January 15, 1942
ANALYTICAL EDITION
nal weight before settling started and no mistake has been made in either weighing, it is proof positive that samples are being taken from too close to the bottom. The graduate should not sit in it position where hot or cold air can strike part of it t o set up convection currents, nor should the temperature change more than 2" C. during the course of the test. Distilled water may increase the accuracy slightly, and if distilled water at 20" c. is used with a weighed quantity of dispersing agent, the first weighing of the specific gravity bottle with the clear solution may be omitted, since under those conditions this weight will always be the same. ,
If care is used, this method is just as accurate and as rapid (probably a great deal more rapid) as with equipment costing hundreds of dollars, where not more than four or five different sizes are wanted from one sample. A 500-ml. graduat,e is not snt,isfactory for more than this number of determinations. Using a larger sample and a deeper settling container additional determinations could be obtained.
13
Great cleanliness of the containers is not necessary, as in methods measuring the transmission of light through the suspension. Calculations of percentages should not be carried into fractions of a per cent, since the accuracy of this method (or any other method) probably does not warrant it. Where some other material than water must be used for t h e settling medium, its viscosity may be determined with a cheap viscometer (Ostwald or similar). The specific gravity is readily obtained with the specific gravity bottle.
Literature Cited (1) Alexander, Jerome, "Colloid Chemistry", New York, D . Van Nostrsnd Co., 1937. (2) Am SOC.Testing Materials, D-422-39. (3) Travis, P. M..A . 8. T.M . BztZl, 102, 29-32 11940)
Recording Color of Opaque Objects MAURICE E. STANSBY' AND JOHN A. DASSOW U. S. Fish and Wildlife Service. Technological Laboratory, Seattle, Wash.
To compare the colors of opaque objects, such as fish fillets, photographic color transparencies are prepared and their spectral distribution curves obtained, using a photoelectric spectrophotometer. Errors due to variations in illumination during exposure and in processing of the film are eliminated by taking pictures of objects to be compared on the same negative.
C
OLOR of transparent liquids can conveniently be recorded by obtaining a spectral distribution curve, utiliz-
ing equipment which is now available in many chemical laboratories for colorimetric analyses. Opaque objects present more of a problem, and no simple yet entirely satisfactory method is available that does not require special equipment for reflectance measurements. I n connection with studies on changes in color of salmon caused by cooking and by cold storage, a method was developed in which color photographs were taken on Kodachrome film, the transparencies were placed in a Coleman spectrophotometer, and spectral distribution curves n ere obtained. While such curves are not absolute records of the color of the original object, they are very useful in recording relative changes in color-for example, pictures of two identical objects taken on the same negative and processed together will give substantially identical curves. If the two objects-e. g., fish fillets-are subjected t o different treatments, such as storage at different temperatures, the relative change in color of the two objects will be recorded and the effect of the treatment upon the color change can be determined. If the change in color takes place over a relatively short period of time, the photographs of the object before and after the color change has taken place can be made on the same roll of film, and if reproducible illumination and expo1 Present address, U. $. Fish and Wildlife Service, Fishery Products Laboratory, Ketchikan, Alaska.
sure conditions are used the curves can be directly compared. If color changes slowly over a period of weeks or months, t h e original and subsequent photographs must be made on separate film, and slight differences in processing will render a direct comparison unreliable. Even in such a case, however, color changes between a control and one or more variable conditions can be photographed on the same negative, and the relative color change recorded. Photogrflphs \yere takeii using equipnient whereby il1uiiiiii:Lt io11 a n d exposure conditions could be readily duplicated. A 35-nm. camern with f 3.5 anastigmatic lens was mounted on ;I rigid upright attached to a large wooden base, which was p1:icc.d inside :{ light-tight box without a top and of such a height tll:it tlie c:inier;i \vas slightly above the open top of the box. Ttvo lights in metal reflectors were rigidly attaclicd above the camera and pointing inward slightly. Five hundred-watt, clcnr, 3200' Kelvin lnrnps were utilized. These lamps arc corrected for Eastman type L3 film which is available only as cut film in the larger sizes. The type oi film employed with the 33 is desipned for use w i t h photoflood lamps, but can be 3200' Kelvin bulbs ivith only a slight difference in r disadvantage is more than compensated by the f A c t t h x t tlie 31'00' Kelvin lamps have a iar more con.itant illumination o \ w tl:eir liie (30 hours) t h n n the photoflood lamps. The voltage to tl.ese lamps was adjusted to 120 volts by means of an autotrm~fvrnicr. Two square. of opal flysh glass were supported on t1.e top oi the box direcrly beneath the lampj but leaving an opening a t the r r n ter for the cnniera. This resulted i n a fairly uniform i1lumiii:~tion of the object to bc phc,topraphed. The camera \\:I+ niounterl .+o t h a t it could be raised or lonered t o obtain a field of varying dimensions. By adjusting the field of the camera properl),, it wsi pos3ible t o obtain a photoarsph of the object of :t sizc vcr?. slightly lfirger than the exit slit o i the spcctrophot[imc.tr.r. Ir' thcse conditions are fulfilled the color of the entire object \vi11 tie measured; this is an important consideration in obtaiiiinr rcprt,ducible results, if the color oi the object is not uniform tlirourliout.
If the pliotograph of the object is considerably larger than the exit slit of the spectrophotometer, only a portion of the image nil1 be in the path of the light beam, and the color of that portion only will be recorded. If there is considerable variation in color from one part of the object t o another, different results will be obtained, depending upon the portion of the photograph placed in the beam of light. On the other hand, it is imperative t h a t the photograph of the object be a t
INDUSTRIAL AND ENGINEERING CHEMISTRY
WAVELENGTH IN M ILLlMlCRONS
FIGURE1
least as large as the light beam; otherwise the color of a portion of the background will also be recorded. Therefore, it is best to make the photograph of the object slightly larger than the beam of the spectrophotometer. I n obtaining reproducible exposure for consecutive pictures, the setting of the lens opening and speed of the shutter are very important. With the particular camera employed (Kodak 35 with f 3.5 lens) the setting of the shutter speed could be duplicated accurately, whereas i t was impossible to reproduce the setting of the lens opening with any degree of certainty. Accordingly, exposures were always made with the lens opened wide and a suitable shutter speed was selected to give the appropriate exposure. A magnifying glass was used in setting the shutter speed, to obtain exactly the speed desired, but even with these precautions it was difficult to obtain reproducible exposures. Each time the shutter speed lever was moved, a number of exposures had to be made before equilibrium was reestablished and the desired speed once more at~ tained. For example, if a picture were taken a t l / second, and the speed lever was moved to another setting and then a t once moved back to exactly second, a second picture would not have exactly the same exposure as the first. Subsequent exposures taken without moving the shutter speed lever gradually approach that of the initial exposure and finally become constant. This error can be reduced by adjusting the shutter speed before winding the shutter or eliminated by making the necessary adjustments, snapping the shutter a large number of times before loading the film, and then not altering the adjustments during the series of pictures on that roll. Examples of this error are shown in Table I, in.'which per cent transmission of the film a t a wave length of 590 millimicrons is used as a measure of the exposure of the
Vol. 14, No. 1
film. This same effect was obtained with two different cameras of the same type and make. The considerable error made if the camera is a t different temperatures for the different exposures (Table 11) can be eliminated by placing the camera in an incubator a t a standard temperature before taking the pictures. After processing, the film can be cut and the photograph of each object mounted separately on a suitable cardboard form containing a hole over which the transparency is carefully centered so as to coincide with the exit slit of the spectro- ' photometer. An extensive series of pictures was taken a t different exposures. The red end of the spectrum was most affected by differences in exposure. It was the purpose of the present work to study gradations from red colors to yellow, and under these circumstances a t least the greatest difference in transmission curves was obtained when the pictures were overexposed with about twice as much light as gave the best picture by visual inspection. I n any case it is better to overexpose than to underexpose, and in order to allow a sufficient margin of safety, doubling the correct exposure is a n adequate precaution. With the particular arrangement of camera, stand, and lights used in these experiments, an exposure of 1/26 second a t f 3.5 gave the best results. Using all the precautions described, a fairly high degree of precision is attainable-for example, a series of pictures of the same object, taken on the same film, gave the following per cent transmission: 23.9, 23.6, 23.8, 23.6, 23.9, 23.6. The same picture taken with one-half the exposure time gave a per cent transmission of 34.7. One example of the application of this method is shown in Figure 1, which gives spectral distribution curves for transparencies of a fresh silver salmon fillet and one which had been frozen and stored for one year. The protracted storage period had caused a discoloration from the normal red color to an orange-red hue. I n the blue and yellow portion of the spectrum (350 to 600 millimicrons) the stored fish had a higher per cent transmission than the fresh fish, while a t the red end of the spectrum the reverse was true. I n the application of this method two points are of especial importance if erroneous conclusions are to be avoided. First, unless the control color is very similar to that of the color
TABLE I. EFFECT OF SHUTTER-SPEED ADJUSTMENT ON &PRODUCIBILITY OF EXPOSURE Shutter Setting Second
No change No change Shutter moved to about 1/27 then back to 1/25 No change No change Shutter set at 1/50 Shutter aet at 1/25 No change Shutter set at 1/10
OF TABLE11. EFFECT
Temperature
c. 5 20 30
Exposure at 590 Millimicrons 70Tranamisaion 28.3 22.4 27.6 28.3 28.3 23.0 28.0 28.0 24.0 27.0 27.4 35.4 22.8 28.1 9.6
TEMPER.4TURE OF CAMERA AND FlLM ON
EXPOSURE Exposure at 590 Millimicrons Picture 1 Picture 2 70Tranamission 40.0 32.3 37.3 31.1 36.9 29.6
ANALYTICAL EDITION
January 15, 1942
under consideration, entirely misleading results will be obtained. For example, if a n artificial, painted color standard were used with some object such as fish, results obtained could only lead t o confusion and misleading interpretation. The second point t o be stressed is the necessity of restricting all comparative data t o results obtained on a single roll of film. Even if i t were possible t o eliminate differences in processing (which ordinarily would be excessive for photometric work), there would still remain the uncertainty of differences in the original rolls of unexposed film. Improvements in
15
color film are constantly being made which undoubtedly would markedly affect the results obtained by this method. Accordingly, the method as described is of value only when a control very similar to the object under investigation is photographed, preferably simultaneously on the same negative b u t at a n y rate within a short period of time on the same roll of film. PUBLISHED with the permission of the Director, Fish and Wildlife Service. Acknowledgment is made t o t h e Works Project Administration 0. P. 76593-3-11, for assistance in carrying out a portion of the work.
The Analysis of Peppermint Oil I,. H. BALDINGER, University of Notre Dame, Notre Dame, Ind.
I
N THE routine examination of a large number of peppermint oil samples, some interesting observations were made on pharmacopeia1 as well as unofficial methods of assay. This paper serves as a summary of these observations and as a supplement to other reports (19) concerning the production, collection, and storage of the oil samples. For general reference purposes, the reader is referred to (9, 10, 16, 17, 62, 64).
Composition of Oil of Peppermint I n the earliest elaborate research of Power and Kleber (18) on the composition of American peppermint oil the following compounds were identified in the oil: Acetic aldehyde Acetic acid Alpha-pinene L im o n en e Menthone Menthyl acetate Cadinene 4 menthyl ester Isovalerianic aldehyde Isovalerianic acid Phellandrene
Historical I n a brief history (6) of the inclusion of oil of peppermint in the United States Pharmacopoeia, it is pointed out that a chemical assay for this product was not included in the monograph until the eighth revision, 1900. I n the sixth revision, 1880, the committee on essential oils included in the official definition the statement that the oil is a “colorless, or yellowish, or greenish-yellow liquid becoming darker and thicker by age and exposure to air”, which indicated that the effects of poor storage conditions had already been noted by investigators. The seventh revision, 1890, advised that the oil be kept in a cool place in well-stoppered bottles. This revision also included a number of qualitative tests which were intended to eliminate the practice of adulterating the oil with foreign substances or with oils from other species of mint. I n the eighth revision, 1900, following the work of Power and Kleber ( I @ , the committee on essential oils defined the product as “the volatile oil distilled from the fresh or partly dried leaves and flowering tops of peppermint, rectified by steam-distillation and yielding, when assayed by the process given below, not less than 8 per cent of ester, calculated as menthyl acetate, and not less than 50 per cent of total menthol, free and as ester. It should be kept in well-stoppered, amber-colored bottles, in a cool place, protected from light.” The process for menthyl acetate involved the saponification of a weighed sample of oil with alcoholic potassium hydroxide solution and subsequent titration of the residual alkali to determine the amount of esters present. The saponified oil was then treated with acetic anhydride and sodium acetate to convert the menthol to menthyl acetate. After washing and drying, the acetylated sample was treated with excess alcoholic potassium hydroxide for 1 hour, the mixture was back-titrated with normal acid, and from the amount of base used, the per cent of total menthol was determined. In this revision a solubility test using 70 per cent alcohol, the degree of rotation, -25” to -33” in a 100-mm. tube a t 25” c.,and a qualitative test for dimethyl sulfide intended to detect nonrectified oils were included. Limits for the specific gravity of the oil were included, 0.894 to 0.914 a t 25’ C. In the ninth revision the ester requirement was lowered to 5 per cent, no change was made in the mentholDcontent, and the optical rotation was changed to -23’ to -33 . Separate portions were taken for the ester and menthol assays, but the committee failed to take into consideration the menthol in the esterified state in the calculation of the total menthol. In the tenth revision, 1920, the formula was changed to include the menthol combined as menthyl acetate, and limits for the refractive index were included.
CHaCHO CHsCOOH CioHia CraHts C;oH;aO CioHio. CzH301 ClSHZ4 CioHlo. CaHiiOz CaHoCHO C4HoCOOH CioHie
I n addition t o these compounds a heavy, resinous material has been observed, particularly in older oils or in those which have been stored improperly. Amyl acetate and dimethyl sulfide, the latter particularly in nonrectified oils, have been detected in traces. Concerning the biogenesis of these compounds in peppermint, the reader is referred to a discussion by Hall (11), who also included a brief history of the theories proposed t o explain the formation of these substances by the plant. Inasmuch as citronellal, an open-chain unsaturated aldehyde obtained chiefly from oil of citronella, is now being used for the production of synthetic menthol, further proof is given t o the theory proposed by Kremers ( I S ) that citronellal serves as t h e precursor of menthol in the plant. CH3
I
C/H
n
CH3CHs I
CHs
I A
CH
CHsCHs I1
CHa
I A
CH
CH3 CHs I11
Menthol (I),menthone (11), and menthyl acetate (111) constitute the major portion of peppermint oil distilled from mature plants. T h a t the other components are necessary for oils of pleasing aroma and taste is apparent when organoleptic tests are applied t o natural as well as t o synthetic oil of peppermint. B y careful removal of the lower-boiling terpene fractions, either by fractional distillation under reduced pressure
