DETERMINATION OF MOLYBDENUM IN PLANTS

Molybdenum occurs in the aerial portions of plants to the extent of 0.5 to 5 mg.per kilogram of dry material, although some may contain as high as 75 to 100 mg. or moreper kilogram. The determination of very small quantities is difficult but largerquantities, such as are found in plants grown in areas of high concentrations in thesoil, are readily determined.

The procedure for the determination is essentially that of Narmoy (1) modified byBarshad (2). Molbydenum is determined as "tiie thiocyanate after reduction of any hexavalentmolybdenum by stannous chloride. The stannous chloride also reduces ferric iron to theferrous state. In the original method where concentrations are very low, molybdenimi hasto be extracted with ether to concentrate it, but, when dealing with larger quantities,the water extraction as used by Barshad is satisfactory. The addition of sodium nitratetends to stabilize the very complex mixture of chemicals and prevents rapid fading thuspermitting a more deliberate procedure and at the same time maintaining accuracy.

Choice and Preparation of Samples. Samples to be analyzed must be chosen andhandled in such a manner as to represent the problem being studied. If you are studyingthe Mo. content of mixed forage, it should be clipped to represent what would be out forhay or what the animal woiild eat in grazing. All species in the forage should be groundtogether and thoroughly mixed. If you are studying "the relative absorption by differentplant species, they should be handled separately and plants should be of similar age.If you are studying the effect of soil management systems on acciimulation, it will bebest to choose one or "two indicator crops, rather than to examine mixed forage.

Chop the samples into short pieces about one half to one inch long with a pair ofscissors or a trim board. Place the chopped samples in paper bags and air dry them for24 to 36 hours. Place in 0"7en at 80 "bo 90 degrees C. for "two to three hours. At theend of this period, remove samples and grind, either by the Wiley mill or food chopper.Place groxmd samples in tight jars. Redry samples at 80 to 90 degrees C. for "two tothree hours just before analysis is performed.

Reagents:

(1) ^drochloric Acid, (141): Add 500 ml. of concentrated hydrochloric acid(36^) to 500 ml. of distilled -water.

(2) Hydrochloric Acid, (14100): Add 10 ml. of concentrated hydrocholoric acid(36^) to 1,000 ml. of distilled water.

(3) Sodium Nitrate, solution; Dissolve 210 grams of sodium nitrate in 500 ml.of distilled -water.

(4) Ferric Chloride, 0.01 N: Dissol"7e 0.54 grams of ferric chloride in 200 ml.of distilled water.

(5) potassium Thiocyanate, 10^ solution: Dissolve 100 grams of potassiumthiocyanate in 900 ml. of distilled -water.

(6) Stannous Chloride, 10^ in 10^ acid: Dissolve 25 grams of stannous chloridein 25 ml. of concentrated hydrocholric acid. Use heat if necessary. Whencompletely dissolved, add 225 ml. of distilled water. Date this solution.If over 5 days old, prepare a new solution, (if analysis does not -warrantthe use of 250 ml. of solution in 5-day period, cut proportions do-wnaccordingly.)

1) Narmoy, F.B.: J. Soc. Chem. Ind. 1939;58,275-2762) Barshad, I. Anal. Chem. (in press}

Reagents: (continued) ^4

(7) Ethyl Alcohol, If this cannot be supplied through some countydepartment with a government permit, we can arrange to secure suppliesthrough the University*

(8) Acid, Nitric, Concentrated, (70^)

(9) Ammonium Molybdate, standard (stock): Dissolve 0.4597 grams of C.P.ammonium molybdate, (NB4)6 Mo7024* 4H2O, in 50-75 ml. of distilled water.Transfer carefully to 500 ml. folumetric flask with several thoroughwashings and bring to volume. Stopper. This standard solution will besupplied by the Berkeley office upon request.

(10) Ammonium Molybdate, dilute steindard: Pipette 10^ ml. of stock ammoniummolybdate solution into 500 ml. volxmetric flask and dilute to volume.This solution will then contain 0.01 mg. Mo. per ml.

Analytical Procedure. Transfer eight, 5.00 gram ground samples in duplicate* toevaporating dishes. Add 10 ml. of ethyl alcohol, 95^^ to each dish. Place the dishes onnon-inflammable platform or on an old wood board. Ignite ihe samples. When flaming hasceased, transfer evaporating dishes to the electric furnace at 450 to 500 deg. C. for3-4 hours, (use Nichrome screens for stacking samples in furnace.) After 3-4 hours,remove the evaporating dishes from the furnace; cool; then add carefully, avoiding lossby effervescence, 10 ml. of hydrochloric acid (l-fl) and 1 ml. of concentrated nitric acidPlace on the steam table or hot plate and evaporate to dryness, (if hot plate is used,place evaporating dishes on clay triangles and evaporate slowly preventing visible boil¬ing of the solution.) Leave the evaporated samples on the steam table or hot plate forone half to one hoiir to insure complete dehydration of ihe silica. If necessaary,transfer to an oven at 100-105 degrees C. for about an hour to insure dehydration.

Remove the samples and add 28 ml.of concentrated hydrochloric acid, (14-1) to eachand return to the steam table or hot plate for a couple of minutes to warm the solutions.Filter the warm solutions through ^ Whatman filter papers collecting the filtrates in100 ml. volumetric flasks. Wash evaporating dishes and filter papers with dilutehydrooholoric acid, (14100) until the volume in the flasks is around 70 ml. If the colorof the filtrates is yellowish, do not add any ferric chloride but if they are not coloredadd 2 to 3 drops of ferric chloride, 0.01 W solution.

-yr SPrepare a distilled water blank containing 2^ml. of concentrated hydrochloric

acid, (1+1) and 2 to 3 drops of ferric chloride, 0.01 N solution.

At this point there should be 17 volumetric flasks in use, 8 unknowns in duplicateplus the blank.

To each volimietric flask add 5 ml. of sodium nitrate solution and shake thoroughly.Now take each flask separately, starting with the blank. Add 6.0 ml. of potassiumthiocyanate, 10^ solution; shake; add 6.0 ml. of stannous chloride, 10^ in 10^ acidsolution; shake. Dilute to volume using the dilute hydrochloric acid, (1+100). Mixby inverting several times. Pour the blank in one tube of the colorimeter. Now take

♦ It will be advisable to continue making determinations in duplicate until analyticalprocedures are well established as demonstrated by uniform, close agreement of duplicates

the next senile and add 6.0 ml. of potassium thiocyanate, 10^ solution; shake; add6.0 ml. of stannous chloride, 10% in lOffo acid; shake and dilute to volimie. Mix byinverting several times. Fill the second tube of colorimeter. Balance the colorimeterusing the blue (425) filter as per instructions received with the colorimeter. Obtainreading of the color density on the 'A* scale and record. Repeat for each of thesamples until all 16 samples are finished, using the blank solution to check on thezero reading for each sample.

By reference to a standard curve, mgs. per 5.00 gram sample may be obtained.Mgs. per 5.00 gram sample x 200 is equal to PPM. (If duplicate samples do not agreewithin 3-4^^, they should be repeated.)

Note: If the color of an unknown is intense enough to give a reading above 50 on the•A* scale, it will be necessary to dilute ilie unknown to get an accxurate determination.To do this pipette directly from the 100 ml. volumetric flask containing the coloredunknown a" 10 ml. aliquot and dilute to 100 mis. in another volumetric flask using dilutehydrochloric acid (14100) as the diluting agent. Then take a reading of "tiie colorintensity. After this dilution the result as read ia in terms of mgs. per 0.5 gramsample. To get PPM, multiply by 2000.

Standard Curve. Pipette 10.0 ml. of dilute standard (O.Ol mg./^o. per ml.) intoa 100 ml. volumetric flask, dilute to volume with distilled water. Mix by invertingseveral times.

Set up a series of 100 ml. volumetric flasks. Pipette samples as follows:1 - Blank (HgO)2 - 10 ml. from 100 ml. volumetric flasks 0.01 mg.

3 - 20 ml. from " " n n 0.02 mg.

4 •• 40 ml. from " " ti n 0.04 mg.

5 - 10 ml. from dilute standard M 0.10 mg.

6 - 20 ml. from " n n 0.20 mg.

7 40 ml. from " !t tt 0.40 mg.

Rtm this series as you would that of blank and unknown in the Analytical Procedure,using 2 to 3 drops of ferric chloride, 0.01 N solution, in each.

Plot the readings of the «A* scale against mgs. on ordinary graph paper. Yourcurve should approximate a straight line.