of the compound to be determined. It is, of course, not possible to avoid such overlapping in the corresponding excitation spectra; it is necessary for radiation transmitted by the single filter used to excite luminescence from both analyte compound and internal standard, so that some overlap in excitation spectra must occur if the internal standard technique is to be used in this manner.1,2,4,5-Dibenzopyrene, 3,4,9,10-dibenzopyrene and 3,4,8,9-dibenzopyrene were deter- mined by measurement of their quasi-linear luminescence in n-octane - cyclohexane at 77 K at the wavelengths employed in order to construct their luminescence growth curves shown in Fig. 13. The quasi-linear emission of 3,4-benzopyrene at 403 nm was used as internal standard for the determination of 1,2,4,5-dibenzopyrene and 3,4,9,10-dibenzopyrene and a standard additions procedure was employed in order to produce the calibration graphs shown in Fig.14 (a) and (b). For the determination of 3,4,8,9-dibenzopyrene in n-octane - cyclohexane the yuasi-linear emission of 1,2-benzopyrene at 387.75 nm was used for internal standardisation ; the calibration graph obtained is shown in Fig. 14 (c). In each instance the ratio of the observed intensities for the luminescence of the analyte and internal standard are plotted against concentration. lo3 3 Concentrationh Fig. 13. Luminescence intensity zlem,ts concentration graphs for some dibenzopyrenes determined a t 77 K in n-octane - cyclohexane: 0, 3,4,8,9-dibenzopyrene; A, 3,4,9,10-dibenzopyrene; 0, 1,2,4,5- dibenzopyrene ; and [7, l12,3,4-dibenzopyrene Although the use of an internal standardisation procedure leads to improved precision by decreasing the effects of random errors in the measurement, an “inner filter” effect occurs in a manner similar to that mentioned above owing to the overlap of the excitation spectrum of the internal standard compound with that of the compound determined ; lower quasi-linear luminescence emission intensities for the analyte compound are thus obtained in the presenceJune, 19741 POLYNUCLEAR AROMATICS BY LUMINESCENCE SPECTROMETRY 353 Concentration x IOVM Working curves for (u) 1,2,4,5-, (b) 3,4,9,10- and (c) 3,4,8,9-dibenzopyrene using the com- Fig.14. bined method (addition method and internal standard) a t 77 K in n-octane - cyclohexane of the internal standard compound. Improvement in precision is obtained over the analytical working range when the internal standardisation procedure is employed, but the decrease in luminescence signal intensity that results leads to some deterioration in detection limit for the compounds studied.Table I1 shows the effect of the presence of equimolar or greater concen- trations of some other PAH compounds on the quasi-linear luminescence of the dibenzopyrene compounds excited at 300 nm in n-octane - cyclohexane. The percentage suppression or enhancement of the luminescence signal in the presence of these compounds is listed. In almost all instances suppression of quasi-linear emission of the dibenzopyrenes occurs owing to the “inner filter” effect or, possibly, by energy transfer. The enhancement of 1,2,3,4- dibenzopyrene emission at 395.5 nm in the presence of 1,2,4,5-dibenzopyrene is caused by the direct overlap of their quasi-linear emission at this wavelength; no effect is observed when the luminescence of 1,2,3,4-dibenzopyrene is measured at 470.5 nm.TABLE I1 EFFECT OF OTHER PAH COMPOUNDS ON MEASURED LUMINESCENCE INTENSITIES AT 77K FOR DIBENZOPYRENE COMPOUNDS STUDIED Results are expressed as change in luminescence intensity, per cent. Interferent 1,2,3,4- 1,2,4,5- 3,4,8,9- 3,4,9,10- 3,4- 1,2- 1,2,5,6- DBP DBP DBP DBP BP BP DBA PERe 1,2,3,4-Dibenzopyrene - +226a; -35%; -35%; -54*0a*b - +22.2a.g; - Nilb Nilb Nilb - 29.6b 1,2,4,5-Dibenzopyrene - 34.5 - -35.0 -57.2 -60.5 - - 22.9’ - 3,4,8,9-Dibenzopyrene - 37.8 - 33.4 - -114.9 -58.0 -45.5‘ - - 22.8 ; - 358d 3,4,9,10-Dibenzopyrene - 17.8 -31-5 -45.8 - -20.6 - -22.8 -31.5 - 56-26 - 32.8d - 82.5d a at 395.5 nm; b at 470-5 nm; C four-fold excess of 1,2-BP; d ten-fold excess: e perylene is not excited with the filter employed; f a t ten-fold excess there is direct interference; g a t ten-fold excess there is direct interference in the emission a t 395.5 nm and suppression (-60 per cent.) a t the 470-0 nm emission.3,4-Benzopyrene, 1,2-benzopyrene, 1,2,5,6-dibenzanthracene and perylene appear as 3,4-BP, 1,2-BP, 1,2,5,6-DBA and PER, respectively, in the table; DBP denotes dibenzopyrene. CONCLUSIONS Our preliminary study reported here confirms that the high selectivity claimed for the identification of PAH compounds utilising the Shpol’skii effect at 77 K is readily attained. The quasi-linear emission spectra can be used for unambiguous “fingerprinting” of these compounds in solution at trace concentrations.The wavelength assignments for the principal quasi-linear luminescence emission maxima observed agree well with those made by other workers. At present, the application of this fingerprinting technique is limited to those354 KIRKBRIGHT AND DE LIMA aromatic hydrocarbons which are soluble in n-alkanes. The ability to obtain quasi-linear emission spectra in tetrahydrofuran, however, suggests that the technique may be extended to compounds that are not soluble in these solvents and that are more polar than PAH compounds. Although it is necessary to match the molecular dimensions of solute and solvent species, thisrequirement can be used to advantage in order to improveselectivity by choice of suitable solvent if spectral overlap at major luminescence peaks is observed for some compounds.The quantitative use of the Shpol’skii effect for trace analysis requires careful calibration and use of a combined internal standard - standard additions technique so as to minimise the effects of energy transfer, the inner filter effect and experimental variables and to attain acceptable precision and accuracy. It has been sh0wn4~~~3 that the corresponding absorption spectra of PAH compounds at 77 K may be quasi-linear in character. The use of a narrow-line excitation source, such as a tunable dye laser, would therefore provide even greater selectivity and sensitivity for the analytical application of the Shpol’skii effect. One of us (C.G. de L.) thanks the University of Brasilia for study leave and UNESCO for the grant of a Fellowship.We also thank those agencies mentioned in the text for the provision of samples for examination. 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. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. REFERENCES Clar, E., “Polycyclic Hydrocarbons,” Academic Press, London, 1964. McKay, J. F., and Latham, D. K., Analyt. Chem., 1972, 44, 2132. Van Duuren, B. L., Ibid., 1960, 32, 1436. Sawicki, E., Hauser, T. R., and Stanley, T. W., I n t . J . A i r Pollut., 1960, 2, 253. Hood, L. V., and Winefordner, J. D., Analytica Chim. Acta, 1968, 42, 199. Sauerland, H. D., and Zander, M., Erdol Kohle, 1966, 19, 502. Shpol’skii, E. V., Il’ina, A. A., and Klimova, L. A., Dokl.Akad. Nauk. S.S.S.R., 1952, 87, 935; Bowen, E. J., and Brocklehurst, B., J . Chem. SOC., 1954, 3875. Shpol’skii, E. V., Zh. Prikl. SpeRtrosk., 1967, 7, 492. Pesteil, L., and Ciais, A., C.R. Hebd. Se’anc. Acad. Sci., Paris, 1959, 249, 528. Bolotnikova, T. N., Optika Spektrosk., 1959, 7, 44. -, Ibid., 1959, 7, 217. Shpol’skii, E. V., Soviet Phys. Usp., 1959, 2, 378. -, Ibid., 1960, 3, 372. Chem. Abstr., 1953, 47, 420513. -, Ibid., 1962, 5, 522. -, Ibid., 1963, 6, 411. Parkcr, C. A. , “Photoluminescence of Solutions,” Elsevier, Amsterdam, 1968, p. 383. m7inefordner, J. D., and Lucasiewicz, R. J., Talanta, 1972, 19, 381. Il’ina, A. A., and Personov, R. I., Geochemistry, Ann Arbor, 1962, No. 11, 1089. Dikun, P. P., J . A@Z. Sfiectrosc., 1967, 6, 130. Gurov, F. I., and Novikov, Yu. V., Gig. Sanit., 1971, 36, 409. Parker, C. A., and Hatchard, C. G., Photochem. Photobiol., 1966, 5, 699. Eichhoff, H. J., and Kohler, N., 2. analyt. Chem., 1963, 197, 272. Personov, R. I., Zh. Analit. Khim., 1962, 17, 506; Chem. Abstr., 1963, 58, 24b. Dikun, P. P., Vop. Onkol., 1961, 7, 42; Chern. Abstr., 1962, 57, 65%. Muel, B., and Lacroix, G., Bull. SOC. Chim. Fr., 1960, 2139. Jager, J., Chemickk L i s t y , 1966, 60, 1184. Personov, R. I., and Teplitskaya, T. A., J . Analyt. Chem. U.S.S.R., 1965, 20, 1176. Florovskaya, V. N., Teplitskaya, T. A., and Personov, R. I., Geochemistry Int., 1966, 3, 419. Danil’tseva, G. E., and Khesina, A. Ya., J . A p p l . Spectrosc., 1966, 5, 196. Fedoseeva, G. E., and Khesina, A. Ya., Ibid., 1968, 9, 838. Dikun, P. P., Krasnistkaya, N. D., Gorelova, N. D., and Kalinina, I. A., Ibid., 1968, 8 9 254. Varshavskii, I. L., Shabad, L. M., Khesina, A. Ya., Khitrovo, S. S., Chalabov, V. G., and Pakhol’nik, Kolyadich, M. N., Khesina, A. Ya., Shefer, S. S., and Yamskova, V. P., Gig. Trud. Prof. Zabol., Shpol’skii, E. V., Klimova, L. A., Nersesova, G. N., and Glyadkovskii, V. I., Optics Spectrosc., N . Y . , Bolotnikova, T. N., and Naumova, T. M., Ibid., 1968, 24, 253. Dokunikhin, N. S., Kizel, V. A., Sapozhekov, M. N., and Soloda, S. L., Ibid., 1967, 23, 42. Ciais, A., and Pesteil, L., C.R. Hebd. Se’anc. Acad. Sci., Paris, 1959, 248, 1311. Bowen, E. J., and Brocklehurst, B., J . Chem. Soc., 1955, 4320. Shpol’skii, E. V., Klimova, L. A., and Personov, R. I., Optics Spectrosc., N . Y., 1962, 13, 188. Teplyakov, P. A., Ibid., 1963, 15, 350. Klimova, L. A., Ibid., 1963, 15, 185. Ruzevich, E. S., Ibid., 1963, 15, 191. A. I., Ibid., 1965, 2, 68. 1971, 15, 3. 1968, 24, 25. Received October 23rd, 1973 Accepted January 2nd, 1974

ISSN:0003-2654    

DOI:10.1039/AN9749900338

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

Analyst, June, 1974, Vol. 99, pp. 355-359 355 Application of the Spectrophotometric Determination of Nickel and Cobalt in Mixtures With Bipyridylglyoxal Dithiosemicarbazone to the Analysis of Catalysts BY J. L. BAHAMONDE, D. PfiREZ BENDITO AND F. PIN0 (Department of Analytical Chemistry, University of Seville, Seville, Spain) Bipyridylglyoxal dithiosemicarbazone forms a complex with nickel(I1) a t pH 5.2, which can be extracted into chloroform (Amax. = 410 nm). A similar complex is obtained with cobalt(II), but is not extractable in this solvent, thus allowing nickel and cobalt to be determined in mixtures. Two procedures are proposed for the accurate analysis of such mixtures in which 1 p.p.m. of one of the ions can be determined accurately in the presence of as much as 5 p.p.m.of the other. One of the procedures has been applied to the determination of nickel and cobalt in industrial catalysts and the results obtained have been compared with those obtained by atomic-absorp- tion spectrophotometry. Satisfactory results were obtained. THE work described in this paper forms part of an investigation into the use of dithiosemi- carbazones as analytical reagents. In a previous paper1 we have studied the reactions between iron(I1) and (111) ions and bipyridylglyoxal dithiosemicarbazone (BGT)- I H2N-C-NH S II In this paper the determination of nickel(I1) and cobalt(II), both separately and in mixtures, with the above reagent and its application to the analysis of catalysts are described. EXPERIMENTAL APPARATUS- S9ectrophotometers-Unicam SP800 and SP600 spectrophotometers, equipped with 1-0-cm quartz or glass cells, were used for ultraviolet and visible-light absorbance measure- ments.A Perkin-Elmer 402 atomic-absorption spectrophotometer was also used. Digital PH meter-A Philips PW 9408 instrument, with glass - calomel electrodes, was used. SOLUTIONS- All solvents and reagents were of analytical-reagent grade. Bipyridylgglyoxal dithiosemicarbazone solution-A 0.1 per cent, m/V solution in ethanol. Standardised solutions of n i c k e l ( I I ) and cobalt(II). Acetic acid - sodium acetate bufer solutiorlz, pH 5.2. The reagent is synthesised from bipyridylglyoxal and thiosemicarbazide.2 PROCEDURE- Determination of nickel-Up to 6 p.p.m. of nickel, 15 ml of 0.1 per cent. m/V bipyridyl. glyoxal dithiosemicarbazone solution in ethanol, 20 ml of acetic acid - sodium acetate buffer @ SAC and the authors.356 BAHAMONDE et al.: SPECTROPHOTOMETRIC DETERMINATION OF NICKEL [Analyst, Vol. 99 solution (pH 5.2) and up to 50 ml of water are poured into a separating funnel. The mixture is shaken briskly, left to stand for 30 minutes and then extracted four times with 5-ml volumes of chloroform. The chloroform extracts are collected in a 25-ml calibrated flask and diluted to the mark with chloroform. The absorbance at a wavelength of 410 nm is measured against a blank obtained by extraction of the reagents (containing no nickel) in the same way. Determination of cobalt-A solution with a concentration of up to 8 p.p.m. of the cobalt is placed in a calibrated flask, 15 ml of 0.1 per cent.m/V bipyridylglyoxal dithiosemicarba- zone solution in ethanol and 20ml of pH 5.2 buffer solution are added and the mixture is diluted to 50ml with water. The flask is shaken vigorously, allowed to stand for 1 hour and the absorbance is measured at 410 nm against a reagent blank. Determination of nickel and cobalt in mixtures: Method A-A neutral solution of nickel(I1) and cobalt(I1) is poured into a separating funnel; the pH is maintained at 5.2 by use of the buffer solution. An excess of 0.1 per cent. m/V reagent solution is added. After 30 minutes the complexes are extracted into chloroform as indicated above. The aqueous and chloroform layers are separated and the absorbance of each is measured at 410 nm, as described. The amount of nickel is calculated from the absorbance of the organic layer, and cobalt from that of the aqueous layer. Determination of nickel and cobalt in mixtures : Method B T w o identical aqueous sample solutions are prepared and in one of them the sum of nickel plus cobalt is determined at pH 5.2.With the other sample nickel is determined alone after extraction into chloroform. A cali- bration graph is plotted for the nickel ion in the aqueous layer and the amount of cobalt is obtained by difference. Determination of nickel and cobalt in industrial catalysts-A 0.5-g amount of catalyst is weighed accurately and is placed in a flask that is suitable for refluxing to which 20 ml of concentrated nitric acid and 60 ml of concentrated hydrochloric acid are added. The mixture is refluxed for 1 hour and is then concentrated to about 25 ml.The solution is adjusted to pH 5 to 7 and diluted to 100 ml with distilled water in a calibrated flask. Aliquots are taken from this solution and the nickel and cobalt complexes are developed and the analysis is completed as described under Method A . RESULTS AND DISCUSSION REACTION OF BIPYRIDYLGLYOXAL DITHIOSEMICARBAZONE WITH NICKEL AND COBALT- Bipyridylglyoxal dithiosemicarbazone forms yellow - green chelates with nickel( 11) and cobalt(I1) in a weakly acidic medium, the wavelengths of maximum absorption being 390 and 410 nm, respectively (Fig. 1). Both complexes are formed slowly and the solutions must be allowed to stand for a time for the development of a stable colour. Reducing agents do not Wavelengthhm Fig.1. Absorption spectra of solutions a t pH 5-2 of complexes formed with bipyridylglyoxal dithiosemi- carbazone: 1, 5 p.p.m. of nickel(I1) in a homogeneous medium; l’, 5 p.p.m. of nickel extracted into chloroform; and 2, 7 p.p.m. of cobalt(I1) in a homogeneous mediumJune, 19741 AND COBALT IN MIXTURES FOR THE ANALYSIS OF CATALYSTS 357 affect the cobalt complex spectrum, but oxidising agents, such as potassium persulphate and hydrogen peroxide, shift the maximum towards the ultraviolet, with a notable hyperchromic effect. The nickel complex is extracted into chloroform at pH 5.2 and the A,. shifts to 410 nm. The cobalt complex is not extracted into this solvent at any pH value. The different behaviour of nickel and cobalt complexes in regard to extraction into chloroform is probably caused by the charge on the cobalt complex.With iron(II), the bi- pyridylglyoxal dithiosemicarbazone - iron( 11) complex is extracted into chloroform both in an acidic and in an ammoniacal medium, giving an emerald-green colour, but the extraction is easier in an ammoniacal medium. The extraction of other ions has not been tested, but their extraction is to be expected as some of them produce positive errors in the study of the inter- ferences of the nickel(I1) and iron(I1) complexes. Iron(I1) ions interfere in the determination of cobalt and nickel, as can be seen from the results of the study of the interferences of both ions. The absorbance - pH graphs for the cobalt and nickel complexes are shown in Fig. 2. Despite its not being the optimum value, a pH of 5.2 has been chosen for the nickel complex because the sensitivity of the reaction is good, the acetic acid - sodium acetate buffer is more readily available, and some interferences are avoided at this pH value.The absorbance of a chloroform solution of the nickel complex remains stable for at least 24 hours. The ethanol (from the reagent solution) extracted with chloroform stabilises the solutions. The stoicheiometry of the complexes has been studied by the continuous variation method (Fig. 3). The reagent - metallic ion ratio found is 1 :1 for the nickel complex and 2:l for the cobalt complex. f i n e " - A - - - - v v - B I I I I I I I I I 1 (u 1.2 1.0 + 0.8 a 0.6 0.4 0.2 0 0 Fig. 2. Absorbance veisus pH graphs of nickel and cobalt complexes of bipyridylglyoxal dithiosemicarbazone : A, nickel complex extracted into chloroform for 6 p.p.m.of nickel (Amax. 410 nm) ; and B, cobalt complex in homogeneous medium for 3.2 p.p.m. of cobalt (Amax. 410 nm). Both graphs have been obtained with use of various amounts of hydrochloric acid and sodium hydroxide ANALYTICAL APPLICATIONS OF THE COBALT AND NICKEL COMPLEXES- Nickel com$Zex-The optimum conditions for the formation and extraction of the nickel complex have been indicated under Experimental. Beer's law is obeyed between 1 and 5 p.p.m. of nickel and the molar absorptivity at 410 nm is 1.17 x lo4 1 mol-1 cm-1. Ringborn's graph shows that 1.4 to 4.0 p.p.m. of nickel(I1) is the minimum range of error. The relative error (P = 0.05) of the method is 5 0.13 per cent.The interferences on 4 p.p.m. of nickel have been investigated: 100 p.p.m. of cobalt(II), manganese(II), chromium(VI), silver, platinum( IV), tungsten(VI), lanthanum, calcium, barium and aluminium and 10 p.p.m. of mercury(I1) ions gave errors below 5 per cent.; 2 p.p.m. of iron(II), copper(II), zinc, cadmium and osmium(1V) ions gave errors between 4 and 8 per cent.; 200 p.p.m. of fluoride, oxalate, citrate, phosphate and thiosulphate did not358 BAHAMONDE et d. : SPECTROPHOTOMETRIC DETERMINATION OF NICKEL [Analyst, VOl. 99 [Me"+] t [BGTI Fig. 3. Stoicheiometry of nickel and cobalt complexes of bipyridylglyoxal dithiosemicarbazone (continuous variations) : A, nickel complex extracted into chloroform a t pH 5.2 (Amax. 410 nm) ; and B, cobalt complex in homogeneous medium at pH 5.2 (Amax.410 nm). The initial solutions of nickel and cobalt were a t a concentration of 1 x M interfere or gave errors below 2 per cent. ; and 5 p.p.m. of EDTA and cyanide gave an error of 10 per cent. Cobalt comjdex-The conditions established in the recommended procedure have been determined empirically. Beer's law is obeyed at pH 5-2 for between 1 and 7 p.p.m. of cobalt and the molar absorptivity is 9.05 x lo3 1 mol-l cm-I. The optimum concentration range, evaluated by Ringborn's method, is 1.5 to 7.5 p.p.m. of cobalt. The relative error (P = 0-05) of the method is & 0.55 per cent. The interferences for 4 p.p.m. of cobalt were investigated: 100 p.p.m. of manganese(II), cnromiumt v 11, piarinumti v j , rungsrent v i j , ianrnanum, calcium, parium, aluminium, lithium, sodium, potassium, magnesium, rubidium and strontium ions did not interfere or gave errors lower than 2 per cent.; 10 p.p.m.of mercury(I1) and cadmium ions, 2 p.p.m. of zinc and osmium(1V) ions and 0-5 p.p.m. of iron(II), copper(I1) and nickel ions gave errors of over 15 per cent.; EDTA and cyanide ions interfered above 5 p.p.m.; and 200 p.p.m. of citrate gave errors of 5 per cent. At this concentration (200 p.p.m.) fluoride, oxalate, per- chlorate, phosphate and thiosulphate do not interfere. TABLE I DETERMINATION OF NICKEL AND COBALT IN MIXTURES BY METHOD A Determination of nickel Determination of cobalt r Nickel added/pg ml-l 0.5 1.0 1.0 1.0 2.0 2.0 3.0 4.0 4.0 3.0 1.8 - Ni :Co ratio 0.05 0.1 0.17 0.2 0.33 0.5 1.0 2.0 4.0 6.0 9.0 -l Nickel found/pg ml-I 0.55 1.05 1.0 1.0 2-05 2-05 3.06 4-06 4.06 3.0 1-8 f Cobalt added/pg ml-l 5.0 5.0 6.0 5.0 6.0 4.0 3.0 2.0 1.0 0.5 0.2 - Co :Ni ratio 20.0 10.0 6.0 5.0 3.0 2.0 1.0 0-5 0.25 0.17 0.1 1 1 Cobalt found/pg ml-1 5.3 5.2 6.0 5.0 6.0 4.0 3.0 2.0 1.0 0.55 0.25June, 19741 AND COBALT IN MIXTURES IN THE ANALYSIS OF CATALYSTS 359 Analysis of nickel and cobalt mixtures-We have applied method A to a series of eleven samples in which the nickel to cobalt ratio varied from 0.05 to 9.The absorbance should not exceed 1. Table I shows the results obtained. It can be deduced from these results that nickel can be determined, within the concentration range given in Table I, in the presence of up to ten times its own concentration of cobalt with an error of less than 5 per cent.It is possible to determine cobalt in the presence of concentrations of nickel up to four times greater with errors below 4 per cent. Table I1 shows the results obtained by use of method B when applied to thirteen samples in which the nickel to cobalt ratio varied from 0.14 to 7. In the presence of up to five times greater concentrations of one ion, the other can be determined with minimal error. TABLE I1 DETERMINATION OF NICKEL AND COBALT IN MIXTURES BY METHOD B Nickel Nickel Cobalt Cobalt added/pg ml-l found/pg ml-f addedlpg ml-l found/pg ml-1 1.0 1.5 7.0 7.5 1.0 1.5 6.0 6.1 1.0 1.0 5.0 5-0 1.0 1.0 4.0 4.0 2.0 2.0 6.0 6 0 2.0 2.0 4.0 4.0 3.0 3.0 3.0 3.0 4.0 4.0 2.0 2.0 6.0 6.0 2.0 2.0 4.0 4.0 1.0 1.0 5.0 5.0 1.0 1.0 6.0 6-1 1.0 1.5 7.0 7.6 1.0 1.5 Amlysis of industrial catalysts-The techniques described above have been applied to the determination of trace amounts of nickel and cobalt on aluminium oxide supports and in process catalysts such as the Unifining catalyst, Isomax-UOP-DHC-2, Filtrol 475-8 and Unifining Petresa.All of these supports have the shape of a ball of about 2 mm diameter, or a small cylinder of similar dimensions. For Unifining catalyst method A was used. The results are compared with those obtained by atomic-absorption spectrophotometry, and are shown in Table 111. It should be noted that neither an excess of aluminium oxide nor the presence of molybdenum, which is present in these catalysts, interferes. TABLE I11 ANALYSIS OF INDUSTRIAL CATALYSTS Sample* Methodt Unifining catalyst . . .. . . AA Isomax-UOP-DHC-2 . . . . AA Filtrol 475-8. . .. .. . . AA Unifining Petresa . . . . . . AA BGT BGT BGT BGT Nickel, per cent. Cobalt, per ccn?-. 0.30 2.20 0.33 2.20 0.82 - 0.81 - - 1-16 - 1.16 2.31 - 2-36 - *Unifining catalyst: nickel 0-30 per cent., cobalt 2.20 per cent., molybdenum 3.40 per cent. Isomax-UOP-DHC-2 : nickel 0-82 per cent., molybdenum 4.60 per cent. Filtrol 475-8: cobalt 1.20 per cent., molybdenum 2.45 per cent. Unifining Petresa: nickel 2.35 per cent., molybdenum 4.50 per cent. (According to standard of UOP.) t AA = atomic absorption ; BGT = bipyridylglyoxal dithiosemicarbazone. REFERENCES 1. 2. Bahamonde, J. I,., Pdrez Bendito, D., and Pino, F., Talanta, 1973, 20, 694. -, -, -, Infcidn Quipn. Analit., 1972, 26, 7. Received Juty l l t h , 1973 Accepted December 28th, 1973

ISSN:0003-2654    

DOI:10.1039/AN9749900355

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

360 Analyst, June, 1974, Vol. 99, $$. 360-366 Ionic Polymerisation as a Means of End-point Indication in Non-aqueous Thermometric Titrimetry Part VI.* The Determination of Thiols BY E. J. GREENHOW AND MISS L. H. LOO (Departmeizt of Chemistry, Chelsea College, University of London, Manresa Road, London S . W.3) Alkyl and aryl thiols have been determined in the presence of carboxylic acids and phenols by means of acid - base catalytic thermometric titrimetry. Two titrations are carried out, with acrylonitrile and acetone as the end- point indicators. With the former indicator, thiol groups are not deter- mined, so that the difference between the titration values obtained by using the two methods of end-point indication is a measure of the thiol content. The thiol content of 2-mercaptothiazoline, 4,6-dihydroxypyrimidine-2- thiol (2-thiobarbituric acid), purine-6-thiol and 2-mercaptobenzimidazole can be determined by the same procedure.In the titration of 2-thiohydantoin, 4-hydroxypyrimidine-2-thiol (2-thiouracil), 2-mercaptobenzoxazole and 2-mercaptobenzothiazole, however, both end-point methods give the same titration value. These apparently anomalous results can be explained if it is accepted that the last four heterocyclic thiols exist in the thione tautomeric form in dimethylformamide solution. Some thioamides also titrate as acids, and differences between titration values obtained by using the two methods of end-point indication can again be attributed to thione - thiol tautomerism. Thiols can be determined conveniently in amounts down to 0.01 mequiv, i.e., about 2 mg of dodecane-l-thiol, with 0.1 M titrants. In instances when the acrylonitrile method can be used for the direct determination of the thiol function, 0.001 M titrant can be used and the lower level of determination is then about 0.0001 mequiv.THE determination of the thiol function is important in connection with the refining of petroleum because thiols are an undesirable impurity in distillate fractions. In other industries, certain thiols find use as additives. For example, dodecane-l-thiol is used to control the degree of polymerisation in the manufacture of thermoplastics, while 2-mercapto- benzothiazole is added to rubber formulations as an accelerator for the vulcanisation stage. The performance of such additives is dependent on their thiol content, and determination of the latter can be used as a method of quality control.Thiols differ from hydroxy compounds in their ability to form insoluble copper, silver and mercury derivatives and in the ease with which they undergo oxidation. These reactions form the basis of the preferred chemical methods for the determination of the thiol function in organic compounds.1,2 Thiols are more acidic than the corresponding hydroxy compounds, thus even ethanethiol, with a pK, of 10.5 at 20 "C, can be titrated as a weak acid.2 Acid - base titration is not, however, generally recommended for the selective determination of thiols because other acidic compounds in the sample would interfere. An attempt has been made to determine thiols iodimetrically in non-aqueous solution by use of catalytic thermometric titration with ethyl vinyl ether as an end-point indicator, but the titration values obtained were considerably lower than those required by a 1 : 1 stoicheiometry.3 Apparently, a high proportion of the thiol was converted into an un- reactive, i.e., unoxidisable, sulphide by addition to the indicator monomer during the course of the titration : -SH + C2H,0CH=CH2 -+ C,H,OCH,CH,S- * For Part V of this series, see Analyst, 1974, 99, 82.@ SAC and the authors;GREENHOW AND LOO 361 In the present paper an analytical procedure is reported in which the acid content of a sample is determined by two catalytic thermometric methods, the acrylonitrile method described in Part 114 and the acetone-indicator method of Vaughan and S~ithenbank.~ With acrylonitrile as the end-point indicator thiol groups that undergo rapid addition to acrylonitrile are not determined, and the difference between the titration values obtained by using the two end-point indicators is taken as being a measure of the content of these thiol groups.This procedure offers an alternative to the two preferred methods, noted above, for the selective determination of the thiol group, and it has been evaluated for a range of alkyl, aryl and heterocyclic thiols and for some thioamides. EXPERIMENTAL REAGENTS- Acetone, propan-2-01 and methanol were analytical-reagent grade materials, and acrylonitrile, toluene and dimethylformamide were laboratory-reagent grade materials. All were dried over molecular sieve 4A before use.Other solvents and reagents, including the thiols, were laboratory-reagent grade materials and were used as received. Potassium hydroxide, 1.0 and 0.1 M solutions in propan-2-02-Standardise these solutions against benzoic acid (analytical-reagent grade) in acetone by the thermometric method using acetone as the end-point indicator. Tetra-n-butylammonium hydroxide, 0- 1 M solution in toluene - methanol-Laboratory- reagent grade material was used as received. Prepare 0.01 and 0.001 M solutions by adding appropriate volumes of toluene - propan-2-01 mixture (3 + 1) to the 0.1 M reagent. Standardise the solutions against benzoic acid (analytical-reagent grade) dissolved at 0.1 or 0.01 N concentration in dimethylformamide by the thermometric method with acrylonitrile as the end-point indicator.APPARATUS- ture, and a 10-ml titration flask with a magnetic stirrer, as described in Part 111.6 Use a motor-driven syringe to supply the titrant, a thermistor to measure the tempera- PROCEDURE A. ACETONE-INDICATOR METHOD- Use the procedure described by Vaughan and Swithenbank6 in the following manner. Add potassium hydroxide titrant solution at a rate of 0.1 ml min-l to a mixture of 1 ml of sample solution and 3 ml of acetone in the titration flask; use 1.0 mequiv of sample with the 1.0 M titrant and 0.1 mequiv of sample with the 0.1 M titrant. Record the temperature and titrant volume on a millivolt chart recorder (50 and 20-mV scales with the 1.0 and 0.1 M titrants, respectively) at a chart speed of 600 mm h-l.B. ACRYLONITRILE-INDICATOR METHOD- Use the procedure described in Part IV7 for the titration of acidic functions. Dissolve the sample in 1 ml of dimethylformamide and use 0.1 mequiv of sample with the 0-1 M tetra-n-butylammonium hydroxide titrant or correspondingly smaller amounts with the 0.01 and 0.001 M titrants; 1.0 or 0.1 M potassium hydroxide solution can also be used as a titrant. The end-point of the titration, when either indicator method is used, is located at the point where the tangent to the main heat rise leaves the curve at its lower temperature end.8 RESULTS AND DISCUSSION Table I lists the compounds titrated and the reaction stoicheiometries obtained by using the acetone and acrylonitrile-indicator methods. Some thioamides that can be titrated as acids are included in the table.Titration curves obtained in the determination of some of the thiols and thioamides arc shown in Figs. 1 and 2, respectively. It can be seen from Table I that the monofunctional alkyl and aryl thiols are not deter- mined by the acrylonitrile-indicator method. Thus, the addition of acrylonitrile to these compounds, i.e., cyanoethylation, must have proceeded to completion before the acid - base362 reaction could occur. GREENHOW AND LOO : IONIC POLYMERISATION FOR END-POINT [Analyst, Vol. 99 Cyanoethylation is catalysed by the alkaline titrant : OH- RSH + CH2=CHCN --+ RSCH2CHCN The carboxylic and phenolic groups of 2-mercaptobenzoic acid and salicylideneaminobenzene- 2-thiol are determined, and the difference between the titration value for each compound and the corresponding titration value obtained by using the acetone-indicator method, which determines the thiol function also, is a measure of the thiol content.TABLE I THIOLS AND THIOAMIDES TITRATED WITH 1.0 M POTASSIUM HYDROXIDE AND 0 . 1 M TETRA-n-BUTYLAMMONIUM HYDROXIDE SOLUTIONS WITH ACETONE AND ACRYLONITRILE, RESPECTIVELY, AS END-POINT INDICATORS Conditions: titrate 1 mequiv of thiol in 3 ml of acetone with 1.0 M potassium hydroxide solution by using the acetone-indicator method, and 0.1 mequiv of thiol in a mixture of 1 ml of dimethylformamide and 2 ml of acrylonitrile with 0.1 M tetra-n-butyl- ammonium hydroxide solution by using the acrylonitrile-indicator method Aliphatic thiols- Heptane-l-thiol (1 : 0.9 : 0) ; dodecane-l-thiol (1 : 0.9 : 0) ; 2,3-dimercaptopropan-l-o1 (2 : 1.7 : 0) ; and mercaptosuccinic acid (3 : 2.5 : 1.9) Toluene-1’-thiol (1 : 0.8 : 0) ; toluene-4-thiol (1 : 0.8 : 0) ; 4-aminobenzenethiol (1 : 0.65 : 0) ; 2-mercaptobenzoic acid (2 : 2 : 1) ; salicylideneaminobenzene-2-thiol (2 : 1.9 : 1) ; and pyridine- 2-thiol (1 : 1 : 0.18) 2-Mercaptothiazoline (1 : 1 : 0) ; 2-thiohydantoin (1 : 1 : 1) ; 4-hydroxypyrimidine-2-thiol (2 : 1 : 1) ; 4,6-dihydroxypyrimidine-2-thiol (3 : 2 : 1) ; purine-6-thiol (1 : 1.7 : 0.8) ; 2-mercapto- benzimidazole (1 : 1 : 0.3) ; 2-mercaptobenzoxazole (1 : 1 : 1) ; and 2-mercaptobenzothiazole Aromatic thiols- Heterocylic thioZs- (1: 1: 1) Thioamides- Thioacetamide (1 : 1 : 0.9*) ; thiourea (l(2) : 0.1 : 1*) ; thiocarbanilide (l(2) : 1 : 0.36 or 0.53*) ; dithiooxamide (rubeanic acid) (2 : 2 : 1) ; thiosemicarbazide (l(2) : 1.1 : 0.9) ; and diphenyl- thiocarbazone (dithizone) (l(2) : 1 : 0-8 or 1*) Figures in parentheses following the name of the compound denote the theoretical number of acidic functional groups in the molecule, the number of groups titrated by using the acetone- indicator method and the number of groups titrated by using the acrylonitrile-indicator method, respectively.* Values obtained in the titration of 1 mequiv of sample by using the acrylonitrile-indicator method and 1.0 M potassium hydroxide titrant. The content of thiols in mixtures with carboxylic acids and phenols can also be deter- mined by using the two indicators. In Fig. 3 calibration graphs are shown for mixtures of dodecane-l-thiol with benzoic acid and 3,5-xylenol with 2-mercaptothiazoline.The irregular shape of the calibration graph for the mixture containing dodecane-l-thiol is due to the low reaction stoicheiometry with this compound. It can be seen from Table I that the titration reactions with several of the thiols are sub-stoicheiometric, and it is necessary to use calibration graphs, or to allow for the sub- stoicheiometry in some other way, when determining these thiols by the suggested procedure. The small but finite titration value obtained when pyridine-2-thiol is determined by using the acrylonitrile-indicator method indicates that the rate of cyanoethylation is influ- enced by the heterocyclic ring. The titration values obtained with the other heterocyclic thiols examined have been found to depend on the position of the thiol group in the molecule, or the presence of reactive functional groups in addition to the thiol group, or both.Thus, while 2-mercaptothiazoline behaves on titration in the same way as do simple alkyl and aryl thiols, the corresponding benzo-derivative, 2-mercaptobenzothiazole does not. The thiol group in the latter compound can, apparently, be determined by either of the two catalytic end-point methods. This anomaly can be explained if it is assumed that, in solution in dimethylformamide, 2-mercaptobenzothiazole exists entirely in the thione t automeric form. In this form it is the acidic imido group and not the thiol group that is titrated. The reactionJune, 19741 INDICATION IN NON-AQUEOUS THERMOMETRIC TITRIMETRY. PART VI 363 stoicheiometry of 2-mercaptobenzoxazole can be similarly explained.This proposed reaction path presupposes that the imido group undergoes cyanoethylation more slowly than it undergoes neutralisation by the titrant. In contrast with its sulphur and oxygen analogues, 2-mercaptobenzimidazole was found to give a titration value corresponding to a sub-stoicheiometric reaction when it was deter- mined by using the acrylonitrile-indicator method. This would suggest that the compound is only partly in the thione form when in solution in dimethylformamide. 2-Thiohydantoin and 2-thiouracil (4-hydroxypyrimidine-2-thiol) were also found to be titrated as monobasic acids irrespective of the end-point indicator used in the thermometric titration.With these two compounds, however, there is the possibility that the hydroxyl group in the enolic form of the molecules rather than the imido group, is being titrated. c f 9 h J I I I I Titrant/rnl (1 division=lml) Fig. 1. Thermometric titration curves obtained in the determination of thiols by the acetone and acrylonitrile-indicator methods Compound/mg . . A, 205.5 B, 1.67 C, 105.9 D, 212.5 E, 7.8 F, 3.7 G, 6.4 H, 7.1 Solvent/ml . . K, 2 D, 1 K, 2 K, 1 D, 1 D, 1 D, 1 D, 1 Titrant/M . . P, 1-0 B, 0.01 P, 1.0 P, 1.0 B, 0.1 B, 0.1 B, 0.1 B, 0.1 Indicator method K P K K P P P P Compounds-A, dodecane-l-thiol; B, 2-mcrcaptobenzothiazole; C, 2,3-dimercaptopropan-l- 01 ; D, salicyliderieaminobenzene-2-thiol ; E, 2-thiobarbituric acid ; F, mercaptosuccinic acid; G, 2-thiouracil; and H, 2-thiohydantoin a* b C* d* e f g h Solvents-K, acetone ; and D, dimethylformamide Titrants-P, potassium hydroxide reagent ; and B, tetra-n-butylammonium hydroxide Indicator methods-K, acetone method using 3 ml of acetone; and P, acrylonitrile method * 1 division of temperature scale = 0.5 "C reagent using 2 ml of acrylonitrile Thiobarbituric acid (4,6-dihydroxypyrimidine-Z-thiol), when determined by the thermo- metric titration, functions either as a dibasic or monobasic acid, depending on whether the acetone or acrylonitrile method of end-point indication is used.Barbituric acid (2,4,6- trihydroxypyrimidine), in contrast, behaves as a dibasic acid whichever method of end-point indication is used, thereby supporting the hypothesis that thiol groups cannot be determined in the presence of acrylonitrile.Purine-6-thiol is similar to thiobarbituric acid with respect to the acidity it displays on catalytic thermometric titration, but the corresponding stoicheiometries, 1.7 and 0.8, are less well defined. Although the thioamides are formally classed as thiones, they can undergo tautomeric change to thienols. The stoicheiometries of the neutralisation reactions in which dithio-364 GREENHOW AND LOO : IONIC POLYMERISATION FOR END-POINT [ A ~ ~ d y s t , Vol. 99 oxamide and thiocarbanilide are determined by using the two methods of end-point indi- cation suggest that, in solution in dimethylformamide, the former compound has the structure S: C(NH,).C(SH) : NH, while the latter is partly in the thienolic form, C6H,NH.C(SH) : NH.- Thioacetamide, thiosemicarbazide and diphenylthiocarbazone (dithizone) would appear to retain the thione structure in solution in dimethylformamide, as a stoicheiometry of 1 : 1, or nearly 1 : 1, is obtained when either method of end-point indication is used.Thiourea behaves in an unusual way in the thermometric titration in that, while the simple 1 : 1 stoicheiometry is obtained by using the acrylonitrile indicator, the neutralisation proceeds to only a small extent when acetone is used to mark the end-point. Both thiourea and thioacetamide show an immediate temperature rise when they are titrated in the presence of the acrylonitrile end-point indicator, but the neutralisation then proceeds and an S-shaped titration curve results (Fig.2). The curves are similar in shape to those obtained in the titration of slightly soluble cat echo la mine^^ but, as thiourea and thioacetamide are readily soluble, the shape must be due to some other factor. A possible answer is that the initial addition of titrant causes a rearrangement of either the thioamide or an unstable addition compound of the thioamide and acrylonitrile. It should be noted (Table I) that, for these two amides, 1 . 0 ~ potassium hydroxide solution was used as the titrant instead of 0.1 M tetra-n-butylammonium hydroxide solution. With the latter titrant the titration curves were rounded and the end-point was difficult to assess. C6H5. Tit ran t/m I (1 division= 1 ml) Fig. 2. Thermometric titration curves obtained in the determination of thioamides by the acetone and acrylonitrile-indicator methods Compound/mg .. A,31.0 A,48-4 B,43.5 CJ4.1 C.Fi.96 D,278.0 D,17.2 E,3.2 E,36.8 F,83.7 F,117.4 Solvent/ml . . D , 1 D , 1 D , 1 K , 1 D , 1 K , l D , 1 D , l K , 2 D , 1 D , 2 Indicator method K P P K P K P P K K P carbazide ; and F, diphenylthiocarbazone a* b c d* e f* g h j k* m Titrant/M . . P, 1.0 P, 1.0 P, 1.0 P, 1.0 B, 0.1 P, 1.0 B, 0.1 P, 0.1 P, 1.0 P, 1.0 P, 1.0 Comfiounds-A, thioacetamide ; B, thiourea ; C, dithiooxamide ; D, thiocarbanilide ; E, thiosemi- Solvents-K, acetone ; and D, dimethylformamide Tztrants-P, potassium hydroxide reagent ; and B, tetra-n-butylammonium hydroxide reagent Ilzdicator methods-K, acetone method using 3 ml of acetone; and P, acrylonitrile method using 2 ml * 1 division of temperature scale = 0.5 "C of acrylonitrile When 1.0 M potassium hydroxide titrant was used in conjunction with the acrylonitrile method for the determination of thiocarbanilide and diphenylthiocarbazone, the stoicheio- metries of the neutralisation reactions differed from those obtained when the tetra-n-butyl-June, 19741 INDICATION IN NON-AQUEOUS THERMOMETRIC TITRIMETRY.PART VI 365 ammonium hydroxide reagent was used, and the possibility of steric hindrance influencing the stoicheiometry must be considered. The precision of the method for the determination of thiols in the presence of carboxylic acids and phenols has been determined for mixtures of dodecane-1-thiol and benzoic acid, and 2-mercaptothiazoline and 3,5-xylenol, by carrying out eight titrations on each mixture dissolved in dimethylformamide against 1.0 and 0-1 M potassium hydroxide titrants, respec- tively.The acetone and acrylonitrile-indicator methods were used alternately as the titrations were carried out. 0 50 100 Thiol, mol per cent. Fig. 3. Calibration graphs for the thermo- metric titration of mixtures of dodecane-l- thiol with benzoic acid, and 2-mercapto- thiazoline with 3,5-xylenol. (u), Dodecane-l- thiol plus benzoic acid, 1 mequiv dissolved in 2 ml of dimethylformamide; and (b), 2-mercapto- thiazoline plus 3,5-xylenol, 0.1 mequiv dissolved in 1 ml of dimethylformamide. 1.0 and 0.1 M potassium hydroxide titrants, respectively, were used in the determinations of mixtures (a) and * A and B are the titration values obtained by using the acetone-indicator method with 3 ml of acetone and the acrylonitrile-indicator method with 2 ml of acrylonitrile, respectively (b).Precisions were calculated in two ways: (a), from the differences between pairs of titration values taken in sequence; and (b), from the precisions of the groups of four titration values obtained by each method of end-point indication. In (b) the required coefficient of variation has been calculated as the square root of the sum of the squares of the coefficients of variation of the two groups of four titrations. Details of the experimental results and the calculated values are shown in Table 11. It can be seen that the direct determination of the coefficient of variation from the differences between pairs of titration values leads to lower values than by calculation using method (b) and there is, therefore, apparently some advantage in carrying out the titrations in the sequence proposed. The method is suitable for the determination of thiols in the presence of carboxylic acids and phenols at precisions of about 1 per cent., and in amounts down to 0.01 mequiv when 0.1 M titrants are used, provided that the thiol function is titrated when the acetone-indicator method is used, but is not titrated in the acrylonitrile-indicator method.This procedure requires that two titrations be carried out with each sample but, of course, gives the content of acidic compounds other than thiols as well as the thiol content. Values for the precisions of the titration values obtained in the direct determination of the thiol function in some other compounds are shown in Table 111, and it can be seen that these values are of the same order, Le., 0.32 to 2-2 per cent., as those obtained previously in acid - base titrations in which acrylonitrile was used as the end-point indi~ator.~ When this366 GREENHOW AND LOO TABLE I1 RESULTS FOR PRECISION FROM THE THERMOMETRIC TITRATION OF MIXTURES OF DODECANE-1-THIOL WITH BENZOIC ACID, AND 2-MERCAPTOTHIAZOLINE WITH 3,5-XYLENOL, AGAINST 1.0 AND 0.1 M SOLUTIONS OF POTASSIUM HYDROXIDE Titration resuZts- Method* .. .. .. .. . . A B A B A B A B Compoundslmgt (i) Dodecane-l-thiol, 81.2 Titre/ml 0.683 0.390 0.681 0.391 0.678 0.388 0.678 0.391 plus benzoic acid, 48.2 } A-B/ml 0-293 0.290 0.290 0.287 1.004 0.492 1.012 0.494 1.016 0.500 1.016 0.496 0.512 0.518 0.516 0.520 Coeficients of variation of the differences in titration values obtained by using methods A and B- Indirectly from the individual coefficients of variation of methods A and B, per cent.[method (b)] Directly from the differences in sequential titration values, per cent. [method (a)] Mixture (i) .. 0.99 1-19 Mixture (ii) . . 0.67 0.90 * A, acetone-indicator method ; B, acrylonitrile-indicator method. t Dissolved in 1 ml of dimethylformamide. end-point indicator is suitable for the direct determination, it is possible to useO.001~ titrants and to determine thiols in amounts down to about 0.0001 mequiv, although this is possible only if other acidic functions are absent. TABLE I11 RESULTS FOR PRECISION FROM THE THERMOMETRIC TITRATION OF THIOLS AND THIOAMIDES WITH 1.0 AND 0.1 M POTASSIUM HYDROXIDE AND 0.1 M TETRA-n- BUTYLAMMONIUM HYDROXIDE REAGENTS Amount/ Thiol or thioamide mg 2-Mercaptobenzothiazole 14.8 2-Mercaptobenzothiazole 0.17 2-Thiohydantoin .. . . 7.12 Mercaptosuccinic acid . . 7.40 2-Mercaptobenzoic acid . . 67.5 4-Aminobenzenethiol . . 163.5 Dithiooxamide . . . . 6.0 Titration method* P P P P A A P Titrantt / Mean B, 0.1 3 0.87 B, 0.001 4 1.31 B, 0.1 4 0.58 B, 0.1 3 0.94 K, 1.0 3 0.84 K, 1.0 3 0.88 B, 0.1 3 0.97 M n$ titrelml Standard deviation 0.004 0.029 0.003 0-003 0.007 0.005 0.005 Coefficient of variation, per cent. 0.46 2.20 0.52 0.32 0.83 0.57 0.50 * P, acrylonitrile indicator; A, acetone indicator. t B, tetra-n-butylammonium hydroxide reagent ; K, potassium hydroxide reagent. $ Number of determinations. 1. 2. 3. 4. 5. 6. 7. 8. REFERENCES Ryland, L. B., and Tamele, M. W., in Karchmer, J. H., Editor, “The Analytical Chemistry of Miller, J. W., in Snell, F. D., and Ettre, L. S., Editors, “Encyclopedia of Industrial Chemical Greenhow, E. J., Chem. 6% Ind., 1973, 697. Greenhow, E. J., and Spencer, L. E., Analyst, 1973, 98, 90. Vaughan, G. A., and Swithenbank, J. J. Ibid., 1965, 90, 594. Greenhow, E. J., and Spencer, L. E., Ibid., 1973, 98, 98. -- , Ibid., 1973, 98, 485. Vauihan, G. A., and Swithenbank, J. J., Ibid., 1970, 95, 890. NOTE-References 4, 6 and 7 are to Parts 11, I11 and IV of this series respectively. Sulfur and its Compounds,” Part I, Wiley-Interscience Ltd., London, 1970, p. 465. Analysis,” Volume 15, Interscience Publishers Ltd., London, 1972, p. 551. Received December 4th, 1973 Accepted January 1 lth, 1974

ISSN:0003-2654    

DOI:10.1039/AN9749900360

出版商:RSC    

年代:1974

数据来源: RSC