33.2.08AOAC Official Method 937.05

Lactic Acid in Milkand Milk Products

Spectrophotometric MethodFirst Action 1937

Final Action

A. Reagents(a) Barium lactate standard solution.Dissolve in ca 10 mL H2O

amount of a pure lactate, such as lithium, zinc, or calcium lactate, con-taining equivalent of ca 300 mg free lactic acid. Transfer material to ex-tractor, Figure 937.05, add 0.5 mL H2SO4 (1 + 1, v/v), and adjustvolume to 50 mL. Extract with ether 3 h. Add ca 20 mL H2O to extrac-tion flask, evaporate ether on steam bath, and carefully titrate with0.05M Ba(OH)2, using phenolphthalein. Transfer neutralized materialto 200 mL volumetric flask, dilute to volume, and mix. Pipet into500 mL volumetric flask amount of this barium lactate solution con-taining equivalent of exactly 250 mg free lactic acid, dilute to volume,mix, and designate as lactate standard solution. (2 mL equivalent to1 mg lactic acid. To plot standard curve, use freshly prepared solution.)Transfer 20 mL lactate standard solution to 100 mL volumetric flask,dilute to volume, and designate as dilute lactate standard solution(10 mL equivalent to 1 mg lactic acid).

(b) Carbon.To 10 g high-grade C (Nuchar C-190-N, Suchar,or Darco G60) in 600 mL beaker, add ca 200 mL H2O and 30 mL 1MHCl, and keep on steam bath 20 min, agitating continuously with airpassed through cotton. Filter on Bchner and suck as dry as possible,tamping with flat-end rod. Transfer cake to beaker, add ca 200 mLH2O, mix thoroughly, and refilter. Repeat washing and filteringtwice, and dry at 100C.

(c) Ferric chloride solution.Dissolve 2 g FeCl3 6H2O in H2O,add 5 mL 1M HCl, and dilute to 200 mL.

(d) Phosphotungst ic acid solut ion .Dissolve 20 gphosphotungstic acid in 80 mL water.

(e) 0.5M Sulfuric acid.Use premade or prepare as in 936.15(see A.1.06).

B. Preparation of Test Portion

(a) Liquid, whole and skim milks.Weigh 50 g into 100 mL vol-umetric flask.

(b) Dried, whole and skim milks.Weigh 5 g into 100 mL beaker,and using heavy stirring rod, make into smooth paste with H2O. Trans-fer mixture to 100 mL volumetric flask with ca 50 mL H2O.

(c) Cream and ice cream.Weigh 20 g into 100 mL volumetricflask and add ca 50 mL H2O.

(d) Sweetened condensed milk.Weigh 25 g into 100 mL beakerand transfer to 100 mL volumetric flask with ca 50 mL H2O.

(e) Evaporated milk.Weigh 25 g into 100 mL volumetric flaskand add ca 50 mL H2O.

To mixtures add 6 mL 0.5M H2SO4 and mix, avoiding vigorous agi-tation. Add 5 mL 20% phosphotungstic acid solution (1 mL for creamand 2 mL for ice cream) and dilute to volume with H2O. Mix, and filterthrough folded paper.

(f) Butter.Weigh 20 g into centrifuge bottle, add 25 mL H2O, andwarm on steam bath. Neutralize with 1M NaOH, usingphenolphthalein. Cool, add 50 mL ether, and mix well, avoiding vigor-ous agitation. Add 50 mL petroleum ether, mix well, and centrifuge.Draw off ether layer as completely as possible by siphon with lower endbent upward. Repeat extraction, using 25 mL of each ether. Place bottleon steam bath to remove remaining ethers. Transfer residue in bottle to100 mL volumetric flask, add 3 mL 0.5M H2SO4, and mix. Cool mix-ture and precipitate proteins with 20% phosphotungstic acid solution,adding reagent dropwise until precipitation stops. Dilute to volume,mix by shaking, and filter through folded paper.

C. Preparation of Standard Curve

Transfer from buret to volumetric flasks, graduated at 50 and55 mL, volumes of standard solutions in left-hand column of Ta-ble 937.05. Right-hand column gives mg of lactic acid in 40 mL fil-trate from each sample after C treatment described below, and that

2000 AOAC INTERNATIONAL

Figure 937.05Liquid extractor.

Table 937.05 Preparation of dilutions for standard curve

Solution to be transferredto 5055 mL volumetric flask, mL

Lactic acid in 40 mLaliquot, mg

Dilute lactate standard solution6.9 0.5

13.8 1.027.60 2.0

Lactate standard solution8.25 3.0

11.00 4.013.75 5.016.50 6.019.25 7.022.00 8.024.75 9.027.50 10.030.25 11.033.00 12.0

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will therefore be read in spectrophotometer. Blank using 40 mL H2Oin place of lactate solution must be included in each series.

To each flask, including blank, add 6.6 mL 0.1M HCl and H2O un-til volume is ca 40 mL. Now add 200 1 mg prepared C, shake, andkeep on steam bath 10 min, mixing frequently. Cool, dilute to 55 mLmark with H2O at room temperature, and promptly filter throughquantitative paper, pouring back until clear.

Transfer 40 mL of each clear filtrate to 50 mL volumetric flask,painted black or wrapped in black paper. As 40 mL filtrate used con-tains only 4.8 mL acid added during C treatment, add 1.2 mL 0.1MHCl. (Total of 6 mL 0.1M HCl is required in flask.) Pipet 5 mL FeCl3solution into one flask at time, dilute to volume, and mix. Pour solu-tion into 1 cm quartz cell and determine wavelength of maximum Aon recording spectrophotometer between 350 and 600 nm, usingbaseline correction at 600 nm. If recording spectrophotometer is notavailable, manually scan region around 365 nm to determine wave-length of maximum A. Read all standard solutions at this wavelengthagainst blank set at 0 A. (On exposure to direct light, color fades, butprotected as provided it is stable for number of h.) From readings ob-tained, prepare standard curve, plotting mg lactic acid as abscissaand scale readings as ordinates. (Large-scale graph paper is recom-mended to permit more accurate interpolations.)

Recheck wavelength of maximum A and standard curve occa-sionally and whenever new batch of C or FeCl3 is used. If different, ad-just spectrophotometer or prepare new standard curve at new wave-length of maximum A.

D. Extraction

Place 50 mL prepared filtrate and 0.5 mL H2SO4 (1 + 1) in innertube of extractor and connect to longest bulb-type condenser avail-able, having outlet 13 mm id to minimize ether regurgitation. RunH2O through condenser at maximum condensation efficiency. Con-nect extraction flask containing 200 mL ether, and lower flaskslowly onto preheated heating mantle or hot plate to prevent super-heating the ether. Protect extractor from heat of hot plate by uprightsheet of fire retardant insulation and extract all the lactic acid.

When ether in extraction flask is kept at rapid boiling and condenserH2O is cold enough to let condensed ether return to extraction flask insteady stream, extraction for 3 h delivers all the lactic acid. When thisrate of extraction cannot be maintained because of high temperature ofH2O passing through condenser, continue extraction until equivalent of7500 mL ether has passed through solution. Time required, T, estab-lished for each set of new conditions, is calculated from 2 factors: x, vol-ume ether necessary to fill extractor to overflowing at side-arm, whichis constant for each apparatus; and y, time in min required for volume xto pass from extraction flask and fill extractor.

To determine x, place 50 mL H2O and 0.5 mL H2SO4 (1 + 1) in ex-tractor. With extractor held upright, carefully pour ether from gradu-ate into inner tube until it just starts passing out of side-arm.

Determine y in ordinary course of starting each determination. Withstopwatch, record interval from time ether first drops from con-denser and falls into inner tube to time first drops return to extractionflask from overflow into side-arm. Time, T, necessary for 7500 mLto pass through apparatus = 7500 y/x. Calculated T holds only if rateof boiling and condensing is constant throughout extraction period.E. Determination

To flask containing ether extract add 20 mL H2O and expel etheron steam bath. Do not let flask remain on steam bath after ether is ex-pelled. Neutralize with saturated Ba(OH)2 solution, usingphenolphthalein. Wash into 110 mL volumetric flask with alcoholuntil volume is ca 90 mL. Heat almost to boiling on steam bath, cool,dilute to volume with alcohol, and filter through quantitative paper.To expel alcohol, evaporate 100 mL filtrate to ca 10 mL, add ca50 mL H2O, and again evaporate to ca 10 mL (or evaporate the100 mL filtrate to dryness on steam bath).

Add, from buret, 6.6 mL 0.1M HCl and transfer contents of beakerwith H2O to 5055 mL volumetric flask until volume is ca 40 mL.Prepare blank containing 6.6 mL 0.1M HCl diluted to ca 40 mL withH2O. Add 200 mg prepared C, mix immediately, and keep on steambath 10 min, mixing frequently. Cool, dilute to 55 mL mark withH2O at room temperature, and filter through quantitative paper,pouring back until clear.

Transfer 10 mL filtrate to 50 mL black volumetric flask. (Total of6 mL 0.1M HCl must be in flask; 10 mL filtrate contains 1.2 mL 0.1MHCl from C treatment; therefore, add 4.8 mL additional acid.) Add5 mL FeCl3 solution from buret or pipet, dilute to volume, and mix.(After color develops, diluting to reduce color intensity is not permis-sible.) Fill 1 cm quartz cell with solution and read inspectrophotometer at wavelength of maximum A against blank set at0 A.

Determine amount lactic acid present in the 10 mL aliquot fromstandard curve. If amount lactic acid in 10 mL portion is