Determination of Nitrogen, Phosphorus, and Potassium in LeaF Tissue Application of Micromethods ROBERT H. COTTON’ Laboratory of Plant Nutrition, Department of Horticulture, The Pennsylvania State College, State College, Pa. REAGENTS
Seeking procedures of approximately the same accuracy as A.0.A.C. macromethods, attempts have been made to duplicate the Lindner and Harley nitrogen determination and eliminate its sources of difficulty. The Firke and Subbarow colorimetric procedure for blood phosphorus has been adapted to leaf analysis. Several methods of determining phosphorus are compared and fundamental data are presented on colorimetric phosphorus determinations i n which molybdic acid is used. The dipicrylamine micromethod for determining potassium i n leaf tissue is more time-consuming than the turbidimetric cobaltinitrite procedure, but gives relatively high precision.
Concentrated sulfuric acid. Xessler’s reagent, prepared preferably according to Vanselow (31) or standard texts (16). Hydrogen peroxide, 30y0. Baker’s analyzed. Sodium hydroxide, 2.5 N . Sodium silicate, 10%. Ammonium molybdate, 5%. 1, 2, 4-Aminonaphtholsulfonic acid (0.5 gram of there crystallized acid). Shake with 30 grams of sodium bisulfite and 6 grams of crystalline sodium sulfite with enough water to make the volume 250 ml. Filter clear and make up every 2 weeks. Lithium dipicrylaminate, 0.67,, saturated with potassium dipicrylaminate.
T
HROUGHOUT the world interest is great and ever-increasing in leaf analysis as a means of studying the effect of varying cultural and fertilizer practices upon the nutrition and yield of plants (11, 18, 19, 86, 29). Leaf analysis is being used also to discover gross mineral deficiencies (36). The method of foliar diagnosis developed by Thomas (26) and Thomas and Mack (27] 88) is extremely valuable in fundamental studies, and being quantitative, requires analyses of considerable accuracy. The objective of the present study was to find procedures of approximately the same accuracy as the traditional A.O.A.C. macromethods, even if their speed should be somewhat less than that of the so-called rapid tests. The samples of leaf tissue analyzed in this study were taken from field experiments of the Department of Horticulture. I n 1942 Lindner and Harley (19) reported the successful adaptation of wet-ashing and subsequent nesslerization to the determination of nitrogen in leaf tissues. The solution resulting from the ashing of 0.1 gram of dried leaf, it was felt, should be of use &s a parent solution from which aliquots could be taken for the analysis of phosphorus pentoxide and the other mineral elements. Toward the end of this study, Lindner (18) reported a scheme for doing this. The present paper deals with attempts to duplicate the Lindiier and Harley nitrogen determination and to eliminate the sources of di5culty in the method as described (19). It also presents an adaptation of the Fiske and Subbarow colorimetric procedure for blood phosphorus to leaf analysis, together with a comparison of several methods of determining phosphorus, and some fundamental data pertinent to all colorimetric phosphorus determinations in which molybdic acid is used. Finally, data are presented which show for the first time the use of dipicrylamine in the determination of potassium in leaf tissue, employing the procedure developed by Amdur ( 2 ) for synthetic solutions. Several “rapid test” procedures developed primarily for soil analysi3 can also be used for leaf tissue. Those of Merkle (80) and Morgan (61) are widely used and are of great value. The degree of accuracy desired and obtained in these procedures is relatively low compared with the requirements of foliar diagnosis, although adequate for soil testing. Wolf (35) has recently described leaf analyses, using as a starting point the Lindner and Harley (19) wet-ashing technique, but here again the agreement with A.O.A.C. procedures is only fair. 1
Present address, Natural Research Corporation, Plymouth, Fla.
734
APPARATUS
A photoelectric colorimeter, calibrated colorimeter tubes,
light filters transmitting a t 470 and 660 millimicrons wave length, volumetric flasks of 100- and 50-ml. capacity, 2 dropping bottles and pipets, and conical flasks of 50-ml. capacity. PROCEDURE
WET-ASHING. Place a leaf sample of 0.1 gram of dried and ground material or the equivalent of fresh material in a 50-ml. Erlenmeyer flask. Two samples can be conveniently run as a unit. Add 2 ml. of concentrated sulfuric acid, swirl the vessel, and allow it to stand about 10 minutes. Then place it upon a wire gauzeasbestos screen through which a hole 2.5 cm. (1 inch) in diameter has been cut. Five centimeters (2 inches) below this is a gas flame, blue in color, 1.25 cm. (0.5 inch) in height. Heat 3 minutes over the low flame; dense fumes similar to cigaret smoke will come off a t this point. Raise the flame to 1 inch in height and continue the heating 2 minutes. Allow the sample to cool; add 0.5 ml. of 307, hydrogen peroxide to the flask. Place clver a 0.5-inch flame until bubbling begins; remove until bubbling ceases and then replace over the flame. Once the hydrogen peroxide has stopped bubbling in the flask, raise the flame and allow the mixture to come to a boil. During this period the solution first becomes somewhat lighter and then much darker. Remove the flask, cool to room temperature, add 5 drops of 30% hydrogen peroxide, and place the flask over a 0.5-inch flame again. The 5 drops correspond to 0.25 ml. Again remove the flask from the flame when bubbling begins and replace it when bubbling ceases. When bubbling ceases raise the flame and heat to boiling. If the solution is yellow add 3 drops of hydrogen peroxide once the mixture has been cooled; if darker add 5 drops, Repeat the heating as before and if the resulting solution is not nearly colorless repeat the cooling, adding of peroxide, and then heating once more. When colorless or nearly so boil for 3 minutes to expel all the peroxide. Cool, add about 10 ml. of distilled water, and a few minutes later rinse into a 100-ml. volumetric flask and make to the mark. This constitutes the parent solution from which aliquots are taken for subsequent analysis. A reagent blank is carried along a t the same time. XITROGEN DETErwINATIoiv. For estimation of total nitrogen which is now in the form of ammonium sulfate, pipet a 5-ml. aliquot into a 50-ml. volumetric flask, add 1 ml. of 2.5 S sodium hydroxide and 1 ml. of 10% sodium silicate, make to volume with distilled water, and thoroughly mix. If a Klett-Summerson photoelectric colorimeter is to be used,. pour out 5 ml. of the above solution in a colorimeter tube, using the 5-ml. calibration on the tube as a guide. Also measure out standard solutions having from 1 to 5 parts per million; two standards will sometimes suffice when operating in a range in which the standard curve is a straight line. When all the samples are measured out add to each 4 drops of Nessler’s reagent, shaking after the addition of each, drop. The Nessler’s reagent is kept in a dropping bottle dehvenng 55 drops
November, 1945
ANALYTICAL EDITION
per ml. Read in about 10 minutes in the colorimeter, using a blue filter. A filter having maximum transmission at 470 millimicrons wave length is satisfactory. The amount of nitrogen present is read from the curve. Colorimeter tubes should be calibrated for light transmittance against each other while containing distilled water and the appropriate corrections applied when reading samples in the colorimeter. PHOSPHORUS DETERMINATION.Phosphorus determinations are carried out upon 5-ml. aliquots of the parent solution which are poured directly into the colorimeter tubes, as are standards having from 1 t o 10 p.p.m. of P106. To the standards add 11 drops of concentrated sulfuric acid from a dropping bottle (55 drops per ml.); t o the leaf sample aliquots add 5 drops. From a 25- or 50-ml. buret add 1 ml. of a 5% solution of ammonium molybdate, shake vigorously, and then add from a 2-ml. buret 0.25 ml. of 1,2,4-aminonaphtholsulfonic acid prepared as per King (13). Shake well again. The two reagents should be made up fresh every few weeks. Allow the tubes to stand about 30 minutes and read in a colorimeter using a red filter; one having maximum light transmission at 660 millimicrons is satisfactory. Usually two or three standards will be adequate, since the standard curve is straight from 0 to 10 p.p.m. of PoOs. POTASSIUM DETERMINATION. While the nitrogen determination is being carried out have 10-ml. aliquots of the parent solution evaporating in 30-ml. Vycor crucibles on the steam cups. When the volume is down to approximately 0.5 ml. set aside the crucibles for determination of potassium or place them immediately on a hot plate or on a wire gauze-asbestos pad over a low flame in a hood. Allow to heat a t a rate sufficient t o produce fuming but not spattering. When fuming has almost ceased, rotate the individual crucibles over a free flame approximatelv 7.5 cm. (3 inches) high having a cone an inch high, to drive off sulfuric acid and ammonium sulfate adhering to the sides of the vessel. Avoid producing more than a dull red heat in the crucible; usually no apparent redness is needed. It is not essential that the residue be white. When the vessel contents are dry and cooled somewhat, add hot distilled water to wash any potassium sulfate down the sides, so that all the potash will be within 0.93 to 1.25 cm. (0.375 to 0.5 inch) from the bottom. A rubber policeman may be helpful here. When evaporated over a steam cup to dryness add exactly 1 ml. of lithium dipicrylaminate solution of Amdur ( 2 ) to each sample and to a series of evaporated standards containing from 0.1 to 0.6 mg. of K 2 0 . Allow to stand 3 hours a t a relatively constant temperature. At the end of this time draw off from each of the standards and samples 0.4 ml. of the clear supernatant reagent and dilute with water to 100 ml. Read in a photoelectric colorimeter, using the same filter used in the nitrogen determinations. When one knows the range of potash content in the samples two standards are usually sufficient. It is essential that there be no ammonia fumes in the laboratory while the nitrogen determinations are being carried out and after the potash determinations have gone as far as the completion of the heating process. Ammonium ion forms an insoluble salt with dipicrylamine, as does potassium.
Teble
73 5
I Comparison of M i c r o and Macro (Kjeldahl) Analyses Kjeldahl
Sample Sweet corn, 7-13-43 Plot 1 Plot 2 Plot 3 Plot 4
Nitrogen,
%
3.83 3.76 3.55 3.53 3.39 3.34 3.70 3.57 3.63 2.24 4.77 2.08 2.00 2.40
3.89 3.77 3.53 3.60 3.37 3.39 3.64 3.53
.. .. .. .. .. ..
Lindner ,and Harley Nitro.sen, % 3.73 3.87 3.63
,.
3:49 3.83 3.60 3.54 2.20 4.80 2.11 2.15 2.42
3:95
.. ..
3:49 3.65 3.61 3.49 2.27
.. ..
2:52
2 minutes should drive off hydrochloric acid which is very volatile, and which arises from the action of the sulfuric acid on the chloride present in the leaf. Its odor is distinct a t the start of the wet-ashing. The procedure outlined above was evolved after examining each step of the Lindner and Harley method. For the sake of brevity only a few observations will be noted. For example, King (IS) in wet-ashing blood with hydrogen peroxide specifies a 3-minute final boiling in connection with subsequent P20s determination. That residual hydrogen peroxide can cause a large error was shown by the addition of one drop to a cooled wetashed solution; the deviation from the Kjeldahl nitrogen value was 40%. The present procedure, used on a corn leaf sample having a Kjeldahl nitrogen value of 3.35%, gave for 10 completely separate replicates a nitrogen percentage of 3.64 with an average deviation of 0.044. This degree of accuracy was deemed adequate for the uses proposed for the rapid micromethod. Table I gives analyses by the rapid method and the corresponding Kjeldahl analyses. The sample designations in the tables conform with those in (89, SO).
The standard ammonium sulfate solution was analyzed by distillation into 5/14 N sulfuric acid, standardized gravimetrically, and back-titrated with 1/14 N ammonium hydroxide in order that the macro and micro results might be on the same basis. The principal modifications of the Lindner and Harley procedure as published were (1) allowing the leaf and sulfuric acid to stand together a t least 10 minutes before heating, ( 2 ) specifyDISCUSSION OF WET-ASHINQ AND XITROGEN DETERMINATION. ing more precisely the initial heating technique including raising The wet-ashing technique and the method of determining nitrothe flame after 3 minutes, (3) taking care to have the hydrogen gen are essentially those described by Lindner and Harley (19). peroxide react and bubble off at the minimum temperature posThe agreement reported by Lindner and Harley between nesssible, (4) following this by heating to boiling each time hydrogen lerization technique, “rapid method”, and the Kjeldahl properoxide is added, and finally ( 5 ) boiling the wet-ashed solution cedure was usually very close, within several per cent of the total, for 3 minutes as the last step in the wet-ashing procedure. Point while an error of 7.47, of the total was the largest they reported. (4) was confirmed by Lindner (17), who also states that dried, In the early stages of the present work, seven replicates of ground leaf takes longer to ash than fresh leaf tissue. tomato leaf, tier 2, plot 5 , 15th leaf, sampled August 1, 1941 (30), Several notes about the colorimetric procedure used in deterKjeldahl nitrogen analysis of which wias 2.87%, gave an avermining the ammonia are pertinent here. age value of 2.48, or a value 10% low, with an average deviation of 0.11. These data were obtained by scrupulously following the A Klett-Summerson photoelectric colorimeter was used with a procedure of Lindner and Harley. The wet-ashing took several blue filter having maximum transmission at 470 millimicrons. hours for six samples or about twice as long as Lindner and The standard curve obtained with pure ammonium sulfate was a Harley specify. straight line between 0 and 4 p.p.m. The colorimeter tubes supplied with the instrument are already Nitrate was estimated a t each step of the procedure v i t h dicalibrated and if the mark placed on them faces the operator, phenylamine prepared according to Merkle (20). These tests variations in readings due t o optical variations inherent in the showed that losses of nitrate, if any, must have occurred at the tubes are supposed to be negligible. An experimental test of start of the wet-mhing. Very cautious heating a t the first stage several tubes revealed differences among them as great as 7 units on the colorimeter scale. Since most determinations are carried of the procedure therefore was adopted. Anderson (3) has shown out between 0 and 200 on the scale, the magnitude of the error is that the driving off of hydrochloric acid a t this point is an adconsiderable. vantage, since ammonia can be oxidized t o nitrogen and driven Calibration is carried out easily as follows: The instrument is off in the presence of an oxidizing agent. The strong heating for set a t 10 with the filter to be used and with a tube filled with dis-
736
INDUSTRIAL A N D ENGINEERING CHEMISTRY
tilled water or the colored solution ordinarily used. The other tubes are read and the correction is written on the tube with a diamond or Carborundum pencil. Corrections were found to be minimized if the Klett marking was ignored and the tube rotated in the instrument until its reading was a minimum. The correction written on the part of the tube facing the operator then allows him always to place it in this position. The corrections change but little if the tubes are handled carefully and not scratched. Work with an Evelyn photoelectric colorimeter also lias shown the necessity for tube calibrations. The Sessler’s reagent was prepared according to Vanselow (51). Four drops from a dropping bottle delivering 55 drops per
ml. wrrr adequate if the tube c o n t h t s xere mixed after each drop. The Klett tubes are calibrated at 5 and 10 ml. One can pour out the 5-ml. aliquot for nesslerization, using the calibration as a guide rather than pipetting out 5 ml. This represents a great saving in time. A few drops more or less than 5 ml. tvere found to make no detectable difference in the reading. The 2.0 ml. of 2.5 ,I’ sodium hydroxide used to neutralize 10 uil. of the xet-ashed solution are not critical; 1.5 ml. gave no difference in the final reading nor in the pH of 11.8 after adding the Sessler’s reagent as measured by a glass electrode and a 1,eeds & Sorthrup potentiometer-electrometer. In a series of more than four determinations the zero setting of t,he instrument for the blank or distilled water may change slightly and this has been observed to cause errors of several units on the scale. I t is a necessary precaution, therefore, to check this after every 4 to 6 readings. PHOSPHORUS DETERMINATION
Vol. 17, No. 11
The concentration of sulfuric acid present in the Lindner and Harley solution was found by titration to be approximately 0.64 N . With this normality no stable readings could be obtained with 5% ammonium molybdate and sulfonic acid prepared according to King. Figure 1 shows colorimeter readings plotted against sulfuric acid normality of the solution being tested. Between 0.8 and 1.65 N the readings are constant: therefore, if one adds enough drops of concentrated sulfuric acid to obtain a normality of 1.3 A;, slight differences in concentration of sulfuric acid obtained will have no effect on the reading, whether caused by differences in amount of acid lost in wet-ashing or by differences in size of drops of sulfuric acid from the dropping bottle. Woods and Mellon (36) found, using aminonaphtholsulfonic acid prepared somewhat differently, that colorimeter readings w r e approximately constant between 0.6 and 1.3 X sulfuric acid. Fontaine (IO) found that variation between 1.7 and 2.1 S in sulfuric acid concentration caused less than 2% difference in light transmittance when stannous chloride was the reducing agent; they recommended 1.9 as the approximate normality. Berenblum and Chain ( 6 )found that a normality of 1.1 was necessary to prevent the appearance of color with no P205 in the solution, and they recommended this normality. The use of sulfonic acid allows a much wider range of acid concentration than does Fontaine’s method, as can be seen from Figure 1. Sulfonic acid reacts at higher concentrations of P20a than does stannous chloride, however, which in the present case coincides with the range found in leaves in this laboratory, approximately 0.2 to 0.6%, or 2 to 6 p.p.m. of Pa05 in the diluted Polution of the leaf prepared by the Lindner and Harley method. When colorimeter readings are plotted against time of standing, after addition of the reagents, for several concentrations of P205 it is seen that the rate of change of colorimeter readings with time varies up to 25 minutes, after which it is constant. For thiq reason, leaf samples may be read in comparison with the standards, which have been standing under the same conditions, at any time betneen 25 and 35 minutes after the reagents are mixed. The results will be identical whether readings are made after 25 or 35 minutes, even though the colorimeter readings have changed, because the standards have changed at the same proportionate rate as has the sample. Table I1 gives a comparison of results from the sulfonic acid micromethod as just described and those from the volumetric
Quantitative colorimetric estimation of phosphorus present in microquantities is carried out almost universally by the molybdenum blue reaction. Denigks ( 7 ) described the method in 1920 and numerous modifications have appeared since then, many of which have been discussed by Zinzadze (37). The probable reaction is as follows: Orthophosphate ions catalyze the reduction of soluble molybdates by stannous chloride or other reducing agents to a blue complex having molybdenum present in 5- and 6valent forms (5, 16). This view conflicts viith earlier assumptions which can be found in textbooks on colorimetry that metallic tin, stannous chloride, hydroquinone, or naphtholsulfonic acid “specifically” reduces ammonium phosphomolybdate to the n,olybdenum blue complex. Zinzadze (5;) has shown that the reduction can take place in the absence of phosphate; Berenblum and Chain (6) contend that any reducing agent \vi11 produce the blue color if given wfficient time. As is pointed out belox, changes in the sulfuric acid normality change markedly both the reaction rate and equilibrium of the reaction. The molybdenum blue‘ reaction was used in this study because it is sensitive to very small amounts of phosphorus and the determination on an aliquot of the viet-ashed material is very rapid. King’s procedure (13) for blood analysis was chosen, in which 1,2,4-aminonaphtholsulfonic acid is used as the reducing agent, as originated by Fisk and Subbarow (9). Fiske and Subbarow showed that this reducing agent is far less sensitive to interfering compounds than is stannous chloride. King used a greater sulfuric acid content and an improved method of making up the aminonaphtholsulfonic acid. \\ Fudge (11) presented data on phosphorus conI 1 1 1 1 1 4 1 I I tent of leaves obtained by means of the King 0 .2 .4 Ib 2.0 2.2 2 4 2.6 2.8 30 3.2 procedure, but without details as to the way in which the method was adapted t o leaf material. Figure 1. Effect of Sulfuric A c i d Normalib on Molybdenum BlueComplex Color
.
ANALYTICAL EDITION
November, 1945 Table II.
Sample Corn 7-13-43 Plot 1 Plot 2 Plot 3
PzO6 Analyses as Percentage of VoluGravimetric metric A.O.A.C. A.O.A.C. 0.570 0.572 0.548
0.551 0.547
....
....
Plot 4
0.499
Plot 5
0,535 0.542
Plot 6 Plot 7 Corn 8-3-43 Plot 3 Plot 7
0.547 0.605 0.602 ,,
..
0.612 0.447
0.449 0 535 0.525
..
. .. . . ,
.. .. ., 0.54; ,,.. ,,
,,
..
0,523 0,517
.., .. .. .. ,. .. . . ,
0,612 0,606
Sulfonic Acid Micromethod 0.574 0.584 0.547 0.550 0.520
0.532 0,532 0,510 0.520
0.525 0.495 0.616 0.629 0.595
....
0.612
0.610
0,453 0.451 0.529 0.525
0.455
....
0.535 0.555
Dry Matter
Stannous Chloride A.O.A.C. MicroMicromethod method
.... .. . . .... ...,
0.587
.. .. 0.54
0.537
0.57 0.55
.... ....
0.560
....
0.530 0.550
0.62 0.63
0.600
0.66
0.608
.... .... ....
....
series of 28 analyses to the tabulation of the percentages and this included the time to plot the standard curve. DETERMINATION OF POTASSIUM
The accurate determination of potassium (expressed here as the oxide, KzO, or potash) in biological material is far more difficult than that of any other major element of the leaf.
....
....
.... ... . ...,
.... ....
137
I...
....
.... .... ,...
....
0 525
,...
procedure of the Association of Official Agricultural Chemists (4). Also included are 10 gravimetric analyses, A.O.A.C. procedure, run as a check on the volumetric method. For eight of the nine samples studied the A.O.A.C. micromethod also was carried out’. This procedure is a colorimetric one, differing from the sulfonic acid procedure primarily in the reducing agent which is hydroquinone. 4 2-ml. aliquot of a dry-ashed leaf solution having d grams of dry leaf in 250 ml. was needed, or 40 mg.; it thus recluires eight times as large a sample as the sulfonic acid method. The sulfonic acid micromethod and the macromethods agreed within 3.5% or less of the total except for leaves from one plot where the former was 6.2% of the total lower than the latter. In five cases out of the nine, the agreement was within 2% of the total. Data are given in Table I1 also for analyses by the Lindner recommendations, which require more manipulation and the use of stannous chloride which is inherently more sensit’ive but less accurate than sulfonic acid. Before controlling the acid concentration the results were erratic. The volumetric and gravimetric methods agreed reasonably well. Standard PzOs solution, 6 p.p.m., whose REPRODUCIBILITY. concentration was checked volumetrically and gravimetrically, was analyzed with 10 replicates; the lowest value was 5.92 and, the highest 6.12 with an average deviation of 0.04 p.p.m. or about 0.7Yc of the total. Two separate solutions were made of a sample of tomato leaf tissue, plot 7, 8-3-43. From each, 10 aliquots were analyzed, giving for the first 0.536 * 0.005 and the second 0.529 * 0.0057c of PzOsin the leaf, or average deviations of less than lyc of the t,otal. The averages differed by 1.1% of the total. Wet-ashed solutions allowed to stand six months to a year in Kimble Exax volumetric flasks were observed to give values from 10 to 20% of the total too high. The use of sodium bisulfite t o reduce arsenates to arsenites, which is advisable if arsenates are known to be present (23),was found to reduce t.his error. Long standing, therefore, probably resulted in dissolving some arsenic from the glass. To reduce the arsenate in the solution t o arsenite, approximately 0.03 gram of crystalline sodium bisulfite was added to D 5-ml. aliquot, heated to 80” C. for a t least 30 minutes, and cooled, and the determination carried out in the usual way. Standard PsO6 solutions were kept in old volumetric flasks which had been aged with hot potassium dichromate-sulfuric acid cleaning solution over a steam bath for a t least 24 hours. The cleaning solution is effective in reducing arsenate interference (37). Once the wet-ashed extracts are prepared, the sulfonic acid t.echnique is rapid. Exactly 2 hours elapsed from the start of a
The Lindo-Cladding method employing platinic chloride, which is the official method of the Association of Ofiicial Agricultural Chemists, is expensive and very time-consuming, and loses accuracy when applied to microquantities. The cobaltinitrite reagent of Adie and Wood (1) is widely used rather than platinic chloride since it is cheaper and quicker. I t suffers from the disadvantage of variability of composition of precipitate formed but can be made to give comparative results under controlled conditions. Perchloric acid sometimes is used as a precipitating reagent. Fales and Kenny (8) state that results obtained are not so precise as those by the platinic chloride method. The possibility of dangerous explosions probably prrvents its more widespread use. In the realm of microchemistry applied to plants and soils the cobaltinitrite reagent is virtually the only method used. Alcohol, usually ethyl or isopropyl, is added t o a vial containing the solution of the sample to be analyzed and sodium cobaltinitrite. This causes turbidity which is compared visually or photometrically with standards similarly treated (6). For more precise work and usually for semimicroquantities, the potassium cobaltinitrite precipitate is separated by filtration or centrifugat)ion and then can be measured colorimetrically by its reaction with a colored oxidizing agent (34) or by titration with an oxidizing agent (14) or with nitroso It salt (26). Klein and Jacobi (14) have described a procedure for determining 0.1 t o 0.2 mg. of potassium by a titration of silver potassium cobaltinitrite. Chloride and phosphate ions interfere and must be removed, a considerable drawback in leaf analysis. Peech and English (22) have recently examined the turbidimetric cobaltinitrite procedure. They point out that “:iuide from variabilit’y in the composition of the precipitate due to possible substitution of potassium and ammonium ions, the apparent turbidity is markedly influenced by particle size and crystti1 form of the precipitate”, and that considerable practice is essential to obtain precision. They did not try their method on leaf tissue. tindner ( I S ) used the turbidimetric procedure in his scheme O F analysis of leaf material. Some measure of the variability t o be expected with the method is given in his Table 11, LLrecoveryof potassium from pure solutions of potassium chloride”. With pure salts he reports variations as great as *S% of the total for a sample of 25 micrograms (0.025 mg.) and *2Yc of the t&:il for 0.2 mg. Percentage recovery of potassium added to leaf niatrrial varied from 94 to 108. Several attempts by the author to obtain a reasonably low average deviation using the cobaltinitrite turbidimetric methods on series of replicates of wet-ashed leaves were failures. O d y a few of the 55 determinations gave values in agreement with thoie obtained by the platinic chloride or macro cobaltinitrite procedure. The reproducibility was too low and the procedure ab abandoned. Many more determinations would have to be made before the accuracy of which the method apparently is capable could be achieved. Kolthoff and Bendix (16) were the first Americans to describe the use of dipicrylamine, hexanitrodiphenylamine, in the quantitative estimation of potassium. They credit Poluektoff (24) with being the first t o use a solution of the sodium salt of dipicrylamine as a reagent for potassium. They say that thallous thallium, beryllium, zirconium, lead, and mercuric mercury have been found to give colored crystalline precipitates with sodium dipicrylaminate. Aluminum, ferric iron, chromic chromium, nickel, cobalt, copper, bismuth, vanadium, titanium, thorium, and mercurous mercury are said to give amorphous precipitates. Kolthoff and Bendix then say: “The reagent has an alkaline reaction and the last-mentioned group of cations should yield a precipitate which may consist of the hydrous oxide or some basic salt”. Amdur (2) attempted to use the Kolthoff and Bendix methods on blood samples and found that an indirect colorimetric procedure using the lithium salt of dipicrylamine gave greater precision with the pure salts and mixtures of salts analyzed. Harrington
738
INDUSTRIAL AND ENGINEERING CHEMISTRY
(12)independently worked out a method using lithium dipicrylaminate for the determination of potassium in blood. Veigle (32) also used lithium dipicrylaminate for the same purpose and gives a procedure which is apparently as accurate as Harrington’s and yet more rapid. Harrington and Veigle used a direct procedure in which the potassium salt of dipicrylamine was precipitated, washed, dissolved in an organic solvent, and read colorimetrically. The procedure of Amdur (2) is relatively simple and rapid and since i t is indirect and the precipitation is carried out in an alkaline medium, interference from the last group of elements listed above by Kolthoff should be eliminated. The method proposed here is fundamentally similar t o that of Harrington (19). The procedure calls for the removal of 0.4 ml. of the supernatent liquid by means of a 0.4-ml. pipet with the tip covered by filter paper. It is best to calibrate 6 pipets carefully for this purpose, so that they will be identical. An alternative scheme for the last step is to use dry Pyrex filter sticks and t o draw the supernatant liquid into a dry tapered test tube b suction. Enough liquid is brought over so that one can rinse d e pipet three times and still obtain 0.4 ml. for dilution to 100 ml. I n this way the same pipet may be used for all samples and standards, eliminating the need to calibrate six pipets.
Vol. 17, No. 11
and 5% of K 2 0in the leaf if one were using a 10-ml. ali uot of the Lindner and Harley solution) showed no appreciable 8ifferences between those having and those not having added salts. The standard curve changes somewhat from day t o day, so that it is essential t o run two standards along with the samples being analyzed. The graph is almost, but not quite, perfectly straight between 0.1 and 0.6 mg. of KzO,within which range came all the leaves examined. For samples having less than 1% K20 one can use either a larger aliquot, say 15 or 20 ml., or make up a more dilute reagent than the 0.6% specified by Amdur. Samples ranging between 1 and 2% KzO were analyzed satisfactorily by the present method, as shown by Table 111. The dipicrylamine micromethod for potash determination in leaf material takes more time for a determination than does the turbidimetric cobaltinitrite procedure. The relatively high precision obtained with it, however, warrants its serious consideration in a procedure for reasonably rapid microanalysis of leaf material. ACKNOWLEDGMENT
Table I11 gives a comparison of results obtained by this procedure and those obtained by the A.O.A.C. Lindo-Gladding platinic chloride method or the volumetric cobaltinitrite procedure of Yolk and Truog (33).
The author is deeply indebted to Walter Thomas for suggestion of this problem and helpful cooperation throughout the course of the study. LITERATURE CITED
Adie, R. H., and Wood, T. B., J . Chem. Soc., 77, 1076-80 (1900).
Table 111. Comparison of Macro- and Microdeterminrtionr of It0 Sample
KzO Macrodethod
%
%
Amdur, Elias, IND.ENG.CHEM.,ANAL.ED., 12, 731-4 (1940). Anderson, A. K., and Schutts, H. S., J . Biol. Chem., 61, 57-61 (1924).
KnO,Micromethod %
Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 5th ed., 1940. Berenblum, K., and Chain, E., Biochem. J., 32, 286-93 (1938). Bray. R. H.. J. Am. SOC.Agron., 24, 312-16 (1932). DeiigBs, G., Compt. rend., 171, 802 (1920). Fales, H. A., and Kenny, F., “Inorganic Quantitative Analysis”, New York, D. Appleton & Go., 1939. Fisk, C. H., and Subbarow, Y., J . Biol. Chem., 66, 375-400
5.09 5.10 Corn Plot 1 , 7-13-43 4.89 Plot 2 4:84 4.89 Plot 3 Plnt 5 .. 4.75 _.__ 4.78 Plot 6 3 : i 5 3.27 Tomato T2P5. 15th leaf, 8-1-41 1.3P Apple York B D2 7-7-43 1.84a.. Apple’ York B’ D2’ ‘2-18-43 * Apple: York B: D2: 8-18-43 1.18s a Volumetric cobaltinitrite procedure of Volk and Truog (33).
.. .. ..
..
(1925).
Fontaine, T. D., IND.ENQ.CHEM.,A N ~ LED., . 14, 77-8 (1942). Fudge, B. R., Fla. Agr. Expt. Sta., Tech. Bull. 331 (1939). Harrington, C. R., Biochem. J., 35, 545 (1941). King, E. J., Ibdd., 26, 292-7 (1932). Klein, Bernard, and Jacobi, Mendel, IND.ENG.CHEM.,ANAL. ED., 12, 687-9 (1940). Kolthoff, I. M.,and Bendix, G. H., Ibid., 11, 94-8 (1939). Latimer, W. M., and Hildebrand, J. H., “Reference Book of Inorganic Chemistry”, New York, Macmillan Co., 1933. Lindner, R. C., personal communication (Oct. 4, 1943). Lindner, R. C., Plant Physiol., 19, 76-89 (1944). Lindner, R. C., and Harley, C. P., Science, 96, 565-6 (1942). Merkle, F. G . , Pa. Agr. Expt. Sta., Bull. 398 (1940). Morgan, M. F., Conn. Agr. Expt. Sta., Bull. 450 (1941). Peech, Michael, and English, Lesh, Soil S c i . , 57, 167-96 (1944). Pett, L. B., Biochem. J., 27, 1672-6 (1933). Poluektoff, N. S., Mikrochemie, 14, 265 (1933-34). Sideris, C. P., IND.ENQ.CHEM.,ANAL.ED.,14, 821 (1942). Thomas, Walter, Plant Phusiol. 12, 571-600 (1937). Thomas, Walter, and Mack, TV. B., J . Agr. Research, 57, 397-
For plots 1 and 2 the average of six replicates is given. The average deviation is approximately 2% of the total. Six replicates of pure potassium chloride containing 0.3 mg. were analyzed, giving 0.3 * 0.0016 mg. of K20; the average deviation was 0.53% of the %tal.
Attempts to carry out the determination in porcelain evaporating dishes, followed by rinsing into crucibles or in porcelain crucibles directly, gave low results in all cases. It was difficult to drive off all the sulfuric acid without causing local overheating. Even Then the crucibles were heated slowly in a muffle furnace a t below 400’ C., losses of KzO were considerable. This loss was probably due to reaction between potassium and the porcelain glaze. With Vycor crucibles in three recovery experiments, 0.2 mg. of KzO added to 5-ml. aliquots of plot 2 gave 98, 101, and 98% recoveries. I t has been said that the ammonium ion also forms a precipitate with lithium dipicrylaminate (15). To 0.2 mg. of KzO, ammonium chloride was added t o correspond with 4% nitrogen in the leaf. The amount of K20 found was 0.49 mg., which fact emphasizes the necessity of removing ammonium ions in the procedure. Information was desired as to the amount of interference to be expected from the calcium, magnesium, and sodium in the leaf.
A solution was made up such that 1 ml. when added to the standard &O samples used in making the standard curve would provide amounts of material corresponding to 1% of NazO, 3.5% of CaO, and 0.75% of MgO. The calcium was in the form of CaHa(POr)2.2Hz0, which provided phosphorus as well. Samples containing 0.1, 0.2, and 0.5 mg. of KzO (corresponding to 1, 2,
414 (1938).
Thomas, Walter, and Mack, W.B., Pa. hgr. Expt. Sta., BdZ. 378 (1939). (29) Thomas, Walter, Mack, W. B., and Cotton, R. H., A m , Fertilizer, 98 (4), 5-7, 26, 28 (1943). (30) Thomas, Walter, Mack, W. B., and Cotton, R. H., Am. SOC. Hort. Sci. Proc., 42, 535-41 (1943). (31) Vanselow, A. P., IND. ENG. CHEM.,ANAL.ED., 12, 516-17 (1940). (32) Veigle, A. J. M.,“Colorimetric Determination of Potassium in
Whole Blood with Dipicrylamine”, master of science thesis, Pennsylvania State College, 1942. (33) Volk, N. J., and Truog, E., J . Am. Soc. Agron., 267, 537-46 (1934). (34) (35) (36) (37)
Wander, I. W., IND.ENO.GEM., ANAL.ED., 14, 471-2 (1942). Rolf, B., Ibid., 16, 121-3 (1944). Woods, J. T., and Mellon, M.G., Ibid., 13, 760-3 (1941). Zinzadze, Ch., Ibid., 7, 227-30 (1935).
THISpaper in fuller form was submitted t o the Graduate School in partial fulfillment of the requirements of the degree of doctor of philosophy.
