Quantitative Determination of Alpha-Pinene - Analytical Chemistry

---

linkage., sodium 1aurJ.i sulfatc is subject

to hj.drolysis in the presence of strong acids but is, reportedlj., stable in alkaline media (I). The surfactant is negatively charged and will adsorb strongiy to acidic surfaces but not to basic or neutral ones. Therefore, acid-washed asbestos (Colorado asbestos) was packed into Gooch crucibles arid washed several times with distilled water. The crucibles were then placed in an 800” C. oven for 1 hour. After cooling, they were washed with 0.1M sodium phosphate (pH 8.4) until the washing was alkaline t o phenolphthalein and rinsed 5 times with distilled water. A dense suspension of bacterial cells (Staphylococcus aureus) was incubated with sodium lauryl sulfate for 12 hours at 30” C. to allow for maximum binding. The mixture was then boiled in an alkaline medium (0.0lM sodium phosphate), cooled, and filtered over the alkalitreated asbestos pads. The pads were then washed with five 5-ml. portions of distilled water. Thc filtrate and washings were combincd, 5 drops of O.lh‘ phosphoric acid were added, the volume was made up to 100 ml., and the sodium lauryl sulfate content was determined by the o-tolidine method. I3uffered sodium lauryl sulfate solutions treated in the same manner served as controls. The results summarized in Table I11 indicate that the asbestos itself was free from interfering materials and that the bacterial cells themselves, or substances therefrom, did not interfere with the recovery procedure. Alkaline

digestion f o r as long as 30 minutes e v e high recoveries, thus validating the basic hypothesis, establishing the feasibility of the proposd mc,thod, and confirming the reported stability of sodium lauryl sulfatv in alkaline medium ( I ) . DISCUSSION AND SUMMARY

At p H 3.5 in 3N sodium phosphatephosphoric acid buffer, sodium hypochlorite and o-tolidine dihydrochloride react to form a haloquinoid compound. When the solution containing this haloquinone is a t pH 3.0, added sodium lauryl sulfate will couple with it to form a complex giving a purple color with a maximum light absorption a t 590 mp. The intensity of this color is directly proportional to the amount of sodium lauryl sulfate added. High sensitivity and accuracy of determination can be obtained with observance of the established optimal conditions for the test. Of all the variables investigated, the final p H of the buffer system seems to be the most critical one for the development of the purple color. As interfering substances were absent from the particular system for which this test was developed, no specific investigation of other interfering substances was made. Howevc.r, attention is called to the obvious interference which would be expected from alkylbenzenesulphonates and other ester sulfates, if present. The presence of oxidizing agents, such as ferric and manganic ions and nitrites, which commonly interfere with the o-tolidine test for available chlorine ( 5 ) ,

Qua nti ta tive Dete rmination of

does not interfere unless the combined effect exceeds that of 7.5 p.p.m. of available chlorine. When in doubt, similar systems free of the surfactant can be analyzed and employed as blanks. The presence of materials which complex with sodium lauryl sulfate, such as protein, will interfere. However, the proposed recovery method, though investigated only for bacterial cells, can be extended to any other system, provided adequate digestion without destruction and subsequent extraction of the sodium lauryl sulfate can be achieved. LITERATURE CITED

(1) I h Pont de Nemours, E. I., & Co., Or anic Chemicals Department, Dyes an2 Chemicals Division, Wilmington 98, Del. Dyes and Chemicals Bull. (2) Harris, J. C., IND. ENG. CHEM.. ANAL.ED.15,254 (1943). (3) Harris, J. C., Short, F. R . , F d Technol. 6, 275 (1952). (4) Kolthoff, I. M., Sandell, E. E;., “Textbook of Quantitative Inorganic Analysis,” 3rd ed., pp. 621, 623, Macmillan, New York, 1946. ( 5 ) Milk Indust;? Foundation, Washington 5, D. C., Method7,of Analysis or Milk and Its Products, pp. 421, 425, 1949. _. ~ -

.

(6) Putnam, F. W.: Advances in Proteir Chem. 4,79 (1948). (7) Scales, F. M., Kemp, Mnriel, Assoc. Bull. (Intern. h o c . Milk Dealers) 31, 187(1939). RECEIVEDfor review October 2, 1958. Accepted June 1, 1959. Paper prepared from data presented in a thesis by Horace D. Graham in partial fulfillment of requirements for the degree of doctor o[ philosophy at the University of Illinois. October 1958.

AI pha-pinene

ROBERT L. KENNEY, TOMMY C. SINGLETON,l and GORDON S. FISHER Naval Stores Research Station, Southern Utilization Research and Developmenf Division, Agricultural Research Service, U. S. Department o f Agriculture, Olustee, No.

.A new method for the quantitative *determination of a-pinene in turpentine and other mixtures is closely analogous to the familiar isotope dilution methods. However, it depends on optical activity rather than radioactivity and hence has been named optical dilution. The ratio of d- to I-a-pinene in a turpentine can be calculated from the optical rotation of a small sample of pure a-pinene obtained from the mixture. Quantitative separation of all the a-pinene is not required. Similarly, the ratio of the concentration of a-pinene of any rotation originally present in a turpentine to the concentration of an added a-pinene ob different rotation can be calculated from the rotation of the pure a-pinene

1676

ANALYTICAL CHEMISTRY

distilled from the mixture of turpentine and added a-pinene. From this ratio and the concentration of added apinene the original concentration can b e calculated. The a-pinene content of known mixtures of a- and p pinene was determined with an accuracy of *0.5% (90% confidence limit). Similar accuracy can be expected with unknown mixtures if proper precautions are taken to asslure the purity of all the a-pinene fractions used.

pukJr,hrd analyses for a-pinene in turpntineb (2, 4 ) or other mixtures are based on anal? tien distillations. This technique is capah!,

of yielding accurate results but is very time-consuming. Further, pure apinene can be obtained in the main fractions but the later fractions are seldom pure and must be analyzed by other means, usually on the basis of physical properties (6). I n the Course of investigating thc oxidation (5) of a-pinene and the uniformity of gum turpentine, a more rapid and more arcuratc method than analytical distillation was required. For turpentines, this need was partially mei. by an infrared spectrophotometric. method, but absorption by the oxidation

EARLY ALL

’ Present address, Monsanto Chemical C O .Texas ~ City, Tex.

products interfered with its application +o oxidates (partially o x i d i d ssmples) of &pinene. This paper describes a novel. moderately rapid, accurate method which will be referred to as optical dilution and discusses its applicaZion to both turpentines and oxidates. Thismethod is, in many respects, analogous to the isotope dilution methods ivhich have been used with great succesa in recent years for analyses in which a pure derivative of the unknown can be x q x r e d but cannot be isolated quanti%tively (8.Aa enantiomorphs cannot he separated by distillation, the optical rotation and hence the isomeric composition of a portion of a-pinene distilled from a mixture will be the same as chat of the a-pinene remaining in the mixture. Separation from a mixture of enough pure a-pinene for determination of optical rotation can be carried out relatively rapidly and easily. If a known proportion of a-pinene having a different rotation is then added to a fresh sample of the unknown mixture and a second portion of a-pinene is distilled from this new mixture, the u-eight fraction of the original a-pinene in the distillate can be caicuiated from the various optical rotations. This ,\.eight fraction is ?quai to the ratio of original a-pinene to total a-pinene in the new mixture. The amount of apinene in the unknown mixture can also be calculated as follows:

w*=

optical rotation of a-pinene originally present in unknown mixture optical rotation of a-pinene added optical rotation of a-pinene &tilled from final mixture weight of sample of unknown mxture weight of a-pinene in W , weight of a-pinene added

=

wi

;Jet a , = xz =

=

21

w.= WI

j':

=

- weight fraction of orig-

w,f w,

inat a-pinene in final distillate then l)r

Pial -t- (1 - P,)a,

(1)

p1 = a ! ? IV1 Ql - a 4 WI wz

(2)

a3 =

~

+

'7, a-pinene in sample = -iv 1 x 100 = W"

EXPERIMENTAL

25latefidS.

d-n-PINENE

W89

Ob-

. % i n 4 by careful fraczionation ;i rexican gum turpentine or wood Gurnentine from western Louisiana. French (Alep) and Greek surpentines also x o v i d e good sources at' d-apinene and most commercial sarnpks x f a-pinene are dextrorotary. Purity

-

Table 1.

.KO.

Data from Optical Dilution Analyses of Turpentines and Known Mixtures

Samde grams

w.,

42.88 42.93 22.62 49.33 12.88 42.84 42.72 42.63 42.89 20.01

a1

a-Pinene nDistilled Pinene in Sampie, yo from Mixture, aa Found Prepared

n-finene Added QZ Wz,grams

- 4.89" +37.17" - 4.99" +36.71' - 5.05' +11.03" +.I 1 .M"

+16.14' 49.84 49.35 21.37 4-37.17" 49.50 49.62 21.12 -38.74" - 0 67" Cd.* 89.87 89.78 21.31 1 3 6 . 7 1 " t 1 6 . 3 5 ' DbJ 9.59 9 98 19.98 - 4'.99' 2.99" LOP. 114.59" 56.92 .. 21.20 +37.'20° +22.17" 67.65 .. TLCd 21.48 +37.20" TLCd 56.95 -10.33" _ ~ _ 21.16 -39.20' 61 37 ZTC' 7.25' 21.36 +37.20" +12.73" 57 18 21.35 137.20" 7.76' SPC' -17.87" MEX* 21.32 - 4.99" f13.41 " 82 I2 +37.28' a Prepared mixture containing21.16 grams oi 1-a-pinene and 21.72 grams of @-pinene. inch i n diameter and 48 inches Fractionated through a vacuum-jacketed column, long, packed with 0.16 X 0.16 inch protruded nickel packing. Prepared mixture containing 21.30 grams of d-a-pinene and 21.63 grams of B-pinene. Fractionated through Podbielniak column as described under Experimentai. Prepared mixture containing20.31 grams of I-a-pinene and 2.31 grams of @-pinene. Prepared mixture containing 5.00 grams of d-a-pinene and 45.11 grams of @-pinene. 8 Analytical distillation indicated 56% a-pinene. Average of four separate analytical distillations indicated 81.5% a-pinene. Aa.b

Rc .d

+

-

+

'

'

Table II.

Data from Optical Dilution Analyses of a-Pinene Oxidates

a-Pinene Distilled No. 1'

2. 3'

4b

*

e

7 . . 94 _7.93 8.09 9.12 8 90

a1

.a-Pinene Added Wp, grams a2

+29 , -_ 8.50

70 0 -

--.?I) .., 370 -.

+29.85" t29.85"

8.0 10.0 3.46

-39.37" -39.37" -39.37" -38 15"

t29.85"

+37.10" 0.101 mole of oxygen per mole. 0.202 mole of oxygen per mole. 0.208 mole of oxygen per mole.

55 a

Sample W,, grams

5.54

of all the a-pinene samples used was checked by direct infrared spectral comparison with a standard sample in the region of 2 to 15 microns. using a Perkin-Elmer 21 Ppectrophotometer and O,.j-mm. cells. Because a-pinene oxi&res readily, the samples were stored a t a low temperature in an inert atmosphere or protected by an antioxidant. Ga-PINEm was obtained in the Same way either from Indian turpentine or from isomerized &pinene (I). The lower rotating I-a-pinene waq obtained from commercial slash gum turpentine. !-&PINENEw a s obtained ny careful iractionation oi gum turpentine or commercial @pinene. Apparatus m d Method. Suitable vacuum stills and an accurate poiarimeter, readable t o *0.0lo, are required. The efficiency of the fractionating column needed to separate substantially pure a-pinene varies vith the mixture being analyzed a n d -ne concentration of zhe a-pinene in the sample. Simple Vigreux roiumns were adequate for the (>xidates cf a-piner?e but a coiumn with + reet oi protruded metal packing or a $-foot Podbielniak Heligrid column was a d for turpentines. The wnerai technique i s illustrsted 5y r;he analysis of .sampie TLC I Table I). A T5-mi. portion of turpentine -.T ,'( I LJb was refiuxed for 0.5 hour in tile odbielniak column a t a boil-up raw of 180 XI. per hour a t 20nim. przssxe. q t B hice-off af 5To,thrw fractions were

a-pinene

from Mixture,

Found,

70

28" -7.550 -13.87' - 1 1 .00" -4 57

86.01 55.83 7 2 . io 72.36 81.18

-11

then coilected. Fractions 2 and 3 were spectroscopically pure. Mi.

R.P., C.

About 4 hours nas required for the complete distillation. Kext, 21.16 grams (W,) of I-a-pinene (a? = -39.20') (ai) w m added tx) 42.72 grams ( W , ) of turpentine TLC, and the mixture was refluxed for 0.75 hour a t 20-mm. pressure and a 180 mi. y r hour 'uoii-up rate. Six fractions were cut at a 5% take-off.

:

MI.

B.P., " c.

.i4

51.3

az_l.5

=

-11.00"

Substitution of these -values in the equation above gives 67.0Yc a-pinene. Another analysis was made similarly on this same sample using u-a-Dinene (a%' = +37.20") as the diluent. This run indicated that TLC contained 57.7% a-pinene. Table h eives the data and results of nndyses o f k h e r turpentines and known mixtures of a- and &pinenes. '401. 31, NO. 10, OCTOBER 1959

* 1m

Table I1 gives the corresponding data for Lhe analysis of oxidized a-pinene. Samples 1 to 4 were prepared by thermal autoxidation in the dark a t 80" C. until the desired volume of oxygen had been absorbed. Sample 5 was prepared by photosensitized oxidation using chlorophyll and incandescent light at 20' C. DISCUSSION

hithough this method is not as rapid as infrared spectrophotometric or gasliquid phase chromatographic methods vould be, it requires less than one half rhe time required for an analytical distillation of comparable accuracy. Its major advantage is its direct applicability to complex natural products or reaction mixtures. Neither sample preparation nor standardization is required. Several factors affect the accuracy uf this method. The two weights, WZ and W8,can be determined readily with more than adequate accuracy; howrver, the accuracy of the optical rotations is dependent not only on that of the polarimeter used but also on the precision of the fractionating column and the distillation conditions. In addition, the magnitude of the optical rotation (a1)of the a-pinene present in the sample as well as that of the added a-pinene fa2) can limit the accuracy of the method. Whenever possible, nearly optically pure @pinene of rotation opposite to cy, should be used for dilution, and when ax ran be controlled, as in oxidation and other chemical reactions, it also should be high. It should be noted that any error in a3 is doubled in the per cent of a-pinene because it appears in the numerator and denominator with opposite sign. Ideally, the weight of added a-pinene W 2 )should equal the weight already present (VI),making a3 - a2 = a1 CYS and minimizing the percentage error due to errors in a],apt and as. However, when the concentration of a-pinene in the unknown is low, the use of larger amounts of added a-pinene may be necessary to obtain pure a-pinene with the available distillation equipment. Thus, the 4foot protruded nickel rolumn failed to separate pure a-pinene from mixture D (Table I) when Wz = W,, and a value of 12% a-pinene was obtained using the rotation of the piirest fraction. Use of W 2 = 4 Wt under the same conditions gave pure a-pinene and a value of 9.59'% a-pinene. This difficulty in separating pure apinene from dilute solutions obviously limits the accuracy of the method for !ow concentrations of a-pinene, as in

1678

0

ANALYTICAL CHEMISTRY

commercial B-pinene. However, Tables I and I1 show that the over-all accuracy and reproducibility obtainable on mixtures containing higher concentrations of a-pinene, such as are present in commercial turpentines, are excellent. It should be stressed that the a-pinene used for dilution must be pure and must be -protected from oxidation if it is stored after distillation. Low concentrations of commercial hydrocarbon antioxidants are satisfactory and storage a t low temperatures under an inert atmosphere is helpful. Obviously, impurities in the added a-pinene will make Wz too large and give a proportionate error in the concentration of apinene found (Equation 4). Also, the value of a2 will be in error if impurities are present. The effect of these errors on calculated a-pinene content will depend on both the rotation of the apinene and that of the impurities. Hence, the purity of the a-pinene for dilution should be checked spectrophotometrically before use. Because the a-pinene present in commercial turpentines may be either dextro or levo (Table I), both d- and Z-apinene of high optical purity should be available for dilution. Xaturzlly occurring @-pinene is nearly optically pure and can be isomerized to a-pinene of high optical purity by refluxing with four times its weight of gum rosin for 16 to 20 hours ( 1 ) . As noted under Experimental, d-a-pinenr of high optical purity is present in some samples of wood tyrprntine from the Louisiana-Mississippi area and in Mexican gum turpentine which is commercially available in the west and southwest. The variation in optical rotation of the a-pinene in commercial turpentine is primarily due to the different species of pine. Thus, sample TLC is from predominantly longleaf (Pinus palustmi) gum while SPC is from predominantly slash (Pinus ellioti) gum. The other samples are from mixed stands. Statistical analysis of the data for known mixtures (Table I) indicates an accuracy of *O.5y0 a t the 90% confidence limit or =!=0.7% a t the 95% confidence iimit. Analytical distillations of similar mixtures in this laboratory (6) showed similar accuracy but took about four times as long as the optical dilution analysis. When applied to the analysis of CYpinene oxidates, a packed column is no longer necessary t o separate pure a-pinene from the mixture, as the oxidation products will have a higher boiling point than a-pinene. Thus, a &inch Vigreux column was adequate for these

distillations in most cases. The close agreement between samples 1 and 2 and between 3 and 4 indicates that the method gives excellent results on these oxidates, so no attempt was made to check the method on synthetic oxidation mixtures. The difference in a-pinene content between sample 5 and samples 3 and 4 is due to the oxidation technique. Sample 5 was oxidized a t room temperature with a photosensitizer and bright light under conditions which gave nearly quantitative yields of hydroperoxide ( 7 ) . Therefore, equimolar consumption of oxygen and apinene was to be expected. The concentration of a-pinene found in this sample corresponds to 81.2% of the original a-pinene or 2% more than predicted from the oxygen absorbed. Samples 3 and 4 were oxidized a t 80' C. in the dark. This thermally promoted oxidation yields a variety of products including a-pinene epoxide (5, 7 ) ; hencr, the stoichiometry of the reaction is not so clearly dcfinrd. In thesr runs only 75.7% of the original a-pinenr \vas found in the oxidatr. Thereforo, niorc than one mole of (Ypinenr is consumed per mole of oxygen absorbrd. Although the method has been applied only to the determination of cy-pinenr in turprntiiies and oxidates, it is anticipatrd that it \vi11 be equally usrful in studying other reactions of a-pinenr. In addition, the general procedure should be usrful in the quantitative determination of other terpenes such R S limoncnc. LITERATURE CITED

( 1 ) Austerweil, G., Bull. soc. chim. France 39, 16-13 (1926). ( 2 ) Goldblatt., L. A., Burgdahl, A. C., I d .Eng. Chem. 44,1634 (1952). (3) Meinke, W. ANAL.C H E M . 28, 740 (1956).

w..

(4)-%0v, N. T., J . Forest Products Research Soc. 4, 1 (1954). (5) Moore, R. N., Golumbic, C., Fisher, G. S., J . A m . C h a . SOC. 78, 1173

(19% j. (6) Oldroyd, D. M., Goldblatt, L. A., IND. ENG. CHEM.,ANAL. ED. 18, 761 (1947). (7) Schenck, G. O., Eggert, H., Denk, W., Ann. 584, 177 (1953).

RECEIVED for review January 9, 1959. Accepted Jane 5, 1959. Southeastern Regional Meeting, ACS, Gainesville, Fla., December 1958. Mention of names of firms or trade products does not im ly that they are endorsed or recommenied by the U. S. Department of A culture over other firms or similar p r s c t a not mentioned.