1827
V O L U M E 2 7 , N O . 11, N O V E M B E R 1 9 5 5 $tarits is accomplished by the use of the tables in the Forziati lxtper. Table 111 lists the values of the constants for the 14 hydro(,:I rhons. DISCUSSION AND CONCLUSIONS
T h e use of a monochromator together with a tungsten filament lamp as a means of obtaining nionochromatic light for index of i.efraction nieasiirements is satisfactory and convenient. The index values obtained hy the monochromator method and those ticlterniined using the sodiiirii or mercury vapor lamp agree within :Ll)oiit & 0,00003 unit. ACKNOWLEDGMENT
T h e authors appreciate the American Petroleum Institute g w i t which made this research possible. This is part of the \\-ark of Project 12. The assistance of B. E. Kuiper with some of the measiiremeiits is nckmwledged. T h e advice of the .\dvisoyy Committee has Ijren helpful: H. Sutherland (chairman),
E, 11.Barber, J. R. Bates, L. C. Beard, Jr., G. H. Denison, R. F. llarschner, C. E. Morrell, and J. H. Ramser. REFERENCES (1) American Petroleum Institute Research Project 45, Ohio State
TJniversitv. Columbus. Ohio. ( 2 ) Drude, P., Ann. p h y s . , 14, 677 (1904). (3) Forziati. A. F., J . Reseaich S a f l . Bur. Standards, 44, 373 (1950). (4) Kurtz, S. S., Jr., and Ward, d. L., J . Franklin Inst., 222, 563 (1936). (5) Schiessler, R. W., and NcLaughlin, 11. L., "Synthesis and Purification of High AIolecular Weight Hvdrocarbons." from "Chemistry of Petroleum Hydrocarbon3," vol. I, Reinhold, New York, 1954. (6) Schiessler, R. W.,and Whitmore, F. C., Ind. E n g . Cheni., 47, 1660 (1955). (7) Sellmeier, W., POQQ. Ann., 1 4 3 , 2 7 2 ( 1 8 i l ) ; 145, 399, 520 (1872); 147, 359 (1872). (8) Weissberger, A , , "Physical Methods of Organic Chemistry," up. 1141-240, 2nd ed., Interscience, Yew York, 1949. R E C E I V Efor D review Alarcli 3 , 1955. i c c e p t e d July 8 , 1955.
Titration of Basic Copolymers of Acrylonitrile in Nonaqueous Solution C. A. STREULI Research Division, Stamford Laboratories, American Cyanamid Co., Stamford, Conn.
'The methods of nonaqueous titration for the determination of organic bases have been extended to include the determinatioii of these bases when copol) merized with acrylonitrile. imines, amine salts, and q~iaternar? amnioniuni salts in the poljmer may be titrated with 0.05.Y perchloric acid when the pollmers are dissolied iii a niixture of nitromethane and formic acid. The salts of p: d i n e deritatives but not those of aliphatic amines niaj he determined in polymers hj titration with 1,3-di-o-tol) lguanidine.
Ma=y
simple organic compounds are readily titrated in nonaqueous solution 1):acid-base technique. Fritz ( 2 )and Pifer, Wollish, and Schmall ( 6 ) have done extensive work i n this field, and have described methods for the quantitative determin:rtion of amineii, amine salts, and quaternary ammonium s ; t l t P . These methods n i t h appropriate modifications hnve ])roved suitable for the n-ork described in this paper. Wheii acrj-lonitrile is copolymerized with an unsaturated organic molecule containing either an amino or quaternary aniriionium nitrogen, the resulting polymer retains the basic char:rcteristic.s of t,he comonomer. This property may be used to tieternline the amount of basic material which has been copolymerized. As mineral acids are uaed in the polymerization procera. the amines contained in the polymer may occur as salts as well as free amine. A n y means of analysis must then be capable of determining both f1,ec amine and amine salt, in order t o give the total amount of amine present. This is also true of quatern:wy ammonium compounds, as the:-, too, occur as salts in the polyniers. SOLVESTS AND TITRANTS
Acrylonitrile copolymers are all insoluble in acetic acid arid acetonitrile, but show complete solubility in dimethyl formamide. Unfortunately, it is not possible to titrate amine salts (with perchloric acid) in this solvent, hecause of t,he basicity of the solvent. The polymers are also partially soluble in nitromethane, a reagent recommended by Fritz (3),and are completely solubilized by addition of small amounts of formic acid, maleic anhydride, or water to the susperisio~i. The addition of acetic acid or acetonitrile to the nitrompth:rnr sliywnsion does not aid m1ul)iliziition.
X 2 to 370 solution of $B70 formic acid in nitromethane s l i o ~ ~ the best solvent properties of the mixtures tested. Solution will also occur if 00% formic acid is used, but the presence of the increased amount of water tends to give poorer end points in the perchloric acid titration. Either 0.1 or 0.055 perchloric acid in dioxane was used as titrant for the nitromethane-formic acid solutions of the po1yniei.s. Amines, amine salts, and qiiaternar>. ammonium salts nrr d l titrated under these conditions, provided that the salt is not :t halide. The use of the more dilute titrant increases the precision of the determinations. The end poirit is detected potentiometrically through the use of a glass-c:iloniel electrode com1jin:tt ion. Sodium methylate, the uswil titrant for the acid portio~iof amine salts, cannot be used to determine the amine salts i n the polymers, as this reagent attacks the acrylonitrilr portion of the S o definite end poiiit c i ~ n polymer, apparently by hydrol) lie obtained in such a titration. 1,8-Di-o-tolylguanidine, designated 117 Davis and Hetzei, ( 1 ) :is one of the stronger substituted guanidine lx~sc+.\vas found c:ipaI~le of titrating heterocyclic amine salts in the presence of the fi,ee srnine, but could not be used successfully with aliphatic amine salts. This is undonbted1)- due to the nearly equal basic strength of t,he amines and the titrant. Thip h s e combines with the acid portion of the salt and is, therefore. useful in determining that part of the compound. I t does riot attack the nitrile portion of the polymer. TITRATION OF QUATERN.4RY A\IMONIURI SALTS
All of the polymers containing quitternary compounds were prepared by the copolymerization of acrylonitrile and a quaternary ammonium chloride in the presence of mineral acids. The chloride ion is not completelj, displaced, so that some of this anion remains in the polymer. The presence of the halide ion necessit,ates the use of Pifer and Wollish's ( 6 )mercuric acetitte modification. Their work was done by dissolving an amine or quaternary ammonium halide in glacial acetic acid and adding R solution of mercuric acetate. The formation of the mercuric halide removes the halide ion froin solution and the excess mercuric acetate, being essentially iinclissociated, does not intwfrre i\-ith the perchloric acid titration.
1828
ANALYTICAL CHEMISTRY
As the polymers were insoluble in acetic acid, it was first necessary to show that this method would be equally successful in another solvent. Accordingly a n essentially pure sample of tetrabutyl ammonium iodide was dissolved in the nitromethaneformic acid mixture, a 6% solution of mercuric acetate in glacial acetic acid was added, and the sample was then titrated with 0.1N perchloric acid in dioxane. Six determinations gave an average value of purity of 98.8 f 0.5%. Four Volhard determinations of the salt run as a check gave an average value of 98.9 =t0.4%. The excellent agreement of results shows that Pifer and Wollish’s method works as well in the nitromethane-formic acid mixture as in acetic acid Other simple compounds analyzed in this manner gave equally accurate results.
lodo
I
1000
I
-
I
S o l v m t ~nltromothanr 98% formic acid
800 0 H
-fF
Figure 2.
I
2 3 mi. 0.05d HCIO,
4
Titration of amine and amine salt in acrylonitrile copolymer
600
Solvent: nitromethane98% formic ocid
400
1
0
I
1
I
I
2 3 4 mi. ao5H HCIO4
I
5
I
Figure 1. Titration of quaternary ammonium chloride in acrylonitrile copolymer
-1number of polymer samples were then analyzed. A typical titration curve is given in Figure 1. Polymers containing from 2 to 5 % of the salts may be anal>.zed by this method with a relative precision within 4%. The accuracy of the method was checked by preparing a polymer which contained chloride salts exclusively. Sonaqueous data for this polymer indicated that 4.T8 f 0.08% quaternary ammonium chloride was present. Bomb combustion of the polymer followed by a Tolhard analysis for chloride gave 4.7 f 0.2y0 as the quaternary ammonium chloride content..
Solvent: dimethylformomide
-I TITRATIO3 OF AMIYES 4 X D AMINE SALTS
These polymer* rvere formed by copolymerizing acrj lonitrile and an unsaturated amine in the presence of a mineral acid; the amines included both tertiary aliphatic amines and pyridine derivatives. As these salts are completely titrated by perchloric acid in nonaqueous solution ( Z ) , the mercuric acetate modification is unnecessary A typical titration c u v e for the amine-acrylonitrile copolymer is shown in Figure 2. Both the aliphatic and heterocyclic aminecontaining polymers give essentially the same titration curve. the When the total amine content of the polymer is about 5y0> relative precision of determination is 4%; the absolute precision 0.2%. The pyridine-containing copal> mers may also be titrated 17 ith aqueous acid and alkali when the polymer is dissolved in concentrated thiocyanate solution. This method, developed by Leus-
Figure 3.
0 I 2 3 4 ml. O . I H 1,3-di-o~~toIyIguonidine
i
Titration of amine salts in acrylonitrile copolymers
sing ( d ) , gave agreement with nonaqueous results within 0.2% when the sample contained about 5.0% base. The titration of the acid portion of the amine salts in the polymers a ith 1,3-di-o-tolylguanidine is illustrated in Figure 3. Curve 1 is typical of the polymers containing ealte of pyridine derivatives and shows a clearly recognizable end point. Curve 2 is representative of salts of aliphatic amine-containing polymers. The titration break is too poor to be of much use analytically. Dimethylformamide makes an excellent solvent for this titration. Glass and calomel electrodes are used to detect
1829
V O L U M E 27, NO. 11, N O V E M B E R 1 9 5 5 the end point. The addition of standard perchloric acid to the solvent corrects the small negative blank. REAGENTS
Perchloric acid, 0.05-\-. Dissolve ca. 4.2 ml. of i 2 y 0 perchloric acid in 1 liter of dioxane and standardize against potassium acid phthalate according t o the method of Seaman and Allen ( 7 ) . Mercuric acetate solution. Dissolve ca. 6 grams of mercuric acetate in 100 ml. of hot glacial acetic acid ( 5 ) . Xitromethane, C.P. Fisher Scientific Go. Formic acid, 98%, Fisher Scientific Go. 1,3-Di-o-tolylguanidine,0.I-\-. Dissolve 24 grams of reagent in 100 ml. of methanol and 900 ml. of methylchloroform. Standardize potentiometrically against 20-ml. portions of standard 0.1-V aqueous hydrochloric acid or 40 ml. of the 0.05.V perchloric acid in dioxane. Methanol is used as sample solvent. Dimethylformamide, E . I. du Pont de Nemours &- Co.
Introduce the glass and calomel electrodes into solution, set the pH meter to mv., and titrate the solut,ion potentiometrically with Ptandard 0.05.V perchloric acid. A microburet should be used. The end point of the titration is the maximum value of ( a E / A V ) . Appropriate plots may be made. A blank must also be titrated and the titer of the sample corrected for the solvent titration. Determination of Heterocyclic Amine Salt. Dissolve a rveighed 0.5-gram sample of polymer in 70 ml. of dimethylformamide. TJsing a glass and calomel electrode combination titrate the solution potent,iometrically with the standardized solut.ion of 1,3-di-o-tolylguanidine. The end point is det'ermined by the maximum value of ( a E / A V ) . The negative solvent blank is detcrrnined by tit,rating 70 nil. of dimet,hylformamide with standard perchloric acid and adding this correction t o the sample tit,er.
+
LITERATURE CITED PROCEDURE
Sample weights are Iiased on an expected base content of the polymer of 5yG. Determination of Total Base. Accurately weigh 0.3 to 0.5 giam of the polvmer into a tared 150-ml. beaker and stir with 20 ml. of nitromethane until the material is well dispersed. Add 2 ml. of 9Syo formic acid, and heat until the suspension dissolves completely. Dilute with 50 ml. of cold nitromethane and allow the solution to come to room temperature. If the amine or quaternary salts present contain halide ion, add 2 ml. of 6 % mercuric acetate solution.
(1) Davis, 11.AI., and Hetaer, H. B., J . Research .\-afZ. Bur. Standa,ds, 48, 381 (1952).
(2) Fritz, J. S.,"Acid-Base Titrations in Sonaqueous Media," G. Frederick Smith Chemical Co. Columbus. Ohio. 1952. (3) Fritz, J. S.,and Fulda, 11.O., h s a ~ CHEX.. . 25, 1837 (1953). (4) Leussing, D. L., unpublished data. (5) Pifer, C. W., and Wollish. E. G., -%N\L. CHEY..24, 300 (1952). (6) Pifer, C . W., Wollish, E. G., and Schmall. I1 , J . Am Phaiin. Assoc., 42, 509 (1953). (7) Seaman, W., and .Illen, E., . i s 4 ~ CHEM., . 23, 592 (1951). RECEIVED for review May 18, 1955.
Accepted July 23, 1933
Fluorometric Determination of 0.1 to 10 Micrograms of Cholesterol R. WAYNE ALBERS
and
OLIVER H. LOWRY
Department of Pharmacology, Washington University School o f M e d i c i n e , St. Louis, M o .
A fluoronietric method is described for the nieasurement of as littleas 0.1 y of cholesterol in animal tissues. The simplicity of the procedure and the stabilitj of the fluorescence facilitate the measurement of a large number of samples. The effect on the reaction of other substances likely to be present in lipide extracts of animal tissues has been studied. No substances have been encountered which seriously affect the results. Free and esterified cholesterol produce nearly the same final fluorescence on an equimolar basis.
I
C O N S E C T I O S with n quantitative histochemical study of brain ( 6 ) a method vas needed for measuring as little as 0.1 y of cholesterol, or 1000 times less than the amount required for example. The reliability of the in the Sperry method (9), colorimetric Liebermann-Burchard reaction prompted an investigation of its fluorescence possibilities. A simple procedure resulted, which is based upon the measurement of a stable fluorescent product and which has the required sensitivity when used x i t h a photomultiplier-type fluorometer ( 5 ) . Glick (3)has recently adapted the colorimetric Sperry method t o the measurement of as little as 0.2 7 of cholesterol. Severtheless, the fluorometric method proposed may have certain advantages including greater ultimate sensitivity. Chen has reported a fluorometric method for hydroxy steroids ( 2 ) ,but no details have been published. ?;
METHOD
Reagents and Equipment. ..icetic anhydride, analytical grade, is used without further purification. Sulfuric acid, analytical grade. If the sulfuric acid contributes to the reagent blank, it may be purified by heating with 5o/c by
volume of 70yoperchloric acid until the solution hecomes colorless and fuming subsides. 1,1,2-Trichloroethane. The solvent obtained from Distillat,ion Products Industries (No. T 8 5 l ) is suitable after furt,her purification. The fraction boiling a t 111-113° C. is washed four times with 0.1 volume of concentrated sulfuric acid, twice x i t h water, and is finally dried over anhydrous sodium suliat,r. Redistilled absolute et.hyl alcohol. St'andard cholesterol solutions are prepxed in trichloroethane a t concentrations of 0.02 to 0.4 mg. per ml. Solutions of 0.01 to 0.1 mg. % safranine 0 (Sational .hiline Co.) or rhodanine B (Distillation Products Industries) in 0.01S hydrochloric acid. These are not essential, but may be convenient t o control instrument, settings during fluorescence measurements. Lang-Levy pipets ( 6 , 7 ) are used throughout,. For the initial extraction the tip must be rather slender ( 7 ) . Fluorometer tubes are selected from 7 X 70 mm. serological tubes (Iiimble, S o . 45060-Sl81, -4.S. .\lot: CJ., St. Louis). Tolerances are kept within 1 or 2y0for 170th inner and outer diameter. The fluorometer tubes need to he very carefully cleaned before use. The folloning procpdure is recommended: two rinses with a det,ergent, two with water follon-rd by heat,ing in half concentrated nitric acid a t 100" C. for 43 minutes, then three rinses n-ith distilled witer The Farrand fluorometer is suitahle for me:isuring the fluorescence after adaptation to hold tubes of 7 miii. in diameter with 15O-pl. volumes ( 7 ) . Centrifuge evaporator (Figure 1 ), The motor, Jf, 280 r.p.m., Type KCI-23, Bodine Electric Co.. 2254 R'est Ohio St., Chicago, is suspended from desiccator plate, P , and is fitted with a 9-inch Lurite rotor, R. The rotor is d d l e d nitli 50 holes 8 nim. in half-inch strap of metal, S , permits diameter a t a 45" angle. easy removal of the assembly, which is mounted in a 10-inch vacuum desiccator. The electrical leads are stripped bare a t the point of emergence and sealed in with a De Khotinsky type of cement. The low speed motor for the small centrifuge constructed within the vacuum desiccator provides centrifugal force to prevent boiling or creeping of t.he solvent. up the vialls of the tubes during evaporation. K i t h the vacuum from a water aspirator and a little heat from an infrared lamp: the samples (alcohol) A\
