assay secondary filters (Corning 3389 and 4308) for the fluorometric determination of chortetracycline. This combination of filters has a transmittance maximum a t about 405 mp, and transmits doivn to 360 mp. They permit the transmittance of scattered incident light, and of any fluorescent light emitted by impurities down to 360 nip, Another combination of Corning filters, Kos. 3359 and 5113, has a transmittance maximum near the emission maximum of isochlortetracycline and transmits down t o about 405 nip. Comparison of the blanks of mash samples obtained using the two filter combinations (Table I) reveals a marked reduction of blanks v i t h the use of So.. 3389 and 51 13. Table I.
Blanks of Mash with Two Filter Combinations
Range of Blank Reading!, hlm., Using Samplc
I3ilutio11 1 :250 1 : 500 1:1,000
1:2,500
1 : 5,000 1 : 10,000
1OS. _-
+
3389 4308 48-60 36-50 26-40 16-22 12-20 10-14
+
3389 5113
10-30
10-14 5-10 5-10
5-6 1-4
Cary recording spectrophotometer curves of the absorption spectra of different sets of Kos. 3389 and 5113 filters showed the existence of differences in the wave length of the minimum, and of the half-peak band u-idths. These
sets gave different fluorescence readings with the same samples. Curves taken on permutations of the combinations revealed that S o . 3389 determined the shape of the curve and the wave length of the minimuni for the combination. The absorbance a t the minimum is determined by No. 5113. Pairs of filters uere then matched to have minimum absorbance or maximum transmittance a t 435 mp, and a half-peak band width of 60 mp. Vatched pairs on different instruments gave identical fluorescence readings for samples, and made the blank readings consistent n ith dilution, and reproducible. Control of pH. The variation of the fluorescence intensity of alkalinedegraded chlortetracycline with pH is shown in Figure 4. A change of 0.1 pH unit near 7.5 will produce a 7 to 8% relative change in fluorescence intensity. This ]vas confirmed in the analysis of mash samples which were diluted n-ith both water and O.1N hydrochloric acid. The buffers in mach did not affect the pH when qamples were diluted about 100-fold with O . l A r hydrochloric acid. Blanks were read within 2 minutes after addition of buffer becauqc of the slow conversion of chlortetracycline to ieochlortetracyclinp a t pH 7.5 and room temperature. There is no evidence that chlortetracycline fluoresces a t this pH. Where calcium ion has been concentrated in some aqueous extract samples, i t may precipitate out during buffer treatmmt as the phosphate and change the pH of the reaction mixture. To prevent this precipitation and the resulting
errors due to light scattering, the calcium was sequestered with Versene. ACKNOWLEDGMENT
Credit must be given to J. Norman Laing for initially adapting the Levine method to the Pfaltz and Bauer fluorophotometer. The authors wish to thank Frank Wilcoxon and Charles 137. Dunnett for their help in planning the statistically designed experiments and interpreting the results. LITERATURE CITED
( 1j Boltz, D. F., ed., “Selected Topics in
Modern Instrumental
Analysis,“
p. 87, Prentice-Hall, Sew York,
1952. (2) Hutchings, B. L., Waller, C. W.. Broschard. R. W..Wolf. C. F.. Fryth, P. ’W., Wilfiams, J. H., J,’ Chem. Ana. Soc. 74,4980 (1952). Hutchings, B. L., \Taller, C. W., Gordon, S.,Broschard, R. W., Wolf, C. F., Goldman, A. A , , Williams, J. H., I b i d . , 74,3710 (1952j. Kelsey, H. S.,Goldman, L., J . Clin. Invest. 28, 1048 (1949). Levine, J., Garlock, E. A., Jr., Fischbach, H., J . Bm. Pharm. Assoc.. Sei. Ed. 38, 473 (1949). Pruess, L. M., Demos, C. H., “Encyclopedia of Chemical Technology,” T’ol. SIII, p. 776, Int,erscience, S e n . I-ork, 1951. Waller, C. W.,Hut,chings, B. I,., Wolf, C. F., Broschard, R. IT., Goldman, A. A , , Williams, J. H., J . Am. Chem. Soc. 74, 49% (1952 1. Kaller, C. IT.,Hutchings, B. L., Wolf, C. F., Goldman, A. -4.,Broschard, R. W.,Williams, J. H., Ibid., 74, 4981 (1952). RECEIVEDfor review June 26, 1956. hccepted June 26, 1957.
Squalane: A Standard KARL J. SAX and FRED
H. STROSS
Shell Development Co., Emeryville, Calif.
b Squalane has been prepared by the hydrogenation of highly purified squalene. The properties of the product were determined and a synthetic squalane sample was prepared for comparison. Squalane was found useful as a standard for carbonhydrogen, molecular weight, and viscosity determinations.
M
ASSLYTICAL PROCEDURES require standard substances of reproducible purity for development of the method and calibration of the instruments. Such generally used pure hydrocarbons as triphenylmethane and n-hexadecane are solids or .melt just below room temperature; no convenient
1700
AST
ANALYTICAL CHEMISTRY
standards with more than 20 carbon atoms are available. During a n investigation of liquid standards of moderately high molecular weight, highly purified squalane (2, 6, 10, 15, 19, 23hexamethyltetracosane) was prepared by the hydrogenation of squalene. A synthetic squalane sample was prepared for comparison. I t s branched structure and the number of diastereomers make squalane one of the few readily available, stable, liquid hydrocarbons in its molecular weight range. The proportions of isomers formed during the hydrogenation of squalene under standard conditions should not vary greatly. No significant variation in properties was found. Tests show that squalane is a useful
and often superior standardizing and calibrating substance in such widely different applications as the determination of molecular weight, of viscosity, of refractive index, of carbon and hydrogen by combustion, and as a stationary liquid in gas-liquid partition chromatography (4). This work suggests the use of squalane in the latter and other applications. EXPERIMENTAL
Purification. A 100-ml. quantity of squalene (Distillation Products, Inc.) was dissolved in 140 ml. of acetone, The solution was cooled in a dry ice bath and seeded with crystalline squalene. The product was collected in a chilled funnel,
nashed with cold acetone, a n d recrystallized from acetone. The product was collected and melted; t h e acetone was removed in vacuum. The slightly yellon- squalene (melting point, -5.8' to -4.2" C., uncorrected; nz;, 1.1959) contained less material giving a carbonyl-type absorption in the infrared spectrum than did the starting material. The product was recrystallized three times more from acetone; 64 grams were recovered (n*;, 1.4960). It was then passed through a Tsn-ett column (0.75 X 6 inches) of silica gel; 54 grams were recovered (freezing point, -5.447' C.; n*;, 1.3961; A H , = 1581 cal. per mole). The infrared absorption spectrum showed no carbonyl absorption. Calorimetric purity (12) was 99.9%. Hydrogenation. Squalene (1551 grams purified by passage through a column of silica gel, 6.5 X 36 cm.) \\as hydrogenated in a rocking bomb n i t h 10 grams of platinum oxide catalyst. The reaction was quite exothermic. With a n initial hydrogen pressure of 1000 pounds per square inch, t h e temperature reached 130' C. T h e reaction was continued a t lower pressures until the theoretical uptake of hydrogen was reached, and qhaking was continued under 500 pounds per square inch of hydrogen. The product mas filtered on Hyflo Super-Cel to remove the platinum and mas passed through a silica gel column (15 X 6.6 cm.) to remove a slight odor. The product was distilled in a high vacuum pot still intwobatchesat 176" C.and0.05 inm. A small amount of forerun was taken; the main fraction was constant in reactive index throughout. Redistillation of the center cuts gave a total of 800 ml. (n*;, 1.4516; die, 0.80% gram per ml.; viscosity a t 100" F., 20.46 cs.). Calculated for C30HBZ(molecular weight, 422.8): C, 85.22; H, 14.78%. Found (corrected for weights in vacuo) : C, 85.22; H, 14.78%. Although squalane has been prepared by the hydrogenation of both q-nthetic and natural squalene (1, 2, 6, 10, 11), the reported constants vary. Physical constants of the squalane prepared in a number of different runs in these laboratories all fell within the limits indicated in Table I. RESULTS
Because of its biological significance squalene has been synthesized by several workers (S, 6 , 7 , 9. IO) The natural squalene used in this n-ork \vas obtained from two different commercial sources. It was purified by a combination of adsorption and low temperature crystallization. The melting point (-5.5" C.) is much higher than previously reported. The calorimetric purity, determined by conventional calculation methods (12)) was 99.9%. Mathematical analysis of the melting curve showed that a small amount of impurities which form solid solutions with squalene were present.
I.
Physical Properties of Squalane n?? 1.4516 =k O.O0Ola dp (gram per nil.) 0.80785 i.0.00003 Table
Viscosity (cs.), 100°F. 20.48 zt0.02 210" F. 4.197 + 0 . 0 0 4 The refractive indes of different preparations was the same to zkO.00003; the absolute value is uncertain to =kO.OOOl. 0
The purified squalene was hydrogenated with a platinum catalyst and the product was distilled in a pot still at 170" C. and 1micron. The main fraction did not vary significantly throughout its range in either refractive index or viscosity. Infrared spectroscopy and carbonhydrogen analysis showed that a C,H6, branched chain hydrocarbon had been prepared. Mass spectroscopy showed clearly the location of the methyl groups. Other physicochemical measurements indicated uniformity of product prepared from the two squalene sources. Synthetic squalane 11-as prepared for comparison ( 8 ) . The infrared absorption spectrum and carbon and hydrogen analyses of the synthetic product mere identical with those obtained from hydrogenated squalene. The refractive index was 0.0003 higher than the reference material and the viscosity was 1% lower. Mass spectroscopy shon-ed the presence of a small amount of CIOHW product. As it was not removed by acid permanganate treatment, it R-as concluded that the impurity was cyclic. Aside from this, the product m s identical with squalane produced by the hydrogenation of squalene. Some separation of the impurity was noted in a thermal diffusion column, but complete separation could not be achieved.
from tlie air, and has a tendency toward incomplete combustion. This last property demands the use of proper techniques and ensures good results on unknown substances which may burn with difficulty. The precision of carbonhydrogen determinations in these laboratories has improved since squalane has been used as a standard. I t has been suggested a t the ASTM committee concerned Kith viscosity methods that squalane could replace or complement water as the priiiiary viscosity standard for oil measurements. Two squalane samples mere prepare11 under conditions which differed coiisiderably. I n one case (sample A ) tlic, starting squalene was percolat,ed through a short silica gel column and hydrogenated a t temperatures as high as 130' C. I n the second case (sample B ) tlic: squalene was also crystallized (W.%%, pure) and the hydrogenation TT carried out at 25' C. After distillatioil the densities and refractive indices of the two samples were identical. Tlir viscosities 11-ere determined in tu-o master and two xorking viscoineters a t 100" F. The results are presented in Table IT, where the viscosit,ies of two other samples are also noted. These data show the maxinlunl variation encountered in four squalanc samples prepared under the differing conditions described. Under standardized conditions of purification it appears that squalane of highly reproducible properties can be prepared. ACKNOWLEDGMENT
The authors wish to thank L. n. TeSelle and F. R. Brooks for the carbon and hydrogen determinations and J. H. Badley and C. F. Lee for purity determinations. LITERATURE CITED
APPLICATIONS
(1) Chapman, A. C., J . Chrm. Soc. 111,
The use of pure hydrocarbons as standards in carbon-hydrogen analysis is coninion. Squalane has several advantages which make it valuable. It is not volatile, does not absorb mater Table II.
r-iscometel Master 1
Viscosity" of Squalane at 100.0' F.
Sample A 20 47 20 41
1Iastei 2
a
56 (1917). (2) Chapman, A. C., Zbid., 123, (1923). (3) Dauben, W. G., Bradlow, H. L., J . Am. Chem. SOC.74, 5204 (1952). (4) Eggertsen, F. T., Knight. H. S
20 46 20 46 Routine 1 20 45 Routine 2 20.46 20.44 Routine 3 20.48 ;Iv . 20.46 Values given in centistokes.
Sample B 20 48 20 49 20 49 20 51 20 51 20 49
Sdmple C
SainIde D
20 49
20.52 20.49 20.50
VOL. 2 9 , NO. 1 1 , NOVEMBER1957
1701
Groennings, S., ANAL. CHEJI. (1956). Heilbron, I. M., Kamm, E. D., Owens, W. M., J . Chem. SOC. 1926, 1631. Mer, O., Riiegg, R., Chopard-ditJean, L., Bernhard, K., Helv. Chim. Acta 39, 897 (195s). 28, 303
(7) Karrer, P., Helfenstein, A., Ibid., 14, 78 (1931). (8) Sax, K. J., Stross, F. H., J . Org. Chew 22, 1251 (1957). (9) Schmitt, J., Ann. 547, 115 (1941). (10) Trippett, S., Chem. & Ind. (London) 1956,80.
(11) . . Tsujimoto, M., Ind. Ena. Chem. 8 .
889 (191s). ' (12) TunniclifF, D. D., Stone, H., ANAL. CHEM.27, 73 (1955).
RECEIVEDfor review January 15, 1957. Accepted July 8, 1957.
Reaction Kinetics in Differential Thermal Analysis HOMER E. KlSSlNGER National Bureau of Standards, Washington, D. C.
,The effects of the kinetics of reactions of the type solid + solid gas on the corresponding differential thermal analysis pattern are explored. Curves of reaction rate vs. temperature for constant heating rates constructed b y analytical methods are used to demonstrate the effect of varying order of reaction. The information so obtained is used to analyze the differential thermal patterns of magnesite, calcite, brucite, kaolinite, and halloysite. The results of the differential thermal study agree with results obtained isothermally except in some specific cases.
+
holders obeys the general heat flow equation (11).
W
a reaction occurs in differential thermal analysis (DTA), the change in heat content and in the thermal properties of the sample is indicated by a deflection, or peak. If the reaction proceeds a t a rate varying with temperature-Le., possesses an activation energy-the position of the peak varies with the heating rate if other experimental conditions are maintained fixed. I n a previous paper ( l o ) ,it was demonstrated that this variation in peak temperature could be used to determine the energy of activation for first order reactions. The present paper extends the method t o reactions of any order, and proposes a method for determining the order of reaction from the shape of the differential thermal analysis peak.
dT -at
-
k -V2T pc
length, with the temperature of the outside given by T = To &, where Q is a constant rate of temperature rise and To the initial temperature, the temperature a t T , a t the center of the reference sample is, by integration of Equation 2 with proper limits,
+
DIFFERENTIAL TEMPERATURE AND REACTION RATE
I n Equation 1, the rate of heat generation is a function of temperature in the active sample. The equation then is a nonlinear partial differential equation, and cannot be solved by known analytical methods. \Ye assume that, if the same boundary condition holds-Le.. that the temperature of the outside of the holder rises a t a linear rate-the solution expressing the temperature a t the center of the sample will be of the form:
I n previous work it was assumed that the temperature of maximum deflection in differential thermal analysis is also the temperature a t which the reaction rate is maximum. Because the proposed method for determining kinetic constants depends on the accuracy of this assumption, a more detailed discussion of its validity is given. The temperature distribution in the differential thermal analysis specimen
where f is a function of the reaction rate which also includes any secondary effects of the reaction, such as changes in volume, density, or thermal properties. The differential temperature is the difference in temperature of the centers of the two samples. The differential
1702
ANALYTICAL CHEMISTRY
e =f
(2)
sample
- (4%)'
reference
(5)
where T is the temperature, t the time, k the thermal conductivity, p the density, c the specific heat, and dq/dt the rate of heat generation due to a chemical reaction per unit volume of sample. No heat effects occur in the reference sample, so the temperature distribution in the reference is given by:
If the sample is assumed to be a cylinder of radius a and of infinite HEK
temperature, 0, is then given by
(f)
and de dt
When 0 is a maximum, d0/dt is zero. From Equation 6 it is seen that when d2q/dt2, the derivative of the rate of heat absorption, is zero, &/dl is also zero. Since the rate of heat absorption is proportional to the rate of reaction, Equation 6 states that the peak differential deflection occurs when the reaction rate is a maximum. This is true only when the heating rate of the reference is constant, as otherwise Equation 6 would contain a derivative of 4. A solution of the form of Equation 3 can be obtained for a sample of any shape. If the reference material is thermally inert, the heating rate will be the same throughout the sample after quasi-steady state conditions have been established. Equation 6 is thus valid for any sample shape.
-
TEMPERATURE OF MAXIMUM DEFLECTION
&lost reactions of the type solid solid gas can be described by an equation
+
where dx/dt is the rate, x is the fraction reacted, n is the empirical order of reaction, and T is the Kelvin temperature. Reactions of this type may take place by one of a number of elementary mechanisms, as well as combinations of these mechanisms. I n most cases which have been studied, the exponent n in Equation 7 is unity or fractional, and remains constant through the greater part of the reaction. The results of studies of solid decomposition mechanisms are summarized by Garner (4). I n the present paper it is assumed that n remains constant.
