Evaporation of Standard Solution from the Tips of Mieroburets A. A. BENEDETTI-PICHLER AND SIDNEY SIGGIA, Queens College, Flushing, N. Y.
HEN working with microburets having graduations on a tube of 2-mm. capillary bore or less (3, 4),it is customary to immerse the tip in the solution to be titrated immediately after filling the buret and reading the position of the meniscus. With the buret of Schwarz (6) and with microburets using remote control (I,$,5 , 7 )evaporation of standard solution may be prevented by keeping the opening of the tip inside the titration vessel and a little above the meniscus of the solution to be titrated. If, however, the tip is left in the open for any length of time, evaporation of standard solution becomes noticeable. The purpose of this investigation was to determine the consequences of such evaporation from the tip of microburets; it was proved that no error results with standard solutions containing a nonvolatile active agent, if some simple rules are followed. The experiments were carried out with U-shaped burets with remote control ( 5 ) ,which may be described as measuring pipets having mechanical regulation of the air pressure in the space above the standard solution. Evaporation from the meniscus in the graduated capillary is highly improbable with these burets, for the air space above the meniscus is very small, of a shape to prevent convection currents, and in addition, is connected to a reservoir in which the air is saturated with water vapor. From the outset it appeared obvious that no serious difficulties should be met with standard solutions containing a nonvolatile active agent, and most of the commonly used standard solutions belong in this category. When evaporation takes place from the tip of the buret, only solvent is lost. The corresponding active agent collects in the solution near the opening of the tip and is expelled with the next drop of standard solution. The decrease of volume is prematurely indicated in the graduated capillary, but no error will result, if the phenomenon is taken properly into consideration. No error will result from evaporation between the time the zero reading is taken and the time a t which the last portion of standard solution is added to the titrated solution. A buret reading, however, must not be taken following a period of significant evaporation. If an appreciable amount of time has lapsed after filling the buret, the solution in the tip must be rejected before the zero reading is taken. At the close of a titration the buret must be read a short time after addition of the last portion of standard solution from that buret. The appropriateness of these measures has been proved by a series of experiments in which evaporation was intentionally permitted to take place to an extraordinary extent. Approximately 0.5 molar solutions of sodium hydroxide and hydrochloric acid were first titrated against one another in such a manner that no significant amount of eva oration could occur, and thus the correct ratio of centimeters o?sodium hydroxide t o centimeters of hydrochloric acid was obtained as the mean of three determinations equal to 1.046. In the following experiments one buret was used in a way to preclude a significant amount of evaporation. The other buret was read immediately after filling, and then was allowed to stand for 20 to 60 minutes, whereupon a second zero reading was taken just before starting the titration, which was conducted with proper precautions. The differences between the volumes corresponding t o the first and second zero readings give the volumes of solvent which eva orated from the tip of the burets. In reading the burets, milfmeter scales were placed behind the measuring capillaries which had a bore of approximately 0.8 mm. Thus, 1 em. on the scales corresponded to approximately 5 cu. mm. of volume.
Table I indicates that even with 0.5 molar hydrochloric acid no significant amount of active agent is lost by evaporation from the tip of the buret. The ratios calculated with the use of the first zero readings are satisfactory, whereas those based on the second zero readings are grossly erroneous. T.4BLE
I. EVAPORATION FRO51 MICROBURETS Amount of Standard Solution Ratio NaOH HC1 NaOH/HCl Cm.
0.5 N Acid and Base Evaporation prevented (mean of 1.044, 1.045, !nd 1.049) EvaDoration Dermitted from tiD of NaOH -buret Zero reading after filling 15.82 2 n d . zero reading after standing 60 minutes 14.82 Evaporation permitted from tip of HCl buret Zero reading after filling 14.90 2nd zero reading after standing 60 minutes 14.90 11
0.1 N Iodine and Thiosulfate Evaporation prevented (mean of 1.131, 1.129, and 1.125) EvaDoration Dermitted from tiD of iodine buret Zero reading after filling 15.70 2nd zero reading after standing 60 minutes 14.05
Cm.
1.046 15.18
1.042
15.18
0.976
14.30
1.042
13.54
1,100 Ratio Iz/NazSzO
NazSzOa
1.128 13.50
1.163
13.50
1.041
As is to be expected, standard solutions containing a volatile active agent are troublesome. If active agent and solvent would volatilize a t rates that caused no change in the composition of the remaining solution, it would a t least be possible to disregard evaporation occurring before the zero reading is taken. A fortunate coincidence of this kind will rarely happen, and in practice it must be expected that, in addition to a change of volume, some change of the composition of the standard solution will occur in the tip of the buret. This change of composition cannot be computed in a simple manner, and it becomes necessary to prevent loss of a significant amount of active agent during the whole time lapsing between last adjustment of meniscus previous to taking the zero reading and final establishment of the end point of the titration. This statement is borne out by the experiment with 0.1 N iodine solution, the result of which is summarized in Table I. The ratio of centimeters of iodine to centimeters of sodium thiosulfate was determined. While care was taken to minimize evaporation from the tip of the thiosulfate buret, the tip of the iodine buret was left exposed to the atmosphere for 60 minutes after reading the zero position of the meniscus. A second zero reading was then taken immediately before starting the titration, and the tahle indicates that neither zero reading permits calculation of a satisfactory ratio. Iodine solution and other standard solutions containing a volatile agent require filling the buret immediately before use or rejecting the contents of the tip immediately before taking the zero reading. The titrations must be started at once, and care must be taken to conduct the titration in a manner which assures that all active agents leaving the tip of the buret in either the dissolved or gaseous state will react with the titrated solution. The final reading must be made without much delay, If the titration is interrupted, a reading must be
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ANALYTICAL EDITION
taken before the buret is removed from the titrated solution. and when the titration is resumed the buret must be read again after rejection of the solution contained in the tip.
(3) Kirk, P. L., I b i d . , 14,1 (1933). (4) Linderstrdrn-Lang, K.,and Holter, H., Compt. rend. trav. lab. Carlsberg, 19,No. 14 (1933). ( 5 ) Llacer. A. J.. and Sozzi. J. A.. see A. A. Benedetti-Pichler. “Introduction t o the Microtechnique of Inorganic Analysis”; New York, John Wiley & Sons, 1942. (6) Schwarz, K., Mikrochemie, 13, 1 (1933). (7) Struszynski, M.,Przemysl Chem., 20,53 (1936). I
Literature Cited (1) Heathy, N. G.,Mikrochemie, 26, 147 (1939). (2) Hybbinette, A,-G., and Benedetti-Pichler, A. A,, I b i d . , Emich Memorial Issue (1941).
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Nicotinic Acid Content of Cereals and Cereal
Products Microbiological Method of Assay JOHN S. ANDREWS, HAROLD M. BOYD, AND WILLIS A. GORTNER General Mills Research Laboratories, Minneapolis, Minn.
T
HE dietary position of cereals and cereal products in relation to their pellegra-preventive attributes has recently been given considerable prominence by the inclusion of nicotinic acid or niacin in the list of required components of enriched flour (3). (The word “niacin” has recently been accepted by the Federal Security Agency as a nontechnical synonym for the term “nicotinic acid”.) This action has focused attention not only on the natural nicotinic acid content of flours designed for enrichment but also on the relative position occupied by the whole wheat from which these flours are derived, together with the other products obtained from milling. TABLE I. NICOTINICACID ASSAYSOF EXTRACTS OF WHOLE WHEATFLOUR Type of Extraction
Nicotinic Acid Irg./g.
37 75 37 72 70
Search for information on this subject has been handicapped by the uncertainty of the suitability of the analytical methods used for estimating nicotinic acid, particularly chemical procedures. Kodicek (4) has emphasized the discrepancies observed in the analysis of cereals and pointed out the adverse role played by “chromogenic” substances of unknown composition. Similar observations have also been reported by Waisman and Elvehjem (IO). Melnick, Oser, and Siegel (6) have applied an adaptation of the MelnickField chemical method (6) to the determination of flour and bread and reported fairly good agreement between the values thus obtained and those resulting from the application of the microbiological method of Snell and Wright (9). This observation lends support to the validity of the assay data and suggests a satisfactory specificity for the two types of procedures when the specified precautions are followed in the chemical method. This compatibility leaves the analyst with some latitude in the choice of methodology, with reasonable assurance that the selection will not introduce serious errors into the final assay results. I n the authors’ laboratory the chemical and microbiological methods have been investigated for the purpose of comparing their applicability to cereal analyses, for both controlling the
manufacture of enriched flours and evaluating other grain products. While in general fairly good agreement has been observed between the two types of procedures, values obtained chemically tend to be high. This is due to the difficulty of ensuring the complete removal of or compensation for “chromogenic” substances which are naturally present or are formed during extraction processes. For this reason microbiological assays have appeared to be more acceptable in instances where large discrepancies are observed. Recent studies of the microbiological procedure have given rise to speculation about the accuracy of the nicotinic acid values thereby obtained. Depending on the procedure employed for extracting the cereal sample, assays varying more than twofold in magnitude can be produced. Snell and Wright (9) in their original description of the micrpbiological method reported that alkaline extraction of cereals, in contrast to animal tissues, gives somewhat higher values than those obtained when water ia employed for the extracting medium. They apparently attributed this to differences in extraction efficiency, since they found that finely ground samples gave comparable values by both extraction procedures. Oser, Melnick, and Siegel ( 7 ) also reported that alkaline extraction gives higher values but attributed this to hydrolysis rather. than extraction since treatment of aqueous extracts with alkali raises the apparent nicotinic acid content to that found by direct extraction with sodium hydroxide. This explanation assumes that hydrolysis imparts greater availability t o the test organism of some watersoluble derivative or precursor of nicotinic acid. The nature of this substance is unknown, since nicotinamide, cozymase, and nicotinuric acid all possess equivalent nicotinic acid activity to Lactobacillus arabinosus 17-5 (9). The remaining derivative, trigonelline, is inactive and is not readily converted to an active form. The authors’ observations have shown even wider differences than those previously reported. These results have necessitated a careful inquiry into the relative behavior of various cereal products and a more extended evaluation of extraction procedures. The data presented in Table I demonstrate the influence of aqueous, acid, and alkaline solvents on the microbiological assay of a typical whole wheat flour.
Extraction Procedures The aqueous extracts were prepared by autoclaving 1-gram samples with 90 ml. of water for 15 minutes at 6.8-kg. (15-pound) pressure. After cooling to about 50” C., 1 ml. of 6 per cent takadiastase solution was added, and the mixture was allowed to cool at room temperature (30 to 40 minutes), made up to 100 ml., and filtered.
