Potentiometric Determination of Exchangeable Hydrogen in Unsaturated Soils KENNETH A. MAEHL, 159 South Western Ave., Saint Paul, Minn.
A
SOIL is considered as being in a state of base unsaturation when its colloidal complex contains hydrogen which can be replaced b y other base metals. Base unsaturation has long been acknowledged b y soil scientists. Robinson (4) states that, under field conditions, unless a soil is in equilibrium with an excess of calcium carbonate, the content of exchangeable bases may fall below t h a t representing saturation. T h e total exchange capacity of a n unsaturated soil, t o be correct, must necessarily include the exchangeable hydrogen expressed as milligram equivalents in the same manner as the other exchangeable base metals.
Curve A , Figure 1, shows the relationship between the volume of acetic acid added and the rise in potential effected before and after the point of neutrality. Curve B shows the potential rise with each cubic centimeter of acid added. The maximum potential rise was obtained during the addition of the fourth, fifth, and sixth cubic centimeters of acid. The p H value of the solution just preceding the addition of acid in each case amounted to 7.19, 7.07, and 6.96, respectively. These data show that the greatest slope with uniform curvature will be obtained if the ammonium acetate solution is adjusted to a p H of 7.07 at the outset. Table I1 and Figure 2 show the rise in potential effected upon addition of known quantities of standardized acetic acid to ammonium acetate solutions of various strengths. I n each case the p H value of the ammonium acetate solution was adjusted to 7.07 with ammonium hydroxide before the titration was started. -4stock solution of 2 N ammonium acetate mas made u p by weighing out the salt and the p H value was adjusted to 7.07. Measured portions of this solution were then diluted with distilled water to obtain the various strengths desired. The p H value was again checked and adjusted to 7.07 as before. A very small quantity of ammonium hydroxide was required in the second case. The curve shown in Figure 3 was dereloped from a portion of the data shown for a normal solution in Table 11. The acetic acid added is calculated to milligram equivalents of exchangeable hydrogen present per 100 grams of soil when leaching a 25- and 50-gram sample. The data shown in Table I1 and Figure 2 present a very interesting study. At the outset, the rise in potential obtained per cubic centimeter of acid added varied in an inverse manner; however, as the titration was continued the rise in potential reached the same value in all cases. It is evident that solutions weaker than 1 Ai will give a larger potential rise per milligram equivalent of hydrogen exchanged. Although a 1 N solution was used by the author for all determinations made thus far, a solution weaker than 1 N can, no doubt, be employed to advantage on sandy soils having a low total exchange capacity. If a group of soils containing a large amount of exchangeable hydrogen are to be examined, only the upper portion of the curve will be used. I n this case a n advantage would be gained by adjusting the initial p H value of the ammonium acetate leaching solution to a point higher than 7.07 (Table I and Figure 1)
Development of the Method I n this method, t h e soil is treated with a solution of ammonium acetate which liberates the exchangeable hydrogen as acetic acid. Advantage is taken of t h e behavior t h a t a neutral ammonium acetate solution exhibits as acetic acid is added t o it. Data were first obtained t o enable the author to observe the difference in potential effected b y adding acetic acid t o a solution of ammonium acetate made alkaline with ammonium hydroxide at t h e outset. The details and operation of the glass electrode used t o measure the electrical potentials are given below. T h e data obtained are shown in Table I and Figure 1. b
TABLEI. BEHAVIOR OF ALKALINE AMMONIUM ACETATESOLD TION TITRATED POTENTIOMETRICALLY WITH ACETICACID [Basis, 500 00. of ammonium acetate solution (pH adjusted t o 7.45) ] Aoetio Aoid Cumulative Added E. M. F. Rise in E. M. F. Rise i n E. hl. F. co. Volt Volt Volt
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 8.00
0.0000 0.0048 0.0053 0.0058 0.0067 0.0070 0.0067 0.0061 0.0052 0.0048
The procedure adopted by the author was t o
digest a 25- or 50-gram sample of air-dried soil with 300 cc. of 1 N ammonium acetate solution having a pH of 7.07 for a period of at least 24
hours. The solution was then filtered from the soil by means of a Buchner funnel and transferred to a 500-cc. volumetric flask. During the filtering operation the Buchner funnel was covered with a watch glass to protect the ammonium acetate solution from the atmosphere of the laboratory. The soil on the filter was then washed with 50-cc. portions of the same ammonium acetate solution until a volume of 500 cc. was reached, care being taken not to allow the washing operation to proceed too rapidly. The solution in the volumetric flask was mixed well by shaking and a small portion used to obtain a voltage reading on the glass electrode. The milligram equivalents of exchangeable hydrogen present in the
24
JAKUARY 15. 1940
ANALYTICAL EDITION
25
solution containing a cation capable of entering the complex other than any present in a n exchangeable state originally. The amounts of exchangeable base metals thus removed are then determined quantitatively and expressed as milligram equivalents per 100 grams of soil. The total exchange capacity is determined by analyzing the soil quantitatively for the amount of cation placed on the complex and this is also expressed as milligram equivalents per 100 grams of soil. The amount of exchangeable hydrogen is then obtained by subtracting the former from the latter. 0.03 2 0
0.0300
a02 80 0.0260 0.024 0
g 0.0220 2
0.0200
'
O.Ol80
4
T
2
p
0,0160 0.0I40
2
0.01 2 0
2
0.0040
2
0.0020
TABLE11. BEHAVIOR OF NEUTRAL SOLUTIOKS OF AMMONIUM ACETATE TITR4TE.D POTENTIOMETRICALLY WITH ACETICACID [Basis, 500 cc. of ammonium acetate solution (pH adjusted to 7.07)] N N Solution 0.5 N Solution 0.1 N Solution Acetic Cumulative Cumulstive Cumulative nee In Rise in nee in Rise in rise in Acid Rise in Added e,m,f. e.m.f. e.m.f. e.m.f. e.m.f. e.m.f. Volt Volt Volt CC. Volt Volt Volt 0.00 0.0000 0.0000 0.0000 0.0000 0.0000 0,0000 1.oo 0.0073 0.0073 0.0125 0.0125 0.0370 0.0370 0.0153 2.00 0.0067 0.0140 0.0093 0.0218 0.0523 3.00 0.0058 0.0198 0.0073 0.0291 0.0097 0.0620 4.00 0.0046 0.0244 0,0057 0.0348 0.0070 0.0690 0.0076 0.0437 6.00 0,0320 0.0089 0.0103 0.0793 0.0067 8.00 0,0060 0.0380 0.0504 0.0072 0,0865 11.00 0.0065 0.0445 0.0078 0.0582 0.0082 0.0947 15.00 0.0072 0.0517 0.0073 0.0655 0.0080 0.1027 22.00 0.0091 0.0608 0.0095 0,0750 0,0098 0.1125 30.00 0.0075 0.0683 0.0075 0.0825 0,0082 0,1207 50.00 0.0130 0.0813 0.0133 0.0958 0.0130 0.1337
soil were then obtained from a curve similar to that shown in Figure 3. Since nothing was added or taken away from the solution being examined, it could be returned to the volumetric flask for use in the determination of the other exchangeable bases.
If a 25-gram sample of soil were treated as described above with 500 cc. of the 1 N ammonium acetate solution used in obtaining the data for the curves shown in Figure 3, and the potential rise obtained amounted to 0.0140 volt, the milligram equivalents of hydrogen exchanged per 100 grams of soil would be 8.0. A new curve should be made for each batch of ammonium acetate solution t h a t is made up, as a slight difference in the inital p H value affects the slope of the curve obtained. This fact is clearly brought out by Table I and Figure 1. A variation in the strength of the solution also has a n effect on the slope of the curve, as is shown b y Table I1 and Figure 2. Discussion Sumerous methods for this determination have been suggested and published in recent soil literature. These methods can be placed in two general classes. 1. The exchangeable hydrogen is determined by difference. The exchangeable bases and hydrogen existing on the colloidal complex are removed by treatment of the soil with a
s
'/
I
While methods of this type are conducive to good results when followed b y a competent analyst, they embody some difficulties and undesirable features. All the exchangeable bases must be quantitatively determined before the determination of hydrogen can be attempted. The excess exchange reagent must be completely removed from the soil without removing any of the ions on the exchange complex before the total exchange capacity can be determined. Any errors incurred in the analytical work are also reflected on the exchangeable hydrogen. Included in the methods of this type are those of Kelley and Brown ( 2 ) and some of those reviewed by Parker ( 3 ) . 2. The exchangeable hydrogen is determined directly. The exchangeable hydrogen together with the other exchangeable cations is brought into solution as before by treatment of the soil with a solution containing a suitable cation followed by a titration. I n a method of this type, the solution containing the exchange reagent must exhibit some buffering properties and hence the titration be carried out by means of a potentiometer and suitable electrodes. If this solution undergoes a marked change in pH through the liberation of hydrogen ions, some of the sesquioxides present may be attacked and the determination will run correspondingly low. If the leaching solution contains a measured amount of free base, the excess of which is to be determined after neutralization of the liberated hydrogen ions, some of the secondary
IKDUSTRIAL AND ENGINEERING CHEMISTRY
26
silica present will be attacked and the results obtained will be correspondingly high. Included in the methods of this type are those of Hissink (1) and Schollenberger (j), and some of the methods reviewed by Parker ( 3 ) . Schollenberger (5) advocates treatment of the soil with a neutral 1 fl solution of ammonium acetate and titration of the acetic acid formed with standardized ammonium hydroxide using a potentiometer and quinhydrone electrodes.
VOL. 12, NO. 1
TABLE111. COMPARISOX OF EXCHANGEABLE HYDROGEN AND PH VALUES Soil Depth Inches 0 to3.5 3.5 t o 9 9 t o 13 13 t o 17 17 t o 21 1 1 t o 25 25 t o 29 29 t o 33 33 t o 37
ic'"'?
Total ExchangeExchange able Capacity Hydrogen .Ifzlligram equivalents p e r
%
100 orams
60.1 41.3 47.0 47.5 47.7 47.3 46.0 45.8 39.0
Exohangeable Hydrogen
2.9 8.5 12.7 11.2 10.2 8.2 5.2 1.l
0.0
pH
4.8
2::
27.1 23.6 21.4 17.3 10.9 3.7 0.0
4.7 4.7 4.8 4.9 5.7 7.1 7.9
20.6
TABLE I V . COMPARISON OF POTEXTIOMETHIC TITRATIONS MADE CALOMEL HALF-CELLS SUBJECTED TO VARIOUSSEASONING PERIODS
WITH
[Basis, 500 cc. of S ammoniuin acetate solution ( p H value adjusted t o 7.07)] Calomel Half-Cell Immersed Calomel Half-Cell Immersed in N Ammonium Acetate in N Ammonium Acetate N Solution for 1 Day Solution for 12 Days Acetic Cumulative Cumulqtive Acid Rise in rise in rise In Rise in Added E.1n.f. e.m.f. e , m . f . E.ni.f. e.m.f. e.m,f. Cc , Volt VOlf Volt Volt Volt Volt 0.0000 0.0000 0.00 0 0390 0.0311 0.0000 0.0000 0,0079 0.0079 0,0587 0.0076 1.00 0 0469 0,0076 2.00 0 0538 0.0069 0.0148 0.0654 0.0143 0.0067 0.0055 0 , 0 2 0 3 0.0712 3.00 0 0593 0.0058 0.0201 0.0045 0.0248 0.0757 4.00 0 0638 0.0045 0.0246 0 , 0 0 7 8 0.0326 0.0834 0,0077 0.0323 6.00 0 0716 0 , 0 0 6 0 0.0386 0,0897 0.0063 8.00 0 0776 0.0386 0.0073 0.0459 0.0849 11.00 0.0966 0.0069 0.0455 0.0073 0.0532 0.1039 0,0922 15.00 0.0073 0.0528
PER
LP -
FIGURE 4. GLASSELECTRODE ASSEMBLY
The method described herein is similar to the latter mentioned in that the same exchange reagent is employed; however, the potentiometric titrations are carried out using a glass electrode and only one potentiometric titration is required for a large number of samples. I n Table I11 are shown the results obtained on the soils of a profile from the Red River Valley region in Minnesota. Note the agreement b e h e e n the p H values and the per cent of the total exchangeable material which was found to be hydrogen. A %-gram sample of soil was treated with a total of 500 cc. of 1iV ammonium acetate solution in each case.
Description and Operation of Equipment This discussion is limited to the glass electrode and potentiometric equipment used. All other equipment required can be found in an ordinary chemical laboratory. Figure 4 shows the glass electrode circuit used to produce the electrical potentials in the various solutions tested. The potentials were measured on a Leeds & Northrup type K potentiometer which was brought into balance with the aid of a Leeds & Northrup type R suspension galvanometer and light scale. The glass electrode was blown very thin and was of such sensitivity that a deflection of five spaces was obtained on the light scale (stationed at a distance of 120 em., 4 feet, from the galvanometer) when the potentiometer was thrown out of balance0.0010 volt. The glass electrode and two calomel half-cells were
mounted on a stand which was so constructed that the danger of breaking the electrode was eliminated. At first the glass electrode was filled with a saturated solution of potassium chloride. As evaporation took place, crystals of potassium chloride dropped t o the bottom. Further evaporation caused a growth of these crystals until eventually they punctured the side of the electrode. A period of about 18 months was the average life of an electrode of this type. The use of agar solution prevents the formation of crystals on the bottom of the electrode and seems to reinforce the thin glass membrane. No polarization difficulties were experienced. The calomel half-cell forming the liquid junction was made up using a saturated solut'ion of potassium chloride and agar, as is shown in Figure 3, but was immersed in a solution of 1 N ammonium acetate for some time before being put into service. In this way equilibrium conditions were established at the liquid junction, and the potentials were constant with no drifting. When not in use, this half-cell was kept immersed in a 1 N ammonium acetate solution. Until this procedure was adopted considerable difficulty was experienced. Table IV shows the potentiometric titration of an ammonium acetate solution when using a calomel half-cell which was immersed in 1S ammonium acetate solution for 24 hours and the same titration carried out on an equal volume of the same solution after the calomel half-cell had been immersed in 1 A; ammonium acetate solution for 12 days. The temperature of the solution was the same for both titrations. When the calomel half-cell was first immersed in the ammonium acetate solution, the potential of the system changed 0.0025 volt on standing for a period of 20 minutes. This change in potential was undoubtedly due to the diffusion of ions across the liquid junction. If any current was drawn from the cell a furbher change was produced. After the calomel half-cell was immersed for 24 hours the potential change on standing for 1 hour was very small and when small amounts of current were drawn from the system there was no apparent change. After the 12-day period the potentiometer could be thrown out of balance as much as 0.5 volt without changing the potential of the system. During the 11-day period between titrations, the initial potential of the system changed 0.0121 volt. The ammonium acetate solution was stored in
JANUARY 13. 1940
ANALYTICAL EDITION
27
permission to use the division’s potentiometric equipment and also to show a portion of the data on a soil profile prior to publication.
a well-stoppered bottle and did not change appreciably, its pH value being checked before each titration was started. K h e n taking readings, the potentiometer was brought into balance in the regular way; however, the zero point was always confirmed by throwing the potentiometer out of balance equal amounts on each side (usually 0.0010 volt) to note if equal deflections on the light scale were obtained. When once the system was set up, readings were obtained with no more effort than is required to weigh a sample of material on a Chain-omatic balance. Acknowledgment is given to F. J. Alway and C . 0. Rost of the Division of Soils, University Farm, Saint Paul, hlinn., for
Literature Cited (1) Hissink, D. G., Trans. Faraday SOC.,20, 551 (1924). (2) Kelley, TT. P., a n d B r o w n , S. SI.,“Proceedings a n d P a p e r s of F i r s t I n t e r n a t i o n a l Congress of Soil Science”, p. 491, 1928. (3) P a r k e r , F. W., Ibid., p. 164. (4) R o b i n s o n , G. W., “Soils, T h e i r Origin, C o n s t i t u t i o n a n d Classification”, 2 n d ed , p. 111, N e w York, D. V a n N o s t r a n d Co., 1936.
(5) Schollenberger, C. J., Soil Sci., 30, 161-73 (1930).
A Method for Determining Glutamine in Plant Tissues GEORGE W.PUCHER AND HUBERT BRADFORD VICKERY Connecticut Agricultural Experiment Station, New Haven, Conn.
Glutamine is converted to ammonia and pyrrolidone carboxylic acid by hydrolysis in neutral solution. Conditions have been ascertained under which this acid can be quantitatively extracted from the mixture of plant tissue components. On being hydrolyzed, pyrrolidone carboxylic acid is converted into glutamic acid; accordingly the increase in amino nitrogen during this operation furnishes a measure of the glutamine amide nitrogen originally present in the tissue. The details of a method to determine glutamine, founded on these reactions, have been developed and it is shown that satisfactory results are secured. The new procedure is not proposed as a substitute for the convenient and accurate amide hydrolysis method, but as a more specific means of estimating glutamine when present in small amounts or in especially unfavorable conditions, such as in the presence of a large excess of asparagine or other substances that might interfere with the simpler hydrolytic method.
T
HE method of determining glutamine, originally proposed
by Chibnall and Westall (1) and subsequently modified in certain details ( S ) , depends upon the complete hydrolysis of this somewhat unstable amide when it is heated to 100” C. for 2 hours at a reaction in the range p H 6 to 7 . The ammonia liberated under these conditions has been shown to be a reasonably trustworthy measure of the glutamine amide nitrogen present, and the method leaves little to be desired in most practical cases on the scores of convenience or precision. Severtheless there are other plant components that liberate ammonia, though usually in small amounts, under the same conditions. Urea and allantoin are two that are found in certain plant species, and asparagine, the other widely
,
distributed plant amide, is not entirely unaffected. Accordingly, if reliance is placed on the determination of the unstable amide nitrogen, the results may be misleadingly high in cases TI-here the proport’ionof glutamine present is unusually smal1,l and especially in cases where the t’issues are unusually rich in asparagine. Furthermore, no account is taken of the possibility that unknown plant components that also interfere with the method may be occasionally encountered. The development of a procedure which depends on a more specific property of glutamine than the hydrolysis of the unstable amide group therefore seemed desirable. The present method is not suggested as a substitute for the convenient amide hydrolysis method, but to be applied as a confirmation both qualitative and quantitative in cases of doubt, and especially when the proportion of glutamine present is unusually small. It is necessary to emphasize that no method of plant analysis short of the isolation of a characteristic crystalline derivative is thoroughly trustworthy in the present inadequate state of our knowledge of plant tissue composition. When glutamine is heated with water at pH 6 to 7, hydrolysis of the amide group and ring closure to pyrrolidone carboxylic acid take place. The liberation of ammonia is, so far as is known, precisely quantitative, but the ring closure may not be strictly so. The loss of amino nitrogen has been shown, horneyer, to be a t least 98 per cent, and the silver salt of pyrrolidone carboxylic acid has been isolated in an amount equivalent to nearly 90 per cent of that calculated from the amount of amide hydrolysis. The hydrolysate obtained was slightly colored, however, and a trace of brown flocculent precipitate separated on the addition of the first drop of silver nitrate. This suggests the presence of a small amount of some by-product of the reaction (3). Consideration of the solubility of the silver salt and the conditions under which i t was isolated indicates, however, that the amount of glutamine destroyed b y side reactions is, for most purposes, negligible. Pyrrolidone carboxylic acid is a relatively strong imino acid (pK = 3 . 3 2 , 6 ) that can be quantitatively extracted with ethyl acetate from aqueous solution buffered in the range p H 2 to 3. Furthermore i t is easily hydrolyzed, when heated for a 1 The petioles of rhubarb leaves furnish a case in point. It is shown in another communication ( 4 ) that the small amount of glutamine present was overestimated by the hydrolytic method since negative values for the calculated asparagine amide nitrogen were obtained. With the present method, the calculated asparagine amide nitrogen values were very emall but positive and there is reason t o suppose that they were in fact zero.
