l 1 WI ' MI H' r i ll} ‘ till 131 180 TH . A PHOTO-METRIC. DETERMENATIC'IV CF CHLOROPHYLL AND CAROTENE EN EXTRACTS OF GREEN TISSUE Thesis fer the Degree cf M. S. MECHIGAN STATE COLLEGE Walter V/oiman 1940 ‘A'PHOTOHETRIC DETERHINATION OF CHLOROPWYLL AND CAROTEYE IN EXTRACTS OF GRE’N TISSWE by Walter Wolman A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial ful- filment of the requirements for the degree of MASTER OF SCIENCE Department of Botany lgho ‘; TABLE OF CON?ENWS Introduction - - - - Review of literature - - - _ Extraction methods - - _ _ Separation of the green and yellow pigments Separation of carotenoids — - - “Quantitative chloroohyll determinations Quantitative carotene determinations - The problem - - _ - Experimental procedure - _ - - Materials and methods - - _ The principles of photometry — Analytical procedure - - _ Results — _ _ - Chlorophyll experiments Carotene eXperiments- - - Discussion and conclusion — - _ Summary - - - _ _ Acknowledgements - _ - - Literature cited - m - _ Tables - - - _ Figures - _ _ _ y...» to at" Hz. ‘ I . .1 ‘1} ‘0 *4 r.) r—J (Di) \N IYTRODVCTI N All life processes are dependent upon the all important green pig- ment in the higher plants. It has been known for many years that this pigment is in some way related to food manufacture. The ready conversion of the energy of the sunlight to energy stored in the food materials of plants and the part chlorophyll plays in the plant has intrigued invest- igators for many years. Quantitative determinations of chloroohyll have been used in numer- ous studies, such as photochemical reactions, chlorophyll in relation to, metabolic processes and other biologically important compounds, genetical studies, effects of pathological conditions, the structure of the chloro- plasts, and a host of other studies. Carotene, too, has been of great interest to workers in the field of vitamins during the past ten years. This yellow pigment accompanying chlorOphyll in the green plastid is known to be the precursor of vitamin A. As such, determinations for carotene in plant materials are of con- siderable value. New methods, then, for the determination of chlorophyll and carot- ene in green tissue are still of great interest to plant physiologists and biochemists. Procedures based largelv on the methods of Willsteetter and Stoll (68) are long and tedious. Most methods lack either accuracy, reproducibilitv or are useless when both pigments are to be determined in the same sample. Although chlorophyll determinations are laterious and time consuming, those for carotene are even more so. Consequently, a new method, which would be rapid, reliable and simple to utilize was sought. -2 The application of the photoelectric colorimeter for chlorophyll and carotene determinations has been suggested although little experimental evidence is available. A number of excellent instruments are now obtain- able, and their use has become widespread. However, the lack of suit- able filters has limited their acceptance. REVIEW OF LITERATURE Until the investigations of Willstaetter, botanical and spectroscopic methods were employed in the study of the occurence, morphology, and de- tection of the pigments. Then Willstaetter and his co-workers worked out technioues for the separation and purification of these chlorOphylls and carotenoids. Meanwhile, studies relative to the physiological significance of all these pigments in plant metabolism have been constantly investigated. During the last decade interest has centered in a study of the physical- chemical constitution of chlorOphyll as it exists in the chloroplast. In addition, new analytical methods for the quantitative extraction, separ- ation, and estimation of the pigments have been devised. The extensive literature on pigments, scattered throughout numerous Journal articles, and the contradictory evidence found therein (at least to 1913) make it a difficult task to attempt to review plant pigment studies. The assembled data that preceeded Willstaetter's investigations were so contradictory and confusing that the conclusions drawn from them are now considered only of historical interest. Following this period the best review is given bV'Palmer (ME), although his book was published eighteen years ago. Willstaetter and Stoll‘s book (68) provides an ex- cellent background for pigment studies, but the review of Priestly's (M7) concerning the work of Lubimenko must not be omitted. L Zechmeister (70) has recently published a very good book on the carotenoids. Extraction methods Berzelius (2) was apparently the first to attempt an extraction of chlorophyll with acids or alkalies. There was little or no success with this method, but for many years his renown as a chemist appeared to influence other investigators. Conseouently, many workers obtained eoually poor results with the same type of extraction. Berzelius did succeed, however, in isolating the yellow pigments from autumn leaves with eighty five percent alcohol, and, thus, extracted water soluble pigments as well as carotenoids. Alcohol was the most popular solvent for the extraction of the pig- ments until the time of Willstaetter. Fremy (12), Sorby (56), Timiriazev (60), Filhol (10), Hansen (18), Immendorf (93), Nonteverde (37), Kohl (26), Monteverde and Lubimenko (38), and, as late as 1932, Deleano and Dick (9) used this solvent for the extraction of the green and yellow pigments. Hansen (18) used ninetysix percent alcohol in the cold after boiling the leaves in water and drying them. Arnaud (1), interested largely in extracting the yellow pigments, found that petroleum ether used on air or oven dried material extracted only a small part of the green pigment and xanthophyll. Tswett (62) adopted a modification of this method for extractions preliminary to his chromatographic separation. J. H. C. Smith (5h) preferred this treat— ment and Russell (M9) used it for the extraction of carotene from dry alfalfa. Willstaetter and Stoll (68) suggested aoueous acetone as an extraction medium for all plastid pigments, and it is now being used extensively, especially where the isolation of both the green and yellow pigments is desired. The extraction methods of Wurmser and Duclaux (69), Schertz (52), Maiwald (33), Sprague and Shive (57), Peterson (M5), Bills and McDonald (M), Oltman (M2), Miller (36), Russell (M9), and Godnew and Kalischewicz (15) are based on Willstaetter's procedure or some modifi- cation. The extraction of carotenoids from seeds was accomplished by Gill ' (1h) with carbon bisulfide. Coward (8) extracted carotenoids satis- factorily with aoueous alcoholic-potassium hydroxide, while Pyke (N8) used methyl alcohol-potassium hydroxide with ether for the same purpose. At the suggestion of J. H. C. Smith,'Smith and Smith (55) extracted the carotenoids with pyridine. The great objection to the use of this sub- stance is its known toxicity. Holmes and Leicester (22) extracted carotenoids with chloroform after soaking the tissue with alkali to get rid of chlorophyll. Kuhn and Brockmann (31) extracted green tissue for carotenoids with a pet- roleum ether-methyl alcohol mixture. In 193M, Guilbert (16) suggested saturated potassium hydroxide ~ methyl alcohol as an extraction mixture for carotene, thus causing decomposition of the chlorophyll. He then extracted the mixture with ethyl ether. Peterson, Hughes and Freeman (#6) in 1937 modified this procedure by substituting petroleum ether for e thyl e the r . Methyl alcohol was employed by Hicks and Panisset (21) and Fleischer (11) for chlorophyll determinations of algal material. ‘ plII'vlnllIf XII. inlnuqtrla lufiEh‘Elloh..!P‘ I... ! .uw . 1:63.:er ...4:l...|‘ 0.5” .(lr/pk. Eurtu. a». .L»..» . . b’ .. 3.4.9: .N o H v... t Separation of the green and yellow pigments The saponification of the chlorOphyll and a subsequent separation from the yellow pigments has been the standard procedure for many years. However, attempts to study the yellow pigments were largely confined to etiolated tissue or autumn leaves previous to the adoption of this method. Berzelius (3) extracted autumn leaves with alcohol but considered the yellow pigment as a fat derived from the green pigment. As early as 186M Stokes (59) separated the green and yellow pigments by removing the chlorOphyll and carotenoids with an aluminum hydroxide preparation. Fremy (13) found that not only aluminum hydroxide but magne- sium hydroxide and barium hydroxide solutions were effective in carrying down both pigments. He found that barium hydroxide solution was the most effective and that by the use of alcohol the yellow pigment was removed from the mixture. Timiriazev (60) and Monteverde and Lubimenko (38) em- ployed the same method in their determinations. Contrary to these reports, it was found in the present work that barium hydroxide does not carry down both pigments. It selectively reacts with the chlorOphyll leaving the carotenoids in solution. Apparently, these early workers added enough of the barium hydroxide solution to dilute the extract with water to such an extent as to precipitate both pigments. Sorby (56) separated the green and yellow pigments by the use of the two immiscible solvents, alcohol and carbon bisulfide. Treub (61) repeat— ed this work and obtained similar results. Arnaud (1) took up the caro- tenoids in carbon bisulfide after the petroleum ether, the extraction medium, was distilled off. Filhol (10) was able to remove the chloroDhyll from carotenoids bv treatment with animal charcoal. Recently, animal charcoal was tried as a selective adsorbent for chlorophyll, but the carotenoids were carried out of solution along with the green pigment. ‘ \ 'rl ,Illsl Ir. . ‘IVI In. ..IFI‘EEv. t.‘ 3.Imusi."tn’l.“r "L .‘.i£!.lfll~§ ....1. IV ..V , .. ... straw..- K1 ..,»......\,.... . ,,.. 6 The method sugzested by Kraus (29) depended on the use of immiscible solvents as eighty five percent alcohol and benzene. Konrad (27) found that the separation was more effective with seventy percent alcohol. Treub (61) suggested that carbon bisulfide be used in place of benzene when chlorophyll was present in a stronger alcoholic solution. Wiesner (66, 67) showed that many vegetable and ethereal oils as well as many aromatic compounds could be used for the separation from alcohol. Tswett (63) separated the pigments by means of a chromatographic technioue. Hansen (18) appears to be the first worker to separate the pigments by alkali saponification, then salting out the soap with NaCl. Kohl (26), Willstaetter and Stoll (68), Wurmser and Duclaux (69), Schertz (52), Maiwald (33), Peterson (M5), Coward (8), Holmes and Leicester (22), Kuhn and Brockmann (31), Guilbert (16), Miller (36), Deleano and Dick (9), and Pyke (M8), used some modification of the alkali saponification treatment. Even in a ouantitative photometric determination, Oltman (”2) used an alkali saponification method, although Godnew and Kalischewicz (l5) and Johnsbn and Weintraub (25) made no separation of the green and yellow pig- ments. They measured the chlorOphyll in the original extract by the selection of the proper filters. Separation of carotenoids. Borodin (5) established a basis for the classification of yellow pig- ments when he found one group more soluble in alcohol than the other. His investigations showed also that the group slightly soluble in alcohol was highly soluble in petroleum ether. Monteverde (37) and Monteverde and Lubimenko (38) used this method of separation in their studies. Since 7 that time practically all workers with few exceptions have used the pet- roleum ether — methyl alcohol or ethyl alcohol method as the basis for carotene — xenthOphyll separation. Among these were Willstaetter and Stoll (68), Henrici (20), Wurmser and Duclaux (69), Schertz (52), Coward (8), Peterson (ME), Deleano and Dick (9). Kuhn and Brockmann (31), Guilbert (16), Miller (36), Pyke (M8), and Peterson, Hughes and Freeman (36). Arnaud (1) found that petroleum ether extracted very little xentho- phyll from dry material. Tswett (63) developed a chrometograohic technioue for the separation'of the carotenoids. This method_has proven to be of great importance in the study of the chemical nature of these pigments. and is being used extensively today. Recently, Clausen and NcCoord (7) have obtained excellent results by substituting diacetone for eoueous alcohol for the carotenoid separations. Pegsted et a1 (19) have adopted this method. Quantitative chloroohyll determinations Actual quantitative determinations of chlorophyll do not seem to have been attempted before 1910. Stokes (59) made optical studies of chlorophyll but no quantitative measurements. Monteverde (37) attempted to measure the relative chlorophyll values by spectocraphic means. The Spectronhotometer has been used irenuently with considerable success. Nalarshi and Harchlemshi (3h) treated chloroohyll with hydro- chloric or oxalic acid and measured the extinction coefiicient of the phaeophytin. Jacobson (2h) used the same treatment and compared the sample against an arbitrary standard. Although Wistert (65) did not act- ually carry on anv work, he pointed out how chlorOphyll might be nuan- titatively estimated by the use of the spectrophotometer. Both Wurmser R -for chlorophyll determinations. ZSCheile (71) was able to measure both chlorophyll "a" and “b“ by this method. Zscheile's work on chlorophyll determinations with the spectrophotometer has been the most precise of any that has been attempted. The photoelectric calorimeter is of great— er practical value and it is more rapid. However. it is by no means as accurate an instrument as the spectrophotometer. Most quantitative methods for the determination of chloronhyll since Willstaetter's work have been colorimetric. Willstaetter and Stoll (68) decomposed the chlorOphyll and compared it against methyl or ethyl chloro- phyllide both treated in the same manner. He also compared the extracted material against a pure chlorophyll standard. Schertz (52) followed the same general procedure as did Hicks and Panisset (21). Henrici (20) com- pared her samples with a solution of crude chlorophyll extracted from net- tle and, consequently. obtained only nualitative results. A mixture of chlorOphyll a and chlorOphyll b was used as a colorimetric standard by Naiwald (33). In 1998 Guthrie (17) suggested an inorganic standard 0‘ definite preportions of copper sulfate (5 molecules of water), potassium dichromate, and ammonium hydroxide. This standard has been used rather extensively. In 1929 Sprague and Shive (57) introduced a dye mixture of a solution of malachite green and naphthol yellow which was modified later by Sprague and Troxler (58). However, their standard has been used very little. Lovibond slides have been employed for chlorOphyll determinations (MS) in numerous cases. Lubimenko (32) used a spectrocolorimeter with a standard of eth,l chloronhyllide and Fleischer (11) made the same tvpe of determination using a No. 2M3 Corning red filter of 3.12 mm. thickness. 9 Oltman (#2) appears to have been the first to use a photoelectric colorimeter for chlorophyll determinations. However the chlorOphyll de- terminations were made after saponification rather than on the original sample as did Godnew and Kalischewicz (l5) and Johnunn and Weintraub (25). Koziminski (28) used an ethyl chlorophyllide standard, and th amount of chlorphyll was calculated by the use of a conversion factor. Quantitative carotene determinations The first attempt to determine carotene was made by Wiesner (67), who compared the total alcoholic extract from an etiolated plant with the yellow alcoholic extract from green plant tissue obtained by the Kraus separation. Arnaud (1) dissolved carotene in carbon bisulfide and using blue glass filters, compared it colorimetrically with 0.001 percent solution of carotene in carbon bisulfide. Kohl (26) compared the unsaponifiable pigb ment colorimetrically with a standard in the same manner as did Arnaud. Monteverde and Lubimenko (38) followed the same method as did Willstaetter and Stoll (68) although the latter made their comparisons with a 0.2 per- cent aoueous solution of potassium dichromate, as well as with a pet- roleum ether solution of pure carotene. Sprague and Shive (57) suggested a dye standard of Naphthol yellow and Orange G. Kuhn and Brockmann (31) suggested azobenzene, while duilbert (16), Russell (M9), and Peterson, Hughes and Freeman (MS) used potassium dichromate. {rogis (30) employed the dichromate solution with a phOSphate buffer. Deleano and Dick (9) oxidized the carotene with a known amount of standard dichromate in excess, and then back-titrated with iodine to ob- tain a measure of the carotene concentration. Recently. Brooke. Tyler, 10 and Baker (6) used a Cenco photelometer for carotene determinations employing a Corning No. 55M and a Corning No. 038 filter. Munsey (Ml) has urged a wide adoption of the photoelectric colori— meter in carotene investigations because of its accuracy and other advantages. THE PROBLEJ Colorimetric analyses are the most common methods of determining chlorophyll and carotene. The old standard methods are unsatisfactory in many phases of the determinations especially when the determinations of both pigments are desired from the same sample. The need of a rapid. accurate, and simplified method of chlorophyll and carotene determine ions becomes evident when many pigment determinations are attempted in the lab- oratory. Consequently, an effort was made to devise a method satisfying these requirements. EXPERIMEVTAL PROCEDURE Materials and methods The principles of photometry Colorinetry implies different meanings to workers in certain other fields (MO). In this paper it refers to the measurement of the concentra- tion of a colored substance by comparing the depth of the color against a standard. The Beer - Lambert law states that the amount of light absorbed by different concentrations of a colored solution is an eXponential function of the concentration when the thickness of the solution is constant: ltlc I - onlO- Where I - intensity of light after passing through the solution ll Io - incident intensity k - molecular extinction coefficient (depending on solute and solvent) 1 thickness of solution c = concentration 10510 Io/I . E (extinction) Then in matching two solutions E a 1 E2 k 1 keg 1 cl 1 ' 2 or cl/c2 . 12/11 Thus, the unknown concentration can be calculated by matching the depth of color against a standard. The above law holds very well where an in- strument like the spectrophotometer is used. Nearly monochromatic light can be obtained with this instrument. The application of this lav to ordinary colorimetric work can not give accurate results since white light is used. The different wave lengths employed introduce complex factors. Photometry is an advance over colorimetry and an approach to the accuracy of spectrophotometry. Filters, as used in photometry, sim— plify the conditions, since many wave lengths are eliminated, and the light is limited to a narrow region of the spectrum. A photoelectric instrument does not depend upon the presence of a standard for each determination as does a colorimeter. A calibration curve is determined and can be used thereafter without any standards. although it is wise to recalibrate occasionally. The most important advantage of the photoelectric colorimeter lies in the objectivity of the instrument. Different observers obtain the same values that are not subject to the vagaries of the human eye in reading H [0 colors. The Slieard-Sanford photelometer is one of the simplest comner— icellv standardized instruments available. The instrument (fig. 1) consists of a light source focused through a variable diaphragm onto a plenoconvex le ens. The lijht then passes throush one of the cells on the cell carrier containin: the nure solvent or solution to be tested. The light emerging from the en ”1y ir n3 cells then passes through the were length filter and from there t the photronic cell. The current generated in the ohotronio cell circuit is me? sured Mir a sensitive galvanometer. )hragm is similar to a comnur ceuere shit ter. The ligh t The varieble die rays are rendered essentiallv parallel by the nlenoconvex lens. Port of ’4. the light reech mg the cell is reflected and part is absorbed. Both cells must be of the seme thickness to eliminate any errors due to differences \ in absorption. tra anew Mi sion, or reflection. The cells were one cm. thick and tron er“ Ht ed ninetv two percent of the l The amount of current H- {7H h! L) C‘- O ght intensitv. The source of .J H. set up by the photronic cell measures the the light is a smell auto hesfilight bulb atteched to the 110 volt AC line throu h a transformer. Although a transformer is used the current is ant to fluctuate due to the use of other electricel eouipnent. A storeze battery may be used to a greeter edventege es a source of steadv current if facilities are available for recharging the hetterv, The greatest linitetion to the use of the photoelectric colorimeter is the lack of suitable filters. An ideal filter is one that would have a maximum transmission in the exact region 0? reyimum absorption 0? the solution th et is bein ng ex arined. Since the ideal one is unavailable, . one whose maximum transmission is neer the naximum absorption should be selected. In nocition, the effective region of trensnission shoulé le as narrow as possible. The spectroohotometer is bv fer tie better instrument 13 to employ. since filters are not necessary, and the region of the spectrum used can be limited by slit adjustments. However. this instrument is not as readily available to the general worker as the photoelectric colori- meter. Two filter systems were used in these studies. The first, that trans- mitted in the red, was used for chlorOphyll determinations., Since the carotenoids do not absorb at all in this region. the chlorophyll could be determined directlv in the original extract without any separation of the pigments. The combination finally chosen was a Corning No. 2M3 red filter (11. MM) and a Corning No. 396 H. R. blue filter. The latter was needed for the purpose of heat resistance since the infra-red has an effect on the photronic cell. The transmission range of this combination lay be- tween 6100 1 and 7500 I in the infra-red. Since carotenoids and chlorophyll have an absorption band in the blue region of the spectrum. it was necessary to remove chlorOphvll in order to determine the carotene content. After the chlorophyll had been removed it was necessary only to choose a filter with maximum transmission in the blue region. A Corning No. 55“ H. R. was finally selected after trying certain other ones. Excellent reviews on photometry have appeared by R. H. Muller (MO) and n. G. Mellon (35)- Analytical procedure One very important step which has not been emphasized sufficiently in most quantitative pigment determinations is that of a complete grind- ing procedure preliminary to extraction. It is imperative that the tissue be thoroughly disintegrated. This applies regardless of the method 114 A. of extraction or the solvent used. The temptation is to give a prelim- inary grinding and then let the solvent do the rest of the work. In general, the procedure used for extraction is somewhat like that of Ulvin (6M). Two grams of fresh tissue are placed in a mortar (glass is preferred since it is easier to clean out) with a small amount of sodium carbonate to neutralize any possible acidity. In this way chlorophyll decomposition is prevented. The tissue is then ground as finely and rapid- ly as possible. If the tissue is too dry it is moistened with pure acetone. Horeover. the acetone prevents oxidation of the carotene to a certain extent. It is then ground more with quartz sand and moistened frequently with pure acetone. After it is finely macerated. about twenty five cc. of pure acetone are added and the grinding continued for a few more minutes. The extract is decanted through a Buchner funnel. and the residue is again macerated and extracted with eighty five percent acetone (twenty five or thirty cc.). The residue and extract are placed in the Buchner funnel and the residue soaked two or three times before drawing off the liquid. The number of grindings depends upon the tissue used. The experienced worker soon knows when extraction is complete. The presence of water soluble pigments in the acetone extract -- particularly when using eighty five percent acetone often gives the impression of incomplete ex- traction. If. when using pure acetone, the liquid comes throuqh clear in— to a test tube, one can be certain of complete extraction. The filtrate collected in a test tube should be shaken with a little petroleum ether when eighty five percent acetone is used, and if no yellow color passes to the petroleum ether layer, carotene is not present. The extract is made up to one hundred cc. or any desired volume with eighty five percent acetone. 15 The chlorophyll content is then readv to be measured. A part of the extract is placed in the absorption cell of the photoelectric colorimeter. and its transmission is compared with that of distilled water. This value is then interpreted as concentration through the use of the standard curve. Fifty cc. of the acetone extract are taken for the carotene determinations. This is placed in an oil flask or one hundred ml. Erlenmeyer flask with the prepared barium hydroxide. The latte is prepared by hydrating one gram of the anhydrous barium hydroxide (J. T. Baker. Barium Hydrate, anhydrous) or barium oxide in an organic solvent miscible wit- water. The solid barium compound is put into five cc. of acetone to which five cc. of water is add- ed while stirring. It may also be prepared in one of two other ways. If there were enough water in the extract (and there should be), no previous activation would be necessary. Again. an activated barium hydroxide re- sults when a soluble barium salt reacts with an alkali in the presence of he organic solvent miscible with water. The acetone extract and barium hydroxide mixture are refluxed for thirty minutes on a hot plate or water bath. The flask and contents are then cooled and filtered. The green sludge remaining on the filter is washed with pure acetone to remove all the carotene. The yellow solution is placed in a separatory funnel to which fifty cc. of petroleum ether (3. P. 30°— 5000.) is added. The solution must never be shaken but the ex- traction can be completed by swirling the funnel with a rotary motion. The acetone is washed out of the petroleum ether layer with water and is re- extracted with two fifteen cc. portions of petroleum ether. The extracts are combined with the rest of the petroleum ether. The petroleum ether is then washed with either eighty five and ninety percent methyl or eighty and eighty five percent ethyl alcohol in order to was out xanthouhylls. A. The alcohol e"tract is re-extracted with petroleum ether. This extract is 16 added to the petroleum ether layer which is washed again with a little alcohol, then once with water and then filtered through anhydrous sodium sulfate into a volumetric flask and made to volume. The concentration of carotene in the petroleum ether solution (largely beta-carotene in green tissue) is then determined in the same manner as in the chlorophyll deter- mination described above. The Corning No. 55h H. R. filter was used. Again the value is interpreted from the standard curve. Correction for the presence of chloroDhyll may be made, but this is rarely necessary. 06- casionally greenish material may be present, but it will be filtered out in the sodium sulfate filtration. Results Chlorophyll experiments The most important single factor in this study was the proper choice of suitable filters. Certain filters and filter combinations were tested, amely, the Cenco N0. 3, and combinations of the Corning n.R. 2h} plus 0.25 N copper sulfate solution, Corning No. 2h} and a Cenco blue glass H. n. filter, and finally the Corning No. 2%} and 396 blue filter. The calibration curve for each filter is represented in figure 2. It is obvious from these data that the Cenco filter is not satisfactory for this particular study. It is desirable that a large change in transmission be induced by a small change in concentration. The Cenco filter shows this characteristic only in an extremely short range. The best curve was (1 .’. obtained with the Corning filter No. PM} plus a solution of O. 5 N Copper . o f sulfate. An excellent curve was also obtained with a 0.107 N cooper sul- fate solution. The advantage of the la ter was that a greater light in— tensity could pass through. However, the inconvenience caused by the prep- 17 aration of these solutions led to their discontinuance. The other two curves, obtained throurh combining the Corning No. 2%} filter with the Cenco blue glass filter; and combining the Corning No. 2M3 with the Cor- ning No. 396 filter. were much alike and readily reproducible. Each could be used effectively and for most of the work the No. 396 was selected be- cause it absorbed more of the infra-red. Both the curves exhibited gentle slapes throughout most of the entire range of concentrations emploved. The glass filters were much more convenient although slightly less satis- factory than copper sulfate. Th calibration curves for these filters were determined in the following manner. Pure chlorOphyll (5x grade obtained from the American Chlorophyll, Inc.) was dissolved in eighty five percent acetone. Sixty two and one-half mg. samples were diluted to a volume yielding a stock solution of one hundred twentv five mg./1. Different concentrations of chlorOphyll were prepared from this stock solution by diluting certain aliquots to definite volumes. The photelometer was adjusted to read 100 with the filters in place and distilled water in the standard cell. The cell with the standard (or unknown) was aligned with the light source of the instrument by shifting the carriage. The ammeter reading was then taken. Each photometric value was plotted against its concentration to obtain the resulting curve. This procedure was followed for every deter- mination whether for chlorOphyll or carotene, known or unknown. The chlorOphyll concentration of any unknown could be determined by inter- preting the photometric reading from these standard curves. Table I shows the data obtained in a study of the accuracy of chloro— phyll determinations upon weighed samoles of chlorophyll with the use of the four filters. It can be seen readily that the percentage error with 18 ‘the Cenco filter exhibits a greater range than with the copper sulfate. The poor curve obtained with the Cenco filter accounts for this large er- ror. The results, in general, seem to be more reliable in that range where h5-75 percent of the light is transmitted and the practice has been to dilute within that range. The absolute error tends to be large though the'percentage error is small where transmission is below #5 per- cent. It became necessary to know exactly what effect the presence of carot- ene would have upon chlorophyll determinations. although theoretically it should have none. Known quantities of chlorOphyll were weighed out and solutions of different concentrations were made. Carotene was added to each of these solutions to make a concentration of twenty seven ppm. The results are shown in table II. The perCent variation is very close to the exnerimental error indicating that there is little, if any, effect. These data agree with the conclusions of JohrSRVIand Weintraub (25) as well as Godnew and Kalischewicz (15). Photometric and colorimetric chlorOphyll determinations were com- pared on certain green materials. A Duboso calorimeter was used for all colorimetric determinations. Guthrie's standard was first used for de- termining the chlorOphyll concentration. then a pure chlorophyll stand- ard. and finally, a photoelectric colorimeter. The data in table III indicate that the values obtained colorimetrically with a pure chlorophyll standard compare favorably with those when the photelometer was used. How- ever large variations at the higher concentrations were secured with Guthrie's standard. 1.9 Carotene experiments The proper selection of filters or filter combinations is also the important factor in the determination of carotene concentrations. Three filters were tried, namely, a Cenco No. l, and a Corning No. 55h H. R. filter with and without a Novial A No. 038 filter. The readings for carotene content could not, however, be made direct- ly upon the crude extract since chlorophyll has an absorption band in the blue region of the spectrum from M000 3 to 5100 K, which overlaps that of the absorption by carotene (#180 1 — H900 5. Values in ether solution). This necessitated a separation of the green pigment from the yellow. The data in table IV show that the transmission values at all con- centretions (approximate) are similar. The Corning No. 55h plus Corning No. 038, a combination used by Brooke, Tyler, and Baker (6) served only to cut down the transmission without increasing the sensitivity. The Corning number 55% served the purpose as well as any filter. Eleven and four tenths milligrams of beta-carotene obtained from the S. H. A. Co. were diluted to 500 ml. in petroleum ether at 26°C. Dilutions were made from this stock solution to give the various con- centrations reouired. Photometric readings were made in the same manner as in the chlorophyll determinations. The resulting values were plotted against concentration and the curve (fig. 3) was thereafter used for inter— preting unknown values. A method of correction for the possible presence of chlorophyll in any carotene sample was worked out. Rarely was chlorophyll found present in the carotene solution with the method of separation used. The inset in figure 3 depicts the curve obtained for chlorOphyll with the filter No. 55% used for carotene determination. With the aid of both curves in 20 figures 2 and 3 the correction for the chlorophyll present is made in the following manner: the amount of chlorOphyll in the petroleum ether solution of carotene is determined by using filters No. 2H} and No. 396. The reading for total percent transmission in the blue region with filter number 55% is then obtained. This is the result for both carotene and chlorophyll. The curve inset in figure 3 indicates the amount of absorp- tion due to chlorophyll if no carotene were present. The actual carotene content is determined by the use of the following formula: Y : TXIOO C Y = actual percent transnission of carotene. T = total percent transmission of carotene and chlorophyll in the blue spectrum. C = percent transmission of chlorophyll concentration in blue Spectrum if no carotene were present (from figures 2 and 3). As reported in the review of the literature. practically -ll methods used at present depend on the separation of the two pigments by an alka- line saponification of the chlorOphyll. This saponification method re- trieves carotene in a petroleum ether solution. The means of separation used in this study is based on a new principle which was devised largely by Dr. H. G. Petering of the Michigan State College Chemistry Experiment Station. This involves the separation of the chlorophyll from carotene by precipitating the green pigment with barium hydroxide octahydrate. The carotene could then be filtered off. Magnesium hydroxide and calcium hydroxide were not as efficient as the barium hydroxide. Ordinary barium hydroxide was activated by first placin: the material in acetone or al- cohol. A subsequent addition of water resulted in a fine, porous mess. (1)This reaction was later shown to be due to the formation of the barium hydroxide octahydrate. El The material was then added to the chlorophyll solution. Several exgera iments were conducted to establish the effect of different volumes of water and acetone upon the activity of the barium hydroxide. It was found that there was little difference in these amounts as long as enough water was present to complete the reaction. It also appears that good activity is obtained with either anhydrous barium hydroxide or the octehydrate.1 The use of eighty five percent acetone seems to be more effective than pure acetone.2 Data were gathered, as shown in table V. to determine just how much barium hydroxide is necessary in order to remove the chlorophyll. It is obvious that there is a close relationship between the amount of bar- ium hydroxide, the time of refluxing, and amount of chlorophyll removed. The general practice has been to use one gm. of barium hydroxide (an- hydrous) five cc. of acetone, and five cc. of water for each fifty cc. of a one hundred ppm. solution of chlorOphyll for a thirty minute reflux period. The amount of Ba(0H)2 used in getting this data is in excess of the amount needed for these pigment concentrations as later shown by Petering and Morgal. Since a quantitative recovery of carotene is imperative the effect of the barium hydroxide upon the carotene was studied. EXperiments were run in which duplicate samples -- one treated, the other not -— were com- pared. A known amount of carotene was added to an unknown sample, and then none added to a duplicate sample. The difference between the samples with and without the added carotene then represented the amount recovered. The data in table VI show the results of these tests. The results indicate that a complete ouantitative recovery of carotene was (2) Petering and horgal, (private communication) have shown that eighty five percent acetone is the best concentration since more water will throw out the pigments. D.) . F0 accomplished. A number of experiments were run in order to determine the accuracy of this method for carotene determinations as compared with the present accepted method (A. O. A. C.). A number of experiments were run in con- junction with Dr. E. J. Benne of the Michigan State College Chemistry EXperiment Station. The comparison was made with the Peterson. Hughes and Freeman method (“6). The data presented in table VII shows remark- ably close agreement between the two methods. Both chlorophyll and carotene were determined on the same extract by this new procedure. The older method does not allow for a chlorophyll determination. hany plant tissues were employed which would present a wide variation in both chloro- phyll and carotene content. The greatest percentage variation occured with yellow wax bean pods, which had an extremely small carotene content. A very small loss of pigment in this case would have produced a large error. Samples of blue grass taken in the fall gave results with the barium treatment twenty to twenty five percent higher than those run by the Peterson, Hughes and Freeman method. This was the only instant of poor agreement between the two methods. Either the acetone extracted more caro- tene than the alcoholic KOH, or the acetone takes up an inpurity other than carotene which is not extracted from the tissue by the Peterson, Hughes and Freeman method. Moon (39) reported that grasses yield red or brown resinous substances which are precipitated during sancnification with alcoholic alkali. This makes the extraction of carotene from the residue by petroleum ether very difficult. This may have been the source of the trouble. Both chromatographic and spectrOphotometric studies revealed no im- purities present in either solution. DISCUSSION AND COHC VSION Although many aspects of the procedure as given in this paper re- semble other methods, it differs in several important respects. There is a growing belief, well substantiated, that the photoelectric colorimeter is a better instrument than the calorimeter. The important error in colori- metry induced by visual impressions of different individuals is absent in photoelectric colorimetry. The values obtained are the result of objective readings, and tints or shadings need not be interpreted by the eye. The short period employed in testing and the almost immediate results obtained, reduce the chances of pigment decomposition. The time saved is a factor in which all investigators are interested. The use of a standard curve which needs to be checked only occasionally is preferable to the freouent preparation of new standards. The standard eouipment employed is an ad— vantage and the spectral filters are more sensitive than those in many other methods. On the other hand, errors are found in the photometric method. The sensitivity of the photoelectric cell to certain wave lengths varies, and the fluctuation of the current from an AC line will cause a deviation on the ammeter which is a source of error. This can be easily eliminated by the use of a storage battery provided a charger is available. The greatest disadvantage may appear to be the cost of the instrument, but this is slight when compared to the spectrOphctometric method. The use of barium hydroxide octahydrate also presents many advantages over the older methods. There is no doubt that its action is more rapid 5 than the alkaline saponification for chlorophyll breakdown. T18 use of a solid material without any special preparation, the small amount of handling, and the ease of separation by filtering, give it an advantage over the alkaline saponification method. Moreover, a complete removal of chlorophyll is obtained. It was found that the filtrate after filter- ing off the green sludge had no barium present. This simplifies the pro- cedure tremendously since numerous washings are not necessary in ridding the solution of alkali as is the case where alkaline saponification is employed. A separation of the pigments is not necessary for the determination of chlorophyll, and although a separation is required for the carotene determination, the same sample can be used for both estimations. An additional advantage lies in the reduction of the number of opera- tions and transfers in this manner reducing the sources of error. Fewer reagents are needed to carry through a determination. The entire procedure takes about one-half the time ordinarily spent with other procedures, even those in which photoelectric colorimeters have been used. wflEmRY l. The literature and methods involved in extraction of pigments, separation of the green and yellow pigments, separation of the carotenoids, and quantitative determinations of chlorophyll and carotene are reviewed. 2. A discussion of the principle of photometry and a description of the photoelectric colorimeter used is given. 3. Chlorophyll was determined photometrically on the crude extract in the red region of the spectrum by means of suitable filters. The preparation of a calibration curve for chlorophyll is described. h. Data for the accuracy and reliability of the method as compar- ed to colorimetric methods are shown. 5. A study of a few potentially available filter systems was con- ducted. 6. A report of the preparation of the extract for the carotene determination, the type of filter used, a method of correction for any chlorophyll present in the carotene sample, and the calibration curve is discussed. 7. A studv of the method for treating the barium hydroxide for chlorophyll decomposition and its effect on carotene is presented. 8. COWparisons of values obtained by this method with those from the generally accepted method are included. 9. Exact details are presented for an analvtical procedure for chlorOphyll and carotene in green tissue. 10. A discussion of advantages, disadvantages and errors of the method is presented. AC KNOWLED GE} TENT The writer is grateful to Dr. R. P. Hibbard and Dr. H. G. Petering for their aid and advice in this study and in the preparation of the manu- script. He also wishes to thank Dr. B. H. Grisby and Mr. C. W. Robertson for reading the manuscript. W) O\ 7. 11. 12. 13. 1h. 15. s) —4 LITERATURE CITED Arnaud, A. Dosage de la cerotine contenue dans les feuilles des_ vegetaux. Compt. rend., 10h: 1293 - 1296. 1887. Berzelius. J. J. Ann. d. chem., 21; 296. 1838. Cited by Willstaetter and Stoll. Investigetions on chlorophyll. page 1. Translated by Schertz and Merz. Berzelius. J. J. Ann. d. chem., 21: 257 - 26 2. 1837. Cited by Palmer. Caroiinoids and relateafbigments s pe.ge 56. 1922. Bills. C. 3., and F. G. McDonald. The cerotene content of ten varieties of carrots. Science. n. s., IE: 108. 1932 Borodin, J. Ueber krystel‘inische Nebenpignente des Chlorophvlls. Boten. Ztg., El; 577 - 579.1883. Brooke. R. 0.. S. W. Tyler, and W.S. Baker. Determination of beta—carotene in alfalfa meals. Ind. Eng. Chem. (Anal. Ed.), 11: 10h - 106. 1939 Clausen, S. W., and A. B. McCoord. The determination of carotene and xanthOphyll by a single distribution between liquid phases. J. Biol. Chem., 113: 89 - 10u. 1936. Covard K. H. Some observations on the extraction and estimation of lipochromes from animal e.nd plent tissues. Biochem. Jour., 18: 111k - 1122.192u. Deleeno, N. T., and J. Dis . Beitrage zur Kenntnis des Carotins. Biochem. 2., 2 m9 110 - 133. 1933. Filhol. E. Recherches sur la chlorOfihylle. Compt. rend., 88: 1218 — 1220. 1868. Fleischer. W. E. The relation between chloronhyll content and rate of photosynthesis. J. Gen. Physiol.. 18: 573 - 597. 193%. Fremv, E. Recherches sur la matiere colorants verte des feuilles. Compt. rend., 5o. h05 - M12. 1860 Fremy. E. Recherches chimiaue sur la metiEre verte des feuilles. Comet. rend.. 61: 188 - 192. 1865 Gill. A. H. The occurence of carotin in oils and vegetables. Ind. Eng. Chem., 19; 612 - 61h. 1918. Godnew, T.N., and S.W. Kelischewicz. Die Quantitative Bestimmung des Chlorophylls vermittels des Lichtolektrischen Kolorimeters von Lenge. Planta.. E2: 19hr - 195. 1935- 16. 17. 18. 26. 97. 28. 29. F8 Guilbert, H. R. Determination of carotene as a means of estima— ting the vitamin A value of forage. Ind. Eng. Chem.. (anal. ed.), .6: M52 - 151. 193M. Guthrie, J. D. A stable colorimetric standard for chlorophyll de- terminations. Am. J. Bot.. _1_§_: 86 - 87. 1928. Hansen, A. Der Chlorophyllfarbstoff. Botan. Ztg., h2: I15 — 320. 1883+. ‘— Hegsted. D. M.. J.W. Porter. and W. H. Peterson. Determination of carotene in silage (an improved method). Ind. Eng. Chem.. 11: 286 ~ 258. 1938. Henrici, M. Chloronhyllqehelt und Kohlensaure - Assimilation bei Alpen - und Ehenen - Pflenzen. Verhandl. Naturf. Ges. Rese1.. 30: M3 - 136. 1919. Cited by r. 11. Schertz. 21m. Physiol” 1: 32h? 192 . Hicks. P. A.. ene T. E. Panisset. The eventitetive determinetion of minute amounts of chlorophyll. New Phytol.. 3?: 199 — 210. 193R. Holmes. H. N., 2rd H. N. Leicester. The isoletion of carotene. J. Am. Chem. Soc., :3: 716 - 7?”. 1932- Immendorf, H. Des Carotin in Pflenzenkorner und einiqes uber den grunen Farbstoff des Chlorouhyllkorns. Landwirt. Jehrb.. 18: 507 — 520. 1889. Cited by Palmer. Carotinoids end related pigments. page 59. 1922. Jacobsen, C. A. A delicate method for determining minute euentities of chlorOphyll. J. Am. Chem. SOC., 33: 1266 - 126_. 1912. Johnston. E. S. and R. L. Weintraub. The determinetion of small amounts of chlorophyll -— apparatus and method. Smithsonian Hisc. Kohl, F. G.‘Untersuchungen,hber das Cerotin und seine physiologische Bedeutung in den Pflanzen. 1902. Leipzig. Konrad, H. Vorlaufige Notiz fiber die Trennung der Chlorophyllfarhstoffe. Flora, 25: 396 - 397. Cited b7 Palmer. Cerotinoids and related pigments. page 32. 1922. Koziminski, Z. Amount and distribution of the chloronhvll in some lakes of Northeastern Wisconsin. Trans. Wisc. Acad. Sci.. 31: hll ~ M38. 1938. Kraus, G. Zur Kenntnis der Chloronhvllfarbstoffe und ihrer Verwandten. Soectralanelvtische Untersuchunzen. neges 1 - 111. Stuttgart. lQfl2. Cited by Palmer. Cerotinoids and related niements. neees 11 — 32. 1922. Cited bv Pelledin's Plent Physiology. edited by Livingston. page 7. 37. 38. 1111, 1+5. Krogis. A. Eine Modifikation des K Bichromatmethode zur kolori- metrischen Answertung des Carotinoide. Biochem. A., 287: 226 - 23M. 1937. Kuhn. R. and H. Brockmann. Bestimmung von Carotinoiden. Zeitschr. Physiol. onen.. 206: M1 - 6M. 1932. Lubimenko. V. Sur la nuantité de la chlorOphylle chez les algues marines. Compt. rend., 179: 1073 - 1076. 192M. Maiwald, K. Wirkung hoher Nahrstof23aben auf den assimilations- asparat. AngeW. Bot.,‘§: 13 - 7M. 19?}. Malarski, H. and L. Marchlewski. Studien in der Chlorophyllgrupne. VI. Bestimmung des Chloronhylls in Pflanzenteilen. Biochem. Zeitschr.. 33: 319 - 392. 1910 Mellon, M. G. Present status of colorimetry. Ind. Eng. Chem.. 11: 8O - 85. 1939. Miller. E. S. A rapid and accurate quantitative method for the determination of the_common carot-.oids; analyses of B - carotene and leaf - xanthoohylls in thirte n plant tissues. J. Am. Chem. Soc.. 21: 337 - 3M9. 1915. Monteverde. N. A. Des Absorptionsspectrum des Chloronthls. Acta Horti Petroplitani 11: 123 - 178. 1891. Cited by Schertz. Plant Physiol.. 2: 393. 1928. Monteverde, N. A., and N. V. Lubimenko. Recherches sur la formation de la chloronhylle chez les plantes. Bull. Acad.-Sci. Petrozrad, Ser. 6. 7: 1907 - 1928. Cited by Palmer. Carotinoids and re- lated piErents. Page 250. 1922. Moon, F. E. The ouantitative extraction of carotene from grass. Muller. R. H. Photelectric methods in analytical chemistry. Ind. Eng. Chem.. 11: l - 17. 1939. Vunsev. V. E. Report on carotene. A. O. A. C.. 21; 626 — 631. 1938. Oltmen, R. E. A new method and instrument for the nuantitative de- termination of chlorouhyll. Plant Physiol.,‘§: 321 - 326. 1933. Palmer, L. S. Carotinoids and related pigments. Amer. Chem. Soc. Ponograoh series. New York. 192?. Peterine, H. G.. B. H. Dug er and F. Daniels. Quantum Efiicienev of Photosynthesis in Chlorelle. II. J. Am. Chem. Soc., 61: 3575 - 3529. 1989. Peterson. P. D. Methods for the quantitative extraction and separa- ration of the olnstid elements of tobacco. Plant Physiol., 5; 957 - 261. 193 . \J'l (E) Peterson, W. J., J. S. Hughes, and H. F. Freeman. Determination of carotene in forare. A modification of the Giilbert method. Ind. Eng. Chem., (Anal. Ed.). 9: 71 - 7?. 1937 Priestlv, J. H., The bioloav of the livinz chloronlast. Fem phy- tologist, 28: 197 - 217. 1929. Pyke, H. A rapid method for the determination of carotene, xanthonhvll. and chloronhyll in artificially dried grass meals. J. 80a. Chem. Ind.. 55: 179 - 1rd. 1936, Russell. W. 0.. H. W. Taylor, and D. F. Chichester. Colorimetric' determination of carotene in plant tissue. Plant Physiol., 1“: 325 - 3uo. 1935. Sanford, A. H., C. Sheard, and A. E. Osterherg. The nhotelometer and its use in the clinical laboratory. Amer. J. of Clin. Path., 1: nos — M20. 1973. Schunck, C. A. The yellow colouring matters accompanying chloro- phyll and their spectroscooic relations. Proc. Rov. Soc. London. 65: 177 - 186. 1899. 68: M71 - Mao. 19o1. 72: 165 - 176. 19oz. laited by Palmer. Carotinoids and related pigments. page 39 - Ml. 1922. Schertz. F. M. The extraction and separation of chloroohyll (a & b) carotin and xanthophyll in Fresh green leaves, prelimin- ary to their nuantitative determination. Plant Physiol.. 3: 211 - 216. 1928. 7 The quantitative determination of carotene by means of the spectronhotometer and the colorimeter. Jour. Agr. 5/ :0: 333 - HQ“. 1993. ------------- The quantitutive determination of chlorophyll. Plant Physiol., 3: 393 - TX”. 1998. Smith. J. H. C. The hvdroaenation of carotenes obtained from dif- ferent sources, o? dihvdrocarotene, and of lvcooin. J. Biol. Chem., 96: 35 - 51. 1972. Smith, L. L. W., and O. Smith. Light and the carotinoid content of certain fruits and vegetables. Plant Physiol.. g: 265 - 275. 1911. Sorbv, H. C. On comnarative vegetahle chromatolozv. Proc. Roy. Soc. London. 21: MM? - 383. 1877. Cited hv Palmer. Carotinoids and related pigments. page 33. 1922, Sprague. H. 3., and J. W. Shive. A stu‘v of the relations between G chloroplast nigments and drv weights of tops in dent corn. Plant / n Physio1.. M: 165 - 192. 19r9. Sprague, H. 3.. and L. B. Troxler. An inuroved color standard f or the colorimetric cetermiration of chlorophyll. Science. n. s., 7 :_ f .0 7"". n 606 " qu. A93.) UN 0 O\ ‘44 69. 70. 71. 11 Stokes, G. G. On the supuosed identitv of biliverdin with chloro-I p1"1l, with remarks on the constitution of chlorophvl . Proc. Roy. Soc. London, 13 : 19M. 186%. Cited by Palmer. Carotinoids and relat ted pi.;ments. page 39. 1922. Tiniriazev, K. A. So 1871. Citedb 3 Pa / Page 0. crt a1 an? .1 'sis of ch1crophy11. St. Petersburg. ad in's Plant m"rsiolomr editedb y Livinjston. :1 P; Treub, H. Zur Chloronhyllfraje. Flora, h: by Palmer. Carotinoids and related filament. .) 55 - 56. 187M. -Cited 3. pa3e 32.. 1922. Tsvett, H. Plysixa1isc h—chemische Stucien uber das Chlo rophyll. Die Adsorptionen. Ber Deut. Botan. Ges., c_h: 316 - 323. 1906. ana 1vse und Chromatoaranhische Methods. U C mi: des Chloro:1vlls. Ber. Dent. Botan. fies., 29: 33” ~ 393. 1926. Ulvin, G. B. Chloroyhyll production under various environmental conditions. Plant Physiol.. 9: 59 - 81. l93u. Wiegert, Fritz. Uber Absorptionssye‘: tren und uber eine einfa che Methods zu ihrer quantitativen Bestinmung. Ber Deut. hen. Ges.. M9: iugo - 1532. 1916. Wiesner, J. Untersuchung uber die Beziehun; des Lichtes sum Chloro- o 'I ' 0 O f " ' phyll, Sitzoer. Axed. W1ss. W1en., 09: 327 ~ 385. 187%. Cited by Palmer. Carotinoids nd related pigments. page 32. 1922 heile deg -----..--- Bemermlnojen uber C 19 Anreblicl :en Be .611 ed va Palmer. a c sta Chlorouhylls. Flora. 18: 278 - 285. 187L. Cit Carotinoids and related pigments. page 32. 197 Willstaetter, R., and A. Stoll. Investigations on Chloro1hV11. Science Press Printing Co. Lancaster, Pa. Trans. _bv Schertz an d Eerz. \ Wurmser, 2., and J. Duclaux. Sur la photosynthese chez les Algues Floridées. Compt. rend., 171: 1231 - 1233. 1920. Zechmeister, L. Carotinoide. Monographien aus dem Gesnmtgebiet der Phsrsiologie der_Pflanzen und der Tiere. Berlin. 193“. Zscheile. F. P. An improved method for the purification of Chloro- phylls a and b. Quantitative measurement of their absorption spectra. Evidence for the er .istence of a third component of chlorophyll. Bot. Sez..|9§: 529 - 562. 193M. IEDBX CF "ABLES Table were I. The accnrecv o” chloroobyll Esterninetions J13 II. The etfect of cerotene on chloroohyll deter- minations ’ 33 III. Connerisens of the colorimetric end photo- metric methods for ne‘erninntion of ch1oroohvi1. 35 IV. The effects of certain filters on photocetric reedinrs of stendnrd erotene solntions 35 V. The emonnt o? berinm hydroxide reonired nnfier certain conditions to revere chloronby11 for the carotene deterrinrtions. 37 VI. Carotene recoveries from acetone solutions folloWing the barium hydroxide treatments 33 '7 _‘ o Vil. Congerison of methods for carotene deter- TAPLE I THE ACCVRACY OF CWLCQOPVYLL DETERVIYATIO"S Pure chloroohyll in acetone solution (mg/1.) ‘- Semple No. Chlorophyll concentration Chloronhyll concentration Percent by weight photelometer with Corning error ‘ 243 2 Aklo 396 1 19.9 23.5 3.0 2 3907 38.0 -]+.2 3 79.9 82.0 1.3 n 99.3 102.5 3.2 5 119.1 129.0 9.1 fi_ 39.? 39.5 ~0.5 _1_ A—Gorning 233 + H/uCu. Sflufilter. B-Corning 2M} + Cenco H. R. blue filter C-Cenco No. 3 filter , . 7A 2fijh ‘2h.8 . 0.6—~ 7s 2h.h 2h.0 -0.6 70 29.9 23.0 -5.7 EA 31.3 33.1 5.7 83 31.3 31.9 0.9 80 31.3 30.6 -2.2 9A 12.0 61,0 h.8 93 92.0 11.0 M.S 90 112.0 1114.0 14.8 101 52.2 55.6 6.5 1GB 52.2 53.7 2.5 100 52.2 M7.2 -9.6 111 66.5 69.0 3.8 113 66.5 70.0 5.u 110 66.5 62.3 -6.3 121 105.0 100.0 5.0 123 105.0 103.0 3.0 120 105.0 93.5 6.5 TABLE II THE EFFECT OF CARVTEVE ON CHLOROPHYLL DETEPHIHATIOYS Expressed as mgtfl. -.-,‘Q - Chlorophyll concentretions. Chlorophyll concentration. containing in solution Percent mole Checks 27 n.p.m. of carotene. verietion 1 20.5 20.9 1.9 2 1214.0 117.0 ~5.6 3 6.9 6.5 ~5.7 1* 6.9 7.3 5.7 5 85.0 85.0 0.0 6 30.7 31.0 1.0 31+ TABLE III COMPARISONS C? THE COLORIHETQIC AVE PHOTCYETRIC METHODS FOR.ET@7RNIXATIOH OF CHLOVOPHYLL Photelometer Calorimeter Guthrie's standard Corning filters (K Cr 0 + CuSOu + Chloronhyll m . on 06 2 9 fi. . Sample Lo.r.3 plus 3, H.40H) stanoard Tomato leaf lhl.0 mg/l. 139.6 mg/l. 1k6.0 ng/l. Tenet» leaf 150.0 179.8 193.0 Nettle leaf 153.0 117.1 156.0 Kettle leef 128.0 11h.h .13?.0 Jettle leef 80.0 75.3 Alfalfa leaf 57.5 57.? 55-1* Celerv leef 33.0 78.5 Celerv leaf 70.0 72.2 0 ~J a ~ 0 Celerv leef 103.- 5 .5 \4“ \J1 TABLE IV TH? EFFECTS OF CERTAIN FILTERS ON PHOTCKWTRIC READI¥GS OF SCAYDARD CAROTETE SOLUTIONS fl. _ ~ ._ , - Ho.553 + Carotene conconfretion Corning No.ESM Noviel A (fl0.038) Cenco Uo.l 9.16 27.1 26.9 29.0 3.33 33.5 39.3 32.2 2.66 91.3 no.7 39.5 9.13 99.0 NS 8 27.5 1.36 62.8 62.3 62.0 1.09 68.8 68.2 68.0 0.59 82.5 83.1 82.5 0.28 92.. 92 3 91.8 0.19 97.0 97.8 98.0 \->~J C \ TABLE V TUE AMOUHT 0F BARIUM HYDRCXIEE REQVIRRD UNDER CERTAIN CONDITIONS T0 RENOVE CVLOROPHYLL FOR THE CAROTE“E DEWERFINATIOYS Time in p.p.m. cc. chl. Weight cc.Ho for min..of Chl. p.p.m. chl. sol. 82(0Hl? 32(OH)2 refluxing Reaéing chl. conc. 100 50 1 gm. 5 30 98.7 0.9 100 50 1 gm. 5 25 92.1 0.5 100 50 1 gm. 5 20 97.2 0.9 100 50 1 gm. 5 15 96.9 1.0 50 50 0.5 8“ 5 30 93.1 2.9 100 50 2.0 g”. 20 97.5 0.8 100 50 2 gm. 5 30 99.2 0.2 100 50 3 gm. 5 20 98.2 0.5 100 50 u gm. 5 10 96.9 1.0 100 50 8 gm. 6 10 100 0.0 100 50 0.5 gm. ll 20 9U.8 1.7 Ba(OH)2 + 0.5 gm. TABLE VI CAROTE"E RECOVERIES FROM .CET "E SOLUTIONS FOLLOWIHG THE BARIVM HYDFOXID? WRCATHENTS Sample ChlorOyhyll concentration Carotene concentration 1 0.0 mg. /1. 0. 89 mg./1. 2 0.0 .0 91+ 3 0.0 1. 90 0"1“E§r' u 0.0 0. 89 5 0.0 0. 63 6 0.0 1.55 1.5? l & M -- 50 cc. acetone and 50 cc. ca.rotene solution 2 & 5 —- 50 cc. acetone and 50 cc. c%1oronhv11 ext rect. 3 & 6 -- 50 cc. carotene solution and 50 cc. 10 roohyll extract. Sum of 1 2nd 2 should eoual 3. Sun of h and 5 should equal 6. Fifty cc. of en acetone solution of carotene oil (5. II.A.) refluxed 15 minutes with 1. 5 grams barium hvéroxioe. Extre ction with petroleum ether. Chec: has no barium hydroxide treetment. Sample Concentration of check ' , Concentration of tr eeted A 1.05 mg./1. 1.00 21 ./1. B 1.12 1.10 c 1.35 . 1.99 D 2.19 2. 21 Fifty cc. of an 2cetone solution of c2rotene refluxed one- Sample half hour with 1.5 gms. barium hydroxide. No barium hydro- xide treatment in check. Acetone- check 0.67 mg. /1. Acetone- treated 0.71 Pet.ether check 0.6M Pet.ether treated 0.67 Agbtr cruqf( meQ h E15,; H. agEWn I‘I A‘A ) "Wyatt... QWL mtafbrufldo Worm mQCmDmU HHb mqmdm MO m2CmHmtoH mxcnowpp< m.::H H.0u x.ux Cr.m m.mg bu.mH 3.3m ow wmlwnmoaccrtpma Hwaamv mobuoa ecaafln c.1umfi c.11a w.m: 11.1 1.u: mm.m c.n om-mu-w A1111 1L0Ho wwofim mmv flomrfiam C.u0HH m.HmH V.M: mu.; “.mm am.: c.0w omnmusm .Cw mospomqv modppeq c.awa 1.31 12.1 21.1 mm.w cm 1 o.~w m11wa-m Awfit1m Hr» msHomm army Amazobvmmoc camp m.oaeswapw sumac . — I! c \40 0‘. O\ x O . u . \J 0.21 1.1 11 H He 1 no 1 H1 1 1 1m m~1afi-1 “11111 uadb mzaoomaxmv Amuse .vmoop camp McE EOHHmw m.1mma w.nc; 0.11 1.:H m.am ~.:H m.1~ mm-~H-m A111111 oomowe1; , amaama< 1.. ... 1 1... \o... . .1 O .\ 1 O J 1 x; \I 4 \ 3 - a. .. C cuwH U w a a: o.uH 0 mg w.ua u :u mmouaou AVHmCm+¢am romv mmcpw oSHm .Luccfix.un .ttcc~\.mfi .fnCcH\.z .mex Efiwpum tmmfimsmxcomampmm INDEX OF FIGVRES Sketch of Cenco-Sheard—Snnford photelometer. Standard chlorophyll curves ob— tained by the use of different f .5. lters. Standard carotene curve. Corréction curve for carotene sol- ution containing chlorOphyll. (Inset) SOURCE SHUT TE R DIAPHRAGM CONDE'NSING LENS 7///// £15523: 1 HEAT FILTER WAVE LENGTH FILTER PHOTRONIC CELL x) MIC ROAMME TE R FIG. I on 0.? 20—mm_2m2IaOmOJIU mOu 3250 295119.20 5.5233011. / vow :u / N Z + MVN 02.2100 —Nm¢ "my—whim / / c/ :/ / / % / / / / / rill! AT--. ll 1 .11.. I 11/ _.._-..+-- __——T>—--'-—-4‘~ .. _ / -....// N .0: 0.. ON 0. m IVUILV OJJ‘IJJIVUJ 0. 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