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Studies of cuticular lipides of arthropods. II. The chemical composition of the wax from Ceroplastes destructor
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 67, 307-319 (1957)
Studies of Cuticular Lipides of Arthropods. IL The Chemical Composition of the wax from Ceroplastes destructor A. R. Gilby’ From
the School of Applied Chemistry, Wales University of Technology,
New
South
Australia ReceivedJune 16, 1956 INTRODUCTION
As mentioned in Part I of this series (14), the chemical composition of the wax of Ceroplastesdestructor (Newst.) has been previously examined by Hackman (1) who suggested that the wax consisted principally of a mixture of esters formed from Cz6 and CB normal para& chain acids and alcohols, together with the acids and alcohols in an uncombined state. Such a composition is typical of insect cuticular waxes and, although the wax is not laid down in a very typical form, it nevertheless appeared a likely material for developing physicochemical techniques
for the study of insect waxes in general. EXPERIMENTAL
The techniquesusedwere as describedin Part I of this series.The wax wasa sampleprovided by Dr. Hackman, Division of Entomology, C.S.I.R.O. It had beenextracted from insectscollectedfrom the branchesof Bursaria spinosa Ca:av. in November, 1951, i.e., at the end of the life span of the insects. The wax was not subjected to any purification procedures. Samplesof the nonsaponifiableand saponifiable fractions also provided.
prepared
by alkaline
RESULTS
hydrolysis
of the crude wax (1) were
AND DISCUSSION
A. Crude Wax 1. Infrared Absorption Spectrum. The general features of the spectrum (Table I) are very similar to those obtained under similar conditions for known long-chain fatty acids, esters, and alcohols by Shreve, 1 Present address: Division
of Entomology 307
C.S.I.R.O.,
Canberra,
A.C.T.
308
A.
R.
GILBY
TABLE I Infrared Absorption Spectrum of Sample W.W.S. I1 Spectrum No. I.R.I. 2-15 p Capillary film Band Cm.-
Intensity0
Assignment
O-H stretching; primary alcohol C-H stretching C-H stretching O-H stretching; carboxyl group 2670 C-0 stretching; eater 1731 1705 C=O stretching; carboxyl group cis C=C stretching (conjugated?) 1645 1469 CHt in-plane bending 1378 CHa in-plane bending 1249 C-O stretching; carboxyl group C-O stretching; ester 1171 1022 C-O stretching; alcohol C-C stretching 981 C-C stretching 910 C-C stretching 888 730 C& rocking a S = strong, M = medium, W = weak, s = sharp, b = broad. Carbon dioxide and water vapor bands present also.
3428
2920 2859
Ms SS MS Wb s 8 ws ws SS Ms Wb Wb Wb Wb Wb Wb MS
Heether, Knight, and Swern (2) and by Sinclair, McKay, and Jones (3,4), whose data, together with those of Stein and Sutherland (5), have been used in interpreting the results. The absence of bands between 740 and 800 cm.+ associated with branched-chain structures (6) discounts the occurrence of branching. The strength of the 1460 cm.-1 band arising from methylene in-plane bending vibrations relative to that at 1378 cm.-1 due to similar vibrations in methyl groups suggests the occurrence of relatively long chains. The wavelength of the relatively weak band at 1645 cm.-’ is rather longer than the C-C stretching frequency of a &-unsaturated double bond, although it agrees well with that of a conjugated double bond (4). Reference to the C-H stretching frequencies around 3000 cm.-‘, using a lithium fluoride prism for optimum resolution, showed no G-H bands corresponding to olefinic bonds. The infrared absorption spectrum, therefore, suggests that the wax consists of n-paraffin chain acids, alcohols, and esters with the possibility of the presence of some unsaturated compounds. 2. Monolayer Properties. Figure 1 shows that the wax gives liquid ex-
ARTHROPOD
LIPIDES.
II
0)
02
0.4
06
WWSlI
pH= 2
I.0
A (Vfn9) FIG. 1. Surface pressure (II) and surface potential (AV) of monolayer8 of W.W.S. II at various pH and of hydrogenated W.W.S. II on 0.01 N HCl. (Top curves Av - A; lower curves II - A).
panded films over the pH range studied. Between 0.3 and 0.4 sq. m./mg. a transition phase appears and the monolayer passesover into a solid condensed film. The differences in the force-area curves are not great, but the significant lowering of surface potential on the alkaline solution confirms the presence of free acid groups in the wax (7). On 1 iV NaOH solution a very rapid increase in area at constant surface pressure is observed, consistent with the hydrolysis of long-chain ester compounds (8). If the wax consisted of saturated long-chain compounds with molecular weights corresponding to the results of Hackman (l), the limiting areas of the expanded state would correspond to 80-90 sq. A./molecule, which are surprisingly large. A clue to the high degree of expansion in the film is revealed by the behavior of monolayers spread on acid potassium permanganate solutions and maintained at a surface pressure of 5 dyne/cm. (see Fig. 2). The initial increase in area with time when spread on 0.0001 N KMn04 and the decreasein 0.05 N KMn04 indicate
310
A.
___--
-
R.
GILBY
----
__-
-300
C-
AV mv -250
A
5m’/m I
\
-06
,,
0.4
03
0.51
AV
20
40
60
80
2
loo
FIG. 2. Oxidation of monolayers of W.W.S. II by KMnO, - time; lower curves A - time).
(I)wwSlt
befofe oxidation. after caklalion after
oxida~lon
solutions.
OCQOI 005N
(Top curves
N KMnO+. KMnO,.
FIG. 3. Surface pressure (II) and surface potential (AV) of monolayers W.W.S. II as B function of area (A). (Top curves AV - A; lower curves II A).
of -
ARTHROPOD
LIPIDES.
311
II
the presence of long-chain unsaturated compounds (9). The properties of the monolayer after oxidation are illustrated in Fig. 3, the considerable residue showing that the wax is not entirely unsaturated compounds. The curves in Fig. 2 may be analyzed to determine first-order rate constants for the reactions (9), and these are given in Table II, together with those for a number of known unsaturated compounds for comparison. Following the convention applied previously, that part of the reaction in which there is an increase or decrease of area with t’ime is labeled the “increasing reaction”or “decreasing reaction,” respectively. The decreasing reaction on 0.0001 N KMn04 is very slow. Its interference with the increasing reaction will be correspondingly slight, and the rate constant calculated will not be affected to the same degree as with the pure compounds (9). It is not possible to deduce the detailed nature of the unsaturated compounds in the wax from these figures alone, since the saturated molecules in the film would probably decrease the rate of reaction by decreasing the accessibility of double bonds to the surface. The presence of conjugated systems would not be revealed in these experiments as their rates of reaction would be too great. 3. Hydrogenated Wax. With the white wax scale, where considerable quantities of wax were available, confirmation of considerable unsaturation could be obtained in other ways. Thus Wij’s method gave an Iodine Number of 100, which is surprisingly high, although a value of 130.5 has been reported for Ceroplastes rubens Mash. wax (10). HydrogenaCon
First-order
KM1304
TABLE II Monolayer Ozidations on KMnO, rate constants [k.,i(min.-l)] for increasing and decreasing Temp. 2526°C. 0.01 N HzSOc II = 5 dyne/cm. concentration
Increasing reac :tion 0.0001 N
0 Pure compounds Oleic acid Oleyl alcohol Me linoleate Me linolenate Erucic acid B. Ceroplastes destructor wax Crude wax Nonsaponifiable fraction 0 From Gilby and Alexander (9). ) First 5 min. of reaction excluded. c v.r. = too rapid to be measured.
Decreasing N
0.0001
reactions
reaction 0 0.5 N
A.
0.039 0.039 0.58 v.r.c 0.025
0.007 0.008 0.050 0.13 0.007
0.029 0.037
0.06 0.16 . .c o:o; 0.033 0.079”
312
A.
R.
QILBY
experiments using palladium on charcoal as catalyst and ethyl acetate as solvent were difficult since the catalyst could not be properly equilibrated with hydrogen, although quantitative measurements showed that the wax took up approximately 0.004 g. moles hydrogen/g. wax. The monolayer properties of the hydrogenated wax are also shown in Fig. 1. The films are still highly expanded, suggesting that considerable short-chain material (co. CU) is present. In all the curves shown in Fig. (necessarily calcu1, there is a continuous fall in the surface moment lated in arbitrary units) over the liquid-expanded region. Since in pure compounds the surface moment is constant over this state, this fall is interpreted as a squeezing out of unsaturated and short-chain material from the film as the surface pressure increases. The hydrogenated wax fihn is stable to higher surface pressures, no doubt due to the greater ease with which unsaturated molecules are squeezed out from mixed monolayers [e.g., Harkins and Florence (ll)]. The oxidized monolayers also are stable to higher surface pressures, consistent with the elimination of unsaturation by the oxidation processes. All results thus point to the presence of a considerable degree of unsaturation in the wax. Assuming the wax to have the structure proposed by Hackman (1) and the unsaturation to be uniformly distributed, it is possible to calculate the number of double bonds per “average molecule.” This calculation may be based on either the hydrogen uptake figure or the iodine value. Both calculations estimate approximately 2.5 double bonds per “average molecule” or 2 double bonds per long (C&v) chain. However, it must be emphasized that a small proportion of unsaturated shortchain material would completely dominate the properties and invalidate any such argument. This has been found to be the case. Owing to the impossibility of precise interpretations on the basis of crude mixtures, an attempt was made to separate the wax into fractions by a graded solvent extraction using successively light petrol (b.p. 6065”C.), benzene, and chloroform. Fractions obtained were investigated by the monolayer and infrared techniques. However, these showed that no effective separation was achieved. B. Hydrolyzed
Wax
A sample of the crude wax was hydrolyzed by Dr. Hackman by means of alcoholic KOH, and the saponifiable and nonsaponifiable fractions, both before and after hydrogenation, were investigated. Insticient material was available for the determination of iodine values or for quantitative hydrogenation.
ARTHROPOD
LIPIDES.
313
II
1. Absorption Spectra. (a) Infrared. No unexpected information was obtained from the infrared absorption spectra of the saponifiable and nonsaponifiable fractions. These were shown to be parafhn-chain acids and alcohols, respectively. The nonsaponifiable fraction was contaminated with a small amount of acid. The weak absorption band at approximately 1645 cm.-I, thought to indicate unsaturation in the crude wax, was present also in the saponifiable fraction and was extremely weak in the nonsaponifiable fraction. Since the background absorption due to water vapor in the atmosphere occurs in this region, the manually operated Perkin-Elmer densitometer attachment, which compensates for the background, was used in studying this weak band. The spectra of the hydrogenated waxes, including the crude, saponifiable, and nonsaponifiable fractions, were similar to those of the original waxes. However, in each case, the 1645 cm.-’ band had disappeared. This absorption is, therefore, due to some form of unsaturated linkage. It has been suggested already that conjugated unsaturated material, present in very small amount, may account for the band. (b) Ultraviolet. Ultraviolet absorption spectra were determined on a Cary recording spectrophotometer. An absorption maximum was observed at 227.5 rnp in each case, as indicated in Table III. The wavelength of the absorption maximum is rather too long for (Y, /3unsaturated acids (204-220 mp). An CY, p-unsaturated ketone cannot be excluded, although this would be expected to be concentrated in the nonsaponifiable fraction. Most likely the absorption arises from conjugated diene systems (12), present in small amount since the values of E :%. are very small. A quantitative estimate is not possible. The main result of this work, therefore, is to show that there is a small TABLE Selective
Absorption
of Wax
WaX
III Samples
-bar.
W.W.S. II
2!T5
W.W.S. II nonsaponifiablefraction
227.5
W.W.S. II saponifiablefraction
227.5
in the Ultraviolet
EL%” Iam.
solvent
5yo chloroform in ethyl alcohol 50.5 5yo chloroform in ethyl alcohol 91.5 5yo chloroform in ethyl alcohol 47
a E: ?$,+ is the optical density of a 1 cm. layer of 1% solution. (Experimental concentrationsof waxesused = 0.Q4~ow/v.)
314
A.
R.
GILBY
FIQ. 4. Surface pressure (II) and surface potential (AV) of monolayers of various derivatives from W.W.S. II, as a function of area (A). (Top curves AV N A; lower curves II - A).
amount of unspecified conjugated material in the wax which could give rise to the weak 1645 cm.-’ band found in the infrared spectra. 2. Monolayer Properties. (a) NonsaponiJiabk Fraction. The monolayer characteristics of this fraction, both before and after (i) hydrogenation and (ii) oxidation on permanganate, are given in Fig. 4. The fact that after hydrogenation the fraction still gives an expanded monolayer indicates that considerable short-chain material is present. However, after the unsaturated compounds are removed from the film by oxidation, the monolayer becomessolid and condensed. At this stage the film must consist of long-chain molecules entirely, and the unsaturated compounds must therefore be present largely in the short-chain material. (1) Estimation of Unsaturated Molecules. It is possible to estimate the approximate chain length and proportion of the unsaturated material in the following manner. Over the liquid-expanded region of the hydrogenated sample, the product of A and AV, which is proportional to the surface moment (P) is approximately constant with parb. = 150. (AAV = 4?r~().Since the hydrogenated fraction consists of saturated alcohols, we would expect the usual value of N = 210 mD. approx. (13). At approximately the point where the film first becomes coherent (i.e.,
ARTHROPOD
LIPIDES.
315
II
A = 0.8 sq. m./mg., although any other point over the liquid-expanded region could equally be chosen), the observed value of the surface potential is AV = 180 mv. From the relation AAV = 4 rp, t’he area at this point is thus calculated to be A = 43.8 sq. A./molecule. Now, if we convert the area in sq. m./mg. to sq. A./molecule in terms of the molecular weight, then 0.8 sq. m./mg.
=
0.8 X M.W. 6.02 ’
sq. A./molecule
= A.
Substituting the value of 43.8 sq. A./molecule calculated above for A, the mean molecular weight of the fraction = 330. This may be compared with a molecular weight of 394 for alcohols of chain-length average Cn as found by Hackman (1). The further assumption must now be made that, after oxidation, only alcohols of average chain length Ct, remain and that the limiting area of the condensed film is Ao = 22 sq. A./molecule. On this basis, the observed value of AV at this point gives p = 200 mD.; hence the assumption appears to be reasonable. Therefore, using a molecular weight of 394, A0 = 22 sq. A./molecule when converted to sq. m./mg. becomes A0 = 0.28 sq. m./mg. where the weight used in the calculation includes unsaturated compounds, at this stage no longer in the monolayer. To render the two values equivalent, 0.34 sq. m./mg. = 0.28/0.835, i.e., from each milligram of nonsaponifiable fraction, 0.165 mg. of unsaturated material is lost on oxidation leaving 0.835 mg. of Cz, alcohols. Let the mean molecular weight of the unsaturated compounds = M. Then in 100 g. of the nonsaponifiable fraction there are 83.5/394 g. molecules of C2, alcohols and 16.5/M g. molecules of unsaturated alcohols. Since the mean molecular weight of the fraction will not be very different whether or not the unsaturated compounds have been hydrogenated, the average molecular weight is equal to 100 8g
= 330
+ Eg
whence M = 18‘2 corresponding to Cl2 approximately. molecular ratio of saturated alcohols to unsaturated is 83.5 16.5 = 2 : 1 approximately. 394%2
Further,
the
316
A.
R.
GILBY
(2) Nature of &saturation. A marked change in the characteristics of the oxidation reaction on dilute permanganate is shown in Fig. 2. Whereas before hydrolysis the wax showed an increase in area with time on 0.0001 N KMnOa , after hydrolysis a decreasing reaction only was obtained with the nonsaponifiable fraction. The rate constants obtained by the usual methods are given in Table II. The initial reaction on 0.05 N KMn04 is very rapid, and the reaction whose rate has been determined is not established until 5-10 min. after the beginning. The behavior is very similar to that shown by methyl linolenate (9) in that no increasing reaction is observed at either concentration of permanganate. The rate constant of the decreasing reaction on 0.0001 N KMnOl is less than that determined for the linolenate (0.13 min.-l), although this could arise, at least in part, from the shielding effect of the saturated molecules in the film. On the other hand, if there were two double bonds in each molecule, the ratio of unsaturation to chain length (two double bonds in Clz) would be similar to that in methyl linolenate (three double bonds in Cl*). Hence the rates of solution of the hydroxy compounds are likely to be similar, and this could give rise to the similar behavior, especially on 0.05 N KMn04 . Since the oxidation reaction follows a different pattern from that of the crude wax under similar conditions, either the unsaturated alcohols were combined in the form of esters in the crude wax or some change has occurred during the hydrolysis. Such a change might be isomerization to conjugated and faster reacting molecules under the alkaline conditions. However, the ultraviolet absorption spectra do not support this latter explanation. Thus it is concluded that the nonsaponifiable fraction consists of long-chain (C& average) alcohols and short-chain (cu. Cl,) unsaturated alcohols in the ratio of approximately 2: 1. The unsaturated alcohols contain at least two, and possibly three, unconjugated double bonds per molecule. In the crude wax, the unsaturated alcohols are combined in the form of esters. (b) SaponiJiabb Fraction. Once again, a liquid-expanded type of monolayer is obtained (Fig. 5) which passes over into a solid condensed film on compression. On hydrogenation there isvery little change in the II-A relation although there is a reduction in AV of about 25 mv. When spread on KMn04 solution, there appears to be almost no reaction, the
ARTHROPOD
LIPIDES.
317
II
250 AV
30-
mv 200
10 SaponMablc 20-
~~Hybogenoted
I50
traction. sapcMioMe
traction.
-Iv dyn cm:’ IO-
FIQ. 5. Surface pressure (II) and surface potential (AV) of monolayer8 ous derivatives from W.W.S. II, as a function of area (A). (Top curves lower curves II - A).
AV
of vari- A;
slight reduction in area being probably due to the trace of conjugated material shown to be present by the ultraviolet spectra. An estimate of the average chain length may be made by similar methods to those used for the alcohols. Thus, taking p = 180 mD. for saturated acids, in the expanded state, at the cohesion point (A = 0.8 sq. m./mg.), AV = 210 mv. whence A = 32.3 sq. A./molecule. Now, as before, 0.8 sq. m./mg. = (0.8 X M.W.)/6.02 sq. A./molecule = A. Hence the mean molecular weight = 250. This may be compared with a mean molecular weight of 410 corresponding to C&r (average chain length) acids as found by Hackman (1). If it is assumedthat the short-chain acids are of approximately the same chain length as the short-chain alcohols, i.e., Clz (and they must be about this to account for their expanding effect on the monolayer), the molecular ratio of C,Z acids to Cz7would be 2%: 1 approximately. Confirmation of these results was obtained by determining the Acid Value of the saponifiable fraction. The calculated value on the above basis would be 216. The experimental result was 201. The calculations
318
A.
R.
GILBY
from the monolayer results, therefore, predict an answer which is of the right order. Since wax tended to precipitate from solution as titration proceeded, the value determined would tend to be low. It is concluded, therefore, that the saponifiable fraction has a large proportion of short-chain saturated acids of chain length most probably approximately Cl2 , in addition to long-chain saturated acids of average chain length CZ7 . CONCLUSION
The wax from Ceroplastes destructor consists of n-paraffin-chain acids and alcohols largely combined as esters. In addition to long-chain acids and alcohols, of average chain length about Cz, , a significant proportion of short-chain (cu. Cl,) acids and alcohols is present, the alcohols being unsaturated with at least two unconjugated double bonds per molecule. The molecular ratio of C27 to CIZ molecules is approximately 1:2.5 for the hydrolyzed acids and approximately 2: 1 for the hydrolyzed alcohols. There also exists a minor fraction of an unknown conjugated unsaturated compound (probably a diene). The present results differ from those of Hackman (1) in that they indicate the presence of shorter chain and unsaturated materials, and this is thought to be due to the preliminary crystallization from chloroformethanol to which Hackman’s samples were subjected. Knowledge of the presence of an appreciable fraction of unsaturated and short-chain compounds is of considerable importance in considering biological aspects of lipides, and the methods used here possess advantages in providing information from crude mixtures, thereby avoiding possible loss of important fractions. REFERENCES 1. HACEMAN, R. H., Arch. Biochem. and Biophys. 33, 150 (1951). 2. SHREVE, 0. P., HEETHER, M. R., KNIQHT, H. B., AND SWERN, D., Anal. Chem. 22, 1498 (1959). 3. SINCLAIR, R. G., MCKAY, A. F., AND JONES, R. N., J. Am. Chem. Sot. 74,257O (1952). 4. SINCLAIR, R. G., MCKAY, A. F., MYERS, G. S., AND JONES, R. N., J. Am. Chem. Sot. 74, 2578 (1952). 5. SUTHERLAND, G. B. B. M., Discussions Faraday Sot. No. 9, 274 (1950). 6. FREEMAN, N. K., J. Am. Chem. Sot. 74, 2523 (1952). 7. SCHULMAN, J. H., AND HUGHES, A. H., Proc. Roy. Sot. (London) A136, 436 (1932).
ARTHROPOD
LIPIDES.
II
319
8. ALEXANDER, A. E., AND SCHULMAN, J. H., Proc. Roy. Sot. (London) A161,115 (1937). 9. GILBY, A. R., AND ALEXANDER, A. E., Australian J. Chem. 9, 347 (1956). 10. WARTH, A. H., “The Chemistry and Technology of Waxes.” Reinhold Publishing Corp., New York, 1947. 11. HARKINS, W. D., AND FLORENCE, R. J., J. Chem. Phys. 6, 847 (1938). 12. GILLAM, A., AND STERN, E. S., “An Introduction to Electronic Absorption Spectroscopy in Organic Chemistry.” Edward Arnold, Ltd., London, 1954. 13. HARKINS, W. D., AND FISCHER, E. K., J. Chem. Phys. 1,852 (1933). 14. GILBY, A. R., AND ALEXANDER, A. E., Arch. Biochem. and Biophys. 67, 302 (1957).
Studies of Cuticular Lipides of Arthropods. IL The Chemical Composition of the wax from Ceroplastes destructor A. R. Gilby’ From
the School of Applied Chemistry, Wales University of Technology,
New
South
Australia ReceivedJune 16, 1956 INTRODUCTION
As mentioned in Part I of this series (14), the chemical composition of the wax of Ceroplastesdestructor (Newst.) has been previously examined by Hackman (1) who suggested that the wax consisted principally of a mixture of esters formed from Cz6 and CB normal para& chain acids and alcohols, together with the acids and alcohols in an uncombined state. Such a composition is typical of insect cuticular waxes and, although the wax is not laid down in a very typical form, it nevertheless appeared a likely material for developing physicochemical techniques
for the study of insect waxes in general. EXPERIMENTAL
The techniquesusedwere as describedin Part I of this series.The wax wasa sampleprovided by Dr. Hackman, Division of Entomology, C.S.I.R.O. It had beenextracted from insectscollectedfrom the branchesof Bursaria spinosa Ca:av. in November, 1951, i.e., at the end of the life span of the insects. The wax was not subjected to any purification procedures. Samplesof the nonsaponifiableand saponifiable fractions also provided.
prepared
by alkaline
RESULTS
hydrolysis
of the crude wax (1) were
AND DISCUSSION
A. Crude Wax 1. Infrared Absorption Spectrum. The general features of the spectrum (Table I) are very similar to those obtained under similar conditions for known long-chain fatty acids, esters, and alcohols by Shreve, 1 Present address: Division
of Entomology 307
C.S.I.R.O.,
Canberra,
A.C.T.
308
A.
R.
GILBY
TABLE I Infrared Absorption Spectrum of Sample W.W.S. I1 Spectrum No. I.R.I. 2-15 p Capillary film Band Cm.-
Intensity0
Assignment
O-H stretching; primary alcohol C-H stretching C-H stretching O-H stretching; carboxyl group 2670 C-0 stretching; eater 1731 1705 C=O stretching; carboxyl group cis C=C stretching (conjugated?) 1645 1469 CHt in-plane bending 1378 CHa in-plane bending 1249 C-O stretching; carboxyl group C-O stretching; ester 1171 1022 C-O stretching; alcohol C-C stretching 981 C-C stretching 910 C-C stretching 888 730 C& rocking a S = strong, M = medium, W = weak, s = sharp, b = broad. Carbon dioxide and water vapor bands present also.
3428
2920 2859
Ms SS MS Wb s 8 ws ws SS Ms Wb Wb Wb Wb Wb Wb MS
Heether, Knight, and Swern (2) and by Sinclair, McKay, and Jones (3,4), whose data, together with those of Stein and Sutherland (5), have been used in interpreting the results. The absence of bands between 740 and 800 cm.+ associated with branched-chain structures (6) discounts the occurrence of branching. The strength of the 1460 cm.-1 band arising from methylene in-plane bending vibrations relative to that at 1378 cm.-1 due to similar vibrations in methyl groups suggests the occurrence of relatively long chains. The wavelength of the relatively weak band at 1645 cm.-’ is rather longer than the C-C stretching frequency of a &-unsaturated double bond, although it agrees well with that of a conjugated double bond (4). Reference to the C-H stretching frequencies around 3000 cm.-‘, using a lithium fluoride prism for optimum resolution, showed no G-H bands corresponding to olefinic bonds. The infrared absorption spectrum, therefore, suggests that the wax consists of n-paraffin chain acids, alcohols, and esters with the possibility of the presence of some unsaturated compounds. 2. Monolayer Properties. Figure 1 shows that the wax gives liquid ex-
ARTHROPOD
LIPIDES.
II
0)
02
0.4
06
WWSlI
pH= 2
I.0
A (Vfn9) FIG. 1. Surface pressure (II) and surface potential (AV) of monolayer8 of W.W.S. II at various pH and of hydrogenated W.W.S. II on 0.01 N HCl. (Top curves Av - A; lower curves II - A).
panded films over the pH range studied. Between 0.3 and 0.4 sq. m./mg. a transition phase appears and the monolayer passesover into a solid condensed film. The differences in the force-area curves are not great, but the significant lowering of surface potential on the alkaline solution confirms the presence of free acid groups in the wax (7). On 1 iV NaOH solution a very rapid increase in area at constant surface pressure is observed, consistent with the hydrolysis of long-chain ester compounds (8). If the wax consisted of saturated long-chain compounds with molecular weights corresponding to the results of Hackman (l), the limiting areas of the expanded state would correspond to 80-90 sq. A./molecule, which are surprisingly large. A clue to the high degree of expansion in the film is revealed by the behavior of monolayers spread on acid potassium permanganate solutions and maintained at a surface pressure of 5 dyne/cm. (see Fig. 2). The initial increase in area with time when spread on 0.0001 N KMn04 and the decreasein 0.05 N KMn04 indicate
310
A.
___--
-
R.
GILBY
----
__-
-300
C-
AV mv -250
A
5m’/m I
\
-06
,,
0.4
03
0.51
AV
20
40
60
80
2
loo
FIG. 2. Oxidation of monolayers of W.W.S. II by KMnO, - time; lower curves A - time).
(I)wwSlt
befofe oxidation. after caklalion after
oxida~lon
solutions.
OCQOI 005N
(Top curves
N KMnO+. KMnO,.
FIG. 3. Surface pressure (II) and surface potential (AV) of monolayers W.W.S. II as B function of area (A). (Top curves AV - A; lower curves II A).
of -
ARTHROPOD
LIPIDES.
311
II
the presence of long-chain unsaturated compounds (9). The properties of the monolayer after oxidation are illustrated in Fig. 3, the considerable residue showing that the wax is not entirely unsaturated compounds. The curves in Fig. 2 may be analyzed to determine first-order rate constants for the reactions (9), and these are given in Table II, together with those for a number of known unsaturated compounds for comparison. Following the convention applied previously, that part of the reaction in which there is an increase or decrease of area with t’ime is labeled the “increasing reaction”or “decreasing reaction,” respectively. The decreasing reaction on 0.0001 N KMn04 is very slow. Its interference with the increasing reaction will be correspondingly slight, and the rate constant calculated will not be affected to the same degree as with the pure compounds (9). It is not possible to deduce the detailed nature of the unsaturated compounds in the wax from these figures alone, since the saturated molecules in the film would probably decrease the rate of reaction by decreasing the accessibility of double bonds to the surface. The presence of conjugated systems would not be revealed in these experiments as their rates of reaction would be too great. 3. Hydrogenated Wax. With the white wax scale, where considerable quantities of wax were available, confirmation of considerable unsaturation could be obtained in other ways. Thus Wij’s method gave an Iodine Number of 100, which is surprisingly high, although a value of 130.5 has been reported for Ceroplastes rubens Mash. wax (10). HydrogenaCon
First-order
KM1304
TABLE II Monolayer Ozidations on KMnO, rate constants [k.,i(min.-l)] for increasing and decreasing Temp. 2526°C. 0.01 N HzSOc II = 5 dyne/cm. concentration
Increasing reac :tion 0.0001 N
0 Pure compounds Oleic acid Oleyl alcohol Me linoleate Me linolenate Erucic acid B. Ceroplastes destructor wax Crude wax Nonsaponifiable fraction 0 From Gilby and Alexander (9). ) First 5 min. of reaction excluded. c v.r. = too rapid to be measured.
Decreasing N
0.0001
reactions
reaction 0 0.5 N
A.
0.039 0.039 0.58 v.r.c 0.025
0.007 0.008 0.050 0.13 0.007
0.029 0.037
0.06 0.16 . .c o:o; 0.033 0.079”
312
A.
R.
QILBY
experiments using palladium on charcoal as catalyst and ethyl acetate as solvent were difficult since the catalyst could not be properly equilibrated with hydrogen, although quantitative measurements showed that the wax took up approximately 0.004 g. moles hydrogen/g. wax. The monolayer properties of the hydrogenated wax are also shown in Fig. 1. The films are still highly expanded, suggesting that considerable short-chain material (co. CU) is present. In all the curves shown in Fig. (necessarily calcu1, there is a continuous fall in the surface moment lated in arbitrary units) over the liquid-expanded region. Since in pure compounds the surface moment is constant over this state, this fall is interpreted as a squeezing out of unsaturated and short-chain material from the film as the surface pressure increases. The hydrogenated wax fihn is stable to higher surface pressures, no doubt due to the greater ease with which unsaturated molecules are squeezed out from mixed monolayers [e.g., Harkins and Florence (ll)]. The oxidized monolayers also are stable to higher surface pressures, consistent with the elimination of unsaturation by the oxidation processes. All results thus point to the presence of a considerable degree of unsaturation in the wax. Assuming the wax to have the structure proposed by Hackman (1) and the unsaturation to be uniformly distributed, it is possible to calculate the number of double bonds per “average molecule.” This calculation may be based on either the hydrogen uptake figure or the iodine value. Both calculations estimate approximately 2.5 double bonds per “average molecule” or 2 double bonds per long (C&v) chain. However, it must be emphasized that a small proportion of unsaturated shortchain material would completely dominate the properties and invalidate any such argument. This has been found to be the case. Owing to the impossibility of precise interpretations on the basis of crude mixtures, an attempt was made to separate the wax into fractions by a graded solvent extraction using successively light petrol (b.p. 6065”C.), benzene, and chloroform. Fractions obtained were investigated by the monolayer and infrared techniques. However, these showed that no effective separation was achieved. B. Hydrolyzed
Wax
A sample of the crude wax was hydrolyzed by Dr. Hackman by means of alcoholic KOH, and the saponifiable and nonsaponifiable fractions, both before and after hydrogenation, were investigated. Insticient material was available for the determination of iodine values or for quantitative hydrogenation.
ARTHROPOD
LIPIDES.
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II
1. Absorption Spectra. (a) Infrared. No unexpected information was obtained from the infrared absorption spectra of the saponifiable and nonsaponifiable fractions. These were shown to be parafhn-chain acids and alcohols, respectively. The nonsaponifiable fraction was contaminated with a small amount of acid. The weak absorption band at approximately 1645 cm.-I, thought to indicate unsaturation in the crude wax, was present also in the saponifiable fraction and was extremely weak in the nonsaponifiable fraction. Since the background absorption due to water vapor in the atmosphere occurs in this region, the manually operated Perkin-Elmer densitometer attachment, which compensates for the background, was used in studying this weak band. The spectra of the hydrogenated waxes, including the crude, saponifiable, and nonsaponifiable fractions, were similar to those of the original waxes. However, in each case, the 1645 cm.-’ band had disappeared. This absorption is, therefore, due to some form of unsaturated linkage. It has been suggested already that conjugated unsaturated material, present in very small amount, may account for the band. (b) Ultraviolet. Ultraviolet absorption spectra were determined on a Cary recording spectrophotometer. An absorption maximum was observed at 227.5 rnp in each case, as indicated in Table III. The wavelength of the absorption maximum is rather too long for (Y, /3unsaturated acids (204-220 mp). An CY, p-unsaturated ketone cannot be excluded, although this would be expected to be concentrated in the nonsaponifiable fraction. Most likely the absorption arises from conjugated diene systems (12), present in small amount since the values of E :%. are very small. A quantitative estimate is not possible. The main result of this work, therefore, is to show that there is a small TABLE Selective
Absorption
of Wax
WaX
III Samples
-bar.
W.W.S. II
2!T5
W.W.S. II nonsaponifiablefraction
227.5
W.W.S. II saponifiablefraction
227.5
in the Ultraviolet
EL%” Iam.
solvent
5yo chloroform in ethyl alcohol 50.5 5yo chloroform in ethyl alcohol 91.5 5yo chloroform in ethyl alcohol 47
a E: ?$,+ is the optical density of a 1 cm. layer of 1% solution. (Experimental concentrationsof waxesused = 0.Q4~ow/v.)
314
A.
R.
GILBY
FIQ. 4. Surface pressure (II) and surface potential (AV) of monolayers of various derivatives from W.W.S. II, as a function of area (A). (Top curves AV N A; lower curves II - A).
amount of unspecified conjugated material in the wax which could give rise to the weak 1645 cm.-’ band found in the infrared spectra. 2. Monolayer Properties. (a) NonsaponiJiabk Fraction. The monolayer characteristics of this fraction, both before and after (i) hydrogenation and (ii) oxidation on permanganate, are given in Fig. 4. The fact that after hydrogenation the fraction still gives an expanded monolayer indicates that considerable short-chain material is present. However, after the unsaturated compounds are removed from the film by oxidation, the monolayer becomessolid and condensed. At this stage the film must consist of long-chain molecules entirely, and the unsaturated compounds must therefore be present largely in the short-chain material. (1) Estimation of Unsaturated Molecules. It is possible to estimate the approximate chain length and proportion of the unsaturated material in the following manner. Over the liquid-expanded region of the hydrogenated sample, the product of A and AV, which is proportional to the surface moment (P) is approximately constant with parb. = 150. (AAV = 4?r~().Since the hydrogenated fraction consists of saturated alcohols, we would expect the usual value of N = 210 mD. approx. (13). At approximately the point where the film first becomes coherent (i.e.,
ARTHROPOD
LIPIDES.
315
II
A = 0.8 sq. m./mg., although any other point over the liquid-expanded region could equally be chosen), the observed value of the surface potential is AV = 180 mv. From the relation AAV = 4 rp, t’he area at this point is thus calculated to be A = 43.8 sq. A./molecule. Now, if we convert the area in sq. m./mg. to sq. A./molecule in terms of the molecular weight, then 0.8 sq. m./mg.
=
0.8 X M.W. 6.02 ’
sq. A./molecule
= A.
Substituting the value of 43.8 sq. A./molecule calculated above for A, the mean molecular weight of the fraction = 330. This may be compared with a molecular weight of 394 for alcohols of chain-length average Cn as found by Hackman (1). The further assumption must now be made that, after oxidation, only alcohols of average chain length Ct, remain and that the limiting area of the condensed film is Ao = 22 sq. A./molecule. On this basis, the observed value of AV at this point gives p = 200 mD.; hence the assumption appears to be reasonable. Therefore, using a molecular weight of 394, A0 = 22 sq. A./molecule when converted to sq. m./mg. becomes A0 = 0.28 sq. m./mg. where the weight used in the calculation includes unsaturated compounds, at this stage no longer in the monolayer. To render the two values equivalent, 0.34 sq. m./mg. = 0.28/0.835, i.e., from each milligram of nonsaponifiable fraction, 0.165 mg. of unsaturated material is lost on oxidation leaving 0.835 mg. of Cz, alcohols. Let the mean molecular weight of the unsaturated compounds = M. Then in 100 g. of the nonsaponifiable fraction there are 83.5/394 g. molecules of C2, alcohols and 16.5/M g. molecules of unsaturated alcohols. Since the mean molecular weight of the fraction will not be very different whether or not the unsaturated compounds have been hydrogenated, the average molecular weight is equal to 100 8g
= 330
+ Eg
whence M = 18‘2 corresponding to Cl2 approximately. molecular ratio of saturated alcohols to unsaturated is 83.5 16.5 = 2 : 1 approximately. 394%2
Further,
the
316
A.
R.
GILBY
(2) Nature of &saturation. A marked change in the characteristics of the oxidation reaction on dilute permanganate is shown in Fig. 2. Whereas before hydrolysis the wax showed an increase in area with time on 0.0001 N KMnOa , after hydrolysis a decreasing reaction only was obtained with the nonsaponifiable fraction. The rate constants obtained by the usual methods are given in Table II. The initial reaction on 0.05 N KMn04 is very rapid, and the reaction whose rate has been determined is not established until 5-10 min. after the beginning. The behavior is very similar to that shown by methyl linolenate (9) in that no increasing reaction is observed at either concentration of permanganate. The rate constant of the decreasing reaction on 0.0001 N KMnOl is less than that determined for the linolenate (0.13 min.-l), although this could arise, at least in part, from the shielding effect of the saturated molecules in the film. On the other hand, if there were two double bonds in each molecule, the ratio of unsaturation to chain length (two double bonds in Clz) would be similar to that in methyl linolenate (three double bonds in Cl*). Hence the rates of solution of the hydroxy compounds are likely to be similar, and this could give rise to the similar behavior, especially on 0.05 N KMn04 . Since the oxidation reaction follows a different pattern from that of the crude wax under similar conditions, either the unsaturated alcohols were combined in the form of esters in the crude wax or some change has occurred during the hydrolysis. Such a change might be isomerization to conjugated and faster reacting molecules under the alkaline conditions. However, the ultraviolet absorption spectra do not support this latter explanation. Thus it is concluded that the nonsaponifiable fraction consists of long-chain (C& average) alcohols and short-chain (cu. Cl,) unsaturated alcohols in the ratio of approximately 2: 1. The unsaturated alcohols contain at least two, and possibly three, unconjugated double bonds per molecule. In the crude wax, the unsaturated alcohols are combined in the form of esters. (b) SaponiJiabb Fraction. Once again, a liquid-expanded type of monolayer is obtained (Fig. 5) which passes over into a solid condensed film on compression. On hydrogenation there isvery little change in the II-A relation although there is a reduction in AV of about 25 mv. When spread on KMn04 solution, there appears to be almost no reaction, the
ARTHROPOD
LIPIDES.
317
II
250 AV
30-
mv 200
10 SaponMablc 20-
~~Hybogenoted
I50
traction. sapcMioMe
traction.
-Iv dyn cm:’ IO-
FIQ. 5. Surface pressure (II) and surface potential (AV) of monolayer8 ous derivatives from W.W.S. II, as a function of area (A). (Top curves lower curves II - A).
AV
of vari- A;
slight reduction in area being probably due to the trace of conjugated material shown to be present by the ultraviolet spectra. An estimate of the average chain length may be made by similar methods to those used for the alcohols. Thus, taking p = 180 mD. for saturated acids, in the expanded state, at the cohesion point (A = 0.8 sq. m./mg.), AV = 210 mv. whence A = 32.3 sq. A./molecule. Now, as before, 0.8 sq. m./mg. = (0.8 X M.W.)/6.02 sq. A./molecule = A. Hence the mean molecular weight = 250. This may be compared with a mean molecular weight of 410 corresponding to C&r (average chain length) acids as found by Hackman (1). If it is assumedthat the short-chain acids are of approximately the same chain length as the short-chain alcohols, i.e., Clz (and they must be about this to account for their expanding effect on the monolayer), the molecular ratio of C,Z acids to Cz7would be 2%: 1 approximately. Confirmation of these results was obtained by determining the Acid Value of the saponifiable fraction. The calculated value on the above basis would be 216. The experimental result was 201. The calculations
318
A.
R.
GILBY
from the monolayer results, therefore, predict an answer which is of the right order. Since wax tended to precipitate from solution as titration proceeded, the value determined would tend to be low. It is concluded, therefore, that the saponifiable fraction has a large proportion of short-chain saturated acids of chain length most probably approximately Cl2 , in addition to long-chain saturated acids of average chain length CZ7 . CONCLUSION
The wax from Ceroplastes destructor consists of n-paraffin-chain acids and alcohols largely combined as esters. In addition to long-chain acids and alcohols, of average chain length about Cz, , a significant proportion of short-chain (cu. Cl,) acids and alcohols is present, the alcohols being unsaturated with at least two unconjugated double bonds per molecule. The molecular ratio of C27 to CIZ molecules is approximately 1:2.5 for the hydrolyzed acids and approximately 2: 1 for the hydrolyzed alcohols. There also exists a minor fraction of an unknown conjugated unsaturated compound (probably a diene). The present results differ from those of Hackman (1) in that they indicate the presence of shorter chain and unsaturated materials, and this is thought to be due to the preliminary crystallization from chloroformethanol to which Hackman’s samples were subjected. Knowledge of the presence of an appreciable fraction of unsaturated and short-chain compounds is of considerable importance in considering biological aspects of lipides, and the methods used here possess advantages in providing information from crude mixtures, thereby avoiding possible loss of important fractions. REFERENCES 1. HACEMAN, R. H., Arch. Biochem. and Biophys. 33, 150 (1951). 2. SHREVE, 0. P., HEETHER, M. R., KNIQHT, H. B., AND SWERN, D., Anal. Chem. 22, 1498 (1959). 3. SINCLAIR, R. G., MCKAY, A. F., AND JONES, R. N., J. Am. Chem. Sot. 74,257O (1952). 4. SINCLAIR, R. G., MCKAY, A. F., MYERS, G. S., AND JONES, R. N., J. Am. Chem. Sot. 74, 2578 (1952). 5. SUTHERLAND, G. B. B. M., Discussions Faraday Sot. No. 9, 274 (1950). 6. FREEMAN, N. K., J. Am. Chem. Sot. 74, 2523 (1952). 7. SCHULMAN, J. H., AND HUGHES, A. H., Proc. Roy. Sot. (London) A136, 436 (1932).
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LIPIDES.
II
319
8. ALEXANDER, A. E., AND SCHULMAN, J. H., Proc. Roy. Sot. (London) A161,115 (1937). 9. GILBY, A. R., AND ALEXANDER, A. E., Australian J. Chem. 9, 347 (1956). 10. WARTH, A. H., “The Chemistry and Technology of Waxes.” Reinhold Publishing Corp., New York, 1947. 11. HARKINS, W. D., AND FLORENCE, R. J., J. Chem. Phys. 6, 847 (1938). 12. GILLAM, A., AND STERN, E. S., “An Introduction to Electronic Absorption Spectroscopy in Organic Chemistry.” Edward Arnold, Ltd., London, 1954. 13. HARKINS, W. D., AND FISCHER, E. K., J. Chem. Phys. 1,852 (1933). 14. GILBY, A. R., AND ALEXANDER, A. E., Arch. Biochem. and Biophys. 67, 302 (1957).