Determination of Asphalt Molecular Weight Distributions by
Gel Permeation Chromatography
L. R. Snyder
Union Oil Co. of California, Research Center, Brea, Calif. 92621
The direct determination of asphalt molecular weight
distributions by gel permeation chromatography
(without obtaining independent molecular weights on
separated fractions) presents a number of problems
which appear to have been overlooked by previous
workers. These include unexpected solution aggrega-
tion effects, adsorption of asphalt on the polystyrene
gel, and a peculiar dependence of RI or UV response
data on fraction molecular weight. Attention to these
various effects allows the rapid, accurate determina-
tion of an asphalt molecular weight distribution at
minimum cost, using high efficiency gel permeation
chromatography in an automated unit.
SEVERAL WORKERS (1-5) have reported the use of gel perme-
ation chromatography (GPC) in the determination of molecular
weight distribution for residual petroleum samples-e.g.,
asphalts. Such data have been used in attempts to define the
molecular structure of these materials and in correlations of
their performance or physical properties. A number of
petroleum laboratories are carrying out these analytical GPC
separations on a more or less routine basis, using one of two
possible procedures. A preparative separation can be carried
out in a large column (usually of low efficiency), the molecular
weights of individual fractions can be determined by an
independent method, and the resulting data then yield a mo-
lecular weight distribution for the original sample (2, 3, 5).
This method is reliable and straightforward, but time consum-
ing and tedious. Alternatively, similar GPC separations can
be carried out on much smaller samples in high efficiency,
automated units (4). The resulting elution curve (usually a
trace of eluate refractive index us. volume) can be translated
into a molecular weight distribution curve, assuming (a) a
constant response factor for the detector (differential refrac-
tometer), and (b) a standard sample calibration curve of elution
volume us. molecular weight. The second procedure is rapid
and applicable to automatic data handling, but its reliability
is limited by assumptions (a) and (b). However, similar pro-
cedures are widely used in the analysis of synthetic polymers,
with generally adequate accuracy.
In the course of setting up a GPC procedure of the second
type (small scale, automated operation) in our own laboratory,
it became apparent that a number of previously ignored com-
plications exist in the GPC analysis of asphalts. The present
paper describes these complications and the measures that
are necessary to insure accurate molecular weight dis-
tributions by GPC. Similar care may be required in the
quantitative GPC analysis of other heterogeneous polymeric
materials, such as reaction tars, certain natural products, shale
oils or coal tars.
~
(1) J. P. Dickie and T. F. Yen, presented before the Div. of Petro-
leum Chem., ACS Meeting, Miami Beach, Fla., April, 1967;
Petrol. Diu. Preprints, 12, NO. 2 B-117, 1967.
(2) K. H. Altgelt, J. Appl. Polymer Sci., 9, 3389 (1965).
(3) K. H. Altgelt, Mukromol. Chem., 88, 75 (1965).
(4) W. B. Richman, Proc. Ass. Asphalt Paving Technol., 36, 106
(1967).
( 5 ) C . A. Stout and S. W. Nicksic, SOC.Petroleum Eng. J., 8, 253
(1968).
EXPERIMENTAL
GPC Separations. All separations were carried out in
the unit described previously (a, using four 4-ft X */&
columns (0.30-in. i.d.) connected together in series. The
columns (i-iv) were packed with Styragel (Water Associates,
Framingham, M!ss.) of varying pore size: (i) a 50/50 mixture
A
of 100 and 250 A gel, (ii) and (iu) lo3 gel, and (iv) lo4 8,
gel. The overall efficiency of the four column unit was equal
to 9000 theoretical plates at the standard solvent flow rate
z
of 2.0 ml/min (0-xylene solute, 5 v methanol-chloroform
solvent). In the procedure eventually used for the routine
determination of asphalt molecular weight distributions,
9 mg of sample was charged as a 20-gram/liter solution
in the elution solvent, elution was carried out with 5 vol z
methanol-chloroform (reagent grade), and 10 ml fractions
were collected. The absorbance of these fractions at 370
nm was determined spectrophotometrically. This manual
procedure has since been replaced by use of a flow-through
spectrophotometer set at 370 nm. Molecular weights were
determined by vapor phase osmometry, using chloroform
solvent.
METHOD DEVELOPMENT
The following discussion is divided between the problems
of setting up a satisfactory experimental system and the
subsequent interpretation of experimental data from that
system.
Experimental Conditions. Previous workers who have
attempted the automated GPC analysis of asphalts and
related fractions ( I , 4) have generally used tetrahydrofuran
(THF) as solvent and a refractometer as detector. In the
present study it was found that T H F as solvent gives incom-
plete recovery of asphalt samples: the total UV absorbance
(370 nm) of collected GPC fractions (0-1 column volumes)
averaged 20-40 z low, relative to the value calculated from
the original sample. The strongly retained asphalt compo-
nents were slowly eluted from the column in several column
volumes of eluate. Several other solvents were investigated,
including chloroform and various mixtures of chloroform,
THF, and/or various alcohols. Of these solvents, only 5 vol
z methanol-chloroform gave sample recoveries which aver-
z
aged higher than 95 (UV absorbance), in agreement with
(2, 3). However, with the latter solvent it proved impossible
to obtain a satisfactory output from the refractometer detec-
tor. Sample chromatograms were ragged and irreproducible,
although baseline stability in the absence of sample injection
was satisfactory. We accordingly changed our detection
system to ultraviolet monitoring at 370 nm.
Some interesting sample size effects were noted in the GPC
analysis of asphalt samples. Earlier it had been reported (4)
that the performance qualities of road asphalts appear to be
related to the relative concentrations of high molecular weight
components present. This is illustrated in Figure l a (THF
solvent, refractometer output) for two asphalt samples from
our laboratory ( A , “bad,” B, “good”). At a sample concen-
(6) L. R. Snyder, ANAL.CHEM.,39,698 (1967).
VOL. 41, NO. 10,AUGUST 1969 1223
 \

en
q

--
500 1000 2000 6000 l0,OOO I . I I I I
MW (uncorrected)
I20 I40 I60
ELUATE VOLUME ( m l )
Figure 1. Sample size effects in GPC separa-
tion of asphalts Figure 2. Detector response (1 gram/
(a) THF solvent, (b) THF solvent, sample B, (c) liter) for narrow GPC fractions as a
5 vol. methanol-chloroform solvent, sample C. function of average elution volume
Refractive index (---) and UV (-) for
samples Cand D.
tration of 30 mg, asphalt B shows a well defined hump in the
4000-6000 molecular weight region, and this hump is much
tion. The relative concentrations of this high molecular
less pronounced in the case of asphalt A . These results
fraction in different asphalts correlated roughly with their
parallel those of (4). Upon decreasing the sample size to 4
asphaltene content. One possible explanation for the effect
mg, however, the high molecular weight hump in asphalt B
of Figure ICis that at very low sample concentrations in 5 wt
is much depressed (Figure lb), and it is then difficult to see
significant differences between “good” and “bad” asphalts.
% methanol-chloroform there is a dissociation of certain
asphalt micelles, with subsequent recombination of the frag-
Similarly, by increasing sample size to 150 mg (Figure lb),
ments into irreversibly bound high molecular weight aggre-
a large hump at very high molecular weights (lO,OOO+)
gates. In any event, the various results summarized in Figure
appears in sample B. The latter sample size effects suggest
1 emphasize the importance of controlling sample size within
increasing association of sample molecules at higher concen-
narrow limits, if the resulting molecular weight distribution
trations, and this is not unexpected (7). Thus the difference
data are to be meaningful. The use of a 9-mg sample in the
between “good” and “bad” road asphalts appears to be more
routine separation avoids the production of a high molecular
a function of intermolecular associations than of relative mo-
weight band as in Figure IC, and avoids normal association
lecular weight distribution. This does not detract from the
effects which occur at still higher sample concentrations.
possible use of GPC in the study of asphalt quality, but it does
Data Interpretation. The reduction of a raw chromatogram
emphasize the importance of sample size in carrying out such
(as generated by the routine procedure of the Experimental
studies.
section) into a final molecular weight distribution requires
At very small sample sizes in 5 vol % methanol-chloroform,
three operations: conversion of the detector output into
another interesting effect was observed (Figure IC). Whereas
mass units, correction of the resulting mass os. volume
a sample size of 10 mg or larger gave a simple monomodal dis-
chromatogram for the band spreading of individual asphalt
tribution curve for all samples, at sample sizes less than 1 mg
components during separation, and a correlation between
many asphalts show a definite bimodal distribution, with a
sample elution volume and molecular weight. We will
new band appearing of very high apparent molecular weight
discuss each of these items in sequence.
(6000-10,000). This effect is not expected on the basis of
In the GPC analysis of typical polymer samples, it can often
simple intermolecular association. The relative size of the
be assumed that detector response-Le., refractometer-for
high molecular weight band is somewhat exaggerated in
different sample components is constant on a mass concentra-
Figure IC, because the UV absorbance of this band is about
tion basis. This is not the case for typical asphalt samples,
five times greater than that of the total sample. In no case
as shown in Figure 2. Here relative weight response for both
did the estimated concentration of the high molecular weight
refractometer (dashed curve) and UV detection (solid curves)
band exceed 10 wt of the original asphalt. This new band at
is plotted us. the elution volume of narrow asphalt fractions
small sample concentrations does not appear to be a chroma-
prepared by preparative GPC. Data on the UV response of
tographic artifact or the result of adsorption on the gel. Re-
two samples (C, a deasphaltened asphalt; and D, a total
peated separation of the sample of Figure I C at low sample
asphalt) are shown. Both detectors give a high response for
concentrations (0.8 mg) gave two fractions, corresponding to
low and high molecular weight components, with minimum
each band. Reseparation of each fraction (0.8-mg charge)
response for molecular weights of 600-1000 (140-150 ml).
gave an elution band that coincided with the band collected
Furthermore, the UV response curve appears to vary from
in the original separation (except for minor tailing). Thus the
one asphalt sample to another. The method of the Experi-
high molecular weight band does not reequilibrate to form
mental section is based on the UV curve for sample D of
lower molecular weight material in the course of GPC separa- Figure 2, but this is seen to be approximate at best. The
preferred solution to this problem is the use of a detector which
(7) R.S.Winniford, J. Inst. Petrol, 49, 215 (1963). responds directly to mass, rather than a physical property

1224 ANALYTICAL CHEMISTRY

 IO00 c‘t
ELUATE VOLUME (ml.)
Figure 3. Calibration curve for molecular
length L , or molecular weight (mw) us. sample
elution volume
0 standard polymer fractions and n-alkanes (15,)
asphalt GPC fractions (mw)
V deasphaltened asphalt GPC fractions (mw)
such as refractive index or spectral absorptivity. Commercial
liquid chromatography detectors based on solvent stripping
and detection by flame ionization come close to meeting this
requirement. An alternative is simple fraction collection, with
a gravimetric finish on the solvent-free fractions (using a
micro balance).
In the case of GPC polymer analyses, band broadening of
individual sample components during separation can be
handled in a variety of ways (8). In the case of asphalt sep-
arations, it can be estimated that normal band broadening as
a result of eddy diffusion and mass transfer effects is of minor
importance. Thus the data of Hendrickson (9) show an
inverse correlation between plate height and sample elution
volume; this is reasonable in terms of the slower mass transfer
of larger sample molecules. Application of this correlation
in the present study suggested that band broadening was of
minor significance, because of the relatively low molecular
weights of major asphalt components (500-2000) and the
high column efficiency (9000 theoretical plates). However, it
was observed that higher molecular weight asphalt components
exhibit slowly reversible adsorption with band tailing, even in
the presence of the highly polar solvent 5 vol z methanol-
chloroform. This effect was shown by separating a typical
asphalt into narrow fractions by GPC, then rerunning each
fraction under the conditions of our routine analysis. In
each case some tailing of an original fraction into later eluted
fractions was evident. Thus 1 8 z of the band initially eluted
between 110-120 ml was found in the 130-140 ml fraction of
the second separation. Even larger overlap into the 120-130-
r d fraction occurred, but this could not be distinguished from
the normal spreading of the 110-120 ml band in the second
separation. Similarly, 18 of the 120-130-ml fraction from
the first separation was found in the 140-150-ml fraction of the
second separation. This band tailing dropped to 6z for the
130-140-ml fraction, and 4z for later fractions. The correc-
tion of the chromatogram for band tailing is important if use
is made of UV or refractive index detection, because the ini-
tially eluting (strongly tailing) fractiocs have higher detector
response factors. Correction for tailing becomes less im-
portant with gravimetric detection of the column effluent.
One approach to the correlation of sample elution volume
(8) K. H. Altgelt, Adu. Chromatography, 7, 3 (1968).
IO0
80 -
60 -
I I
40 -
20 -
I I I I
400 600 1000 4000
MOLECULAR WEIGHT
Figure 4. Experimental asphalt molecular weight
distribution
- routine GPC method (iFnequal 768)
___
0
routine GPC method ( M nequal 837)
preparative scale GPC with molecular weight deter-
minations on each fraction
in GPC with molecular weight is to measure a calibration
curve for available standards, in terms of effective molecular
length L, us. elution volume V. Such a curve is shown in
Figure 3 (closed circles, solid line), based on commercial poly-
styrene and polypropyleneglycol standards (Waters Asso-
ciates) and the n-alkanes (9). We can assume a propor-
tional relationship between t, and the molecular weight
( M W )of a sample component:
MW = CL,
Cis normally constant for a narrow range of sample molecular
structures-e.g., synthetic polymers of a given type-so that
a measurement of GPC elution volume for a narrow molecular
weight sample fraction provides a value of C from a plot of
the type shown in Figure 3. Equation 1 seems to adequately
describe the relationship between molecular weight and elution
volume for narrow GPC fractions from a single asphalt, as
shown in Figure 3 (open squares and triangles, for two differ-
ent original asphalts). However, the value of the constant C
can vary by a factor of three among different asphalt samples,
particularly for solvent extracted asphalts. This reflects the
heterogeneity of typical asphalts in terms of the types of
molecular components present. Bulky molecules have smaller
molecular lengths per unit molecular weight, and give larger
values of C in Equation 1. Consequently, it is necessary to
calculate C for each asphalt to be analyzed by GPC. This
can be done easily, providing we measure the number-average
molecular weight of the original asphalt sample in each case:
Lvn. Vapor phase osmometry (VPO) was used in the present
study, as described in the Experimental section. Use of a
“standard” value of C (equal to 20, from Figure 3) provides
an initial, uncorrected molecular weight distribution, from
which an initial estimate of &Vncan be calculated. The true
value of C is then equal to 20 (A%‘n)t,,,/(L%‘n)est. With a final
value of C, the true molecular weight distribution can be
calculated. This is most conveniently expressed as an accu-
mulative plot of wt zof sample us. molecular weight, as in
Figure 4, or as a tabulation of maximum molecular weight us.
accumulative sample percentages (Table I).
(9) J. G . Hendrickson and J . C . Moore, J . Polymer Sci., A1(4), 167
(1966).
VOL. 41, NO. 10,AUGUST 1969 1225
 DISCUSSION OF RESULTS
Table I provides some typical asphalt molecular weight dis-
tribution data, as calculated by the present procedure. These
are based on UV detection, and are to some extent inaccurate
because of the variable UV response curves for different
asphalt samples-e.g., C and D in Figure 3). However it
should be noted that the procedure for calculating a true
value of C in Equation 1 partially compensates for this in-
accuracy, because n, for the calculated molecular weight
distribution is set equal to the experimental value of a,.
Some internal consistency checks given below suggest that
this is not a serious practical problem. The data of Figure 4
and Tables 1-111 are corrected for normal band broadening and

Table I. Typical Asphalt Molecular Weight Distributions as Determined by GPC

x
Wt of sample
“Good” asphalts
Molecular weight
“Bad” asphalts
falling below
indicated mol wt. E F G H asphaltenes
5 450 400 380 506 1110
10 535 490 430 455 1310
20 680 660 515 550 1660
30 815 820 595 635 1990
40 945 lo00 660 715 2300
50 1090 1190 730 810 2700
60 1280 1380 835 930 3150
70 1540 1640 995 lo00 3700
80 1930 2050 1260 1330 4500
90 2950 3 300 1810 1900 6200
95 4900 5300 2900 2850 8900
Rso,io 5.1 6.7 4.2 4.2 4.7
(C/20) 1.os 0.82 0.93 0.95 2.3
-
M n 994 986 720 768 2400

Table 11. Comparison of Molecular Weight Distributions on Isopropanol Extracted Fractions with Original Sample Distribution
Molecular weight
Wt Z of sample
falling below Fractions
indicated mol wt. 1 2 3 Composite Original asphalt
5 415 530 560 510 490
10 465 580 640 565 555
20 540 655 760 655 670
30 605 720 860 735 770
40 640 785 945 800 860
50 685 845 1040 870 960
60 725 915 1190 960 1050
70 785 1000 1380 1100 1160
80 860 1130 1640 1330 1360
90 1020 1390 2100 1750 1760
95 1160 1690 2600 2200 2300
Reom 2.2 2.4 3.3 3.1
c/20 0.98 1.06 1.06
E n 653 834 1009

Table 111. Repeatability of Present GPC Asphalt Analysis

Wt of z
sample
falling
below
indicated Molecular weightD (sample E of Table I)
mol wt. 1 2 3 4 5 6 Av.
5 450 450 450 440 440 450 447 f 5
10 535 540 535 530 530 535 534 f 4
20 690 685 680 670 675 670 678 f.8
30 825 820 820 805 820 795 814 &12
40 965 940 920 935 955 930 946 +13
50 1090 1080 1080 1100 1110 1100 1093 f.12
60 1260 1260 1270 1300 1310 1290 1282 f21
70 1500 1500 1520 1590 1570 1540 1537 *37
80 1850 1850 1940 2030 1970 1940 1930 f.70
90 2750 2560 3020 3350 3000 3000 2940 f270
95 4500 4200 4900 6000 4900 5000 4900 f600
a Numbers 1-6 represent run numbers, uncertainty figures are std. deviations.

1226 ANALYTICAL CHEMISTRY

the tailing of high molecular weight fractions into lower mo-
lecular weight fractions.
Figure 4 compares a molecular weight distribution curve
(solid line) obtained by the present GPC method with corre-
sponding data (squares) from preparative GPC separation and
molecular weight determinations on individual fractions.
The differences in these two sets of data arise mainly from
errors in the corresponding molecular weight determination
by VPO. If the VPO value of for the original sample is
obtained from the composited GPC fractions (squares), a
value of 837 is obtained, us. a value of 768 in the original
sample. Use of the 837 value (rather than the 768 value) in
the routine GPC calculation yields the dashed curve of Figure
4. This is seen to be in reasonable agreement with the data
by preparative scale GPC. A series of fractions from the
successive extraction of an asphalt at two temperatures with
isopropanol are shown in Table 11. Comparison of the
original feed analysis with the composited fraction data shows
good agreement. These two examples indicate that the
present method is reasonably reliable, despite the limitations of
UV detection. The repeatability of the present GPC method
is illustrated in Table 111, for replicate analyses of the same
sample using a single experimental value of ATn.
The resulting molecular weight distributions from the
present GPC procedure furnish three useful parameters for
characterizing the sample. The ratio of molecular weights at
Rapid Analysis of Ribonucleosides and Bases
at the Picomole Level Using Pellicular Cation
Exchange Resin in Narrow Bore Columns
Csaba Horvathl and S . R. Lipsky
Section of Physical Sciences, Yale University School of Medicine, New Haven, Conn.
The separation of the four major bases and nucleosides
of RNA has been investigated under conditions utilizing
wide ranges of pH, temperature, and flow rates. Small-
bore columns packed with pellicular cation-exchange
resin were used in a high-pressure liquid chromato-
graph equipped with a micro UV detector. Dilute
acidic potassium or ammonium phosphate solutions
served as eluents. The four bases or nucleosides
were analyzed at the subnanomole level in less than
6 minutes using 3-meter colunms and high-flow veloc-
ities. With a shorter column operated at relatively low-
flow velocities, a few picomoles of ribonucleosides can
be separated and determined without resorting to
radioactive techniques.
THE DETERMINATION of the base composition of nucleic acids
is one of the most important steps in sequence analysis.
Further progress in this field largely depends on the develop-
ment of highly sensitive and rapid techniques for quantitative
analysis of nucleic acid constituents. High sensitivity is
often required for the minute quantities of nucleic acids or
nucleic acid fragments that may be available-e.g., by zonal
centrifugation or single cell isolation. On the other hand,
the need for a large number of analyses can only be overcome
by using a fast and reliable method that is amenable to auto-
mation. Thus far, attempts to utilize gas chromatography
to analyze nucleic acid hydrolyzates, although promising
1 Also Department of Engineering & Applied Science, Yale
University.
90 and 10 wt % ( R W , ~is~a) good measure of the molecular
weight spread of the sample. The quantity C/20 provides a
measure of the deviation of the sample molecular weight from
the calibration of Figure 3, and is an indicator of average
molecular shape. Large values of C/20 correspond to rela-
tively bulky molecules, while small values of C/20 mean long,
narrow molecules. The number-average molecular weight is
the third characteristic parameter of interest. These various
parameters are tabulated in Table I and I1 for the samples
shown there. The difference between these particular “good”
and “bad” asphalts seems to be related to molecular weight
spread (Roo,lo)and to mean molecular weight, but these may
be accidental correlations based on different boiling ranges
in the original crude oils. The molecular shape factor C/20
does not appear to be related to asphalt performance. As-
phaltenes have higher average molecular weights and large
values of C/20. As expected, asphaltene molecules are
bulkier and more compact than other asphalt components.
ACKNOWLEDGMENT
Several asphalt GPC fractions (triangles in Figure 3,
sample C of Figure 2) were kindly supplied by S. W. Nicksic of
the Chevron Research Corp., La Habra, Calif.
RECEIVED
for review April 4, 1969. Accepted June 2, 1969.
06510
( I , 2), have not been wholly successful. Thin-layer and paper
chromatography (3, 4) are widely used, but quantitative
analysis is time consuming and generally inaccurate below the
nanomole level (5). Using an elaborate radioactive tracer
technique, however, the Randeraths (6, 7) recently achieved
base composition determination at the picomole level.
Higher sensitivity has been obtained only in the quantitation
of ATP by the luciferin-luciferase enzyme system (8).
Various column chromatographic methods with gels
(9, IO), with a ligand exchange resin ( I I ) , with a liquid ion
(1) C. W. Gehrke and C . D. Ruyle, J. Chromatog., 38, 473 (1968).
(2) M. Jacobson, J. F. O’Brien, and C . Hedgcoth, Anal. Biochem.,
25,363 (1968).
(3) K. Randerath and E. Randerath, in “Methods in Enzymology,”
Vol. XII, “Nucleic Acids,” Part A., L. Grossman and K. Moldave,
Eds., Academic Press, New York, 1967, pp 323-50.
(4) G. R. Wyatt, in “The Nucleic Acids Chemistry and Biology,”
Vol. I, E. Chargaff and J. N. Davidson, Eds., Academic Press,
New York, 1955, pp 243-65.
(5) J. M. Gebicki and S . Freed, Anal. Biochem., 14,253 (1966).
(6) K. Randerath and E. Randerath, ibid., in press.
(7) E. Randerath, J. W. Ten Broeke, and K . Randerath, FEBS
Letters, 2, 10 (1968).
(8) G. E. Lyman and J. P. DeVincenzo, Anal. Biochem., 21, 435
(1967).
(9) I. Mezzasoma and B. Farina, Boll. SOC.Ital. Biol. Sper., 42,1449
(1966).
(10) M. Carrara and G. Bernardi, Biochim. Biophys. Acta, 155
(1968).
(11) G. Goldstein, Anal. Biochem., 20,477 (1967).
VOL. 41, NO. 10,AUGUST 1969 1227