AN APPARATUS FOR THE EXTRACTION OE GAS EROM SMALL AMOUNTS OE PLANT SAP OR TISSUE BY W. G. BURTON AND W. T. SPRAGG Low Temperature Research Station., Cambridge (With 2 figures in the text) An apparatus for the extraction of gas from plant tissue may be designed to remove the whole of the gas, dissolved as well as intercellular (Denny, 1946), or to sample only intercellular gas (Kidd, 1937; Wardlaw & Leonard, 1939; Smith, 1947). The apparatus described below is of the former type and was designed for use with sap or tissue of the order of i ml. in volume. The principle of the method extraction of the gas in a vacuum is simple and has often been used (see, for example, Magness, 1920; Brooks, 1937; Denny, 1946), but the use of small amounts of material necessitated modifications which avoided various sources of error which were not serious when the volume of extracted gas was large. Thus, for example, stopcocks were excluded from positions such that they might cause leaks into the evacuated chamber. A leak of the order of o-oooi ml./hr. might appreciably affect the results obtained. DESCRIPTION OF APPARATUS The apparatus is illustrated in Fig. i(a). It consisted of a tube about 20 cm. long and 1-2 cm. in internal diameter, sealed on to an extraction chamber {E) about 15 cm. long and 2-5 cm. in diameter. The top of the extraction chamber was closed by a valve (^4) immersed in mercury contained in a funnel (C) sealed on to the stem of the valve by Everett's wax (Z)). The lower end of the tube was bent smoothly at right angles and immersed in mercury (L) contained in a trough (A'^) scooped out of a I lb. block of B.D.H. paraffin wax. The hollow scooped in the block was just large enough at one end to contain the tube. At the other end it was made wider and deeper to permit the ready manipulation of cores of tissue under the surface of the mercury. The apparatus was connected to a mercury reservoir by means of pressure tubing attached to a side arm (7) leading into a trap (//) designed to avoid the possibility of air bubbles (which might leak through the pressure tubing) being carried into the apparatus with the mercury. In Fig. I the side arm is shown, for clarity, at right angles to its actual position which should be depicted perpendicular to the plane of the paper. The con- I. E.xtraction apparatus. struction of the valve is shown more clearly in Fig. \{h). A piece of capillary tubing (external diameter 6 mm.; bore i mm.) was drawn out to a thickness of about 2-5 mm.. New Phytol. 47, i ^
i8 W. G. BURTON AND W. T. SPRAGG bent over sharply at the constricted part, and cut in two (in the line OP) at the bend. The part retained for use as a valve was then ground down to the plane RS, on a No. 109 'Carborundum' sharpening stone, to give a smooth semi-cylindrical face. The capillary tubing as thus prepared was sealed on to the top of the extraction chamber. PREPARATION FOR USE The apparatus was cleaned with chromic acid, well rinsed with tap water and distilled water, and clamped in a vertical position with the bottom end under mercury in the trough {N), the stopcocks G and K being open. The open end was then closed with a rubber bung (M) and the mercury reservoir attached to the side arm (7). Clean mercury was allowed to run in slowly from the reservoir until it overfiowed from the open end of the capillary tubing at the top, stopcock G being turned off when the trap H was completely full of mercury. Mercury was then poured into the funnel C to the level B and stopcock K closed. A i cm. length of bicycle valve tubing was squeezed between the thumb and finger under the mercury in the funnel, and then slipped over the end of the capillar}' tubing, thus forming a valve similar in principle to a bicycle valve. The extraction chamber, and as much of the tube below it as possible, were rapidly evacuated and filled three or four times by lowering the reservoir with stopcock K open, closing the stopcock, raising the reservoir, and opening the stopcock to allow mercury tofiowinto the apparatus as quickly as possible until it squirted from the valve A. The extraction chamber was finally evacuated and left in this condition for at least as long as the time which the extraction of gas from the tissue or liquid was expected to take. During this period the stopcock K was closed and the reservoir placed in the 'raised' position to lessen the danger of air leakage through the pressure tubing. In practice we found it convenient to prepare the apparatus in the evening and leave it evacuated overnight ready for use the next day. METHOD OF GAS EXTRACTION A. From borings of potato tissue The reservoir was lowered and the stopcock K opened, thus transferring to the trap H any air which had leaked into the stopcock. The latter was then partially closed and the reservoir raised, the apparatus being thus slowly filled with mercury. The stopcock was then closed, and the bung M removed. A cork-borer (internal diameter i cm.),filledwith boiled water, and with no air bubbles adhering inside, was plunged through the tuber, held under boiled water and with no air bubbles adhering. The boring was expelled under mercury in the trough N, its ends were rejected, and a section about 1-1-5 cm. long was inserted into the open end of the apparatus and propelled up the tube by a length of steel wire, being prevented from going right to the top by the indentations F. The bung was then replaced, the stopcock K opened, and the mercury reservoir lowered until the extraction chamber was evacuated, after which the stopcock was closed and the reservoir raised. The time taken in our experiments between first plunging the cork-borer into the tuber and evacuating the extraction chamber was 45-60 sec. From the results given in Table i it will be seen that about 95% of, for example, the nitrogen was extracted in half an hour, and extraction was practically complete after 55 hr. The gas extracted was ejected from the apparatus by slightly opening the stopcock
An apparatus for extraction of gas frotn plant sap or tissue 19 K, and collected in a mercury-filled tube (Fig. i(c), T) held over the valve at an angle such that the valve tubing (f7) was pushed away from the orifice of the valve (see Fig. i (c)). It was then transferred over mercury to a Bonnier-Mangin gas analysis apparatus. Table i. Nitrogen extracted from potato tuber borings, held in vacuum for varying periods of time, expressed as a percentage of the volume extracted after 23-5 hr.* Time in vacuum (hr.) O'S 1-5 3-5 S-5 23-5 Boring no, I 948 965 979 99'S 1000 Percentage of volume Boring no, 2 94-7 95-7 97-1 99-6 loo-o Boring no. 3 95-3 96 3 98-1 996 ioo-o * Extraction for longer than 23-5 hr. produced no measurable increase in the volume. B. From liquids Before filling the apparatus with mercury and clearing the air from it, as under 'Preparation for use' above, two 4x1 cm. pieces of a 'Whatman' ashless filter tablet were inserted into the open end of the tube and pushed into the extraction chamber with a length of steel wire. Apart from this the apparatus was prepared as previously described and before use the stopcock K was cleared of air and the apparatus filled with mercury as under 'A' above. The stopcock was then closed, the valve tubing removed from the valve, and the mercury reservoir lowered to an intermediate position at about the level of the stopcock G. The liquid from which the gas was to be extracted was placed in a i ml. burette to the delivery end of which, held under the surface of part of the residue of the liquid, was attached a i cm. length of valve tubing. The tap of the burette was opened until a drop formed at the end of the valve tubing, then closed, and the free end of the tubing slipped over the valve of the extraction apparatus, slightly off centre to leave clear a channel from the burette to the valve orifice. The burette tap was opened and the desired amount of liquid drawn into the apparatus by manipulation of stopcock K. Burette and tubing were then disconnected with the burette tap still open. The stopcock K was opened sufficiently to draw a little mercury into the extraction chamber from the funnel C, thus clearing the liquid from the upper part of the valve, over which the original length of valve tubing was finally slipped, care being taken to expel all the air from it first. The mercury reservoir was then completely lowered and the stopcock K opened until the extraction chamber was emptied of mercury, when the stopcock was closed and the reservoir raised. The gas extracted was ejected as under ' A' above, but the liquid was retained by the filter tablets in which it had been absorbed when it was drawn back into the extraction chamber. Re-solution of the extracted gas and the introduction of liquid into the Bonnier-Mangin apparatus were thus avoided. We found an extraction period of 2 hr, to be adequate for the extraction of oxygen and nitrogen from the liquids we used (distilled water and potato sap). METHOD OF GAS ANALYSIS The gas extracted could be analysed with sufficient accuracy for our purpose in a Bonnier- Mangin apparatus (Aubert, 1891; Thoday, 1913). We used a tube with a bore of the order of I mm. (i cm. in length was equivalent to a vouimc of 0-00827 ''''' ) ^''"-' *"'j'' readings
20 W. G. BURTON AND W. T. SPRAGG with the aid of a watchmaker's lens. The oxygen-absorbent employed was a io% alkaline solution of 1-2-4 triacetoxybenzene. The results of analyses of small volumes of air obtained during routine calibration of the apparatus are given in Table 2. With such small volumes of gas the accuracy of the analysis is of course not so great as with larger volumes (see Thoday, 1913). Table 2. Analyses of small volumes of COo-free air in the Botinier-Mangin apparatus Vol. of sample (ml.) '"O= 1 (bs-dw.) Vol. of sample (ml.) 0 0 ' '^ " % N2 (by difr.) 0-075 0-043 0-039 0-037 0-031 0-029 20-S 79-2 20-9 79-1 20-4 79-6 20-7 1 79-3 20-6 1 79-4 20-4 1 79-6 0-028 0-025 0-025 0-020 0-017 0-016 20-9 20-8 20-4 20-8 20-7 21-2 79-1 79-2 79-6 79-2 79 3 78-8 ACCURACY OF THE METHOD The degree of accuracy obtained can be seen from Fig. 2, in which the circles represent the volumes of gas, corrected to N.T.P., extracted from i ml. of distilled water saturated with air at various temperatures. The continuous lines represent the values given in Landolt-Bornstein (1923), in the International Critical Tables (1928) and in the Handbook of Chemistry and Physics (1945). They are largely based on the results of Winkler, who used about 2 1. of water. 5 10 15 20 Temp. 'C. Fig. 2. Volumes of gas, at N.T.P., extracted from 1 ml. of distilled water saturated witb air at various temperatures. This work forms part of the programme of the Food Investigation Board of the Department of Scientific and Industrial Research.
An apparatus for extraction of gas from plant sap or tissue 21 REFERENCES AUBERT, M. E, (1891). Nouvel appareil de Mm. G. Bonnier et L. Mangin pour l'analyse des gaz. Rev. gen. Bot. 3, 97. BROOKS, C. (1937). An apparatus for the extraction of internal atmospheres from fruits and vegetables. Proc. Amer. Soc. hort. Sci. 35, 202. DENNY, F. E. (1946). Gas content of plant tissue and respiration measurements. Contr. Boyce Thompson Inst. 14, 257. Handbook of Chemistry and Physics (1945). Ed. C. D. Hodgman. Cleveland, Ohio: Chemical Rubber Publishing Co. 29th edn. 2640 pp. International Critical Tables of Numerical Data, Physics, Chemistry and Technology (1928). Ed. E. W. Washburn?f a/. Vol. Ill, 444 pp. New York: McGraw-Hill. KiDD, F. (1937). The internal atmosphere of potatoes. Rep. Food Invest. Bd., Lond., 1937, p. 179. LANDOLT-BORNSTEIN (1923). Physikalisch-Chemische Tabellen. 5th edn.. Vol. i, 784 pp. Berlin: Springer. MAGNESS, J. R, (1920). Composition of gases in intercellular spaces of apples and potatoes. Bot. Gaz. 70, 308. SMITH, W. H. (1947). A new method for the determination of the composition of the internal atmosphere of fleshy plant organs, Ann Bot., Lond. (N.S.), 11, 363. THODAY, D. (1913). On the capillary eudiometric apparatus of Bonnier and Mangin for the analysis of air in investigating the gaseous exchanges of plants. Ann. Bot., Lond., 27, 565. WARDLAW, C. W. & LEONARD, E. R. (1939). Studies m tropical fruits. IV. Methods in the investigation of respiration with special reference to the banana. Ann. Bot., Lond. (N.S.), 3, 27.