11-1. REEXAMINATION OF ANALYTICALMETHODS FOR A~ ANO Mo IN PLANTMATERIAL

March 20, 1980

JUNJI ISHIZUKA 1MANOEL V. DE MESQUITA FILHO

1. Consultor de Curta Duração (NIAS, MAFF/JAPAN)2. Pesquisador do CPAC

11-1. REEXAMINATION OF ANALYTICAL METHOOS FORAt ANO Mo IN PLANT MATERIAL

A. Analysis for AI in plant materialAluminon is a kind of pigment which

easily dissolves into water and shows weakacidtiv. Under weak acid of neutral condi-tion, it combines with a trace amount ofaluminium and develops red color. Everyaluminon colorimetric method has thedisadvantage that the AI-aluminon complexis unstable. Some researches intend toimprove the method. Authors carried outreexaminations of AI analysis according to"Saibai Shokubutsu Bunseki Sokuteiho"(2) and checked the following points

1. Heating time;2. Time course of discoloring;

3. Recovery of AI added to plant ma-terial.

Heating timeColor development of aluminon-AI

complex is promoted by heatinç. Excessiveheating may result in discolorinq. In orderto investigate the relation between heatingtime and color intensitv, the volu metricflasks containing the reaction mixture wereput into boiling water for 2, 4, 6,8,10 and15 min, and the absorbanceswere measured120 min after the heating began. From theresuIts shown in Table 1, it is concludedthat the reaction mixture should be heatedfor 6 mino

Table 1. Relation between color intensity and heating time.

~eNC? of (rnin.)2 4 6 8 10 15measure

absorbance (520 min)1 0.195 0.199 0,201 0.203 0.200 0.1962 0.192 0.193 0,201 0.202 0.189 0.203

average 0.194 0.196 0,203 0.203 0.195 0.199

Time course of discoloring

1 9 of the standard sarnple of soybeanwas ashed in a muffle furnace and extrac-ted with 50 ml of N Héi. Mixtures of 5 mlof the extract and the reagents were heatedfor 6 min, and the changes in color intensi-ties were investigated.

As shown in Fig. 1., the rate of disco-loring is very h igh up to 60 min after colordevelopment and is decreased with increa-sing of time. The rate 120 min after colordevelopment is less than 1 percent perhour. Therefore, it is concluded that the

0.35

Eco~ 0.33

'"ocjg(;1l 0.31'"

O.ü1T ~ =_-w 1W 100

mlnetter calor dcvetorxnent

FIG. 1. Relation between color intensity ofaluminon-AI complex and timeafter color development.

211

aosorbance shou Id be measured 120 rni-nutes after colo development.

Recovery testIn order to check the recovery of A I

added to a standard sample of soybean,authors determined AI concentrations insamples, to which were added 1 mg of AIor noto

From the results shown in Fig. 2 andTable 2, the average recoveries are 97 -99%, and thus it is concluded that this me-thod is applicable for analyzing AI con-centration in soybean plant material.

From the facts described above, it wasdecided to use the following method:

Table 2. Results of recovery test (AI)

Concentration of AI (mg/g) at 520 nm

N<? of measure Standard samp le Standard sample + 1 mg of AI

120 mino * 150 mino * 120 min.* 150 mino *

1 1.34 1.30 2.29 2.282 1.28 1.29 2.28 2.263 1.32 1.28 2.29 2.314 1.30 1.35 2.25 2.275 1.29 1.24 2.28 2.27

average 1.31 1.29 2.28 2.28

C.V.(%) 1.6 2.7 0.7 0.8

* Time after color developmentRecovery

120 min.* 2.28 -1.31 = 0.97 97%150 min.* 2.28 - 1.29 = 0.99 99%

0.3

~ 0.2oNLD

13iõ~ 0.1{!

20 30 ug·AI/50ml10

FIG. 2. Calibration curve

212

Reagent1.0.22% aluminon solution (in distil-

led water)2.1% mercaptoacetic acid solution (in

disti fled water)3.20% ammonium acetate buffer pH

4.84. AI standard solution

2 ml of AI stock solution (1000ppm) is transfered to 1 liter-volu-metric flask and made up to volumewith distilled water.

ProcedurePut 1 9 of dried ground sample into a

30 ml pyrex beaker and ash in a muffle

furnace at 5000C for 5 hours. After cool inq,transfer 5 ml of 5N NCI of 5N H2S04 tothe beaker and boil for 2 min on a hotp late. T ransfer the ex tract to 50 m I - vo lu-metric flask, make up to volume and shake.Put 1 ml of the sample solution into a 50ml - volumetric flask, add 10 ml of arrimo-nium acetate buffer, 20 ml of distilledwater and 1 ml of mercaptoacetic acidsolution and shake. Heat in boiling waterfor 6 min., and make up to volume withdistilled water. Measure absorbance at 520nm and compare with the absorbances ofAI standard.

Prepare cach AI standard by placingthe amount of 2 ppm solution and waterindicated below, 1 ml of 5N HCI of 5NHCI or H2S04, 10 ml of ammonium ace-tate buffer and 1 ml of mercaptoacetic acidsolution in a 50 rnl volumetric flask.

AI standardhg/50ml

O4812162030

AI solutionml (2 ppm)

O24681015

disti Iled waterml30282624222015

B. Analysis for Mo in plant materialMo in plant material was ànalyzed

routinely with the dith iol method by oneof the authors in Japan. However, the expe-rimental condition in CPAC (water, reagentsand instruments etc.) might possibly differfrom NIAS(l) in Japan. In particular, 50ml-volumetric flask had to be used in placeof separating funnels due to a carelessoversight by one of the authors and themethod was modified as follows:

Reagents1. dithiol reagent: Transfer 1 ml of

dith iol to a brown colored bottle

contarrunq 500 ml of O 5N NaOHsolution warmed to about 300C.Agitate using glass rodo When mostof dithiol is dissolved, add rnercap-toacetic acid until a slight precipi-tation appears. Store the bottle in arefrigerator.

2. 50% su Ifuric acid (V N)3. ferric am mon iu m suIfate so lu tion:

Dissolve 9.1 9 of NH4Fe(S04)2 in100 ml of 2% H2S04 solution.

4. 50% tartaric acid (W/V)5.50% potassium íodide solution

(W/V)6. 50% ascorbic acid solution7. 10% thiourea solution

reagents 5, 6, 7 should be preparedjust before the determination.

8. Mo standard solution (1 pprn}: Di-lu te the stock solu tion (100 ppm)to 1 ppm with disti Iled water.

ProcedurePut 1 - 5 9 of dried ground plant mate-

rial into a 30 rnl-pvrex beaker. Ash in amu ffle furnace at 5000C for 5 hou rs. A ftercoolinq, transfer about 2 ml of distilledwater and 10 ml of 50% sulfuric acid to thebeaker. Boil on a hot plate for 3 minoTransfer the extract to a 50 ml-volumetricflask and add distilled water until the volu-me of solution reaches about 30 rnl.Trans-fer 0.3 ml of ferric ammonium sulfate solu-tion and 0.3 ml of KI solution to eachflask. Shake occasionally for 10 - 15 mi nu-teso Add 4 - 5 drops of ascorbic acid solu-tion. The brown color disappears cornple-tely. Transfer 0.3 ml of tartaric acidsolution and 2 ml of thiourea solution,rnix , and add 4 ml of dithiol reagents.Stopper and shake vigorously and thenlet stand for 30 rninutes. Then transfer 5ml of isoamyl acetate to the f lask. Shakevigorously and let stand for 20 minutes.Add distilled water until the interface

213

between the water and the isoamy I acetatereaches to the neck of flask. Transfer theisoamyl acetate phase to a centrifugetube with a pipette and centrifuge at 2000r.p.rn. for 20 minutes. Measure the absor-bance of the isoamyl acetate layer at 680nm and compare with the absorbance ofthe Mo standard.

Prepare each of the Mo standards byplacing the amount of 1 ppm solution andwater indicated below and 10 ml of 50%H2S04 in a 50 ml-volumetric flask.

Mo standard Mo solution Waterug/50ml (1ppm)ml ml

O O 202 02 184 4 166 6 148 8 1210 10 10

Recovery Test

I n arder to confirm that the modifiedmethod is applicablê to estimate Mo con-centration in p lant material, the recoverywas examined. The calibration curve shownin Fig. 3 seems to be almost linear. Theaverage recovery summarized in Table 3is 99% and amost complete. Therefore. theauthors conclude that the method descri-bed above ís su itable to determ ine Moconcentration in plants.

Reference

1) ANNUAL Report ai tne National lns-titute of Agricu Itural Sciences.Divi-sion of Plant Nutrition. Tokvo. 1972.

2) HIRAMINE, J. Aluminium. In: SaibaiShokubutsu Bunseki Sokuteiho. To-kyo, Yokendo, 1976. p. 146-8.

214

Table 3. Results of recovery test (Mo)

N? ofContent of Mo g/2g at 680 nm

measure Standard Standard samp le

I sample + 6 9 of Mo

1 0.26 6.142 0.28 6.243 0.22 6.184 0.22 6.195 0.27 6.20

average 0.25 6.19

Recovery6.19 - 0.25 = 5.945.94 - 6.00 x 100 = 0.99%

o.•

o.,

e.aÊco..~

0.2C>U.,oeo..D 0.1o

• 10,. ""'1/10.'

Fig. 3. Calibration curve (Mo)

Final reportJunj i Ish izu kaApri12,1980

I - Reexamination of analytical methodsfor AI and Mo in plant material.a. Aluminum

According to "Saibai ShokubutsuBunseki Sokuteiho ", analytical me-thod for AI w'is carried out and thefollowing pint were checked.Results1. Heating time

The reaction mixture should beheated for 6 mino

2. Time course of discoloringThe rate of discoloring was veryhigh up to 60 min after color de-velopment and was decreased withincreasing of time. The rate 120mino after color development wasless than 1% per hour. Therefore, itwas concluded that the absorbanceshould be measured 120 minutesafter color developments.

3. Recovery of AI added to plant ma-terialAverage recoveries were 97 - 99%,and thus it was concluded that th ismethod was applicable for analusingAI concentration in scvbean plantmaterial.

From the facts shown above, it wasdecided to use the method describedin the other sheets in detail.

According to annual report of divi-sion of plant nutrition (NIAS), dithiolcolorimetric method was examined.The resuIts of recovery test showedthat the modified method was applica-ble to analysis for Mo in plant material.The method was· described in theother sheets in detail.

tion and their mobil ities with in thesoybean plants grown in experi-mental field, concentrations of Ca,Mg, Fe, Zn, Mn, P, Mo and AI weredetermined and the resuIts weresummarized as follows:

1. Plant growth·Liming and P-fertilization influ-enced beneficial effects on plantgrowth.

2. CaThe concentrations were increa-sed with increasing of both limeand P-application.

3. MgThe concentrations were increa-sed by liming, but effect of Pwas not clear.

II - Effects of liming and phosphorus fer-ti lization on status of mineral nutrientsin soybean plants grown in the cerradosoi I.

In order to investigate the effects ofliming and P- fertilization on themacro - and micronutrients absorp-

4. "FeThe concentrations were de-creased with increasing of. li-ming and P-fertilization.

5. ZnThe concentrations in stemsand roots were decreased byliming and P-fertilization, but inleave, their effects on Zn werenot clear.

6. CuLiming and P-fertilization donot affect on Cu concentration.

7. MnThe concentrations were de-creased by liming and P-fertili-zation except in leaves, and theeffects of P-fertilization werenot clear.

8. PP concentrations were increasedwith increasing of P-fertilizatiOr1however the effects of limingis obscure.

215

9. MoWhen 4t;ha of lime was applied ,the Mo concentrations were in-creased by P-fertilization. Butwhen 1 ,5t/ha and °,5t;ha oflime were app Iied, they weredecreased. Liming was seem toincreased the ability of -soil tosupply Mo.

10. AIThe concentrations were de-creased by liming and P-fertili-zation. The plant growth nega-tively correlate with the AI con-centration in leaves and stems.

This work was described in the othersheets in detail.

Acknowledgement

The author stayed in Brasil ia fromFeb. 7 to April 2, 1980, and worked on thesubjects described above. This work wasgreatly assisted by continued efforts ofM.V. Mesquita. Ouring his stav, he receivedguidance and encouragement from Or. W.J. Goeder t and got dose relationsh ip withali members of CPAC, especially Dr . E.Wagner. This work could not have beenperformed without helpfull discussion withthe Japanese experts. He would like tothan k ali of thern.

RELATIONSHIP BETWEENNUTRITIONAL OISOROERS CAUSEO

BY AI ANO MICRONUTRIENT STATUS.

(Junji Ishizuka)

Nutritional disorder should not consi-dered to be caused by excess or deficiencyof a sole nutrient, but influenced by inte-raction of various nutrients. For example,Zn deficiency is stimulated by P-fertiliza-tion; Mo uptake by plants is reduced bysulfate addrtion and increased by limingand P-fertil ization; Cu of Mn induce Fedeficiency. In my experiments with riceplants, I found that disorder by heavy me-tais is promoted by N-deficiency. In rela-tion to AI, we should be interessed in theinteraction among other nutrients such asP, N, Mn, and Mo, etc.

Mesquita et ai intend to clarify therelationship amogn AI tolerance of 15soybean varieties, PAertilization and liminq.

216

Recently, I observed the soyvean plants inhis experimental field, and I recognized theeffects os P-fertil ization and I iming onplant growth and differences among varie-ties. On the other hand, I was interested innodule formation.

General lv , root nodules appear up tofirst trifol iage-Ieaf emergence date. Thesoybean plants grown by Or. Milton werenodulated, but the main root were no t.From these phenomena, I supposed thatthe facto r repressed nodu lation in earlygrowth stage in cerrado soi I. Therefore, theeffect of various elements other than AI onnodulation should be examined, but I shallnot be able to solve the problem because ofmy short-terrn stav.

RELATIONSHIP BETWEENNUTRITIONAL OISOROERS CAUSEO

BY AI ANO MICRONUTRIENT STATUS.

(Junji Ishizuka)

(Feb.21-22)Analysis for nutrients in soybean

plants grown in Mesqu its experimentalfield.

Sampling:- varieties. 3 (toler ant. moderate,

sensitive)- liming levei. . . .3 (500kg/ha,

1500kg/ha,4000kg/ha)- P-fertilization levei ... 3 (160kg/ha,

778kg/ha, 1374kg/ha).. 3 (leaf . stern, root)

.. 3x3x3x3 = 81- Plant part-Total ..

(Feb.25-26)Sample propar ation

(Mar.34)Ree'xamination of analv tical mo thodsfor AI and Mo.- AI - Aluminon colorimetric rncthod- Mo - Oithyol colorimetric method

(as SOOI1 as rcagents and irnp lc-ments arr ivc from Japan)

(Mar. 5-21)AnalvsisN. P K. Ca. Mg. AIFc. Mn Zn. Mo. Cu. Zu .

A) Visit to Araxá Expcrimcnts and farmIII São Pau 10

B) Preparation of Report.

217