making its facilities available and rendering assistance when needed. LITERATURE CITED
(1) Allen. C. F. H., Edens, C. O., Van Allan, J. A., Org. Syntheses 26, 34 (1946). (2) Baer, J. E., Lockwood, R. G., J . Am. Chem. SOC.76, 1162 (1954). (3) Berger, J., Acta Chem. S a n d . 8, 427 (1954). (4) Brown, E. L., Campbell, N., J . Chem. SOC.1937, 1699. (5) Cheronis, N. D., Entrikin, J. B., “Semimicro Qualitative Organic An-
alysis,” 2nd ed., p. 455, Interscience, Xew York, 1957. (6) Easton, N. R., Hlynsky, A., Foster, H., J. Am. Chem. SOC.73,3507 (1951). (7) Knott, E. B., Morgan, J., U. S. Patent 2,514,650 (July 11, 1950). ( 8 ) Levy, W. J., Campbell, N., J. Chem. SOC.1939, 1442. (9) Ma, T. S., New York University (present address, Brooklyn College, Brooklyn, N. Y.), personal communication. (10) Markunas, P. C.. Riddick, J. A., ANAL.CHEM.23,337 (1951). (11) Puetzer, B., U. S. Patent 2,156,193 (April 25, 1939).
(12) Schacht, W., Arch. Pharm. 235, 441 (1897) [Chem.Zentr. 1897 11,1941. (13) Shriner, R. L., Fuson, R. C., Curtin, D. Y., “Systematic Identification of Organic Compounds,” 4th ed., pp. 243-4, Wiley, New York, 1956. (14) Wilson, W.,J. Chem. SOC. 1955, 1389. RECEIVEDfor review August 24, 1959. Accepted December 7, 1959. Taken from a dissertation presented by Morton Meadow in 1956 to the Graduate Facult of New York University in partial fulfiz ment of the requirements for the degree of doctor of philosophy.
Characterization of Alkyl Halides P. M. G. BAVIN Chemistry Department, The University, Hull, East Yorkshire, England
b Methyl fluorene-9-carboxylate anion reacts rapidly with a wide range of alkyl halides to give good yields of the methyl 9-alkylfluorene-9-carboxylates, many of which are easily obtained in crystalline form. Saponification to the 9-alkylfluorene-9-carboxylic acid and determination of the equivalent weight enable the size of the alkyl group to b e determined. The alkylation succeeds with some tertiary halides and polymethylene diha lides; some hyd roxya I ky I halides also yield characteristic products. Table
1
2 3 4 5 6 7
8
9 10 11 12 13 14 15 16 17
I.
T
HE alkyl halides have been commonly characterized by their physical properties and by the formation of derivatives, particularly S-alkylisothiourea picrates, alkyl 2-naphthyl ethers and their picrates, N-alkylphthalimides, N-alkylsaccharins, and N-alkyl-p-bromobenzenesulfon - p - anisidides. I n addition, derived Grignard reagents have been allowed to react with isocyanates or mercuric halides t o give, respectively, anilides or alkyl mercuric halides (’7). These methods are not universally applicable, are not always
Melting Points of Methyl 9-Alkylfluorene-9-carboxylates and 9-Alkylfluorene-9-carboxylic Acids
(X, preparation in a crystalline condition not attempted.) Melting Points, O C. Methyl Alkyl Group Halide Used ester Acid CH, 170-1 Br, I 108-9 Methyl Br, I 81.5-2 0 Ethyl Br 85.0-5,5 Propyl 79-80 Br Isopropyl 745 132.0-2.5 Br Allyl Br 34.0-4.5 115-16 Butvl 70-1 Br sec-Butyl 68-9 Br Isobutyl 112-13 233-5 tert-Butyl c1, Br 125-6 x Br Pentyl 200-1 56-7 c1 tert-Pentyl 117-18 x Br Hexyl c1 53-4 3-Methyl-3-pentyl c1 66.5-7 .O 3-Ethyl-3-pentyl X 120-2 Br Crotyl 182-1 117-19 Br Propargyl 20 1-2 c1 74-5 Benzyl
18 2-Phenylethyl
Br
(yCClzCH2
G7-8
113 5-4.0
‘Q
c1
19 Cinnamyl
s
162-3
*CH=CHCHz
L.5J 554
ANALYTICAL CHEMISTRY
(Continued)
well suited t o characterizing small quantities of compounds, and generally fail with weakly reactive halides. The formation of Grignard reagents and subsequent reaction with isocyanate8 have been reported to yield rearranged products in some instances (6).
q coocr, I
Methyl fluorene-9-carboxylate anion (I), easily generated from the ester with methanolic sodium methoxide, reacts rapidly with a wide range of alkyl halides t o give good yields of the corresponding 9-alkyl derivatives (11) (1,
R COOCH3
I1
3). [Wislicenus prepared several derivatives of the ethyl ester (8), but these have lower melting points than the methyl homologs.] Many of the products are easily obtained in crystalline form and have sharp melting points. Although isomeric derivatives may have similar melting points, this is a defect common to many methods of characterization; suitable mixed melting point determinations show large depressions. EXPERIMENTAL
Fluorene-9-carboxylic acid may be prepared from benzilic acid (6). The following has proved to be more convenient on a large scale. Fluorene (166 gram, 1 mole) in dry ether (500 ml.) was added with stirring
t o ethereal phenyllithium (from bromobenzene, 240 grams, 1.5 moles). After a n hour the orange solution was poured as rapidly as possible onto powdered solid carbon dioxide (approximately 500 grams) previously slurried with a little d r y ether. Next day 10% hydrochloric acid (200 ml.) was added and ether removed by steam distillation. After cooling to 35' to 40' the solid was collected and dissolved in a solution of potassium carbonate (80 grams) in water (100 ml.), Norite (5 grams) was added and the mixture filtered through a layer of Celite to give a clear pale yellow solution. Pouring slowly into excess 30y0 hydrochloric acid precipitated fluorene-9-carboxylic acid as a white. easily filtered crystalline solid which was collected, washed, and airdried. The methyl ester was best prepared by suspending the acid (1 part) in dry methanol ( 5 parts) and saturating the mixture with d r y hydrogen chloride. When boiled gently under reflux for 4 hours, the acid passed into solution. On cooling, the bulk of the ester separated as colorless prisms, (melting point 64-5'). The yield was generally over 70% and occasionally reached 90%. Alkylation Procedure. Methyl fluorene-9-carboxylate (1.12 grams, 5 mmoles) was dissolved in d r y methanol (10 ml.) containing sodium methoxide (from sodium, 0.23 gram, 10 mmoles), giving a yellow solution with a pronounced pale blue fluorescence. The alkyl halide (3 mmoles, although 1.1 to 1.2 mmoles is sufficient with methyl iodide or allyl bromide) was added and the solution left until the pale yellow color had been discharged. With very reactive halides such as methyl, allyl, and benzyl, reaction was complete in less than 5 minutes; cyclohexyl and tertiary halides required several hours, but less if the mixture was warmed on the steam bath. I n the case of alkyl chlorides, sodium chloride invariably separated; bromides occasionally yielded sodium bromide in the same manner. T h e product was obtained b y dilution with water. followed bv extraction into methylene chloride. Evaporation and stripping of high boiling substances if present were followed by crystallization from methanol or hexane. The 9-alkylfluorene-9-carboxylic acids were obtained by adding a solution of potassium hydroxide (1 gram) in water (3 ml.) to the alkylation mixture, additional methanol was required to render the mixture homogeneous. The solution was warmed on the steam bath and the course of the hydrolysis followed in the usual way by diluting small samples. The time required for saponification to approach completion varied from a few minutes (9-methyl) to several hours (9-krt-butyl). The acids were isolated by evaporating the bulk of the methanol at 20 mm. of mercury, diluting with water (30 ml.), and removing insoluble material by washing with hexane, followed b y filtration with Norite through Celite. The filtrate was poured slowly and with stirring into 20% hydrochloric
Table
I.
Melting Points of Methyl 9-Alkylfluorene-9-carboxylates and 9-Alkylfluorene-9-carboxylic Acids (Continued)
(X, preparation in a crystalline condition not attempted.) Melting Points, O C. Methyl Alkyl Group Halide Used ester Acid Br 106-7 20 Benzcyclobutenyl
21 Cy clopentyl
Br
83.04.5
22 Cyclohexyl
Br
120-1
c1
23 3-Cy clopentenyl
93-4
Br
24 3-Cyclohexenyl
X
c1
104-5
c1
119.520.5
c1 c1 c1
Br Br
129-31 166-7 9 1-2 90.5-1 .O 184-5
Br
215-16
33 9-Methosycarbonyl-9fluorenyl
Br
227-8
34 Br omomethyl
Br
112-13
25 2-Thien ylmethyl 26 3-Thienylmethyl 27 hlethoxymethyl 28 hfethoxycarbonyl 29 Methoxycarbonylmethyl 30 4Cyanobutyl 31 Phenacyl
32 9-Fluorenyl
Table 11.
jjSy2 S CH3OCHz CHaOzC CH302CCHz NC( CH2)aCHZ
184-6 168-70
aco-cH2
Melting Points of Lactones Obtained from Hydroxyalkyl Halides
(Structures assigned are based on carbonyl stretching frequencies in infrared spectra) Alkyl Group Halide Used Product Melting Point, O C. 184-5 35 2-Hydroxyethyl Br
36
3-Hydroxypropyl
c1
37
2,3-Dihydroxypropyl
c1
acid (30 ml.) and left overnight, prcferably a t 00 t o 50. T h e acids crystallized well from benzene-hexane. After drvinn. their eauivalent weights m a v be d&er;ined b$ well-know; metgods
(4)
Esters not obtained crystalline may be hydrolyzed to the more easily crystallized acids (HI), but these are not entirely suitable for characterization pur-
qy T?
232-4
180-1
Table 111. Melting Points of a,w-Di(9-~ethoxycarbonyI-9-fluorenyI)alkanes PreDared from a,w-Dibrornoalkanes
Meltinog Alkyl Group Points, C. 38 Dimethylene 261-2 39 Trimethylene 194-5 40 Tetramethylene 221-2 VOL. 32, NO. 4, APRIL 1960
* 555
poses, as their melting points may not always be reproducible (1).
qp R COOH
The compounds listed in the tables were prepared in t h e course of other work (1, 9 ) , a few being described in the literature. All new compounds have given satisfactory analytical figures for carbon and hydrogen. Their description and a discussion of the theoretical aspects of the anion (I) will be published elsewhere (2, 3). This method of characterizing alkyl halides should prove of value because fluorene-9-carboxylic acid is readily prepared from either benzilic acid or
fluorene, and is easily esterified by methanolic hydrogen chloride; the alkylations are rapid, give good yields, and result in a large increase of molecular weight. As little as 50 mg. of the ester may be successfully alkylated. The alkylations succeed with chlorides as well as with bromides and iodides. Tables I, 11, and I11 illustrate the wide variation possible in the alkyl radical. Hjrdrolysis of the alkylation product. which need not be isolated, to the 9alkylfluorene-9-carboxylic acid and measurement of the equivalent weight enable the size of the alkyl group to be estimated. ACKNOWLEDGMENT
The author is indebted to K. Clarke for his interest and criticism of the manuscript. The a v a r d to the author
of a n I. C. I. Fellowship by The University, Hull, is acknowledged. LITERATURE CITED
(1) Anet, F. A. L., Bavin, P. 11.G., Can. J . Chem. 34, 991 (1556). (2) Bavin, P. 11. G., Zbid., 38, 2023 (1509), and subsequent papers. (3) Bavin. P. AI. G.. unoublished data. ( 4 ) Linetead, R . P.: Eividge, J. A ,
Whalley, AI., “Course in Modern Techniques of Organic Chemistry,” pp. 153-60, Butterrorthe, London, 1955. (5) Richter. H. J., Org. Syntheses 33, 3 i (1953). (6) Schwartz, A. AI., Johnson, J. R., J . Am. Chem. SOC.53, 1063 (1931). (71 Kild. F.. “Characterisation of Organic kompoundE,” pp. 34-46, Cambridge University Press, 19-17. (8) Kislicenus, W.,Ruthing, A., Ber. 46,2770 (1913). \
,
RECEIVEDfor reviex August 28, 1959. ilccepted January 4, 1960.
A Modified Method for Hydroxyproline Determination FERENC HUTTERER and EDWARD J. SINGER Deparfmenf of Pathology, The Mount Sinai Hospital, Fifth Ave. and 100fh St., New York
b The chromogen produced b y the reaction of the oxidation product of hydroxyproline with p-dimethylaminobenzaldehyde has been stabilized by increasing the concentration of npropyl alcohol in the reaction mixture to 45.5 volume %. To eliminate nonspecific interference, differential spectrophotometry a t 500 and 560 mp has been employed, and a suitable correction formula devised. The improved reaction and measurement conditions permit precise and accurate microdetermination of hydroxyproline in complex materials, such as tissue hyd rolyzates.
C
analytical methods for hydroxyproline determination are, for the most part, based on Lang’s work (5), the method of Neuman and Logan (8, 9) being most widely used. Several modifications of this method have been developed (2, 4, 6, 7). The general lack of precision and accuracy in these methods is attributable to instability of the specific chromogenic compound under the conditions at present used, impurity of p-dimethylaminobenzaldehyde (p-DAB), incomplete removal of the oxidizing agent (hydrogen peroxide), and loss of reaction specificity in the presence of various tissue components. I n the method 556
URRENT
ANALYTICAL CHEMISTRY
described, these sources of errors were eliminated. EXPERIMENTAL
Reagents. L-Hydroxyproline (Nutritional Biochemicals Gorp., Cleveland, Ohio) was purified by precipitation from methanol-ethyl alcohol
(1).
p-Dimethylaminobenzaldehyde (pDAB) (Eastman Kodak Co., Rochester, N. Y.) was dissolved in ethyl alcohol (100 grams of crude material per 200 ml. of ethyl alcohol) by heating a t 70” C. Charcoal was added with continued heating and stirring for 5 minutes and the solution was filtered by suction. More water than was needed to precipitate the p-DAB was added to the solution; then the precipitate was washed on a Buchner funnel with water and dried in vacuo. The procedure was repeated until a white product, giving a colorless solution with propyl alcohol, was obtained. A 5% solution in npropyl alcohol was prepared. Hydrogen peroxide (Superoxol Merck) was standardized by titration with potassium permanganate. A 4% solution was prepared. Recommended Procedure. Place 1.0 ml. of a solution containing 1 t o 10 y of hydroxyproline in a suction tube (Fisher Scientific Co., Catalog Xo. 14-941). Add 0.2 ml of 0.05M CuS04.5H90, 0.5 ml. of 2.5N NaOH, and 0.5 ml. of 4.0% H202. After shaking, let it
29, N. Y .
stand for 5 minutes a t room temperature. Place in a 70” C. water bath for 10 minutes. After the first 5 minutes, apply vacuum and agitate the tube for about 30 seconds to complete removal of the H20z. After cooling in ice, add 0.8 ml. of 8.ON HzS04and 2.5 ml. of 5.0% p-DAB in propyl alcohol. Shake vigorously. Place in a 70” C. water bath for 40 minutes. Cool and read the absorbance measured against a blank determination in a photometer a t 560 mw using a 1-cm. cell. A standard curve was prepared employing this procedure with five replicate determinations of each point (Table I). I n routine practice, duplicate determinations of the standard need be made a t only two points to define the curve. Effect of Solvent. T h e oxidation product of hydroxyproline and the red chromogen obtained by the condensation of this oxidation product with p-DAB are both decomposed during heating in acid (IO, 1.2). The decomposition of red chromogen results in a decrease of absorbance at 560 mp (Figure 1). I n aqueous systems the reaction proceeds rapidly, but the color yield is low and the decomposition of the red compound is rather rapid. Both increased concentration and lengthened alkyl chain of the alcohol (lower di-
