677
S E P T E M B E R 1947 Table 11. Titration of Hydrochloric Acid by Electrolytic Hydroxyl Concn. of NaBr M 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.30 0.30 0.30 0.30 0.30 0.60 0.60 0.60 0.60 0.60
Theoretical Base Total Acid Produced Used Meq. Meq. 0.133 0.139 0.148 0,145 0.153 0,151 0.0713 0,0738 0.0738 0,0746 0.0786 0,0803 0,0895 0.0910 0.123 0,124 0.120 0.119 0.122 0.123 0.126 0,128 0.130 0 126 0.130 0.130 0 0353 0,0373 ' 0.0746 0 0779 0,112 0.113 0.149 0.149 0.193 0.197 AIean Average deviation from mean Standard deriation
E.M.F.
Current
Volts
Ma.
6 6 6 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
7.5 7.8 8.1 3.9 4.0 4.3 4.8 6 9 6.4 6.6 6.9 7.0 7.0 2.0 4.0 6.0 8.0 10 0
Difference
% -4.3 +2.l f1.3 -3.4 -1.1 -2.1 +1.7 -0.8 +O 8 -0 8 -1.6 -3.1 0 0 -5 4 +4 4 +0.9 0.0 T
X
-0.6% 2.0% 2.5%
Table 111.. Potentiometric Titration of Hydrochloric Acid by Electrolytic Hydroxyl "21 Added Micrograms 29.9 59.8 149.5
No. of Determinations 29 19 11
Average Recovery
DeTViation from Mean
%
%
91.7 86.3 90.5
3 5 2 5 3 1
StandRrd Deviation q 4 3 2 9 3 8
what lower than the theoretical. This is shown in Table 111, which gives the results of a series of titrations of known amounts of hydrochloric acid by this method. DISCUSSION
Accuracy. As may be expected, the potentiometric titration is not stoichiometric. This is of little consequence for the projected
use of this system, since calibration is easily accomplished empirically.~ bv maintaining a constant current and meawiing the time necessary to titrate known quantities of added acid. I n actual practice the electrolysis time has been recorded on a drum rotating a t constant speed. The quantity of acidity was then directly proportional to the length of the recorded line. Precision. Using the standard deviation as a measure of precision, the chances for a determination to be within * l o 5 of the mean calibration are better than 96 out of 100. For the purposes of this laboratory this precision is satisfactory. The precision is shown to be practically unaffected by the quantitl- of acid determined for the range of experimental amounts, ea. 30 to 150 micrograms. Sources of error are the measurement of current and time and the Pinkhof titrationitself. Since thissystem depends on constancy of current, precautions must be taken to prerent polarization. Usually, adequate stirring by the air stream is sufficient. The milliammeter and the stopwatch should be calibrated. The Pinkhof system is known to be not too precise but its advantages in speed and simplicity are in its favor when a *lo% precision is allowable. Applications. This method can have considerable application in the sampling of air contaminated by nosious gases, since it can be made continuous, and the cell is portable, comparatively nonbreakable, and above all remotely controlled. Because both the generation of ions and the observation of the end point are accomplished electrically, it can be readily adapted to automatic control by simple electronic devices. Examples of such instrumentation have becn developed by S o d h r o p (3:and have been i n use in this laborctory for several years. LITERATURE CITED
(1) Kolthoff. I. 11..and Furman. N. H., "Potentiometric Titrations." pp. 115-52. New York. John Wiley & Sons, 19%. (2) Lingane, J. J., J . Am. Chsm. SOC.,67,1916-22 (1945). ( 3 ) Northroo. J. H.. uersoiial communication. (4) SzebellcdL L., and Somogji. Z , 2 . anal. Chem 112, 313, 323, 332, 385 391, 395, 4'10 (1938).
Determination and Nature of Leaf Sterols RfOYROE E. W-ALL A ~ EDWiRD D G. KELLET Eastern Regional Research Laboratory, L . S . Department of Agriculture. Philadelphia 18, Pa. Methods for the determination of unsaturated and saturated sterols in leaf meals are described. Alternati\e procedures using micro- or macrosamples and colorimetric or graJimetric techniques are giten. The close relationship hetw een the time us. density curtes, absorption curies, and E of the color reaction product is demonstrated.
L
EAF sterols may have potential value a; sources for the
preparation of vitamin D-active compounds and sex hormones. I n vie!v of that fact it seemed desirable to determine the sterol content of a number of leaf meals and extracts. No method has been published for the quantitative determination of leaf sterols, although many procedures are available for the determination of cholesterol in blood and animal tissue s n d sterols in vegetable oils. Sterols in such materials can be determined by macro gravimetric methods or micro colorimetric procedures. The former are all based on the work of Windaus ( l 7 ) , who shoved that on the addition of digitonin, cholesterol or phytosterols in alcohol solution form a characteristic insoluble digitonide. The sterol digitonide is then filtered and 'Lveighed. All the plant sterols isolated to date can be precipitated by digibDin.
The macroprocedures devised for cholesterol are accurate but have the disadvantages of being time-consuming and requiring
large samples and considerable quantities of expensive digitonin. Microprocedures devised for cholesterol eliminate these difficulties. Of these, the Liebermann-Burchard reaction, i n iyhich the sterol is t,reated with acetic anhydride and sulfuric acid, has been the most extensively studied. Bloor (2) adapted the reaction to determination of cholesterol in blood and many workers have introduced modifications. Schoenheimer and Sperry (1.2)precipitated cholesterol as the digitonide and, after dissolving the compound in acetic acid, carried out the colorimetric reaction in the presence of digitonin. Kelsey ( 7 ) also precipitated cholesterol as the digitonide, which he then decomposed with boiling benzene and, after removing the insoluble digit,onin, carried out the color reaction on the free sterol. Sperry (13) has shown that determination of cl!olesterol by the Schoenhcimer-Sperry microprocedure has an accuracy comparable with that of macromethods. By utilizing a number of the foregoing procedures and intro-
678
V O L U M E 19, NO. 9
T a b l e I.
Essential Steps Involved in D e t e r m i n a t i o n of Total, Free, and Saturated Sterols Petroleum ether extract of dried plant materials Free Sterols
Total -two18
Saturated Sterols
Super-Cel in a small sintered Hirsch funnel and washed thoroughly with previously cooled Skellysolve B. T o remove excess iodine the filtrate containing the sterols is transferred to a separatory funnel and shaken vigorously with 5 ml. of aqueous 10% sodium thiosulfate solution. The thiosulfate layer is drawn off, and the extract is shaken several times with 10 to 20 ml. of water and finally with 10 to 20 ml. of 90% methanol. The solution, which should be colorless or light yellow., is run into an Erlenmeyer flask, evaporated on a water bath to approximately 10 ml., and made to 25 ml. with Skellysolve B. PRECIPITATIOS A N D COLORIMETRIC E s r I x i r I o s O F FREE STEROL.The following steps are essentially a combination of
the procedures of Kelsey ( 7 ) and of Schoenheimer and Sperry ( 1 2 ) and are shorter and simpler than either of the originals:
I
Remove
