摘要:

1062 Analyst, November, 1979, Vol. 104, PP. 1062-1069 Determination of Mercaptoacetic Acid in Hair Waving and Depilatory Products N. Goetz, P. Gataud and P. Bore L’Oreal Research Laboratories, 1 Avenue de Saint-Germain, 93601 A ulnay-sous-Bois, France Identification of mercaptoacetic acid in hair waving and depilatory products containing other mercapto acids is possible by using thin-layer chromato- graphy. However, gas-chromatographic methods are to be preferred for accurate quantitative -determination. - Four gas-chromatographic methods are pro- posed; the first and the second involve derivatisation of the mercaptoacetic acid with diazomethane to the methyl ester, methyl thioether derivative. In the third method, the mercaptoacetic acid is converted into its methyl ester by use of p-toluenesulphonic acid.The last method involves the direct chromatography of underivatised mercaptoacetic acid. Keywords : Mercaptoacetic acid; hair waving products ; depilatory Products ; gas - liquid chromatography Mercaptoacetic acid (MAA), often named thioglycolic acid, is the most frequently used compound in hair waving and depilatory products and it has been evaluated by acidimetry,l bromimetry,, c~lorimetry,~-~ polarography7 and potentiometry.8 The method usually employed up to now has been direct iodimetric determinationg-12; however, this method is only valid when the MAA is the only reducing agent present in the sample to be analysed. Methods have been developed for the determination of MAA in the presence of other reducing agents. These methods include iodimetric determination in the presence of sulphite, thiosulphate and ~ulphidel~,~* and in the presence of sulphite alone,15 and conductimetric determination in the presence of sulphite.ls However, we have not found any method in the literature that could be used to determine MAA in the presence of other mercapto acids (MA) such as 2-mercaptopropionic acid (MP,A), often named thiolactic acid, and S-mercapto- propionic acid (MP3A), often named @-mercaptopropionic acid.These mercapto acids are frequently mixed with MAA in the formulation of cosmetic products. Thin-layer chromatography quickly leads to identification of the acids and affords an approximate determination of the MAA content of a product. However, with mixtures, gas - liquid chromatography appears to be the best method for the accurate determination of MAA.This method is easier to apply than a method using high-performance liquid chromatography. In this paper we propose a thin-layer chromatographic method for the identification of MAA in mixtures, and four gas - liquid chromatographic methods for its accurate deter- mination. Chromatographic methods allow the desired separations to be carried out. Thin-layer Chromatographic Identification of Mercaptoacetic Acid Principle The sample to be analysed is acidified in order to transform any MAA salts present into free MAA, which is then subjected to analysis by thin-layer chromatography. Elution permits the separation of MAA from MP,A and MP,A, as well as 3-mercaptopropane-l,2-diol (MPD), often named thioglycerol, which is still used in certain countries.There are two methods of detection: the first, using potassium iodoplatinate, detects spots with a good sensitivity but is not specific; the second, using ammonium molybdate, is less sensitive but is more specific to t hiols. Reagents Unless otherwise stated, all the reagents are of analytical-reagent grade.GOETZ, GATAUD AND BORE 1063 Solutions 0.4% m/V in de-ionised water were prepared from MAA, mercaptoacetic acid Standard solutions disulphide (MAA),, MP,A, MP3A and MPD. Silica plate (Schleicher and Schull F1500 or similar). Ready-made 20 x 20 cm plastic plates, with a layer thickness of 0.25 mm, are used Develo@ment solvent Chloroform - glacial acetic acid (9 + 2 V/V). Detection reagents Reagent A .Mix, volume to volume, just before use 1% m/V potassium iodide solution in de-ionised water and 0.1 yo m/ V hexachloroplatinic acid hexahydrate solution, also in de- ionised water. Reagent B. This reagent consists of a 10% m/V solution of ammonium molybdate in de-ionised water. Apparatus Standard laboratory apparatus and thin-layer chromatographic materials are used. Procedure Treatment of samples (the use of indicator paper is sufficient). sample must be carried out. Acidify the sample with a few drops of concentrated hydrochloric acid until the pH is 1 In some instances a 10% dilution of the analysed If necessary, filter the resulting solution. Thin-layer chromatographic determination The solution to be analysed is spotted on to the plate, as are the different standard solutions.Drying should take place under nitrogen and all possible precautions should be taken to avoid oxidation of the thiol. The chromatogram is run to a solvent height of about 10 cm. Then spray the first plate with reagent A and the second with reagent B. The development with reagent B will be satisfactory only if the drying time has not exceeded 30 min. Results Compare the R, values and colours given by the standard solutions with those of samples. The R, values given below for guidance have only a comparative value. They depend on the activation state of the layer just as chromatography is carried out, the ambient tempera- ture and the saturation time of the chromatography tank. Mercapto compound R, MAA 0.80 0.35 0.95 MP,A (MAA), Mercapto compound R, MP3A 0.95 MPD 0.35 With a mixture, it is possible to determine roughly the amount of MAA present by operating at a dilution giving a weak spot and comparing the intensity of that spot with a standard scale.Gas - Liquid Chromatographic Determination of Mercaptoacetic Acid Principle and Reactions on the chromatogram that interfere with those of mercapto acids. acids have been isolated by precipitation as cadmium mercaptides. Hair waving and depilatory products are complex formulations; they often show peaks Accordingly, these last The uptake of the1064 GOETZ, GATAUD AND BORE : DETERMINATION OF AnaZyst, Vd. 104 mercaptide in methanol, in the presence of hydrochloric acid, is generally followed by methylation with diazomethane. This methylation gives, under the experimental con- ditions used, the dimethylated derivative ; the latter is then chromatographed by using methyl octanoate as the internal standard.The reactions are as follows. Mercaptide formation (HS-R-COOH);! where R = -CH2-(MA), precipitate. S-R-COO, YZ Cd --+ Cd' + 4CH3COOH 'S-R-COO, '/2 Cd I -CH-(MP,A) or -CH,-CH,-(MP,A). MPD mercaptide does not 8 Diazomethane formation Diazomethane is formed from N-methyl-N-nitroso-N'-nitroguanidine. NH-NOz I NH-NO;! I - '-'\N-C=NH __* HO-C=NH + CHZN2 CH,/ Diazomethane formation Diazomethane is formed from N-methyl-N-nitroso-p-toluenesulphonamide. y 3 CH3 I I ON-N-CH3 OR where ROH = diethylene glycol monoethyl ether. Methylation with diazomethane HS-R-COOH + 2CHZNz -+ CHS-S-R-COOCH, + 2N2 The first three proposed methods proceed by means of mercaptide precipitation and re-dissolution in an acidic medium.The first method, for which we give the full scheme for the analysis and relevant comments, uses diazomethane formed in sit% in a specific apparatus from N-methyl-N-nitroso-N'-nitroguanidine, as was described by Fales et aE.17 The second method differs from the first only in the use of diazomethane prepared in diethyl ether from N-methyl-N-nitroso-9-toluenesulphonamide, according to Fieser and Fieser.18 The third method employs MA methyl ester formation (without methylation of the thiol, by methanol, in the presence of 9-toluenesulphonic acid. Finally, the fourth method suggests the use of direct MAA chromatography, without methylation or isolation from the mixture.Derivatisation Using Diazomethane Formed in situ (Method I) Apparatus is used, with an LTT, Model 8733, integrator. the preparation of diazomethane, as described by Fales et aZ.17 A gas chromatograph equipped with a flame-ionisation detector, such as a Packard A7400, Semi-micro scale apparatus is required forNovember, 1979 MERCAPTOACETIC ACID IN HAIR WAVING AND DEPILATORY PRODUCTS Reagents 1065 All reagents are of analytical-reagent grade. Mereaptoacetic acid, 2-mercaptopropionic acid and 3-mercaptopropionic acid. Concentrated hydrochloric acid, 35%. Methanol. Cadmium acetate [Cd(CH,C0,),.2H20], 10% m/V solution. Methyl octanoate, 2% m/V solution in methanol. Methyl decanoate, 2% m/V solution in methanol. Acetate bufler solution, PH 5. Methanolic hydrochloric acid, 3 N.Freshly prepared. N-Methyl- N-nitroso-N '-nitroguanidine . Sodium hydroxide solution, about 5 N. Iodine solation, 0.1 N. Diethyl ether. The con- centrations of these acids were checked by iodimetry. This is prepared from sodium acetate (77 g), acetic acid (27.5 ml) and water (1 000 ml). Procedure The sample.should be taken from a new container, which has not previously been opened. Into a 50-ml centrifuge tube, weigh accurately an amount of sample containing 50-70 mg of MAA. Acidify the sample with a few drops of concentrated hydrochloric acid until the pH is about 3. Next add 5 ml of de-ionised water and 10 ml of acetate buffer solution and check with indicator paper that the pH is about 5. Then add 5 ml of cadmium acetate solution, wait 10 min and centrifuge for at least 15 min at 4000 g.Separate the supernatant liquid. This may contain insoluble fatty material, which should not be mistaken for the cadmium mercaptide collected in a compact precipitate at the bottom of the tube. Check the amount of reducing agents in the supernatant. If there is no reducing agent other than MA in the formulation, the result of this iodimetric determination should not exceed 643% of the previous determination from the whole formulation. Introduce 10 ml of methanol into the centrifuge tube containing the precipitate. Next, disperse the precipitate completely with a glass rod and centrifuge the solution again for a t least 15 min a t 4000g. Pour off the supernatant liquid and check that there has been no MA loss, as was described above; carry out a second washing, also as described above.Then add to the contents of the centrifuge tube 2 ml of the methyl octanoate solution and 5 ml of the methanolic hydrochloric acid solution. Dissolve the mercaptide completely (a slight amount of insoluble material, owing to the excipient, may remain), thus obtaining solution S. To the methylation apparatus containing 1 ml of diethyl ether transfer 50 p1 of solution S by use of a syringe. Carry out methylation according to the method of Fales et a1.l' with about 300 mg of N-methyl-N-nitroso-N'-nitroguanidine. After 15 min check that the ethereal solution is yellow (evidence of an excess of diazomethane) and transfer it into a 2-ml hermetically closed flask. After methylation, inject 3 p1 of this solution R into the chromatograph.Meanwhile, check the MA content of the remaining solution S by iodimetry; this must reach at least 90% of the value obtained previously. Carry out five assays on every methy- lated sample and two methylations simultaneously. MAA standard preparation. Into a 50-ml calibrated flask, accurately weigh about 1500 mg of MAA. Make up the volume to 50 ml with de-ionised water; the resulting solution is solution E. Introduce, with a pipette, exactly 2 ml of solution E into a 50-ml centrifuge tube. This solution contains m't mg of MAA per millilitre. Sample preparation. Place it in a refrigerator overnight. M ' x 2 x T 60 m't = where T is the volume of MAA titrated and M' is the amount of MAA taken. Carry out the precipitation, determinations and methylation as previously described.Stainless-steel column, 2 m in length, Q in in diameter, packed with 10% didecyl phthalate on Chromosorb W AW, 80-100 mesh. The carrier gas is Chromatographic details.1066 Analyst, Vol. 104 nitrogen at a flow-rate of 35 ml min-l. The injector and detector temperatures were 200 O C , and the column temperature was between 100 and 110 "C. The new column was con- ditioned by successive injections of the standard mixture until reproducible peaks were obtained. GOETZ, GATAUD AND BORE : DETERMINATION OF Peak areas were measured with an integrator (Fig. 1). Time --+ Fig. 1. Chromatogram of mercaptoacetic acids deriva- tives after methylation with diazomethane. Methyl octa- noate is the internal standard. 1, CH&CH,-COOCH,; 2, CHa-CH(SCHJ-COOCH,; 3, CH+CH&H,-COOCH3 ; and 4, C,H,,COOCH,.Calculations MA A proportionality coeflcient. The coefficient is calculated with respect to methyl octanoate, from the standard mixture. If a is mercaptoacetic acid, k , is its proportionality coefficient, m', its mass (in milligrams) in the mixture and Sfa its peak area, and if o is methyl octanoate, m', is its mass (in milligrams) in the mixture and So its peak area, then m', Sf0 k a = - 7 x - T mo s , Amount of MAA in the sample. If a is mercaptoacetic acid, ka is its proportionality coefficient and Sa its peak area, and if o is methyl octanoate, mo is its mass (in milligrams) in the mixture, So its peak area and M the initial mixture mass, then the percentage (m/m) of MAA in the sample is given by Derivatisation Using a Solution of Diazomethane in Ether (Method 11) Methylation, by use of an excess of diazo- methane (the solution remaining yellow overnight), gives good results. It is easier to perform this procedure than that using the semi-micro scale apparatus, but as diazomethane is a toxic and very unstable gas it is necessary to carry out all of the experiments in a powerful fume cupboard, and also to avoid the use of apparatus with ground-glass joints.This method is a variation of the first one. Exfierimental Fieserl* from N-met hyl-N-nitroso-9- t oluenesulphonamide . Diazomethane solution in diethyl ether is prepared according to the method of Fieser and The met hylat ion of solution SNovember, 1979 MERCAPTOACETIC ACID IN HAIR WAVING AND DEPILATORY PRODUCTS 1067 (see Sample preparation) is carried out by adding 1 ml of diazomethane solution to 50 p1 of solution S contained in a test-tube.The mixture is left to stand overnight in a refrigerator. For the continuation of this procedure, see the method using diazomethane formed in situ (Method I). Derivatisation Using p-Toluenesulphonic Acid (Method 111) precipitation but avoids the use of diazomethane. presence of p-toluenesulphonic acid, methylates only the carboxylic acid function. This method is also a variation of the first one in that it involves the use of mercaptide Methylation with methanol, in the Exfierimental Following mercaptide precipitation (see Method I) and washing of the precipitate with methanol, add a 20y0 methanolic solution of P-toluenesulphonic acid in sufficient amount to dissolve the precipitate, that is release MA, forming solution S'.To 2 ml of solution S' add the standard solution containing methyl decanoate (see Method I) and maintain the mixture at 65 "C for 90min (solution M). Then inject the solution according to the procedure described for Method I. NOTE- We carried out an assay by injecting solution M on to a 0.3 mm diameter and 40 m long glass capillary column, wall-coated with FFAP stationary phase (Carbowax 20M modified by nitroterephthalic acid). The oven temperatures were adjusted to 160 "C for the injection port, 200 "C for the detector and 150 "C for the column. The helium carrier gas flow-rate was 3 ml min-l. The results are shown in Fig. 2. 1 2 1 Time -b Fig.2. Chromatogram of mercaptoacetic acid methyl ester, after methylation with methanol, in presence of p - toluenesulphonic acid. Methyl decanoate is internal standard. 1, HS-CH,COOCH,; and 2, C,H,,COOCH,. This system has been selected to minimise adsorption due to the polarity of the free thiol It avoids contact between the solute and the injector, the column metal and the group. column support. Direct Gas - Liquid Chromatography of Underivatised MAA (Method IV) The determination of MAA as free acid is possible, using the following chromatographic conditions: a stainless-steel column, 2 m in length and Q in in diameter, packed with 15%1068 GOETZ, GATAUD AND BORE : DETERMINATION OF Analyst, VOZ. 104 diethylene glycol polysuccinate (DEGS) and 2% orthophosphoric acid on Chromosorb W AW, 80-100 mesh; nitrogen as the carrier gas at a flow-rate of 35 ml min-l; injector and detector temperatures of 200 "C; and a column temperature of 170 "C.The column is conditioned by a flow of carrier gas loaded with formic acid vapour. It is to be noted that this treat- ment changes the retention characteristics and the efficiency of the column. However, after this treatment, the MAA is no longer adsorbed. Results and Discussion A few results are summarised in Table I. TABLE I RESULTS FOR THE DETERMINATION OF MERCAPTOACETIC ACID IN HAIR WAVING AND DEPILATORY PRODUCTS BY GAS - LIQUID CHROMATOGRAPHY Recovered, % m/V (2 f amt*) Expected, f A > Sample % miv Method I Method I1 Method I11 Method IV Waving lotion (a) 7.30 7.09 + 0.04 7.25 & 0.11 (20 assays) (10 assays) Depilatory cream (a) 3.30 3.32 f 0.06 3.25 f 0.05 (b) 10.00 (20 assays) (10 assays) (5 assays) (5 assays) (5 assays) (5 assays) 3.00 3.10 f 0.05 2.71 f 0.05 6.20 6.20 f 0.06 6.00 f 0.08 (b) (4 * omt calculated with Student - Fisher tables; P = 0.05.9.8 & 0.10 The good reproducibility of the MAA proportionality coefficient with respect to methyl octanoate has been checked: The use of an internal standard eliminates the uncertainty regarding the injected amount. The validity of the four methods has been tested on samples prepared for this purpose. The results proved consistent with those expected and in agreement with iodimetric determinations when MAA was the only reducing agent present. For depilatory cream (b) the differences between methods I and I11 can result from a variation in the proportionality coefficient (there is, in this instance, an excess of methyl octanoate in relation to MAA) or, more probably, from the discrimination of the splitter used for the capillary column.Concerning sample preparation, the following observations are made. Cadmium mercaptide precipitation has the advantage of isolating the MA from a complex medium, of obtaining a non-oxidisable compound and of eliminating water, which hinders methylation ; however, this methylation is necessary in classical chromatography in order to reduce the polarity of MA and to avoid irreversible adsorptions. The experimental procedure must be closely adhered to. It is impossible to avoid a loss of about 5% of MAA in the supernatant, but treatment under the same conditions of a standard MAA solution eliminates this source of error.It has been shown that the mercaptide is stable for 1 h, as is the MAA regenerated by mercaptide acidification before methylation. Thiol regeneration by use of methanolic 3 N hydrochloric acid has proved to be the most suitable technique; for example, the use of trifluoroacetic acid leads to a set of chromato- graphic peaks and the use of 3 N hydrochloric acid in methyl acetate does not effect the total release of MA. Diazomethane allows a complete methylation of carboxylic acid and thiol functions (checked by use of nuclear magnetic resonance spectrometry). Its formation in situ in the specialised semi-micro apparatus avoids safety problems. All of the methyla- tion assays carried out with boron trifluoride in methanol led to mixtures of monomethylated (-COOH derivatives only) and dimethylated compounds (Fig. 3).We have shown that the reducing thiol function was maintained for the first peak (HS-COOCH,) by interposing a splitter (9 : 1) before the flame-ionisation detector; the collected fraction reduces a drop of a 10% aqueous solution of ammonium molybdate deposited on paper (molybdenum blue formation). It also bleached a drop of 0.01 N iodine solution deposited on paper. No reduction was observed for the second peak (H,C-S- COOCH,). & omt = 2.11 & 0.036 (20 assays).November, 1979 MERCAPTOACETIC ACID IN HAIR WAVING AND DEPILATORY PRODUCTS 1069 2 Time __+ Fig. 3. Chromatogram of mercaptoacetic acid deriva- tives after methylation with COOCH,; and 2, CHaS- BF, - CH,OH.1, HS-CHS- CH&OOCH 8. Conclusion The four procedures described have been successfully applied to the analysis of mercapto- acetic acid (MAA) in hair waving products and depilatories even when thiol mixtures are present. If the laboratory uses classical apparatus and columns, the methylation with diazomethane in semi-micro apparatus (Method I) will be preferred. The choice of the method depends on the laboratory equipment. The authors express appreciation to P. Lasserre and G. Kaba for their technical assistance and thank Mr. G. Kalopissis for permission to publish this work. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Larsson, E., 2. Analyt. Chem., 1929, 79, 170. Hellstriim, N., Chem. Zentbl., 1933, 11, 3889. Shinohara, K., J . Biol. Chem., 1936, 112, 671. Hirsh, F., Seifen-Ole-Fette- Wachse, 1951, 77, 457. Walker, G. T., and Freeman, F. M., Mfg Chem., 1955, 26, 11. Walker, G. T., Inds Parfum. Cosmet., 1955, 10, 236. Liberti, A,, and Cervone, E., Annuli Chim., 1951, 41, 95. Vandeputte, M., Analusis, 1975, 3, 500. Mayr, C., and Gerauer, A., 2. Analyt. Chem., 1938, 113, 198. Hoshall, E. M., J . Ass. 08. Agric. Chem., 1940, 23, 737. Walker, G. T., Soap Perfum. Cosm., 1954, 27, 1050. Forster, H., and Meyer, A., Mitt. Lebensmittel Hyg., 1954, 45, 490. Bucci, F., Annuli Chim., 1952, 42, 193. Strache, F., and Mierau, H. J., Dt. ApothZtg, 1955, 95, 55. Wronski, M., Chemia Analit., 1962, 7, 851. Freytag, H., 2. Analyt. Chem., 1953, 137, 331. Fales, H. M., Jaouni, T. M., and Babashak, J. F., Analyt. Chem., 1973, 45, 2302. Fieser, L. F., and Fieser, M., “Reagents for Organic Synthesis,” John Wiley, New York, 1967. Received February 19th, 1979 Accepted May 2nd, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790401062

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

1070 Analyst, November, 1979, Vol. 104, fip. 1070-1074 Analytical Methods Committee REPORT PREPARED BY THE METALLIC IMPURITIES IN ORGANIC MATTER SUB-COMMITTEE Determination of Small Amounts of Nickel in Organic Matter by Atomic-absorption Spectrometry The method for the determination of nickel in organic matter is based on the atomic-absorption procedure already shown to be applicable to the deter- mination of lead; both nickel and lead can therefore be determined on the same analytical sample. Organic matter is destroyed by wet oxidation in the presence of sulphuric acid. After dilution, the nickel (and lead) are recovered from the aqueous acid solution by extraction of the complexes formed with ammonium tetramethylenedithiocarbamate complexes into 4- methylpentan-2-one. The nickel content is determined by aspirating the extract into an air - acetylene flame, measuring the atomic absorption at 232.0 nm and comparing the resulting signal with a calibration graph prepared by submitting standard nickel solutions to an identical procedure.It is stressed that practice in sample preparation must be thorough before results can be reliable. Keywords Nickel determination ; wet oxidation ; ammonium tetramethylene- dithiocarbamate ; atomic-absorption spectrometry The Analytical Methods Committee has received and approved for publication the following Report from its Metallic Impurities in Organic Matter Sub-committee. Report The constitution of the Sub-committee responsible for the preparation of this report was : Dr. L. E. Coles (Chairman until July 1978), Mr.C. A. Watson (Chairman from July 1978), Mr. W. Cassidy, Mr. P. N. Coleman (Honorary Secretary from September 1977), Mr. R. E. Collier, Dr. W. H. Evans, Mr. M. T. Friend (appointed December 1977), Mr. S. Greenfield, Mr. W. H. Hill, Mr. B. E. Pearce, Mr. W. L. Sheppard (retired July 1976) and Dr. J. M. Skinner (appointed February 1977), with Mr. P. W. Shallis (until September 1977) and (the late) Dr. N. W. Hanson (from October 1977) as Secretaries. Introduction This Report describes the investigation of the applicability of the method for the deter- mination of leadl to the determination of nickel in organic matter, primarily as a means of obtaining data on lead and nickel together in the analysis of food. One member of the Sub-committee, with previous experience in the use of ammonium tetramethylenedithiocarbamate (ammonium pyrrolidine dithiocarbamate, APDC) for the determination of nickel, reported good recoveries when using the procedure published by the Sub-committee for the determination of lead.The Sub-committee felt, therefore, that both lead and nickel could be determined in the same analytical sample using the one extract of the combined APDC complexes. Experimental Sample preparation, prior to the measurement of nickel, consisted of wet oxidation using sulphuric acid and, where possible, soy0 hydrogen peroxide. 293 Some members, however, used either 30% hydrogen peroxide or nitric acid as the oxidant. Details of the method are set out in the Appendix. Two members of the Sub-committee reported on the possible interferences in the analysis by copper and iron.As the levels of these elements required to cause the interferences were higher than normally found in samples likely to be analysed by this method, the investigationANALYTICAL METHODS COMMITTEE 1071 proceeded with the cautionary note that recoveries of nickel from samples containing more than about 0.2 mg of copper and/or 2 mg of iron could be low. Another member investigated the effects of varying sulphuric acid concentrations on the recovery of nickel, but found no differences when extracting from acid concentrations of between 1 and 10% (V/V), or from acid buffered with sodium acetate up to pH 10. The same member also reported no difference in response in the air - acetylene flame set either fuel rich or lean. Initially a collaborative exercise, using full cream condensed milk, was carried out on a sample as received and on a sample containing 0.55 mg k g l of added nickel.Most of the Sub-committee members repeated the exercise when experience in the wet-oxidation tech- nique had been acquired. The results obtained are given in Table I. TABLE I DETERMINATION O F NICKEL I N FULL CREAM CONDENSED MILK The results were not corrected for any reagent blanks. Laboratory Run A .. .. 1st 2nd B .. . . 1st 2nd c .. . . 1st 2nd D .. . . 1st 2nd 3rd 4th - E .. .. Nickel found in sample/ <0.05, (0.05, <0.05 0.04, 0.0, 0.04 0.10, 0.09, 0.08, 0.04 0.07, 0.10, 0.07 0.22, 0.21 0.04, 0.04 0.07, 0.09 0.0, 0.02, 0.03 0.04, 0.05 mg k g ' 1 <0.01 - Mean/ ng kg-1 < 0.05 0.03 0.08 0.08 - - 0.22 0.04 0.80 0.02 0.05 Nickel found in sample + 0.55 mg kg-1 of added nickel/ mg kg-l 1 0.48, 0.44, 0.48 0.56, 0.53, 0.60 0.62, 0.59, 0.60 0.58, 0.60, 0.62 0.30, 0.31, 0.27 0.53, 0.57 0.70, 0.70 0.51, 0.51 0.60, 0.64 0.53, 0.51, 0.61 0.63, 0.65 Mean/ ng kg-l 0.47 0.56 0.60 0.60 0.29 0.55 0.70 0.51 0.62 0.55 0.64 Average recovery, % -85 96 95 95 53 100 87 85 98 96 107 The second collaborative exercise was carried out on three samples of detergent, all as received and with no added nickel.The results obtained are given in Table 11. TABLE I1 DETERMINATION OF NICKEL IN DETERGENT SAMPLES Nickel found/mg kg-l A r 1 Sample 1 Sample 2 Sample 3 v L z i z z - - * Laboratory results Mean results Mean results Mean B .. . . 2.2, 2.3, 2.4 2.3 3.4, 3.3, 3.2 3.3 2.3, 2.4, 2.3 2.3 D ... . 2.5, 2.4, 2.3, 2.1 2.3 3.5, 3.4, 3.1 3.3 2.4, 2.5 2.5 E .. . . 2.3, 2.4 2.4 3.1, 3.2 3.2 2.7, 2.8 2.8 It should be noted that the detergent samples used in this exercise were historical samples, which could well have had higher nickel levels than current commercially available materials. The third collaborative exercise was carried out on Standard Biological Kale samples, kindly supplied by Dr. H. J. M. Bowen of Reading University; only nine analysts had determined nickel, the range being 0.76-1.1 mg k g l by atomic-absorption spectrometry and neutron-activation analysis, with an outlier of 2.6 mg kg-l by atomic-emission spectro- s c o ~ y . ~ The results obtained by the members are given in Table 111. The fourth collaborative exercise was carried out on three samples of olive oil: oils as received, oil with 2.0 mg k g l of added nickel and oil with 1.2 mg kg-l of added nickel.Nickel was added to the olive oil as a solution of nickel 4-cyclohexylbutyrate in 2-ethyl- hexanoic acid. The results obtained are given in Table IV.1072 ANALYTICAL METHODS COMMITTEE : DETERMINATION OF AnaZyst, VoZ. 104 TABLE I11 DETERMINATION OF NICKEL IN KALE Laboratory Nickel found/mg kg-l Meanlpg k g l A .. . . 0.73, 0.75, 0.80 0.76 B .. . . 0.75, 0.80, 0.90 0.82 D .. . . 0.79, 0.81, 0.82, 0.84 0.82 F .. . . 0.73, 0.76, 0.76 0.75 The sample with 1.2 mg kg-l of added nickel was issued without the participants being aware of the level of addition, whereas they were informed of the 2 mg kg-l addition.The percentage recovery on the blind spike was calculated after subtracting the level found in the oil as received. TABLE IV DETERMINATION OF NICKEL IN OLIVE OIL It should be noted that some of the results were not corrected for reagent blanks. Laboratory Run - A .. .. ,. B .. ,. .. 1st 2nd 3rd 4th - c .. .. .. D .. .. .. 1st 2nd 3rd E .. .. .. F .. .. .. 1st 2nd - Nickel found in oil + Nickel found in oil/ Mean/ 2.0 mg kg-l of added Mean/ mg kg-l mg kg-l nickel/mg kg-l mg kg-l 0.0, 0.04 0.02 2.44, 2.27 2.36 0.08, 0.09 0.09 1.82, 1.92 1.87 0.0, 0.05 0.03 1.65, 1.75 1.70 0.63, 0.67 0.60 2.54, 2.39 2.47 0.05 1.87, 1.83 1.85 0.06, 0.03 0.05, 0.06, 0.08 0.06 (2.0) 6 results 0.08 6 results 1.82 0.19, 0.24, 0.20 0.21 2.35, 2.38, 2.15 2.29 1.02, 0.70 0.86 3.11, 3.03 3.07 0.12, 0.04 0.08 2.30, 2.25 2.28 - - (2.0) - (0) 0.06, 0.06, 0.05 0.06 (2.0) - Nickel found in oil + 1.2 mg kg-l of added Mean/ Recovery, nickel*/mg kg-l mg kg-l '$(, 1.27, 1.27 1.27 104 0.13, 0.11 0.12 3 1.10, 0.89 1.00 81 1.55, 1.34 1.47 73 90 1.14, 3 - 88 1.05 - 1.10, 1.17, 1.11 1.13 89 1.20, 1.28, 1.24 1.24 98 6 results 1.17 91 1.25, 1.51, 1.40 1.39 98 1.98, 2.21 2.10 103 1.31, 1.33 1.32 103 *Level of addition not known by participants. The results shown in Table IV from laboratories C and D were obtained by members when the oil with 2 mg kg-l of added nickel was used to calibrate their instruments at the 2 mg kg-1 level.Both of these members also made their atomic-absorption measurements by direct aspiration of the oils after straightforward dilution with 4-methylpentan-2-one, again using the 2 mg k g l addition of nickel as the standard for calibration.Laboratory C recovered 1.2 mg kg-1 from the third oil sample, whilst Laboratory D recovered 1.15 mg k g l , an average result of six determinations. Clearly, the greatest difficulty in sample preparation was experienced with the olive oil samples, which were new to most members. The members with experience in dealing with oil samples achieved good results in the first run. It cannot be urged strongly enough, therefore, that practice in sample preparation of organic materials for trace-metal analysis must be thorough before results can be reliable, and analysts should conduct recovery experi- ments to verify the methods in their own laboratories. Recommendation The Sub-committee recommends that the method described in the Appendix be used for the determination of small amounts of nickel in organic matter. APPENDIX Method for the Determination of Nickel Principle methylenedithiocarbamate. this solution is aspirated into the burner chamber of the atomic-absorption spectrometer.After destruction of the organic matter, the nickel is complexed with ammonium tetra- The complex is then extracted into 4-methylpentan-2-one andNovember, 1979 SMALL AMOUNTS OF NICKEL IN ORGANIC MATTER BY AAS 1073 Reagents 4-MethylPentan-2-one. Analytical-reagent grade. Ammonium tetramethylenedithiocarbamate solution. Place approximately 1.5 g of APDC in a porosity 4 sintered-glass crucible, wash with 20 ml of acetone and suck dry by means of a water pump.Weigh 1.Og of the washed and dried APDC, dissolve it in 1 O O m l of water containing 0.1 g of mercaptoacetic acid and adjust the pH of the solution to between 6 and 7. Standard nickel solution. Dilute commercial stock 1 000 pg ml-1 solution to give a working standard of 10 pg ml-l. Alternatively, dissolve 1.000 g of pure nickel in 100 ml of 10% V/V nitric acid, cool, and dilute to 1 1 with distilled (or de-ionised) water to give a stock 1000 pg ml-l solution for further dilution. Sulphuric acid, concentrated. Analytical-reagent grade. Hydrogen peroxide, 50% m/V. Nitric acid, concentrated. Analytical-reagent grade. Analytical-reagent grade. Apparatus cathode lamp. Use an atomic-absorption spectrometer with an air - acetylene burner and a nickel hollow- Procedure Preparation of the sample solution Destroy the organic matter in an appropriate amount of sample (see Note), using the recommended methods of the AMC.2,3p5 When using 50% hydrogen peroxide it is essential that the instructions g i ~ e n ~ , ~ are closely followed for the particular type of sample being anal ysed .About 5ml of concentrated sulphuric acid will usually be necessary for the digestion, to give the optimum concentration of 10% (V/V) for the ultimate determination of lead, should lead and nickel be determined together. Analysts wishing to determine lead are recommended to refer to the previous report of the Sub-committee on that spbject.l If hydrogen peroxide is used in the oxidation, either sulphur dioxide gas or sodium sulphite solution, 10% m/V, is used to destroy excess of the oxidant.Excess of nitric acid can be destroyed either by two or three successive evaporations to fuming sulphuric acid between small additions of water, or by the addition of a small amount (about 0.1 g) of ammonium oxalate with a small volume of water to the reaction vessel prior to the final evaporation to sulphuric acid fumes. It is essential, for the stability of the APDC complexes, to destroy all excess of the oxidant. When oxidation is complete, dilute the cold solution to approximately 50 ml with water. Transfer the solution into a separating funnel by means of the minimum volume of water. Add 2 ml of the APDC solution, shake and set aside for 5 min. Accurately add 10.0 ml of 4-methylpentan-2-one and shake vigorously for 1 min. Allow the layers to separate, discard all of the aqueous layer and filter the organic layer through a small dry filter-paper into a suitably stoppered vessel. Calibration Prepare standard solutions, to cover the required range of nickel contents, by adding portions of the standard nickel solution to 5 ml of sulphuric acid and diluting each standard to 50 ml with water; proceed as described for the sample solution beginning with the addition of APDC reagent.Measurement Set up the spectrometer with a nickel hollow-cathode lamp and the monochromator adjusted to 232.0 nm, using the manufacturer’s recommended procedures. Aspirate 4- methylpentan-2-one into the flame when setting the zero of the instrument and between all readings. Care must be taken with some burner assemblies, particularly the three-slot1074 ANALYTICAL METHODS COMMITTEE Boling type, when changing solutions, as the removal of the ketone renders the flame weak, with a tendency to flash back. Prepare a calibration graph by aspirating the standard solution extracts, and plotting the mean signal response against the nickel content. Linearity of the calibration graph will depend on the instrumentation employed, but the relationship should be linear up to about 20-50 pg of nickel, i.e., 2-5 pg ml-l in the solution that is aspirated. It is recommended that frequent checks on the zero and calibration be made when handling large numbers of samples. NOTE- As stated in the section on Measurement, the upper limit of the nickel content will be dictated by the Therefore, the sample size taken should be chosen so as not to exceed that type of instrument used. limit. References 1. 2. 3. 4. 6. Analytical Methods Committee, Analyst, 1975, 100, 899. Analytical Methods Committee, Analyst, 1967, 92, 403. Analytical Methods Committee, Analyst, 1976, 101, 62. Bowen, H. J. M., personal communication. Analytical Methods Committee, Analyst, 1960, 85, 643.

ISSN:0003-2654    

DOI:10.1039/AN9790401070

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, November, 1979, Vol. 104, p p . 1075-1082 1075 Analytical Methods Committee REPORT PREPARED BY THE ANTIBIOTICS IN ANIMAL FEEDINGSTUFFS SU B-COM M ITTEE Microbiological Assay of Avoparcin in Animal Feeds and Pre-mixes A microbiological method for the determination of avoparcin in animal feeds (10-20 mg kg-l; lowest limit of determination 2 mg kg-l) and pre-mixes (20 g kg-1) is described. The method has been submitted to collaborative study by a UK Committee and six other EEC Member States, and gave mean recoveries of 92.8, 104.2 and 101.8~o with standard deviations 17.6, 14.0 and 14.6% absolute from chick mash, pig meal and pre-mix, respectively. In the method, the sample is extracted with a mixture of acetone, water and hydrochloric acid, and the antibiotic activity of the clarified extract is deter- mined by measuring the diffusion of avoparcin in an agar medium inoculated with Bacillus subtilis.Keywords : Avoparcin assay ; antibiotics ; animal feeds ; microbiological assay The Analytical Methods Committee has received and approved for publication the following Report from its Antibiotics in Animal Feedingstuffs Sub-committee. Report The constitution of the Sub-committee responsible for the preparation of this Report was: Mr. R. S. Hatfull (Chairman), Mr. M. Bentley, Mr. R. Goodey, Mr. H. L. Hatfield, Mr. A. F. Lott, Mr. G. S. Meadows, Mr. G. H. Palmer, Mr. R. Ryden, Mr. R. Smither and Mr. L. D. Ward, with (the late) Dr. N. W. Hanson as Secretary. Introduction Avoparcin is an antibiotic produced by a strain of Streptomyces candidus and is used as an antibiotic growth promoter for improving growth rates and feed conversion efficiency of broiler chickens and pigs.The normal levels of inclusion for avoparcin in feeds are in the range 5 4 0 mg kg-l. Very little published information on the assay of avoparcin was available when the Sub- Committee began its work. A member of Cyanamid of Great Britain Limited, the company that manufactures avoparcin in the UK, was co-opted to the Sub-Committee with the result that access was obtained to a Company method. Experimental and Results The Company method referred to above was a microbiological agar diffusion assay using Bacillus sztbtilis. It appeared to be straightforward but its design was judged unlikely to be favoured by the Commission of the European Economic Community (EEC), which had published official methods for certain other antibiotics1y2 and was known to be preparing further methods.Furthermore, the Company method involved the preparation of standard solutions in an autoclaved extract of the feed sample, an idea that had proved unsuccessful during the Sub-Committee’s work on another antibiotic. Accordingly, before any serious practical work was undertaken the method was edited into the now established EEC form. Preliminary trials yielded good results using standard solutions made up in plain buffer solution. Initially the method was assessed in five laboratories on a pig feed already medicated at 20 mg kg-l and broiler feed already medicated at 20 and 10 mg k g l . Some laboratories used the more readily available B.suubtilis ATCC 6633 in place of the B. subtilis ATCC 11774 originally specified in the method. As the results (Table I) were generally1076 ANALYTICAL METHODS COMMITTEE : MICROBIOLOGICAL Analyst, VOl. 104 satisfactory, the method was submitted to the EEC Expert committee on Microbiological Determination of Antibiotics in Feedingstuffs. A larger collaborative trial followed in which the laboratories of the Sub-committee were joined by laboratories in six other EEC Member States. The method detailed in the Appendix (except for the culture strain) was applied to a pre-mix (nominally containing 20 g k g l of avoparcin) supplied by the manu- facturer and to a centrally supplied pig feed and poultry feed medicated to 20 and 10 mg kg1, respectively, by the addition of the pre-mix in each laboratory.The results presented in Tables II-IV include, by agreement with the EEC Expert Committee, those from the seven overseas laboratories together with those from the Sub-committee members. TABLE I DETERMINATION OF AVOPARCIN I N ANIMAL FEEDS BY THE RECOMMENDED METHOD: FIRST UK TRIAL Avoparcin was added to the feeds by the manufacturer. Avoparcin found/mg kg-1 I A I Laboratory Broiler feed a t 10 mg kg-l A 9.4, 11.0 20.0, 23.8 B 9.6, 10.6 19.6, 19.2 C 11.3 22.6 D* 8.9, 8.7 21.0, 20.4 E* 11.0, 11.0 21.0, 21.6 Over-all mean .. .. 10.1 21.0 Standard deviation . . .. 1.02 1.47 Pig feed a t 20 mg kg-1 * B. subtzlis ATCC 6633 used. ATCC 11774 used by remainder. A questionnaire circulated to all participants in the latter trial enabled a few points of detail to be clarified.(i) Both B. subtilis cultures appeared to be satisfactory. A few workers with experience of both cultures preferred ATCC 6633, which additionally has the advantage of being com- mercially available as a spore suspension. (ii) Centrifuge tubes must be well stoppered to prevent loss of acetone by evaporation. (iii) Commercially available assay media from Difco and Oxoid behaved satisfactorily, although a similar product from another manufacturer did not in one laboratory. A small proportion of assays produced results that were considered to be invalid owing to non-parallelism of response between sample and standard. These results have been excluded from the tables. The avoparcin pre-mix currently marketed in the UK contains 5% of avoparcin and not 2% as used in the trials, but its composition is otherwise unchanged. The Sub-committee recommends that the method in the Appendix should be used for the determination of avoparcin in animal feeds and pre-mixes.It will be noted that the Evaluation section differs from that in the method published for monensin.3 The difference is attributable to protracted discussions within the EEC Expert Committee ; in practice both calculation procedures lead to closely similar results. APPENDIX Recommended Method for the Microbiological Determination of Avoparcin Scope and Field of Application stuffs and pre-mixes. The lowest limit of the determination is 2 mg k g l . The method is for the determination of the concentration of avoparcin in complete feeding-November, 1979 ASSAY OF AVOPARCIN IN ANIMAL FEEDS AND PRE-MIXES TABLE I1 DETERMINATION OF AVOPARCIN IN CHICK MASH BY THE RECOMMENDED METHOD : EEC TRIAL Avoparcin pre-mix was added to the feed within each laboratory.Amount of avoparcin/mg kg-1 Laboratory F* G H* Jt K* L* M* N O* P* Q* R S* T* Over-all mean .. .. .. .. . . .. .. . . .. .. .. .. .. .. .. Standard deviation . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. Added 10 10 10 10 10 10.08 10.52 10.50 10 10 10 10 10 10 10 10 10 10 10 10 10.04 10.04 10.32 10 10 10 20 10 10 10 10 10 10 - Found 7.75 8.15 8.13 10.00 8.33 10.20 9.13 8.90 7.42 8.52 7.79 9.16 9.10 9.13 8.76 9.40 9.20 8.86 10.69 11.83 8.38 8.41 6.42 9.11 9.43 8.71 17.38 12.30 13.40 14.20 9.40 9.10 9.40 - Recovery, % 77.5 81.6 81.3 100.0 83.3 101.2 86.8 84.8 74.2 86.2 77.9 91.6 91.0 91.3 87.6 94.0 92.0 88.6 106.9 118.3 83.6 83.8 62.2 91.1 94.3 87.1 86.9 123.0 134.0 142.0 94.0 91.0 94.0 - Mean recovery, % 80.1 91.7 90.9 79.7 86.8 91.0 90.3 112.6 76.5 92.7 87.0 133.0 93.0 92.8 16.34 1077 * B.subtilis ATCC 6633 used throughout. t Both ATCC 6633 and ATCC 11774 used. ATCC 11774 used by remainder. Principle After centrifuging, the antibiotic activity of the clarified extract is determined by measuring the diffusion of avoparcin in an agar medium inoculated with Bacillus subtilis. Diffusion is shown by the formation of zones of inhibition of the micro-organism. The diameter of these zones is taken to be in direct proportion to the logarithm of the antibiotic concentration over the range of antibiotic concentrations employed.The sample is extracted with a mixture of acetone, water and hydrochloric acid. Micro-organism The micro-organism is Bacillus subtilis ATCC 6633 (NCIB 8054). Maintenance of the parent strain overnight at 30 "C. the agar slopes every 4 weeks. Inoculate B. sztbtilis on to an agar slope prepared from the culture medium and incubate Store the culture in a refrigerator at about 4 "C and re-inoculate on to1078 ANALYTICAL METHODS COMMITTEE : MICROBIOLOGICAL Analyst, Vd. 104 TABLE I11 DETERMINATION OF AVOPARCIN I N PIG MEAL BY THE RECOMMENDED METHOD : EEC TRIAL Amount of avoparcinlmg kg-l Laboratory F G H JK L M N 0 P QR S T Over-all mean .. .. .. . . .. .. .. .. .. .. .. .. .. .. I . .. .. .. .. .. .. .. .... .. .. .. .. .. .. r Added 20 20 20 20 20.20 20.52 20.08 20 20 20 20 20 20 20 20 20 20 20 20 20.52 20.52 20.72 20.72 20 20 10 20 20 20 20 20 20 - Standard deviation . . .. Preparation of the Spore Suspension* 1 Found 21.62 22.11 19.69 20.38 21.00 17.42 21.70 18.02 19.30 19.14 19.90 22.49 23.19 23.27 21.54 21.54 16.66 15.63 20.98 22.00 21.44 21.73 20.94 21.38 19.23 20.14 10.28 30.80 21.60 28.70 19.80 19.70 17.20 - Recovery, 108.1 110.5 98.4 101.9 105.0 86.2 105.8 89.7 96.5 95.7 99.5 112.5 116.0 116.4 107.7 107.7 83.3 78.2 104.9 110.0 104.6 105.9 101.1 103.2 96.2 100.7 102.8 154.0 108.0 143.5 99.0 98.6 86.0 % - Mean recovery, 105.7 % 103.5 93.9 96.5 97.6 115.0 94.2 107.5 103.7 98.5 102.8 135.2 - 94.5 104.2 14.61 Harvest the growth from a recently prepared agar slope with 2-3 ml of sodium chloride solution.Inoculate the suspension into 300ml of culture medium contained in a Roux flask. Incubate for 5-7 d at 30 "C and then confirm by examination under the microscope that sporulation has occurred. Harvest the spores in 10 ml of sodium chloride solution and centrifuge, discarding the supernatant liquid. Re-suspend the spores in 10 ml of sodium chloride solution and heat at 65 "C for 20 min. This suspension can be kept for at least 6 months in a refrigerator at about 4 "C. Make preliminary tests on the assay plates using the culture medium to determine the amount of inoculum needed to obtain the largest possible clear zones of inhibition with the different concentrations of antibiotics used. Inoculate the culture medium at a temperature between 50 and 60 "C.Culture Medium? Peptone . . .. .. .. . . 6.0 g Tryptone . . .. .. .. . . 4.0 g Yeast extract * . .. .. . . 3.0g Meat extract. . .. .. .. . . 1.5g Glucose . . .. .. .. . . 1.og Agar (according to quality) .. . . 10-2og Water .. .. .. . . . . l000ml * Other methods, or a commercially prepared suspension of spores, can be used. t Any commercial culture medium of similar composition and giving the same results can be used.November, 1979 ASSAY OF AVOPARCIN IN ANIMAL FEEDS AND PRE-MIXES Adjust the pH so that after sterilisation for 15 min at 121 "C it is 6.6. 1079 Reagents Sodium chloride solution, 0.8-O.9%, sterile. Hydrochloric acid, concentrated. Sodium hydroxide solation, 2.0 M. Phosfihate bufer solution, pH 4.5. Dissolve 13.6 g of potassium dihydrogen orthophosphate (KH,PO,) in water and dilute to 1000 ml.Adjust the pH to 4.5. Acetone - water - concentrated hydrochloric acid (65 + 32.5 + 2.5). Standard substance. Avoparcin of known activity. Cyanamid of Great Britain Limited, Gosport, Hampshire. Standard Solutions Dissolve an accurately weighed amount of approximately 10 mg of the standard substance in phosphate buffer solution and dilute with the buffer solution to give a stock solution containing 100 pg ml-l of avoparcin. When stored in a stoppered flask at 4 "C, this solution is stable for up to 7 d. TABLE IV DETERMINATION OF AVOPARCIN IN A 2% AVOPARCIN PRE-MIX BY THE RECOMMENDED METHOD: EEC TRIAL Laboratory Standard deviation . . F L M N 0 P Q R S T Over-all mean .. .. .. .. .... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. Amount of avoparcin found, yo 1.96 1.91 2.12 2.12 2.04 1.75 2.00 2.02 1.71 1.86 1.85 1.85 1.85 2.05 2.01 1.97 1.92 2.04 1.96 2.01 2.28 2.01 2.30 2.69 3.00 3.11 1.91 1.92 1.87 1.73 1.95 1.92 2.08 1.94 1.90 1.80 2.00 Recovery, 98.0 95.5 106.0 106.0 102.0 87.5 100.0 101.0 85.7 93.0 92.5 92.5 92.5 102.5 100.3 98.4 95.9 102.0 98.0 100.5 114.2 100.5 115.2 129.7 150.0 165.5 95.5 96.0 93.7 86.7 97.7 96.0 104.0 97.0 96.0 90.0 100.0 % Mean recovery, 99.8 % 104.0 93.8 93.4 92.6 99.3 100.0 105.1 137.6 95.8 90.2 97.7 99.0 95.0 101.8 14.821080 ANALYTICAL METHODS COMMITTEE : MICROBIOLOGICAL Analyst, VoZ. 104 For concentrates and $re-mixes Prepare, from the stock solution by dilution with phosphate buffer, a standard working solution s g containing 4 pg ml-l of avoparcin.Then prepare solutions having the following concentrations of avoparcin by means of successive dilutions (1 + 1) with phosphate buffer solution: S,, 2 pg ml-l; S,, 1 pg ml-1; and S,, 0.5 pg ml-l. For f eedingstufs S,, 1 pg ml-l; S,, 0.5 pg ml-1; and S,, 0.25 pg ml-l. Prepare standard solutions as above but with the following concentrations: S,, 2 pg ml-1; Procedure Extraction (a) Pre-mixes and concentrates. Weigh, to the nearest 10 mg, sufficient sample to contain 100 mg of avoparcin. Transfer the sample into a 100-ml calibrated flask with 60 ml of the acetone - water - hydrochloric acid mixture. Shake for 16 min on a mechanical shaker. Check the pH and adjust to pH 2, if necessary, with concentrated hydrochloric acid. Make up to volume with the mixture and mix well.Filter a portion through suitable filter-paper (e.g., Whatman No. l), discarding the first 5 ml of the filtrate. Dilute an aliquot with buffer solution to obtain an expected avoparcin concentration of 4 pg ml-l (= Us). From this solution prepare solutions U, (expected content : 2 pg ml-l), U, (expected content : 1 pg ml-I) and U, (expected content: 0.5 pg ml-l) by means of successive dilution (1 + 1) with buffer solution. (b) Feedingstufs. Weigh, to the nearest 0.1 g, 50 g of the sample and blend with 100 ml of acetone - water - hydrochloric acid mixture for 30 min on a mechanical shaker. Clarify the extract by centrifugation (using stoppered centrifuge tubes), take an aliquot of the clarified extract (see table below) and adjust the pH to 4.5 with 2 M sodium hydroxide solution.Dilute this aliquot with buffer solution to provide U, (see table below). Presumed level of avoparcinlmg kg-l . . 5 7.5 10 15 20 40 Mass of samplelg ( f 0 . l g) . . .. . . 50 50 50 50 50 50 Volume of acetone - water - hydrochloric acid mixture/ml . . .. .. . . 100 100 100 100 100 100 Volume of clarified extractlml . . . . 20 15 20 15 20 10 Final volume/ml (U,) . . .. . . . . 26 25 50 50 100 100 Expected U, concentration/pg ml-l .. 2 -2 2 -2 2 2 From this solution prepare solutions U, (expected content: 1.0 pg ml-l), U, (expected content : 0.5 pg ml-l) and U, (expected content : 0.25 pg ml-1) by means of successive dilution (1 + 1) with buffer. Determination Diffusion through agar is carried out in plates, all four concentrations of the standard solution (S8, S,, S, and S,) and of the sample extract (S8, U,, U, and U,) being used.The four concentrations of sample extract and standard solutions must be used in each plate. For this purpose, select flat-bottomed plates that are large enough to allow at least eight holes of 10-mm diameter, with at least 30mm between centres, to be punched out of the agar medium. Inoculate with the spore suspension, at a temperature between 50 and 60 "C, an amount of the melted assay medium sufficient to give a layer approximately 2 mm thick in the assay plates to be used. Swirl to mix thoroughly, and pour into accurately levelled sterile assay plates (complete the pouring of one plate before proceeding to the next).With a sterile cork borer, remove agar plugs to form holes as described above. Pipette into each hole an exactly measured and equal volume (0.10-0.15 ml) of, respectively, solutions s g , s,, s, and S, and ug, U,, U, and U,. Apply solutions of each concentration four times so that the determination is subject to an evaluation of 32 zones of inhibition. Incubate the plates for approximately 18 h at 28-30 "C.November, 1979 Evaluation For each zone two measurements at right-angles should be made. Calculate the mean diameters (s8, s4, s2 and sl and u8, up, u2 and u,) for each of the concentrations of standard and sample. Plot the mean diameters against the logarithms of the concentrations for both standard’ and sample solutions. ASSAY OF AVOPARCIN IN ANIMAL FEEDS AND PRE-MIXES 1081 Measure the diameters of the zones of inhibition, if possible to the nearest 0.1 mm.Determine the “best fit” lines for the standard and sample as follows. Determine the “best fit” point for the standard low level (SL) from the equation Similarly, determine the “best fit” point for the standard high level (SH) from the equation Similarly, calculate the “best fit” points for the sample low and high levels (UL and UH) by substituting ul, u2, u4, ug for sl, s2, s4, s8, respectively, in the above equations. Plot SL and SH on the same graph paper and join them to give the “best fit” line for the standards. Similarly, plot UL and UH to give the “best fit” line for the sample. In the absence of interference, the lines should be parallel.For practical purposes, the lines can be considered parallel if the values SH - SL and UH - UL do not differ by more than 10% from their mean value. If the lines are found to be non-parallel either ul and s1 or 248 and s8 may be discarded and SL, SH, UL and UH calculated, using the alternative equations, to give alternative “best fit” lines : - (4) 5s4 + 2s2 - s1 6 5s8 + 2s4 - s2 6 .. .. or SH = and similarly for UL and UH. The alternative “best fit” lines should be checked for paral- lelism as before. The fact that the result has been calculated from three levels should be noted on the final report. When the lines are considered as being parallel, calculate the logarithm of the potency ratio (log A ) by means of one of the following equations: For fozlr ZeveZs- For t h e e LeveZs- or Calculate the high-level concentration of the sample from the relationship Sample high-level concentration = standard high-level concentration x potency ratio1082 ANALYTICAL METHODS COMMITTEE When the lines are considered as not being parallel, repeat the determination. If paral- lelism is still not achieved, calculate the logarithm of the potency ratio (log A) by means of equation (5). The result obtained must, however, be considered as approximate and this should be noted in the final report. References 1. 2. 3. Third Directive of the Commission of 27 April 1972 Establishing Community Methods of Analysis for the Official Control of Feedingstuffs (72/199/EEC). Off. J . Eur. Commun., 1972, No. L123/6, 74. Eighth Directive of the Commission of 15 June 1978 Establishing Community Methods of Analysis for the Official Control of Feedingstuffs (78/633/EEC), 08. J . Eur. Commun., 1978, No. L206, 43. Analytical Methods Committee, Analyst, 1977, 182, 206.

ISSN:0003-2654    

DOI:10.1039/AN9790401075

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, November, 1979 SHORT PAPERS 1083 Fast and Simple Polarographic Method for the Determination of Free and Total Sulphur Dioxide in Wines and Other Common Beverages P. Bruno, M. Caselli, A. Di Fano and A. Traini Istituto di Chimica Analitica dell’Universit2, Via Ame.Pzdola 173, 70126 Bari, Italy Keywords : Direct-pulse polarography ; sulphur dioxide determination ; wine analysis The employment of sulphur dioxide as a preservative in wines and fruit juices has an ancient origin. At present, only sulphur dioxide, or salts that yield sulphur dioxide in acidic solution, and sorbic acid or sorbates, may be used as chemical additives in wine production and their amounts are strictly controlled. The sulphur dioxide in wines may be bound for the most part with acetaldehyde, aldose (such as glucose), glyoxylic, pyruvic, or-ketoglutaric and galacturonic acids, some unsaturated compounds and phenolic compounds, such as caffeic and 9-coumaric acids, while the remainder is present as free sulphur dioxide. It is important to control the amount of sulphur dioxide present in wines for many reasons.Some studies1 indicate that sulphur dioxide and sulphites may be mutagenic and that the toxicity of sulphur dioxide and dietary sulphites (dietary preservatives) may be exacerbated by the occurrence of sulphite oxidase deficiency. The added sulphur dioxide content may decrease during ageing and must be replaced; an excess of free sulphur dioxide is responsible for the disagreeable sulphurous odour of some wines. One important reason for controlling sulphur dioxide levels in wines is that the EEC limits are only slightly above the level that is technically necessary to stabilise the wine.The determination of sulphur dioxide in wines is generally based on an oxidation - reduction reaction : In the determination of free sulphur dioxide, the wine is first acidified to prevent the dissociation of bound sulphur dioxide and to reduce the oxidation of polyphenols by iodine, and then titrated with iodine using starch as the indicator to detect the end-point. This method is not exact as wines that contain no sulphur dioxide will still consume some iodine owing to certain non-sulphite iodine-reducing substances. Red wines, because of their higher tannin and colouring matter content, may consume an appreciable amount of iodine. The sulphur dioxide bound by aldehydes, sugar and other compounds makes it difficult to measure accurately the free sulphur dioxide as it is subject to change.Despite this, direct iodine titration is almost always used commercially for free sulphur dioxide determination in white wines but it is unreliable for red wines because of the lower free sulphur dioxide levels and the very difficult end-point detection. The errors involved in the iodine method were reviewed by Joslyn and Braverman.2 Modified methods were proposed by Kielhofer and Aumann,3 Paul4 and Burroughs and Sparks6 In these methods the free sulphur dioxide is removed from acidified wine at room temperature by a stream of air, absorbed in neutralised hydrogen peroxide and the acid formed is titrated with sodium hydroxide solution or the mass as barium sulphate deter- mined, after precipitation by barium chloride.This method must be employed with red wines because the colour does not permit an exact evaluation of the end-point of the titration with iodine. In order to determine the total sulphur dioxide content the acetaldehyde-or-hydroxy- sulphonate must be hydrolysed. This may be carried out by using a strong alkali and then acidifying, and titrating directly with iodine or back-titrating the excess of iodine with1084 SHORT PAPERS Analyst, Vol. 104 standardised sodium thiosulphate solution. In a more accurate procedure the sample is hydrolysed with a strong acid and the sulphur dioxide is distilled from the wine into hydrogen peroxide solution.The sulphuric acid produced is then titrated with standardised sodium hydroxide solution.s It is well known that sulphur dioxide gives a polarographic wave in an aqueous medium.7 This reaction has been used for the determination of the sulphur dioxide in foodst~ffs.8~@ However, this method has some disadvantages : the low solubility of the sulphur dioxide in aqueous media gives rise to losses during bubbling with nitrogen to remove any oxygen, and the standard addition of sulphite can produce erratic results owing to combination with carbonyl groups present in the wine. Dimethyl sulphoxide (DMSO) is a very good solvent for sulphur dioxide. A well defined polarographic wave is obtain&, which is not very susceptible to interferences10 (e.g., up to 10% of water does not interfere).The sensitivity is very high if differential-pulse polaro- graphy is employed. Currents are very stable over a large range of pH values and are not affected by bubbling with nitrogen. We have found that this method is suitable for measuring, in a fast and simple way, the free and bound sulphur dioxide in wines and other beverages. Experimental Reagents and Apparatus DMSO. Reagent-grade material (Carlo Erba) was employed without further purification. Lithium chloride solution, 0.1 moll-l. A calibration graph was obtained using two standard solutions, sulphur dioxide in DMSO, 1.35 x 10-2 moll-1, and sodium sulphite, 1.67 x mol 1-1 in water. No significant differences were found between the two series of values. The operating conditions were as follows: initial potential -600 mV against Hg I Hg2C12 I LiCl 0.1 mol 1-1 DMSO electrode, modulation amplitude 50 mV and scan rate 2 mV s-l.This was employed as the ground electrolyte. A PAR 174 A polarograph with mercury-drop timer was employed. Procedure Although the sulphur dioxide reduction in DMSO is a complicated process involving adsorption and kinetics,ll this is not relevant for the analytical discussion. The only effect that must be taken into account is the effect of pH. In Fig. 1 the height of the differential- pulse polarographic peak of sulphur dioxide is plotted against sulphuric acid concentration and against pH. The pH values were measured by an Orion 901 microprocessor ion analyser. rn - a - - (a) I 10 30 50 H2 SO4 concentration/rnrnol I-' 2 9 2, a 2.0 3.0 4.0 PH Fig.1. Current, i, of the differential-pulse polarographic peak of sulphur dioxide versus (a) sulphuric acid concentration and (b) pH.November, 1979 SHORT PAPERS 1086; Acetic acid - acetate and salicylic acid - salicylate buffers were used for the standardisations.12 As can be seen the current is constant in the range 10-50 mmol l-l. The calibration graph in this range is a straight line presented by the relationship i (PA) = 0.0060 + 0.0893~ where c is the concentration of sulphur dioxide in the polarographic cell in pmoll-l. It is perfectly linear in the range 0-20 pmol 1-1 of sulphur dioxide. The analytical procedure was therefore carried out as follows: 10 ml of wine are treated in a flask with 5 ml of sodium hydroxide solution (3 moll-l) and left to stand for 30 min; 10 ml of a solution of 0.1 mol 1-1 of lithium chloride in DMSO are placed in the polarographic cell with 2 0 0 4 of 0.5mol1-l sulphuric acid and de-gassed by bubbling with nitrogen for 5 min.The base line is then recorded [Fig. 2 (A)]; A 1-11 of untreated wine are added to the cell and the polarogram is started [Fig. 2 (B)]. The concentration of sulphur dioxide can be derived from the calibration graph or the standard-additions procedure cari be used [Fig. 2 (C), (D) and (E)]. This procedure is illustrated better in Fig. 3. The free sulphur dioxide is calculated by the equation where c9 mgl-1 is the concentration of sulphur dioxide in the wine, a pmol 1-1 is the absolute value of the intercept with the x axis (see Fig.3), VTot. ml the total volume in the cell and A p1 the volume of untreated wine added. An additional 50p1 of sulphuric acid and 50p1 of the alkali-treated wine are then added and the polarogram is recorded [Fig. 2 (F)]. In this way the total sulphur dioxide content is measured by the difference between curves (F) and (E) in Fig. 2. The procedure is the same for white and red wines and no more than 1 h is needed for the whole analysis of a single sample; several samples may be hydrolysed simultaneously. F 2 1.5 a 3 1 0.5 0 EImV Fig. 2. Differential-pulse polarograms : A, the base line, 0.1 moll-' lithium chloride in dimethyl sulphoxide; B, free sulphur dioxide; C, D, E, standard additions of sulphur dioxide; and F, total sulphur dioxide.1086 SHORT PAPERS Analyst, Vol.104 -4.92 0 5 10 Added SO, /pmol I-' Fig. 3. Standard-additions method for free sulphur dioxide. Sulphur dioxide concentration in wine = 16.3 mgl-l. The absolute value of the negative intercept on the x-axis gives the sulphur dioxide concentration in the cell. Results and Discussion The precision of the method can be evaluated using statistical methods and the calibration graphs (Table I). TABLE I RESULTS FOR EVALUATION OF PRECISION c/pmol 1-1 100t,s,/c ( p = 0.05) 2 14 3 9.14 4 6.68 5 5.22 6 4.27 7 3.60 8 3.11 c/pmol 1-1 9 10 12 14 16 18 20 lOOt,S,/c ( p = 0.05) 2.72 2.41 2.06 1.80 1.65 1.54 1.46 The confidence limits at a 5% significance level are calculated for the concentrations of These limits can be calculated by the relation- sulphur dioxide in the range 1-20 pmol l-l.ship where t, is Students t-value at the 9 level of significance and AC = t,S, where i is the current corresponding to the concentration c, 2- = %/m, C = Cc/m, m is the number of data for calculating the regression line i = i, + bc and From Table I it is evident that the error becomes greater than loo/, for a concentration of approximately 3 pmo11-1 in the cell. This corresponds to a current of about 0.3 pA. Should the current be less than this value it may be advisable to increase the volume of wine added. Five replicate analyses on a sample of commercial wine gave a value for the free sulphur dioxide of 16.6 6 0.37 mg I-l, in accordance with the calculated precision, taking into account that in our procedure this value corresponds to 5 pmol l-1 in the cell.In order to test the accuracy eight samples of white and red wines were tested by the classical and polarographic methods for the total sulphur dioxide content. We found that by directNovember, 1979 SHORT PAPERS 1087 iodimetric titrations values of sulphur dioxide were 7-10 mg 1-1 higher than by the polaro- graphic method described in this paper. This is due to the presence of non-sulphur dioxide iodine-reducing substances in the wine.13 This method is readily extendible to other beverages such as lemon or orange juice. Further, as the same calibration graph is obtained by adding a solution of sulphur dioxide in DMSO or small amounts (microiitres) of an aqueous solution of sulphites to DMSO, the method is suitable for determining sulphites or other sulphur-containing compounds that can be chemically transformed into sulphites. 1.2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. References Hickey, H. J., Cleeland, R. C., Bower, E. J., and Boyce, D. E., Archs Envir. Hlth, 1976, March/April, Joslyn, M. A., and Braverman, J . B. S., Adv. Fd Res., 1954, 5, 97. Kielhofer. E., and Aumann, H., Mitt. (Klosterneuburg) Rebe u. Wein. Obstbau u. Friichtverwert., Paul, F., Mitt. (Klosterneuburg) Rebe u. Wein. Obstbau u. Friichtverwert., 1958, 8A, 21. Burroughs, L. F., and Sparks, A. H., Analyst, 1964, 89, 55. “Official Methods of Analysis of the Association of Official Analytical Chemists,” Eleventh Edition, Kolthoff, U., and Miller, C. F., J . Am. Chem. Soc., 1941, 63, 2818. Prater, A. N., Johnson, C. M., and Prol, M. F., I n d . Engng Chem. Analyt. Edn, 1944, 16, 153. Demair, W., Koch, J., and Hess, D., 2. Analyt. Chem., 1961, 5, 321. Bruno, P., Caselli, M., Della Monica, M., and Di Fano, A., Talanta, in the press. Bruno, P., Caselli, M., and Traini, A., to be published. Kolthoff, I. M., Chantooni, M. C., Jr., and Bhowmik, S., J . Am. Chem. SOC., 1968, 90, 23. Joslyn, M. A., Am. J . Enol. 1955, 6, 1. 108. 1957, 7A, 287. Association of Official Analytical Chemists, Washington, D.C., 1970, pp. 191. Received November 62h, 1978 Accepted April 19th, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790401083

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

November, 1979 SHORT PAPERS 1087 Direct Differentia I = pu Ise Pola rog rap hic Determination of Mixtures of Food Colouring Matters, Chocolate Brown HT, Tartrazine and Green S A. G. Fogg and K. S. Yo0 Chemistry Defiartment, Loughborough University of Technology, Loughborough, Leicestershire, LE 11 3T U Keywords : Food colouring matters ; diflerential-pulse polarography ; Chocolate Brown H T ; tartrazine; Green S The direct determination of the following mixtures of food colouring matters by differential- pulse polarography has been reported previously1 : Sunset Yellow FCF - tartrazine, tartrazine - Green S and amaranth-Green S. The procedures that were developed were applied to determination of these colouring matter combinations in orange, lime and blackcurrant drinks. The marked effect of the addition of tetraphenylphosphonium chloride on the differential- pulse polarograms of some colouring matters was clearly illustrated, and was used in some procedures.The effect on the differential-pulse polarograms of tartrazine was most marked, the polarographic peak being doubled in height and shifted 70mV to a more negative potential on the addition of tetraphenylphosphonium chloride ; this allowed the direct determination of both tartrazine and Sunset Yellow FCF in orange drink, the peak height of the Sunset Yellow FCF being halved but remaining at the same potential. The differential- pulse polarographic peak of Green S is suppressed almost completely at pH 9 in the presence of tetraphenylphosphonium chloride.1088 SHORT PAPERS Analyst, VoZ.104 This short paper describes the direct polarographic determination of a mixture of Chocolate Brown HT, tartrazine and Green S, which is commonly used commercially in colouring sparkling dandelion and burdock drink. Experimental Differential-pulse polarograms were obtained using a PAR 174 polarographic analyser (Princeton Applied Research) with two-electrode operation (dropping-mercury electrode and saturated calomel electrode). A forced drop time of 1 s, a scan rate of 2 mV s-1 and a pulse height of 50 mV were used, except where otherwise indicated. Procedure A typical dandelion and burdock drink was considered to contain 60 p.p.m. of Chocolate Brown HT, 20 p.p.m. of tartrazine and 2 p.p.m. of Green S. The procedure developed for the determination of this mixture is as follows.Pipette 5 ml of sparkling dandelion and burdock drink into a 30-ml beaker. Add 3 ml of 1 M tetramethylammonium chloride solution and 5 ml of Britton - Robinson buffer (pH 1.9, 0.04 M in boric acid, acetic acid and orthophosphoric acid). Adjust to pH 4 with 0.2 M sodium hydroxide solution and dilute to 25 ml in a calibrated flask. Deoxygenate a portion of this solution in a polarographic cell, and obtain a differential-pulse polarogram between 0.0 and -0.8 V versus the saturated calomel electrode (S.C.E.). Carefully re-adjust the solution in the cell to pH 9 with 4~ sodium hydroxide solution and add 10 mg of tetraphenylphosphonium chloride. Pass nitrogen through the solution to aid the dissolution of the solid and to deoxygenate the solution.Obtain a differential-pulse polarogram between -0.6 and -1.0 V versus S.C.E. Determine the Chocolate Brown HT using its first differential-pulse peak at -0.18 V (pH 4) and Green S using its peak at -0.6 V (pH 4). Determine tartrazine using its differential-pulse peak at -0.8 V (pH 9). Compare the peak heights with standards con- taining the other two colouring matters at the levels normally found in the drink. Preparation of Calibration Graphs Prepare a stock sparkling dandelion and burdock drink to which no colouring matter has been added by diluting 15ml of basic syrup to 100ml with distilled water, previously carbonated with dry-ice. Prepare blank solutions for use in obtaining calibration graphs as follows. Blank solutions for determination of each colouring matter Chocolate Brown H T .Mix the stock drink (10 ml), tetramethylammonium chloride solution (1 M, 5 ml), Britton - Robinson buffer (pH 1.9, 10 ml), tartrazine solution (200 p.p.m., -1.2 -0.9 -0.6 -0.3 -1.2 -0.9 -0.6 -0.3 PotentialN Fig. 1. Differential-pulse polarograms of the colouring matters: (a) at pH 4 and (b) a t pH 9. A, Chocolate Brown HT; B, tartrazine; and C, Green S.November, 1979 SHORT PAPERS 1089 1 ml) and Green S solution (40 p.p.m., 0.5 ml), adjust the resulting solution to pH 4 and dilute it to 50 ml with water in a calibrated flask. Mix the stock drink (10 ml), tetramethylammonium chloride solution (1 M, 5 ml), Britton - Robinson buffer (pH 1.9, 10 ml), tartrazine solution (ZOO p.p.m., 1 ml) and Chocolate Brown HT solution (630 p.p.m., 1 ml), adjust the resulting solution to pH 4 and dilute it to 50 ml with water in a calibrated flask. Mix the stock drink (10 ml), tetramethylammonium chloride solution (1 M, 6 ml), Britton - Robinson buffer (pH 1.9, 10 ml), tetraphenylphosphonium chloride solution (0.01 M, 5 ml), Chocolate Brown HT solution (630 p.p.m., 1 ml) and Green S solution (40 p.p.m., 0.5 ml), adjust the resulting solution to pH 9 and dilute it to 50 ml with water in a calibrated flask.In order to obtain a calibration graph for a particular colouring matter, pipette 20 ml of the appropriate blank solution into a dry polarographic cell, deoxygenate it and then polaro- graph it. Add aliquots of a concentrated solution of the colouring matter using a 100-pl syringe. Obtain polarograms after each addition; deoxygenate the solution for 1 min after each addition before obtaining the polarogram.Green S. Tartrazine. The polarogram obtained is the blank. Results and Discussion Differential-pulse polarograms of the three colouring matters at pH 4 and 9 under the solution conditions recommended in the procedure described above are shown in Fig. 1. Clearly, at pH 4 the second differential-pulse peak of Chocolate Brown HT coincides with the peak of tartrazine, whereas the first peak of Chocolate Brown HT is free from interference and can be used to determine this colouring matter. In polarograms of samples of dandelion and burdock drink the Green S peak is superimposed on the high base line produced by the Chocolate Brown HT, but nevertheless it can be used for determining Green S.At pH 9 in the presence of tetraphenylphosphonium chloride the differential-pulse peaks of Chocolate Brown HT and Green S are suppressed, leaving high base lines, particularly with Chocolate Brown HT. In samples the tartrazine peak is superimposed on this base line but it can be used to determine tartrazine. -0.6 -0.3 0. PotentiaVV Fig. 2. Diff erential-pulse polarograms obtained to produce a calibration graph for Chocolate Brown HT. Chocolate Brown HT concentration: A, 0; B, 3.1; C, 6.2; D, 12.3; E, 18.3; F, 24.1; and G, 29.9 p.p.m. Modulation amplitude, 50 mV; scan rate, 2 mV s-1; drop time, 1 s.1090 SHORT PAPERS Analyst, Vol. 104 a 3 c 3 1 t 1 -0.6 -0.3 0.0 PotentiaW Fig. 3. Diff erential-pulse polarogram obtained to produce a calibration graph for Green S.Green S concentration: A, 0; B, 0.20; C, 0.40; D, 0.78; E, 1.16; F, 1.54; G, 1.90; and H, 3.80 p.p.m. Modulation ampli- tude, 50 mV; scan rate, 2 mV s-l; drop time, 1 s. a i t c aJ 6 1 1 -1.2 -0.9 -0.6 -0.3 PotentialN Fig. 4. Differential-pulse polaro- grams obtained to produce a calibra- tion graph for tartrazine. Tartrazine concentration: A, 0; B, 1.00; C, 2.98; D, 4.88; E, 6.76; and F, 8.61 p.p.m. Modulation amplitude, 50 mV; scan rate, 6 mV s-l; drop time, 1 s. Calibration graphs for the three dyes were rectilinear in the range studied. The differential- pulse polarograms obtained to produce calibration graphs for Chocolate Brown HT, Green S and tartrazine, respectively, in the presence of the other two dyes are shown in Figs.2 4 . The procedure was tested with 10 determinations, using a carefully prepared sparkling dandelion and burdock drink containing Chocolate Brown HT (62.8 p.p.m.), Green S (2.0 p.p.m.) and tartrazine (20.0 p.p.m.). The Chocolate Brown HT content was determined to be 61.7 p.p.m. with a coefficient of variation of 3%. The Green S and tartrazine contents were determined to be 1.96 p.p.m. with a coefficient of variation of 4% and 20.3 p.p.m. with a coefficient of variation of 2yo, respectively. The work described in this paper illustrates again the fact that differential-pulse polaro- graphy can be used to determine certain mixtures of dyes without the need to separate them from each other first. In this application to soft drinks they were determined directly without t?e need to separate the dyes from the matrix. The authors thank Mr. B. A. Saturley and Mr. D. Hicks of Beecham Products and Dr. N. T. Crosby of the Laboratory of the Government Chemist for the provision of samples and for helpful discussion. One of us (K. S. Y.) thanks the Ministry of Overseas Development (UK) and Ulsan Institute of Technology (Republic of Korea) for financial assistance. Reference 1. Fogg, A. G., and Yoo, K. S., Analyst, 1979, 104, 723. Received April 3rd, 1979 Accepted May 31st, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790401087

出版商:RSC    

年代:1979

数据来源: RSC

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November, 1979 SHORT PAPERS 1091 3-(2'-Thiazolylazo)-2,6-Diaminotoluene as a Selective and Sensitive Reagent for the Spectrophotometric Determination of Palladium F. Garcia Montelongo, V. Gonzblez Diaz and C. R. Tallo Gonzhlez Department of Analytical Chemistry, University of La Laguna, Tenerife, Canary Islands Keywords : 342 '-Thiazolylazo) -2,6-diaminotoluene reagent ; palladium deter- mination ; spectrophotometry Although many reagents have been used for the spectrophotometric determination of palladium,l the selectivity is rarely satisfactory. In an effort to improve the sensitivity and selectivity of this determination, o-amino heterocyclic azo dyes have been introduced and studies have been made of 4-(5'-chloro-2'-pyridi1azo)-l,3-diaminoben~ene~ and 5-(2'- thiazolylazo)-2,6-diaminopyridine .3 As part of an investigation on the uses of o-thiazolylazodiamines as analytical reagents, this paper describes a highly selective and sensitive procedure for the spectrophotometric determination of palladium with 3-(2'-thiazolylazo)-2,6-diaminotoluene (2,6-TADAT).This compound forms a blue complex with palladium in strongly acidic media, even in the presence of chloride ions. HZN CHS 2,6 - TADAT Experimental Apparatus The apparatus used included a Beckman 25 recording spectrophotometer with silica cells with 1-cm path length, a Radiometer PHM28 pH meter with glass and saturated calomel electrodes, a Pye Unicam 1900 atomic-absorption spectrophotometer and a Sartorius MPR35 balance. Reagents unless otherwise stated. Analytical-reagent grade chemicals were used throughout, without further purification, 3-(2'-Thiazolylazo)-2,6-diaminotoluene solutio.n, Palladium(I1) chloride solution, Palladiurn(I1) perchlorate solution, The two palladium solutions were standardised gravimetrically using quinolin-8-01.M in 1 M perchloric acid. M in 1 M hydrochloric acid. M in perchloric acid. Synthesis of 2,6-TADAT 2-Aminothiazole (5.0 g) was dissolved in 40 ml of 6 M hydrochloric acid, ice-cooled and slowly diazotised with a solution of 3.5 g of sodium nitrite in a small amount of water. The solution of the diazotate was then poured slowly, while stirring, into a well cooled solution of 2,6-diaminotoluene (5.56 g) in 100 ml of 4 M hydrochloric acid. The mixture was left in the ice-bath for 1 h, with stirring, and then sodium acetate solution was added until a pH of 6 was reached.A dark red precipitate began to settle immediately. The solution was filtered, and the precipitate washed with water and dried in air. The crude product was purified by column chromatography on silica gel, 70-230 mesh (Merck), with benzene - ethyl acetate mixtures of increasing polarity as the eluent. TheAnalyst, Vol. 104 yield was 40% and the melting-point 186-187 "C. The elemental analysis gave the following results: C 51.51y0, H 4.86y0, N 29.21% and S 13.060/; calculated for C,,H,,N,S: C 51.50%, H 4.72%, N 30.04% and S 13.73%. 1092 SHORT PAPERS Properties qf the reagent 2,6-TADAT is sparingly soluble in water, moderately soluble in methanol and ethanol and very soluble in acidic solutions.The mass spectrum shows a parent molecular ion (M+) at m/e 234. The NMR spectrum, [(CD,),SO], 90MHZ was as follows: 8 = 1.92 (3H, s, -CH,), 6.30 and 7.26 (each lH, d, J = 9 Hz, H, and H,), 7.39 and 7.73 (each lH, d, J = 5 Hz, H,' and H5'). The infrared spectrum (potassium bromide pellet), Vmax. cm--l was as follows: -NH- stretching (3420 m, 3380 m, 3340 m), -CH, arcffhatic (2930 w, 2860 w), -NH, aromatic (1620 s), -N=N- (1 420 s), N aromatic (1 295 s). Reactions of 2,6-TADAT with metal ions The reactions of 2,6-TADAT with 50 metal ions at various pH values were investigated. Only cobalt(I1) (5 p.p.m., pH < 5 ) , copper(I1) (5 p.p.m., pH 4-5) and palladium(I1) (1 p.p.m. pH 0-12) gave coloured compounds, showing the high sensitivity and selectivity of 2,6-TADAT.Recommended Procedure for the Determination of Palladium( 11) To a solution of palladium(II), as the chloride or perchlorate, in a 25-ml calibrated flask, add 2-8 ml of 60% perchloric acid and 5 ml of a 1.0 x lo-, M solution of 2,6-TADAT in 1 M perchloric acid, and dilute to volume with distilled water. After 30 min measure the absorbance of the solution at 590 nm against a reagent blank, in 1-cm path length cells. Determination of Palladium in Different Materials dissolve it in perchloric acid, hydrochloric acid, aqua regia or EDTA. solution with distilled water to 100 ml in a calibrated flask. (2-5 ml) as described previously. Weigh accurately about 10 mg of a catalyst or palladiated or platinated dental gold, and Dilute the cooled Analyse suitable aliquots Results and Discussion 2,6-TADAT behaves as a tri-basic substance, with protonation of the ring nitrogen and the two amino group nitrogen atoms; ionisation constants will be reported at a later date along with other aspects of the complexation reaction.The reagent is stable in 1 M 0.9 2 0.7 3 0.5 m -e : 0.3 0.1 400 500 600 700 Wavelenyth/nm Fig. 1. Absorption spectra of solutions a t H, = -0.96 of the palladium complex : A, 1 p.p.m. of palladium(I1); B, reagent alone (water blank). 4 2 0 -Ho Fig. 2. Influence of the acidity on the formation of the palladium(I1) complex.November, 1979 SHORT PAPERS 1093 perchloric acid solutions, reducing agents do not affect it, but oxidising agents, such as periodate and persulphate, destroy it. TABLE I DETERMINATION OF PALLADIUM(II) IN THE PRESENCE OF VARIOUS IONS The amount of palladium taken in each determination was 0.861 p.p.m. Molar ratio, Ion ion : Pd SO,*- ... . 600 NO,- . . . . 376 c1- . . .. . . 600 Br-. . .. . . 126 F- .. .. . . 260 I- .. . . 0.2 C,H,O,*- . . . . 187 c,o,*- . . . . 660 Cu(I1) . . . . 600 Fe(I1) . . . . 136 CO(I1) . . . . 376 Ni(I1) . . . . 926 SCN-‘ .. . . 0.2 Pd(II), found, p.p.m.* 0.849 0.847 0.862 0.862 0.849 0.819 0.806 0.846 0.847 0.869 0.848 0.866 0.869 Ion ::[::)If Th(1V) Ir( IV) Ga( I1 I) Bi(II1) Au(1 I I) Zn(I1) Mo(V1) Y(II1) OS(1V) Pt(I1) Molar ratio, Pd(I1) found, 100 0.847 180 0.863 120 0.861 112 0.848 26 0.848 20 0.866 130 0.866 369 0.865 120 0.863 126 0.862 382 0.863 130 0.862 ion : Pd p.p. m. * * Mean values of three determinations.t Wait 3 h before reading the absorbance. Reaction with Palladium 2,6-TADAT forms a blue chelate with palladium(II), which is soluble in perchloric acid of up to 5-6 M and has an absorption maximum at 590 nm (Fig. 1). The colour develops in 30 min and its absorbance remains stable for at least 48 h. The absorbance versus Ho graph for the palladium complex (Fig. 2) shows that the optimum hydrogen ion concentration range for the complex formation lies between 1 and 3.5 M as perchloric acid, because at higher pH values a dark solid begins to settle out. Therefore, a 1 .O-2.5 M perchloric acid concentration is proposed for the determination, because at this acid concentration copper(I1) and cobalt(I1) complexes do not form at all. The stoicheiometry of the complex, determined by the continuous variation and the molar ratio methods, is shown to be 1 : 1 metal to ligand.Analytical Applications In solutions where Ho = -0.95 and palladium is present as the chloride ([Pd]/[Cl] = 1 : 50) Beer’s law is obeyed between 0.1 and 1.9 p.p.m. The molar absorptivity is 5.35 x lo* 1 mol-l cm-l at 590 nm. The optimum concentration range, as evaluated by Ringborn’s method, is 0.4-1.3 p.p.m., and the relative error of the method is 0.14% (95% confidence TABLE I1 DETERMINATION OF PALLADIUM IN DIFFERENT MATERIALS Material Pd - asbestos . . Pd - charcoal . . Pd-CaSO, .. Pd-BaSO, .. Au - Pt - Pd (dental) Au - Pd (dental) Pd - A1808 . . Mass of sample/mg* .. . . 10.916 .. . . 10.180 .. . . 10.420 . . . . 10.106 .. . . 10.472 .. . . 16.430 . . .. 16.290 Palladium found, % 2,6-TADAT A tomic-absorption A I > method method 4.34 4.41 4.14 3.99 9.01 9.07 6.91 6.84 10.02 9.92 0.16 0.14 2.20 2.10 * Sample dissolved in perchloric acid, hydrochloric acid, aqua regia or EDTA and made up to 100 ml.1094 SHORT PAPERS Analyst, Vol. 104 limits) ; starting with palladium perchlorate solutions the same results are obtained. As shown in Table I the method is highly selective as only thiocyanate and iodide seriously interfere. Among the other ions of the noble metal group, only osmium(1V) andiridium(1V) can interfere if present in amounts greater than 20 times the actual concentration of palladium. Determination of Palladium in Different Materials The technique described above has been applied to the determination of palladium in small samples of catalysts, and palladiated and platinated dental gold. The results are shown in Table 11, where they are compared with those obtained by atomic-absorption spectrophot ome try. We thank M. A. de las Casas who supplied us with samples of dental gold. References 1. 2. 3. 4. Beamish, F. E., and van Loon, S. C., “Recent Advances in the Analytical Chemistry of the Noble Shibata, S., Ishiguro, Y., and Nakashima, R., Analytica Chim. Acta, 1973, 64, 305. Ivanov, V. M., Busev, A. I., and Usama a1 Dbik, Vest. Mosk. Gos. Univ., Ser. Khim., 1970, No. 1, Slavin, W., “Atomic Absorption Spectroscopy,” Interscience, New York, 1968, p. 138. Metals,” Pergamon Press, Oxford, 1972. 88. Received March 6th. 1979 Accepted April 19th, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790401091

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

1094 SHORT PAPERS Analyst, Vol. 104 Rapid Spectrophotometric Method for the Determination of Arsenic(ll1) in Borate Glasses Saryoo Prasad Singh, Ram Pyare, Gur Prasad and P. Nath Department of Ceramic Engineering, Institute of Technology, Banaras Hindu University, Varanasi-22 1006, India Keywords ; A rsenic(III) determination ; spectrophotornetry ; borate glasses A titrimetric method for the determination of arsenic(II1) in glass, using iodine mono- chloride, has been reported by Pau1.l Sandhu2 developed a spectrophotometric method for its determination in potable water, using the chemical reaction of arsenic(II1) with potassium iodate in the presence of sulphuric acid; this reaction can be accelerated considerably by using hydrochloric acid instead of sulphuric acid.3 Based on this observation, a rapid spectrophotometric method for the determination of arsenic(II1) in glasses has been developed.Experimental Sodium and potassium aluminoborate glasses containing different concentrations of tri- and pentavalent arsenic were prepared by melting the glasses in platinum crucibles in an electric furnace. All chemicals were of analytical-reagent grade. Determination of Arsenic(II1) Arsenic(II1) oxide (0.1320 g) was dissolved in 10 ml of doubly distilled water containing 1 g of sodium hydroxide. The solution was acidified with 10 ml of concentrated hydro- chloric acid and the volume was made up to 11 with water; this solution contained 0.1 mg ml-l of arsenic(II1). An aliquot was then diluted with water to give a stock solution containing 10 pg ml-l of arsenic(II1).Different volumes of the stock solution were taken and the acidities adjusted to be 3 M in hydrochloric acid. Iodine monochloride (1 ml) wasNovember, 1979 SHORT PAPERS 1095 added to each solution, which were then shaken with 20ml of carbon tetrachloride in a separating funnel for nearly 1 min, to extract the liberated iodine. A second extraction with 20ml of carbon tetrachloride was carried out to ensure complete removal of iodine from the aqueous phase. The volumes of the organic phases were made up to 50 ml and the absorbances of the pink solutions measured at 520nm using a Sicospec-100 spectro- photometer. A calibration graph was constructed and Beer’s law was obeyed in the con- centration range 400-800 pg of arsenic(II1) in 50 ml of carbon tetrachloride.Powdered and finely ground glasses containing different amounts of trivalent arsenic were decomposed by shaking with 3 M hydrochloric acid in calibrated flasks until clear solutions were obtained. These are pure glass solutions and a suitable portion of each was taken and made to react with iodine monochloride, and measurements were carried out using the procedure described above. Determination of Arsenic(V) A 0.4160-g mass of sodium arsenate (Na2HAs0,.7H,O) was dissolved in 11 of doubly distilled water. This solution contained 100 pg ml-l of arsenic(V) ; 20 ml of this solution were then diluted to 100ml with water to give a stock solution containing 20pgml-1 of arsenic(V). Different volumes of this stock solution were taken, and by adding 1 M hydrochloric acid the acidity was adjusted to be 0.5 M in hydrochloric acid.A blue colour was developed in butan-2-01 using a molybdenum blue method.44 The volume of the solution was adjusted to 50 ml, which then contained 40 ml of butan-2-01 and 10 ml of ethanol. The absorbances of the solutions were measured at 740 nm, and the calibration graph showed that Beer’s law was obeyed in the concentration range 40-100 pg of arsenic(V) in 60 ml of butan-2-01- ethanol. Powdered and finely ground glasses containing different amounts of pentavalent and trivalent arsenic were decomposed in 50 ml of 2 N hydrochloric acid giving clear solutions. These solutions were diluted to 100ml with water. The concentrations of pentavalent arsenic in the solutions were determined by the molybdenum blue method,4-6 in the presence of trivalent arsenic.TABLE I COMPARISON OF RESULTS FOR DETERMINATION OF ARSENIC(II1) I N SODIUM ALUMINOBORATE GLASSES BY THE IODINE MONOCHLORIDE AND MOLYBDENUM BLUE METHODS Mass of arsenic, % r 7 XAs 0.224 0.196 0.176 0.174 0.171 0.167 0.166 0.214 0.183 0.170 0.166 0.160 0.140 0.138 0.224 0.196 0.178 0.160 0.147 0.141 Molybdenum blue method As(V) XAS - 0.067 0.078 0.098 0.107 0.127 0.132 0.122 0.066 0.072 0.082 0.088 0.098 0.091 0.089 0.062 0.073 0.079 0.076 0.073 0.070 1 As(V) = As(II1) 0.167 0.118 0.078 0.067 0.044 0.036 0.034 0.169 0.111 0.088 0.068 0.062 0.049 0.049 0.162 0.122 0.099 0.076 0.074 0.071 3 Iodine monochloride method : As (111) 0.166 0.114 0.076 0.066 0.043 0.034 0.036 0.166 0.110 0.092 0.072 0.063 0.060 0.060 0.160 0.120 0.100 0.076 0.074 0.072 Standard deviation : 0.0021096 SHORT PAPERS Analyst, Vol.104 Determination of Total Arsenic Trivalent arsenic was oxidised to pentavalent arsenic by chloramine-T solution and the total arsenic (in the pentavalent form) was determined employing the above molybdenum blue method.- In glasses melted under normal conditions arsenic is found to be present in only two oxidation states, namely the tri- and pentavalent states. Thus, the difference between the amounts of total and pentavalent arsenic present in a particular glass gives the amount of arsenic present as arsenic(II1). Results and Discussion The concentrations of pentavalent arsenic determined by the molybdenum blue method have been reported and agree well with the standard values.8 The concentrations of trivalent arsenic, found by the difference between total and pentavalent arsenic, were in good agreement with those determined by a titGmetric rneth~d.~ In the series of glasses investigated the concentrations of arsenic(II1) determined by the iodine monochloride method were compared with those obtained by the difference between the total and pentavalent arsenic determined by the molybdenum blue method.4-6 The results are shown in Tables I and 11.The data obtained from both methods agree well. The standard deviation is 0.002% , which shows that the variation in results obtained by the two different methods is small. TABLE I1 COMPARISION OF RESULTS FOR DETERMINATION OF ARSENIC(III) IN POTASSIUM ALUMINOBORATE GLASSES BY THE IODINE MONOCHLORIDE AND MOLYBDENUM BLUE METHODS Mass of arsenic, yo r A * Molybdenum blue method Iodine monochloride r A > method : XAs As(V) XAS - As(V) = As(II1) As (111) 0.198 0.063 0.146 0.146 0.186 0.076 0.109 0.110 0.180 0.092 0.088 0.089 0.160 0.103 0.067 0.067 0.137 0.097 0.040 0.041 0.130 0.092 0.038 0.040 0.188 0.060 0.128 0.124 0.186 0.080 0.106 0.106 0.169 0.089 0.080 0.082 0.160 0.096 0.066 0.064 0.160 0.090 0.060 0.060 0.130 0.078 0.062 0.063 0.194 0.063 0.141 0.144 0.182 0.072 0.110 0.116 0.166 0.076 0.090 0.092 0.148 0.072 0.076 0.076 0.132 0.063 0.069 0.070 Standard deviation: 0.002 In the presence of a high concentration of hydrochloric acid (3-9 M), iodate is ultimately reduced to iodine monochloride, which forms a stable complex ion with chloride i0n.3 The over-all half-cell reaction is as follows3: 103- + 6H+ + 2C1- + 4e- + IC1,- + 3H,O (EO = 1.23 V) .. (1) Sandhug utilised the following reaction for the determination of arsenic(II1) : SAsO,* + 210,- + 2H+ -+ 1, + 6 A ~ 0 , ~ + H20 (Eo = 0.64 V) . . (2)November, 1979 COMMUNICATIONS 1097 As the reduction potential of reaction (1) is higher than that of (2), it is expected that reaction (1) would take place faster than reaction (2); our observations confirm this con- clusion. Further, the pink colour of the iodine - carbon tetrachloride extract is stable for up to 15 d and the reaction is quantitative, even below a 3 M hydrochloric acid concentra- t i ~ n . ~ References 1. 2. 3. 4. 5 . 6. Paul, A., Glass Technol., 1965, 6, 22. Sandhu, S. S., Analyst, 1976, 101, 856. Vogel, A. I., “A Text Book of Quantitative Inorganic Analysis,” Third Edition, Longmans, London, Daniels, M., Analyst, 1957, 82, 133. Singh, A,, PhD Thesis, Banaras Hindu University, 1974. Sandell, E. B., “Colorimetric Determination of Traces of Metals,” Third Edition, Wiley-Interscience, 1961, p. 374. New York, 1959, pp. 284 and 299. Received January 29tk, 1979 Accepted May llth, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790401094

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

November, 1979 COMMUNICATIONS 1097 Communications Material for publication as a Communication must be o n a n urgent matter and be of obvious scientijic importance. Rapidity of publication i s enhanced i f diagrams are omitted, but tables and formulae can be included. Communications should not be simple claims for priority: this facility for rapid publication i s intended for brief descriptions of work that has progressed to a stage at which it is likely to be valuable to workers faced with similar Problems. A fuller paper may be offered subsequently, if justijied by later work. Manuscripts are not subjected to the usual examination by referees and inclusion of a Communication i s at the Editor’s discretion. An Improvement in the Determination of Available Lysine in Carbohydrate-rich Samples Keywords : Lysine determination ; carbohydrate-rich samples ; 2,4,6-trini- trobenzenesulphonic acid The chemical determination of available lysine in carbohydrate-rich materials is seriously affected by the formation of what Booth1 called the “humin” artifact produced during the hydrochloric acid hydrolysis of dinitrophenylated protein following reaction of the sample with l-fluoro-2,4- dinitrobenzene.2 Low and variable recoveries of edinitrophenyllysine added to carbohydrate feeds were obtained.Similar difficulties were reported by Hall and co-workers3~* in their use of 2,4,6-trinitrobenzenesulphonic acid (TNBS) as the protein coupling reagent. The presence of carbohydrate during the hydrolysis causes the production of a dark brown compound, which, depending on the amount of carbohydrate, may form a heavy black precipitate. Hall et al.3 described this interference as a “caramelisation” of the carbohydrate causing the formation of carbon particles which may adsorb the etrinitrophenyllysine (E-TNP-lysine) and concomitantly imparts a colour measurable at 415 nm, together with the yellow c-TNP-lysine.Carpenter (with Booth)5 has also drawn attention to the adverse effects of starches in the determination of available lysine. This communication describes an improvement in the standard procedure3 employed in this laboratory for the determination of available lysine with TNBS, which it is believed removes most of the difficulty with carbohydrate samples. It consists essentially of what is probably a chlorination to bleach the interfering “humins,” at the same time changing the chemical structure of the TNP products to make the application of the method more specific for lysine and more versatile for a wider range of samples.Crown Copyright.1098 COMMUNICATIONS Analyst, Vol. 104 Experimental Determination of Available Lysine The procedures employed for the preparation of the samples and for the determination of available lysine using TNBS were as described in previous papers3p4 i.e., 0.5-ml aliquots were taken of sample suspensions in 0.1% m/V of agar, containing 10, 20 and 40 mg ml-l of sample, ground in a porcelain ball mill to pass a 200-mesh BS sieve. Samples of 5 mg mass were reacted with TNBS for 30 min at 30 "C (short coupling) and lysine was determined as rep~rted.~ Samples of 5, 10 and 20 mg mass were reacted with TNBS for 75 min at 40 "C (long ~oupling).~ A 4-ml aliquot ( ~ 2 - 8 m g sample mass) of the 10ml of hydrolysate was diluted to 7ml with water and extracted twice with 5ml of diethyl ether, the extracts being discarded.The aqueous solution containing 6-TNP-lysine was treated with 0.2- 2.0 ml of 5% V/V sodium hypochlorite solution (10-14y0 m/V available chlorine), mixed and the volume made up to 10 ml with water. After centrifuging at 3000 rev min-l for 10 min the absorbance of the supernatant solution was measured at 415 nm in an optical cell of 10-mm light path. i. ii. Determination of total lysine Total lysine was determined in hydrochloric acid hydrolysates (0.3 g sample mass refluxed at 100 "C for 16 h with 200 ml of 6 M hydrochloric acid) by the method of Moore et al.6 using a Locarte bench-model amino acid analyser.Lysine was eluted from a 6-cm column (for basic amino acids) with a sodium citrate buffer of pH 5.28 and reacted with ninhydrin. Results In an attempt to apply TNBS to the determination of available lysine in carbohydrate-rich materials, Hall et aL4 proposed reacting a 5-mg sample for 30 min at 30 "C and obtained good correlation with the total lysine values for a number of plant materials, but showed that for some types of sample, an "available" lysine figure could be measured that was actually greater than the total lysine content. The problem was highlighted when samples of pea husk meal (Pisurn sativurn) "charred" so severely during hydrolysis that the measurement of the e-TNP-lysine was impracticable.In order to overcome the interference due to the highly coloured products of hydrolysis, it was decided to study the effects of bleaching and reducing agents on the hydrolysed sample. Dilute solutions of ascorbic acid, sodium sulphite, hydrogen peroxide and sodium hypochlorite were added to the hydrolysate after ether extraction of the TNP-amino compounds. Only sodium hypochlorite had any decolorising action but this was extremely effective, reducing the interfering absorbance by 70-90% or more. The introduction of the hypochlorite resulted in the absorbance for the control or method blank (i.e., the sample treated with hydrochloric acid before the addition of TNBS and then heated and extracted with diethyl ether as for the deter- mination) from most carbohydrate samples being less than the absorbance due to €-TNP-lysine.Before bleaching, the reverse situation pertained, the method blank having a much greater absorbance than that due to e-TNP-lysine, and this was analytically undesirable. Table I shows TABLE I EFFECT OF HYPOCHLORITE ON ABSORBANCE OF E-TNP-LYSINE AND Results are absorbance values at 415 nm. PEA HUSK MEAL HYDROLYSATE Treatment with 5% V / V hypochlorite/ml 7 L * Sample 0 0.2 0.5 1 .o 2.0 DL-Lysine, 100 pg . . Method control* . . .. . . .. Pea husk meal, 2 mg . . . . .. Control . . .. .. . . .. Pea husk meal, 4 mg . . . . .. Control . . .. . . .. .. Pea husk meal, 8 mg . . . . .. Control . . * . .. .. .. (corrected for method control) 0.412 0.010 0.428 0.393 0.694 0.560 1.080 0.865 0.393 0.011 0.166 0.091 0.274 0.169 0.627 0.362 0.394 0.014 0.134 0.082 0.241 0.127 0.461 0.250 0.405 0.009 0.107 0.055 0.213 0.103 0.422 0.208 0.405 0.013 0.109 0.059 0.213 0.094 0.360 0.141 * Sample and reagents subjected to the analytical procedure but with 6 M HCl acidification followed by addition of TNBS before incubation for 75 min.November, 1979 COMMUNICATIONS 1099 the effect of various amounts of hypochlorite on the absorbance using the pea husk meal and it can be seen that the absorbance difference between the test and the control became constant for 0.5-2.0 ml of hypochlorite and was stoicheiometric within experimental limits for 2-8 mg sample mass.Absorbance values for standard samples of c-TNP-lysine and -taurine were hardly affected.A comparison of the available lysine values of 14 carbohydrate-rich samples obtained by the two procedures (long and short TNBS couplings) indicated that the anomalous situation of the “available” lysine being higher than the total lysine content had been resolved ; without exception the hypochlorite-treated reactions resulted in lower available lysine values than were measured from unbleached reactions and correlated closer with the total lysine levels (Table 11). TABLE I1 TOTAL AND AVAILABLE LYSINE IN CARBOHYDRATE-RICH SAMPLES Lysine, yo mlm A I 7 Available Sample Cassava . . . . Barley . . .. Field beans . . Flaked maize . . Maize cobs . . Oats . . .. Pea meal . . .. Potato waste . . Poultry mash . . Rice bran . . . . Turkey crumbs .. Pig meal . . . . Tapioca . . . . Wheatings. . . . . I . . .. . . . . . . .. . . .. . . . . . . . . . . Total “Bleached ’’ * “Unb1eached”t . . 0.08 0.08 0.28 . . 0.36 0.21 0.36 . . 1.21 0.95 1.14 . . 0.26 0.25 0.42 . . 0.21 0.19 0.48 . . 0.34 0.27 0.40 . . 0.50 0.25 0.27 . . 0.48 0.44 0.53 . . 0.23 0.21 0.21 . . 0.53 0.47 0.52 . . 0.44 0.37 0.45 . . 0.02 0.02 0.21 . . 1.34 1.25 1.55 . . 0.36 0.35 0.61 * Long coupling, treated with 1 ml of 5% V/V hypochlorite. f Short ~oupling.~ Discussion As reported earlier,4 the determination of true nutritionally available lysine in animal feed- stuffs has been far from simple. Experience has shown that although 2,4,6-trinitrobenzene- sulphonic acid has proved very useful as the protein coupling reagent, it is necesary to be aware of its limitations.Apart from the interference of carbohydrates (which applies equally to the use of l-fluoro-2,4-dinitrobenzene) it has been shown in this laboratory that several naturally occuring amines such as agmatine, spermine, spermidine and taurine (unpublished findings), and also ornithine, hydroxylysine, galactosamine and gluc~samine,~ react with TNBS as does lysine and can be measured as lysine. During this work it was additionally observed that the action of the hypochlorite, which may well be that of a chlorination, changed the physical properties of the E-TNP-amino compounds formed from synthetic reagents, and from animal and plant samples. Thus, TNP-lysine and some of the TNP derivatives of the compounds already mentioned became soluble in diethyl ether ; TNP-agmatine and TNP-taurine remained in the aqueous phase whilst TNP-galactosamine and TNP-glucosamine were converted into colourless products.I t seems, therefore, that by selective ether extraction of the “chlorinated” c-TNP-lysine, the hypochlorite treatment of the TNP reaction should lend itself to a more accurate differential determination of available lysine in the presence of these interferences. Such a step would be invaluable in the analysis of fish meals that contain variable and significant amounts of taurine. Indeed, some evidence in this respect has already been demonstrated. Using the standard TNBS methodJ3 the apparent available lysine of a sample of krill (a small shrimp of the Euphausiacea) was 6.01% m/m (8.62 g per 16 g of N) but the total lysine and total taurine levels, measured with an amino acid analyser, were 4.05% m/m (5.81 g per 16 g of N) and 2.29% m/m, respectively. When the hypochlorite-treated c-TNP-lysine was further extracted into diethyl ether and the residual absorbance, assumed to be due to TNP-taurine left in the1100 COMMUNICATIONS Analyst, Vol.104 aqueous phase, was measured against a TNP-taurine standard, the values for available lysine and taurine were 3.45% m/m (4.95 g per 16 g of N) and 2.08% m/m, respectively. The corresponding results for an ordinary feeding fish meal were as follows: apparent available lysine, 4.68% m/m (6.68 g per 16 g of N) ; total lysine, 4.29% m/m (6.12 g per 16 g of N) ; “true” available lysine, 3.15% m/m (4.49 g per 16 g of N); total taurine, 0.93% w/m; and taurine measured spectrophotometrically, 1.33% m/m. It is believed that this further modification of the use of TNBS makes the determination of available lysine much more reliable and applicable to a wider range of materials. I t should be a significant advantage in evaluating protein quality for what is regarded as the most important amino acid for growth purposes. It is hoped to submit a full report of this work in a later publication. References 1. 2. 3. 4. 5. 6. Booth, V. H., J . Sci. Fd Agric., 1971, 22, 658. Carpenter, K. J., Biochem. J . , 1960, 77, 604. Hall, R. J., Trinder, N., and Givens, D. I., Analyst, 1973, 98, 673. Hall, R. J., Trinder, N., and Wood, M. R., Analyst, 1975, 100, 68. Carpenter, K. J. (with Booth, V. H.), Nutr. Ab:itr. Rev., 1973, 43, 423. Moore, S., Spackman, D. H., and Stein, W. H., Analyt. Chem., 1958, 30, 1185. Received August 31st, 1979 Ministry of Agriculture, Fisheries and Food, Agricultural Development and A dvisory Services, Analytical Chemistry Department, Government Buildings, Kenton Bar, Newcastle upon Tyne, NEI 2YA R. J. Hall K. Henderson

ISSN:0003-2654    

DOI:10.1039/AN9790401097

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

1100 COMMUNICATIONS Analyst, Vol. 104 Calibration of Proposed Wet-combustion Procedure with Dry-combustion Method for the Determination of Total Carbon in Soils Keywords Chromic acid digestion; soil organic carbon determination ; dry combustion Dalall described a simple, rapid and relatively cheap method for the determination of organic carbon in soils and plant materials. The samples containing organic carbon were digested, in an autoclave, at 121 "C and approximately 105 kPa for 1 h, with chromic acid digestion mixture [Zb g of chromium trioxide in 100 cms of concentrated sulphuric acid (2 volumes) - 85% ortho- phosphoric acid (1 volume)] in a McCartney bottle containing alkali solution in a test-tube for the absorption of carbon dioxide. The proposed method estimated 21% more soil organic carbon than that obtained by the Walkley and Black method.' However, the proposed method was not calibrated with the dry-combustion methoda for the determination of total carbon in soils.The soil carbon determination by the dry-combustion method was carried out in a Fisher Induction Carbon Apparatus according to the procedure described by Jacksoas The carbon contents of 17 soils (pH range 4 . 4 4 8 ; clay content 9-68%) determined by the proposed wet-combustion procedure and the drycombustion method are given in Table I. The soil carbon values obtained by the two methods were similar. The regression equation C% (wet combustion) = -0.06 + l.OlC% (dry combustion) r = 0.99, P (0.001, showed that the proposed wet-combustion procedure can be used to determine total carbon in soils.November, 1979 COMMUNICATIONS TABLE I 1101 CARBON CONTENTS OF SOILS OBTAINED BY THE PROPOSED AND DRY-COMBUSTION METHODS Sample No.1 2 3 4 5 6 7 8 9 Carbon content, % A f -7 Proposed Dry-combustion Sample method method No. 0.353 f 0.012 0.37 f 0.02 10 1.049 f 0.018 1.04 f 0.02 11 2.297 f 0.021 2.46 f 0.07 12 4.102 f 0.029 4.36 & 0.13 13 1.502 f 0.010 1.52 f 0.04 14 1.728 f 0.012 1.69 f 0.02 15 3.319 f 0.052 3.64 & 0.07 16 2.237 f 0.020 2.32 & 0.04 17 1.428 f 0.018 1.36 f 0.02 Carbon content, yo I h > Proposed Dry-combustion method method 1.272 f 0.028 1.13 f 0.05 1.673 f 0.012 1.70 f 0.03 2.155 & 0.015 2.14 f 0.04 0.859 0.005 0.80 f 0.03 2.196 & 0.015 2.26 f 0.04 2.940 & 0.019 3.02 f 0.06 1.385 f 0.015 1.46 f 0.07 16.90 f 0.55 17.148 f 0.32 I am grateful to Mrs. Rosslyn Henderson for soil carbon determinations by the dry-combustion method. References 1. 2. 3. Dalal, R. C., Analyst, 1979, 104, 151. Walkley, A., and Black, I. A., Soil S c i . , 1934, 37, 29. Jackson, M. L., “Soil Chemical Analysis,” Prentice-Hall, Englewood Cliffs, N. J., 1958, p. 208. Received September 24th, 1979 Department of Soil Science, Waite Agricultural Research Institute, Glen Osmond, South Australia 5064 R. C. Dalal

ISSN:0003-2654    

DOI:10.1039/AN9790401100

出版商:RSC    

年代:1979

数据来源: RSC

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1102 BOOK REVIEWS Analyst, Vol. 104 Book Reviews ULTRATRACE METAL ANALYSIS IN BIOLOGICAL SCIENCES AND ENVIRONMENT. Edited by TERENCE A Symposium Sponsored by the Division of Analytical Chemistry at the 174th Advances Washington, DC: American Chemical Society. H. RISBY. Meeting of the American Chemical Society, Chicago, Illinois, August 29-30, 1977. in Chemistry Series 172. 1979. Price $36.50 Pp. x + 264. This book is more than an illustration of the most productive areas for collaboration between analytical, biochemical and environmental scientists as was modestly claimed by the Editor. Many chapters in all three disciplines provide interesting and stimulating reading. A wealth of data on the sources and concentrations of a wide range of trace metals in the environ- ment (Chapters 5, 6 and 14) is tabulated in an easily accessible form that will provide useful refer- ence material.An excellent chapter on the role of zinc in biochemical processes by Auld provides a good basis for the review of the nutritional and clinical aspects of this metal. There are also competent reviews of the clinical and nutritional importance of copper and other trace metals. A very interesting preliminary report on the Ni,S, induced cell transformation in tissue culture experiments indicates a promising new laboratory technique for testing the carcinogenic potency of metal compounds. With a few exceptions the analytical chapters are somewhat disappointing. There were interes- ting applications of electrothermal atomisation and atomic-absorption spectroscopy (ETA - AAS) ; one was a study of matrix interferences in the analysis of sea water and a means of overcoming these problems, and the other was the determination of molybdenum in serum and erythrocytes, but with no apparent consideration given to molecular-absorption interferences.Inductively coupled plasma - optical-emission spectroscopy (ICP - OES) was used to analyse feeds and other biological matrices with success because of the relatively high analyte concentrations (Chapter 2) and accurate analyses of standard reference materials were reported. The analysis of urine, however, was less satisfactory with many important elements particularly cadmium, cobalt, copper, vanadium and nickel having concentrations in urine from non-occupationally exposed persons below the detection limit of the technique.This is all the more disappointing because of the considerable experience in ICP - OES of the authors from Iowa State University. It was also disturbing to see these workers quote a “model” (presumably normal) concentration of cadmium in urine as 70 p g 1-I. Notwithstanding these criticisms, the book will be useful for all workers concerned with trace metals in biological and environmental samples. This is at least one and possibly two orders of magnitude too high. H. T. DELVES ASBESTOS. VOLUME 1. PROPERTIES, APPLICATCONS AND HAZARDS. Edited by L. MICKAELS and S. S. CHISSICK. Pp. xiv + 553. Chichester, New York, Brisbane and Toronto: John Wiley. 1979. Price i25. This book is stated to be the first attempt to bring together (in two volumes) the fundamental and essential information about asbestos.I t has 16 chapters, each by a different author or authors. Chapter 1 is an introduction by the editors giving points of note in the development of the British asbestos industry together with a list of terms used and some of the equipment and services that are available commercially. Chapter 2, by J . Zussman, describes the mineralogy of asbestos and includes crystal structures and optical properties. X-ray diffraction data. and electron-optical characteristics are also in- cluded. The third chapter, on the chemistry and physics of asbestos, is by A. A. Hodgson, a notable worker in this field. He deals most interestingly with the occurrence and production of the raw material and the various physical, thermal and surface properties of the fibres.Some infrared spectroscopic data are also provided. S. S . Chissick, one of the two editors, contributes the fourth chapter by discussing the attitudes to asbestos of government, industry, trades unions, the pressure groups, the media and the general public. Appendices are given illustrating record keeping of exposure to asbestos.November, 1979 BOOK REVIEWS 1 103 Non-occupational asbestos emissions and exposures are reviewed by B. E. Suta and R. J . Levine in the chapter following, attention being paid to re-distribution by air and water. Tables given include concentrations of asbestos in the air of office buildings and in drinking water in the USA. The next 2 chapters deal with the monitoring and identification of airborne asbestos, by S.T. Beckett, and the identification of asbestos in solid materials, by A. P. Middleton. Light and elec- tron microscopy methods are described in both and the us’: of infrared spectrophotometry and X-ray diffraction methods are described in the analysis of solid materials. Thermal and elemental methods are mentioned briefly. Chapter 8, by J. D. Cook and E. T. Smith, deals with the problems and control of asbestos in working environments. A good bibliography is given reviewing the publications available from the Asbestosis Research Council, the Asbestos Information Committee, the Health and Safety Executive, legislative documents and others. Risks to health are discussed. Details of alternatives to asbestos in industrial applications are given by A.M. Pye. Thermal and mechancial properties are considered and health hazards of the substi- tute materials are suggested. P. Warren contributes one of the shorter chapters and covers the literature relating to asbestos. The toxicity of asbestos to man is illustrated by human and animal studies and information on the hazards caused by the use of asbestos-containing materials in industry is given. They are: an intro- duction by W. D. Buchanan; pathology and experimental pathology of asbestos dust exposure by J. S. P. Jones; asbestos associated thoracic disorder - clinical features by L. H. Capel; epidemiology of asbestos-related disease by M. Newhouse; and the prevention of asbestos-related diseases by R. J . Levine, M. D. Gidley, M.Feverstein, M. Chesney, P. M. Giever and J. S. Felton. Numerous illustrations are included , showing the effect of asbestosis and mesothelioma on the body. Each chapter has its own contents page and references. It is factual and generally achieves a sensible approach to the subject without resort either to scaremongering or dismissive tactics. The book is thought likely to be purchased by libraries for use by a wide range of professions. The section devoted to applications seems a little short in comparison with the rest of Volume 1 ; no hint is given as to the possible contents of Volume 2, and it may be that this will cover applications in greater detail. R. Derricott next describes the use of asbestos and asbestos-free substitutes in buildings. The final 5 chapters of the book are given over to asbestos-related diseases. As a whole the volume is written clearly.D. SIMPSON HANDBOOK OF ANALTYCIAL CONTROL OF IRON AND STEEL PRODUCTION. By T. S. HARRISON. Ellis Hormood Series in Analytical Chemistry. Pp. 602. Chichester : Ellis Horwood. Distributed by John Wiley in Australasia, South-east Asia, Canada, Europe and Africa and by Halsted Press in North and South America and the rest of the world. Price d37.50. 1979. The author is faced with a dilemma in tackling this subject at the present time. Historically, analysis was by wet-chemical methods, which were primary techniques based on stoicheiometric reactions and are enshrined today in British Standard Handbook No. 19. However, as modern steel making practice evolved these methods became too slow to provide the rapid results required for control of the liquid melt.The introduction of direct reading spectrometric procedures re- solved the bottleneck by producing results in 2 - 3 min but these procedures required accurately analysed standard samples for calibration purposes. This in turn implied that a primary (i.e., chemical) method was available to analyse the standards. Here is the author’s dilemma: to decide how much emphasis to place on chemical methods compared with the practical applications based on spectrometric techniques. The dilemma is solved by virtually combining two books into one and the result is a complete handbook for the chemist and spectrographer in one volume. For the smaller steel making plant where spectrometric methods have not been introduced and for all laboratories acting in a test- house role the authoritative treatment of chemical procedures will be welcome, extending to some 30 alloying and trace elements.In a modern integrated iron and steel works the analyst is required to examine a wide range of materials in addition to steel and these are fully described in the book. A specially contributed chapter on the determination of gases in steel by Dr. J . D. Hobson is a valuable addition by a recognised authority and summarises the present position of this subject.1104 BOOK REVIEWS Analyst, Vol. I04 In Part 1 a comprehenisve treatment of sampling is followed by theoretical descriptions of the techniques used in the book and these are listed under the headings Chemical, Physico-chemical and Physical.Each chapter carries a generous list of references that is a praiseworthy feature of the book. Some of the sections have a surer touch than others, probably reflecting the author’s own interest, and in this respect emission spectroscopy leads X-ray fluorescence but with the up-to-date bibliography provided, the reader should find no difficulty in using the book as a primary source of information. Brief mention is made of the use of lasers and plasmas as sources in emission spectroscopy and they are described as developments that could be useful to the iron and steel industry. The author’s prediction has already been fulfilled to such an extent that, certainly for plasmas, they are already front page news. Part 2 opens with detailed working methods for 30 constituents of iron and steel followed by a short section on atomic-absorption spectroscopy that describes the determination of some 15 elements in concentrations ranging up to approximately 1% in steel. This is a conservative use of the technique that according to some workers may also be used for major alloying constituents with coefficients of variation of 0.5% or even better.Methods are given for the analysis of 13 ferro alloys by chemical procedures followed by chapters on refractory materials and an extensive range of topics collected under the heading miscellaneous. In this section there is the important subject of environmental pollutants together with welding rods, pickling acids and non-ferrous alloys. Part 3 consists of 3 chapters on fuels and coke-oven byproducts plus a full treatment of analytical methods for waters and effluents and finally lubricants, oils and greases.With the one exception mentioned above it is agreed that “the procedures in this handbook coupled with the many refer- ences should cover most requirements in the foreseeable future.” W. R. NALL METAL /3-DIKETONATES AND ALLIED DERIVATIVES. By R. C. MEHROTRA, R. BOHRA and D. P. GAUR. Pp. viii + 382. London, New York, San Francisco: Academic Press. 1978. Price k20.50. There can be few classes of coordination compounds that have been the subject of such extensive research as the metal p-diketonates. In recent years their applications in gas chromatography and solvent extraction] in laser technology and in nuclear magnetic resonance spectroscopy have made them familiar to scientists of varied disciplines and interests. This new monograph deals with the chemistry of these /?-diketone complexes, and concentrates on four main aspects : synthesis, bonding and aromatic character, chemical reactions and physical properties] including spectroscopic, magnetic and structural characteristics. After a brief introduc- tion, the text deals in turn with oxygen-bonded, carbon-bonded and thio-p-diketonato complexes. Only brief discussions of applications are given, the authors considering that these have been the subjects of other recent monographs. The book concludes with an extensive reference list con- containing 1890 citations and author and subject indexes. Although this book covers a rather specialised area of chemistry for the majority of analytical chemists] its existence is well worth noting, for it is the most complete survey of these interesting compounds yet prepared. Anyone using or contemplating the use of metal /3-diketonates in analytical chemistry will find this comprehensive account of the subject worthy of more than just a cursory glance. W. I. STEPHEN

ISSN:0003-2654    

DOI:10.1039/AN9790401102

出版商:RSC    

年代:1979

数据来源: RSC