Apparatus Cor the Rapid Determination of Chlorophyll and Carotene R. B. GRlFFlTH
R. N. JEFFREY
AND
Kentucky Agricultural Experiment Station, Lexington, Ky.
I
through D into the adsorption column, C'. A second three-way stopcock, E, is also connected with the adsorption column. One arm of this stopcock is connected to the air supply, while the other releases the pressure in C. D and E should be equipped with plug locks. The adsorption column consists of a 22-mm. (outside diameter) glass tube drawn out a t one end and sealed to 4-mm. (outside diameter) tubing which is bent to run parallel to the large tubing and with two additional bends to bring the drawnout end approximately 3 cm. from the larger tube. The tip of the smaller tube should be a t or above the level of the top of the packed adsorbent.
S A number of the methods for determining chlorophyll and
the carotenoids in plants the sample is extracted with acetone and the pigments are transferred to ether by washing with water a number of times in separatory funnels (f-4). This procedure was followed in the earlier work in this laboratory ( 3 ) . The time required for these analyses can be decreased considerably by means of the apparatus herein described. The pigments are transferred to ether by passing an acetone extractether mixture through a vertical column in which water is moving in the direction opposite to t,he flow of the mixture. As the mixture bubbles through the water, the acetone is removed by the water and the ether solution collects at the top of the column. By changing the water level in the column, the washed ether solution is driven into a cooling coil which decreases the solubility of water in ether and dries the solution sufficiently for immediate pigment determinations. A new type of adsorption column which retains solvents until pressure is applied and apparatus for delivering solvents under pressure into the adsorption column are also described. Using this equipment, the speed of carotene and xanthophyll separation is greatly increased.
PROCEDURE
PREPARATION OF ETHERSOLUTION.I n preparing the washing apparatus for operation, the water supply connected to tube 13 is turned on and regulated so that some water overflows through tube 9 a t all times. Stopcock 7 is opened to tube 8 and stopcock 6 adjusted to deliver between 20 and 40 ml. per minute. Tube 2 is
filled with water by opening stopcock 14 to tube 12. After the ice bath, 17, has been filled with ice and the space around the ice has been filled with water and about 5 grams of A t have been added, the apparatus is ready for operation. Fifteen milliliters of diethyl ether are added to funnelaandthen 10 to 20 ml. of an 85% acetone extract of normal green leaves, prepared as previously described (3),are added, so that thorough mixing occurs. Funnel 3 is then connected to tube 2 by opening stopcock 14 enough to allow the ether-acetone extract mixture
APPARATUS
WASHINGAPPARATUS. A diagram of the apparatus used in transferring the pigments from an acetone extract to an ether solution is shown in Figure 1. The most important part of this apparatus is tube 1. Water enters from tube 8 through stopcock 7 at 4, and flows out through tube 5 a t the base, the rate of flow from the column being regulated by stopcock 6. The water level in tube 8 is controlled by overflow tube 9 and should be adjusted so that the water level in tube 1 will be 3 cm. above the inlet tube when the water is flowing through the column a t the desired rate, which is between 20 and 40 ml. per minute. Stopcock 7 is also connected to tube 10. The water level in this tube is controlled by overflow tube 11, which extends into tube 8. Water enters tube 10 through tube 13, which is connected to a water tap, and to tube 12, which connects t o stopcock 14. The water level in tube 10 should be a t least 5 to 6 cm. above the level of stopcock 14, so that liquid may flow through the stopcock from tube 12 into capillary tube 2. The end of tube 2 enters the base of tube 1 and extends about 3 cm. above the base. Since the acetone extract-ether mixture flows by gravity from funnel 3, stopcock 14 must be high enough above the water level in tube 1 to overcome the pressure created by the water column when tube 2 contains ether. The top part of tube 1 consists of a 7-mm. (outside diameter) curved tube, constricted at the highest point of the bend. Just beyond the constriction, vertical air inlet tube 15 is sealed in t o prevent siphoning. The end of tube 1 is drawn out and is inserted in the enlarged end of the cooling coil, 16, which passes through an ice and salt bath, 17. This bath may be drained by means of stopcock 18. The lower end of the coil extends into receiver 19, which is provided with a three-way stopcock, 20. This apparatus is suited to the preparation of 20- to. 30-ml. ether solutions. The large part of tube 1 has an outside diameter of 25 mm. and a length of 52 cm. A scale is provided on the side of Figure 1 from which the other dimensions can be estimated. (It has since been found that more satisfactoSy results can be obtained by decreasing to about 13 cm. the diameter of tube 1 from 6 cm. above point 4 to about 10 cm. from the bottom, and increasing the diameter of the bottom part of tube 1 to 33 cm.) ADSORPTIONCOLUMN APPARATUS. A diagram of the adsorption column apparatus is shown in Figure 2. This consists of an ether reservoir, A , and a reservoir for an ether-ethanol mixture, B , these two being connected to a 3500 kg. per sq. meter (5 pounds per sq. inch) air supply by glass tubing that enters the reservoirs above the solvents. Tubes extending to the bottom of the solvent reservoirs are connected to the same three-way stopcock, D. Thus, when air pressure is applied, the solvents may be forced
I Figure 1.
448
Washing Apparatus
July, 1945
ANALYTICAL EDITION
to flow from the funnel in about 5 to 10 minutes. The characteristics of the individual apparatus, particularly the type of tip a t the end of tube 2, will determine what rate of flow can be used without loss of pigment in the wash water. The ether solution of the pigment collects a t the top of the column, while the acetonewater mixture passes through the outlet a t the base of tube 1. When the last of the ether-acetone extract mixture passes into tube 2, stopcock 14 is closed, the sides of the funnel are washed with 2 ml. of fresh ether, and the wash ether is passed into tube 2. By changing the position of stopcock 7 to connect with tube 10, the water level in tube 1 is raised and the pigment solution is driven into the cooling coil. When the ether solution has passed the constriction a t the bend in tube 1, stopcock 7 is turned to connect with tube 8 and the water column returns to its former level. An additional 3 ml. of wash ether are added to the funnel and passed into tube 2. This ether is driven out of the tubing into the water column by turning stopcock 14 so that water flows from tube 12 into tube 2. The surface of the glass a t the top of the water column is washed pigment-free by changing the water level in tube 1 so that the wash ether a t the top of the column contacts the same surface that the pigment solution contacted. This may be accomplished by closing stopcock 7 until the wash ether ia below the lowest point contacted by the pigment solution. The wash ether is then driven out of tube 1 into the coil by turning stopcock 7 to connect with tube 10 as before, care being taken to limit the amount of water passing into the cooling coil. Stopcorh 7 is again connected to tube 8, another sample may be introduced in funnel 3, and washing started before continuing with thv fir-t sample. By introducing fresh ether from a wash bottle into tube 15 and the mouth of coil 16, the last traces of pigment are removed from the coil. The temperature of the ether solution decreases a i ~t passes through the coil, thereby decreasing the solubility of wartar in ether, and when the solution passes into reservoir 19, two layeis are formed. The water layer is drained off through one arm of stopcock 20 and the ether solution is drawn off into a 25-ml. volumetric flask placed a t the tip of the other tube. The reservoir and the tips of the coil and stopcock are washed with ether. When the ether solution in the volumetric flask is made to volume, it is ready for pigment determinations. Chlorophyll determinations are made on a dilution of this ethri solution by measuring the light absorbed a t the chlorophyll a and b maxima in the red end of the spectrum. The chlorophyll a and b contents are calculated by means of simultancou. equations as previously described (3). SEPARATIOX OF CAROTENOIDS. The adsorption column used i l l separating carotene from the xanthophyll fraction is prepared a5 follows: A piece of cotton, F , is tamped into the constricted portion ot the adsorption tube, C . The tube is filled to within 3 cm. of the top with petroleum ether (Skellysolve F)! 8 grams of a n adsorbent mixture composed of equal parts by weight of magnesium oxide (Micron Brand KO.2641, Westvaco Chlorine Products Co., Newark, Calif.) and Hyflo Super-Cel (Johns-Manville, S e w Tork) are added, and adsorbent remaining on the sides of the tube is pushed into the solvent. Air is gently stirred out of the adsorbent mixture and the top of the tube is connected to a 3500 kg. per sq. meter (5 pounds per sq. inch) air pressure line. The air pressure packs the adsorbent to the position shown in Figure 2, G. The tube is disconnected when the petroleum ether above the adsorbent is approximately 1 cm. in depth. A 5-ml, portion of the ether solution prepared in the washing apparatus is added to the column and the top 0.3 to 0.5 cm. of the adsorbent is stirred with a stirring rod. Stirring the top of the column results in a more even movement of the pigment bands through the column. After the stirring rod is washed with petroleum ether, the column is connected to the adsorption column apparatus shown in Figure 2. Air pressure is applied by means of stopcock E to force the pigment solution into the adsorbent. Pure ether in small portions is then added under pressure by opening stopcock D to A until the last of the pigments is washed into the adsorbent. Larger quantities may then be used, care being taken to keep a solvent layer above the adsorbent a t all times. The movement of liquid through the column can be stopped instantly by releasing the pressure using stopcock E. Carotene is unadsorbed and is collected in a 25-ml. volumetric flask placed below the outlet tip of the column. The carotene is completely separated when colorless solution comes through following the colored carotene solution. If no colorless solution is obtained, the separation is incomplete and another column is necessary. Separation may be incomplete if the adsorbent has lost its adsorbing properties or if some substance is present in the pigment solution or ether which elutes the xanthophyll. In-
Air
449
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I-
-
-I I
- -11
Figure 9 .
I /
I
Chromatographic Apparatus
creasing the quantity of adsorbent or the depth of petroleum ether above the adsorbent prior to the addition of the pigment solution is helpful in improving the separation if difficulty is encountered. The carotene is determined spectrophotometrically a t its highest absorption maximum after the solution is made to volume. This chromatographic method is applicable to plant extracts in which the carotene is of the beta form. If other carotenes were present, the method would need modification, although the apparatus might still be useful. If xanthophyll is to be estimated, its elution may be started by adding a n ether-ethanol mixture to the top of the column by opening stopcock D to B before the collection of the carotene fraction is complete. Shortly after the carotene solution is removed, the yellow xanthophylls come through the column, and are collected in a second 25-ml. volumetric tlask. If a group of very similar samples is to be analyzed for xanthophyll, the mixture in B can be regulated to the proper concentration, although it is usually better to have the mixture stronger in alcohol than necessary and to open stopcock D to A and B alternately until the mixture added to the column completely elutes the xanthophyll. So set procedure has been found applicahle to all plant materials tested. Even when grown under similar conditions, different varieties of a species must be treated differently. At times difficulty is encountered in completely eluting the xanthophyll and a t other times part of the chlorophyll comes through with the xanthophyll. With some varieties of tobacco the individual xanthophylls can be quantitatively separated. Each plant material presents a different set of problems which must be worked out as required. The xanthophyll solution is measured spectrophotometrically a t the wave length of maximum absorption and the data are reported as the extinction coefficient, K . If the proportions of the different xanthophylls remain constant, the value arrived a t is nearly proportional to concentration, but it is not an absolute concentration value. The xanthophyll values obtained by this method are low because there is some loss of xanthophyll in the wash water and this xanthophyll is not recovered as it was in the method forherly used (3). This apparatus was designed primarily for chlorophyll and carotene analyses and for these components more reproducible results are obtained. It is possible for one person to determine chlorophyll and carotene and to estimate.xantho-
450 Table
1.
Portion No.
>lean, Z Standard deviatidn s Coefficient of variaiion, a/;, a
Vol. 17, No. 7
INDUSTRIAL AND ENGINEERING CHEMISTRY Reproducibility of Results Chlorophyll Total a
Ma./l.
%
5.13 5.12 5.12 5.13 5.15 5.13 0,015 0.29
73.29 73.44 73.44 73.68 73.20 73.41 0.19 0.24
Carotene Mg./l. 6.72 6.56 6.58 6.52 6.63 6.60 0.08 1.21
Xanthophyll
K 0.407 0.397 0.393 0.310 0.381 0.3944 0.0110 2.80“
Sample 4 not included,
phy11 in a tobacco leaf within an hour from the time the sample is removed from the plant, by means of a Waring Blendor, the apparatus herein described, and a spectrophotometer. Less time is required if the xanthophyll estimation is not included. RESULTS
Typical results which may be expected when using the apparatus and procedure described above are given in Tables I and 11. The reproducibility of results obtained using this procedure on five 10-ml. portions of an acetone extract of tobacco leaves is shown by the data presented in Table I. Fifteen-milliliter portions of ether were mixed with the acetone solutions each time, and the final ether volumes were adjusted to 25 ml. The chlorophyll and carotene contents are expressed in milligrams per liter in the measured solution, and the xanthophyll is expressed as the extinction coefficient, K , in the measured solution. The chlorophyll, carotene, and xanthophyll solutions were not diluted to the same extent; therefore the reported concentrations of the differentpigments are not comparable. The standard deviation from the mean of the five samples \yas 0.015 mg. per liter for total chlorophyll, 0.187 for percentage chlorophyll a, and 0.08 mg. per liter for carotene. (“Percentage chlorophyll a” is the percentage of the total chlorophyll which is chlorophyll a.) The xanthophyll content of sample 4 was obviouzly low, probably because of incomplete removal of xanthophyll from the adsorption column. The standard deviation from the mean of the four other determinations was 0.011 K or 0.039 K if sample 4 is included. The correqponding coefficients of variation are given in Table I. Table I1 presents the results of a test to determine the ratios of acetone, ether, and plant extract concentrations at which the washing apparatus functions properly. An approximately 85% acetone extract, as concentrated in pigments as possible, was prepared by extracting very green tobacco ieaves with pure acetone. Portions of this extract, as indicated in column 2 of Table 11, were pipetted into flasks as promptly as possible to prevent changes in concentration due to evaporation and 85y0 acetone in the quantities given in column 3 was added to the flasks. Each acetone solution was mixed with 15 ml. of ether prior to washing. After washing, the ether solutions were made to volume (25 ml.) and chlorophyll readings were made immediately on appropriate dilutions. The samples were washed by one person and chlorophyll readings were made by another. The ten determinations of total chlorophyll and percentage chlorophyll a were completed in 2 hours. A 5-ml. portion of each ether solution was then chromatographed and carotene and xanthophyll were determined. A total of 4 man-hours was required for chromatographic and spectrophotometric determination of carotene and xanthophyll on the ten samples. The average time spent on each sample for washing, chromatographic analysis, and spectrophotometric determination of carotene, xanthophyll, chlorophyll, and per cent chlorophyll a was 48 minutes. Results were multiplied by 2 in samples 2 and 4 and by 4 in samples 3 and 5 when necessary to make all results comparable. The reproducibility of these results a t all five concentration ratios used was better than that of most of the determinations reported in the literature, especially the values obtained for per-
centage chlorophyll a. This degree of reproducibility IS obtained only if the whole analysis is completed promptly. These data were subjected to an analysis of variance to determine whether any of the results a t different concentration ratios were significantly different. The differences in means of the various concentration ratios which are statistically significant a t the 5 % and 1% levels are shown, for each analysis, a t the bottom of Table 11. The No. 3 samples appear to be significantly low in total chlorophyll, indicating that too much acetone in proportion to the pigment present may result in some loss of chlorophyll. The finding of higher values for percentage chlorophyll a in samples where more acetone in porportion to pigment was used, indicates that the loss of chlorophyll b during washing is about the same as the loss of chlorophyll a in spite of the smaller initial concentration. This would indicate that the acetone extract should be as concentrated in pigment as possible. The mean of the total chlorophyll results of samples l a and b is significantly lower than that of samples 4s and b, in which the ratio of pigment to acetone is the same but the ratio of acetone to ether is lower The means of samples 2a and b and 5a and b also differ in the same direction, though not enough to be statistically significant. This indicates that the volume of acetone extract should be as low as practical with respect to the volume of ether to prevent loss of chlorophyll. This is probably true with respect to the previous method of washing. The carotene values are also significantly higher on sample 4 then on sample 1 and somewhat higher on sample 5 than sample 2, indicating a loss of carotene when the ratio of acetone extract to ether is too high. When different ratios of pigment to acetone are considered, the intermediate concentration is higher by an amount which is statistically significant, although no reason for this is apparent. None of the differences in the xanthophyll values is statistically significant. I n spite of the variations mentioned in the preceding two paragraphs, if the ten samples are all considered as a group, the reproducibility obtained was better than that obtained with any other method yet tried in this laboratory. This was probably due to (1) the short time alloxed for deterioration of the pigments and (2) the decreased opportunity for loss on glassware or in transfers. When the chlorophyll determination is carried out as quickly as it can be done with this apparatus, the coefficient of variation of the per cent chlorophyll a is about 0.37, However if a t any stage of the proces3 the pigment solutions are allowed to stand for several hours before the chlorophyll readings are made, the variations are much larger, even if the 3olutions
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Table II. Effect of Acetone and Plant Extract Concentration Used in Washing Chlorophyll CaroSanthoSample Acetone 85% No. Extract Acetone Total a tene phyll MI. M1. Ma. % Ma. K la
20
b
hlean 2a
0
10
10
3a b
5
15
4s
10
o
b
Mean Mean
b
Mean
163.1 163.9
74.31 24.33
163.5
14.55
162.8 162.6 162.7 157.2 157.4 157.3 166.8 167.3
74.81 74.72 74.77 74.74 75.02 74.88 74.64 74.50 74.57 74.90 74.81 74.86 74.68 0.28 0.43 0.23 0.31
167.1
5s 5 5 167.0 b 162.7 Mean 164.9 163.1 Mean of all samples 5 3.6 Significant differend 5’7 ) 5.6 Signiecant difference [1%) Standard deviation a 3.6 Coefficient of variat‘ion, 6, % 2 2
8.41 8.29 8.35
24.9 24.7 24.8
8.64 8.58 8.61 8.31 8.41 8.36 a.58
24.0 24.5 24.3 ?5.8 14.5 25.2 23.3 24.8 24.1 36.5 25.9 26.2 24.9 3.0 4.8 0.95 3.8
8.60 8 59 8.84
8.70 8.77 8.54 0.18 0.28 0.18 2.1
ANALYTICAL EDITION
July, 1945
are kept in the dark at 7 " C. The atandard deviations and coefficients of variation for all samples are shown a t the bottom of Table 11. The apparatus for separating the carotenoid pigments has several advantages over that used previously. The outlet tip of the adsorption column is high enough so that it will not go dry unle.;s pressure is tzpplied. Solvents can be added to the column as required while continuous pressure is maintained. This increase? the speed of the determination and decreaseq the tendency for pockets to develop in the adsorbent. SUMMARY
Uding apparatus described i t was pos$ible to determine total chlorophyll, percentage chlorophyll a, @-carotene, and to estimate xanthophyll concentration within an hour from the time a leaf sample was removed from the plant. The standard deviation of the total chlorophyll and carotene results obtained by
45 1
this method was less than 2% of t,he means and thht of the percentage chlorophyll a less than 0.3% of its mean. Total xanthophyll determinations were less accurate than those previously reported. The apparatus eliminates possible loss from the many transfers in the older procedure, decreases t,he amount of apparatus required, increases the speed of dfattmninations, is easily constructed, and is self-cleaning. LITERATURE CITED
(1) Coniar, C. L., IXD. ENG.CHEM., ANAL.ED., 14, 877-9 (1942). (2) Coniar, C. L.. and Zscheile, F. P., Plant Physiol., 17, 198-209 (1942). (3) Griffith, R. B., and Jeffrey, R. N., IND.ENG.CHEY.,- h . 4 1 , . ED., 16, 438-40 (1944). (4) Schertz. F. >I Plant ., Physiol., 3, 211-16 (1938). THEinvestigatioii reported in this paper is in connection with a project of the Kentucky Agricultural Experiment Station and is puhlishecl hy perniiasion of the director..
Thermostatic Bath for Low Temperatures E. L. RUH, G . E. CONKLIN, AND J. E. CURRAN Oil Development Co., Bayonne, N. 1.
Standard Inspection Laboratory, Standard
A thermostatic bath, primarily for low-temperature viscosity determinations, operates effectively at temperatures ranging from +40° to -70" F. It consists of a three-stage installation, one bath containing dry ice and isopropyl alcohol, a second bath maintained under rough automatic control, and a third bath under close control. The last is a vacuum-jacketed glass jar, filled with acetone, and equipped with sccessories for mounting viscometers. The development described superseded one involving only two stages. This was effective, but the first stage required time-consuming manual control of temperrture. The three-rbge installation is mounted in a special cabinet which augments its operating efficiency.
THE
laboratory with n-hich the authors are associated ninkcs niiiwrous viscosity determinations at temperatures ranging fro111 +10" to -70" F. The bath originally used contained wetone which was kept chilled by periodic immersion of a cylindrical, perforated basket filled with dry ice. Reasonably good temperature control was obtained by this method, but it was t iine-consuming. Development and construction of a bath with automatic temperature control were therefore undertaken. PRELIMINARY EXPERIMENTATION
q u a n t i h of dry icc from time to time. This necessitated experience on the part of the operator, and a considerable waste of his time. G E N E R A L FEATURES OF F I N A L D E S I G N
Experience with the two-bath unit pointed to the desirability of a three-bath system, which is now in service. It consists of one jar held a t a suitable low temperature, containing solid carbon dioxide and isopropyl alcohol: a second jar under rough thermostatic control; and a third, Dewar-type jar under close control. The temperature-control system of the second jar is identical with that used in the earlier two-bath unit. The third bath is chilled continuously by circulating its acetone through the coil in the aecond bath which is held a t a temperature approximately 10" F. lonw than that desired in the third bath. Temperature control in the latter is effected by a conventional circuit consisting of a 125-watt knife-blade hcater, a bimetallic thermoregulator, and a suitable relay. The three-bath design provides the d e h e d close temperature control (within *0.1" F.) in the Dewar jar with a minimum of attention from the operator. After the initial adjustments have been made, all that is necessary is to add to the first bath enough dry ice to keep its temperature at least 30" F. below the temperature required in the Dewar jar. This requires much less attention than holding the temperature of the same bath between maximum and minimum limits.
Trials were made of an apparatus consisting of a large Dewartype jar filled wit,h acetone, chilled by circulation through a copper coil immersed in an insulated glass jar containing isopropyl alcohol and dry ice. Motor Motor The pump was operated continuously to stir the acetone bath. The coil in the cooling- bath Maanetic was by-passed part of the time by a magnetically operated three-way valve connected with a suitable relay, actuated by a bimetallic regulator in the acetone bath. The regulator also turned on an immersion heater, whenever the flow through the cooling coil W M interrupted. This was found to help in maintaining close control. This installation gave satisfactory results when the temperature of the cooling bath bore the correct relation to the temperature desired in the -. acetone bath. BathNo.3 Both No.1 Bath No.2 I t was necesbary, . . hoivcver. to give the coolDiagram of Three-Bath Thermostatic Installation for Low Temperatures ins bath much attention, putting in the proper Figure 1 ,
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