Lactic Acid Determination 1

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THE DETERMINATION OF LACTIC ACID.*BY THEODORE E. FRIEDEMANN, MARGHERITA COTONIO, ANDPHILIP A. SHAFFER.

(From the Department of Bio logic al Chemistry, Wash ington UniversitySch ool of Medicine, St. Louis.)

(Received for publication, February 26, 1927.)

As the result of some experience in the determination of lacticacid in blood, muscle, and solutions of sugar derivatives, we haveintroduced various modifications in the process by which it gainsconsiderably in simplicity, speed, convenience, and reliability.The basis of the method is the oxidation of the lactic acid inboiling sulfuric acid solution to acetaldehyde by permanganate(l), aeration of the aldehyde into large excess of sodium bisulfitesolution, and titration of the combined bisulfite as described byClausen (2). The chief modifications which we introduce are:(1) the form of apparatus used for the oxidation and for the collec-tion of the aldehyde, (2) the addition of manganous sulfate tocatalyze the oxidation by permanganate, and (3) more accuratelydefining the conditions for the titration of the sulfurous acid boundby the aldehyde. We have tested separately the several stagesof the method and have checked it on known amounts of lacticacid in pure solution and after addition to blood. A number ofother substances have been tested as to the extent of their inter-ference in the determination of lactic acid.By the procedure as herein described the yield of aldehyde isconsistently 96 to 98 per cent of the theoretical; and routineresults are, in our hands, not subject to the variability and un-certainty commonly experienced with lactic acid determinationsby older methods. The process s also much shorter in time and

* This paper includes data to be presented by one of the authors (M.C.)in a dissertation for submission to Washington University.335

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336 Determination of Lactic Acidis to a considerable extent free from certain bothersome detailswhich heretofore have imposed serious handicaps in the deter-mination of this substance. The oxidation, collection of aldehyde,and titration may be completed in from 15 to 20 minutes, or aboutone-third to one-fifth the time required by older methods.2 Withfour sets of apparatus two persons have made more than 60determinatiqns in 8 hours. The technique is readily adapted tothe determination of widely different quantities, and is to thisextent. rather generally applicable. Besides being a usefulanalytical method, the reactions involved present several interest-ing theoretical points which are considered.

Apparatus.The apparatus, which is simple and inexpensive (Fig. 1)) consists

essentially of a boiling flask fitted to a reflux condenser throughwhich an air current carries the very volati le aldehyde into anabsorbing tube or tower containing bisulfite. Instead of the beadtower shown in the figure two large test-tubes in tandem may beused for the absorption. The whole apparatus may readily beconstructed from ordinary flasks, test-tubes, and glass tubing.Besides being a convenient form in which to conduct the reaction,the apparatus accomplishes two main purposes, (1) the rapidremoval of the aldehyde by the air current without disti llingliquid into the receiver, thereby keeping the volume small for thesubsequent titration, and (2) a fairly efficient separation ofacetaldehyde from other less volati le sulfite-binding productswhich may result from the oxidation of substances other than lacticacid. The number of interfering substances is thus reduced andthe method made more nearly specific for lactic acid (or sub-

1 See for example the precautions advised by Embden (2. physiol. Chem.,1925, cxliii, 297) who prescribes that the oxidation of 3 mg. of lac tic acidshould require 16 hours; (also Hirsch-Kauffmann, Z. physiol. Chews., 1924,cxl, 30,45).

2 With sma ll amounts of lactic acid, as in blood analysis, it is quitepos sible to obtain equally high yields without added MnS04, provided therate of KMnOr add ition be very slow and we ll regulated , as show n for ex-ample by the work of Hirsch-Kauffmann. In such circum stance s the effectof MnSOa merely permits more rapid oxidation without the sacrifice o faccuracy which otherwise occurs.

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I I

Stole- Inches012345

3FIG. 1. A is a reflux condenser of the Hopkins type, connected with a

300 cc. Kjeld ahl flask by a stopper which carries also a dropping funnelthrough which enter the air current and KMnOd solution. The flask is SUD-ported from the condenser and needs no separate clamp . For most effi-cient operation the spac e between the insid e condense r tube and the innerwall of the jacket should be only 1 to 3 mm. and the rate of water flow suffi-cient to keep the air in A and B cold. The condenser may be made of largePyrex test-tubes . C, D, E is a tower of glas s beads, connected to a 150 cc.wide mouth extraction flask which contains the bisu lfite solution, the wholebeing conne cted to the condenser by a rubber tubing at B. The tower isconstructed from a large Pyrex test-tube by sealin g on tubes, and makingindentations at C to hold a perforated glas s or porcelain plate at D, whichsupports an 8 cm. colum n of beads. A tube from a water pump connected atE provides the air current. The entire apparatus is mounted permanentlyby burette clam ps A and E. The clamp at A (Arthur H. Thom as Companycatalogue No. 3220) is kept lose at its base so as to permit the condenserbeing swung sidewise when the Kjeldahl flask is put in place. At the endof the oxidation the aeration is stopped by removing the stopper from thetop of the tower, the extraction flask is detached and lowered to the shelfbelow it, and the tower and beads washed with five to seven 5 cc. portionsof water. Th e flask is then removed for the titration. If many determina-tions are to be run it is convenient to set up a series of these units; we usesix, which may be operated by one person.

337

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338 Determination of Lactic Acidstances which yield under the conditions very volatile bisulfite-binding products).

Action of Manganese Sulfate.The presence of an excess of manganous salt at the start of the

oxidation of lactic acid has a remarkable effect in greatly increasingthe rate of oxidation by permanganate, and in increasing theyield of aldehyde. The effect of increasing the rate of oxidationis doubtless the same as that often observed in the titration ofoxalic acid, and is of interest apart from its use in the lactic acidmethod. In both cases the oxidation, in the absence of manganoussalt, proceeds at first very slowly and rapidly increases after someof the added permanganate is reduced; but if manganous salt beadded before the permanganate the latter reacts almost instantlywith the result that the lactic acid is virtually titrated (as withoxalic acid), an excess of permanganate consequently does notaccumulate until toward the end, and the danger of further oxida-tion of aldehyde by excess permanganate is thus minimized.Rapid removal of the aldehyde from the boi ling liquid by the aircurrent is an additional protection.

The action of manganous salt thus appears to be ideally suitedto the purpose at hand. Besides increasing markedly the rate ofthe oxidation, it tempers or restrains the reaction so as to avoidsecondary oxidations which result in products other than acetalde-hyde. This latter effect considerably reduces the importance of avery slow addition of permanganate which is otherwise so essentialto obtain correct results.

The effect first named, that of increasing the rate of oxidation,is very evident in the actual process of determination if one com-pares the rat,e of permanganate consumption by lactic acid in thepresence and absence of added manganous sulfate; and is moresimply demonstrated by noting the rate at which the color fadeswhen a few drops of dilute KMn04 are added to acidified solutionsof lactic (or oxalic) acid, with and without added MnS04. Atroom temperat,ure the pink color of the solution containing man-ganous salt fa.des at once with oxalic acid, or after a few minuteswith lactic acid, while without the MnS04 the pink persists muchlonger.

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Friedemann, Cotonio, and ShafferThe second effect, that of increasing the yield of acetaldehyde

is shown by analytical data. Under comparable conditions 5 to20 per cent more aldehyde may be obtained with MnS04 thanwithout. As an illustration of this effect the following resultsmay be cited. From the oxidation of 109 mg. of lactic acid inN H&304 by dropping into the boiling l iquid 0.1 N KMn04 andsimple distillation of the acetaldehyde into bisulfite solution, theaverage yield without added MnS04 was only 67.5 per cent,while with the addition of 5 to 50 cc. of 5 per cent MnS04 theyields rose to 85 per cent of the theoretical value. The low resultseven with protective action of manganous salt also illustrate thefact that simple distil lation is much less effective than aerationplus distillation, which with the aid of manganous salts yieldsconsistently 96 to 98 per cent.2

The explanation of the action of manganous salt seems fairlyclear. It is due to the reaction bet,ween permanganate andmanganous salts which results in the formation of manganiteor MnOz which is the effective oxidizing agent of lactic acid.

MnzO, + 3MnO = 5MnOz or 2XIn7+ + 3?\In?+ = 5Mn4+In this reaction by an exchange of electrons, the permanganate(Mn7+) is transformed to a lower intensity level (Mn4+), which isstill high enough to oxidize the lactic acid,3 but not so intense asto oxidize rapidly the acetaldehyde. The reaction betweenMn2+ and Mn7+ takes place with the separation of hydrated

manganite or MnOs if no oxidizable substance is present, but inthe presence of lactic acid or other oxidizable substance the solu-tion remains clear due to reduction of the Mn*+ to soluble saltsofMn?+. In either case the Mn + is removed and oxidations whichmight result from its action are thus prevented. If one adds adrop of dilute KMn04 to a solution of acetaldehyde in N H2S04,and the same amount also to another portion of aldehyde solutioncontaining MnS04, the color fades more rapidly in the solutionwithout MnSOa, while a precipitate forms in the other whichpersists. This evidently means that Mn+ (KMn04) oxidizesacetaldehyde while Mn4+ (MnOn) does not do so, or but slowly.

3 As observed long ago by Boas (1) MnOn oxidizes lactic acid.

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340 Determination of Lactic AcidThe MnS04 therefore protects the aldehyde from oxidation, byremoving the Mn7+ (MnOa-).4

Why the MnOb- alone oxidizes the lactic acid (or oxalic acid orother substance) more slowly than does the lower MnOz is notevident, though presumably it is due to the electronic condition ofthe molecule containing Mn + (Mn04-).5 That the general expla-nation advanced above is correct seems to be indicated by a con-sideration of the electric potentials of the systems. The follow-ing values are given (Kolthoff (5)) as the normal potentials,referred to the normal hydrogen electrode, for the electrodereactions stated.

Mn02 (so lid) + 2HrO = MnO; + 4H++ 38 . . . . . . . . . . . . 1.63 v.Mn++ + 4 Hz0 % MnOr + 8H+ + 5 0 . . . . . . . . . . . . . 1.50 3 2Cl- = Cla + 20... . . . . . . . . . . . . . . . .._.. 1.36 Mn++ + 2H20%Mn02 (solid) + 4H+ + 20.. . . . . . . .1.35

A few observations of our own of the chainHg Hg,C& saturated saturated KC1 0.01 N KMnOl in Pt (or Au)

KC1 bridge N H:SO.,gave values at 25C. varying in different trials from 1.18 to 1.26v, sometimes the Pt and sometimes the Au being the higher.Converting these values with reference to the normal hydrogenelectrode we have + 1.426 to 1.507 v. The addition of approxi-mately 0.05 M MnS04 caused a drop of potential to 1.11 (or

4 A somewhat related phenomenon is probably that long ago noted inthe titration of ferrous iron by permanganate in the presence of chlorides(for disc uss ion see Treadw ell, F. P., Quantitative analysis, New York,2nd edition, 1910). In the presence of chlorides (and many other substa ncesalso) more KMn04 is required than corresponds to the oxidation of ferrousto ferric salt, due to oxidation of Cl- to Clz, induced by the peroxide-likeform of iron which is the primary product (3, 4). T his induc ed oxidationof Cl- i s however prevented if manganous salt be added before the per-manganate. Here one may suppos e with Manchot (3) and others that theMn++ is oxidized by the iron peroxide (as we ll as by MnOr) thus removingthe latter and thereby preventing its actio n on Cl-.5 If, as given by Lew is, the electron orbits of the Mn7+ atom, contain 2,8, and 8 electrons respectively, this cond ition should be a more stablearrangement (being that of A, K +, Ca++, etc.) than that of Mn*+, withorbits of 2, 8, and 11 electrons. (Lewis, G. N., Valen ce and the structure ofatoms and mole cules, New York, 1923.)

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Friedemann, Cotonio, and Shaff er 341+ 1.357 to N H+ electrode) both electrodes coming into agreementwithin a few millivolts. These figures therefore demonstrate thedrop in oxidation intensity which results from the addition ofMnff salt.

Although the oxidation intensity or potentials required tooxidize lactic acid or acetaldehyde are unknown, it may perhapsbe inferred from the observations mentioned earlier that lacticacid begins to be oxidized rapidly under our experimental condi-tions, below + 1.35 v, while to oxidize acetaldehyde at a com-parable rate under the same conditions requires a higher potential.It seems probable that the potential for the oxidation of acetalde-hyde (in HzS04 solution) like that for the oxidation of Cl- to Cl2lies somewhere between the potentials characteristic of Mn7+and Mn4+. According to the value cited above the potential ofCl- =-l-G l, is 1.36 v, which is exceeded by Mn7+ but not by Mn4+.Quite in accord with expectations based on their relative poten-tials, although the addition of KMn04 to boiling N HzS04 con-taining HCl rapidly liberates Clz, no chlorine is formed if MnS04be added before the KMn04. After making this experiment wefind that the same facts were noted by Kessler in 1863.From the above observations we may arrange the apparentoxidizing intensity of these systems in the following order.

Incr&singoxidstionintensity.

KMn04. . . . . . + 1.50 v.T Required to oxidize acetaldehyde, les s than + 1.50 (about + 1.35 (?)).

Cl- % Cl , .,,,.............................. + 1.36~ .MnOa + Mn++. . . .$ 1.35 Required to oxidize lac tic acid, les s than + 1.35

Titration of Xul$te Bound in Acetaldehyde-Xulfite.The equilibrium in the reversible reaction between acetaldehydeand sulfurous acid

Aldeh yde + H,SOz k a.ldehyd e-sulfurous ac iddepends upon the reaction of the solution as well as upon theconcentration of the components.In acid solutions nearly all of the aldehyde (or sulfurous acid)

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342 Determination of Lactic Acidis combined as aldehyde-sulfurous acid in which form both com-ponents are stable. The combined sulfurous acid is not oxidizedby iodine. Furthermore the rate of dissociation of the aldehyde-sulfurous acid is, in acid solution, so slow that any excess of freesulfurous acid may be removed without appreciably disturbing theequilibrium during the short period occupied by the titration.This permits the determination of aldehyde by its addition to aknown amount of alkali bisulfite and titration of the excess un-combined sulfurous acid by iodine, as in the Ripper (6) method.At the end-point of this titration there is left in the solution thecombined aldehyde-sulfurous acid, HBS04 (from the oxidation ofthe excessHBSOJ and HI (from the reduction of L).Clausen (2) noted that the addition at this point of weakalkali such as NaHC03 causes the disappearance of the bluestarch-iodide color and permits the titration of the combinedsulfite by the further addition of IZ, due to the gradual dissociationof the aldehyde-sulfurous acid, which although too slow fortitration in acid solution takes place with greater speed in lessacid or alkaline solution. If sufficient alkali be present to neutral-ize the HI and NaHS04 formed, the dissociation is relativelyrapid and continues to completion if sufficient IZ be added andsufficient time allowed. Under suitable conditions the Ripperand Clausen titrations may therefore be performed on the samesolution, thus giving two determinations, one by difference andone direct, on a single sample.

The Clausen plan of titrating the combined sulfite is preferable tothe Ripper method for several reasons. (1) It is a direct titrationrather than by difference; (2) it allows the use of a large excessofbisulfite to insure complete absorption of the aldehyde and itsrapid conversion to the compound; (3) it avoids the errors causedby air oxidation of the uncombined sulfite and loss by volatiliza-tion of SOZ, both of which may cause serious errors in the Rippertitration; (4) it permits the use of aeration to remove the aldehydefrom the oxidation mixture, which is impractical with the Rippertitration because of (3) and (2). As already noted, aeration isvery desirable because t aids in rapidly sweeping the aldehyde outof contact with permanganate; and when used with a refluxcondenser, as in our type of apparatus, the aeration allows aseparation of acetaldehyde from other lessvolatile bisulfite-binding

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Friedemann, Cotonio, and Shaff er 343or iodine-reacting substances which sometimes arise from theoxidation of substances other than lactic acid.

In order to secure these advantages it must, however, be estab-lished: (1) That at the first end-point (the start of titrating thebound sulfite) substantially al l of the aldehyde is bound. Forif, as might be expected, the combination were incomplete or ifany considerable dissociation of the aldehyde-sulfite were tooccur in acid solution during removal of excess bisulfite in thefirst titration, that portion would be lost in the subsequenttitration. (2) That the dissociation of the combined sulfite iscomplet,e during the second titration; and (3) that the liberatedsulfite may be determined correctly by titration with iodine in thepresence of the added alkali, without the occurrence of side reac-tions to affect the results. Under suitable conditions thesepoints cause no error, and the titration can be made quite accurate.We shall consider the above points in order.

Proportion of Aldehyde Bound by BisulJite.In acid solution the combination of acetaldehyde and sulfurous

acid or alkali bisulfite in excess, although by no means instan-taneous, is substantially complete i f sufficient time be allowed toattain equilibrium. In the presence of large excess bisulfiteequilibrium is reached in 10 minutes or less at room temperature,a period well within the time taken for the oxidation of lactic acidand aeration of the aldehyde. The reverse reaction, the dissocia-tion of the compound, is so slow in acid solution that the equilib-rium is not perceptibly disturbed during the removal of excessbisulfite by iodine titration. Kerp and Kerp and Baur (7) foundvalues of about 2 X low6 for the equilibrium constant (Ii) in theequation for this reaction

[H$SO3] x R = [A] x [HSO ,]in solutions of M to ~/30 sodium acetaldehyde-bisulfite at 25C.The values of K were not very different in the presence of anequivalent excess of aldehyde or M/30 HCl or both.The amounts of lactic acid frequently present in solutions beinganalyzed are from about 20 to 0.2 mg.6 or from 0.2 to 0.002 mM and

6 The amounts which may be analyzed are by no means limited to thesequan tities; see Tab le III.

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344 Determination of Lactic Acidat the time of titration the corresponding amounts of aldehydeare contained in about 50 cc. of solution which is therefore 4 X10-S to 4 X 1O-5 molar as to total aldehyde. With only equivalentconcentrations of bisulfite the calculated percentage of uncombinedaldehyde at these concentrations would be 2 per cent and 20 percent. Solutions of pure crystalline acetaldehyde bisulfite showthis order of dissociation. But in the presence of 5- and 500-foldexcess of bisulfite respectively (10 cc. of 0.1 M solution) thesecalculated fractions are reduced to 0.01 per cent. With 1 equiva-lent of aldehyde in excess Kerp found only 0.04 per cent of thesulfite uncombined at 25C. We have obtained similar results.With 100 cc. of a fresh solution (approximately 0.005 M) of purecrystalline acetaldehyde bisulfite to which were added about 2equivalents of excess acetaldehyde, after standing at room tem-perature a few minutes, the free sulfite was titrated with 0.01N 12. 2 drops gave a distinct blue end-point, which persisted forseveral minutes. Making allowance for a blank, the free sulfiteamounted to not more than 0.06 per cent of the total present(calculated value about 0.02 per cent). An excess of bisulfite hasthe same effect in decreasing the uncombined aldehyde as shown bythe data in Table II. It is evident therefore that combination issubstantially complete.

In acid solution the rate of dissociation after oxidation of theexcess bisulfite is so slow that it may safely be ignored. Experi-ments to test this point showed that in about 0.002 M solutionafter titration (and oxidation) of the excess free sulfite, thealdehyde-bisulfite dissociated (in the presence of an excess ofiodine, which oxidized the sulfite as fast as liberated) only at therate of about 1 per cent in 30 minutes at 25. From this we infert,hat not more than 0.1 per cent of the total will dissociate in the3 or 4 minutes required for the first titration and adjustment ofthe first end-point. This conclusion is confirmed by the datacited later which show almost theoretical results for the tit-ration,under optimum conditions, of the bound sulfite in solutions ofpure acetaldehyde-bisulfite. (Table II.)

Dissociation of Bound Bisuljite by Alkali.The next step in the Clausen titration is the addition of alkali

to liberate the combined sulfite. Clausen states ((2) p. 265) merely

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Friedemann, Cotonio, and Shaffer 345that, Sufficient saturated sodium bicarbonate solution is thenadded to discharge the blue color, and cautions against too highalkalinity from too much bicarbonate, or from carbonate in thebicarbonate, which leads to secondary reactions, such as theformation of iodoform. These directions need amplification,especially if more than very small amounts of aldehyde arepresent. The rate at which the compound dissociates dependsupon the pH of the solution, which is constantly becoming more

TABLE I.EQect of Alkali upon Titration of Sodium Bisul$te Solutions by Iodine.The solution s were titrated in a total volume of about 50 cc.

Alkali added. Titration.Cc.of 120.1x Error.

A. NaHS03 solution.per cent

No adde d alka li.. . . . . . . . . . . . . . . . 22.20Exc ess so lid NaH C03.. . . . . . . . . . . . . 21.35 -3.8Slight excess Na&Os.. . . . . . . . . 20.10 -5.0NaOH to neutralize acid formed.. _... 20.50 -3.1

B. NaHS03 solution.No added alka li.. _. _. . . . . . . . . . . . . . . . . . . . . 40.10+glycerol, 0.5 per cen t.. . . . . . . . . . . 40.10 0NaHC03 + glycerol.. _. . . . . . . . . . . . 39.30 -2.0NaC03 + . . . . . . . . . . . . . . . 39.85 -0.6

C. NaHS03 solution.No added alka li ._. . . . . . . . . . . . . . . . 43.85Added excess acetaldehyde.Aldehyd e + NaHC03. . . . . . . . . . . . . . . . 43.85I + NalHP04.. . . . . . . . . 43.90

0$0.1

acid during the titration due to the HI and HBSO, formed. Tosecure complete dissociation (within short time) a higher pH isrequired than to cause partial dissociation, due doubtless to theinfluence of the increasing free aldehyde concentration.If an insufficient amount of NaHC03 or other alkali be added thecolor may at first be discharged, yet the rate of liberation of sulfiteand the consequent rate at which the titration with iodine maybe carried on may be tediously slow, and may even reach anapparent end-point long before dissociation is complete. If on

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346 Determination of Lactic Acidthe other hand too much alkali be added, with consequent toohigh pH during the titration, the sulfite is liberated rapidly butlou rather than high results are obtained. This error is due mainlyto air oxidation of sulfite, which occurs more rapidly in alkalinethan in acid solution, and is much more serious than the danger ofhigh results from the formation of iodoform noted by Clausen;although loss of iodine by conversion to hypoiodite may becomeimportant if the 1, be added very rapidly in the presence of toogreat excess of a stronger alkali, i.e. too high pH. Obviously theoptimum amount and strength of alkali to be added must besuch as to avoid the two ext,remes, to permit complete liberation ofthe sulfite within a convenient period of titration without produc-ing a dangerous alkalinity.

Without aldehyde the titration of sulfite by I2 in presence ofexcess NaHCO$, Na2C03, or other alkali yields low results (seeTable I); and that these low results are due to air oxidation ofsulfite we have shown by determination of sulfite before and afteraeration of the alkaline sulfite solutions. The presence of thealdehyde markedly protects the sulfite from air oxidations, byforming the stable compound. But this protective action of thealdehyde is in part lost when as a result of too high alkalinity thedissociation takes place much faster than the I2 is added and thefree sulfite is thus exposed to air. The ideal would therefore beto add gradually throughout the titration parallel with the 1%an amount of alkali just sufficient to neutralize the HI and HBS04formed in the oxidation by I2 and thus to liberate the sulfite atabout the rate at which it is titrated with 12.

Bearing in mind the limitations stated above and the fact that13 equivalents of acid (1 HI + 0.5 HBS04) are formed for each

7 A curious fact for which we are not able to offer an explanation is thefollowing. After titrating the excess bisu lfite of an aldehyde-sulfite SO~U-tion, a drop of 2 or 0.1 N NaOH instantly discharge s the blue color of thestarch end-point, in spite of the fact that the solution is stil l strongly acidfrom the HI and HBSO a formed in the first titration. Further add ition of IIrestores the blue, to be discharged again by a few drops of dilute alkali.The addition of 10 or 20 cc. or more of 0.1 N HCl to the solution, thus furtherincreasing its acidity, does not prevent this immed iate disappearance ofthe blue on subsequ ent addition of a drop or 2 of 0.1 N alkali. The effectcan therefore hardly be due simply to pH change. (In the absence of alde-hyde-sulfite the alkali, of course, has no such effect on the blue iodide ofstarch.)

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Friedemann, Cotonio, and Shaffer 347normal equivalent of I, used in the titration, one may add thecorresponding amounts of alkali in various forms, provided thatthe alkalinity in the early stages of the titration is sufficient onlyto allow dissociation at a rate approximately that of the rate oftitration, thereby avoiding the presence of excess free sulfite tobe oxidized by air, and providing that toward the end the residualalkalinity is high enough to allow complete dissociation.

Analysis of Acetaldehyde Sodium BisulJite.The criteria for choice of alkali are obviously correct results and

convenience. By the use of pure aldehyde-sodium bisulfite wehave a means of accurat.ely testing the titration. Data are givenbelow for the analysis of a very pure sample of this compound forthe preparation of which we are indebted to Mr. Earle Adler.*

The figures given for sodium and sulfur content agree well withthe calculated values for the compound containing $ mol of waterof crystallization, and indicate the purity of the preparation.Under optimum conditions the results of titration of the com-bined sulfite (and thus the aldehyde) with standard iodine by theClausen principle also agree very well, as shown by the results of

* Beca use of the purity of the product as well as becau se of failure inseveral earlier attemp ts we record the suc ces sful procedure. -4 fresh con-centrated solution of sodium bisu lfite is made by pass ing SO*, generated bydropping 125 cc. of 1: 1 HzS04 into a solution of 150 gm. of Na2S03 in 250 cc.of water, into a solution of 60 gm. of pure Ka&Os.HaO in 150 cc. of water.The solution must contain an excess of SOS, indicated by a greenish color.The bisu lfite solution in a 2 liter flask is well cooled in ice-sa lt mixture; to it isadded, very slowly and with shaking, 45 gm. of very cold acetaldehyde.(Mallinckrodts preparation containing 9 per cent alcoh ol wasused.) After

standing several hours in the freezing mixture about 1500 cc. of chille d95 per cent alcoh ol are added in sm all portions over a period of an hour. Themixture is kept thoroughly chille d and is stirred or shaken at intervals.Soon after the last portions of alcoh ol are added the mixture sets to a vis-cous mas s of crystals, which is then filtered rapidly by suction on a pre-viously chille d Buchn er funnel and sucked fairly free from mother liquid .The crystalline material is then dissolved in the smallest possible amountof water (75 to 100 cc.), the solution is well cooled in freezing mixture, andabout a liter of alcoh ol is added in portions as before. After crystallizationthe mixture is filtered on a cold funnel, and again recrystallized. The prod-uc t is finally dried in air at room temperature to constant weight. Yield ,after two recrystallizations about 135 gm. The material is free from su l-fates and as shown by the data cited has the com position NaS03C,H,0.+H?0.

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348 Determination of Lactic Acidsuch titrations, calculated as equivalent per cent of aldehyde in thesubstance, given under aldehyde. The accuracy of the Clausentitration is thus demonstrated.

The preparation was substantially free from sulfate. Deter-minations of sodium, sulfur, and aldehyde (combined sulfitedetermined by iodine titration) gave the following results.

Sodium. Sulfur. Aldehyde.per cent

Theory for NaHS 03.C&H,O.f HsO.. 14.64Found................................... 14.60

pet cent per cent20.40 28.0220.52 27.9020.48 28.0020.70 28.10

Average found. .......................... 14.60 20.57 28.00Per cen t of theory ....................... 99.73 100.7 99.93

In Table II is given a summary of a number of titrations ofbound sulfite (or aldehyde) in solutions of widely different con-centrations, and with different alkalies to liberate the sulfite.Al l of the titrations on solutions of the pure compound weremade under conditions simulating those in the lactic acid deter-mination. Weighed amounts of the substance were dissolved toknown volumes and aliquots of the solutions pipetted into sodiumbisulfite solutions of such concentration as to make about 0.02 to0.05 M excess bisulfite after dilution. The solutions titrated thuscontained large excess of bisulfite which prevented dissociation ofthe aldehyde compound and which also liberated acid in the firsttitration (of the excess free sulfite) thereby regulating the reaction,as noted below, when the alkali was subsequently added. With-out attention to these points the results for bound sulfite are lowfor the reasons already stated. The volume of the solutionstitrated was 40 to 80 cc. As indicated by the data in Table II,the results vary by not more than about f 1 per cent or less atwidely different concentrations, except at great dilut ion when theresults are 4 to 8 per cent too high.

Choice of Alkali.Of the alkalies tried, sodium bicarbonate, used by Clausen,

yields correct results and is very convenient. With it the danger

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Friedemann, Cotonio, and Shaffer 349of too great alkalinity is slight and with liberal amounts thetitrations may be quite rapid unless the temperature of the solu-tions is too low. When, as occurs under conditions here describedfor the lactic acid determination, the large excess of bisulfitegives rise in the first titration to HI and HBSO,, this acidprevents dangerous alkalinity from the subsequent addition ofeven large excess of solid bicarbonate. When the first titration is5 to 10 cc. of 0.1 N I2 sufficient of the liberated CO, remains dis-

TABLE I I .Res ults of Titration of Solu tions of Pure Acetalde hyde-B isuljite (with Exces s

of Bisu ljite) after Addition of Various Alkalies .20 or 25 cc. of concen trations stated were titrated in total volume of

about 50 cc.

Alkali usedt$ ;~fte bound

NaHC 03.. . . . . . . . . . . . . . . . . . . . . . + glycerol..KzHPO a + . . . .Borax + .NaHC08.. .I

+ glycerol... . . . . . . . . . . . . . .I + glycerol..

Borax + glycero l..( + ( . . . . . .

-concentrationot aldehyde-bisulfite byweight.

M8 x 10-z55552 x 10-x1.8

4 x lo-112 x 10-s

wi;pnt I%used,.ldehydeitrated.

mg . N180.0 0.1

11.0 0.0111.0 0.0111.0 0.0111.0 0.01

4.5 0.0024.0 0.010.9 0.0020.225 0.0020.225 0.0020.045 0.002

A ldehyde equivalen tof bound SO ?.Per cent of errorfrom theory byweight.

-0.3-0.4$0.1 to -0.4-0.4$0.1 to -0.4+1.0f1.5$0.5+7.0+5.6+4.0 to +s.o

solved to keep the reaction around pH 8.4. Without the pro-tective action of the aldehyde this reaction leads to considerableerrors from air oxidation in the titration of pure bisulfite, but? asthe data in Table II show, the titration of acetaldehyde-bisulfite(in presence of the excess acid mentioned above) yields approxi-mately correct results with even large amounts of bicarbonate.The simplest plan (under the conditions stated) is merely to add afew tenths to + gm. of solid bicarbonate and more if the rate ofI, consumption becomes slow. When the room temperature is

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350 Determination of Lactic Acidhigh and that of the solutions 25 or more, less bicarbonate isneeded than when colder; but under the conditions describedwide latitude is permissible without significant differences in theresult, provided enough be added to insure the end-point beingreached. To avoid this uncertainty more bicarbonate may beadded at the end to test the end-point.

Another equally satisfactory alkali is borax in the presence of anexcess of glycerol. The glycerol so increases the strength ofboric acid that under the conditions used the mixture provides asuitable reaction (pH about 8) for the rapid liberation of thebound sulfite. The use of borax without glycerol gives too greatalkalinity and low results. A suitable amount of borax is thesame volume of a saturated solution as the volume of 0.1 N Izused in the first stage of the titration, and half that volume of 20per cent aqueous solution of glycerol.Sodium or potassium phosphate (B2HPOd 0.6 to 1 cc. of Msolution or equivalent for each cc. of 0.1 N Iz) is also satisfactory.

Sodium carbonate alone, and to less extent with glycerol,ggives low results unless the solutions are quite cold; its use is notrecommended.Behavior of Acetone and Formaldehyde.

The combination of bisulfite with acetone is much weaker thanwith acetaldehyde, and of formaldehyde much stronger. Becauseof this difference we hoped to be able to titrate acetaldehydecorrectly in the presence of acetone and formaldehyde, but thisappears to be impracticable. In very dilute solutions a veryconsiderable fraction of the acetone-bisulfite will be dissociatedat the first end-point if it be adjusted slowly. But the amount ofdissociation so depends upon time and temperature that it is

9 About one-fourth of the bisu lfite is lost during the aeration by volatil-ization of some SO:, and by air oxidation to sulfate. Th is lo ss is of no con-sequen ce so long a s enough is left to hold the aldehyde; but to insure suchexcess a conside rable margin is allowed. The los s by oxidation is very mark-edly decreased and the stock solution is more stable if glycerol be addedto the sulfite solution. Th is device cannot be used however becaus e duringthe aeration, or on standing exposed to air, some of the sulfite becom esbound as though aldehyde were formed. Th is is probably due to an oxida-tion of sm all a mounts of glycerol to glyceric aldehyde or other product,the oxidation being induce d by the air oxidation of sulfite.

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Friedemann, Cotonio, and Shaff er 351difficult to make quantitative statements about it. It is certainhowever that in the determination of lactic acid in blood or urinethe error due to the formation of acetone from oxidation ofhydroxybutyric (or citric) acid will be much less than correspondsto the amount of acetone formed. As indicated in Table IVI-hydroxybutyric acid interferes only to 1 per cent or less of theamount present, and acetone preformed and from acetoacetic acidmay be removed by preliminary boiling. Except when relativelylarge amounts are present, it is our impression that the acetonemay be neglected without serious error. The aldehyde may beoxidized and removed by disti lling the mixture after addingalkali and sodium peroxide, and the acetone determined in thedisti llate (8). We know of no way to accomplish the reverseseparation.

If formaldehyde is present, however, it interferes to a greaterdegree. At the slight alkalinity resulting from the addition ofNaHC03 or borax + glycerol, the bound sulfite of formaldehyde-sulfite is liberated slowly as compared with that of acetaldehydecompound. But when both are present the second end-pointis fleeting and uncertain, and the results are high for acetal-dehyde, due to slow dissociation of the formaldehyde compound.With equal amounts of the two aldehydes one may perhaps guessat the correct end-point for the acetaldehyde with a plus error of10 to 30 per cent. So far as possible substances which giveformaldehyde on oxidation must therefore be removed. Sugarsappear to be the most common source and these are removed bycopper and lime (9).

Description of Method.Solutions.

1. Sodium Bisulfite.-1 per cent (0.1 M) solution. 5 to 10 cc.(or more for large amounts of lactic acid) are used for each deter-mination, and enough water added to cover the beads in thetower. There should be an excess of about 5 cc. or more of 0.1M over the amount required to combine with the aldehyde.

2. Potassium Permanganate.-Approximate 0.1 N stock solution(3.1 gm. to liter) diluted by cylinder as needed to 0.01 or 0.002 N.

3. sulfuric Acid.-Approximately 10 N (285 cc. of concentrated

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352 Determination of Lactic AcidH,SOd to liter) containing also 0.5 M or 10 per cent MnS04. Use10 cc.4. Standard Iodine.-0.1 N standardized at intervals againstthiosulfate, which in turn is standardized with KH(IO&. (3.25gm. of KH(IO& to a liter gives a solution which is 0.1 N IZ whenreacting with excess KI and H&40,.) The 0.1 N I, is diluted to0.01 or 0.005 N strength, preferably fresh each day, and protectedfrom direct sunlight. The 0.1 N I, is used for the first stage of thetitration, the end-point being accurately adjusted with the diluteiodine solution (and back titrated if overrun, with thiosulfate).The 0.01 or 0.005 N 1~ s used to titrate the bound sulfite afteradding NaHC03 or other alkali to liberate it.5. Starch Solution.-5 gm. of arrowroot starch suspended incold water, poured slowly into 500 cc. of boiling water and boiledfor 20 minutes. After standing the supernatant liquid is decantedfor use.6. Alkali to Liberate the Bound &l&e.-Saturated solution orsolid NaHC03*K2HP04 or borax + glycerol may be used instead,but no advantage is gained.7. Talcum powder.

Procedure.The lactic acid solution (after suitable preliminary treatmentfor the removal of interfering substances) is placed in a 300 cc.Kjeldahl flask with 10 cc. of the sulfuric acid-manganous sulfatesolution, and sufficient water to bring the volume to about 80 to100 cc. is added. A pinch of powdered talcum is added to promoteeven boiling. The flask is connected to the condenser, and thesuction pump started (see Fig. 1). The flask is heated with amicro burner and the solution kept boiling vigorously. Rapidaeration for a minute or 2 into an empty receiving flask (beforeadding KMn04) removes any volatile bisulfite-binding substances,such as acetone, if any such be present. Then the flame is re-moved and the aeration is stopped while another receiving flask

containing the bisulfite solution is put in place, when the aerationand heating are resumed. (Th e re iminary distillation may be1omitted in case no volatile interfering substances are present.)The permanganate solution is now dropped into the funnel tubeat such a rate that the solution is kept colorless or nearly so.

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Friedemann, Cotonio, and Shaffer 353The oxidation proceeds rapidly and if the permanganate is notadded too fast the pink color fades immediately after each dropuntil most of the lactic acid is gone. Although the manganoussalt greatly reduces the danger from excess permanganate it iswise to regulate its addition to the rate of consumption so as toavoid excess. When the pink color begins to fade slowly theaddition of permanganate is discontinued for a few moments untilthe color fades, then resumed slowly until the pink persists forabout 1 minute, and manganese dioxide has separated. Thisoccurs about 10 minutes after starting the oxidation. Theboiling is continued for 5 minutes more to sweep out al l the alde-hyde, when the flame is removed, the air current stopped, thereceiving flask lowered, and the tower rinsed five to seven timeswith 5 cc. portions of water. The excess bisulfite is titrated with0.1 or 0.05 N iodine using starch indicator, and the end-pointcarefully adjusted to a delicate blue with the dilute iodine to beused in the titration of the bound bisulfite. The bound bisulfiteis then set free by adding NaHC03 either solid or in solution, andthe bound sulfite is titrated with the dilute iodine solution. If indoubt as to the final end-point more bicarbonate is added; if theend-point has been reached the blue color persists after suchaddition for 15 seconds or longer; otherwise it fades quickly.

A blank should be run on the reagents and should be made so asto include any sulfite-binding material drawn into the apparatusby the air current. The blank will not be high unless the roomair contains aldehyde or acetone vapor, as will happen if thesesubstances are used near by. It is wise to exclude the use of allsuch material from the room and neighboring portions of thebuilding when determinations are run. By slightly modifyingthe apparatus the ingoing air may be washed through bisulfiteand this error avoided, but it is simpler to exclude the sources of al lsuch interfering vapors from the room, and to test their absenceby blank determinations on the reagents alone. The titrationof the blank is subtracted as a correction before calculating theresults. Each cc. of 0.01 N Iz represents 0.5~~. of 0.01 M acetalde-hyde or lactic acid and is equivalent to 0.45 mg. of lactic acid.In analyses of blood filtrates it is preferable to use 0.002 N 12, eachcc. of which represents 0.09 mg. of lactic acid.

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354 Determination of Lactic AcidAnalysis of Zinc Lactate.

The accuracy of the method as a whole is indicated by thercsu1t.s of determinations on solutions of pure zinc lactate shown inTable III. Within optimum ranges the extreme variations areabout 5 per cent, and the average of more than a hundred deter-minations on from 0.04 to 180 mg. of lactic acid is 97.4 per cent,the maximum deviations being - 10 and +5 per cent.

TABLE III.Summw-y of Determinationr on Pure Zinc Lactate, Ulocing Minimum,

Maziwkum, and Avcragc Results.T,actic acid insolution oxidized. No. ofardyscs.

m g.0.045 to 0.5 22 95.0 105.0 98.70.5 to 1.0 15 95.8 100.6 97.61.0 to 5.0 35 94.0 99.0 96.55.0 to 15.0 29 94.5 97.8 96.445 2 97.2 99.3 98.3

180 4 89.8 97.0 96.7--97.4

Minimum Xlaximum. Average.

With amounts above 15 mg. more MnSOJ (20 cc. of 0.1 M up to 20 sm.)was used, and stronger solution s of bisulfite to colle ct the aldehyde. With45 mg. of lac tic acid the yields with 5 cc. and 20 cc. of 0.1 M MnS04 were 97.2and 93.3 per cent respectively. With 180 mg. the yields with varyingamounts of MnS04 were the following: with 20 cc. hr, 89.7 per cent; with2 gm., 93.7 cent;er with 10 96.4 cent;m., per with 20 gm., 97.0 per cen t.

Interfering Xubstances.Many substances, especially hydroxy acids and sugars, as noted

by Clausen, may yield bisulfite-binding substances on oxidation.The rcflux condenser reduces this effect by allowing only the morevolatile products to be aerated over; this is true, however, onlywhen the condenser is kept cool.In Table IV are listed a number of substances with their yieldof bisulfitc-binding or iodine-reacting products when analyzed asdescribed above for lactic acid. The results are stated in terms ofper cent of the yield realized from the same weight of lactic acid.

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TABLE IV.Maximum Yield of Bisuljile-Binding Substances from Various Compounds.

The se data were obtained with apparatus having efficient we ll cooledcondense rs. The results in general are higher with le ss efficient or warmconden sers. The results from the oxidation of substan ce are expressed inper cent of the bound sulfite from an equal weight of lacti c acid. 5 to 10 mg.of each substan ce were analyzed.

None or less than 1 per cent.Glycollic aldehydeGlyoxylic acid.Glycol.Glyoxal.Glycerol.Sodium glycerophosphate.Succinic acid.Levulinic Saccharic Glycocoll.Alanine. *Phenylalanine.a-Aminobutyric acid.Glutamic acid.Aspartic Glycosamine.Leucine..Urea.Uric acid.Allantoin.Creatine.Creatinine.Hippuric acid.Barbituric acid.Glycollic acid.Pyruvic acid.Methylglyoxa1.tMalonic acid.Menthol glycuronic acid.Mucic acid.Arabonic acid.1B-Hydroxybutyric acid.5

1 to 5 per cent.Glucose.Galactose.Tartaric acid.Maleic acid.Tyrosine .

5 to 10 percent.Fructose.Xylose .Arabinose.Dihydroxyacetone.Glyceric aldehyde.Fumaric acid.Erythritol.

10 t,o 15 per cent.Glyceric acid.Malic acid.

Greater than 15 per cent.Cystine (15 to 22 percent).

Rhamnose (25 to 29per cent).

Citric acid (18 percent).

r-Hydroxyisobutyricacid (86 per cent)./

* Th is was true only with a well cooled condenser. With a warm con-denser a yield of 4.6 per cent was obtained.t Solu tions of methylglyoxal always contain some preformed lact ic

acid. Th is amount is iden tical before and after oxidation of the glyoxal byalkaline HzOl.

t Les s than 0.1 per cen t.0 From l-calciu m zinc salt, isolated from diabetic urine, the yield

varied. Two preparations yielded 0.7 and 10.4 per cent respectively. Th epresence of lactate as an impurity is not unlikely. The synthetic d&s altgave o nly 0.8 per cent.

11The conversion into acetone was quantitative.355

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356 Determination of Lactic AcidThe figures permit convenient estimate of the degree of interferenceof these substances if present in solutions being analyzed for lacticacid. The results were obtained under conditions duplicatingthose described for the lactic acid determination with apparatusha:-ing efficient, well cooled condensers. With warm condensersthe interference is in general greater. The data indicate that themain nitrogenous constituents of urine and of muscle extractsyield, under the conditions, little or no products which might bemistaken for lactic acid. Fructose, pentoses, and the trioseshave the effect of less than 10 per cent of the same amount oflactic acid, glucose less than 5 per cent, while cystine, citric, andhydroxyisobutyric acid have much greater effect. Glycol, glycerol,glyoxal, P-hydroxybutyric acid, and pyruvic acid have practicallyno effect.. The fact that the last named substance yields noacetaldehyde indicates that pyruvic acid is not, as might be ex-pected, an intermediate between lactic acid and aldehyde underthese conditions. So far as most of the substances listed are con-cerned it seems unlikely that they would lead to grossly erroneousresults for lactic acid with our method unless present in relativelylarge amounts. It is nevertheless desirable that interfering sub-stances be removed whenever means are available, since the pres-ence of other oxidizable substances uses up more permanganate,prolongs the osidation, and leads to uncertainty as to when theoxidation of the lactic acid is finished.

The interfering substance formed from oxidation of the sugarsis probably formaldehyde, since Rosenthaler, Klein (lo), andothers observed its formation under similar conditions. Thecharacter of the last end-point in the titrations gives an indicationof the presence or absence of substances other than acetaldehyde;and with solutions from the oxidation of sugars it is often slowand fading, just as is the case when formaldehyde is known to bepresent.

Removal of Interfering Substances.The removal of volatile substances such as acetone preformedand from acetoacetic acid is accomplished by preliminary boilingand aeration as described under Procedure. We can confirmthe statement of Clausen and others that lactic acid is not pre-cipitated or lost in the treatment with CuSO4 and milk of lime for

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Friedemann, Cotonio, and Shaff er 357removal of sugar, nor in the Folin-Wu precipitation of proteinsby tungstic acid. Blood and tissue extracts are therefore firstprecipitated with one or both of these procedures and the filtratesare analyzed for lactic acid.

The results with tungstic acid filtrates, with and without sub-sequent copper hydroxide treatment, differ but lit tle, though whensugar is not removed the oxidation proceeds more slowly and lesssmoothly and the end-point in the titration is less sharp.

One of the best means of removing proteins and other nitrog-enous compounds from blood and tissue extracts is the Patein-Dufau precipitation with acid mercuric nitrate and bicarbonate;but this treatment unfortunately causes considerable loss oflactic acid. The Schenk treatment with HgCl, in acid solutiondoes not cause loss. This aspect of the pretreatment of blood andtissue extracts for lactic acid determination has been speciallystudied by Dr. E. Ronzoni, and will be considered by her in aforthcoming paper.

In dealing with sugar solutions, such as after conversion of sugarto lactic acid by alkali, we find but little difference between resultsfor lactic acid in the filtrates from copper hydroxide treatmentand in solutions of the ether-extracted material. In both casesthe results appear to represent fairly accurately the true lacticacid present, and either method of pretreatment is therefore satis-factory. Data on this point will be included in a later paper byone of us (F.).

SUMMARY.A procedure is described for the determination of lactic acid,

based on oxidation to acetaldehyde and titration of sulfite boundby the aldehyde. The steps in the process were separately investi-gated. The use of manganous salt markedly increases the rate oroxidation and also increases the yield of aldehyde. The mannerof its action is discussed. The effect of manganese salts togetherwith the form of apparatus used for the oxidation and collection ofaldehyde simplifies and shortens the method as well as makes theresults more nearly quantitative and less variable. The conditionsfor the Clausen titration of bound sulfite were studied and itsaccuracy established. The extent of interference of about 50substances was determined.

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Determination of Lactic AcidBIBLIOGRAPHY.

1. Boas, I., Deutsch. med. Woch., 1893, xix, 940. von Ftirth, O., andCharnass, D., Biochem. Z., 1910, xxvi, 199. Embden, G., in Abder-halden, E., Handbuch der biochemischen Arbeitsmethoden, Berlin,1912, v, 1256. Hirsch-Kauffmann, H., Z. physiol. Chem., 1924, cxl, 25.Long, C. N. H., Proc. Roy. Sot. London, Series B, 1924, xcvi, 444.Tsukasaki, R., Tohoku J. Exp. Med., 1925, v, 429. Embden, G.,Z. physiol. Chem., 1925, cxliii, 297.2. Clausen, S. W., J. Biol. Chem., 1922, lii, 263.3. Manchot, W., Ann. Chem., 1902, cccxxv, 105,125.4. Goard, A. K., and Rideal, E. K., Proc. Roy. Sot. London, Series A,1924, cv , 148. Moureau, C., and Dufraisse, C., Chem. Rev., 1926,iii, 113.5. Kolthoff, I. M., and Furman, N. H., Potentiometric titrations, NewYork, 1926, 326.6. Ripper, M., Monatsh. Chem., 1900, xxi, 1079.7. Kerp, W., Die schwefligen Saure und ihre Verbindungen mit Aldehydenund Ketonen, Berlin, 1913, pt. 1. Kerp, W., and Baur, E., ibid.,Berlin, 1913, pt. 2.8. Shaffer, P. A., J. Biol. Chem., 1908, v, 211.9. Van Slyke, D. D., J. Biol. Chem., 1917, xxxii, 455. Salkowski, E.,Z. physiol. Chem., 1879, iii, 79.10. Rosenthaler, L., Arch. Pharm., 1913. celi, 581. Klein, G., Biochem,Z., 1926, clxix, 132. b

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Lactic Acid Determination 1