SS S. V. SUI~YAigAE, AYA~NA I~AO, U . VEEI%ESWARA ~ A O , and G. GOPALAX~,AO :

Wie ma n aus Tab. 3 ersehen kann, s t immen die 2 Eichkurven sehr gut iiberein. Die in Tab. 4 gefundenen Wer te sind durchschnit t l ich um 0,66/~g h6her als die zugesetzte Menge, d. h. dab dies derjenige Gehalt ist, der in 25 ml der synthet ischen HeulSsung trotz Anwendung yon p.a.-Chemikalien enthal ten ist.

Tabelle 4. Pri~/ung der Methode an einer synthetischen Heul6sung. Hierzu wurden zu jewefls 25 ml der Heul6sung die nachstehend angegebenen

Mengen Cr III zugesetzt.

Gefunden lxg Cr Angewandt pg CrIIJ !

S~Os ~- : NaOH I R~O2 -F .N~OH i

1 1,s I 1,7 3 3,7 3,6 6 6,6 6,8

10 10,5 10,7 15 15,6 1.5,8 20 20,5 20,6

Bei Anwesenheit gr5Berer Gehalte an oxydierbaren Fremdelementen ist die Methode mittels Wasserstoffperoxyd insofern vorzuziehen, als die ErhShung des Ammoniumpersulfa tzusatzes auch die Zugabe grSi~erer Laugemengen erfordert, wodurch sich der Salzgehalt der LSsung in un- erwiinschtcr Weise vermehrt . Andererseits kann Wasserstoffperoxyd in jeder beliebigen Menge zugesetzt werden.

Literatur. 1 CAZn~CEVvE, A.: Bull. Soc. chim. Paris (3.) 23, 701 (1900); 25, 761 (1901); vgl.

diese Z. 41, 568 (1902). - - 2 HA~sE~, A. : diese Z. 134, 427 (1951/52). - - a LAeROIX, S., und M. LA~A~ADE: Anal. chim. Acta 3, 262 (1949); vgl. diese Z. 131,213 (1950). __ a NYDAHL, F. : diese Z. 116, 315 (1939). - - 50ELSC]~L-AGEI~, W. : diese Z. 144, 27 (1955) . - 6 SA~DELL, E. B.: Ind. Engng. Chem., anal. Edit. 8, 336 (1936); vgl. diese Z. 114, 59 (1938). - - 7 SA~CDEr,L, E. B. : Colorimetric Determination of Traces of Metals, S. 192 (1944). - - s U~o~E, P. F., and I-I. K. A~DE~S : Analyt. Chemistry 22, 1317 (1950); vgl. diese Z. 185, 320 (1952).

Dr. W. OELSC~ILXGEtr Stuttgart-ttohenheim, ~[nst. f. Tierern/~hrungslehre d. landwirtsehaftliehen Itochschule.

Department of Chemistry, Andhra University, Waltair (South-India).

Reaction between Mercuric Chloride and Aseorbie Acid. Estimation of Ascorbic Acid.

By S. V. SURYANARAYANA RAO~ U. VEERESWARA RAO, and G. GOPALA ~A0.

(Received December 17th, 1954.)

Ever since its discovery and isolation in the pure s tate by SZENT- G~6~aw, ascorbic acid (Vit. C) has been a substance of absorbing interest both to chemists and biochemists alike. The most fundaraental

P~eaction between Mercuric Chloride and Ascorbic Acid. 89

chemical proper~;y of ascorbic acid is its powerful reducing action. I t reduces readily iodine, iodate, permanganate, ferrieyanide, vanadate , complex heteropoly molybdic and tungstie acids, silver salts, gold salts, mercuric salts, ferric salts etc. Ascorbie acid also reduces various dyes which are often used as oxidation-reduction indicators such as 2,6-di- chlorophenol indophenol, methylene blue, thionine etc. When ascorbic acid is oxidised in dilute acid solution by a reagent like iodine, the first product of the reaction is dehydroascorbie acid which can be quanti- tatively converted back into aseorbie acid by a suitable reducing agent like hydrogen sulfide. Dehydroaseorbic acid may be regarded as having been produced from ascorbic acid by the loss of two hydrogen atoms. The reaction with iodine may be represented as

O = ~ i O = -

CH~OH " CHOH. CH--C = C--C = 0--2 H ~ CH~0H �9 CROH. CH--C---C--C - : O

OH OH 0 0 In alkaline solutions, especially or on long standing dehydroascorbic acid undergoes further oxidation (passing through a number of intermediate stages) to oxalic acid. These latter reactions of dehydroascorbic acid are irreversible. Methods have been developed for the volumetric estimation of aseorbic acid making use of iodine, very dilute permanganale ~ and iodate i. With the two latter reagents starch-potassium iodide indicator has been used. The reactions of ascorbic acid with the heteropoly molybdic and tungstic acids have been used for the colorimetric deter- mination of ascorbic acid. Among the heteropoly acids used are phospho- tungstic acid, phosphomolybdic acid, arsenotungstic, phosphomolybdo- tungstic, arsenotungsto-mo]ybdie, silicomo]ybdic acid and germano- molybdic acid. The reduction of ferric salts by ascorbic acid has been used by SCHULEK and FLO])EaER 7 for ~he colorimetric estimation of aseorbic acid. The method consists in the quantitative reduction of Fe +++- to Fe++-ions and the photometric determination of the resulting ferrous ions by n].eans of the ~,~'-dipyridyl complex.

PITTARELLI and PITTAI~ELLI 8 have found that the hydrochloric acid formed during ~he reaction of mercuric chloride with ascorbic acid can be used to measure the progress of the reaction. I~]~NA:rO and FEDELE 5 state that the reaction

2 HgCl~ + C6KsO ~ -~ C6HdO 6 -t- I-Ig2C12 ~- 2 HC1. (I) is complete in thir ty six hours at the room temperature. They have employed the conductivity method for studying the kinetics of the reaction and state that the reaction is of the third order. ROSE~THM~F,a 6 prescribed a very drastic procedure for the reduction of mercuric chloride to mercurous chloride by aseorbic acid. His method consists in heating mercuric chloride solution with ascorbie acid in 0.1 N hydrochloric acid

90 S.V. SIJI~YA.NAI~AYANA ~:~AO, U. Vt:EI~ESWAI~h. Rs and G. GOeAL~ [~.ao:

medium at 100~ for four hours. Then decinormal sodium hydroxide solution and 10~/o sodium nitrate solution are added and the mercurous chloride is separated by filtration through asbestos and washed with 10% sodium nitrate solution. The mercurous chloride is weighed gravi- metrically. I t appeared to us tha t it should be possible to achieve the reduction of mercuric chloride even at the room temperature more rapidly than what the above investigations suggest. Aseorbic acid has a sufficiently low oxidation-reduction potential varying from --0 .012 volts at p~ 8.67 to -~ 0.326 volts at PE 1.05 with respect to the normal hydrogen electrode. The mercuric/mercurous potential is ~-0.91 volts. In view of this, ascorbic acid should be able to reduce mercuric chloride easily. Perhaps the hydrogen ions derived from the hydrochloric acid liberated in the reaction (equation I) inhibit the reduction so tha t it appears to be slow in reaching completion. I f a suitable buffer is present in the system, to neutralise the hydrochloric acid liberated, the reaction will go to completion more quickly than has been hitherto observed. In the present investigation, we have therefore studied the effect of varying the hydrogen ion concentration. During the preliminary experiments, it was noticed tha t the mercurous chloride precipitate turned grey or black due to the formation of metallic mercury. This is possibly due to the mercurous chloride first formed being further reduced to metallic mercury. T o ascertain the conditions under which this further reduction occurs, a suspension of mercurous chloride was freshly prepared by the method recommended by P~AM,~cm~nv and GOt~ALA ]~AO ~. 10 ml. of this suspension is t reated with 10 ml. of 0.002 M ascorbie acid in media of varying hydrogen ion concentration making use of McILvAI~ buffers. The results are tabulated below.

Table 1. Reduction o] mercurous chloride by ascorbic acid.

3ICII, VAINE buffers p ~

2'0 3"0 4"0 6"0 7-0 8"0

Observat ion

No reduction even after 26 hours. No darkening of the mercurous chloride. Progressive darkening of the mercurous chloride after 10 minutesl The mercurous chloride turns black in one minute. The mercurous chloride turns black immediately. The mercurous chloride turns black immediately.

From the above results, it can be ,concluded tha t if mercuric chloride is taken in relatively lower concentration than ascorbic acid, the asc0rbie acid not only reduces mercuric ehl0ride to the mercurous chloride but carries the reduction further to the metMlie mercury stage, if the p~ of the solution is kept at 4 and above. Below this Pm there is no risk of the mercurous chloride being reduced to metallic mercury by aseorbic

~eaction between Mercuric Chloride and Ascorbic Acid. 91

acid. E v e n if tlhere is the slightest excess of the ascorbie acid over the

mercuric chloride, there is the danger of the reduct ion proceeding to the

mercury stage, when the pE of the solution is 4 and above. On the other hand, we found tha t even at pH 8, there is no mercury formation, ff the mercuric chloride is t aken far in excess of the ascorbic acid. Hence experiments have been carried out to see if ascorbie acid can be quant i - ta t ive ly est imated through its react ion with excess of mercuric chloride

a t controlled condit ions of pH.

Experimental. The mercuric chloride employed in our experiments is Merck's extra pure quality

material A 0.01 ~ solution was made by weighing the required quantity of the salt and dissolving in triple distilled water. The ascorhic acid used in this investi- gation is the synthetic crystalline vitamin C manufactured by the Hoffmann- La Roche Company Ltd., Basle, Switzerland. A solution of the requisite strength was prepared by dissolving a weighed quantity os the substance in triple distilled water. This precaution is necessary in view of the fact that the autoxidation of ascorbic acid solu';~ions is markedly catalysed by traces of copper ions. The solution was standardized with a standard solution of 0.01 N potassium permanganate according to the method of MV~TH~ and VlSWA~AD~aH ~ or with a 0.01 N sohition of potassium iod~,te according to the method of BALLENTINE 1. Sodium acetate- acetic acid buffers (3.8 pH to 4.98 p~) of IYALPOLE and citric acid-disodium phos- phate buffers (2.21 to 8.0 pu) of ~CILVAIICE have been used. The experimental procedure adopted is as follows.

To 10 ml. of the buffer mixture taken in a centrifuge tube are added 10 ml. of 0.01 M mercuric chloride solution and 5 ml. of freshly prepared ascorbic acid (0.002 M to 0.001 M). After allowing the reaction to proceed for a period varying from 30 min to 90 min., 2 ml. of 2 N barium chloride solution is added to facilitate the complete coagulation (settling) of the precipitate. Then the supernatant liquid is drawn off with the help of a fine capillary tube applying mild suction. The precipitate is washed twice with triple distilled water in this way. Then the precipitate w~s treated with a known excess of say 15 ml. of 0.01 N iodine solution containing excess of potassium iodide. The iodine oxi .dises mercurous chloride to mercuric chloride which dissolves in excess of potassium iodide. The unreacted iodine is then estimated by titrating with standard thiosulphate. From this, the amount of iodine reacted with the mercurous chlorid(~ is known and consequently the amount of ascorbie acid taken is estimated. The results are given below.

Table 2. Estimation o/ascorbic acid with mercuric chloride. 10ml. of W~LeOLE buffer mixture + 10 ml. of 0.01 k T mercuric cMoride solution +

5 ml. of ascorbic acid solution.

A m o u n t of ascorbic acid found in rag. A m o u n t of ascorbie

Pt~ acid t aken in rag, 30 rain, ] 60 rain. 90 rain.

4.6 ' 3.887 3.852 3.852 3.852 5.0 5.170 5.123 5.123 5.123 5.7 4.317 4.306 4.306 4.306

From the above results, i t is seen t ha t the react ion is complete wi th in 30 minutes . Similar results have been obta ined with the MCInVAINE buffers (T. 3, P. 92).

92 SUI~u RAO, VEERESWAt~A RAO, GOI~AL_a RAO : Ascorbic Acid.

Acidities corresponding to a p~ lower t han p~ 2 progressively inhib i t the reaction. Hav ing ascertained t ha t the react ion between ascorbic acid and mercuric chloride reaches quan t i t a t ive complet ion wi thin

Table 3. 20 ml. of MeILvAI~E buffer ~- 10 ml. 0.01 M. mercuric chloride ~ 10 ml. of ascorbic acid

solution.

Amount of Amount of P~ ascorbic acid taken aseorbic acid found

in mg. in rag.

Table 4. 20 ml. of MCILVAI~E buffer ~ 10mL of 0.01 M.mercurie chloride + 10ml.

of aseorbie acid.

Ascorbic acid taken Aseorbic found in mg. in rag.

F

2.0 1.78 t.74 2.080 ! 2.076 4.0 1.78 1,74 7.040 7.020 6.0 1.78 1.74 1.840 1.830 8.0 1.78 1.74 3.536 3.522

30 minutes a t a controlled pH vary ing from 2.0 to 8.0, yielding mercurous chloride, we have carried out several es t imat ions of ascorbic acid through

its react ion with excess of mercuric chloride a t p~ 4.0. Some typical results are given in Table 4.

S u m m a r y .

1. The condit ions for the quan t i t a t ive reduct ion of mercuric chloride to mercurous chloride by ascorbic acid have been improved over those

described in the l i terature. The new procedure requires only 30 minutes for quan t i t a t ive complet ion as compared with 36 hours previously

prescribed. 2. A simple technique has been evolved for the es t imat ion of ascorbie

acid through its react ion with mercuric chloride.

Re[erences. 1 BAr,Ln~TINE, R.: Ind. Engng. Chem. Anal Edit. 13, 89 (1941). - -2 ~V~T~Y,

K. S., and C. R. VISWANAD~A~: Proc. Nat. Inst. Sci. India 10, 217 (1944).- 3 ~:)ITTAICELLI, E. , and M. PITTA~ELLI: Biochim. Terap. sperim. 2~, 100 (1938). - - a RAMACH~LU, P. T., and G. GOl'M~A RAO: 1)roe. Nat. Inst. Sci. India 8, 383 (1942). - - 5 INDOVI~A, t~., and F. M ~ o I : Gazz. Chim. ital. 69, 117 (1939). - - 6 ROSE~T~LEn, L.: Z. Vitaminforseh. 9, 342 (1939). - - 7 SCttVLEK, E., and I. FLODEI~E~: Angew. Chem. 52, 615 (1939).

Prof. G. GoeM~A RAo, D. Sc., Head of the Department of Chemistry, Andbra University, Navroji Road, Visakhapatnam-2 (India).