Characterization and Analysis of Waxes by Gel Permeation Chromatography D. E. Hillman Quality Assurance Directorate (Materials), Royal Arsenal East, Woolwich, London, SEIB, England Gel permeation chromatography has been applied to the characterization of hydrocarbon and ester waxes, to the determination of chain length of major ingredients and the overall carbon number range. Higher molecular weight materials can be examined more directly than is possible by gas chromatography, while the use of milder operating conditions avoids possible sample degradation. The resolution of ingredients is inferior to gas chromatography but both high and low molecular weight ingredients are detectable in a single analysis. The quantitative determination of polyethylene in microcrystalline wax is described.
METHODS FOR THE ANALYSIS of waxes have been reviewed by Robinson and Johnson ( I ) who concluded that a combination of column chromatography, gas chromatography (GC), and infrared spectrometry is probably the best for a complete analysis. However a less complete and lengthy system may be desirable for many purposes. Thus infrared analysis is used for the rapid fingerprint identification of wax types although similar types may be scarcely distinguishable-e.g., hydrocarbon waxes in admixture. The high resolving power of gas chromatography has been applied t o the direct examination of hydrocarbon and ester waxes-e.g., Ludwig (2, 3) was able t o elute hydrocarbons up to C68. Several papers describe the detailed examination of paraffin waxes-e.g., Levy ( 4 ) who used mass spectrometry in conjunction with gas chromatography to quantitatively determine normal, iso-, cyclohexyl, and cyclopentyl hydrocarbons up t o Cas. The examination of ester waxes, however, normally involves saponification and the identification of the component acids as methyl esters and alcohols as hydrocarbons. Downing and coworkers examined beeswax (9, carnauba and wool wax (7) while Edwards, Kipping, and Jeffery (8) examined the fatty acids of montan wax. Gel permeation chromatography (GPC) was introduced by Moore (9) in 1964 as a technique for the examination of high molecular weight substances in solution by a sophisticated form of column chromatography in which molecules are separated essentially by molecular size. By choosing the appropriate porosity range of the Styragel column packing, lower molecular weight substances can be examined, most waxes being covered by the range Cls-Cloo. There appears to be n o published information on the analysis of waxes by GPC and relatively little on the quantitative analytical use of this technique.
(a,
(1) B. Robinson and R. M. Johnson, Lab. Practice, 15, 854 (1966). 37, 1732 (1965). (2) F. J. Ludwig, ANAL.CHEM., (3) F. J. Ludwig, Soap Chem. Spec., 42 (3), 70 (1966). (4) E. J. Levy, R. R. Doyle, R. A. Brown, and F. W. Melpolder, ANAL.CHEM., 33, 698 (1961). ( 5 ) D. T. Downing, K. H. Kranz, J. A. Lamberton, K. E. Murray, and A. H. Redcliffe, Aust. J. Chem., 14, 253 (1961). (6) D. T. Downing, K. H. Kranz, and K. E. Murray, ibid., p 619. (71 Ibid.. 13. 80 (1960). (8) V. A. Edwards, P. J. Kipping, and P. G. Jeffery, Nature, 199, 171 (1963). (9) J. C. Moore, J. Polyni. Sci., A2, 835 (1964).
EXPERIMENTAL
Samples, Apparatus, and Procedure. SAMPLES. The range of samples examined is shown in Table I. GELPERMEATION CHROMATOGRAPHY. A Waters Model 200 gel permeation chromatograph was used. The operating conditions are summarized in Table I1 below. Columns were supplied ready packed by Waters Associates Inc. or were packed with Styragel using the method described by Peaker (10). CALIBRATION OF G P C UNIT. Samples (sizes as in Table 11) , c36, of 0.1 % solutions of n-hydrocarbons (c16, CZO,C Z ~c32, C48), esters (methyl palmitate, trilaurin), n-acids ( C ~ Zc ,16, CZ2,C30), n-alcohols (CI2, CI8, C?& and narrow molecular weight range polystyrenes (No. 25168, 25169, 25171 from Waters Associates Inc.) were injected. Retention times to peak maxima were measured in counts (1 count = 5.0 ml of elution volume). Retention times were plotted against log carbon number, the carbon number for the polystyrenes being calculated by the effectivs carbon number concept of Hendrickson (lZ)-i.e., 1.25-A chain length being equivalent to one carbon number. A typical calibration plot is shown in Figure 1. QUANTITATIVE DETERMINATION OF POLYETHYLENE IN BLENDS WITH MICROCRYSTALLINE WAX. Solutions of wax blend samples in o-dichlorobenzene 1.O% w/w were accurately prepared by heating under reflux at 80 "C. When homogeneous, a 2.0-ml sample of the hot solution was injected into the GPC unit (Column system series 111). Standard solutions equivalent to 1.0% solutions of 5 % , lo%, and 15% blends of polyethylene in microcrystalline wax were prepared and injected into a similar way. Complete dissolution required a lengthy period or use of a higher temperature. Calibration curves were prepared by plotting the polyethylene peak height or area against weight of polyethylene injected. The areas were measured by planimeter, the boundary between the polyethylene and microcrystalline wax peaks being taken as the perpendicular from the trough minimum t o the base line. GASCHROMATOGRAPHY. Samples were analyzed using a Pye series 104 gas chromatograph fitted with a heated dual flame detector and temperature programming unit. Two matched 1-m X 0.6-cm 0.d. stainless steel columns were packed with Diatoport S coated with OV-1 (stationary phase of high temperature stability). OV-1, 1 gram, was dissolved in 100 ml 1 :1 v/v chloroformtoluene by allowing to stand overnight. Thirty-three grams of 80-100 mesh Diatoport S was added, the resultant slurry was filtered under suction, allowed to suck dry before ovendrying at 100 "C. The packed columns were conditioned overnight at 250 "C with n o carrier gas flow, then for 4 hours at 350 "C with carrier gas flowing, and finally for 1 hour a t 375 O C . The column temperature was programmed from 175 to 375 "C at 3 "C/min with the detector oven at 375 "C. Oxygenfree nitrogen was used as carrier gas at a flow rate of 25 ml/ minute. Samples were injected (0.5-1 pl) as hot solutions (1-2 %) in 2,2,4-trimethylpentane using on-column injection. (10) F. W. Peaker and C . R. Tweedale, Nature, 216,75 (1967). (11) J. C. Hendrickson and J. C . Moore, J. Polym. Sci.,A4, 167 (1966). ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
1007
Reference No.
Wax Paraffin Microcrystalline
A
B
C D E F
J
K
Table I. List of Wax Samples Examined Classification Classification by source by chemical type Natural Hydrocarbon Natural Hydrocarbon
Microcrystalline Microcrystalline Microcrystalline Microcrystalline
Natural Natural Natural Natural
Polyethylene Polyethylene/ microcrystalline blends Beeswax Beeswax Chinese insect wax Shellac Japan Carnauba Candelilla
Synthetic
Natural (insect) Natural (insect) Natural (insect) Natural (insect) Natural (berry) Natural (leaf) Natural (plant)
Montan Chlorinated paraffin
Natural (fossil) Synthetic
Abril I Abril 10 DS
Synthetic Synthetic
.
.
Hydrocarbon Hydrocarbon Hydrocarbon Hydrocarbon
Remarks Poor quality-high exudate Poor quality Good quality Good quality Good quality Later batch of D
Hydrocarbon Hydrocarbon
I
Ester Ester Ester Ester Ester Ester Ester/hydrocarbon Ester/acid Chlorinated hydrocarbon Ester Ester
Dark color Pale color
Table II. Gel Permeation Chromatograph Operating Conditions I I1 Column porosities (A) Column temperature Solvent Inhibitor Flow rate Sample concentration Sample size Sensitivity Inlet heater Syphon heater Detector heater
loo, loo, loo, 500
loo, 400,5oo,108 80 OC
Nil 1 . 0 ml/min
30 "C Tetrahydrofurane Quinol 1 .0 rnl/min
1 .O% 2 ml
1 .O% 2 ml
x2
x2
70 "C
30 "C
3 x 104 70 "C To1uene
Attenuation was normally 5 X lo2 using a Speedomax I-mV recorder. NUCLEARMAGNETICRESONANCE (NMR). NMR spectra were obtained using a Varian A60 instrument (60 mHz) with a melted sample at approximately 100 "C. MOLECULAR SIEVESEPARATION OF BRANCHED PARAFFINS. Two grams of wax was dissolved in 75 ml of warm iso-octane in a 250-ml conical flask. Twenty-five grams of fully prepared molecular sieve 5A pellets was added and the mixture refluxed for 8 hours and allowed to stand overnight. The warm mixture was filtered through a G 2 sintered glass filter into a weighed flask. The residue was well washed with boiling iso-octane. The filtrate was evaporated to low bulk before stripping to constant weight with a stream of nitrogen while heating on a water bath. The increase in weight of the flask gave the branched chain content.
RESULTS AND DISCUSSION Figure 1 shows the GPC calibration curve obtained by plotting retention time against log carbon number for column series 111-Le., odichlorobenzene as solvent. The plot is essentially linear over the range of interest but shows the normal marked deviation at high molecular weights where the molecular exclusion limit of the column system is approached. The continuation of the linear region, corre1008
I11
loo, 500, 108,
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
o-Dichlorobenzene Stavox C P (5 glgall) 0.5-1.0 ml/min 1 .O% 2 ml X 2 for fingerprinting X 4 for polyethylene 80 "C
sponding to the n-hydrocarbons, passes through the point for the lowest molecular weight polystyrene standard. Esters also give points on this line showing that the calibration is satisfactory for relatively nonpolar compounds. Acids and alcohols, however, are eluted at considerably higher retention volumes showing that adsorption is also occurring. The calibration is therefore not universal but is applicable t o the majority of waxes examined and which contain mainly hydrocarbons or esters with only small amounts of acids and alcohols. The effect of chain branching is shown t o be small since squalane (2,6,10,15,19,23-hexamethyltetracosane) gives a peak which scarcely deviates from the n-hydrocarbon plot. NMR suggests that the degree of branching in the hydrocarbon wax is rather less than in squalane. Hendrickson (11) found that methyl branching of lower hydrocarbons caused little difference in retention time. A number of workers (11-15) have studied the behavior of (12) W. W. Schulz, G. P. C. Elution Behavior of Branched Alkanes, 9th International G. P. C. Symposium, Miami, October 1970. (13) E. G. Sweeney, R. E. Thompson, and D. C. Ford, J. Chromatogr. Sci., 8,76 (1970). (14) J. Cazes and D. R. Gaskill, Separation Sci., 2 , 421 (1967). (15) A. Kollmansberger and W. B. Smith, J. Phys. Chem., 69, 4157 (1965).
CARBON
I
400
l
l
I l l
60 SO
200 100
RETENTION
io
a',
i4
a'b
io
ao
31
34
$1
30
NUMlCR
40
I
30
I
20
VOLUME I C O U N T S I
Figure 2. GPC separation of hydrocarbon waxes
RETENTION VOLUME lCOUNTSl
Figure 1. GPC calibration. Carbon number as retention volume o-Dichlorobenzene 80 "C. 100 400 500 400 A X Hydrocarbons 0 Polystyrene standards 8 /z-Alcohols 0 n-Acids . n-Esters
+
+
+
small molecules in a GPC system. The fundamental parameter for this type of separation appears to be molar volume but adsorption on the column or hydrogen bonding between solvent and sample may modify this behavior for strongly polar compounds. The single plot of loglo carbon number us. elution volume is justified for the examination of waxes where a limited number of functional groups are present. In addition, the need to separate substances in the wide higher, restricts the carbon number range of C ~ O - C ~orO O resolution of the column system and, therefore, obscures small effects reported by other workers who generally examined a more restricted carbon number range. The moderate resolution of the GPC system is a drawback to precise studies in the wax field. Bombaugh (16) separated triglycerides on a 160-ft column and discussed the factors affecting resolution, and the new recycle technique (17) may also be useful in future work but both techniques require lengthy analysis times. The carbon numbers corresponding to the retention times of the maxima of the gel permeation chromatograms for ester and hydrocarbon waxes were read off the calibration curve. The overall carbon number range was less easily determined since correction is required for peak broadening during elution. Computer systems have been described (18-21) in the literature for correction of polymer chro(16) K. J. Bombaugh, W. A. Dark, and R. F. Levangie, Application of Gel Chromatography to Small Molecules, 11. 5th International Seminar on GPC, London, 1968. (17) J. L. Waters, J. Polym. Sci., A.2, (8) 411 (1970). (18) H. E. Pickett, M. J. R. Cantow, and J. R. Johnson, J. Appl. Polym. Sci., 10, 917 (1966). (19) M. Hess and R. F. Kratz, J. Polym. Sci.,A.2 (4) 731 (1966). (20) L. H. Tung, J. Appl. Polym. Sci., 10, 375 (1966). (21) W. N. Smith, ibid., 11,639 (1969).
ELUTION V O L U M E (COUNTS1
Figure 3. GPC comparison of microcrystalline waxes
-
_. _. ._ ._ . .
Wax B Wax E Wax C (D Similar)
matograms. Since computer facilities were not available, the retention times of the base extremities of the chromatograms were reduced by adding or subtracting twice the standard deviation of the gaussian peak obtained by injecting pure Caa hydrocarbons. This correction is justified at the lower molecule weight ranges by the agreement with the gas chromatography results where the resolution of adjacent carbon numbers is complete. The correction is less precise at high carbon numbers since the discrimination of the column is less. The overall carbon number range is dependent on the sensitivity of the technique to the extreme ingredients and is therefore itself not precise. Hydrocarbon Waxes. Figure 2 shows typical GPC chromatograms obtained with parafin, microcrystalline, and polyethylene waxes using column system 111. The separation of each is largely complete. ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
1009
Wax A B C D
E F
Table 111. Carbon Number Range of Hydrocarbon Waxes by GPC and GC (Column System 111) GPC Maximum Range Range Type Paraffin wax 26 18-34 21-34 36 18-79 Microcrystalline wax 13-> 60 Microcrystalline wax 45 22-84 22-> 60 Microcrystalline wax 42 26-87 2 6 > 60 Microcrystalline wax 23-90 44 22-> 60 Microcrystalline wax 45 27-100 25-> 60 Exudate from F 41 24-90
GC
Maximum 27 34 50 42 42 42
Table IV. Quantitative Determination of Polyethylene in Microcrystalline Wax
z
poiyethylene added 13.96 14.00 9.34 Wax C
Found, % Peak height Peak area 14.314.214.2 14.0 13.9 13.8 13.9 13.9 9.6 9.7 0.8 0.8
1.1.013.914.1 13.9
Standard deviation for 13.96% standard
Mean Height
found Area
14.1
14.0
13.9 9.7 0.8 = 0.16
(height)
0.12 (area) Relative standard deviation for 13,96% standard = 1 . 1 % (height) 0 . 8 (area)
Column system I1 (tetrahydrofurane solvent) is satisfactory only for paraffin wax, the others being insoluble. System I (toluene solvent) gives satisfactory peaks but the sensitivity to polyethylene is very low. Waxes B, C, D, and E are commercial microcrystalline waxes from different sources, and these are differentiated by GPC (Figure 3) except for waxes C and D which are closely similar although not identical. Table I11 compares the GPC and G C results. In general there is good agreement between the two techniques but the upper limit of carbon number range of microcrystalline waxes can be measured only by GPC since the GC limit is at about C S ~ .Wax C differs from B, D, and E in showing no preponderance of even carbon number hydrocarbons around the maximum of the gas chromatograph peak envelope. Determination of Polyethylene. Polyethylene of molecular weight 2000-4000 is waxy in character and is added to other hydrocarbon waxes to improve the melting point and hardness. The quantitative determination of polyethylene is difficult since all three types of hydrocarbon wax have the structure CH8-(CH2)n-CH3 with variations in n or in degree of branching. Methods have been proposed based on differences in solubility (22). GPC permits the direct determination based on differences in molecular weights. Calibration plots of peak height and peak area against concentration of polyethylene were found to be linear. Peak heights were normally used for convenience. Quantitative results are given in Table IV. The precision of the method depends primarily on the noise level of the instrument. Sample C is shown to contain a small amount of polyethylene which was suspected from its physical properties.
(22) TAPPI Special Technical Association Publication No. 2, 1963. 1010
*
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
ELUllON VOLUME ( C O U N l S )
Figure 4. CPC comparison of exudate and parent wax __ Parent wax Exudate . . . . . . With addedgil(1Oz)
----
Exudate from Microcrystalline Waxes. Microcrystalline waxes tend to exude on long storage and an empirical specification method is used to test for this defect. A wax pellet is heated at 75 “C for seven days while standing on frequently changed filter paper pads which absorb the exudate. A maximum limit is set for the weight loss of the pellet. Initially it was suspected that the presence of low molecular weight material (“oil”) would cause this effect. The exudate was extracted from the filter papers by solvent extraction and after evaporation of the solvent, the recovered solid was examined by GPC (Systems I and 111). Figure 4 compares the chromatograms obtained from one wax and the corresponding exudate. The latter shows a general but small shift to the lower molecular weight. Detailed results for the other waxes are not shown but in each case the results are similar. Added oil is clearly indicated by the appearance of a shoulder in the 25-26 count region. The total branched hydrocarbon content of waxes A to D (Table I) was determined by molecular sieve absorption of the straight chain material. About 8 5 % of these waxes were shown to be branched but the figures were not repeatable. The value of this test is uncertain for very long chain hydro-
c 4 3 ALKYL E $ T l R S
SOLVlNf
ALKYL ESTERS
c4b HYDROCARIONS
n
HYDROCARIONI
I
D\
I
I
60
fLU71ON T I M €
Imlnl.)
0
Figure 5. Cas chromatogram of beeswax 3 ft = 4 m m OV-l,175-375,"C,3 'C/min carbons, and the only valid conclusion is that the waxes are largely composed of branched hydrocarbons. Wax B failed the exudation test, the whole pellet virtually melting. This wax is clearly differentiated by its low molecular weight range and therefore low melting point. Wax E however gave very little exudate although it contains an appreciable proportion of lower molecular weight ingredients in the shoulder of the chromatogram (at 26 counts retention time). Exudation therefore appears not to be related to total branched hydrocarbon content or to carbon number range alone. NMR was used to investigate the nature of the branching and showed that waxes B, C, and D had very similar spectra corresponding to essentially linear chains with terminal C
I
tert-butyl groups C-C-(C),-C-C.
I
I
Wax E however con-
C C tained terminal ethyl groups C-(C),-C. The latter would be expected to be of higher melting point than those with tertbutyl groups and therefore to give less exudate. All four waxes showed no apparent unsaturation. One exudate was examined and shown to contain an appreciably higher CH3 content confirming that the exudation properties are related to degree of branching in the higher molecular weight waxes and to carbon number range in those of lower molecular weight. Beeswax. Two samples each of good quality but differing in color were examined. Gas chromatography gave a chromatogram (Figure 5 ) in which the earlier peaks corresponded to hydrocarbons and the later and larger peaks to esters. Carbon numbers were identified by addition of known pure substances and noting which peaks increased in size when chromatographed again. The compositions of the ester and hydrocarbon fractions (Table V) were separately calculated by internal normalization assuming equal detector sensitivity. Palmitic acid which is normally the main free fatty acid gave no peak when chromatographed using a sample size equivalent to 10% free acid present in the wax. The results showed the two samples to be very similar. GPC was possible with all three column systems. When T H F was used as solvent (System II), the two waxes gave identical chromatograms, one being shown in Figure 6.
I
14
I
I
I
I
I
I
11 10 !8 I
