Activation of Dicalcium Phosphate for the Chromatographic Determination of Carotene L. A. AIOORE', 3Iichigan Agricultural Experiment Station, East Lansing, Mich.
A
PREVIOL-S publication (1) demonstrated that in methods of carotene determination that utilize the Rillstatter-Stoll principle of separating xanthophyll from the carotene portion of the extract some noncarotene pigments are not removed and are determined as carotene. Dicalcium phosphate was proposed as a n adsorbent for the chromatographic removal of noncarotene pigments in the determination of carotene from plant materials. It was shown that this compound removed those noncarotene pigments which usually develop in stored hays, silages, and fecal matter. Shortly after publication of the above paper, however, dicalcium phosphates of the brand specified were encountered which were not good adsorbents, and it was thought desirable to find some simple method of activating an inactive phosphate. Such a method is presented herein, together with some of the data collected in the study.
TABLE
I.
Lot So. 21,940a 67 b 68 69 70 10,440 53 54 55 56
TREATIXG DICALCICM PHOSPH.4TE DISODIUM PHOSPHATE
EFFECT OF
Sa2HPOI
Chlurophyll per Gram of Adsorbent
Grams
.Mu.
0
5 10 15 20
0 10 15
20
2.4 9.8 13.8 16.2 17.8 1.4 6.2 9.8 14.0 17.4
Filtration Rate .Miii. Sec. 20 7 00 17 52 11 40 9 30 9 28 2 6 8 49 12 27 4 37
WITH
Height of 8.0-Gram Column Before After vacuum vacuum
Cm. 8.0 9.5 15.5 17.0 16.5
Cm
5 0 6 0
9.0 10.5 10.0
10.0
6.0
14.0 17.3 24.0
10 6 15.2
Manufacturer's number. b Smaller numbers are author's laboratory lot numbers
0
Procedure and Results I n order to arrive at some conclusion as t o the effect of various procedureb on the activation process, the ability of the treated phosphate to adsorb chlorophyll was studied.
While it is not contended that the ability of the treated phosphates to adsorb chlorophyll is proportional to their ability to adsorb other pigments in extracts froin plant material, it does indicate the extent of the activation. For practical purposes the activity of the phosphate can be determined roughly by the use of a dehydrated alfalfa leaf meal known to contain the noncarotene pigments. Meal which has been exposed to the air at laboratory temperature for 2 or 3 Tveeks, so that the noncarotene pigments will have developed, is suitable for this purpose. The meal is extracted by placing about 10 grams in a 200-ml. Erlenmeyer flask, adding about 50 ml. of petroleum ether, and shaking for a few minutes. About 20 ml. of the extract are then decanted onto a column of the phosphate being studied. If the phosphate is properly activated the carotene will pass through, while the chlorophyll will be held to within 3 to 7 mm. from the top of the column and a lower yellow band will form 12 to 25 mm. below, with a shade of red above. The lower yellow band should be so held that it does not pass into the filtrate after considerable washing with petroleum ether. The rate of filtration through the adsorbent with a vacuum of 65 cm. of mercury was determined by noting the time in seconds required for 100 ml. of petroleum ether to pass through a column 2.2 cm. in diameter containing 8 grams of the adsorbent packed with vacuum and slight tamping. While there was some variation in the results obtained by the procedure, an average of several trials gave a fairly reliable figure. The height of an &gram column 2.2 cm. in diameter hefore and after application of vacuum was also recorded, in order to gain some information of the effect of the activation prowdure on the weight-volume relationship of the adsorbent. I t was found, after trying many procedures which might produce activation, such as heat applied under various conditions, that treatment with disodium phosphate, trisodium phosphate, sodium hydroxide, or potassium hydroxide increased thc activity of an inactive dicalcium phosphate. The effects of adding different quantities of disodium phosphate to two different lots of Baker's c. P. dicalcium phosphate n-ere first studied (lots 21,940 and 10,440). One hundred-gram portions of each lot were placed in 2.0-liter flasks and different quantities of disodium phosphate, dissolved in 1 liter of water, were added. The mixtures were boiled on a hot plate for 0.5 hour and then decanted into a 20-cm. (&inch) Buchner funnel, washed with 1 liter of water, and dried in an evaporating dish in an oven for 24 hours at 100" C. The phosphate was then broken up in a mortar, after which various data were collected on its properties (Table I).
The effects of treating one lot of Merck's reagent dicalcium phosphate with disodium phosphate, pchassium hydroxide, and trisodium phosphate were next studied. The results are shown in Table I1
For this purpose a petroleum ether solution (Skellysolve B, used throughout this investigation), containing 1 mg. of crystalline chlorophyll per ml., was prepared. The same lot of petroleum ether was used in the adsorption studies throughout this investigation. One-half gram of the adsorbent was placed in a 30ml. test tube graduated at the 15-ml. mark, 1 to 10 ml. of the chlorophyll solution were added to the test tube, depending upon the expected activity of the adsorbent, and petroleum ether was added to the 15-ml. mark. The tube was then corked, shaken vigorously, and centrifuged. If all the chlorophyll was removed from the solution, an amount of petroleum ether was removed equal to the volume of chlorophyll solution to be added and the procedure was repeated. This procedure was repeated until the solution remained green, at which time 2 ml. were pipetted into a microcell of an Evelyn photoelectric colorimeter with an M-440 filter. -4n end-point reading of 60.0 for the galvanometer was arbitrarily chosen. If the galvanometer reading was above 60.0, more of the chlorophyll solution was added and the procedure was re eated until the reading fell below 60.0. From the amount of chTorophyl1 solution added, the milligrams of chlorophyll adsorbed per gram of adsorbent were calculated.
TABLE11. EFFECT OF TREATING DICALCIUM PHOSPHATE WITH DISODIUM PHOSPHbTE, POTASSIUM HYDROXIDE, AND TRISODIUM PHOSPHATE
Lot No. 32,689 71 72 73 74 80 81 82 83
76
Present addreas. University of Maryland Agricultural Experiment Station, College Park, Md. 1
707
XalHPOd Grams 0 5 10 15 20 KOH 1 3 5 10
NasPOl 5
Chlorophyll per Gram of Adsorbent
MU. 1.6 2.4 6.8
9.8 13.2 3 6
Filtration Rate Min. Sec. 1 10 4 30 4 0 7 25 6 3
Heieht of 8.0-Gram Column Before After vacuum vacuum
-
Cm.
Cm.
11.0 7.5 10.5 12.0 13.2
6.5 4.5 5.7 7.5 8.0
5.0 6.3
3.5 4.5
21.4
4 4 4 3
30 45 10 0
8.0 11.5
8.6
4
20
6.5
6 6 18.0
9.0 4.0
The effect of boiling the mixture for varying lengths of time was next studied (Table 111). Ten grams of disodium phosphate were used throughout for the activation. One lot was just brought t o a boil, one was boiled for one-half hour, and another was boiled for one hour. The effect of adding various quantities of water to the mixture was next studied, using 10 grams of disodium phosphate as the activating agent and boiling for 15 minutes. The results are shown in Table IV.
TABLE111. EFFECT OF TIMEOF BOILING Lot No. 57 58 59
Vol. 14, No. 9
INDUSTRIAL AND ENGINEERING CHEMISTRY
708
Treatment Heated t o boiling Boiled 0.5 hour Boiled 1 hour
NazHPO4 Grams
Chlorophyll per G r a m of Adsorbent
Me.
Filtration Rate M i n . See.
Height of 8-Gram Column Before After vacuum vacuum Cm. Cm.
10
10.4
7
19
18.0
10.1
10
11.4
7
35
15.4
8.8
10
11.6
6
36
15.5
9.1
Discussion of Results Table I indicates that increasing amounts of disodium phosphate markedly increased the activity of both lots of phosphates, as shown by the increasing amounts of chlorophyll adsorbed. This observation was further confirmed by the fact that both original lots only partially active nom held the chlorophyll, xanthophyll, and the lower yellow band in the upper portion of the column. Lot 21,940 was originally slightly more active than lot 10,440, and equal amounts of disodium phosphate produced more activity in lot 21,940 as shown by the milligrams of chlorophyll adsorbed. The filtration rate was decreased by the activation procedure. For instance, lot 10,440 had an original filtration rate of 2 minutes, 28 seconds, but after activation with 20 grams of disodium phosphate the filtration rate mas 5 minutes and 37 seconds, or half as rapid. I n both experiments the activation produced with 5 and 10 grams of disodium phosphate decreased the filtration rate more than the larger amounts. The cause of this alteration is not apparent and was not investigated further. It apparently does not lie in the weight-volume relationship, since the height of an 8-gram column increased almost progressively with increasing quantities of disodium phosphate. The results in Table I1 show that potassium hydroxide and trisodium phosphate are much more effective in the activation procedure than disodium phosphate. This would indicate that alkalinity mas the primary factor of the activation process. Although no data are given in this report, sodium hydroxide mas also very effective. Experiments should have been performed under controlled pH conditions with the various alkaline salts and hydroxides, but were not deemed necessary to the scope and purpose of this report. Other adsorbents may be more highly activated in respect to their ability to adsorb chlorophyll by treatment with alkali-for instance, the activity of barium hydroxide and aluminum oxide can be increased as much as ten times by treatment with alkali. I n comparing the rate of filtration between the phosphates used in Tables I and 11, it will be noted that the one used in Table I1 gave a much more rapid rate of filtration for equal activity than the phosphates used in Table I. The phosphate in Table I1 was Merck’s reagent with a coarse granular crystalline texture. Because this particular brand can be highly activated and yet retain a fairly rapid rate of filtration, it is used almost exclusively in this laboratory for carotene determination work.. Some grades of phosphates have
been ground, which makes them especially unsuitable for carotene determinations because of the slow rate of filtration. A rapid rate of filtration is desirable not only from the standpoint of saving time, but also because slow filtration promotes some slight destruction of carotene. A column 7.5 t o 10 cm. (3 to 4 inches) deep should permit 100 ml. of petroleum ether t o pass through it with application of vacuum in 4 to 5 minutes. The time should not exceed 6 minutes a t the most. If a filter aid is needed t o speed up the filtration rate, Dyno, a commercial dextrose, has been found effective. (Dyno is made by the Corn Products Refining Company, 17 Battery Place, New York, S. Y., and can be obtained a t some grocery and drugstores.) It is as effective as a siliceous earth commonly used for this purpose and does not cause destruction of carotene when used in conjunction with the phosphate. Table I11 s h o w that the length of the time of boiling apparently had little effect on the activity of this particular phosphate. However, owing to the variability of various phosphates, boiling for 15 to 30 minutes has been adopted as a general procedure. Apparently little is gained by boiling for longer than 30 minutes. Table IV indicates that water used in amounts of 500 to 1500 ml. had no noticeable effect on the activation process. The following procedure was finally adopted : One hundred grams of the inactive dicalcium phosphate (preferably Merck’s reagent) are weighed out and placed in a 2-liter Erlenmeyer flask. A quantity greater than 100 grams usually causes considerable bumping. Five grams of potassium hydroxide are added in 1 liter of water. The contents of the flask are boiled on a hot plate for 15 t o 30 minutes, transferred to a Biichner funnel, and washed with 0.5 t o 1 liter of water. The phosphate is placed in an evaporating dish and dried for 24 hours at 100’ C., and is then broken up in a mortar, after which it is ready for use. The activated phosphate will sometimes hold up the carotene on the column when first removed from the oven, especially where more than 5 grams of potassium hydroxide are used. This property has been utilized in making chromatographic separations of extracts of yellow corn. However, after a few days’ exposure to room temperature this particular property is lost. The use of dicalcium phosphate as an adsorbent for the chromatographic determination of carotene ( I ) along with a rapid extraction procedure previously reported ( 2 ) offers an efficient and accurate method for the determination of carotene in plant materials. TABLEIV. EFFECT OF VARIOUS QUANTITIES OF WATER Lot No. 64 65 66
Water Added t o Mixture M1. 500 1000 1500
KalHPOI Grams 10 10 10
Chlorophyll per G r a m of Adsorbent Mg. 10.2 11.0 11.0
Height of 8-Gram Column Filtration Before After Rate vacuum vacuum Min. Sec. Cm. Cm. 14 11 14.2 8.7 13 50 14.7 8.7 14 35 14.2 8.0
Summary Inactive dicalcium phosphate for the chromatographic determination of carotene in plant material can be activated by proper treatment with various strong alkalies. A procedure using potassium hydroxide is described. A commercial dextrose i s suggested as a filter aid.
Literature Cited (1) Moore, L. A , , IND.ESG.CHEM.,ANAL.ED.,12, 726-9 (1940). (2) Moore, L. A , , and Ely, Ray, Ibid., 13, 600-1 (1941). PAPERA-22, scientific journal series of t h e Maryland Agricultural Experiment Station, Maryland Agricultural Experiment Station contribution 1829. Journal article KO,584, n a. Michigan Agricultural Experiment Station.
