2
AS.4 LY TICAL EDI T I O S CC. K O H
E . m.f.
~ ~ ~ s K - 1 2 CC. 5
BUTYL
A E I A cc.
SOLVENT (CURVE -0,185 0.0 -0.124 0.1 -0.031 0.2 0,002 0.3 0.024 0.4 0.049 0.6 0.095 1.0 E n d point, 0 . 1 5 cc. 0.n610 G R A M BENZOIC ACID .. . ~
-0,206 9.0 -0,201 9.2 -0.196 9.4 -0,190 9.8 -0,181 10.0 -0.174 10.1 -0.168 10.2 -0.163 10.3 -0.145 10.5 -0,129 10.6 -0,112 10.7 -0,086 10.8 -0.047 10.9 -0,024 11.o 0.004 11.1 0.026 11.2 0.060 11.4 0.073 11.6 0.086 12.0 E n d point, 1 0 . 9 5 cc. KOH, 2 , 6 0 3 mg. per cc
ALCOHOL
-4)
0.610
0,930 0,330 0,220 0.120 0,115 (CVRVSB )
0.025 0.025 0,020
0.045 0.070 0.060 0.050 0,090 0.160 0.170 0.260 0,390 0.230 0,280 0.220 0.170 0.065 0.030
T a b l e I-Titration Data A E / A cc. Cc. K O H E. m. I. A E / A cc. 0.607 GRAM ROSIN OIL (CURVEC ) 5.735 GRAMS PEANUT OIL (CURVEE ) 0.0 -0.250 10.0 -0.156 0.0 -0,250 11.0 -0.144 0.012 -0.252 0.1 11.6 -0,139 0,008 0.2 -0,254 12.2 -0,135 0.007 -0,254 0.3 13.2 -0.125 0.010 0.4 -0,253 0,010 13.8 -0.118 0.010 0.5 -0,251 0.020 14.2 -0,108 0.016 0.6 -0.249 0,020 14.6 -0.095 0,022 0.7 -0.242 0,070 14.8 -0.085 0,050 0.8 -0.220 0,220 15.0 -0.071 0,070 0.9 -0.130 0,900 15.2 -0.050 0,100 1.0 -0.050 0.800 15.4 -0.010 0,200 1.1 -0,005 0,450 15.6 0.050 0.300 1.2 0.030 0,350 15.8 0.068 0.090 1.3 0.050 0.200 16.0 0,075 0 035 1.5 0.060 0,020 16.4 0.086 0.022 E n d point, 0 . 9 0 cc. End point, 1 5 . 6 cc. KOH. 2.920 mg. per cc. KOH, 2 603 mg. per cc. Blank, 0.50 cc. Blank, 0 . 1 8 cc. Acid number, 0 . 2 0 (colorimetric, Acid number, 6 6 . 7 (colorimetric, 0.17) 69.5) 3.29 GRAMS NEAT'S-FOOT OIL 6,110 GRAMS LARD OIL (CCRVE D) (CURVE F ) 5.0 -0,206 0,012 8.5 -0 198 0.008 l5,O -0.161 0.01s 15.5 -0.156 0.010 0.015 16.0 -0.149 0.014 0.022 17.1 -0,131 0.016 0.050 17.4 -0,121 0.033 0 052 18.0 -0.098 0,039 0.105 18.2 -0,083 0,075 0.155 18.4 -0.058 0.125 0.240 18.6 -0.025 0.165 0.390 18.8 0 012 0.185 0.250 19.0 0.044 0,160 0,210 19.2 0.074 0.150 0,200 19.4 0,089 0.075 0.150 19.6 0.096 0.035 0.050 20.0 0.104 0.020 0,050 E n d point, 18.8 cc. KOH, 2.920 mg. per cc. cc. 0.15 cc. 1 6 . 5 (colorimetric. Acid number, Blank,
Cc. K O H
E . m.f.
An approximately 0.05 11; solution of potassium hydroxide was prepared by dissolving 4.0 grams of powdered solid in 1 liter of the alcohol. I n each determination 123 cc. of the solvent were used, with the addition of 0.05 gram of quinhydrone. All titrations were made in an atmosphere of nitrogen. A blank titration was made first on 125 cc. of the solvent, voltage readings being taken a t 0.1-cc. intervals. The voltage a t the start was about 0.2 volt, with the silver electrode negative, and decreased as the base was added, passing through zero after the end point was reached. Voltage readings with the silver electrode negative are here designated as negative. The potassium hydroxide was standardized from time t o time by electroinetric titration against 0.0610 gram of benzoic acid. I n the determination of the acid numbers of oils the weight of the sample used varied, depending on the acidity.
Cc. K O H
E. m . f .
A E I A cc.
4.60 G R A X S TRAXSPORMEROIL
0.0 2
(CURVEG) -0.230 -0,240
0.4 0.8 1.0 1.3 1.5 1 . 67
-0,218 -0.200 -0,188 -0.146 -0,097 - 0 , 0 47 72
1.8 1.9 2.0 2.2 2.6
-0 028 -0,014 -0,002 0.021 0.059
0,050 0.060 0.048 0.060 0.143 0.240 0.250 0.250 0.190 0.140 0.120 0.115 0.095
orimetric, 2.87 G R A M S
0.0 0.2 0.2
BUTTER
FAT (CURVE
11)
-0.248
-0,248 -0.245 0 1 -0,233 0.9 -0.211 1.0 -0.164 1.1 -0.118 1.2 -0,081 1.3 -0.046 1.4 -0,024 1.5 -0.005 1.7 0.031 1.9 0.043 E n d point, 1.05 cc. KOH, 2 . 6 3 0 mg. per cc Blank. 0 . 1 8 cc. Acid number, 0.80
0.015 0.033 0.120 0.470
0.460 0,370 0,360 0.210 0.190 0.189 0 060
Determinations
The data for all the titrations are given in Table I. Curves for voltage against cubic centimeters of base added and for change in electromotive force per cubic centimeter of base added (AE/ 1 cc.) against cubic centimeters of base are shown in Figures 1 and 2. It is apparent that the end points in these titrations can be determined from the curves. In each case the concentration of the potassium hydroxide and the solvent blank are stated. Some colorimetric values (by standard methods) are given for conipari3on. 16.7)
Literature Cited (1) Noyes and Ellis, J. A m . Chem. Soc., 39,2532 (1917). (2) Seltz and McKinney, IND.ENG CHEM, 20, 542 (1928).
Modified Methyl Red and Sodium Alizarin Sulfonate Indicators' Arnold H. Johnson and Jesse R. Green ~IONTANAQGRICULTURAL
EXPERIMENT STATION, BOZEYAN, MONT.
vv
'HILE a t the Carlsberg Laboratory in Copenhagen the senior author found a solution of methyl red and methylene blue in use as an indicator in work on proteins. This combination and some others have been investigated for the purpose of obtaining indicators that will give greater contrasts of color a t the end point. The indicators commonly used for the protein determination give good results when light and other conditions are favorable, but there are times when a great deal of care must be taken to estimate the end point. Several combinations of indicator and dye are described 1Received August 21, 1929. Director.
Published with the approval of t h e
in the literature. A mixture of ethyl orange and indigo was used by Hallstrom (3) in titrating certain yellow solutions, and a combination of methyl orange and indigo carmine indicator was used by Kirschnick (5) for the same purpose. These investigators obtained more satisfactory results with the combination than with the simple indicator. According t o Luther (6),such mixtures appeared to give a sharper end point in colorless solutions. Later ILIoerk (7') worked out the best proportions of methyl orange and indigo carmine to use in titrating colorless solutions. Also a mixture of methyl orange with xylene cyanole FF was described by Hickman and Linstead (C), and one of methyl orange with acid green and acid blue by Salle (8). The use of mixtures
January 15, 1930
I-VDGSTRIAL B S D EKGINEERING CHEMISTRY
of methyl red and methylene blue was mentioned by Cohn (a)as a suitable indicator for protein work. At a hydrogenion concentration greater than that equivalent to p H 4.2 this indicator gives a violet color, while a t a hydrogen-ion concentration lower than that equivalent to p H 6.3 it gives a bright green color. The indicators considered here are methyl red and sodium alizarin sulfonate, commonly used in protein work. Methyl red is recommended by the American Association of Cereal Chemists (1) and sodium alizarin sulfonate is used by a large number of cereal chemists. T a b l e I-Proportions of I n d i c a t o r a n d Dye Used CO\IBINATION I ~ D I C A T OOR R DYE^ QVANTITY USED
Grams p e r lrter hfethyl red 1 250 hfethylene blue 0 825 2 Methyl red 0 750 Guinea green 0 625 3 Methyl red 0 500 Xylene cyanole FF 1 250 4 Methyl red 0 750 Alphazurine 0 625 5 Methyl red 0 730 Indigo carmine 0 937 6 Sodium alizarin sulfonate 5 000 Methylene blue 0 625 7 Sodium alizarin sulfonate 5 000 Guinea green 0 625 8 Sodium alizarin sulfonate 5 000 Xylene cyanole FF 1 250 9 Sodium alizarin sulfonate 5 000 Alphazurine 0 325 10 Sodium alizarin sulfonate 5 000 1 250 Indigo carmine a T h e methyl red group was prepared in 90 per cent alcohol and the sodium alizarin sulfonate group in >rater. 1
The color changes of all the mixtures here considered are based on a common principle. It is well known that yellow and blue produce green and that red and blue produce violet. Nethyl red is yellow on the alkaline side of the p H range 4.2 to 6.3 and if a blue dye is present a bright green color results. On the acid side of this p H range the indicator has a red color and in the presence of the same blue dye a violet color is produced. On the other hand, as sodium alizarin sulfonate is red on the alkaline side and yellow on the acid side of approximately the same p H range, in the presence of a blue dye a solution on the alkaline side of the p H range will have a violet color and a solution on the acid side will have a green color. The striking difference between violet and green and the intermediate gray a t the end point gives such an indicator a decided advantage in acid and alkali titrations. Hickman and Linstead ( 4 ) call attention to the well-known fact that the eye is less sensitive to a change in intensity than to a change in hue, especially when the change in hue is toward a neutral color. Xow when using the mixture of methyl red and dye as an indicator in titrating from the violet (acid side of the p H range 4.2 to 6.3) to the green (alkaline side of the same p H range) we pass through the end point a t which the solution is gray or colorless. On the basis of Hickman and Linstead's observation, therefore, it is easier to note this color change than when using methyl red alone, in which case the change is from color composed of much red and little yellow t o one made up of less red and more yellow. The indicator solutions containing methyl red were made by dissolving the methyl red and the various dyes in 90 per cent alcohol and those containing sodium alizarin sulfonate were made by dissolving the sodium alizarin sulfonate and the various dyes in water. The dyes used mere methylene blue, guinea green, xylene cyanole FF, indigo carmine, and alphazurine. When dissolved alone these dyes give blue or greenish blue solutions. Table I shows the proportions of indicator and dye that were found to give best results in acid-alkali titrations.
3
I n making up these combinations definite proportions of the constituents should be used, since, as suggested by Moerk (?), the hydrogen-ion concentration for any given color change depends to some extent on the relative concentration of the dye. For example, if a high concentration of blue, as compared with yellow, is present, the green color will appear earlier, or if the yellow predominates the green color will be retarded. The proportions listed in Table I are such that the p H ranges of the mixture of indicator and dye are approximately the same as those of the indicators alone. The end points of the titration or the hydrogen-ion concentrations a t which the solutions were gray were found to be a t approximately p H 5.0-5.4, whether methyl red or sodium alizarin sulfonate was the indicator. On comparing the p H ranges of the mixtures of indicator and dye and the indicators alone by the use of buffer solutions it appeared that about the same difference in p H existed between the violet and green for the mixtures as between the red and yellow for the simple indicators, although the mixed indicators had much sharper end points when used for acidimetry. The greater sensitivity of the end points for the mixtures as compared with those of the simple indicators is due to the operation of the phenomena already discussed. I n conducting the titrations about 0.5 cc. of the indicator solution was commonly used for a volume of 250 cc. By preparing more concentrated indicator solutions the quantity required for titration obviously could be reduced. The mixtures of methyl red and dye were found to be slightly more sensitive than those of the sodium alizarin sulfonate and dye, but the difference in sensitivity was not great. The combinations of methyl red with guinea green and with methylene blue and those of sodium alizarin sulfonate with guinea green and n-ith indigo carmine gave the best results. The differences, however, were very small, and it is believed that the use of any of these mivtiires is an improvement over the use of simple indicators. I t is possible that the use of any blue or green dye with methyl red or sodium alizarin sulfonate would give satisfactory results. It should be mentioned, however, that the mixture of the indicators with xylene cyanole FF appeared to be the least sensitive. T a b l e 11-Protein D e t e r m i n a t i o n s on G r a m P o r t i o n s of a S i n g l e S a m p l e of F l o u r U s i n g Different C o m b i n a t i o n s of I n d i c a t o r a n d Dye 0.1N A C I D EQUIVALENTPROTEIN 'ro NHIOH (.V X 6 . 7 ) COMBIS ATION cc. Per c e n l Methyl red and methylene blue 13.60 10.86 13.59 10.85 Methyl red and guinea green Methyl red and xylene cyanole FF 13.62 10.88 13.59 10.85 Methyl red and alphazurine Methyl red and indigo carmine 13,tjl 10.87 13 5 1 10 79 3lethyl red Sodium alizarin sulfonate and methylene blue 13.55 10.82 Sodium alizarin sulfonate and guinea green 13.59 10.85 Sodium alizarin sulfonate and xylene cyanole FF 13.62 10.88 Sodium alizarin sulfonate and alphazurine 13.65 10.90 Sodium alizarin sulfonate and indigo carmine 13.61 10.87 13.60 , 1 0.86 Sodium alizarin sulfonate 10.86 Average
A11 the mixtures of indicator and dye, as well as the simple indicators, were used in the titration of Kjeldahl distillates obtained from the protein determination of a single sample of flour. The results obtained are given in Table 11. The values in the second column are the averages of two closely agreeing determinations. The data show that practically identical results were obtained whether the indicators were used alone or in mixture with a dye. -4s has been stated, the use of the dye gives a sharper a d more easily determined end point. No attempt has been made to determine whether or not these dyes and indicators are stable a t rathpr high concen-
A S d L YTICAL EDI T I O S
4
trations of acid and alkali, so that the colors given cannot be recommended for such conditions. The mixtures of indicator and dye were developed for use in titrating Kjeldahl distillates and hence were not subjected to hydrogen-ion concentrations more acid than that equivalent to pH 1.0 or more alkaline than that equivalent to pH 7.0.
J-ol. 2 , s o . 1
of the mixture of indicator and dye from that of the indicator alone. Methylene blue or guinea green gave the best results Tvith methyl red. Guinea green or indigo carmine gave the best results Lvitli sodium alizarin sulfonate.
Conclusions
Literature Cited
The use of several blue greenish blue dyes methyl red and with sodium alizarin sulfonate has been found to effect an increase in the sensitivity with which the end point can be determined in titrating acids and bases. The most satisfactory proportions in which to combine indicator and dye are given, the criteria being to obtain the sharpest end point possible and not to change the pH range
(1) Am. Assocn. Cereal Chem., “hfethods for the Analysis of Cereals and Cereal Products,” p . 19, Lancaster Press, 1928. (*) Cohn, J , Gen, Physiol,, 6, 697 (1922), (3) Hallstrom, Ber., 38, 2288 (1905). (4) Hickman and Linstead, J . Chem. SOL.,121, 2502 (1922). (’) Kirschnick7C ’ z e m ~ - Z t s318. ~ 960 (lgo7). (6) Luther, I b i d . , 31, 1172 (1907). ( 7 ) hfoerk, A ~J , ,Phar,n,, 93,675 (1921), (8) Salle, J . Injec/ecliozis Diseases, 38, 293 (1926).
Measuring the Toxicity of Insect Fumigants’,’ A. L. Strand3
A review of the methods used for establishing the H E search for new mac i e n t s , the ratios Letween relative toxicities of insect fumigants is presented. terials useful as insect the toxicities of fumigants in It is shown that the greatest error in these methods fumigants h a s p r o c o m p a r i s o n with sulfur dirises from the attempt to determine minimum lethal gressed rapidly during the last o x i d e taken as a standard. concentrations. The same method was used few years. Much valuable inA method of measuring relative values by comparing by Moore (12) but with some formation has been a s s e in concentrations which kill 50 per cent of the test insects bled and from this have come variations. The concentrain a period of 5 hours has been investigated. These several compounds which are tion of a fumigant, expressed concentrations may be designated as the 5-hour median in gram molecules, required proving of economic imporlethal concentrations. The method appears to possess to kill 5 house flies in 400 tance. Although the methgreater possibilities for accurate work on fumigants minutes was taken by Moore ods for d e t e r m i n i n g t h e than those now in general use. a s t h e measure of toxicity. relative toxicities of chemical compounds have served The experiments were carried very well for pointing out those of outstanding value, the out in liter flasks a t room temperature. Tattersfield and use of many of the data for establishing the general Roberts (23) followed the method of Moore, but also used principles underlying the study of t,he subject is question- a “mean toxic concentration,” the mean of the upper and able, because of the differences in these methods and the lower concentration (death and recovery) values. The same method, but with still greater increase in the selection of criteria for the indication of toxicity. One of the factors measured, and one on which the great majority time of exposure was used by Neifert and his eo-workers of workers seem to agree, is the very one that may coiitribut’e ( 1 4 ) , mho express their criterion of toxicity as “the minimost to inaccuracies in interpretation. Hence a compari- mum percentage concentration which consistently causes son of t,he results of several workers is impossible if close 100 per cent mortality after exposure of 24 hours.’’ The estimates are desired. It would seem worth while, then, to experiments were performed in glass vessels under temperareview briefly what these methods have been and to discuss tures varying from 21” to 32” C. Fleming’s method ( 7 ) possible improvemenB based mostly on findings in the meas- is similar, but his experiments were made a t 26.5” C. Roark and Cotton (6, 18, 19) used the minimum lethal concentraurement of toxicity already made. tion, but with the variation of having the test insects in the Previous Work flasks covered by 250 cc. of wheat. With one exception, then, all of these investigators determined the minimum Among the first important papers on insect funiigant,s amount of a substance required to kill 100 per cent of the having to do with the establishment of relative values is insects in a fixed period of time. that of McClintock, Hamilton, and Lowe (10). These Holt ( 9 ) took the average time to kill 100 per cent of the workers determined the Concentrations of subst,ances required test insects a t varying concentrations. Barnes and Grove ( 2 ) to kill various insects in 1 hour. Exceptions were made in determined the time, temperature, and gas concentration the case of pyridine and nicotine for which %hour exposures necessary to produce death, calling the time so taken t o kill were allowed. They also expressed their results as coeffi- insects the “lethal period.” Keifert and Garrison (13) made * Received June 24, 1929. Presented before the Division of Agricul- comparisons on the basis of the time to obtain 100 per cent lethal doses a t different concentrations. Bertrand and tural and Food Chemistry at the 75th Meeting of the American Chemical Society, St. Louis, Mo., April 16 to 19, 1928. Rosenblatt ( 3 ) compared the vapors of several substance* 2 Published with the approval of the Director as Paper 843 of the by exposing insects to different concentrations, usually for Journal Series of the Minnesota Agricultural Experiment Station. periods of 1 hour, and taking into account the time for the 8 This paper constitutes one section of a thesis presented t o the Graduinsects to recover. Strand (21) used 100 per cent kills a t ate School, University of Minnesota, in partial fulfilment of the requirements different intervals of time and plotted these against temfor the P h . D . degree.
T
