V O L U M E 27, NO. 4, A P R I L 1 9 5 5
623
(173) Stolyarova. I. b.,J . Anal. Chem., r . S . S . R . , 8 , 270 (19531. (174) Strange, E. E., XN.AL.CHEM.,25, 650 (1953). (175) Stross. IT,,Shornik M e r i n d r o d . Polarog. Sjezdii Prare, 1 st Cor,gr.. 397 (1951). (176) Strosa. IT.,and Stros;J,G., Metallurgia, 45, 315 (1952). (177) Suzuki. S.,.I. Chern. Soc. Japan,73, 89 (1952). . 25, 119 (1953). (178) Theurer, K., and Sweet,. T. K., A s a ~ CHEM., (179) Thompson. J. SI., Ibid.,24, 1632 (1952). (180) Ibid., 25, 1231 (1953). (181) Tighe. .J. J . , Gerds. .\, F..Center, E. J., and 1Iallett. SI. W,, Battelle Memorial Inht.. BMI-799 11952). (182) Torrisi. -A. F.. Kernahan, J. L., and Fryxell, R. E.. AXAL. CHEM..26, 733 (1954). (183) Treadwell, IT,D., Guyer, H., Hauser, R., and Rixhofberger, G., H d c . Chim. Acta, 35, 2248 (1952). (184) Tschanun, G . B., .4rch sci., 6 , 101 (1953). (185) U. 9. .ktoniic Energy Commission, AECD 3457 (1952;. (186) Urech. P.. and Suleberger, R., Aluminium,30, 163 (1954:. ( 1 8 i ) Yinci, F. -1,. ; ~ N A LC. H E x . , 25, 1580 (1953). (18%) Yolpi. A., Ricerca sci.. 17, 1405 (1947). ~I
JOHN R. M A T C H E T T and H A R R Y W.
VON
., Sci. Repts. Research Insts. Tbhoktc C'niu., 4, 81 (1952). Wengert. G. B., XN.AI..CHEM.,24, 1449 (1952). Wengert, G. B., Walker. R. C., Loucks, AI. F.. and Stenger. V. A , . Ibid., 24, 1636 (1952). White, J. C., Ross, W.J., and Rowan. R., Jr.. Ibid., 26, 210 (1954). Wiedmann, H., Z . Metallkunde, 44, 565 (1953). Wiedmann, H., and Xufer, H., Ibid.,44, 389 (1953). Yokosuga, S., and Tanaka, 3I., X i p p o n Kinzoktc Gakkai-Sh;. 16, 291 (1952)). Yoneeaki, S., Ibid.,B15,498 (1931). Ibid., 16, 581 (1952). Yoshimura, C., J . Chenz. Soc. Japan, 73, 785 (1952). ZBbranskB. Z., Cheni. Listy, 48, 617 (1954). Zirnmerman, J. B., Rabbitts, F. T., and Kornelsen. E. D., Can. Dept. 3Iines and Tech. Surveys, Mines Branch, Tech. Paper 6 (1953). Ziranovic. D.. B i d . soc. chivz.. 16, 151 (1951).
LOESECKE
Washington Utihzation Research Branch, Agrico/tura/ Research Service, United States Department o f Agriculture, Washington, D. C.
HIS reyieiy covers some 2 years that have passed since the previous one was completed (190), and includes papers published during this time. MOISTURE
Kesearrh on moisture determination by nuclear magnetic resonance has been continued, and a recent paper (263) reports that this method has shown promise for rapid routine use. One important advantage is that it does not require essential modification of the test specimen. The method is effective in moisture ranges from a fevi per cent to as high as 100%. Moisture determinations with the Karl Fischer reagent have been critically evamined and discussed in three papers (57, 152, 281). Efforts have been made to substitute other reagents for methanol, and formamide has been found to be more versatile than methanol for moisture in such foods as beans, cahe mix, dried whole eggs, egg noodles, rolled oats, rice, and wheat ( 1 7 7 ) . Another modification makes use of a solution of bromine and sulfur dioxide in anhydrous chloroform instead of methanol ( 1 4 ) . I n the determination of moisture in honey using different methods, the Karl Fischer technique gave the highest values. while the refractive index method of the A40ACgave the lowest value ( 1 ) A method has been proposed (290) for estimating moisture in sugar-containing products, such as honey, jams, and fruit juices, based on the property of absolute alcohol to form an azeotropic mivture TTith n-ater of the sample, and upon the use of a special colloidal silicate which dries easilv. I n the case of dry sugar, it was found ( 9 4 ) that drying the sugar a t 130' C. for 45 to 60 minutes gave results identical with those obtained by drying a t lower temperatures for 4 hours or longer. 1Ioisture determinations in cheese have been carried out by placing the cheese between two previously weighed sheets of unlacquered aluminum 15 x 19 X 0.0016 em. (268). This is somewhat similar to the glac;s rod technique of the AOAC (@), in which the cheese is smeared on a glass rod. This method results in an improvement in the precision of the vacuum oven method for determination of moisture in both process cheese and cheese food, in that there is less burning of the sample. It has been reported (163) that loss of moisture in cheese is most
rapid when final drying in a water or vacuum oven is preceded by heating in a water bath for 1 hour. An apparatus for determining moisture in grains and flour by use of electric conductivity has been patented in Japan (136). Application of the old toy used to predict the weather (not too accurately) has been utilized to measure moisture in milled products (230). Dried filter paper is impregnated with cobalt chloride, which changes from blue to red when exposed to moisture. An infrared method has been proposed for measuring moisture in starch ($94). A 10-gram sample is placed in an aluminum dish and irradiated with a 250-watt infrared lamp a t 10 em. After 15 minutes the sample is cooled in a desiccator and weighed. Several methods have been suggested for determining moisture in fats and oils. A procedure using calcium carbide ( 5 8 ) has been suggested, as well as a modification of the Roberts and Levens technique using the Karl Fischer reagent (193). The thermal balance Buhler moisture apparatus allows weighing of oilseed samples without removal from the oven (87). At 115' to 125' C. results are obtained in 5 to 10 minutes; they are slightly higher, however, than the official oven method. I t has been suggested ( 7 2 ) that good results can be obtained by drying the fat a t 70" to '75" C. in a desiccator which is being continuously evacuated and flushed with carbon dioxide. A quick method for determining water in sausages involves the use of equations with correction factors (22). PROTEIYS AYD A M I S O ACIDS
The Kjeldahl method is still the subject of considerable research. KO general Kjeldahl method seems to be satisfactory for all types of nitrogen-containing compounds (25). Condition3 of digestion as related to temperature, catalysts, and oxidizing agents have been studied (182, 241). A modified method has been reported (226) in which digestion should not take more than 20 minutes for easily digested materials. Good results have been obtained in destroying organic matter with a mixture of sulfuric acid, potassium peroxydisulfate, and hydrogen peroxide (300). I n regard to catalysts, a study (108) has shown that best results are obtained by using a mixture containing selenium and
ANALYTICAL CHEMISTRY
624
mercuric oxide. The addition of hydrogen peroxide is recommended for high-fat materials. I n a semimicromethod (107), sodium hydroxide and sodium thiosulfate are used in place of sodium monosulfide. Khere the conventional sodium sulfate is used, it has been s h o m that 18 grams of the material, rather than the usual 10 grams, gives better results (26'). Of interest are tn-o papers dealing with amino acid deterniination in foods by chromatography (96, 257). One method uses a Dowex 50 column by means of which nearly all the common amino acids can be determined ( 2 5 7 ) . The other method ( 9 6 ) uses a circular paper chromatography technique, and is said to be ideally suited to the study of amino acid changes brought about by the processing of foods. Also of interest is a review article on chromatographic ion exchange separation of amino acids, purine and pyrimidine bases, nucleotides, sugar derivatives, aarboxylic acids, and proteins ( 4 6 ) . Circular paper chromatography has many advantages in the small laboratory, owing to the simplicity of the apparatus and techniques employed. h paper dealing with a systematic study of the physical factors influencing the Rf for a simple amino acid system is of interest (248). The Rf value of some 21 amino acids and 5 peptides has been determined by circular paper chromatography (256). Separation of glycine, serine, and aspartic acid from each other, and of glutamic acid and threonine, has been achieved by tn-o-dimensional chromatography (98). Lack of reproducibility, irregularly shaped spots, discoloration of the paper, and incomplete separation have been overcome by using a solvent system of butanol, acetic acid, water, cresol, and phenol with a borate buffer ( I ? ? ) . Isatin as a color reagent in amino acid circular chromatography has been described in two papers (247, 270). A paper describing a modified procedure for the photometric determinations of amino acids with ninhydrin reveals t h a t 10 of the naturally occurring amino acids give theoretical yields of color a t room temperature (292). At higher temperatures all amino acids except tryptophan and lysine react quantitatively. -4rapid, simple quantitative biuret test for millr serum proteins has been reported ( l i b ) ,but inasmuch as the color response of casein is different from that of serum proteins, mixtures of these proteins cannot be analyzed by the biuret test. Formol titration has also been suggested (149) as a rapid and dependable method for determining protein in millr. Albumins and globulins in wheat flour can be estimated quantitatively by a method developed from the characteristic tryptophan and amide nitrogen contents of the albumin, globulin, and gliadin protein of the flour (824). Results are reliable to within Glutamic acid in wheat gluten is esapproximately *lo%. timated by determining total dicarboxylic acids (glutamic and aspartic) by adsorption on Amberlite 1R-4B a t p H 3 to 4 with subsequent elution rrith IN hydrochloric acid (203). A method has been proposed for determining ammonia, amide, nitrite, and nitrate nitrogen in plant extracts by using a single portion of the extract (297),and a systematic procedure has been reported ($22) whereby water-soluble extractives of plant and animal tissues are separated into groups for identification or quantitative estimation by paper chromatography. METALLIC IONS
A critical revielv of existing procedures for the preparation of biological material for the determination of trace metals has been presented ( 196). Spectrochemical methods have been suggested in five recent papers (109, 118,191, $29, 309). I n one procedure (309) the minor elements, such as cobalt, zinc, and copper, are first separated in the form of dithizonates from the plant material. Spectrochemical analysis of plant material using spark excitation has the important advantage that all major inorganic elements as well as the usual minor elements can be determined simultaneously from one exposure (191).
Quite apart from spectrochemical technique for determining trace elements is a suggested microbiological method (208) employing such fungi as dspergzllus nzger and Penicillium glaucurn for estimating iron, copper, zinc, manganese, and molybdenum in biological materials. Although organic insecticides have largely replaced arsenical sprays in the fruit industry, appreciable amounts of lead arsenate are still used for apples and other deciduous fruits The bromide distillation method of Magnuson and Watson has been adapted for the determination of arsenic spray residues on fruits and vegetables ( I S ) . Interference of tin (in the case of canned foods) is overcome by extracting tin with cupferron and chloroform before distillation. A colorimetric method more accurate than the Gutzeit test has been suggested for the determination of arsenic in gelatin (71), and estimation of arsenic in eggs involves distilling to obtain arsine and then reading the developed color in a spectrophotometer ( 7 7 ) . Colorimetric determination of copper in plant material has been suggested by means of both the dimethyldithiocarbamate complex ( 8 6 ) and rrith sodium diethyldithiocarbamate (43), which is said to be one of the most sensitive chemical tests for copper. Copper in beers and ales acts to accelerate oxidation changes, adversely affecting flavor and shelf life. A colorimetric procedure for copper determination which requires no ashing of the sample and gives results in about an hour has been suggested (280). Reagent used is zinc dibenzyldithiocarbamate which gives a brownish copper complex subsequently measured in a photometer. A method for estimating lead in foodstuffs is based on the extraction of the dissolved ash with a solution of diethylammoniumdiethylcarbamate in chloroform a t p H 7.0 (175). The method is more accurate than the usual sulfide technique and more convenient than the absorptiometric method with dithizone solutions. Ashing of the material must, of course, be carried out with care and ignition of the sample in a nickel oxygen-filled bomb has proved satisfactory for burning of foodstuffs prior to lead determination by any suitable technique (78). .4 titration method for calcium and magnesium in milk, in n-hich interfering phosphates are first removed by a simple anion exchange technique, has been suggested ( f 4 2 ) , while a method for determining calcium in plants is based on measuring the resulting turbidity when a reagent containing ammonium oxalate, citric acid, ethanol, and butanol is added to a buffered aqueous extract of the material (135). From 0.05 to 0.50 mg. of calcium can be determined. A micromethod (115) which can estimate as little as 4 y of calcium is based on the precipitation of calcium molybdate and subsequent formation of molybdenum thiocyanate, which is determined photometrically. I n the determination of phosphates in biological materials, a micromethod (range, from 1to 70 y of phosphorus) makes use of an absorptiometric technique based on the conversion of phosphorus to phosphomolybdic acid and reduction of molybdenum blue by stannous chloride (117). Procedures for determining total, inorganic, acid-soluble, phosphatide, and phytin phoephorus in plant materials are described in detail in another paper (251). A photocolorimetric method has been proposed for tin in canned foods (e?'),while a suggested procedure for zinc is based on the fact that diantipyrylmethylmethane produces a white turbidity in the presence of zinc (103). The turbidity is measured nephelometrically. Lead and ferric iron interfere and must be removed prior to analysis. Sulfur in plant materials is estimated turbidimetrically in amounts ranging from 0.01 to 2.5%, using as little as 0.1 to 0.3 gram of material (276), while a flame photometric method for potaspiurn gives good agreement with results obtained by the platinic chloride procedure (146). Methods generally used for determining fluoride in vegetation
V O L U M E 27, NO. 4, A P R I L 1 9 5 5 give low results with samples high in silica A modified procedure (244) eliminates the need of a double distillation and evaporation by using alkali fusion of the ash and collection of 500 ml. of distillate. Estimation of 0x1-gen in fruit juices is of importance in studies on quality changes in juice pioducts during storage An apparatus and polarographic method are described n-hich should be useful to those concerned with this field of food research (240). A preashing apparatus, used for highly foaming sugar solutions, is also of interest (52, 53). FATS AKD OILS
IncreaTed production of vegetable oil “ice cream,” n-hich amounted to some 22,500,000 gallons in 1953, has aroused interest in methods for the detection of vegetable and animal fats in dairy products. One method suggests a rapid chromatographic determination of molar concentrations of butyric acid in the fat of dairy products (112). The amount of fat substituted can, however, be estimated only semiquantitatively using this method. Another procedure measures the refractive index of the alcoholinsoluble triglycerides to determine foreign fat addition (17). TKOmethods have been offered for improving peroxide number determinations. One suggests adding a smaller quantity of potassium iodide and carrying out the determination in the same container from beginning to end (106). The other, of particular value for dairy products, when the thiocyanate method for peroxide number is used, utilizes a nonionic detergent to break the emulsion (278). It has been reported ( 9 9 ) that the thiocyanate method s h o w good correlation n-ith values obtained by the Swift stability procedure. The thiobarbituric acid test for oxidative rancidity, which has been used in the past for milk fats, has been modified for use with butter, Cheddar cheese, and powdered whole milk ( 1 8 ) and for frozen pork (295). The Babcock test for butterfat has been reviewed (119, 193). The Bureau of Dairy Industry’s detergent test for butterfat (846), in a limited number of comparisons, shon-ed close agreement with the Roese-Gottlieb and Babcock tests. Another paper reports results 10 to 40% below those of the Gerber method when B D I technique was used (2). It has also been suggested that a detergent be added to the sulfuric acid (9 grams of a 50% quaternary ammonium compound to each 1000 ml. of sulfuric acid) and glymol before measuring the fat volume (303). I n measuring the fat content of half-and-half, which contains 8 t o 13y0fat, the usual Babcock technique mny result in charred fat. It is suggested that ice cream test bottles be used n-ith the addition t o the sample of 9 ml. of a saturated solution of trisodium citrate and 2 ml. of propyl alcohol prior to the addition of sulfuric acid (192). The use of hydrochloric acid in determining butterfat gives slightly lower results than those obtained by the RoeseGottlieb method (206). It has been reported that results from the Schain butterfat test are statistically closer t o those of the ether extract method than is the case Kith the Babcock test (148). d modified butyrometer method for fat in cream compares well with the Roese-Gottlieb procedure ( 1 3 2 ) . An apparatus previously dewribed for determining oil in soybeans can also be used for estimating oil in flaxseed (134). Two other methods of estimating fats in oil-bearing seeds have been suggested (9). One is a Sonhlet extraction technique, while the other depends upon the uptake of oil by sodium sulfate, which is subsequently treated n-ith a solvent. An improvement in the method of determining the oil content of olives pertains to the introduction of a F a r i n g Blendor into the process (147). Riethods are a150 given for the detection of the addition of other oils to peanut oil (298) and determination of total fat, milk fat, and coconut oil in chocolate products (107, 254). By the semimicro butyric acid number, milk fat in chocolate products can be easily determined even in the presence of coconut oil as high as 3% of the mixtures of fat (254).
625
il few minor changes have been recommended for the .4merican Oil Chemists’ Society’s tentative spectrophotometric method of the analyses of fats and oils ( 2 7 7 ) , and a study of spectrophotometric methods for polyunsaturated fatty acids in cottonseed oil makes a comparison with chemical methods (215). A% Tvet-ash colorimetric procedure has been offered for estimating lipide phosphoric acid in eggs and egg noodles ( 6 1 ) and a simplified procedme for determining the approximate lecithin-nitrogen and phosphoric acid content of macaroni serves as the basis for estimating the egg content of the product (122). h spectrophotometric method for grading green soybean oil correlates spectral absorption and visually determines green grades of oil as a suggested basis for a simple, rapid spectrophotometric method for grading (194). The induction period of butter is measured (312) by spreading on a glass plate and exposing the sample a t room temperature to normal sunlight or ultraviolet Periodic weighings and determination of the aldehyde content, as well as observations of color and odor, are made The appearance of aldehyde corresponds to the point of increase in weight-ie., end of the induction period. I n determining aldehyde values of slightly oxidized butterfat by Schibsted’s method, interference of butterfat color is eliniinated if measurement is carried out in a photoelectric colorimeter a t 510 mp (60). F a t t y acids are determined by running paper chromatograms in butanol-ammonium hydroxide mixtures and then spraying with a mixture of methyl red, and bromothymol blue indicators in formalin (YO), and by a potentiometric method in which a common glass electrode serves as a titration vessel for titrating with potassium hydroxide, using potassium acid phthalate and benzoic acid standards (101). The methoxyl content of lard involves saponification, recovery of methanol by distillation, oxidation, and reaction with chromatropic acid ( 6 ) . Absorbance (optical density) of the colored solution a t 5700 A. is proportional to the methoxyl content. A shortening consistometer (44) provides a simple and rapid means of obtaining numerical measurement of the consistency of shortenings, margarine, and butter, and is particularly suitable for production control purposes. 4 report dealing n-ith the estimation of ergosterol in yeast is also of interest (264). ENZYMES
Some batches of filter papers used for the phosphatase test for pasteurized milk contain a reacting substance in sufficient quantities to cause a properly pasteurized milk filtered through such a paper apparently t o fail to satisfy the test ( 4 7 ) . Details of a modified phosphate procedure are given in another paper (252). T h e phosphatase test has also been suggested as a means of control in heat-treated meat products (267). Pink center dewlopment in frozen Brussels sprouts, probably due to inadequate blanching, has caused some concern to freezer. of this vegetable. It has been suggested (189) that instead of using the peroxidase test as proof of adequacy of blanching, a dilute solution of hydrogen peroxide be added to Bruvels sprouts halves to obtain an orange color. Development of this color roughly parallels peroxidase content. Another report of interest is a method of measuring enzyme activity in frozen food. (186) by a spectrophotometric technique Paper chromatographic behavior of eight enzymes has been described (163). Solvent systems used were mivtures of aqueous buffers and organic liquids, among which acetone and methanol were found most suitable -4review of methods (24) witable for evaluation of protease activity in doughs will be of interest to cereal chemist?, and it has been suggested that activity of proteolrtic enzymes can be estimated b j the biuret reaction (668). h method (156) for estimating lipase in cereals involves the
626
ANALYTICAL CHEMISTRY
use of a butterfat emulsion as a substrate buffered a t p H 7.9 with either phosphate or borate. The material is maintained a t 36" C. and electrometrically titrated with standard alkali. CARBOHYDR 4TES
The green color produced when carboh) drates are heated with anthrone in acid solution was firfit used about 8 years ago t o estimate carbohydrates. -4recent paper (310) reports that the anthrone method for soluble sugars in plant extracts yields results comparable with those obtained with the copper reagent, but includes the sugars of stable glycoPides which may constitute a large proportion of the soluble carbohydrate in some plant tissues, Use of the anthrone reagent has been extended to the quantitative determination of pentoses, by employing a simple modifica1 if tion of operating techniques (28). Hexoses ~ ~ 1 1interfere, present. Pentoses will also interfere with determination of hexoses, depending largely on the composition of the anthrone reagent. Methods have been described ( 1 $ 4 ) for reducing this interference. .$ monograph with 120 references dealing a i t h crude fiber determination suggests slight modifications in the present method (128) It has also been pointed out that the percentage figure of crude fiber, especially when calculated on a dry basis, is a far more useful criterion in estimating food value than iq nitrogenfree extract (219). Two variations in estimating sugar by Fehling's solution are of interest. In one (296) the filtrate obtained from the cuprous oxide precipitate is examined colorimetrically using a comparison solution of invert sugar. I n the second (129) the cuprous oxide is dissolved 111 hot 4-11 nitric acid and diluted, and an aliquot is mixed with 1.11 ethylenediamine to obtain the copper-eth) lenediamine complex, which is examined spectrophotometrically a t 550 mp. Of interest to thocc concerned with sugar production is a paper giving the characteristic p H values of all types of beet-sugar factory juices, together with temperature roefficients of p H change for each tvpe of juice (10). Equally of interest are British specifications for four sizes of graduated fla-ks for sugar analyses (29). -1carbon column adsorption procedure iq suggested for the separation of sugars of honey into monosaccharides, disacchmides, and higher sugars (302). Caranielization of the bugars in flour nhen heated with alkali is used to measure total and reducing sugar (56). These figures, along with gluten quality, permit estimation of baking quality. refractometric method has been ieported for edimating sugars in ice cream ( 2 7 1 ) The readingq obtained must be corrected by nieanq of suitable tahleq for vaiiouq brands of ice cream. Quaternary ammonium and culfonir acid-type reeinq have been u s e d in paper chroniatogiaphy of qugars ( 3 0 8 ) ; a 0,:3% ethanol solution of p-aniinohippuric acid has been found to be very sensitive and stable, and spots pioduced bv reducing sugars do not fade (251) There iQalqo an a l n i o ~ imperceptible t background discoloration. Glucose dehydrogenase ha. been employed for the direct stoichiometric determination of D-glucose in a variety of corn Girups (301). I n the presence of oxxgen the enzyme oxidizes 1,-glucose to D-gluconolactone, n-hich is determined quantitativelv t)y titration n i t h standard alkali. Eggs desugared i-iv glucose oxidase and catalase are identified bv the presence of gluconic acid, which give. a color reaction n-ith buffered ferric chloride solution (259). -4 reliable and accurate method ha. been reported (220) for the analvsiq of a milture of glucose, maltose, and fermentable oligo~accharides,using a modification of a technique puhh-hed eallirr. By measuring the ammonia expelled from hydro1 hydrin, a method hac heen deviceti for ectimating g l u r o ~ eand other aldoqe wgars (49). K h e n a solution of fructose or w r b o v
is heated with Folin-Denis reagent and trisodium phosphate, a blue color is produced, and t.his caolor is used in determining the amount, of sorbose and fructose (11.3). -4 method of determining pentose3 and hexoses when prc together is based on the fact that XThcn such a misture is heatrrl with orcinol and acid, colors produced can be measured :it suitable wave lengths and the results calculated directly froin the colorimetric readings by mean? of a noniograni ( 8 0 ) . I t has been suggested that lactose in cheese and other dairy produrts can be determined by a technique baaed on Somogyi's blood sugar method (284);Tvith the anthrone reagent ( ? 9 ) , results of which agree well with copper reduction method;.; and hy chromatographic analyses (1.91 ) involving direct application of milk to the strip and subscqurnt determination of lartocr on the chroniatostrip. -1revien. of methods of testing f o l , nonfat solids in milk is also of interest (125). Pectin in citrus juices is estimated rapidly by a method involving the use of the carbazole-hesuioiiic acid-sulfuric acid reactioii (68). Results are report,ed in terms of anhydrogalacturonic acid (the hapic structure of the pectin molecule), and the procedure is suitable for routine control ~ ~ o r ak ,complete analysis taking 3.5 hours. Pectin methods have a h o been reported (165, 169, 181,286).
-4 method for estimating water-soluble cellulose derivatives, sometimes used in foods as thickening agents, has been reported by German investigators (171). The 11IacJIichael viscometer method with lactic acid for determining the alkaline viscosity test for soft wheat flours ha? been modified t o an alkaline viscosit,y test with sodium bicarbonate (61). +$pplication of the Corn Industries Viscograph to potato starch has been reported (65). Several modifications in design were made while retaining the more important parts of the installation ( 6 4 ) . rch sirup in foods has been of-4simple qualitative test foi fered (15), and several methods for determining starch in flour and baked goods have been reviewd and compared (104). VITA3113S
Biological, microbiological, chemical, and phy~icochemicnl methods for determining vitamins have been reviexed in a recent paper (211), and limitations of the degign usually employed for microbiological assays have been rather t,horoughly discussed (307). Total carotene in tomatoes is estimated by an improved, rapid colorinietric method (176), and in another method, vitamins -4 and E are separated by chromat,ography on filter paper impregnated with 10% silicone st,opcork grease in dimethyl chloride using an ethanenitrile-water em ( 3 1 ) . The vitamins are located on the strip with an automatic spect.rophotometric arrangement which pyovides a graph of hand position ~8s.strip length. -4 standardization procedure for adsorbants used in chromatographic columns for vitamin -1 separation has been described (304). .4 rapid method (259) for calculating the llorton-Stubbe correction in vit>aminA assays is based on the fact that usually analysis i,? based on absorbance ( A ) readings a t 310, 325, and 334 nip. The corrected absorbance is T ( d 3 y , - .4310)4.375 (/lal0 -4314)and may he solved with a .slide rule. Direct application of the Morton-Stubbs correction to vitamin r\ samples containing appreciahle quantities of t,ocophwols yields high results for vitamin .$ potenc4y. The two ritmiins must first be separated chromatographically, folloaed by mkiFequent quantitative recovery of vitamin A , t o which the ?\Iorton-Stuht)s correction can then he applied (262). It has been suggested that 1,2-&chlororthane can be used in place of chloroform in the preparation of the Carr-Price antimony trichloride rtaagent (1?8), and this method has been proposed for
+
V O L U M E 27, NO. 4, A P R I L 1 9 5 5 estimating vitamin A in several dry food product? (3.T). Titamin .4is extracted from the food with ether, and the residue left after evaporation of t'he ether is taken up in chloroform. It has been buggested that the ether extract he stahilizrd r i t h purified hydroquinone (202). Chemical niet'hods of detrrniining free and phosphorylated thiamine, riboflavin, and ascorhic acid have been discussed (238). -4niet'hod that offers ready separation and independent determination of nicotinic acid and nicotinamide is described, which makes possible the analysis of mixtures containing only one part of one form of the vitamin in the presence of 100 parts of the other (286). -4gravimetric method for determining choline in eggs is based on its precipitation as reineckate ( l o b ) , and choline and thiamine have been separated quantitatively by chromatography on starch (124). The possible errors inherent in the U.S.P. and Int'ernational Pharmacopoeia thiochrome method for thiamine have been discussed in a French publication (61), and estimat,ion of thiamine in bread involves removal of the vitamin bj- extraction wit,h v-arm water containing sulfuric and acetic acids (260). A rapid assay method for riboflavin in milk has heen reported ( 2 7 3 ) and a chromatographic technique for folic acid has been new microbiological determination of the suggested (158). individual components of the vitamin B6group involves extracting them from natural materials by hydrolysis with 0.055-V sulfuric acid a t 20-pound pressure for 3 hours (90). Tot,al Be is assayed by using Saccharomyces carlsbergensis 4228. Other aliquots are assayed by cation-anion rePins, followed by the use of S. carlshergensis. .I modified chemical method for pantothenic acid in n-heat consists of extracting pantothenates v-ith methanol instead of water (237). I n every other respect the method is the same as that of Szalkowski el al. A microbiological t,echnique has been suggested for pantothenic acid in foods (291). Phosphataseliver enzymes are used for preparing the sample estracte and ordinary laboratory equipment can he utilized. Suggested methods for determining vitamin Bn have been hased on the use Euglena gracilis var bacillaris (121, 127, 159). Response curves obtained with Lactobacillus leichmannii using titrimetric method for BIB usually yielded straight the U.S.P. lines when log dose \vas plotted against log response, which makes possible the use of a 6-point (3-dose) design (185). r s e of L. (actis for t,he plate method and L . leichmannii in the acidimetric method has been suggested ( 2 3 ) . -4niicrobiological method using L. leichtnannii and measuring response in t,erms of turbidity has heen adopted by the AOAC (161 ). Essentially the same method has been included in the fourth supplement of the U.S.Pharmacopeia (296). A rapid Ppectrophotometric procedure for R12 involves extraction of the vitamin with benzyl alcohol or npropyl alcohol, follovied by estimation of the vitamin in t'he extract by differential spectrophotometric measurements (83). il rat-growth method of assay for HIz using a highly purified diet with casein has been further studied and improved (89). Use of 2,4-dinitrophen?-lhgdrazinefor vit,amin C in milk has been suggested (110)) and a method based on the reduction of picric acid to picranic acid b?- vitamin C permits a colorimetric estimation of this vitamin (66). Cacotheline hae heen found suitable for the detection of less than 5 y of vitamin C on paper chromatograms (289), and another method involves the reaction of vitamin C with diazotized 4-methoxy-2-nitroaniline in an acid medium, followed by development of a blue color in alkaline solution (266). This color, maximum absorhancy a t 5T0 mp, is compared with st,andards in a photoelectric colorimeter. I n the chick assay of vitamin D, precision of analyses n-as pignificantly greater with selected chicks for uniformity of response (160), and improvement in the line t,est was obtained by giving the bones a preliminary wash in an ether-acetone mixture, thus making for more complete staining with silver nitrate (266). Methods have been suggested for determining vit'amin D in
627 homogenized milk (299) and in various oil$ containing vitainin .4(256). Almost all met.hods for determining the individual t,ocopherok in natural products have the disadvantage that the concent,ration of a-tocopherol, the most, biologically active member of the vitamin E group, is estimated hy difference. A suggested method involves paper chromatography for quantihtive estimation of the individual t'ocopherols, a-, 8-, y-, and 6- (30). A rapid method for vitamin E in foods and fodder is based on Emmerie-en gel'^ color reaction (812), and a modified paper chromatographic technique has also been described ( 7 3 ) . -4paper giving the R, values of a nunillei- of flavonoid conipounds is of interest (41). ACIDS
Two reports describe circular paper chromatography for determination of organic acids (.5,97),a technique that in general has been found to be more direct and l e v time-consuming than other chromatographic methodq. Citric acid is estimated by utilizing the conversion of this acid to pentabromoacetone as recommended in earlier methods, h u t substituting a direct polarographic measurement for the subsequent petroleum ether extraction, oxidation-reduction ti eatnient, and final titration procedure ( 7 4 ) . llicrodetermination of citric acid is based on a rapid colorimetiic method (288) Citric, malic, and tartaric acids are separated by ion exchange chromatography and then determined by oxidation with permanganate (263). The method is applicable to fruit, fiuit juices, and fruit products. Organic acids in carlot and apple tissue have been determined by paper chromatograplij (34). Oxalic arid determination in plants is hased on esterifying the material in the presence of sulfuric acid, evaporating the eetei, and collecting the condensate, which is suhrequently saponified : oxalic acid is titrated with potas-ium permanganate (168;. The chief difficulty is that distillation, nhich must be carried out in special apparatus, requires 6 hours. .4n ion exchange resins technique for detei mining organic acids in sugar beets makes it possible to folloir the effect of processing variety and other agronomic factor5 on the organic acid content of the beets (218). Sonnitrogenous oiganic acids in sugar cane are estimated by the application of partition chromatography on silicic acid columns of large capacity (243), and partition chromatography has also been recommended for determination GI organic acids in sorghum sirup (184). Chromatographic methods have heen proposed for estimating volatile acid. in cheeqe (111, 1 7 9 ) . COLOR 4 \ D T4STE
-4rapid spectrophotometric method for measuring the alcoholestractable color of capsicum spices employs a 10-cm. sample cell and involves transmittance readings a t two wave lengths (188). -4ccuracy of the method surpasses that of Loviboncl glasses. Further reports have been issued relative to color grading by the Purdue color ratio meter ( 6 2 ) , and a photoelectric reflectance meter for measuring the darkening of food purees has been evaluated ( 1 7 4 ) . A comparative study of the validities and precisionq of three inptruments t o estimate color grade of Marshall and Sorthwest strawberries gives correlation data between the instruments (261) Chromatographic methods for colo~ing matter in foods have been reviewed ( 6 3 ) One such method uses glass plates (253; on nhich a thin layer of aluminum oxide is spread. The method permits two-dimensional chromatography. The Rf values of 36 food dyes have heen determined by partition filter paper chromatography ( 8 ) . Other chromatographic techniques for de-
ANALYTICAL CHEMISTRY terniining added colors in foods have also been described (42, 86, 138, 201, 221). Those concerned with taste teqt methods will find of value a paper on the comparison of the triangular and t n o-sample tastetest methods (39). A sensory taste method has been suggested (59)that can be used for estimation of levels of significance for replicated judgment of two or more samples that represent no more than one difference. Of interest is a report on the aroma number (155), which is a measure of the potassium bichromate reduction produced by volatile material from 2 ml. of fruit juice as determined under specified conditions in a special apparatus. Those concerned with quality control will find of interest a paper dealing with a brief discussion of methods used for the quantitative evaluation of fruit products (45). INSECTICIDE RESIDUES
Vapors of perchloroethylene have been found useful in controlling rot-producing molds in strawberries. A method has been suggested (187) for determining residual fungicide, involving eytraction of perchloroethylene with ether, followed by evaporation in the presence of ethylbenzene, and determination of the chloride with a nephelometric technique. An improved procedure for determining bromine residues has been offered (267)and techniques for determination of residues of the herbicide 3-( p-chlorophenyl)-1,l-dimethylurea have been suggested (20, 21, Sf 1) Parathion residues on tomatoes are determined b y removing the residues with alcohol, oxidizing the plant pigments with hydrogen peroxide, and hydrolyzing the parathion to p-nitrophenol, rrhich is determined colorimetrically (36). Microquantities of benzene hexachloride can be estimated by an absorptiometric technique (228),and residues of the herbicide K-1-naphthylphthalamic acid by hydrolysis of the amide to give 1-naphthylamine, which is steani distilled from an alkaline solution (269). The amine is coupled with diazotized sulfanilic acid to give a red azo dye IThich can be determined colorimetrically. Procedures for the determinations of residual highly chlorinated pesticides such as aldrin and dieldrin have been reported (4,55, 137, 138, 216). Crag herbicide 1 [sodium 2-(2,4-dichlorophenoxy)-ethyl sulfate] is estimated by measuring the intensity of the colored complex formed with methylene blue chloride (130), and residues of malathion are determined by rapid decomposition of the material by alkali to form dimethyl dithiophosphoric acid, followed b y subsequent determination of this decomposition product b y a colorimetric procedure (214). CONTAIIINATION AND SPOILAGE
Considerable research hap been accomplished on detection of insect infestation of foods. Differentiation of bran and germ from insects and larvae fragments in white flour consists of suspending the specially prepared sample in a dilute crystal violet solution (164). By transmitted light, insect fragments appear bright orange-red, while bran and germ particles are staincd deep violet. Staining with ruthenium red is suggtsted for detecting dung and plant fragments in milk (260) Internal or hidden insect infestation of grains is detected h y means of a low noise level audioamplifier microphone and loudspcalier (3). It has also been suggested that insects in stored grains csn be identified by their mandible characteristics ( 1 6 2 ) , and a recent series of papers dealing with insect rontamination in foods iq particularly timely (151, 179, 209). Improved techniques have been offered for mold and rot in tomato catsup (180) and in tomatoes (38). Difficultirs in determining rodent hairs and insects in oatincal have h e m surmounted (116). Procedures suggested for determining penicilli t i Inti othcr antibiotics in milk include a cup method on agat i 1 suspension of BaczIIus subtilis (100) anrl i n x itrains of
Streptoccccrrs lactis having extreme sensitivity to the particular antibiotic being detected (170). Biphenyl is used for controlling decay of citrus during shipment and storage. d method of estimat,ing biphenyl in treated fiberboard cartons is based on using the biphenyl absorption peak a t 14.34 microns (154). Quaternary ammonium compounds are determined in milk by titration with standard anionic surfaceactive agents using eosin as an indicator (92). Abnormal quantities of free galacturonic acid in fruit and vegetable products have been propoped as evidence of decomposition by microbial activity. I met,hod for detecting galacturonic acid is based on a modificat,ion of Tollen's reaction of uronic acids with naphthoresorcinol ( 7 ) . A direct count method for bact,eria in frozen citrus concentraks has been offered (306); a rapid procedure for identifying bacteria isolated from off-flavor juice is based in part upon cell morphologv (f6), and rapid colorimetric methods for detecting Lactobacillus and Leuconostoc are based on estimating diacetyl, one of the end products of bacterial metabolism (40,75, 126). Improved media for the grorvth and isolation of spoilage organisms from canned foods have also been present,ed (84). A column chromatographic method is used for the detection of added lemon juice to orange juice ( 2 @ ) , making it possible to detect the addition of 570 lemon juice. Techniques for determination of Salmonella in dried eggs h a r e been reported ( 1 2 ) , and a modified DU spectrophotometer is used a t wave lengths of near 5 i 5 mG for detecting blood in white shell eggs ( 2 7 ) . RI values on filter paper Chromatograms on a number of coumarins and related cinnamic acids in various solvents have been determined (283). A chromatographic method, using a specially prepared aluminum oxide, has been suggested for the separation of vanillin from coumarin (2f0), and ethylvanillin in vanilla extract is estimat,ed by using a freshly prepared 1% p-aminophenol solution, which, in an alkaline medium, gives a dark purplish color in the presence of ethylvanillin (140). A fermentation test is suggested for detecting preservatives other than benzoic or sulfurous acid in beverages (905)and in meat products (204). The diazotization method of Edwards has been used to detect benzoic acid and its derivatives in foods (93).
Chicory in amounts of 1% or more can be detect,ed in coffee by the Seliwanoff test for fructose (goo), and detection of adulteration in vinegar is based on its albuminoid ammonia value (199).
The proposal to employ dimethyl dichlorosuccinate as an antimycotic in packaging such foods as cheese, bread, fresh raspberries, and tomatoes created need for a method for its detection and determination. A colorimetric met,hod sensitive to 1 p.p.ni.. based on the det,ermination of degradation products of dimethyl dichlorosuccinate has been offered (235). A colorimetric method, based on the action of t'he curd of the milk sample, with cert~ainsolvents, is used for detecting reconstituted milk in normal milk (238). Cow's milic added t o goat milk is detected hy a serologieal test (Z72). It has been reported (Sfsj that use of tetrazole saks instead of methylene blue in testing the 3anitary condition of fluid milk has n o adrantages. Detection of adulterat,ion of meat wit,h horse meat depends upon t'he fact that horse fat contains a higher percentage of linolenic ncid than does the depot f a t of normal beef animals, sheep, or pigs ( 5 . J ) ;serological methods are also recommended for horse meat detection as well as the addition of pork to hamburger ( 2 1 7 ) . BZISCELLANEOUS
Further viorlc has been carried out on determination of et,hylene in air (27'9). Methods for sampling Cheddar cheese far commercial analysis have been described (234). Various methods of measuring the
V O L U M E 27, NO. 4, A P R I L 1 9 5 5 consistency of tomato puree have been evaluated (60), and a method for sampling tomatoes for grading has been reported (160). The Tenderometer for measuring texture of cooked frozen peas has been correlated n i t h orgmolcptic evaluations of texture ( 1 S 6 ) , and it has been suggested that maturity in frozen peas can best he measured by specific gravity and aleoholinsoluble solids ( 1 6 6 ) . Terpenes in citrus oils liave been determined using filter paper impregnated IT-ith silicic acid (213) or glass plates coated with silicic acid (239); R f Pnlues of many terpene: znd oxygenated terpenes found in essential oils have been reported (196), and methods of analyses of lemon oil h a r e bern the subject of a review (249, 305). h apparatus and proccdurr for wparating and estimating volatile constituents ill fruit prodii.cfs have been reported (102), giving fairly reproducible results on a varirtJ- of fruits. A paper chromatographic method is svggested for determining caffeine and trigonelline in coffee ( 1 6 7 ) ; chemical proccdures for estimating caffeine in coffee ( l l - i j2 4 2 ) and in soft drinks (133), and theobromine in cocoa residues (95) have been proposed. .4 statistical approach to measurement of particle size of dried milk has been presented (141), along n-ith a seKiautomatic cryoscope for rwJtine determination of the freezing point of milk (665),a method for measuring the wettability of povidered milk (11)) and suggested techniques for estimating total solids in milk (50, 120, 167). One of the nevier methods for enumerating bacteria is the molecular or millipore filter technique, which removes bacteria from aqueous solutions and permits groiyth directly on the filter itself (37). Improved techniques for thermal process evaluation (223, 227, 276, Psi?) will be found helpful for those engaged in this field. -4nerv apparatus, made from ordinary laboratory equipment, makes possible a rapid niethod for determining alcohol in raisin residues (33). Benzoyl peroxide in flour and bread is determined absorptionietrically by Mohler’s reaction (153), and testing of antifoam agents involves registering the foam Tyithin a n enclosed standpipe through Jyhich air is bubbled a t a determined rate (86). K i t h comparatively simple apparatue, apparent density of meats can be approximated so that, t’he data may be used t o calculate piece w i g h t s in packing plants (88). Of interest, too, is a dilatometric method for control of f a t hydrogenation ( 9 1 ) useful in hydrogenation plants. Quality of active dry yeast is compared by using the methylene blue staining procedure, plate counts, and baking tests (625). High voltage gaseous discharge phenomena have been employed for measuring the vacuum levels in commercial glass containers (7’6) T h e method is especially adaptable to measurement of relatively rapid gas leakage rates. An apparatus for sampling and analyzing swell gases from cans is also of interest (274). A suggested procedure for measuring gas permeability of sheet material is based on the principle of measuring a change in volume of constant pressure and temperature corditions ( 5 2 ) . Infrared absorption spect,ra of several water-soluble gums have been presented (ROY), useful for the identification of gums isolated from foods. A rapid method for determining the solubility of dried n-hole egg pori-der involves dispersion of the sample in 0.9% saline, centrifugation of saline-insoluble solids, and precipitation of the saline-soluble and colloidal fraction with Esbach reagent, followed by centrifugation of the precipitate and reading the volume of the precipitate as the soluhility index (19). LITERATURE CITED
(1) dbramson, E., Mitt.Gebiete Lebensm. u. Hyg..,44, 469 (1953). (2) Adamo, G., Boll. lab. chznz. prozinciali (Bologna),4, 95 (1953). (3) Adams, R. E., Wolfe, J. E., Mdner, AI., and Shellenberger, J. A,. Sczence. 118. 16
