I
Essential Oils and Related Products ERNEST GUENTHER and EDWARD E. LANGENAU Fritzsche Brothers, Inc., N e w York,
N. Y.
T
H I S fifth review of analytical procedures for esbential oils and related products follom the general outline established in the previous reviews (56-59). The present paper covers the literature from October 1952 to October 1951. There is evidence of further use of infrared and ultraviolet absorption procedures, and of a marked emphasis on chromatographic separations. However, relatively few adaptations of these methods, applied specifically to essential oils, have been reported.
.A second and enlarged edition of Siggia’s viell-knoLm work, “Quantitative Organic -4nalj-sis via Functional Groups” (113), appeared in 1954. .4 select and thoroughly adequate listing of texts, journals, compendia, and reports dealing with the literature of the field of essential oils was compiled by Leidy (7‘5). An excellent publication by the Syndicat Sational des Fabricants et Importateurs d’Huiles Essentielles et Produits riromatiques Saturels of Grasse, France ( 1 2 4 ) , Tyas released. It was prepared under the direction of Igolen, Crabalona, Daumas, Benezet, and Teisseire. This volume describes methods of analysis and gives anall-tical constants for commercial essential oils. This represents a praiseworthy attempt to maintain the quality of commercial oils. The British Standards Institute ( 1 2 ) described methods of testing essential oils, and gave procedures for the determination of physical and chemical properties. The Scientific Committee of the Essential Oil -4ssociation of the United States ( 4 1 ) published specifications and standards for the following:
OFFICIAL CORZPEVDI4
T h e British Pharmacopoeia 1953 ( 1 1 ) was published and became official as of September l, 1953. Monographs are included for the following products of interest to the essential oil industry: Volatile bitter almond oil Anise oil Benzoin Benzyl alcohol Benzyl benzoate Cade oil Camphor Chenopodium oil Cinnamon oil Citric acid Clove oil Colophony Coriander oil Dibutyl phthalate Dill oil Dimethyl phthalate Ethyl oleate Eucalyptol Eucalyptus oil Glycerol
Lavender oil Lemon oil Menthol Xutmeg oil Oleic acid Peppermint oil Rosemary oil Saccharin Saccharin sodium Santonin Prepared storax Tar Terpineol Thymol Balsam of Tolu Turpentine oil Vanillin
Page 26 46 78
79 SO
100 114 134 149 150 I53 165 168
Oil Ocotea Cymbarum Oil guaiac wood Oil Amyris Oil balsam of Peru Oil balsam of TOIU Oil opoponax Oil olibanum Camphor oil 1070 Oil linaloe wood Oil sage, Spanish Oil pennyroyal Oil pimento leaf Isobornyl acetate Oil cananga Oil sweet marjoram Oil clary sage
182
194 197 227 227 228 248 301 304 323 368
No. 62 Yo. 63 S o . 64 S o . 65 S o . 66 S o . 67 KO. 68 No. 69 K O . ‘io KO. 71 No.
72
KO.
73
KO.
74
No. 75 No. 76 s o . 77
Revisions for existing specifications of aromatic chemicals are given in Xo. R-1: for essential oils, in R-2. The EO.$ cassia flask (previously dexribed in E0.4 Apparatus 1) is now available as a stock item from IGmble Glass under the number 28068. This is a flask of verv convenient size for the determination of aldehydes and ketones as vel1 as phenols. B collaborative study of the reproducibility and application of infrared absorption techniques has been inaugurated by the Scientific Committee of this association. S o published findings have appeared as yet. Several new journals of interest to the essential oil field have been issued: Analytical dbst?acts ( Z ) , published monthly for the Society for Analytical Chemistry (England); the J o u r n a l df Agricultuial and Food Chemistry ( 6 8 ) ; and Conserze e deriuatz agrumarz (Palerrno) (24). S o n e of these journals deals exclusively with essential oils, b u t they frequently carry articles of interest. Dawson and Harris ( 3 0 )prepared a bulletin on sensory methods for measuring differences in food quality, n-hich includes a selected bibliography of some 300 papers.
375
406 471 473 47 6 479 519 558 560 571 574 590 597
Appendix IV, page 702, gives detailed procedures for the determination of physical properties; Appendix X, page 754, for the determination of chemical properties. According to TThat little information is available, it appears that a new edition, the seventh, of the Russian Pharmacopoeia (104) was published in 1952. NEW TEXTS AND PUBLICATIONS
ANALYTICAL PROCEDURES FROTI SCIENTIFIC AND TECHNICAL LITERATURE
Volume I11 of “Colorimetric Methods of Analysis” by Snell and Snell (117’) was published in 1953. This volume describes colorimetric methods as applied to organic compounds; Volume IV, as yet unpublished, will conclude the series.
Acids. LIethods employing circular paper chromatography were described for the separation and identification of certain
672
V O L U M E 2 7 , NO. 4, A P R I L 1 9 5 5 organic acids by Giri, Murthy, and Rao (54)and by Airan, Joshi, Barnabas, and Master (1). Paper chromatography, using an ascending method, was used by Van Duuren for the determination of some nonvolatile acids ( 1 2 9 ) . Using a paper chromatographic technique, Jones, Domling, and Skraba ( 6 7 ) reported Rf values for a number of common acids in several solvent combinations. Duncan and Porteous (34) used paper chromatography for the identification and determination of lower straight-chain fatty acids. Burton (18)described the separation of volatile acids by paper chromatography. Micheel and Schweppe (81)used a cellulose acetate paper for the separation of the higher fatty acids; they also reported on a separation of isomeric phthalic acids by this same technique. Lindqvist and Storgards ( 7 6 ) made a comprehensive study of several variables encountered in the separation of volatile fatty acids by paper chromatography, and recommended a detailed procedure, based on this study. Xijkamp ( 9 1 )described a simple chromatographic method for the determination of Clo to Czosaturated straightchain fatt> acids, using a long column packed x i t h silica gel. Kawamura, Momoki, and Suzuhi ( 7 0 ) reported a novel micromethod for the coulometric titration of salicylic acid. For the potentiometric titration of substituted fatty acid, Radell and Donahue ( 9 7 ) recommended as solvent a mixture of benzene and methanol containing lithium chloride; as a titrant, a 0.1S solution of sodium methoxide. Alcohols and Phenols. Ott ( 9 5 ) investigated a method using a mixture of pyridine and acetic anhydride for the determination of tertiary alcohols in essential oils; results v-ere found to be reproducible but slightly Ion-er than the values obtained by the classical Glichitch method. For primary alcohols, Petrova ( 9 6 ) employed as the acetylating agent a miyture of acetic acid (?) and pyridine in the ratio 1 to 3; other minor modifications of the method of Verley and Bolsing viere suggested. Brown and Hafliger (14) described a convenient technique for carrying out micro-Zerewitinoff determinations; several modifications were prepared to give consistent and reproducible results. Rao, Shah, and Pansare ( 9 8 ) used the lithium aluminum hydride method (originall!. proposed by Higuchi) for the microdeterminations of active hydrogen in organic compounds; they described an apparatus and gave a detailed procedure. For the separation and identification of normal aliphatic alcohols and methyl carbinols, Van Duin (128) used as reagent a 4dialkylamino-3,5-dinitrobenzoyl chloride; this gave colored esters which were separated by partition chromatography. Yarborough (134) reported on the mass spectrophotometric analysis of alcohols containing six and seven carbon atoms; of the mixtures studied, analytical results R ere obtained agreeing within =t5% of the true values. Chang (23) compared the separation of menthol from essential oils by fractional distillation with that by chromatographic adsorption; results obtained by using a column packed with activated carbon (100- t o 200-mesh) were good. Rosenthaler and Tegezzi (101) studied the influence of sulfuric acid concentration on the color which developed when aliphatic alcohols were treated with aromatic aldehydes-e.g., vanillin, p-h~-droxybenzaldehSde, salicylaldehyde, and ethylvanillin. Such color reactions must be interpreted with caution when applied to mixtures as complex as essential oils. Bohnie and Opfer ( 9 ) found that pseudosaccharin chloride gave crystalline products n ith some 25 glvcol and glycol derivatives R hich they studied; this reagent had been suggested previously by Meadoe and Reid ( 8 0 )for the identification of alcohols. X chromatographic separation of normal aliphatic alcohols having one to five carbon atoms was reported by Dal Sogare (28); columns of silicic acid and of a mixture of silicic acid and Celite a e r e used. Such columns were not adequate for the separation of the is0 form from mivtures n i t h the normal alcohols. The same technique I n s suitable for the separation of glycols having two to four carbon atoms.
673 Bergner and Sperlich ( 7 ) recommended chromatography for the determination of glycols in foods, cosmetics, and similar mixtures. Griffin (55) reported on the determination of glycerol and propylene glycol in desiccated coconut by a distillation technique using Decalin and a Dean and Stark type of receiver. The high boiling point of Decalin should make this compound useful in determinations in the essential oil industry. hlathers and Pro (7‘9) described an elaborate method for the determination of propylene glycol and glycerol in foods and medicinals. Fritz and Keen (60)suggested a technique for direct titration of phenols in a nonaqueous medium. Dimethylformamide m-as studied as a solvent with azo violet as indicator. For alkylsubstituted phenols, ethylenediamine was recommended as solvent with o-nitroaniline as indicator. If such a method could he adapted for use with essential oils, it would prove very useful in special cases. Fischer and Hall (47’) studied critically the method of the Cnited States Pharmacopoeia for the determination of eugenol in clove oil. They recommended that the flask he thoroughly shaken at least three times during the heating period and be permitted to stand for 20 hours before the reading is taken; a modified cassia flask was also suggested. Analysts experienced in the analysis of clove oil have found that shaking is necessary to assure adequate mixing of the oil and alkali for complete saponification of the eugenyl acetate. The Essential Oil Association flask, now available from Kimble Glass, gives more accurate results than the flask proposed by Fischer and Hall. Esters. For the determination of the saponification value of low boiling esters Davis ( 2 9 ) suggested the use of a balloon attached to the side arm of the reflux tube; this results in a closed (but expandable) system and prevents loss of the volatile esters. Fischer and Hall (45)reported on the assay methods of the United States Pharmacopoeia for esters in volatile oils, and proposed modifications. Dullaghan and Xord (33) described an iodometric procedure for the determination of glycidic esters. The method is based on Darzen’s contention that glycidic esters react quantitatively Kith hydrogen iodide to yield acyclic esters and free iodine. For ascertaining the cinnamein content of Tolu balsam, Rosenthaler (102) suggested triturating a 1-gram sample with 5 grams of sand before extracting with ether; this assures complete extraction. For the separation of methyl esters of fatty acids having 12 to 22 carbons atoms, Cropper and HeyFyood ( 2 6 ) used a vapor phase chromatographic technique. Columns packed with equal parts of Celite and high vacuum silicone grease were employed. Sakabayashi ( 8 6 ) determined R, values for several coumarins using paper chromatography. To detect benzoates and hydrox) benzoates in foods and pharmaceuticals, Gakenheimer (51)used the diazotization technique of Edwards et al. (Si’). Aldehydes and Ketones. Montes and Grandolini (84)studied the hydroxylamine method of Bryant and Smith for aldehydes and ketones, and applied it to 1-20 compounds. Feuell and Skellon (43)proposed the addition of sodium acetate (or ammonium acetate) to the blank to permit a more accurate color match; this proved advantageous in the determination of carbonyl compounds vihen hydroxylamine or semicarbazide n’as used. For the determination of aldehydes in the presence of acids, ketones, acetals, and vinyl ethers, Siggia and Segal (114) developed a method using a modified Tollens reagent; the excess ionic silver was titrated potentiometrically with potassium iodide solution. Such a method for determining aldehydes in the presence of acetals and aldehydes in the presence of ketones should prove of much value to the essential oil industry. Fischer and Hall (46)studied the official method of the United
674 States Pharmacopoeia for the assay of cassia oil and suggested several much needed modifications. The use of the new Essential Oil Association cassia flask would prove more advantageous than 4 novel the use of the special flask proposed by the authors. . method for the determination of aldehydes using bisulfite was proposed by Salomsa (106). A measured amount of a solution of sodium bisulfite containing 5 to 10% of ethyl alcohol was made t o react \I-ith the aldehyde and the excess bisulfite titrated with a 0. I N iodine solution. T o prevent oxidation, the reaction mixture must be covered with a layer of toluene. For the determination of aldehydes and ketones with phenylhydrazine, Terent'ev and Zabrodina (126) described the use of a solution of the hydrochloride in pyridine; the excess reagent was titrated with a 0.1 to 0.2*V copper acetate solution. Kivoli ( 9 2 ) described the preparation of alumina columns for the separation of vanillin and coumarin from toluene solutions. Sjostrom (116) utilized an ion exchange column (iimberlite IRA 400) for the separation of ketones as the bisulfite addition compound. Newombe and Reid ( 9 0 ) suggested treatment of filter paper with sodium bisulfite to separate carbonyl compounds on the basis of degree of reactivity. Lagneau ( 7 4 ) used paper chromatography in a study of vanilla extracts to separate vanillin, ethylvanillin, and heliotropin; R, values were reported. S9kora and ProchAzka (123) identified carbonyl compounds by a paper chromatographic separation of the 2,4-dinitrophenylhydrazones. Kramer and Van Duin ( 7 2 ) separated and identified the 2,4-dinitrophenylhydrazonesof normal aliphatic aldehydes and ketones having 18 carbon atoms or Iess by partition chromatography using silica gel. A micromethod for the determination of carbonyl compounds using nitro-substituted phenylhydrazines was reported by Schoniger ( 1 0 9 ) ; the excess reagent was reduced with a titanium trichloride solution and the excess of this latter solution in turn determined by titration. The procedure appears too complex for use as a routine method. Siggia and Segal (115) proposed the use of primary amines for the determination of aldehydes. An excess of a 2M solution of lauryl amine was used, and was determined potentiometrically by titration with a salicylic acid solution. Methyl ketones cause some interference, but other ketones in moderate concentrations have little or no effect. Onoe ( 9 4 ) studied factors affecting results of chromatographic separation of 2,4-dinitrophenylhydrazonesof alphatic compounds, using the glass chromatostrip technique of Kirchner and Miller. Rf values were sensitive to variations in the moisture content of the strip, so that the extent of desiccation must be controlled in preparing such chromatostrips. Isomeric forms of the individual aliphatic aldehydes could not be separated from the normal compounds. Howard and Tatchell(63) separated milligram quantities of the 2,4-dinitrophenylhydrazones using a reverse phase partition chromatographic column. Pouchay and Graizon (119) reported a polarographic technique for the determination of aldehydes and ketones using semicarbazide. Hasselbach (61) described the polarographic determination of citral and citronellal in essential oils. For determination of a-diketones, Modiano and Pariaud (83) used bromine in the presence of mercuric sulfide. Englis, Fiech, and Bash (38) measured absorption of dimethylglyoxime a t the peak near 226 mp for determining as little as 0 t o 10 p.p.m. of diacetyl; results compare well with the gravimetric method in which the nickel complex of dimethylglyoxime is precipitated and weighed. Ross (103) triturated the 2,4dinitrophenylhydrazones of ketones with mineral oil and determined infrared absorption spectra in the range of 6 t o 15 microns and gave spectra for many such derivatives. Iiaves and Lecomte (89) reported the infrared absorption spectra of ionones, irones, and several of their derivatives.
ANALYTICAL CHEMISTRY Karrer and Blass (69) described a color reaction for a-ionone and 5-oxy-a-ionone. The addition of a few drops of 20% sodium hydroxide solution gives a red color showing a maximum a t 510 mp; the color gradually fades, however. As p-ionone does not show this color reaction, these authors suggested the test could be used t o ascertain the presence (or absence) of a-ionone in p-ionone preparations. Ensminger ( 3 9 ) reported the ultraviolet absorption spectra of vanillin, ethylvanillin, and coumarin in alkaline and acidic solutions for the determination of these compounds in imitation vanilla extracts. Ensminger ( 4 0 ) reported on a collaborative study of the official gravimetric method of the Association of Official Agricultural Chemists for the determination of vanillin and coumarin in the vanilla flavors; low and variable recoveries were found. For the detection of ethylvanillin in imitation vanilla extracts, Janovsky and Filandro (66) suggested the use of a color reaction with a 1% freshly prepared solution of p-aminophenol in alcohol. Broderick (13) questioned the value of the lead number in the evaluation of vanilla extracts; he concluded that as an index of quality, the lead number is useless. Hon-ever, as an index of purity the lead number cannot be ignored. Cartwright (21) reported the solubility and volatility of vanillalike synthetics-propenyl guaethol, bourbonal, vanillin, and coumarin. Castiglioni and Bionda (22) detected heliotropin in a mixture JTith vanillin by a condensation ivith cyclohexanone; separation of the precipitate and subsequent treatment with sulfuric acid yielded a violet color characteristic of piperonylidenecyclohexanone. As a reagent for the identification of aromatic aldehydes in essential oils, Klosa ( Y l ) proposed the use of 4-hydroxpcoumarin. He gave melting points for derivatives of several aromatic aldehydes; aliphatic aldehydes other than formaldehyde and acetaldehyde do not react. For the determination of small amounts of acetone Etienne ( 4 2 ) proposed spectrophotometric measurement of the condensation product with furfural. Costa and Cardosa do Vale ( 2 5 ) applied the method of Garratt for the detection of furfural to essential oils. For the determination of tert-butyldimethylacetophenone and musk ketone, Bogdanov (8) developed a gravimetric method based on the condensation of those compounds with anisaldehyde in the presence of a 40% sodium hydroxide solution. Johnston (66) offered a modification of the hydroxylamine method for the determination of aldehydes; tert-butyl alcohol is used as a solvent to prepare the solution of hydroxylamine. The use of a tertiary alcohol precludes the possibility of acetal formation. A p H meter is used t o establish the end point. These modifications eliminate the two basic problems in the use of an ovimation method for the determination of aldehydes. This procedure should be thoroughly investigated by other laboratories, as it offers much promise. Terpenes. A method employing paper chromatography was described by Schenck and Fromming (107) for the determination of azulene in the essential oil of chamomile. For the determination of azulene in chamomile flowers, Fischer and Resch ( 4 8 )suggested a simple method: distillation of the oil with steam and subsequent measurement with a photoelectric colorimeter. Savier, Verghese, and Yeddanapalli (133) estimated p-cymene by oxidation to terephthalic acid; they reported that rapid oxidation gave better results than slow oxidation. Sutherland (122) published a refractive index-density (n-d) chart for 25 naturally occurring terpenes and described its use. Nitrogen-Containing Products. Freeman ( 4 9 ) described three methods for the determination of Schiff bases by titration in nonaqueous media. For the determination of hydrogen cyanide in drugs, Torres (126) proposed a simple distillation technique wherein the hydro-
V O L U M E 27, NO. 4, A P R I L 1 9 5 5 gen cyanide is distilled through a condenser, the tip of which is immersed in a 1% solution of sodium hydroxide; the resulting sodium cyanide is determined by titration with a solution of silver xiitrate. \Vojahn (132) described a novel method for the determination of kothiocyanates by oxidation with hypoiodite. A s a routine tc’st, the method of the eighth edition of the National Formulary appears to be more useful. Schultz, Gmelin, and Kellcr ( 1 1 2 ) isolated niust,ard oil glucosides viith the aid of anion exchange resins. Schultz and Gmelin ( 110) also investigated a method using paper chromatography for w c h isolation. For the quantitative estimation of mustard oil glucosides these authors (111) proposed a color reaction with a n anthrone reagent,; the color int,ensity was measured photometrically. Interfering substances must first be removed, preferably b!- paper (or ion exchange) chromatography. AIcKenzie and Kallace ( 7 7 ) made a critical study of the Iijeldahl method for the determination of nitrogen; digestion conditions, temperature, cat,alysts, and oxidizing agents were investigated. Peroxides. Beckett and Dombrow (4)used a polarographic; niethod for the det,erniinat,ion of ascaridole in Chenopodium oil and in castor oil solutions of Chenopodium oil. I n a subsequent paper, Becliett and Jolliffe (5)compared thepolarographicniethod \\-ith the iodometric method of the British Pharmacopoeia. They criticized the official method because of the use of a n incorrect factor and because the amount of iodine released is not directly proportional t o the weight of ascaridole. I n spite of the obvious deficiencies of the official method, i t has received acceptance because of the lack of a more adequate procedure that can he used as a routine test for the commercial evaluation of this oil. Maruyama ( 7 8 ) investigated the polarographic determination of ascaridole and reported a n accuracy within i l % . Garratt and Phipcrs (58) assayed ascaridole in cast,or oil solutions of Chenopodium oil by a distillation with ethylene glycol, separation of the oil from the distillate, and final determination of ascaridole by the official method of the British Pharmacopoeia. For the determination of organic peroxides, Sullj- (121) suggested a modified iodonietric determination. Torres and de Diego ( 1 2 7 ) proposed the introduction of the “oxidation factor” as a cheinical property for the evaluation of essential oils. The oxidation factor was defined by these authors as the number of milligrams of essential oil oxidized by 1.0 ml. of a 0.1.V dichromate solution; t,he value increases with age. Determination of Essential Oil Content. Schirm (108) made a study of distillation methods for the determination of the essential oil cnontent in drugs. H e reported that the addition of sodium chloride decreased the wat,er solubility of certain components of the oil and shortened the time required for distillation. T h e use of a measured amount of xylene m-as also recommended to lessen the tendency of t,he oil to dissolve in the aqueous distillate and to aid in the xashing down of the condenser. Stahl (f20)described a new apparatus using continuous steam distillation for the determination of small amounts of essential oil. The introduct,ion of 0.5 ml. of pentane in the receiver was rcconimended. Briickner (15) developed a microapparatus for wtimating the content of essential oil in a 2-gram sample of peppermint leaves. The receiver is a capillary graduated in 0.001-ml. units. Bournot ( 1 0 ) compared this apparatus wit,h others and rrported satisfactory results especially if only a small sample of the botanical was available. An oxidation method was proposed by Hadorn (60) for the dctermination of essential oil in spices. This method cannot be recommended as a general procedure for the essential oil industry. Carson (19) reported a collaborative study of the official method of the .4ssociation of Official Agricultural Chemists; wide variations were noted in the results obtained for nutmegs and allspice (Pinienta berries). The use of a special receiver,
675 applicable for oils heavier than water as well as for oils lighter than water, was investigated; in a subsequent paper, Carson ( 2 0 )reported that this receiver proved unsuccessful for allspice. Determination of Water Content. Duvall and McBay ( 3 5 ) applied the Karl Fischer method successfully to some 34 official drugs. The use of methylene blue (in absolute alcohol or pyridrne) was proposed by Fischer ( 4 4 ) as an aid in the visual detI. E., and Sord. F. F., Mikrochim. Acta, 1953, 17. Analyst, 78, 041-6 Duncan, R. E. B., and Porteous, ,J. K., (1953). Durall, R. X., and lIcBay, A. J., Drug Standards, 20, 143 (1952). Eberius, E., Chem. Tech., 4 , 291 (1952). Edwards, F. IT., Kanji, H. R., and Hassan. 11. I-of ieagents to be employed should be specified (IZ),t h a t stability of reagents should he indicated (8), t h a t t h e allowable percentage deviation of duplicates for each and that qualitative as ne11 as method should be specified (8), quantitative tests should be included ( 3 6 ) . I t is important, however, t h a t the start has been made. It is inevitable t h a t the choice of method. is not acceptable to all. Tn the present volunie each selection was made on the basis of the judgment of a relativelv small nuinhei of clinical chemists. Subsequent volumes nill gain in value and recognition as an
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accepted compilation of standard methods, to t h e extent to which other clinical chemists effectively voice their constructive criticism regarding the selection of methods and manner of presentation, and t h e editors and contributors weigh and act on the suggestions of their colleagues, voiced either in personal communications 01 in the book reviews which follow the appearance of each volume. As the eyperience of a n ever-increasing number of clinical chemists is brought to bear on t h e subject, the work nill acquire greater prestige as a standard compilation until it beconies regarded lyith an esteem comparable to t h a t in which the AOAC publication is held. Wherever the contributors and editorial board c h o x e not to follow suggeitions made by a n appreciable number of readers, it would seem desirable to state in subsequent editions the Ieasons nhy, in the judgment of the board, it is deemed unnise to comply. T h r reading public will then be able to neigh the choices in the light of the reasons given, and to comment furthei if necessary. T n e evolution of such a collection of standard methods n oi t h y of almost universal acceptance 1% ill be a n arduous and continuing but n-orth-n-hile task. It lvould appear to this reviener t h a t it nould be helpful t o have in such a manual not only the directions for t h e procedures to be followed, but also numerous notes, in small print, to indicate the probable source2 and causes of trouble and means of preventing or overcoming difficulties when thece arise X good evample of this may be the type of notes in small print in -A. A. Soyes’s s j stem of quantitative t of t h e American hssociation of Clinical Chemists is t h e launching of its official journal, Clznzcal Chenizstry. A moie coniprehensive coverage of methods of clinical chemistrj ha. been presented clearly and concisely by Hiller ( 2 7 ) for the guidance of technicians. Hepler’s book (26) gives more attention to the nonchemical procedures of the clinical laboratory.
