V O L U M E 23, NO. 9, S E P T E M B E R 1 9 5 1

High residual currents were also obtained a t 0" C. in the presence of mineral acids. Upon removal of oxygen with nitro- gen, these currents decreased to less than 0.1 la .

That the titration of very dilute chloride solutions should be more accurate and precise a t low temperatures than a t room temperature is illustrated in Fig- ures 3, 4, and 5 .

Titration at Low Temperatures.

18

v) 16

2 14

I 4 1 2

W

s 10

5 6

v_ I 8 c'

P

$ 4

2

0

1309

chloride in 0.01 J barium nitrate containing 0.02% gelatin was calculated a t different temperatures. The following values were found: LS.+~CI X 1010: 0.3 a t O", 0.5 a t 5", 4 to 5 a t 28".

The diffusion current of silver ions in neutral medium decreases with increasing concentra- tion of gelatin. This is clearly seen from the slope of the excess of reagent lines in Figure 6. Thus, the accuracy and precision decrease with increasing concentrations of gelatin. In general, a concentration of 0.02oj, of gelatin is satisfactory in the titration of 0.001 S or more dilute chloride solutions.

At room temperature the solubility of silver chloride calculated from the current a t the equivalence point in the presence of 0.02% (to 0.05%) gelatin is of the order of 50 to 80% greater than that calculated from the solubility product a t the same temperature in the absence of gelatin (see Figure 7 ) . Apparently, the difference between the two values is not due to a super- saturation effect caused by the gelatin, because the current a t the equivalence point remained unchanged even after a few days of standing. If the gelatin was addcd at the equivalence point (Figure 7 ) , the current became practically equal to the value calculated from the solubilit,y product. On the other hand, when the gelatin was added when 80% of the chloride had been ti- trated, the current a t the equivalence point was about the same as when the gelatin had been present from the very beginning of the titration. I t seems that either the extremely small colloidal particles of silver chloridc formed in the presence of gelatin have a greater solubility than coarse silver chloride or the colloidal silver chloride has a slight depolarizing effect on the electrode.

Effect of Concentration of Gelatin.

0.2 0.6 1.0 1.4 1.8 2.2 10 - 2 N SILVER NITRATE, ML.

Potassium Nitrate Figure 7. Titration of 0.0001 'V Chloride in 0.1 .V

I .

11. I l l .

Without gelatin; at 4 gelatin added to 0.02% concen-

O . O Z 7 0 gelatin present from beginning of titration Calculated titration line in absence of gelatin

tration

.St 0" and 5' C . a 0,0001 chloride solution in 0.01 to 0.1 LV nitrate solution (0.02% gelatin) could be titrated n-ith an ac- curacy and precision of 1 to 2% when the excess of reagent line was drann aftcr 25% excess of silver had been added. The titration gave equall\- good results in 0.1 nitric acid when varried out in a nitrogen atmosphere. In neutral medium in t'he presencc of air, a 0.00005 S chloride solution could be titrated a t 0" C. with an accuracj- and precision of 3Oj, (Figure ti), when the reagent line was dravm after more than 50% excess of silver had been added. The exact temperature is immaterial, as long as it is below approximately 5' C.

From wrious tit ration lines the solubility product of silver

ACKVOW LEUGMEYT

Ackno\i ledgnient is made to the Graduate School 01 the university for a grant which enabled the authors to carry out this work.

LITERiTURE CITED

( I ) Kolthofi, I. If., arid Kuroda, P. K., ANAL. CHEM., 23, 1.304

( 2 ) Laitinen, H d., Jennings, W. P., and Parks, T. D., IND. ENG.

(3) Laitinen, H. .1., and Kolthoff, I. M., J . Phys. Chem., 45, 1079

(4) Owen, B. B., J . Ani. Chcm. Soc., 60, 2229 (1938). RECEIT LD February 19. 1951.

(1961).

CHEM., A s ~ L . ED., 18, 355 (1946).

(1941).

Rapid Method for Determination of Betaine €1. G. W..ILKER, JR. , 4 N D ROBERTA ERLCVDSEY Western Regional Research Laboratory, Albany, Calif.

T H.4S been known for many years that betaine [carboxy- I methyl trimethyl ammonium salt] constitutes one of the principal noncarbohydrate impurities in sugar-beet processing liquors, and recent interest in feed-supplemrnt use and by-product recovery has made the estimation of this compound of especial interest to the sugar beet industry. At present, no Association of Official Agricultural Chemists (1 ) method is available for betaine, and the present practice (4,5, 8) follows essentially the procedure first described by Stanek (9), which involves precipitation of the betaine with potasRium triiodide (periodide method), followed by titration with thiosulfate or determination of nitrogen by the Kjeldahl method. The method is subject to serious error, he- rause other naturally occurring nitrogenous substances are also

precipitated as complex iodides, especially in acid medium. Thus the preliminary removal of interfering materials, including sucrose, makes the method laborious. Phosphotungstic arid as a precipitant followed by Kjeldahl analysis has been used (6), but the method is nonspecific and somewhat time-consuming.

Strack and Schwaneberg (10) suggested that compounds re- sembling betaine could be determined gravimetrically as betaine reineckates in acid solution, but gave no quantitative analytical data for the determination of betaine itself. The use of Reinecke salt for the determination of choline (3, 11) and other substituted amino compounds (2) is well established. In the authors' ex- perience, however, the gravimetric or colorimetric determination of betaine as the reinmkate has not been successful because no

1310 A N A L Y T I C A L C H E M I S T R Y

is collected in a clean beaker when the suction ia turned on. If necessary, this procedure can be repeated (not over about 10 ml. of acetone solution should be used), and the filter ia finally washed with distilled water. Water is added to the combined washings to make a volume of about 20 ml. Then 10 ml. of silver nitrate solution and a small amount of filter aid are added and the re- sulting suspension is stirred and then filtered through a small Buchner funnel nrith the aid of suction. The silver precipitate is washed thoroughly n-ith water and the combined filtrate and washings (volume about 40 to 45 ml.) are titrated with 0.02 X sodium hydroxide to the disappearance of the acid color of methyl red. The number of milliequivalents of base required for the titration is equal to the number of milliequivalents of betaine in the sample.

For use on sugar-beet diffusion juices, a preliminary prepara- tion of the sample was found necessary. A sample of juice is heated quickly to 60" C. with a small amount (2 to 3% by weight) of calcium oxide, filtered, and cooled to room temperature. A 20-ml. aliquot of this filtrate is acidified to pI3 1 with concen- trated hydrochloric acid and made up to a volume of 25 ml. .4nalyses may be run on aliquots of the solution thus prepared. The processing liquors which had already been treated with lime gave satisfactory results when acidified and made to volume direct 137.

way could be found to isolate betaine reineckate quantitatively, and, a t the same time, analytically pure. In contrast to choline reineckate, betaine reineckate is slightly soluble in cold water or alcohols. Ether, benzene, chloroform, and similar organic sol- vents, on the other hand, fail to dissolve either betaine reineckate or the accompanying coprecipitated reineckates. In order to achieve quantitative removal of the betaine, the precipitation must be carried out in the presence of a large excess of reineckate as common ion, even a t a temperature of 5' C., so that the isola- tion of a pure precipitate represents a serious difficulty.

Because betaine salts are strong acids, they can be titrated with sodium hydroxide in the presence of methyl red indicator. The authors have found it satisfactory to precipitate crude betaine reineckate from hydrochloric acid solution and wash it free of inorganic acid with ether, on a sintered-glass filter. After the washed precipitate has been dissolved in aqueous acetone, the reineckate ion can be removed by addition of excess silver nitrate, and the soluble betaine nitrate determined by titration with so- dium hydroxide with methyl red as indicator. Even though choline or other nitrogenous materials might be precipitated a t the same time, they are not acidic enough to be titrated with alkali a t pH 6. Most of the amino acids do not give precipitates with the Reinecke reagent under the conditions of this procedure, although interference from proline, if present in sufficient quan- tity, might be expected.

REAGENTS

A saturated solution of Reineclce salt (ammonium reineckatr,, Eastnian No. 3806) is prepared by shaking an excess of the solid with distilled water, using a mechanical Bhaker and allowing at least 0.75 hour to ensure saturation. The authors have found no particular advantage from the use of recrystallized material, although results obtained from using a bottle which had been standing open in the laboratory for several months were slightly low. The saturated solution is filtered and adjusted to pH 1 with concentrated hydrochloric acid. The solution should he used the same day as prepared.

Reagent grade ether is used, and should be free of alcohol. Reagent acetone is used. A solution of 70 ml. of acetone and

30 ml. of water is used to dissolve the crude betaine reineckate. Conventionally prepared 0.02 A' sodium hydroxide is used for

titration. A solution approximately 0.1 JV with respect to both silver

nitrate and sodium nitrate was found satisfactory for the re- moval of reineckate ion, and is prepared by weighing the desired amount of both solids and dissolving them in the calculated amount of water.

A standard solution of betaine chloride is prepared by dis- solving 260.2 mg. of the pure, solid, nonhygroscopic chloride in 100 nil. of water. This solution contains 2.00 mg. of betaine per ml., and may be checked by titration with the sodium hy- droxide after dilution of an ali uot with water and a small amount of acetone to produce the con%itions encountered experimentally in the final titration.

PROCEDURE

A sample containing from 4 to 20 mg. of betaine, in a volume of not over 10 ml. of solution which has been acidified to pH 1 with hydrochloric acid, is placed in a small beaker. Ten milli- liters of acidified reineckate solution are added a t room tem- perature, and the sample is cooled to below 5' C. in an ice bath, after which it is filtered through a clean sintered-glass crucible (medium porosity) as rapidly as possible with suction. A rubber policeman aids in the transfer of the precipitate and stirring in thr subsequent washing operation. The original beaker is washed with a small amount of ether, and the washings are poured over the precipitate and sucked through the filter. The suction is turned off and another small portion of ether is poured into the filter, mixed well with the suspended solid, and finally sucked through the filter. The procedure is repeated until about 30 ml. of ether have been used. All droplets of water re- maining on the sides of the filter crucible must be washed out at this stage.

After the precipitate has been allowed to dry in air a few minutes, it is dissolved in acetone-water by pouring a few milli- liters of this solution on the filter without applied suction. The precipitate is stirred to produce an intensely red solution, which

RESULTS

By the described technique, it was found possible to get re- producible analyses on known samples of betaine. The samples varied in size from 2 to 20 mg. in 10 ml. of water and the betaine was precipitated by 10 ml. of saturated ammonium reineckate which had been acidified to pH 1. The values shown in Table I are representative of those obtained over the range of betaine concentration of interest to this investigation. The precision between duplicate determinations run a t the same time by two different operators was good, and the accuracy for analyses run a t different times was within about 4%.

Table I. Analpis of Known Betaine Samples _ _ _ _ _ ~ _ _ _ Betaine Found, M g .

Betaine in Sample, llg, Ana1y.t h Analyst B 2 00 4 00

6.00

8 00

10 00

16 0

2 0 . 0

26 0

30

1 . 8 7 4 03 3 8''

7.80 8 . 0 2

4 03 3 81 6 20 6 12

15,s 1 6 . 2 19.6

1 9 . t 14. E! 19 5 18.7" 14 6 24 4

. . .

19 .6 19 5 19.1

23. ti 2 4 . 1 2 4 . 0

'1 . ina lp is run at room temperature

These typical results were obtained from a pure solution of betaine chloride in water, following the procedure described. The variation in the rwults of differelit determinations is indica- tive of the accuracy obtainable. Samples containing more than 20 my. of betaine xere apparently not completely precipit,ated under the conditions of this analysis, as the results were always low. Similar low values xere obtained when the .wIution con- taining betaine reineckat.e was not chilled to 5" C.

As an example of the application of this method to extracts from natural sources, some d a h are presented on sugar-beet processing liquors.

V O L U M E 23, N O . 9, SEPTEMBER 1 9 5 1 1311

‘Iahle 11. Analysis of Sugar-Beet Diffusion Juice, Showing Effect of Preliminary Lime Treatment

Sample MI. Alg./arl. Sample Sire. Betaine Found,

No. 1. treatrd 3 . 0 0 1 . 3 2

N n . 2 “ , iintreated 3 00 1 . 5 5

1). 00 1.33 10.00 1.32

.5 00 1.34 10.00 1 . 1 2

1 0‘3

u Value of I .3? nig, ~ P P 1111. ohrained u lien raiiiliir ~ i i i rr.r%trd witti l i i i ic.

Table I1 illustrates the difficulty arising irom colloidal mate- rial in the solution analyzed. Sample 2 consisted of a raw dif- fusion juice which had had no preliminary treatment; the filtra- tion was extremely slow and the recipitate discolored. Sample 1 had been treated with lime andPthen analyzed; the figures in- dicate that consistent results could be achieved which were inde- pendent of sample size, within the limits already 3tated.

‘I’ablc 111. Recovery of Added Betaine from DifTttsion .Juice Total Betaine Found, MS. .-

After Addition of 4.00- .idded Betaine Sanlilli. Originally llg. t o Satni,lt, Accoilntod for, llg.

2 60 , 19 10.52 1 1 . 2 2

3,!J2 1 0 3

Table I11 show that when a kuown amount of betainc is added to a suitably prepared diffusion juice, it can be accounted for satisfactorily in the determination of total M a i n e in the sample.

Table IV. Analysis of Johnstown Waste Molasses Reported to Contain 9.0% Betaine Chloride

Betaine Chloride Found, h a i t i l ~ i ~ Size. 311. Weight %

, 8 94 J 9 03

10 S 86

-~

Table I V shows the result of a comparative analysis of Johns- town wast,e molasses. This sample had previously been ana- lyzed (4) by a modified periodide procedure and the result cor- rected for accompanying purines. The authors’ analysis of the material, after dilution, but with no chemical rrnioval of impuri- ties, indicates good agreement with the values ohtuined ti!, the other procedure.

DlscussIo~

Some time was spent trying to tlevolop a graviiiietiic tlrtw- niiiiutioii of betaine ivit,h Reinecke salt us a precipitimt. The use of ice wiiter, methanol, ethyl alcohol, isopropyl alcohol, n- or tert-butyl alcohol, dioxaiic, or ethyl acetate for wash SolutionS gave. washings w-hich ere int,erisely colored a t first, but 311 ooii- tiiiued to give slightly colored solutions HY washing 1irocwded, inciic:itiiig that the betaine reiiic?ckat,e was txing di~solvetl :IS the motliw liquor \viis washed out. Tiit. addition of of hydrocshloric acid failed to decrease the SOIUI reinrck:itc suitably in these solvents. I-arious niistures ol ether and 1jut:tnoIs failed to wash out the coprecipitatetl subatances, or else dissolved so much of the hitaiiic reineckate a t the same tinie that low :tnalytical values were obtained. The results varirtl from 10 to 15% high to 5 to 20% loa-, depciiding on the amount of washing and the solvent mixture used. The coridi- tions necessary to establish the optimum washing conditions for a particular sample size were so specific that the :rnalysis of an

unknown sample was practically impossible. The authors have confirmed the observat,ion (10) that ammonium rhodanilate is not as satisfactory as animoniuni reineckate for the precipitation of betaine. Pure Reinecke acid was not as satisfactory as an acidified solution of the ammonium salt.

The difficulties encountered in the gravimetric deterniincttion of betaine are also inherent iii :Lny colorimetric procedure, be- cause the red color of the solutions is apparently due to the reineckate ion alone. Consequently, unless a pure precipitate of betaine reiiieckate cui1 be obtaiiied, the colorimetric values will be just as erroneous :is the gravimetric values.

The bet,aine det.errnination reported here appears to h&vc cer- tain advantagrs over pi,eviously proposed methods, principally because most, of t,he iritcrfcring substances do not have to br re- moved prior t,o t8he precipit,ation of the betaine; this reduces the time required for analysis. Thc presence of sugar (in t8his study, the sugar concentration w t s a h u t 12%) in the solut,ion to be analyzed does not appc:rr to affect t,he results. The over-all time required for a detcriuiri:iticin of duplicate samples, including preparation of the snmplc, is :tl)out one hour, which is minewhat faster than thv periodide procedure reported in the lit,rrature. lteifer reports that an accuracy of 2% is possible with the “per- iodide method” o n pure I)etaiiie solutions (8 ) but that, “the pot,xs- sium triiodide also yields precipitates with dimethylamine, tri- methylamine, certain cycalic t)uscs, :ind many alkaloids.’’ The need for est,ensive prelimin I rc~iiiov:tl o f impurities wing this method is therefore uiitli.r?it:iiiclal,le.

If it is assumed t,h:it tht, clisaociittion constant, of Ixtaiiic iii-

trate is of thc s:tme o r t l c ~ of m:igiiit,urle as that of the chloride (ahout 1 X 10-9) ( 7 ) , : t i id that tlie c:oiicentratiori of silver ion in the final solution is leas th:tn 0.01 .V, it, can be calculated that the betaine nitratcb (’a11 tw titjr:ttctd qu:mtitatively to utmut pH 6 n-it,hout, interference from t h silvctr io!i. Ehperimcnta Ily deter- mined titratioii curvi’; iii11ic:~tc. that, these assumptioiiu itre a p parcntly justificd, even i i i the :Lqut’trus solutions containing some ac(,tone obtained froni tlir c~\-pc~riiiient:il procedurci. The clirect titration of t,hc Ixt,airic. wincck:itc, in :mitone-water with sodiuin hydroxide, Kith a glass olwtrotlc t o follow pH, was not satis- factory, presumahly I)c~r:tuxc~ tlic Ixttairie reineckat,P is a t’uirly weu k :wid.

KKNOU- Lm(;\mwr

The authors w-ish to cspress their ap1)reciation to 11. 5. Owens for his interest and :lid on thi? prol)lem, They also wish to ex- tend thanks to the 1nternation:tl Ilinerals and Chemicnl Gorp., Amino Products Division! for :i supply of hetainc chloritic, and to the Great Western Sug:ii, Co. for Y sample of waste mol:isses of known composition.

1.ITEKiI’IJRK CITED

(1 j dssoc. Oftic. Apr. Chcniista, “Officio1 and Tentative SIe t lda of

(2) Randelin, F. J., Slifer, I<. I>., and Paiikratz, R . E , , ,I. A m .

(3) Heattie, F. J . I<., f3iochr.m. .J. . 30, 1554 (1936). (4) Bennett, N.. private con~n~unicntion. ( 5 ) Blood, J . W,, and Crantield, H. T. , A n a l y s t , 61, 829 (193fi). (6) navies, W. L., and 1 3 ~ 1 ~ d c i i . H. C., J . Soc. Chcm. I u d . , 55, 175’1‘

(7) Gustafsson, C., Lit.,., 77B, 66 (1944). (8) Reifer, I., Scus Zwlnrrd J . .Sei. Techno?., 22B, 111 (1941). (9) Stanek, V., Z. physiol. C h p m . , 47, 83 (1004); Z . Zucker i i~r l .

J . Cheiii . Soc., Abst,, 86, 11, 790

(10) Strack, E., and Schwaticberg, H., 2. physiol. C h m . , 245, 11

(11) Wilson, J. B., and Kcenan, G . L., J . dusoc. Ofic . A g r . Chemists,

Analysis.” 6th ed., 1945.

fhnrin. Assoc., 39, 277 (1950).

(1936).

Bolmen . , 28, 578 r1902); (1904).

(1936).

21, 474 (1935).

RECEIVED November 25, 1950.