1016
I N D U S T R I A L A N D ENGINEERING CHEMISTRY
Vol. 16, No. 10
Ph ot osyn t h esi s1 By E. C. C. Baly UNIVERSITY OF
LIVERPOOL,
LIVERPOOL, ENGLAND
HE evidence advanced by Willstatter may be accepted as a definite proof that the first product of the photoassimilation of carbon dioxide by the living plant is formaldehyde. The experimental realization of this reaction in the laboratory is of great importance, and Moore and Webster were the first to show that traces of formaldehyde can be detected in aqueous solutions of carbonic acid after exposure to ultra-violet light. These authors, however, stated that the reaction takes place only in the presence of certain inorganic catalysts, and they laid great stress therefore on the important role that salts, such as those of iron, must play in the living leaf. This statement, however, is incorrect, since traces of formaldehyde can always be found in pure aqueous solutions of carbonic acid after exposure to ultraviolet light, provided that the exposure is not longer than about 12 hours, that the solution is stirred, and that the quartz reaction vessels are placed at least 8 inches from the quartz mercury lamp. The tests employed for the detection of the formaldehyde are two-namely, the Schryver reaction with Phenylhydrazine hydrochloride and potassium ferricyanide, and the orcinol test. I n the former it is important to note that the ferricyanide must be added immediately after the phenylhydrazine hydrochloride and that the mixture after shaking must stand for at least one minute before the hydrochloric acid is added. If this period of time does not elapse the test loses greatly in sensitiveness. It may be noted that both these tests are given by hexylic aldehyde and glucuronic acid, as well as by formaldehyde. The quantity of formaldehyde formed in the exposed solutions is very small, being from two to eight parts in one million, but the concentration may be increased by fractional distillation.
A second method of decreasing the action of the harmful light rays is to introduce into the solution a substance which absorbs them and therefore protects the formaldehyde. Compounds of this type were employed by Moore and Webster and wrongly called catalysts since their function is merely to protect the formaldehyde and not to catalyze its formation. It has been experimentally proved that each substance used in this way, such as beryllium chloride and colloidal ferric hydroxide, does in fact absorb the short wave ultra-violet light.
1Abstract of paper presented before Section B (Chemistry) at the meeting of the British Association for the Advancement of Science, Saskatoon, Saskatchewan, August 22, 1924.
When ordinary formaldehyde in not too dilute aqueous solution is exposed to ultra-violet light, it is converted into sub-
T
EXPERIMENTS WITH
QUARTZ
TESTTUBES
A most noteworthy phenomenon may here be mentioned which was the origin of many disappointments in the earlier stages of these experiments. When a quartz test tube is exposed to the rays from the quartz mercury lamp continuously for several days, it develops an amethyst color exactly like that of the natural amethystine quartz. When in this condition the quartz has become almost opaque to ultraviolet light and is therefore useless for any of this work on photosynthesis. If the discolored quartz tubes are heated in a powerful blast flame, they exhibit a very striking green phosphorescence. The heating is continued until the phosphorescence ceases, and on cooling the tubes are found to be again quite colorless and to be transparent to ultra-violet light. The opacity to ultra-violet light has been found to commence a t the short wave end of the ultra-violet and with continued exposure to the rays from the lamp to extend through the ultra-violet into the visible region. Long before any visible color has developed, the quartz tube becomes opaque to rays of wave length 220pp and is therefore useless for the experiments on the conversion of carbonic acid into formaldehyde. The quartz test tubes should be strongly EFFECT OF WAVELENGTH ON FORMALDEHYDE heated at least once a week, if in continuous use; otherwise Formaldehyde in dilute aqueous solution is readily de- they will give negative results. These observations have considerable bearing on the type stroyed by short wave ultra-violet light, and therefore the of quartz mercury lamp which gives the best results. A amount present a t any moment can only be the excess of that lamp working on a 100-volt circuit is liable to become informed over that decomposed. Therefore, the optimum use owing to a change in absorptive experimental conditions will obviously be those in which efficient after continued power such as was described above. The higher power the intensity of the short wave ultra-violet light is decreased lamps operating on a 250 or 500-volt circuit do not deteriorate relatively to that of the light which photochemically converts in this way because of the high temperature developed. carbonic acid into formaldehyde. The wave length of the I n the experiments described above quartz test tubes 9 x 1 light that produces the formaldehyde is about 220pp, while inches were filled with pure conductivity water and a stream rays of wave length less than 190pp destroy it. The shorter of very small bubbles of carbon dioxide was maintained rays are absorbed to a considerable extent by oxygen, and this is the reason why a minimum distance of 8 inches between through each. The gas was prepared from pure marble and synthetic hydrochloric acid and washed with potassium bithe carbonic acid and mercury lamp is necessary for the pres- carbonate solution. Eight of these tubes were mounted in a ence of formaldehyde to be detected. stand at from 8 to 12 inches from a mercury lamp operated by Since all light rays of wave length shorter than 215pp are a current at 250 volts. Perhaps the best yield of formaldeabsorbed by calcite, the quantity of formaldehyde formed is hyde is obtained in the following way: A very fine spray of increased if a screen of this material is interposed between soda water is made by an atomizer in the neighborhood of the the mercury lamp and quartz test tube containing the car- quartz mercury lamp. The spray is collected by means of a bonic acid. This experimental arrangement, however, has large glass funnel screened from the rays of the lamp. The the disadvantage that the screen absorbs some of the light presence of formaldehyde can readily be proved in the solution rays which act on the carbonic acid, and, further, that there thus collected. is an added loss due to the natural imperfections of the calcite plates. PHOTOSYKTHETIC PRODUCTION OF SUGARS
October, 1924
ILVDUSTRIAL AND ENGINEERING CHEMISTRY
stances which reduce Benedict’s solution, and among these the presence of hexose sugars has been proved. It would seem probable, therefore, that when formaldehyde is photochemically produced from carbonk acid in aqueous solution it a t once polymerizes to give the reducing sugars. Indeed, it may be suggested that the first recognizable products of the photosynthesis are the reducing compounds and that the formaldehyde found is a degradation product of the sugars produced by the action of the short wave length light. If this suggestion is sound, reducing sugars should be found in the carbonic acid solutions after exposure to light, and tests with Benedict’s solution have definitely proved the presence of reducing compounds in these solutions. Since the amount found, about 0.005 per cent, is on the limit of sensitivity of this test as ordinarily carried out, it may be mentioned that the delicacy of the test can be very greatly enhanced if the test tube containing the heated mixture is illuminated with the light from the quartz lamp. Small amounts of cuprous oxide in suspension are readily recognized by a red fluorescent effect which is caused by the mercury spectral rays. This formation of reducing sugars from carbonic acid has been proved beyond all question and supports the suggestion put forward above. Furthermore, the production of redwing sugars as the first recognizable product of the action of ultra-violet light on carbonic acid offers a possible explanation of the total absence of ordinary formaldehyde in the living plant, since those rays which in the laboratory degrade the reducing sugars to ordinary formaldehyde are not present in nature. This suggested explanation of the photosynthetic operation differs from the view originally put forward, according to which the formation of reducing sugars takes place in two stages-first, the formation of ordinary formaldehyde from carbonic acid, and then the conversion of this formaldehyde into reducing sugars by the further action of light of wave length 280pp. This explanation is cumbersome and unnecessary since the direct formation of reducing sugars from carbonic acid has been established. The direct formation from carbonic acid is proved by the fact first noted by Moore and Webster and since amply confirmed, that reducing rugars are not formed by the action of light on formaldehyde a t smaller concentrations than 0.25 per cent, the concentration in the carbonic acid solutions after exposure not being greater than 0.001 per cent.
FORMALDEHYDE BY OTHERPHOTOSYNTHETIC METHODS Evidence of the formation of a highly reactive form of formaldehyde by the action of ultra-violet light on aqueous solutiona of carbonic acid has been obtained by an independent method. The same compound, formhydroxamic acid, is produced by the action of ultra-violet light on aqueous solutions of carbonic acid containing potassium or calcium nitrite and on aqueous solutions of formaldehyde containing potassium or calcium nitrite, no reaction whatever taking place in the absence of light. Further, the same compound is formed when solutions of potassium nitrite in aqueous methanol or of calcium nitrite in absolute methanol are illuminated, it being well known that the action of ultra-violet light on methanol gives formaldehyde. I t is of some importance to note Lhat in the case of carbonic acid solutions containing potassium or calcium nitrite no ordinary formaldehyde is produced on exposure to light, the whole of it reacting to give the formhydroxamic acid-a fact which affords some additional evidence in favor of the direct production, not of ordinary, but of highly reactive formaldehyde from carbonic acid. Collateral evidence in support is also to be found in Loeb’s observation that by the action of the silent discharge on mixtures of hydrogen and carbon monoxide, compounds are
1017
formed that reduce Benedict’s solution. These observations have recently been confirmed in Liverpool, and it has been found possible, by the action of the silent discharge on mixtures of equal volumes of hydrogen and carbon monoxide, to prepare several grams of a thick sirup which strongly reduces Benedict’s solution. Although as yet no investigation has been made of the nature of these reducing compounds, their formation is strongly suggestive of the polymerization of formaldehyde as the first transient product of the action of the silent discharge. THEORIES OF CONVERSION BY PLANTS There still remains the question as to how the plant operates the process of conversion of the carbonic acid to sugars, etc., through formaldehyde in the absence of those ultra-violet rays employed in the laboratory. The suggestion has been made that if the carbonic acid is in a state of loose combination with a visibly colored base, the conversion into formaldehyde will be caused by visible light. The formation of formaldehyde in solutions of malachite green, saturated with carbon dioxide, in visible light was brought forward in support of this theory. It has been found, however, that malachite green itself gives ordinary formaldehyde on exposure to visible light, and consequently this experiment can no longer be used as an argument. On the other hand, this principle of what has been called photocatalysis has been established by other experiments involving nitrogen, but since this paper deals solely with the photosynthesis of carbohydrates no advantage would be gained by discussing these experiments in detail. EXPERIMENTAL DIFFICULTIES ENCOUNTERED Before entering on the next stage of this work, emphasis may again be laid on the experimental difficulties caused by the photochemical degradation of the photosynthesized products and also by their oxidation by the hydrogen peroxide formed by the action of ultra-violet light on water. The first difficulty is almost impossible to avoid, since, in the case of the reducing sugars, for instance, degradation is caused not only by short wave ultra-violet light with wave length 19Opp, but also by longer waves. I n general, the greater the complexity of a compound the longer the wave lengths that cause its decomposition. Thus, sugars are more readily decomposed by ultra-violet light than formaldehyde, as has been experimentally proved, and the smallness of the amount of reducing compounds found in exposed solutiops of carbonic acid can therefore be understood and appreciated. Until some effective method of selectively screening the light has been devised, this difficulty will always beset experiments on photosynthesis. The second difficulty-namely, the oxidation by hydrogen peroxide-has recently been partly overcome by immersing in the solutions during exposure an aluminium-platinum couple, which considerably reduces the effective concentration of the hydrogen peroxide.
FURTHER INVESTIGATION OF PROPERTIES OF FORMALDEHYDE AND ITS POLYMERIZATION PRODUCTS I n order to investigate more thoroughly the properties of the highly reactive formaldehyde, and in particular the products of its polymerization, concentrated aqueous solutions of formaldehyde have been exposed to ultra-violet light and considerable quantities of the reducing compounds have been prepared. Exhaustive experiments have been carried out to determine the best conditions for this polymerization, and one of the most important factors has been found to be
1018
INDUSTRIAL A N D ENGTNEERING CHEMIXTRY
the hydrogen-ion concentration. Little or no reducing sugars are formed if the solution is acid, and the yield of these compounds is also very much reduced in the presence of alkali. The optimum condition is obtained when the free acidity usually present in the commercial formaldehyde is neutralized by means of precipitated magnesium carbonate. During the photosynthesis of the reducing sugars the solution becomes acid, and it is necessary therefore to add from time to time during the operation quantities of the carbonate so that it is always present in excess. I n practice, however, it has been found that when the reducing power of the solution reaches about 2 per cent calculated as glucose, a secondary reaction sets in and magnesium carbonate is deposited on the walls of the quartz vessel on the side exposed to the light. This necessitates frequent washing of the apparatus and removal of the magnesium carbonate by acid, an operation which occupies considerable time. Although the yield of reducing sugars in a given time is less, it is more satisfactory to use precipitated calcium carbonate in place of the magnesium salt, since calcium carbonate is not photochemically deposited at any concentration of sugars yet reached. The hydrogen-ion concentration actually employed, therefore, is that of a saturated solution of calcium bicarbonate. The temperature of the mixture during photosynthesis is also an important factor, and it has been found that the optimum temperature is about 37" C. It is hardly necessary to say that blank experiments have been carried out with formaldehyde solution containing either magnesium or calcium carbonate. No reducing substance whatever is formed in the dark a t temperatures varying from 15" to 50" C.
LARGERSCALEEXPERIMENTS The optimum experimental conditions having been determined, it has been found possible to carry out the photosynthesis on a larger scale than in the test tube. For such experiments a rectangular glass tank 15 inches square and 8 inches deep with a circular hole 2l/4 inches in diameter cut in each of the four sides is used, the centers of these holes being about 21/2 inches above the bottom of the tank. By means of India rubber rings a quartz tube 2 inches in diameter and 6 inches long is fitted in each circular hole so that each tube projects into the tank. The tank is filled with 40 per cent formaldehyde solution, the quantity required being about 20 liters, and an electrically driven stirrer is mounted centrally in the tank. Precipitated calcium carbonate is then added and the whole is agitated until neutrality is established. A quartz mercury lamp is then pushed into each of the quartz tubes and by means of cooling coils the temperature is maintained a t 37" C. During the photosynthesis gentle stirring is maintained to prevent the liquid being rendered cloudy by the calcium carbonate. A very important factor is to be found in the absorptive power of the air layer within the quartz tubes. A considerable amount of ozone is formed by the rays of the lamp, and this is detrimental for two reasons. I n the first place the absorption band of ozone lies a t nearly the same wave length as that of formaldehyde, with the result that the ozone decreases the yield of reducing sugars. Furthermore the formation of ozone decreases the concentration of oxygen, with the result that the very short ultra-violet rays are transmitted and photochemically decompose the reducing sugars. The yield of sugars is materially increased by a draft of air forced into each quartz tube, and care must be taken not to maintain too rapid a current of air since in this case the efficiency of the lamps is markedly decreased by cooling. Reference has already been made to the development of acidity during the photosynthesis. This has been found to
Vol. 16, No. 10
be due to the production of glucuronic acid, but it is not known whether this is a direct product of photosynthesis or a secondary product arising from the hexoses. For the reasons previously given there is a limit to the reducing power that can be reached, in spite of the apparent optimum of experimental conditions. With an initial concentration of 40 per cent formaldehyde the maximum reducing power is 8 per cent calculated as glucose, and with 20 liters of formaldehyde this can often be reached after 14 days of continuous illumination. The working up of the exposed solutions is a somewhat lengthy procedure, and the most satisfactory method is as follows: The solution is evaporated under reduced pressure at 60' C. to as small a bulk as possible, and during this process excess of calcium carbonate must be added in order to neutralize the formic acid produced by the formaldehyde undergoing the Cannizaro reaction. Upon addition of absolute alcohol to the concentrated solution, the greater part of the calcium salts is precipitated and collected on a filter. The filtrate is once again concentrated to as small a bulk as possible under reduced pressure and again mixed with absolute alcohol, this process being several times repeated. Since the calcium salt of glucuronic acid is soluble in alcohol a special treatment is necessary. An equivalent amount of zinc sulfate in aqueous solution is added and after removal of the calcium sulfate the filtrate is again concentrated under reduced pressure. Several volumes of absolute alcohol are added when zinc glucuronate separates out. The filtrate is again Concentrated and again taken up with alcohol, this process being repeated three or four times. The last stage consists in the precipitation of the reducing compounds as a thick sirup by the addition of chloroform to the solution in absolute alcohol. The sirup is redissolved in absolute alcohol and reprecipitated until the product is free from formaldehyde. A minimum of three such operations is necessary. The final sirup when freed from chloroform is very thick and viscous, and has a marked sweet taste. It is yellow to pale brown in color, quite transparent, and fluorescent. I n reducing power different preparations vary, some having as much as 34 per cent and others only 25 per cent reducing power calculated as glucose. The most careful tests have established the complete absence of pentoses and trioses. On the other hand, as shown in the following paper, Principal Irvine and Mr. Francis have proved the presence of hexoses, both by glucoside formation and by exhaustive methylation. ~
Registration a t Ithaca Meeting of the American Chemical Society Alabama Arizona California Connecticut Delaware District of Columbia Florida Georgia Hawaiian Islands Illinois Indiana Iowa Kansas Kentucky Louisiana Maryland Massachusetts Michigan Minnesota Mississippi Missouri Montana Nebraska New Hampshire New Jersey New York North Carolina North Dakota Ohio
Men Women 1 1 5
11 14
43
1 2 3 15
1 1 41
12 13 1 2 9 22 64
21 5 2 9
5
1 3 2 1
6
8
3
1
3 2 70 329
4 1 79
2 18
54
1 6
Men Women 1 1 2 73 18 5 1 1
Oklahoma Oregon Philippine Islands Pennsylvania Rhode Island South Carolina Tennessee Texas Utah Virginia Washington West Virginia Wisconsin Belgium Brazil Canada China Denmark England Holland India Japan Russia Scotland Siam
4
4 1 5
2 4
5
3 15
3
1
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1 6 1 1 9 1 1 4
1 1 1
1 1 3 1
_ 163 . . . . ,, . . . .918 . 1081
TOTAL,.
