Determination of lactic Acid in Heavy Corn Steep liquor R. J . SiMITH, George M . Moffett Research Laboratories, Corn Products Refining Co., Argo, 111. HE production of cornstarch in the wet milling industries Tinvolves a preliminary soaking or steeping of whole grain in a dilute solution of sulfurous acid a t a temperature of about 125' F. (6). This process results in the solubilization of some inorganic material, phytin, proteins, and carbohydrates which are partially fermented to lactic acid. Subsequent incubation of the steep liquor results in conversion of a large portion of the carbohydrates t o lactic acid. The light liquor is evaporated to a dry solids content of about 50 to 55%, in which form it is known as heavy steep liquor. It has had wide acceptance as a nutrient in media for the production of antibiotics. Although many methods have been developed for the determination of lactic acid in blood, urine, culture media, milk, and similar materials, comparatively little work has been done on its estimation in agricultural products. An extraction procedure has been described by Leach and Winton (8) for determination of lactic acid in tomato products. Friedemann ( 2 ) utilized an oxidation technique for lactic acid in sugar solutions decomposed by alkali, and a similar method was proposed by Troy and Sharp ( 1 2 ) for the estimation of lactic acid in dairy products. The oxidation procedure was studied further by Friedemann and Graeser ( 3 ) and improved with respect to precision and accuracy. The method was tried in this laboratory on pure solutions of lactic acid and recoveries of 99.5% (h0.5) were obtained (Table I). Colorimetric, gravimetric, and other methods for determining lactic acid in blood have been summarized by Elgart and Harris ( 1), who also have improved the colorimetric (veratrole) method of Mendel and Goldscheider (9). These authors have shown that the maximum and most stable color is obtained by reaction a t 0' C. for slightly more than 1hour. Inasmuch as a more rapid method was desired for control testing, the colorimetric and time-consuming gravimetric procedures (6, 7, 16) were not used. Results obtained by Friedemann and Graeser ( 3 ) were confirmed in this laboratory with respect to both recovery and reaction time. It was found that the time required for complete analysis, including pretreatment of sample, could be reduced to l ~ s than s 30 minutes and that several samples could be run simultaneously, depending on the number of distillation units in operation and the capacity of the centrifuge. Pretreatment of the sample to remove interfering substances was found necessary. Samples of corn steep liquor treated with copper sulfate and calcium hydroxide (IS)and then analyzed by the oxidation-distillation technique were found to have the same lactic acid content as those acidified and extracted with ether for a prolonged period. The general procedure of Van Slyke ( I S ) nas used and the time of treatment was controlled, as lengthy treiitnients resulted in lowering, to the same extent, the recoverjof lactic acid from pure solutions and from the material in question. The treated samples were oxidized (using 0.01 N potassium permanganate), distilled, and titrated according to the Friedemann and Graeser ( 3 ) procedure.
distilled water and add 25 ml. of sirupy (85%) phosphoric acid. Cool this solution and dilute to 1liter. Potassium permanganate, 0.01 N aqueous solution Sodium bisulfite, 1.25% aqueous solution Sodium bicarbonate, saturated aqueous solution Sodium carbonate, 10% aqueous solution Iodine, strong, 2% in aqueous potassium iodide solution Iodine, 0.01 N standard solution Sodium thiosulfate, 0.1 N aqueous solution CO per sulfate, 20% a ueous solution C a b u m hydroxide, 20% aqueous suspension Talcum, finely powdered PROCEDURE
Dissolve 2 grams of heavy corn steep liquor in distilled mater and dilute to 200 ml. Pipet 25 ml. of this solution into a 200-ml. centrifuge cup and add 8 ml. of copper sulfate solution, 8 ml. of calcium hydroxide suspension, and 159 ml. of distilled water (total volume 200 ml.). Shake the reaction mixture for 1 minute and immediately centrifuge for 5 minutes a t 2000 r.p.m. Pipet an aliquot of the clear supernatant solution (usually 20 nil.), containing not more than 5 mg. of lactic acid, into the distillation flask of the apparatus. Add 10ml. of the phosphoric acidmanganese sulfate reagent and sufficient distilled water to bring the total volume to 100 ml. Conduct the oxidation, distillation. ant1 titration according to the outline of Friedemann and Graeser ( 3 )described below. Add a pinch of talcum to the reaction flask to prevent bumping during the oxidation and then connect the flask to the apparatus. Fill the dropping funnel, having a capacity of about 50 ml., with 0.01 N potassium permanganate. Add 10 ml. of sodium bisulfite solution to the receiver and attach to the condenser in such a manner that the inlet tube is submerged in the bisulfite absorbing solution. Heat the reaction flask with an electric heater or with a microburner, so that boiling begins in about 3 minutes (height of flame 3 to 3.5 inches). When boiling begins, add the permanganate solution dropwise a t a rate such that 40 & 5 nil. will be added in 15 minutes while the boiling is continued. The acetaldehyde formed during oxidation is distilled through the trap and condenser, and absorbed in the receiver containing the bisulfite solution. -4fter the oxidation and distillation have proceeded for 13 minutes, lower the receiver so that subsequent distillate will rinse down the tube of the condenser. After an additional 2 minutes (total reaction time 15 minutes), discontinue the addition of permanganate and remove the heat source. Wash the outside tip of the condenser tube with a fine stream of water into the receiver. The total distillate and washings should not exceed 75 ml. -4dd 2 ml. of starch indicator solution to the bisulfite solution, and lace this solution in an ice bath for about 5 minutes. Remove From ice bath, add strong iodine solution until a deep blue color is obtained, and discharge this color with a drop or two of thiosulfate solution. Wash down the walls of the receiver with a fine stream of water from a wash bottle and add 0.01 N standard iodine solution until the blue starch-iodine end point is obtained. Add 15 ml. of sodium bicarbonate solution and titrate the liberated bisulfite with the standard 0.01 N iodine solution. As the end point is approached-observed by the slower consumption of iodine-add 1 ml. or more of sodium carbonate solution and complete the titration. The blue end point should persist for a t least 2 minutes. Conduct a blank determination on all reagents and apply a suitable correction t o all determinations as needed. Each milliliter of 0.01 N standard iodine is equivalent to 0.45 mg. of lactic acid.
REAGENTS
REMOVAL OF INTERFERING SUBSTAWCES
All reagents used in the determination are described adequately by Friedemann and Graeser (3). A 0.5% solution of corn amylose ( 1 0 ) in half-saturated otassium chloride was used as indicator to improve precision o f t h e end point over that obtained with the usual starch indicator. STARCHINDICATOR. Dissolve 1 gram of recrystallized corn amylose ( A fraction) in 100 ml. of boiling distilled water. Cool the solution to room temperature, add 30 grams of crystalline potassium chloride, and dilute to 200 ml. This solution is stable over long periods of time and is immune to mold growth. PHOSPHORlC ACID-MANGANESE SULFATE REAGENT.Dissolve 100 grams of manganese sulfate tetrahydrate in 500 ml. of warm
Because it has been found necessary to remove proteins from materials such as blood and milk in order to obtain accurate analyses by the distillation method, it was thought that it would be essential to remove them from steep liquor before analysis. Protein precipitation IT ith trichloroacetic acid bv thc tcchnique of Elgart and Harris ( 1 ) caused no appreciable lowering of the apparent lactic acid. Precipitation with zinc hydrouide by the method of Somogyi ( 1 1 ) yielded variable results on purc lactic acid, depending upon the length of treatment (Table I). Similar results were obtained on steep liquor treated with zinc h\ droxicte
505
ANALYTICAL CHEMISTRY
SO6 -i.e., the apparent lactic acid was not lowered appreciably by treatment. From these results it was concluded that the proteins removed by pretreatment did not interfere with the determination of lactic acid in steep liquor by the distillation technique. Incubated as well as unincubated steep liquor contains carbohydrates which may interfere with the estimation of lactic acid by the oxidation method. Bccording to Van Slyke (IS), glucose and other carbohydrates are removed quantitatively by treatment with calcium hydroxide and copper sulfate. The technique was applied to steep liquor and the apparent lactic acid content was lowered significantly. Treating with the Van Slyke reagent for l minute before centrifugation lowered the lactic acid values as much as 6%, whereas the recovery of lactic acid from pure solutions treated in a similar manner was about 98.5y0 Prolonged treatment (30 minutes) with copper sulfate and calcium hydroxide lowered the lactic acid recoverable from pure solutions to about 97.5%. As this treatment lowered the lactic acid obtained from steep liquor without appreciably affecting the recovery from pure solutions, indications are that interfering substances (probably carbohydrates) were removed (Table 11). Other experiments xere conducted using both protein and carbohydrate precipitants-e.g., calcium hydroxide and copper sulfate preceded by zinc hydroxide. Recovery of lactic acid from steep liquor treated in this manner was lowered, and the recovery from pure solutions of lactic acid treated in an analogous fashion for 30 minutes was of the order of 92 to 94%. The apparent lactic acid content of the steep liquor was lowered considerably by this treatment, but when corrected for the recovery of lactic acid from pure solutions treated in similar manner, the results were in good agreement nith those obtained on samples treated only with copper sulfate and calcium hydroxide. This observation further substantiates the conclusion that the proteins present in corn steep liquor do not interfere with the determination of lactic acid by the o~idation-distillationtechnique.
Table 11. Determination of Lactic Acid by OxidationDistillation Material Lactic acid
Re agent Kone CuSOa-Ca(OH)?
Time. Inin.
1 1
Zn(OH)r CuSOa-Ca(OH)p
30 30
1 1
1 30
30 30 30 S t r e ; , liquor
Kone
CuSOa-Ca(0H)t
.. .. .. .. 1
1 1 1
Zn(OH)n CuSOd-Ca(OH)r
30 30 30 30
7 Lactic Acid Observed 99 98 98 3 97 7 97 0 97 0
,
96 5 92 0
92.0 25 6 25 6
25 ZJ
z i
24.2 24 1 24 n 21.1 21.8 21.9
evaporated to a volume of about 25 nil. on a hot water bath. This procedure is repeated twice for a total of three evaporations to remove volatile acids which would interfere with the final titration of the extracted lactic acid. The residual solution is transferred to a modified liquid-liquid Soxhlet extractor and extracted with ether until all lactic acid is removed. The ether solution contained in the extraction flask is evaporated to dryncss and the residue dissolved in 50 ml. of distilled water. This solution is boiled for 5 minutes and titrated hot with standard alkali t o a phenolphthalein end point. Each milliliter of 0.1 iV alkali required for titration is equivalent to 9.00 mg. of lactic acid. The lactic acid contents of corn steep liquor samples have been determined in the above manner and plotted as a function of ex-
LACTIC ACID BY EXTRACTION
The lactic acid content of heavy steep liquor determined by oxidation and distillation on samples treated with Van Slyke’s reagent agreed well with values obtained b r prolonged extraction with ether.
Table I.
Determination of Lactic .icid in Pure Solutions
Lactic Acid Used, Rlg. 4 50 4.50 2.25 2.25 3.25 3.25 3 25 3.26 3 25 3 25 3 25 3 25
Pretreatinent Xone Kone Xone None None Sone Zn(OH)r, 1 min. Zn(OH)r, 1 min. Zn(OH)?,15 min. Zn(OH)g, 15 m!n. Zn(OH)x, 30 min. Zn(OH),, 30 min.
% Lactic Acid Found 99.2 99 5 100.0 99.6 99.4 99 2 103.8 102.5 99.8 99.1 98.3 98.2
Leach and JVinton (8) describe a method for the estimation of lactic acid in tomato products which utilizes extraction n ith ether after pretreatment n ith lead acetate. The extract is then evaporated to remove ether, and the residue dissolved in distilled water and oxidized with an excess of standard permanganate. The excess permanganate is determined by titration with oxalate. I n this way the permanganate conmmed in the oxidation is related to the lactic acid content of the extract. This method has been modified in the Corn Products laboratories so as to avoid the permanganate oxidation. A sample of suitable size (1 to 2 grams) of heavy steep liquor is acidified with 2 to 3 dro s of concentrated sulfuric acid and diluted to about 150 ml. witg distilled water. This solution is then
Figure 1. Effect of Steep Liquor Pretreatment on Extractability of Lactic Acid A. B.
Acidified and evaporated before extraction Acidified but not evaporated before extraction
C. Neither acidified nor evaporated before extraction
50?
V O L U M E 25, NO. 3, M A R C H 1 9 5 3 Table 111. Lactic Acid Content of Heavy Steep Liquor as Determined by Ether Extraction for 58 Hours % Lactic Acid (Dry Basis) Conditions 22.8 Acidified and evaporatedbefore extraction 24.0 Acidified but not evaporated before extraction 12.4 S e i t h e r acidified nor evaporatedbefore extraction
traction time. Similar extractions have been conducted on steep liquor which was acidified but not evaporated, and also on samples which were neither acidified nor evaporated (Figure 1, Table 111). CONCLUSIONS
Lactic acid present in heavy steep liquor must exist partially as a salt which may or may not be extracted with ether; in either caqe it would not be included in the final alkali titration. Direct extraction of heavy steep liquor after acidification with sulfuric acid yields results which agree well with values obtained by the oxidation-distillation technique on pretreated samples. On the other hand, values obtained by the standard extraction twhnique were about 1% lower. It does not seem likely that thii. is due to loss of volatile acids during evaporation, as heavy stwp liquor is produced by a concentration operation which should rcmove such volatiles. Friedemann and Kendall(4) have shown that determination of lactic acid in urine by ether extraction alv ays results in a loss of lactic acid due to oxidation by organic
peroxides formed during evaporation and extraction. There also exists the possibility of lactic acid condensation during evaporation, which would result in lowering the apparent lactic acid content determined by extraction. Although the extended ether extraction of acidified heavy steep liquor (not evaporated) yields reliable results which are in agreement with the values obtained by the modified Friedemann technique, the oxidation-distillation procedure is recommended because of the short time required for analysis. LITERATURE CITED
(1)
Elgart, S., and Harris, J. S I IND. ENG.CHEV.,ANAL. ED., 12” 758 (1940).
Friedemann, T. E., J . Biol. Chem., 76,75 (1928). Friedemann, T. E., and Graeser, J. B., Ibid., 100, 291 (1933). Frjedemann, T. E., and Kendall, A. I., Ibid., 82, 23 (1929). Fries, H., Biochem. Z., 35,368 (1911). Kerr, R. W., “Chemistry and Industry of Ptarch,” 2nd ed., Chap. 11,New York, Academic Press, 1960. ( 7 ) Kondo, K., Biochem. Z., 45,88 (1912). (8) Leach, A. E., and Winton, A. L., “Food Inspection and Analysis,’’4th ed., New York, John Wiley & Sons, 1936. (9) Mendel, B., and Goldscheider, I., Biochem. Z., 164, 163 (1926). (10) Pigman, 11’. Ti’., and Wolfrom, M. L., Advances in Carbohydrate (2) 13) (4) (5) (6)
Chem., 1, 257 (1944). (11) Somogyi, M., J . B i d . Chem., 90,725 (1931). 112) Trov. H. C.. and Sharp. P. F., Cornell Cnic., Agr. Ezpt. Sta.. iGemoirs, 179 (1935): (13) Van Slyke, D. D., J . B i d . Chem., 32, 455 (1917). (14) Wolf, C. G. L.,J . Physiol., 48, 341 (1914).
RECEIVED for review
Accepted October
January 17, 1952.
27, 1952.
Calibration of the Rolling Ball Viscometer H. W. LEWIS Bell Telephone Laboratories, Murray Hill, N. J . N 1943, Hubbard and
Brown ( 1 ) carried out a systematic
1 experimental calibration and dimensional analysis of a rolling
ball viscometer. They determined a dimensionless calibration curve, which enables one to design a viscometer of this type to measure any given range of viscosities. It is the purpose of this note to show that one can, to good approximation, derive this calibration curve from a simple approximate treatment of the problem in terms of the hydrodynamics of viscous fluids ( 2 ) . IVe consider a cylindrical tube of diameter D, inclined a t an angle 29 to the horizontal, and filled with a fluid of viscosity L,A, and density p,. SVe suppose to be rolling down the tube with velocity V, a spherical ball of diameter d, and density pa. We will use a cylindrical coordinate system, with Z-axis parallel to the axis of the tube, with polar angles referred to a vertical plane through the 2-auk, and with the origin a t the center of the sphere. We Tvill also make the approximation that the gap between the sphere and the tube is small compared to the diameter of either one, so that the sphere nearly fills the tube-this is the usual, practical situation, and makes the calculation possible. We will systematically neglect higher powers of ( D - d ) / D . Now, if we call the distance betn-een the sphere and the wall of the tube u (79, Z ) , a little consideration of the geometry will show that
a narron- channel of this sort gives rise to a parabolic velocity profile a t any given point. In particular, if L is the mean velocity of the fluid in the gap, then d 2 i l b r 2 = 12; /u2, where r is our radial cylindrical coordinate. But the longitudinal gradient of pressure is given by p LPu/dr* = 12 p u/u2, so that our problem may now be stated as follows: rye have to determine the distribution of G as a function of 9 and 2, so that the total difference of pressure is enough to balance the reight of the sphere, and the total flow across any plane Z = constant is equal to D2V. This will determine p as a function of V , n.hich is the problem we have set ourselves. K e will use the f o l l o ~ ~ i nprocedure: g for each value of 2, we will distribute the flow velocity, as a function of 9, in such a way that the longitudinal pi essure gradient is independent of $-this implieb that we x-ill neglect all components of velocity perpendicular to the axis of the tube. Thus, we write
3
u (79,
a
(2)
The total flux through any plane perpendicular to the axis of the tube \vi11 then he flux =
=
So we have to consider the flow of a viscous fluid through a narrow channel of width given by ( 1 ) . The total flow through this channel will be D2V, since we will, everyn-here except in the expression ( D - d ) , neglect the differencebetu-een D and d. Now, it is well known that the flow of a viscous liquid through
Z) = B ( 2 ) uy9, Z )
a fT
L(79, Z )
flux
x Fz
fx
D ( D - d ) 3B ( Z ) 16
u(8,
Z ) X D d9 (3)
;B(2)
Substituting the expression for ing the integral, we find
x
u3(9,
u
Z ) d8
from Equation 1, and perform-
[5
+ 18a + 24aa + 1 6 4
(4)
