ISSN:0003-2654    

DOI:10.1039/AN96186FX041

出版商:RSC    

年代:1961

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN96186BX043

出版商:RSC    

年代:1961

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN96186FP201

出版商:RSC    

年代:1961

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN96186BP215

出版商:RSC    

年代:1961

数据来源: RSC

摘要:

OCTOBER, 1961 Vol. 86, No. 1027 THE ANALYST PROCEEDINGS OF THE SOCIETY FOR ANALYTICAL CHEMISTRY ORDINARY MEETING AN Ordinary Meeting of the Society was held on Thursday and Friday, October 5th and 6th, 1961, at The Institution of Mechanical Engineers, Birdcage Walk, London, S.W.l. The Chair was taken by the President, Dr. A. J. Amos, F.R.I.C. The following papers were presented and discussed: “Studies of the Boron - Curcumin Complex and its use in Trace Boron Analysis,” by M. R. Hayes, A.R.I.C., and J. Metcalfe, B.Sc.; “Applications of Micro-coulometry,” by R. G. Monk, A.R.C.S., D.I.C., Ph.D., K. C. Steed and G. C. Goode, B.Sc., A.R.I.C.; “The Gravimetric Determination of Uranium as the Phosphate,” by J. S. Wright, J. A. Ryan, A.R.I.C., and T. J. Hayes, A.R.I.C.; “A Study of the Determination of Oxygen in Beryllium by Vacuum Fusion,” by M.R. Everett and J. E. Thompson; “The Application of the Conductimetric Method for the Determination of Carbon to Highly Alloyed Steels and the Less Common Metals,” by J. E. Still, B.Sc., F.R.I.C., and I. R. Green, A.R.I.C.; “The Identification and Determination of Foreign Phases and Constituents in Metals, with Special Reference t o Beryllium,” by H. P. Rooksby, BSc., F.Inst.P., and I. R. Green, A.R.I.C. ; “Developments in Emission Spectrography Arising from the Routine Determination of the Isotopic Abundance of Uranium,” by R. Franklin and J. R. Hartley, B.Sc. , A.R.I.C. ; “Suspension Scintillation Counting of Carbon-14 Barium Car- bonate,” by H. J. Cluley, Ph.D., F.R.I.C.; “The Use of Isotope Dilution for the Determination of Hydrogen in Metals, with Particular Reference to Alkali Metals,” by C. Evans, B.Sc., Dip.Chem.Eng. , and J. Herrington, B.Sc. ; “Reverse-phase Partition Chromatography,” by T. J. Hayes, A.R.I.C., and A. Hamlin, BSc., F.R.I.C.; “Gas Chromatography in the Analysis of Inorganic Systems,” by T. R. Phillips, Ph.D., B.Sc., and G. Iveson; “The Analysis of Nuclear Reactor Carbon Dioxide for Gaseous Impurities,” by R. M. S. Hall, M.A., A.R.I.C., A.Inst .P. DEATH Thomas Cockburn. WE record with regret the death of 629

ISSN:0003-2654    

DOI:10.1039/AN9618600629

出版商:RSC    

年代:1961

数据来源: RSC

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630 PROCEEDINGS [Vol. 86 Obituary HAROLD WRIGHT HODGSON HAROLD WRIGHT HODGSON died on May 13th last, in his forty-eighth year. His sudden and untimely death, a t the peak of his career, was a severe shock to his many friends. Hodgson spent his early life in the North-West of England and was educated a t Arnold House, Blackpool. His chemical training was obtained a t Liverpool Technical College, and he received his first experience in industrial analysis in the laboratories of William Crawford and Sons Limited, Liverpool. In 1940 Hodgson joined the staff of Roche I’roducts Limited, a t Welwyn Garden City, where he stayed until 1946. He developed a keen interest in pharmaceuticals during this time, which he maintained throughout his career. He joined the newly formed Research and Development Department of The British Oxygen Company Limited in 1946, to start the Analytical Laboratory.This he developed into one of the most modern laboratories in the country, and latterly his duties involved the co-ordination of Analytical Services for the whole of The British Oxygen Organisation. Hodgson took a special interest in gas analysis and represented the Association of British Chemical Manufacturers on the B.S.I. Committee on “Sampling and Analysis of Gases”; he represented our Society on the B.S.I. Committees Ion “Paramagnetic and Infra-Red Methods” and on “Gas Chromatography.’’ He was a keen member of the Society and served on the Council in 1953 and 1954. Hodgson had a love for cars and motoring; his mechanical skill was considerable, and he was a willing helper at motor racing meetings. This interest no doubt contributed to the great success of the first Rally held by the London Section of the Royal Institute of Chemistry, of which he was one of the organisers. Harold Hodgson was unmarried; he had a very wide circle of friends, all of whom knew him as a most generous and hospitable man. J. H. GLOVER

ISSN:0003-2654    

DOI:10.1039/AN9618600630

出版商:RSC    

年代:1961

数据来源: RSC

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October, 19611 MORGAN 631 The Identification of Small Amounts of Bases in Urine by Infra-red Spectrophotometry BY P. J. MORGAN (Department of Pharmacology, University of Melbourne, Australia) A method is described for the rapid extraction of organic bases from urine, in amounts of the order of lOOpg, in a sufficiently pure condition for identification by infra-red absorption spectrophotometry. THE analytical significance of all organic compounds having highly characteristic absorption spectra in the wavelength range between 2.5 and 15 p has, in recent years, resulted in the availability of instruments that record spectra in this range and yet do not call for a pro- hibitively large capital outlay in view of the useful work they can perform. An objection often made when considering the application of infra-red absorption to substances extracted from biological sources is that any associated impurities will contribute absorption bands similar in intensity to those given by the substance being studied. The extraction procedure described here is sufficiently selective when applied to urine to permit this disadvantage to be overcome.The procedure is an adaptation of the acid-spot extraction method previously describedl for detecting organic bases in urine at the 1 p.p.m. level. In this method, bases are trans- ferred from a chloroform extract of the urine to spots of dilute sulphuric acid supported on strips of Whatman 3MM filter-paper and are detected by paper chromatography of the strips in acidified solvents after exposure to ammonia vapour.The procedure described here supplements this method in that a larger amount of any base detected on the paper chromatograms can be rapidly isolated in a relatively pure condition. It also facilitates the rapid detection of bases, such as morphine, not readily soluble in organic solvents, when these are present in small concentrations in the urine. APPARATUS- A modified form of the acid-spot extraction apparatus described previously is used (see Fig. 1). It consists essentially of an inner jacket conveniently made from a Liebig condenser 14 inches in diameter and cut off square near one end. It should be made as long as possible, but should still maintain reasonable rigidity of the central tube; a length of 12 inches was found to be satisfactory. The tip of the central tube is constricted in a flame to give an opening about $ inch in diameter. The open end of the Liebig jacket fits into a cylindrical beaker 18 to 12 inches in diameter and 74 inches tall, which is supported in a water bath.The outlet at the top of the Liebig jacket is connected by means of an 18-inch length of polythene tubing to a test-tube fitted with a side-arm. This serves as a trap for condensed solvent vapours and may be cooled if desired. An acidified solution of chromous sulphate, concentrated sulphuric acid and a U-tube of silica gel may be used as previously describedl to provide a dry stream of nitrogen. If the gas is passed through a U-tube of fresh self-indicating silica gel, this will change in colour from pink to blue if the gas is being satisfactorily dried.ACID-SPOT EXTRACTION PROCEDURE- A chloroform extract of the urine is prepared by gentle agitation of the specimen with 100ml of chloroform after adjustment of pH to a suitable value with 20 per cent. sodium hydroxide solution as previously described.l The chloroform extract is introduced into the beaker, which is supported in the water bath at a temperature at least 20" C above that of the room. Spots of 0.25 and 0-5 N sulphuric acid - thymol blue reagent1 are placed about 3 cm from one end of a strip of Whatman 3MM filter-paper having the same width as the Liebig jacket and of any desired length. When the spots have dried somewhat, the strip is inserted into the Liebig jacket, which is then clamped in the beaker at a slight angle to the vertical.The angle of tilt and the arrangement of the polythene tubing should be such that any solvent METHOD632 MORGAN: THE IDENTIFICATION OF SMALL AMOUNTS OF [Vol. 86 condensing in the polythene tube does not run back into the chloroform extract. The acid spots should be below the surface of the chloroform extract, which is agitated by passing a brisk stream of dry oxygen-free nitrogen through the central tube of the jacket. Two filter- paper strips may be inserted if so desired, one on each side of the central tube. Extraction of bases proceeds on to the acid spots, which are examined from time to time for any change in colour of the thymol blue indicator. If the red colour of the spot of 0.25 N acid shows any change towards neutrality (yellow) when compared with the spot of 0.5 N acid, the strip is immediately removed and replaced by a fresh strip.Generally, any early tendency to neutralisation is rapidly overcome as volatile bases, such as ammonia and amines having low molecular weights, are swept out of the chloroform extract by the stream of gas. The tendency of chloroform to emulsify in the urine generally makes a second extraction with a further portion of chloroform desirable; further, for a base not particularly soluble in chloroform, several successive extractions may be needed to extract most of the base. These portions of solvent can be added via the side of the beaker from time to time as chloroform evaporates into the stream of nitmgen and serve to replenish base that is being removed from the solution on to the acid spots.\ extract- To solvent c.- 0 - i - trap Solvent iner jacket ilter paper strip *Beaker ,Acid spots Fig. 1. Acid-spot extraction apparatus Any thick emulsion remaining after the final extraction with chloroform can be broken as follows. The emulsion is run, in small portions, from a separating funnel into a flask and then shaken vigorously. If the proportion of non-aqueous phase is sufficiently high, the shaking will break the emulsion. When this procedure is no longer effective, small amounts of cyclo- hexane are added to the emulsion, and the vigorous shaking is repeated; any further organic phase that separates is added to the main extract in the acid-spot extraction apparatus (with filtration through a dry filter-paper if it is turbid).As a last resort, vigorous shaking, together with the addition of small amounts of anhydrous sodium sulphate, will result in further breaking of the emulsion. Since cyclohexane is a much poorer solvent than chloroform and is also appreciably less volatile, its introduction into the extraction system will lead to an increase in the rate of extraction of most bases on to the acid spots in the final stages. The lack of solubility of some extremely weak bases, such as caffeine, in cyclohexane can be utilised to achieve acid- spot extraction of these bases when they occur in the urine at concentrations of about 1 p.p.m.October, 19611 BASES IN URINE BY INFRA-RED SPECTROPHOTOMETRY 633 It is preferable to pass nitrogen until the volume of chloroform has been decreased to about 50 ml and then to dilute with cyclohexane to about 200 ml.The passage of gas is continued until the chloroform content of the extract has been decreased to less than 1 per cent. by volume; this operation is conveniently carried out overnight at 45" C. The chloroform content is easily determined by recording an infra-red spectrum in a cell 0.05 mm thick, when the intense chloroform absorption band at 13-1 p can be used to determine concentrations of chloroform of less than 1 per cent., the weaker band near 8.3 p in the range 1 to 10 per cent. and the weak cyclohexane band near 8-0 p when the chloroform content is more than 10 per cent. by volume. PAPER CHROMATOGRAPHY OF ACID-SPOT EXTRACTS- Chromatography of the strips removed from the acid-spot extraction apparatus can be carried out as previously describedl by exposing them to ammonia vapour for a short time to neutralise the excess of sulphuric acid on the spots and then developing in either the n-butyl alcohol - hydrochloric acid or the isobutyl methyl ketone - acetic acid solvent. These solvents were chosen for the rapid detection of bases because they give good chromatograms without the need for any equilibration; however, for purposes of isolation, it is preferable to allow some time for equilibration, particularly if the thick Whatman 3MM filter-paper is used.Detection of typical alkaloids is achieved, as before, by spraying the chromatograms with the iodoplatinate reagent. For the detection of the weak base caffeine it is essential to add about 6 per cent.of concentrated hydrochloric acid to this reagent. Amines of lower molecular weight, which may not react satisfactorily with the iodoplatinic acid to give the coloured precipitate on the paper, are detected by spraying with bromocresol green in ethanol after development in the isobutyl methyl ketone - acetic acid solvent. A base may be recovered in good yield from chromatograms that have been sprayed with either or both of these reagents by cutting out the coloured spots, placing them in a small Erlenmeyer flask (Quickfit & Quartz Ltd.) and adding sufficient concentrated ammonia solution just to moisten the paper completely. The stoppered flask is shaken in a mechanical shaker for 30 minutes with sufficient chloroform just to cover all the pieces of paper (the shaking should not be so vigorous that the paper disintegrates). If this condition is observed, it is possible to check for completeness of extraction of base by drying the pieces of paper and re-spraying them with the iodoplatinate reagent after decantation of the chloroform extract.If any cellulose fibres are detached by over-vigorous shaking, or if too much ammonia has been used, then the extract must be filtered, and completeness of extraction checked by re-shaking the residue plus the filtering medium with a further amount of chloro- form. Prolonged shaking of the initial extract should be avoided, as it may lead only to a higher level of impurities in the extract. When this procedure is adhered to, it has been found that bases are obtained in a sufficient degree of purity to give a useful infra-red spectrum ; it is best to aim a t the collection of a few hundred micrograms of base from about ten chromatographic spots.INFRA-RED SPECTROPHOTOMETRY OF EXTRACTS- Spectrophotometry is best carried out in solution in an organic solvent in micro cells having sodium chloride windows. The chloroform extract from the chromatographic spots must be evaporated to dryness before double-beam spectrophotometry can be attempted, even if chloroform is the solvent of choice. This is conveniently carried out in a glass tube having a narrow tip, such as a centrifuge tube, by passing a stream of nitrogen through the extract at 40" C. Vapour condensing on the cooler sides of the tube eventually washes all the solute down into a small region a t the narrow tip, so that, when the amount of solvent required to fill the micro cell is added, the solute is totally covered.Solution may be assisted by the application of gentle heat to the tip of the tube if precautions are taken to avoid loss of solvent by evaporation. After a spectrum has been recorded, some sort of purification technique should be applied to the sample. This purification may take the form of a further paper-chromatographic stage if the level of impurities Appears to be high. It is preferable to use a solvent system different from that used for developing the first chromatograms of the acid spots. Infra-red absorption bands that disappear or are decreased in absorption relative to other bands in634 MORGAN: THE IDENTIFICATION OF SMALL AMOUNTS OF [Vol.86 the spectrum after the purification can then be ascribed to the impurities present. When paper chromatography is used, it is never possible to get rid of the last traces of absorption (due to organic impurities from the paper) in the carbon - hydrogen stretching and bending regions of the spectrum. Sublimation in vacuo is often a most effective final procedure for purification. The use of a double-beam spectrophotometer recording from 2-5 to at least 1 5 p is essential for the satisfactory study of the small amounts of material being examined, Instru- ments that cover this range are usually fitted with a sodium chloride prism for dispersing the radiation ; however, an instrument having a diffraction grating for dispersing the radiation is generally capable of giving better results in the shorter-wavelength portion of the spectrum.A choice of two widely different recording speeds is desirable, as this permits more careful coverage of spectral regions in which absorption from the solvent is high. To cover regions in which absorption from the solvent is extremely high, the use of an alternative solvent is necessary. A laboratory-reagent grade of tetrachloroethylene (fractionated before use, with rejection of the initial and final fractions) was found to be most satisfactory for work on the micro scale; this solvent keeps well in a dark-glass bottle. When a fairly large amount of material has been isolated in a substantially pure condition, simple washing of the sample with small portions of tetrachloroethylene may remove sufficient of the impurities present to permit detection of the absorption bands associated with the substance under investigation.I t is recommended that micro cells about 0-3 and 1.0 rrim thick should be used for work with tetra- chloroethylene. It should be noted that the extraction method described is biased in favour of substances readily soluble in organic solvents and that thicker cells may be required for some solutes. When lack of solubility in organic solvents is associated with the presence of hydroxyl groups in the molecule, it is sometimes expedient to improve the solubility by partial acetylation of the substance. For example, it was found that conversion of morphine into its monoacetyl-derivative permitted a much more detailed spectrum to be obtained in a cell 0.3 mm thick than could be obtained from morphine itself in a cell ten times as thick.The fact that hydroxyl groups can forrn intermolecular hydrogen bonds, even in fairly dilute solution, should be kept in mind when two spectra are being compared, as, when such bonds are formed, the sharp absorption band due to free hydroxyl is replaced by a much broader band at a longer wavelength. Some general aspects of infra-red solvents have been discussed by Cole.2 The use of tetrachloroethylene has been discussed elsewhere.3 DISCUSSION OF THE METHOD The extraction method described is based on a 500-ml sample of urine, but it can be applied as it stands to larger or smaller samples, within reason. Up to 5 per cent.of ethanol may be added to the chloroform used for extraction of the urine in order to improve the rate of extraction of substances, e.g., morphine, soluble only with difficulty, but it is doubtful whether this offers any real advantage, since not only are more impurities extracted from the urine, but difficulty may also be caused by emulsification of solvent in the urine. In order to minimise loss of thymol blue indicator from the acid spots, it is advisable that they should be allowed to dry in air before introdiiction into the chloroform extract ; however, it should be realised that the spots will pick up any volatile bases present in the laboratory atmosphere if they are left exposed for too long a period. Since the thymol blue indicator is retained near the centre of the spot when the acid spots are applied to the paper, it is possible to follow the progress of extraction by adding a drop of an extremely dilute solution of the basic dye methylene blue in ethanol to the chloroform extract.The extracted dye also serves as a standard of R, value when the extracts are subjected to chromatography. Since the colour of the thymol blue indicator may occasionally not be visible when the strips are removed, a fairly concentrated solution of thymol blue in ethanol should be kept at hand and applied to the spots with a loop of platinum wire if there is any doubt. Unless the spots have retained their acidity to thymol blue, there is no guarantee that weak bases initially extracted on to the spots have not been displaced by stronger bases.If long strips of paper are used in the apparatus, it is essential to keep the chloroform extract at a temperature substantially higher than that of the room so that some condensation of solvent vapour will occur on the portion of the strip not immersed in the solvent. This serves to keep the upper part of the strip washed free of solutes that may otherwise accumulateOctober, 19611 BASES IN URINE BY INFRA-RED SPECTROPHOTOMETRY 635 there. If short strips completely immersed in the extract are used, it is not necessary to apply heat, and the risk of loss of any labile material is therefore substantially decreased; however, the time needed for extraction is increased, and chromatography of short strips presents some difficulties, as it is necessary to place the spots of acid close to the end of the strip, and this results in too fast a flow of solvent over the spots when chromatography is begun.The end of the strip may be cut into a serrated pattern, with the tips of the serrations just touching the solvent when chromatography is begun, or wire staples may be used to attach another piece of paper to the end of the strip to serve as a “lead-up” for the solvent. Alternatively, if the highest possible purity is to be sought from the chromatogram, a small rectangle containing the spots This defect may be overcome in three ways, as shown in Fig. 2. Two-phase system S ingle-phase system Fig. 2. is cut out and placed centrally between two glass plates. One edge of the assembly is grasped between thumb and fore-finger so that the other edge opens out slightly, with the edge of the paper as fulcrum, and a short “lead-up” length of paper is inserted to meet the rectangle, with a slight overlap.The pressure is then transferred to the other side of the assembly to permit similar insertion of a longer piece of paper on the other side of the rectangle. The assembly is then clamped together with clothes pegs of the spring-type, and chromatography is carried out by the ascending-solvent technique. Some methods for paper chromatograms TABLE I LIMITS OF DETECTION OF VARIOUS BASES IN URINE Base Limit of detection (as concentration in original sample) in- f A \ solution after concentration cyclohexane solution initial chloroform of several extracts containing 1% v/v extract at pH 9, to 50 ml, of chloroform, p.p.m.Strychnine . . . . 2 Cocaine. . .. .. 2 Amphetamine . . . . 5 Morphine .. . . 8 Caffeine .. . . 100 p.p.m. 1 1 2 2 40 p.p.m. ( 1 1 1 2 <1 In the final stages of the acid-spot extraction from the cyclohexane-rich solvent, some solid matter may be deposited near the tip of the gas-delivery tube. This can be easily re-dissolved by placing the delivery tube for 1 or 2 minutes in a few drops of chloroform in a test-tube; the solution is then washed into the main extract with a little cyclohexane. Table I gives the approximate limits of detection for some bases at various stages of the procedure; the limits shown are based on practical experience of the method. The limit636 FEW: A VANILLIN - PERCHLORIC ACID REAGENT FOR DETECTING [Vol. 86 of detection attained depends largely on the skill and experience of the operator and involves factors such as the technique of spraying the chrornatograms. For instance, if both sides of a chromatogram are sprayed with the acidified iodoplatinate reagent, it is usually found that the sensitivity is greatly increased. Under these conditions, ammonium ion may develop as a grey spot on the pink background at a low R, value. It is sound practice initially to carry out some blank extractions in order to ascertain whether or not (a) the solvents or paper used make any contribution and (b) the ammonia introduced just before development of the chromatogram can be detected. I acknowledge that this work was made possible by grants from the Victoria Racing Club and the Anti-Cancer Council of Victoria. REFERENCIE S 1. 2. 3. Morgan, P. J., Analyst, 1959, 84, 418. Cole, A. R. H., Rev. Pure AppZ. Chem., 1954, 4, 118. Ard, J. S., Anal. Chem., 1953, 25, 1743. Received May 29th, 1961

ISSN:0003-2654    

DOI:10.1039/AN9618600631

出版商:RSC    

年代:1961

数据来源: RSC

摘要:

636 [Vol. 86 FEW: A VANILLIN - PERCHLORIC ACID REAGENT FOR DETECTING A Vanillin - Perchloric Acid Reagent for Detecting Pregnanetriol and Related Compounds on Paper Chromatog:rams BY J. D. FEW (Department of Chemical Pathology, Charing Cross Hospital Medical School, London, W.C.2) The conditions under which a vanillin - perchloric acid reagent can be used for detecting pregnanetriol on paper chromatograms are described. As well as with pregnanetriol, the reagent produces distinctive colours with several other closely related steroids. Other aromatic aldehydes in conjunction with perchloric acid have been investigated as colour-forming reagents ffor steroids, and the results are reported. DURING a study of the urinary steroids excreted in cases of congenital adrenal hyperplasia, the need arose for a simple and sensitive method of detecting pregnanetriol (5p-pregnane- 3a,17ccJ20a-triol) on paper chromatograms.Of the reagents previously used for this purpose, molybdophosphoric and phosphotungstic acids1 are sensitive and simple to use, but have the disadvantage of reacting with a wide range of stieroids to form blue spots only. Antimony trichloride, in either chloroform2 or nitr~benzene,~ :reacts with fewer steroids and is sensitive to pregnanetriol, but is highly toxic and easily affected by traces of water or alcohols. Bush4 described the oxidation of steroidal 17,2O-glycols with periodic acid and detection of the generated 17-oxosteroids with alkaline m-clinitrobenzene. Although this technique has the requisite sensitivity and is specific for 17,2O-glycols, it is neither simple nor rapid to carry out.The use of trichloroacetic acid as st reagent for pregnanetriol was described by de Courcy5; this reagent forms distinctive colours visible in the visual and ultra-violet regions with pregnanetriol and a few closely related compounds. Satisfactory development of colour, however, depends on careful control of the heating of the papers, during which process unpleasant fumes are evolved. Godine described a vanillin - perchloric acid reagent for locating sugar alcohols and ketoses (but not aldoses) on paper chromatograms, and this was later used to locate a wide range of deoxy sugars7 Initial experiments in which this reagent was applied to the steroid field showed it to be of value for detecting 17-hydroxy-20-ketones and 17,2O-glycols.Aromatic aldehydes in the presence of sulphurjc or orthophosphoric acid have been used for locating a wide range of s t e r o i d ~ , ~ ~ ~ J ~ J ~ but the subject has not apparently been systematic- ally investigated. This paper describes the result:; of a detailed study of the use of vanillin and other aromatic aldehydes in the presence of perchloric acid for locating steroids on paper chromatograms, with particular reference to the pregnanetriol group.October, 19611 PREGNANETRIOL AND RELATED COMPOUNDS ON PAPER CHROMATOGRAMS 637 EXPERIMENTAL PAPER CHROMATOGRAPHY- This was carried out with use of the system iso-octane - toluene - methanol - water in the ratio 50 : 150 : 160 : 40 (as recommended by de Courcy6) and 80 : 120 : 160 : 40.Whatman No. 1 chromatography paper was used throughout. In exploratory experiments, spots of solutions of steroids were placed on test strips of paper to give a concentration of 5 p g of steroid per sq. cm, and these strips were treated with the colour-forming reagents without chromatographic development. COLOUR-FORMING REAGENTS- These were prepared in aqueous solution so as to contain 1 per cent. w/v of the aldehyde and 10 per cent. v/v each of perchloric acid and either toluene-$-sulphonic or orthophosphoric acid. For aldehydes sparingly soluble in water, up to 20 per cent. v/v of acetone was included in the mixture. A typical reagent solution consisted of 1 g of vanillin, 10 g of toluene-$- sulphonic acid and 15 ml of 60 per cent. perchloric acid $us water to 100 ml.TREATMENT OF CHROMATOGRAMS- The dried papers were dipped in the reagent, excess of solution was removed by blotting, and the papers were dried in a stream of warm air (75" to 85" C) from a laboratory air blower or an electric convection heater; the spots of steroids appeared when the paper was dry. With the reagent containing vanillin, the coloured spots appeared on a yellow background, which rapidly faded. Excessive heating of the paper was avoided, as it led to charring (as did heating in a closed oven at 100" C). RESULTS COMPOSITION OF COLOUR-FORMING REAGENT- In preliminary experiments on a small range of steroids, a 1 per cent. w/v solution of vanillin containing different proportions of perchloric acid was used; it was found that the intensity of colour was dependent on the concentration of acid present.The concentration of 10 per cent. v/v of perchloric acid was chosen because it led to an adequate colour without causing serious damage to the paper. Concentrations of perchloric acid greater than 15 per cent. v/v were found to produce faint grey spots with almost all steroids, thereby decreasing the specificity. When perchloric acid was replaced by other acids, pregnanetriol produced little colour except when toluene-+-sulphonic or orthophosphoric acid was substituted (and even then the colours were much less intense than those formed in presence of perchloric acid). If toluene-9-sulphonic or orthophosphoric acid is added to the vanillin - perchloric acid reagent, then the colour formed by pregnanetriol (and several other steroids) is considerably intensified, without the disadvantages associated with an increased concentration of per- chloric acid.COLOURS PRODUCED WITH PURE STEROIDS- When the mixture of vanillin, toluene-$-sulphonic acid and perchloric acid, subsequently referred to as the vanillin - perchloric acid reagent, was used, 5 pg of any of the compounds listed in Table I could be readily detected on paper chromatograms and 2 p g were just detectable; on the test strips, 1 pg per sq. cm was detectable. The colours produced initially with most of these steroids change during about 30 minutes when exposed to air at room temperature and then gradually fade. The paper is attacked by the reagent, but only disintegrates after a few days, by which time most of the spots have faded.Derivatives of 3,4-dihydroxybenzaldehyde other than vanillin react similarly with this group of steroids ; 2,4-dihydroxybenzaldehyde and p-hydroxybenzaldehyde also react with the steroids listed in Table I, but the colours formed differ slightly from those produced by vanillin, As well as with this group of compounds, the vanillin-perchloric acid reagent also reacts with other steroids, although the colours formed are less intense. These steroids, which are listed in Table 11, generalIy produce more intense colours with p-hydroxybenzalde- hyde than with vanillin; with 2,4-dihydroxybenzaldehyde they react either not at all or only to produce colours very low in intensity. If the concentration of perchloric acid in the reagent638 FEW: A VANILLIN - PERCHLORIC ACID REAGENT FOR DETECTING [Vol.86 is increased to 15 per cent. v/v, then p-hydroxybenzaldehyde with the addition of 10 per cent. v/v of orthophosphoric acid is a suitable reagent for detecting most of the steroids listed in Table 11, 10 p g being easily detectable on paper chromatograms. A blue-grey colour is produced by most of these compounds, but the sapogenins appear as yellow spots. The corticosteroids cortisone, cortisol and cortexone and the saturated 17-oxosteroids not substituted in the 16-position do not react with any of the aldehyde - perchloric acid reagents at concentrations of less than 20 pg per sq. cm on test strips and give mostly negative results at 50 pg per sq. cm. Other steroids not reacting with these reagents include testo- sterone, 3a-hydroxy-SP-pregnan-20-oneJ 3cc,16cc-dihydroxy-5P-pregnan-SO-one and lithocholic acid.Deoxycholic acid reacts with 9-hydroxybenzaldehyde to produce an intense colour, but not with any of the other aldehydes tested. In many chromatographic systems, solvents having elevated boiling-points are used as stationary phase, and these are often difficult to remove completely from the paper chromato- grams. Several of these solvents have been tested for reactivity with the vanillin - perchloric acid reagent by placing spots of a (1 + 1) mixture of solvent and methanol on test strips of paper, dipping the strips into the reagent, drying and heating in the usual way after evapora- tion of the methanol. 2-Ethoxyethanol (ethyl Cellosolve) produced an intense brown colour immediately after drying; propylene glycol formed a red colour after prolonged heating, and formamide inhibited formation of all colour, including the yellow background.Ethylene glycol and glycerol were inactive. TABLE I STEROIDS REACTING TO PRODUCE INTENSE COLOURS Steroid Colour produced* 5P-Pregnane-3a, 17a,20a-triol . . .. .. .' .* ] Purple changing to blue 6P-Pregnane-Sa, 16a,20&triol . . .. .. . . Mauve changing to blue 3a-17a-Dihydroxy-5 8-pregnane-1 1,20-dione . . ' * 5a-Pregnane-Sa, 17a,20a-triol . . .. .. 5#?-Pregnane-3aJ17a,20/3-triol . . .. .. .. 3a, 17%-Dihydroxy-5 fl-pregnan-20-one . . .. 3a, 17a-Dihydroxy-5a-pregnan-2O-one . . .. .. Pregn-5-ene-3 #?, 17a,20a-triol . . .. Pregn-Fi-ene-3/3,17a,20#?-triol .. .. .. .. Diosgenin . . . . : : } Orange changing to colourlesst ' .> Green changing to blue Pregn-5-ene-3/3,16a,20$triol . . .. ,. . . Grey 3 #?,16a-Dihydroxypregn-5-en-20-one . . .. , . Orange changing to brownt .. .. .. .. . . Yellow * The changes in colour take place during approximately 30 minutes when the chromato- t This change can be reversed by re-heating. gram is exposed to air a t room temperature. TABLE I1 STEROIDS REACTING TO PRODUCE LESS INTENSE COLOURS Steroid Colour produced Cholic acid .. .. .. .. Hecog enin .. . . . . .. Tigogenin . . .. .. .. .. 5a-Androstane-3 /3,17 &diol . . .. 3 /3, 16a-Dihydroxy-6a-androstan-17-0ne Pregnane-3,20-diol (various isomers) . . 3a, 17aJ21-Trihydroxy-6~-pregnan-20-one 3 &Hydroxyandrost-5-en-l'I-one . .3 /3-Hydroxypregn-5-en-20-one . . .. Androst-5-ene-3 8,17 p-diol . . .. Pregn-li-ene-3/3,20fl-diol . . . . .. . . .. Mauve Yellow .. * * .. * . > .. .. : :} Blue .. .. Dull purple Grey-brown .. .. . . . .. .. .. Among the other aldehydes tested, phthalalaerly de (without the addition of orthophos- phoric or toluene-fi-sulphonic acid) was useful, as it produced an intense blue colour with the epimeric pregn-5-ene-3/?,17a,20-triols and only a pale brown with pregnanetriol. This reagent also reacted with all the other A6-3/3-hydroxy-steroids listed in Tables I and 11,October, 19611 PREGNANETRIOL AND RELATED COMPOUNDS ON PAPER CHROMATOGRAMS 639 giving a characteristic olive colour that permitted the easy detection of 5 pg of any of these steroids on paper chromatograms. It did not react with any of the other steroids tested.9-Dimethylaminobenzaldehyde and pyridine-3-aldehyde (also without the addition of orthophosphoric or toluene-9-sulphonic acid) appear to be fairly specific for the A6-3/?-hydroxyl grouping, but neither of these aldehydes produces such an intense colour as do the other reagents, and, especially with pyridine-3-aldehyde, the spots fade rather rapidly. Although other aromatic aldehydes, including benzaldehyde, furfural, 2,3-dimethoxy- benzaldehyde and anisaldehyde, form colours with some steroids in the presence of perchloric acid, they were not found to be of value in this type of analytical work, owing to lack of sensitivity, often combined with lack of specificity and instability of colour developed.Many aldehydes, including all the mononitro- and monochlorobenzaldehydes, m-hydroxybenzalde- hyde and various hydroxynitrobenzaldehydes, do not form colours with any of the steroids tested. APPLICATION TO EXTRACTS OF URINE- Extracts of urines hydrolysed with /3-glucuronidase were fractionated on columns of alumina by the method used by Stern12 in the determination of pregnanetriol. The “preg- nanediol” and pregnanetriol fractions were submitted to paper chromatography, and the chromatograms were treated with the vanillin - perchloric acid reagent. The “pregnanediol” fraction always showed a number of spots, of which that produced by 3a,17a-dihydroxy- 5p-pregnan-20-one was usually prominent. It is only in urine from pregnant subjects that pregnanediol occurs in amounts sufficient to be detected in Stern’s “pregnanediol” fraction with this reagent.When the pregnanetriol fraction was examined, pregnanetriol was generally found to be the major compound reacting with vanillin. With urine from several cases of congenital adrenal hyperplasia, pregnanetriol was the only compound reacting with vanillin that was detectable in this fraction. In extracts from normal subjects, and particu- larly after stimulation with adrenocorticotropic hormone, many other compounds were present besides pregnanetriol. Mostly, these other compounds were not chromogenic with sulphuric acid and hence did not interfere with the determination of pregnanetriol with this reagent. When paper chromatograms of many extracts of urine are treated with the vanillin- perchloric acid reagent, some of the spots formed do not correspond in position or colour to those produced by any of the available reference compounds.At present, several of these substances are being investigated, although at this stage there is no definite evidence that they are steroids. DISCUSSION OF THE TECHNIQUE The initial aim of this work was to devise a reagent suitable for locating pregnanetriol and related compounds on paper chromatograms. When this aim had been achieved, with the vanillin- perchloric acid reagent, it seemed at least possible that, if other aldehydes were used, this type of reaction could be extended to other groups of steroids. Unfortunately, examination of thirty other aromatic aldehydes has led to the conclusion that this type of reagent has little application outside the pregnanetriol group, except when low sensitivity is acceptable or unavoidable.Noteworthy exceptions to this conclusion are the sapogenins, which produce intense yellow colours with the vanillin - perchloric acid reagent. Recently, Slack and Mader13 have utilised a yellow colour formed by diosgenin and 70 per cent. perchloric acid as the basis of a colorimetric determination of diosgenin in the presence of saturated sapogenins (which are not chromogenic with perchloric acid). In contrast to the reaction described by Slack and Mader, the reaction with the vanillin-perchloric acid reagent is given by saturated sapogenins as well as by diosgenin, although the latter compound produces a colour rather more intense than that formed by hecogenin or tigogenin. At present, it is not possible to explain the mechanism of this colour reaction with any confidence.Experiments are in progress to adapt it for quantitative work, and this should facilitate the elucidation of the mechanism of the reaction. Several reagents previously used for detecting pregnanetriol on paper have to be applied as a spray, and, during this process or subsequent heating, highly toxic fumes may be evolved. In 2 years’ experience with the vanillin - perchloric acid reagent, I have never found it to be in the least objectionable when used as described above.640 DANIEL : THE DETERMINATION OF AROMATIC AMINO-COMPOUNDS [Vol. 86 I am grateful to Professor J. Patterson for his interest in this work, REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Kritchevsky, D., and Kirk, M. R., Arch. Biociiem. Biophys., 1952, 35, 346. Bloch, H. S., Zimmermann, B., and Cohen, S L , J . Clin. Endocrinol., 1953, 13, 1206. Rosenkrantz, H., Arch. Biochem. Biophys., 1953, 44, 1. Bush, I. E., Biochem. J . , 1955, 59, xiv. de Courcy, C.. J . Endocrinol., 1956, 14, 164. Godin, P., Nature, 1954, 174, 134. Maclennan, A. P., Randall, M. M., and Smith, D. W., And. Chew., 1959, 31, 2020. Neher, R., and Wettstein, A., Helv. Chim. Acta, 1951, 34, 2278. Sannide, C., Heitz, S., and Lapin, H., Compt. li'end., 1951, 233, 1670. Callow, R. K., Dickson, D. H. W., Elks, J., Evans, R. M., James, V. H. T., Long, A. G., Oughton, McAleer, W. J., and Kozlowski, M., Arch. Biochem. Biophys., 1956, 62, 196. Stern, M, I., J . Endocrinol., 1957, 16, 180. Slack, S. C., and Mader, W. J., Anal. Chem., 1961, 33, 625. J. F., and Page, J . E., J . Chem. Soc., 1955, 11966, Received June 13th, 1961

ISSN:0003-2654    

DOI:10.1039/AN9618600636

出版商:RSC    

年代:1961

数据来源: RSC

摘要:

640 DANIEL : THE DETERMINATION OF AROMATIC AMINO-COMPOUNDS [Vol. 86 The Determination of Aromatic Amino-compounds BY J. W. DANIEL (Imperial Chemical Industries Ltd ., Industrial Hygiene Eesearch Laboratories, The Frythe, Welwyn, Hevts. The efficiencies of five coupling reagents used for the colorimetric determination of aromatic amines and amino-compounds have been compared. Although N-sulphatoethyl-m-toluidine was perfectly satisfactory for reactions involving short periods of coupling, W-diethyl N- 1-naphthylpropylenediamine is recommended for routine application. AROMATIC amines are of considerable industrial importance, and a health hazard is associated with the manufacture of many of them, e.g., aniline, 2-naphthylamine and the toluidines and xylidines. The control of industrial exposure necessitates frequent analyses of the atmos- phere, and a sensitive analytical procedure for determining these compounds has obvious advantages.Such a method should also be applicable to the study of the ilz vivo metabolism of those organic compounds (e.g., nitrobenzene, “azo” food colours and nitrophenols) excreted as the corresponding amines, such as aniline and $jUlpha.dic acid, the determination of which in biological fluids is important in metabolic investigations. Various procedures have been described for determining amino-compounds.1 When the amine is present in microgram amounts, the favoured procedure involves diazotisation with nitrous acid and then reaction with a suitable coupling reagent to give a highly coloured azo-compound.The intensity of the colour can then be related to the concentration of the mine by using a photoelectric colorimeter or some other convenient method. Since the sensitivity depends on the coupling component, an assessment has been made of the efficien- cies obtained with a variety of coupling agents in the determination of a representative group of amines, viz., aniline, the isomeric chloroanilines, toluidines and xylidines and sulphanilic and metanilic acids. The coupling reagents selected included those that have been widely used, namely, 3-hydroxy-2-naphthoic acid,2 2-naphthol-3,6-disulphonic acid (R salt): N-sulphatoethyl-m-toluiding and N-l-naphthylethylenediamine,4 and a new reagent, “-diethy1 N-1-naphthylpropylenediamhe (“Prolabo,” Rhbne - Poulenc) . EXPERIMENTAL The amine (0-100 g) was dissolved in a little dilute hydrochloric acid, and the solution was diluted to 100 ml with water; further dilutions were made from this solution as required.DIAZO TISATI o N- A 1-ml portion of solution, containing 10pg of amine, was placed in each of a series of glass-stoppered tubes, and to the contents of each tube were then added 1 ml of 2 N hydro- chloric acid and 1 ml of a 0.26 per cent. solution of sodium nitrite. Stoppers were insertedOctober, 19611 DANIEL : THE DETERMINATION OF AROMATIC AMINO-COMPOUNDS 641 in the tubes, and the contents were mixed by inversion. A reagent blank was prepared simultaneously, and the tubes were set aside at room temperature for 15 minutes before proceeding with the coupling stage. COUPLING- The various reagents were added to the diazotised amine, and coupling was allowed to proceed until maximum development of colour had been attained.The wavelengths at which the colours absorbed maximally were established in preliminary experiments, and standard graphs were plotted relating optical density of the coloured solution to the concen- tration of amine present. From the slopes of these graphs, the optical density (10-mm cell) equivalent to 1 pg of amine per ml of final solution was calculated for each amine, and the figure so obtained was used as the basis for comparing the efficiencies of the individual coupling reagents. All optical-density measurements were made with a Unicam SP600 spectro- photometer. REAGENTS AND TECHNIQUES 2-NAPHTHOL-3,6-DISULPHONIC ACID- From 15 to 16 g of the disodium salt of the reagent were dissolved, with heating, in 500 ml of water, the solution was made just alkaline to brilliant yellow paper with M sodium carbonate, cooled, filtered and made up to 1 litre with water.To each portion of diazot sed amine were added 10 ml of M sodium carbonate and 0-5 ml of the reagent solution, and the volume was adjusted to 25 ml with water. N-SULPHATOETHYL-~-TOLUIDINE- To each portion of diazotised amine were added 2 ml each of 3 M sodium acetate and a 1 per cent. w/v solution of the reagent. The azo colour was developed by adding 0-5 ml of concentrated hydrochloric acid, and the volume was made up to 10ml with water. For coupling periods in excess of 60 minutes, 1 ml of a 2.5 per cent. w/v solution of ammonium sulphamate was added before the 3 M sodium acetate.3-HYDROXY-2-NAPHTHOIC ACID- To each portion of diazotised amine was added 1 ml of the 2.5 per cent. w/v solution of ammonium sulphamate, and, 1 minute later, 1 ml of a 0-60 per cent. solution of the reagent in M sodium carbonate was added. The solution was transferred to a separating funnel, and 50 per cent. v/v sulphuric acid was added until the solution was just acid to Congo red paper. The solution was successively extracted with five 5-ml portions of chloroform, the extracts were combined in a 25-ml calibrated flask, and this solution was diluted to the mark with chloroform. N-~-NAPHTHYLETHYLENEDIAMINE AND N'-DIETHYL N-1-NAPHTHYLPROPYLENEDIAMINE- Each of these reagents was used in 1 per cent.w/v solution, and the procedure adopted A. To each portion of diazotised amine was added 1 ml of the 2.5 per cent. solution of ammonium sulphamate, and, 1 minute later, 1 rnl of the reagent solution was added; the solution was then diluted to 10 ml with water. B. To each portion of diazotised amine was added 1 ml of the ammonium sulphamate solution, and, 1 minute later, 2 ml of 3 M sodium acetate and 1 ml of reagent solution were added. The azo colour was developed by adding 0.5 ml of concentrated hydrochloric acid, and the solution was diluted to 10ml with water. for coupling was one of those described below. DISCUSSION OF RESULTS The results of the tests are summarised in Table I, from which it is clear that N-l-naphthyl- e t h ylenediamine, "-diet h yl N-1-naph thylprop ylenediamine and N-sulphatoet h yl-m-t oluidine provide the same order of sensitivity and in this respect are much superior to 2-naphthol- 3,6-disulphonic acid or 3-hydroxy-2-naphthoic acid. The last-named reagent is also limited in application to those amino-compounds forming a chloroform-soluble azo-derivative.It is, however, the only reagent adequate for determining both 2,6- and 2,5-xylidine, which couple slowly with the other reagents; maximum development of colour with the xylidines isTABLE I DETAILS OF COLOURS PRODUCED WITH VARIOUS AMINES Amine Aniline . . o-Toluidine . . m-Toluidine p-Toluidine 2,6-Xylidine 2,4-Xylidine 2,3-Xylidine 3,IXylidine 2,5-Xylidine o-Chloroaniline m-Chloroaniline p-Chloroaniline Sulphanilic acid Metanilic w.id Colour formed with iV- 1-naphthylethylenediamine r Optical density x lo3 .... 44* .. .. 25* .. .. 33% .. .. 38* .. .. <1* .. ,. 13* .. .. 4* .. .. 22* .. .. 5* .. .. 30t .. .. 35f .. .. 36t .. .. 27t .. -- 95f Wavelength of maximum absorption, mP 545 540 545 545 530 545 535 550 535 540 540 545 535 535 \ Colour- development period, minutes 15 30 15 30 60 120 60 30 60 15 15 15 15 ?5 Colour formed with "-diethy1 N- 1-naph th ylprop ylenediamine r A 1 Wavelength Colour- density x 10s absorption, period, mP minutes Optical of maximum development 45" 540 15 34* 535 30 34" 540 15 43" 545 30 1* 545 60 18* 540 120 8" 530 60 22* 555 30 8* 535 60 535 15 535 15 30 t 540 15 35t 530 15 37t i 6 27t zat r o c d3iJ Colour formed with N-sulphato- ethyl-m-toluidine A r U Wavelength Colourl 3- Optical of maximum development $ F-) density x lo3 absorption, period, minutes mP 13 505 60 20 505 10 18 505 180 40 515 45 11 500 120 30 500 33 500 10 27 505 15 32 500 10 g 27 505 10 # 2 6o w 2 515 60 5 2 6o 1; 33 505 10 g (1 505 Z + 0 * - - - Colour formed with 3-hydroxy- Colour formed with 2-naphthol- 2-naphthoic acid 3,6-disulphonic acid A f- A \ r \ Wavelength Colour- Wavelength Colour- Rmine density x lo3 absorption, period, density x lo3 absorption, period, mP minutes mP minutes Optical of maximum development Optical of maximum development Aniline .. . . .. 32 500 30 6 485 30 o-Toluidine .. .. 2 505 30 11 485 60 m-Toluidine .. .. 5 505 30 9 485 30 $-Tohidine. . .. .. 9 505 30 9 495 30 2,g-Xylidine . . .. <1 500 60 4 485 60 2,4-Xylidine .. .. 5 515 60 7 495 60 2,3-Xylidine . . .. 1 510 60 3 485 60 3,4-Xylidine . . .. 7 515 30 4 510 30 o-Chloroaniline . . .. 5 500 30 4 485 15 .in-Chloroaniline . . .. 6 500 30 4 485 15 pChloroaniline . . .. 6 500 30 5 485 15 2,g-Xylidine . . .. 3 505 60 7 485 120 * Coupling carried out by procedure B (p. 641). Coupling carried out by procedure A (p. 641).October, 19611 DANIEL : THE DETERMINATION OF AROMATIC AMINO-COMPOUNDS 643 achieved more quickly with this reagent and with 2-naphthol-3,6-disulphonic acid, although the sensitivity obtained is low. The time taken to attain maximum development of colour is approximately the same for N-sulphatoethyl-m-toluidine, N-l-naphthylethylenediamine and ”-diethy1 N-l-naphthylpropylenediamine. The effect of the buffering action of sodium acetate (pH 9) on coupling is pronounced for those amines not containing an electro-negative substituent, e g ., aniline and the toluidines and xylidines. The effect of this buffering action on the rate of coupling was found to be greater with ”-diethy1 N-l-naphthylpropylenediamine than with N-l-naphthylethylenediamine. N-Sulphatoethyl-m-toluidine does not couple in absence of sodium acetate. The magnitude of the reagent blank is important, as low values permit greater analytical sensitivity at low concentrations of amines. Experience in this laboratory with N-l-napthyl- ethylenediamine has demonstrated wide variation in the quality of different batches of this reagent, with subsequent high blank values. The single batch of “-diethy1 N-l-naphthyl- propylenediamine so far examined gave a blank value much lower than that hitherto obtained with N-l-naphthylethylenediamine. For reactions involving short periods of coupling, N-sulphatoethyl-m-toluidine is a satisfactory reagent, but, for the reasons outlined, N’-di- ethyl N-l-naphthylpropylenediamine has been adopted in this laboratory as the most satis- factory reagent for routine use. I thank Mr. G. R. Norton for assistance during part of this work. REFERENCES 1. 2. 3. 4. Wild, F., “Estimation of Organic Compounds, ” Cambridge University Press, 1953. Strafford, N., Strouts, C. R. N., and Stubbings, W. V., Editors, “The Determination of Toxic Substances in Air: A Manual of I.C.I. Practice,” W. Heffer & Sons Ltd., Cambridge, 1956. Rose, F. L., and Bevan, H. G. L., Biochem. J., 1944, 38, 116. Bratton, A. C., and Marshall, E. K., J. Biol. Chem., 1939, 128, 557. Received April 4th, 1961

ISSN:0003-2654    

DOI:10.1039/AN9618600640

出版商:RSC    

年代:1961

数据来源: RSC

摘要:

October, 19611 DANIEL : THE DETERMINATION OF AROMATIC AMINO-COMPOUNDS 643 The Identification of Substances of Low Volatility by PyrolysislGas - Liquid Chromatography“ BY G. C. HEWITT AND B. T. WHITHAM (“Shell” Research Ltd., Thornton Research Centre, P.O. Box No. 1, Chester) The use of pyrolytic degradation for the identification of substances of low volatility and also for more fundamental studies of the break-down mechanisms of polymers is reviewed. Particular emphasis is placed on the advantages of gas - liquid chromatography for the analysis and identification of products of pyrolysis. A description is given of a glass pyrolysis unit that can be connected to an analytical gas - liquid chromatographic column. Solid and liquid samples can be readily introduced into the heated pyrolysis zone of this unit, and typical chromatograms of polymer-degradation products are shown.It is concluded that polymers can be readily classified according to type from their pyrolysis chromatograms and that, often, individual polymers among a given type can also be classified. THE analyst is often presented with the problem of identifying the polymers and other complex substances that occur in many natural and synthetic products. In most instances, degradation processes of some type have to be used in an attempt to isolate discrete fragments of these substances, which can be identified by their chemical and physical properties. The evidence obtained by characterisation of these fragments, together with a knowledge of the properties of the original material, leads to the identification of the complex substance.* Presented a t the joint meeting of the North of England Section and the Physical Methods Group of the Society on Thursday, September 29th, 1960.644 HEWITT AND WHITHAM: THE IDENTIFICATION OF SUBSTANCES OF [vol. 86 In the study of polymeric substances, pyrolytic degradation has been extensively used. As long ago as 1862, VSVliamsl used pyrolysis to isolate the basic isoprene unit from natural rubber, and several publications have described the use of pyrolysis as a method of obtaining information about the structures of substances. The mechanism of thermal degradation of polymers has been studied by Madorsky and Straus,2 who have shown that the types and relative amounts of the products of pyrolysis are functions of the molecular structure and the kind and frequency of side-groups. These workers have shown how, for instance, the thermal stability and break-down products o’btained on pyrolysis can be related to the strengths of the C-C bonds in the polymer chain, i.e., secondary > tertiary > quaternary.C I c-c-c > c-c-c > c-c-c I C I C In recent years, indirect “finger-printing” methods have been used in conjunction with pyrolytic methods. Infra-red3 and mass ~pectrornetry,~,~ for example, have been used to “finger-print” the total products of pyrolysis from various polymers. This type of approach often involves compilation of a library of reference spectra obtained from pyrolysis of sub- stances of known structure, and the identity of an unknown is obtained by referring to these spectra.The approach is generally successful, as the products of pyrolysis obtained under given conditions are specific for each type of substance. With some substances, such as linear homopolymers, direct identification is possible, as the conditions of pyrolysis can be selected so that the monomer is obtained. The usefulness of pyrolytic degradation has been greatly enhanced since the advent of gas - liquid chromatography,6 which is basically a rapid method for separating small amounts of volatile substances. In this technique, the substances are carried through a small packed column by means of a stream of inert gas, and separation efficiencies of many thousands of theoretical plates can be achieved. The co1um:n packing invariably consists of a powdered porous solid phase coated with a non-volatile liquid.Generally, separation depends largely on the relative volatilities of the substances, ‘but considerable variations in the order of separation of different types of substances can be obtained with different non-volatile liquid phases. In recent year^,^^^ the range of gas - liquid chromatography has been extended to the separation of substances having boiling-points as high as 550” C by using columns operated at temperatures up to 300” C. Further extension of the technique to substances even less volatile is limited by lack of suitable stationary phases and by the possibility of decomposing the substances a t the high column-operating temperatures required. A combination of pyrolysis with gas - liquid chromatography offers an alternative approach to the analysis of substances having such low volatility.The important features of gas - liquid chromato- graphy that make it an excellent analytical technique for use in conjunction with pyrolytic degradation are as follows: (i) the products of pyrolysis are rapidly separated, (ii) the separa- tions can easily be recorded automatically, (iii) only a few milligrams of sample are required, (iv) the products of pyrolysis can be identified by the time each constituent takes to pass through the chromatographic column (the retention time), (v) further identification of the products of pyrolysis can be obtained by collecting fractions at the outlet from the column, (vi) the chromatogram of the products of pyrolysis of a substance is in itself a “finger-print” and can be used in a similar manner to infra-red spectra for identification purposes and (vii) the pyrolysis unit can be connected directly to the inlet of the chromatographic column (this is particularly important, as all the products of pyrolysis can be swept into the column by the carrier gas as soon as they are formed; there is little chance of re-combination of the primary products of pyrolysis with this arrangement).APPLICATIONS OF THE TECHNIQUE IDENTIFICATION OF POLYMERS- The earliest work on pyrolysis combined with gas - liquid chromatography was carried out by subjecting the sample to pyrolysis, collecting the products in a cold trap and trans- ferring them to the chromatographic column for analysis.Davison, Slaney and Wraggg applied this principle to natural rubber, copolymers of butadiene with acrylonitrile or with styrene, poly(methy1 acrylate), polyisobutene, poly(viny1 acetate) and poly(ethy1 acrylate).October, 19611 LOW VOLATILITY BY PYROLYSIS/GAS - LIQUID CHROMATOGRAPHY 645 Pyrolysis was carried out at 650" C in a stream of nitrogen at atmospheric pressure, and the products were collected and then examined by gas - liquid chromatography on a column containing dinonyl phthalate as liquid phase and operated at 111" C. "Finger-print" chromatograms were obtained for those products of pyrolysis sufficiently volatile at the latter temperature. Subsequently, Haslam and JeffslO de-polymerised copolymers of methyl methacrylate with styrene, methyl acrylate and ethyl methacrylate.The de-polymerisation was carried out at 350" C in vacuo, under which conditions the monomers were obtained, and these were identified from their retention times. Direct coupling of the pyrolysis unit with the gas - liquid chromatographic column was first described by Radell and Strutz,ll who subjected acrylate and methacrylate polymers to pyrolysis by immersing a metal loop containing the sample (about 5 mg) in a bath of Wood's alloy at 500" C for 30 seconds. The products of pyrolysis were swept by the carrier gas directly into a 2-metre column containing di-n-decyl phthalate at 100" C; chromatograms I I I 15 10 5 0 Time, minutes Fig. 1. Pyrolysis chromatogram ob- tained by Radell and Strutzll for poly- (methyl acrylate) : S, sample intro- duction; A, air; B, methanol; C , ethanol; D, methyl acrylate; E, methyl meth- acrylate.(Reprinted from Analytical Chemistry, 1959, 31, 1891) characteristic of each polymer were obtained. Under these conditions, acrylate polymers were degraded to mixtures of the monomer, short-chain alcohols and various fragments (see Fig. 1); methyl, ethyl and n-butyl methacrylate polymers were degraded primarily to their monomers. The analysis of polymethacrylates has also been carried out by decomposing the polymer coated on a heated filament at the inlet of the gas - liquid chromatographic column and identifying the resulting monomers by their retention volumes.12 COMPOSITION OF COPOLYMERS- The compositions of copolymers have been determined by coating or placing the sample on a filament that can be rapidly brought to any specified temperature for a brief period.Samples are degraded at a particular temperature, and the composition of the copolymer is determined by measuring certain peaks on the chromatogram characteristic of the individual monomers. In this way, vinyl chloride - vinyl acetate copolymers have been analysed by means of the peaks for hydrogen chloride and acetic acid.13 Similarly, the compositions of copolymers of methyl methacrylate with methyl acrylate have been determined by com- parison of the heights of the peaks recorded for methyl methacrylate monomer and for646 HEWITT AND WHITHAM: THE IDENTIFICATION O F SUBSTANCES OF [Vol. 86 methanol plus methyl acrylate monomer with those obtained by pyrolysis of samples of known composition.14 Fig.2 shows a typical calibration curve obtained in this quantitative work, and it is claimed that the components (of these copolymers can be determined with a precision of 3-0.5 per cent. BREAK-DOWN MECHANISMS OF HIGH POLYMERS- Lehrle and Robb13,16 have also used the technique with the heated wire spiral for more fundamental studies of the break-down mechanisms of high polymers by recording chromato- grams of the products of degradation from the same sample of polymer maintained for a few seconds at each of a series of temperature:; up to 1000" C. Information about the im- portance of the various degradation processes throughout the range of temperatures can be obtained by measuring the relative proportions of the products of degradation at each temperature.In this way, it has been shown that the principal process in the degradation of poly(viny1 acetate) is the production of acetic acid at intermediate temperatures and that the polyacetylene skeleton formed is stable and only degraded a t the highest temperatures. By measuring the rate of production of monomer at a specified temperature, the energies of activation and the rates of degradation of de-polymerisation processes can be determined. It is possible to differentiate between random and block copolymers by means of the differences in their rates of degradation determined in this manner.15 4'0 3-a .- U m L U L W 0 .- $ 2'C m 0- I .c C I I I I I 10 20 30 40 I Methyl acrylate in mixture, yo Fig. 2. Curve plotted by Strassburger, Brauer, Tryon and Forziati14 for the peak-height ratios of methanol plus methyl acrylate to methyl methacrylate in a series of polymer mixtures.(Reprinted from Analytical Chemistry, 1960, 32, 456) IDENTIFICATION OF COMPOUNDS OTHER THAN POLYMERS- Janak16~17 has shown that it is possible to identify various barbiturates, plant oils (e.g., olive, linseed, castor and coconut oils), amino acids and alkaloids by pyrolysis of a sample on a heated filament in the inlet gas stream of a gas - liquid chromatographic column. Un- known substances were generally identified by reference to chromatograms recorded after pyrolysis of substances of known composition. Janak gave an example to illustrate the possibilities of this analytical technique. A child had unfortuntely consumed tablets of an unknown nature, and examination of an ether extract of the child's urine by pyrolysis/ gas - liquid chromatography yielded a chromatogram almost identical with that of veronal (diethylbarbituric acid) ; the chromatograms obtained are shown in Fig.3. The structures of certain compounds added to petroleum, such as organic phosphates and thiophosphates, are difficult to characterise by normal methods, owing to the chemicalOctober, 19611 LOW VOLATILITY BY PYROLYSIS/GAS - LIQUID CHROMATOGRAPHY 647 reactivity of these substances, their resistance to hydrolysis and their poor thermal stability. Legate and Burnham18 have described how gas - liquid chromatography of the products of pyrolysis can be successfully used for identifying the organic radicals in such compounds. Their pyrolysis unit, which was attached directly to the chromatographic column, consisted of a stainless-steel tube packed with glass-wool and heated in a furnace; two examples of the type of pyrolytic reaction they obtained are shown below.[ S ] 315°C [ s ] Pb -S-P(-O-CH2-CH2-CHz-CH2-CHJ 2 2 + Pb -S-P(-OH)s 2 + CH2= CH-CH2-CHz-CH3 Lead salt of 00-di-n-pentyl Hypothetical product 1-Pentene thionothiophosphate (not identified) (identified) S II f [ FH3 ] 300°C K-S-P -0-CH-CHS 2 --+ K-S-P(-OH), + CH2=CH-CH3 Potassium salt of 00-di-isopropyl thionothiophosphate Propene Since pyrolysis/gas - liquid chromatography alone cannot distinguish between a n-propyl and an isopropyl group, it would be necessary to obtain the infra-red spectrum of the C,- thionothiophosphate to prove the presence of the iso-configuration.Time, minutes Fig. 3. Identification of diethylbarbituric acid (veronal) in urine. Chromatograms obtained by Janbkl7 for (a) veronal stan- dard and (b) extract from urine: S, sample introduction. (Reproduced by permission of the author and Butterworths Publica- tions Ltd.) Variations of the technique have recently been described,lg in which the pyrolysis tube is replaced by a small heated reaction chamber containing palladium on activated alumina. Sulphur compounds introduced into this reaction chamber were de-sulphurised, and the hydrocarbon fragments were identified from their retention volumes or by mass-spectrometric examination of the collected fractions. It was possible to arrive at the identity of the original sulphur compound from the identities of the fragments.DESCRIPTION OF APPARATUS So far, the main emphasis of our work has been on the pyrolysis of polymers as a means of identification. The pyrolyses have been carried out in the glass unit shown in Fig. 4. This unit is coupled directly to the chromatographic column, and both liquid and solid648 HEWITT AND WHITHAM: THE IDENTIFICATION OF SUBSTANCES OF [Vol. 86 samples can be conveniently handled. Solid samples are introduced by means of a capillary tube containing an iron core; after pyrolysis of the sample has been completed, the tube can be withdrawn by means of a magnet. A hypodermic syringe is used to introduce liquid samples via the rubber serum cap. The pyrolysis chamber is heated electrically by means of windings on the outside of the tube, and the temperature inside the unit is measured by a thermocouple.The few centimetres of column packing before the chromatographic column prevent any tarry matter formed during pyrolysis from contaminating the analytical column. The pyrolysis unit can be readily detached from the column for cleaning or connecting to another column of different length or containing a different packing. The chromatographic 1 gas in A = Taps (extra wide bore) 6 = Serum cap C = Thermocouple inlet D = Level of top of chromatography furnace E = Top of gas-chromatographic column F = Glass-to-metal seals G = Layer of stationary phase H = Lightly packed layer of glass-wool K = Heated pyrolysis zone L = Capillary tube for introducing solid samples M = Iron (' slug " Fig.4. Demountable pyrolysis unit and sample- introduction system (all dimertsions in centimetres) column was generally packed with 52- to 60-mesh Sil-0-Cel firebrick impregnated with 26 per cent. w/w of silicone grease (E301), and two columns of lengths 6 and 12 feet have been used. The detector was a thermal-conductivity cell, and the outlet was connected to a manifold and trap system20 for the recovery of fractions for further examination. The carrier gas was nitrogen, and at the rates of flow normally used the residence time of vaporised substances in the pyrolysis chamber would be about 1 second. RESULTS FOR POLYMERS We have used the apparatus described above €or examining several reference polymers. The pyrolysis chamber was generally maintained at 378" C, and substances that did notOctober, 19611 LOW VOLATILITY BY PYROLYSIS/GAS - LIQUID CHROMATOGRAPHY 649 decompose at this temperature were successfully decomposed at 435" C.The weight of sample used was approximately 10 mg. In general, all the substances examined have given pyrolysis chromatograms sufficiently characteristic for identification purposes ; for some substances, such as polyisobutylene and polystyrene, the monomer was the only product (see Fig. 5). The methacrylate polymers studied were degraded down to the olefin corresponding to the alcohol originally forming the methacrylate ester. The chromatogram from a C,,, Cl!, C,, C,, and C, methacrylate copolymer is shown in Fig. 6; this copolymer had been previously analysed by hydrolysing and then recovery of the alcohol constituents for identification.The direct pyrolytic tech- nique is a considerably shorter method of identifying these alcohols. COOR F E - D C - I L S I I I I I I I 15 10 5 0 Time, minutes Fig. 5. Pyrolysis chromatogram for polystyrene: S, sample introduction; A, styrene. Pyrolysis temperature, 378°C; 6-foot column of 52- to 60-mesh firebrick impreg- nated with 25 per cent w/w of silicone E301 grease; column- operating temperature 105" C; katharometer temperature, 60" C; carrier gas (nitrogen) flow rate, 2.9 litres per hour I I I I I I I I I650 HEWITT AND WHITHAM: THE IDENTIFICATION OF SUBSTANCES OF [VOl. 86 1 1 I I I I I I 30 20 10 0 Time, minutes Fig. 7. Pyrolysis chromatogram for poly(n-butyl acrylate) : S, sample introduction; A, butene; B, butanol; C, monomer.Pyrolysis temperature, 378°C; column as for Fig. 5, but 12 feet long; column-operating temperature, 150" C; katharometer temperature, 60" C; carrier gas (nitrogen) flow rate, 1.4 litres per hour c:"' -CH-CHz-O- In[ -CHz-CHl-O- 1, "U I I I 10 5 0 Time, minutes Fig. 8 I I I I 5 0 Time, minutes Fig. 9 Figs. 8 and 9. Pyrolysis chromatograms for a propylene oxide -ethylene oxide polymer (Fig. 8) and a nitrogen-containing propylene oxide polymer (Fig. 9) : S, sample introduction: A, ethylene; €3, propylene; C, acetaldehyde; D, propionaldehyde. Pyrolysis temperature 378" C; column as for Fig. 5 ; column-operating temperature, 102" C : katharometer temperature, 60" C; carrier gas (nitrogen) flow rate, 2.9 litres per hourOctober, 19611 LOW VOLATILITY BY PYROLYSIS/GAS - LIQUID CHROMATOGRAPHY 651 A number of propylene oxide - ethylene oxide polymers have been subjected to pyrolysis; these also gave distinctive chromatograms, and an example is shown in Fig.8. A nitrogen- containing polymer of this type gave a closely similar chromatogram (see Fig. 9), but differ- ences between the relative proportions of the various peaks were sufficient to distinguish it from the propylene oxide - ethylene oxide polymer. Poly(viny1 acetate) is another type of polymer examined; for this, it was necessary to use the higher temperature of pyrolysis to obtain degradation. The chromatogram obtained (see Fig. 10) showed one "tailing" peak, indicating a polar compound (probably acetic acid).Our results confirm that polymers can be readily classified according to type from their pyrolysis chromatograms, and, often, individual polymers among a given type can also be identified. I I I I 20 10 5 0 Time, minutes Fig. 10. Pyrolysis chromatogram for poly(viny1 acetate) : S, sample introduction; A, acetic acid. Pyrolysis temperature 435' C; column as for Fig. 7 ; column-operating temperature, 53" C; katharo- meter temperature, 50" C ; carrier gas (nitrogen) flow rate, 2.1 litres per hour CONCLUSIONS The requirements for the successful identification of unknown substances by pyrolysis/ gas - liquid chromatography can be summarised as follows. The elemental composition of the substance should be known. It is important to ensure as far as possible that the substance is free from inhibitors, solvents and other impurities.The pyrolysis of the substance should then be studied at two or three temperatures for which reference chromato- grams are available. The chromatographic column should be about 10 feet long and should contain a stationary phase that can be used over a wide range of temperatures. This will ensure that good separation of the most volatile products of pyrolysis is achieved and that the temperature of the column can be increased to elute any lessvolatile products. The technique of increasing the temperature of the column during separation (temperature programming) is ideal for this work because of the wide range of products that may be obtained. If the resulting chromatograms cannot be matched with any reference chromato- grams, the fragments obtained must be identified by means of their retention volumes or spectroscopic examination of recovered fractions, or both.Gas - liquid chromatography has become so widely used in recent years that any extension of its range of application is greatly welcomed. The rapid identification of substances having low volatility and thermally unstable materials is now possible by the combination of pyrolysis with gas - liquid chromatography, and this technique has clearly become another powerful tool at the disposal of the analyst. Numerous applications in research laboratories can be expected in the future.652 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. CROMPTON : THE DETERMINATION OF ALKOXIDE [Vol. 86 REFERENCES Williams, J. C. F., J . Chem. SOG., 1862, 15, part 10. Madorsky, S. L., and Straus, S., J . Res. Nut. Bur. Stand., 1954, 53, 361. Harms, D. L., Anal. Chem., 1953, 25, 1140. Zemany, P. D. Ibid., 1952, 24, 1709. Bradt, P., Dibeler, V. H., and Mohler, F. L., J . Res. Nut. Bur. Stand., 1953, 50, 201. James, A. T., and Martin, A. J. P., Biochem. J., 1952, 50, 679. Adlard, E. R., and Whitham, B. T., in Desty, D. H., Editor, “Gas Chromatography,” Butterworths Khan, M. A., and Whitham, B. T., J . Appl. Chem., 1958, 8, 549. Davison, W. H. T., Slaney, S., and Wragg, A. L., Chem. & Ind., 1954, 1356. Haslam, J., and Jeffs, A. R., J . Appl. Chem., 1957, 7 , 24. Radell, E. A., and Strutz, H. C., Anal. Chem., 1959, 31, 1890. Guillet, J. E., Wooten, W. C., and Combs, R. L., J . Appl. Polym. Sci., 1960, 3, 61. Lehrle, R. S., and Robb, J. C., Nature, 1959, 183, 1671. Strassburger, J., Brauer, G. M., Tryon, M., and Forziati, A. F., Anal. Chem., 1960, 32, 454. Lehrle, R. S., Informal Symposium of the Gas Chromatography Discussion Group, London, 1960. JanQk, J., Nature, 1960, 185, 684. -, in Scott, R. P. W., Editor, “Gas Chromatography 1960,” Buttenvorths Scientific Publications, Legate, C. E., and Burnham, H. D., Anal. Chem., 1960, 32, 1042. Thompson, C. J., Coleman, H. J., Ward, C. C . , and Rall, H. T., Ibid., 1960, 32, 424. Whitham, B. T., in Desty, D. H., Editor, “Vapour Phase Chromatography,” Buttenvorths Scientific Received March 9th, 1961 Scientific Publications, London, 1958. London, 1960, p. 387. Publications, London, 1957, p. 194.

ISSN:0003-2654    

DOI:10.1039/AN9618600643

出版商:RSC    

年代:1961

数据来源: RSC