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Chemical instrumentation feature
Edited by GALEN W. EWING, Seton Hall University, So. Orange, N. J. 07079
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LII. Current Trends in Gel Permeation
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Chromatography.
Part Two: Methodology
JACK CAZES, Mobil Reseorch and Development Corp.,
Paulsboro, N. J. 08066
VI. Calibration
A variety of more or less "well charao-
terized" polymers is now available for
calibrating GPC columns for the estima-
tion of molecular weight distributions of
polymeric substances. Table 4 is a list of
these commercially available substances.
Some &re ~ l a t i v e l ymonodisperse (mea-
sured M J M , approaches 1.O) while others
have somewhat broader distributions.
The molecular weight averages given in
the table were supplied by the respective
manufacturers and were determined by a
variety of techniques, including light-
scattering, membrane osmometry, ultrsr
centrifugation, vapor pressure lowering,
etc.
Additionally, the Polytechnic Institute
of Brooklyn, Polymer Sample Bank has
made available a wide variety of polymer
samples together with "selected properties
which will clea~lydifferentiate those com-
pounds of similar type." These data
while sparse, often, at least, can serve as a
starting point for the construction of a re-
liable calibration curve. Distillation Prod-
ucts Industries, Div. of Eastman Kodak
Co., Rochester, N. Y. 14603, serves as de-
pository and distributor for this project
and should he contacted for further details.
Direct calibration of GPC columns in
terms of molecular weight of a given poly-
mer type remains the method of choice;
this is achieved by recording the GPC re-
sponse for a series of narrow MWD
standards of the polymer of interest whose
peak molecular weights have been deter-
mined independently and then platting
their GPC retention volumes versus
res~ective~ e a kmolecular weiehts. For
suffirienlly itamow 3IWD polym~rsit i.r
often H . I P U ~ I ~ Qthat
~ the p ~ a kmoleeular
weight i c equivalent t o an average value
such as M.
I ~
Alternatively, the widely used "Q-
factor" calibration technique described
earlier (1) can be used t o determine
molecular weight averages for many poly-
meric systems with a cdibration curve
constructed with narrow MWD poly-
styrene standards. The method works
well only if the GPC column response for
the polymer of interest is related t o the
polystyrene response by a Constant factor.
Several techniques have been suggested
for constructing a calibration curve for a
polymer of interest for which there are
only available well characterized broad
MWD samples. One such method that
has been used with great success in the
present miter's laboratory, even where
..
the "Q-factor" method is inamlieable. is
that which has berln d~.wriheclby Purdon
a n a t 1 nrirfly, vhromntop:tms
for b r o d hnIKI) amplrs of the polymer of
interest whose molecular weight averages
are known ere used in conjunction with a
calibration curve constructed with narrow
MWD polystyrene standards t o yield a
plot of retention volume versus log (molec-
ular weight) for the polymer of interest.
Some slight manual adjustment of the
rUNC thw ohtnined is grncrnlly rK?CQ3%3ry,
but the result io n mlilmtiort r m w thnt
vields relinl,le molcrulnr weight svernges.
- Another potentially useful approach
utilizing two or more known molecular
weight averages for one or mare samples of
a polymer of interest involves the use of a
digital. computer program developed at
McMaster University (18). (The pro-
gram is available from Dr. A. E. Hamielec,
McMaster Univ., Hamilton, Ontario,
Canada.) A linear calibration is as-
sumed:
GPC ret. vol. = C, - Cnlog,, (mol. wt.)
where C, and CSare constants t o he found
Volume 47, Number 8, August 1970
via a Rosenbrock search technique such
that the difference between known and
computed molecular weight averages for
the polymer of interest is minimized.
Two approaches are suggested for situa-
tions where a linear calibration does not
adequately describe the true column re-
sponse:
(a) Modify the program t o compute
polynomial expressionssuch as:
GPC ret. vol. = C, - Cnllog,~(mol. wt.)]
- Cs[log,~(mol. wt.)]' ...
-C,[log,~ (mol. wt.)Jn-1
and have the computer search for n con-
stants, given n pieces of data for n / 2 to n
broad MWD polymer standards.
( b ) Search for linear segments that can
he put together t o yield a non-linear cali-
bration curve (see Fig. 2).
Figure 2. Calibration curve condructed from
lineor regrnenb
The most basically sound and uni-
versally applicable approach should have
as its foundation that parameter on which
GPC separatiansare based, namely, effec-
tive molecular volume in solution. It has
been shown, for s m d , non-polymeric
molecules (19) that molar volumes corre-
late well with GPC retention volumes.
Similarly, GPC retention volumes of a
variety of polymers have been correlated
with their hydrodynamic volumes as
measured viscametrically ($0); that the
hydrodynamic volume is, indeed, a uni-
versal parameter regardless of chemical
type or molecular shape was admirably
shown with such suhstanees as linear. star-
shaped, and comb-shaped polystyrenes,
polymethylmetbacrylate, polyvinylchlo-
ride, graft copolymers, polysilotanes, and
more. Thus, a plot of the logarithm of the
hydrodynamic volume (mol. wt. X in-
trinsic viscosity) versus GPC retention
volume yields a single line regardless of
polymer type with only minor deviations.
This universal calibration apparently
"corrects for" such solution phenomena as
polymer-solvent interactions, hetero-con-
tact interactions of copolymers, etc., since
the measured hydrodynamic volumes of
polymers in solution include the effects of
such interactions.
VII. Estimation of Molecular
Weight Distribution
Conversion of raw GPC data (typically,
(Continued on page A606)
/ A505

Chrnkal Instrumentcrtlon
integral equation that describes the effect
of instrumental spreading:
solution by minimization
herip.: of equation, 11191 nrr u d u l for m t l -
v r r l i n ~I:!'(: dnts to the variou. nwrnaurt
of tlw r d t c u l n r wrinhr di~triburiontry the
'<Q -fador method are:
11

the height of the GPC curve at several solution by Fourier transform

points dong the molecular size, or weight,
axis) t o meaningful molecular weight
averages sometimes first requires correc-
tion of the dat,a for that part of the axial
dispeYsion of the sample that occurs in the
column that is not due t o the GPC molecu-
lar size fractionation process. Many
attempts have heen made t o mathematic-
ally correct for such axial dispersion and
imperfect resolution in GPC columns.
None have been universallv auulicable or
yielded consistently satisfac&y results.
There are a t least three methods that have
been used by various workers t o salve the
solution by polynomial represent*
tion of the chromatogram.
A detailed discussion of these correction
techniques is beyond the scope of the
present paper. The reader is referred, for
further details, t o the work of Tung (&I),
Pierce and Armonas (&&), Hess and Krati:
($39, and others ($4-88).
For many polymer systems of interest,
however, sufficiently accurate and usable
molecular weight averages can be calcu-
lated without the necessity of resorting t o
correction of axial dispersion effects. A

Table 4. Available Polymer Standards

a= a" a" Remarks Distributor

where
am
= numbwaverage
- molecular
weight
aW= weight-average molecular
weight
= viscosity-average molecu-
lar weight
as= "z"-average molecular
weight
as,,= "z+l"-average molecular
weight
H~ = height of the GPC curve
from the baseline a t a
given value of A;.
D. Linear Hgdrogenated Polybutadicne A i = size, in angstroms (based on
82,000 ... #NL-14095-21 polystyrene calibration)
141,000 ... #NL-140!13-16 of polymer molecules elut-
126,000 ... #NL-14005-11 ing from the column a t 8.
15X,000 ... #NL-14091,-25 retention volume. This
is an extended chain
Peak M.W. = 7,000 "(I& from GPC) length.
Peak M.W. = 17,900 (Data from GPC) K and a = Mark-Houwink viscometric
Peak M.W. = 27,700 (llsta, from GPC) constants.
Peak I\I.W. = 32,000 (Data from GPC) [,I = intrinsicviscosity.
Penk M.W. = 3f,,700 ( I l n t ~irom
, GPC)
Penk h1.W. = 41.700 ( l h t a from GPC) If a direct calibration based on molecular
F. Pol~m~thylrncthacrylatc weight is available for the polymer whose
49,200 19,400 ... DPI #603X; R&H #MS-1034 (6)(7) molecular weight averages are t o be com-
11.5,000 48,600 ... 1)PI #6036; Il&H XMS-1030 (6)(7) puted, then one can replace Ai in the equa-
267.000 12.5.000 ... D P I #6041; H&R #MS-1036 (6)(7) tions with Mi (molecular weight of poly-
mer molecules eluting from the column at
-700 a given retention volume) and then elimi-
-1, ,500 #700-2 nate the "&" factor from each equation,
-3,650 @&'<1.4 #700-3 sinceMi = AiQ.
-4,830 Mw/ACn<1.3 $7004 A computer program has been written
>6,000 .., g700-5 far both the G.E. and Com-Share time-
Prwsnre Chem. Co., 3419-25 Smallman St,., Pittsburgh, Pa. 15201 sharing computer sysbems which computes
(1) molecular weight averages according t o
I l o y Chem. Co., Midland, Xlch. 48640
(2) the above equations. Additionally, it
(A) Nahonal Bureau of Sl,andards, Wnshingt,on, 1). C. 20234 converts these to average degrees of poly-
(4) Ar-llo Lahs, Inc., 1107 W. Jefferson St., Jaliet,, Ill., 60434
( 5 ) Phillips Petroleum Co., P. 0.Box 968, Phillips, Tex. 78071 merization (DP) by dividing the molecular
(6) Rohm and Haas, Independence Mall, Phil*., Pa. 19105 (Continued a page A608)
(7) The Polymw Bank, Distillation P~.odoctsIndustries, Rochester, N.

A506 / Jovrnol o f Chemical Education

 *** RESULTS OF GEL PERMEATIBN CHRBMATBGRAPHIC A N A L Y S I S ***
6PC CURVE NUMBER 7 0 0 1 9

weight averages by the formula weight of a SAMPLE CALCULATIBhl**I30 MARCH I 9 7 O t t l J . CAZES

single repeating monomer unit. A sample
tabular output, via Teletype, is given in C A L I B R A T I B N NO.: 1-20-69
Figure 3. The computer also provides a. SBLVENT: THF FLBW: 2 ML./MIN. T E M P - I 4 0 DEG. CENT.
normalized differential plot and s. normsl- CPLUMNS: 1 0 1 3 ~ 1 . 5 X I O t 4 ~ 1 0 1 5 ~ 5 X I 0 ~ 6
ired integral plot on the teletypewriter PBLYSTYRENE STANDARDS
whenrequested t o do so.
A more extensive computer program has
been described (92.9) which first applies MBLEC'JLAR DEGREE RF
mathematical smoothing to the data.paints WEIGHT + PBLYMERIZATIBN
and then computes molecular weight
averages from the smoothed data. I n
addition to the tabular output this pro-
gram also produces several plots and har-
graphs. A useful feature of the plotting
routines of this program is the ability to
compute the plotting scales so that the f e e
tures of interest in the normalized GPC THE I N D I C E S ARE:
curves completely fill the page. Details M Z + l /MZ
are then clearly observable. 3 I 1.98258 0
The validity of the molecular weight in-
VISCBMETRIC CBNSTANTIKSI 4.0000E- 5
formation estimated from GPC data de- VlSCBMETRlC EXPBNENTIASI 7.5000E- I
pends, at least in part, upon the reliability INTRINSIC VISCBSITY = 5.3429E 0
of the calibration curve employed, the Q FACTOR = 4.1400E I
complexity of the polymer system being MBNBMER MBL WT.= I.0400E 2
studied-complexity both in terms of
chemical composition and physical poly- * I F Q s l r T H E N MOL WT AVGS SHOWN ARE ACTUALLY MBLECULAR S I Z E S
dispersity, and upon whether axial disper-
sion corrections have been applied. Often,
the presence of very polar moieties in poly- MOL. MOL. DIFF. CUM. DEGREE 0 F
mer molecules results in complication of HEIGHT SIZE WEIGHT PCT. PCT. PBLYMER.
the separation process by observable ad-
sorptive effects (through solutesolute,
solvenholute, and solute-column suh-
strate interaction). For "well behaved"
system, however, the data given in Table
5 are typical. Accuracy of the GPC-de-
rived higher moments of the MWD is
quite good. Deserving of special notice is
the excellent agreement of the GPC-de-
rived intrinsic viscosities with values de-
termined viscometrically. This observrt-
tion would lead one to suspect that the
availability of GPC data would eliminate
the need for carrying out viscometric mea-
surements rtt several concentrations t o
calculate an intrinsic viscosity. The pres-
ent writer has found this, indeed, to be
the case for several different polymers.
This is especially convenient when GPC
fractionation data are to be obtained for
other purposes anyway.
VIII. Fractionationof Low Molecular
Weight Substances
The current rebirth of liquid chromatog-
raphy has resulted in an awareness that Figure 3. Computer tabular output of molecular weights from GPC input doto.
GPC can satisfy a need that is not satisfied
by other liquid chromatographic methods bio-medical origin under the name "gel of-carbon chain length" based on a C-C
(e.g., classicd approaches to adsorption, filtrtttion." However, relatively few at- bond length of 1.54 angstrom. Thus, if
liquid-liquid partition, and ion exchange). tempts have been made t o put such obser- the contributions of all of the atomic
It offers a. new "handle" for separating vations on a theoretically and predictably groupings (C-atoms, hetero-atom, phenyl,
molecules-size and shape! The early sound basis. More often than not,! gel hydroxyl, carbonyl, etc.) are added to-
development of GPC as a polymer frac-, filtration column response curves (callbra- gether, the resultant effectiue chain length
tionation tool came about because it fdled Lions) have been reported on the basis of should he related to the GPC retention
a specific need, and a det,ector was avail- molecular weight. Such correlations hold volume for any given substance.
able that produced a response that could true only for substances of similar chemical Molar volume was later suggested (51)
he equated to amount of polymer and composition and molecular shape. as a more suitable and generally applicable
could readily be converted to molecular Hendrickson (SO), in 1966, presented a paramet,er for correlation with GPC data.
weight information. This is not generally study of GPC fractionation of 130 low After dl, since the basis for the separation
true for low molecular weight compounds molecular weight compounds with cross- is the molecular volume and not a linear
and development here has been slow, that linked polystyrene gels. He suggested dimension for a molecule, one should not
is, until recently. that the retention volume of a given eom- be surprised by such a suggestion. Molar
Of course, numerous sizebased separa- pound could be predicted on the basis of volumes can he readily calculated from
tions h w e been reported for substances of an empirically derived "effective number- (Cmtinwd un page A510)
 Chemical Instrumentation
Table 5. Validity of GPC Data for Pol~stvrener
Pressure Chem. Co., Pittsbrw_h ., Pa. 15201.
L. A. Papasian, private communieatian.
' At 40°C with four columns (lo3, 1.5 X 10: lo6, and 5 X
2 ml/min. flow-rate.
experimentally measured densities (SZ)
or estimated at any tempereture of interest
via,critical properties (33).
Solutesubstrate adsorptive effects are
often observed with small molecules, even
when the comparatively nonpolar poly-
styrene gels are used. Interestingly, GPC
data for the homologous series of mona-
carboxylio acids from acetic to stearic
show that, as molecular weight increases,
apparent adsorptive effects diminish; this
is as one would expect, since, as methylene
groups are added, the resultant carboxylic
acids hecome more "hydrocarbon-like"
and less "acid-like."
Solventaolute interactions can readilv ,
be i,hcenwI, sirtcc the species thnt result
Imm such interactions have largcrcflrcrivr
volumes than either of thr 'rcncrnntr"
and are, in fact, observed to emerge from a
GPC columnearlier than thereactants.
The molar volume concept can be fur-
ther used to make stereochemical awign-
ments. For example, one would predict,
an the basis of estimated molar volumes,
that the trans-1,Zdimethyl-cyclohexane
should emerge from the GPC column
sooner than the &-isomer. This is what
is actually observed.
GPC can he used as a. "yardstick" for
directly measuring the effective volttmes of
small molecules in solution. I t has been
called a "liquid-phase size spectrometer"
( 4 ) . With it, one crtn directly study the
fundamental forces involved in interm-
tions of the type:
( a ) dipoledipole
( b ) dipoleinduceddipole
A510 / Journal of Chemical Education
(c) ion-pairs
(d) complexes (including n-complexes)
lo61)in series, with THF,
and others, where the attractive forces are
strong enough t o survive entering and
leaving the gel pores (56).
IX. Recycling Chromatography
Ofrm, w e is confronrrd w i ~ ha ~ituation
whew adequntc reiolutir~~ of u given mix-
turr mnl.ot be o b f a i ~ ~WWI
d thotqh C:IPC-
ful attention has been given to the proper
selection of column substrate, instrument
design, and operating conditions. A
common answer, at this point, is to in-
crease column length by adding more col-
umns in series with the original one. This
approach works well only up to a point.
I t is sometimes not a pracbicsl approach
either because of the prohibitive expense
of adding more columns or because the de-
sign features of the instrumentrttion will
not permit it (possibly because of space
limitations or because of the unusually
high pressure that would be encountered
when trying to achieve a. reasonable flow
rate through avery long series of columns).
Another, more practical and generally
applicable approach, would be to pass the
sample several times thmugh the same cob
umn. This technique, first reported by
Porath (S6), in called "recycling chroma-
tography!'
Following its introduction into the
chromatographic system, a sample is
passed repeatedly (recycled) through the
same column set until the components of
interest have been separated. It is con-
venient to direct the partially resolved
(Continued on page A618)

ChemicaI Instrumentation
mixture through the detector after each
cycle for direct observation of the extent
of separation. Components of the sample
mixture can be removed as they are re-
solved while the remainder is further r*
cycled. This cyclic process can only be
continued until
l the fastest moving component catches
up to the slowest moving component from
the previous cycle ("the snake bites its
own tail"), or,
t h e band widths increase to the point
at which resolution suffers (the dl-too-
familiar law of diminishing returns!).
Utility of the recycle technique to GPC
separations has been discussed by Bom-
baugh (57). Figure 4 shows the utility
of recycling chromatography in the frac-
tionation of a mixture of two hydro-
carbons. At least two manufacturers
offer equipment designed for the applica,
tion of recycling (Waters Associates and
LKB).
X. Applications
The GPC applications that have been
described we far too numerous and varied
to be covered here, and a detailed discus-

c11
RECYCLE SEPARATION OF ANALIOPER. CONOlTlONS
HYDROCARBONS I C l i 6 Cl4I COLUMNS: FOUR.LFWT
STYRAGEL. SmA

SAMPLESIZE: IOMlCROLlTEAS

c14
c14

I I I I
150 315 480 630 8W
ELUTION VOLUME. MILLILITERS
Figure 4. Recycle reparation of hydrocarbonr.

A512 / Journal o f Chemical Education

 Chemical 1nrh.urnentcrtion

sion of them could easily serve as the sub-

ject of a rather large monograph. GPC
has permeated (no humor intended here)
virtually every facet of the chemical indus-
try and has provided a. new "handle" by
which molecular mixtures can be manipu-
lated; it has been extended t o the separa-
tion of very large and very small mole-
cules. Table 6 is a listing of some of the
kinds of miltorials that have been frsc-
tionilted with GPC together with s n indi-
cation of applicable solvents.
XI. Conclusion
The past few years have seen GPC ini-
tially become accepted sr a "way of life"
for the fractionation of polymers and for
the estimation of their molecular weight
distributions. I t has more recently been
applied t o the solution of problems involv-
ing low molecular weight, nonpolymeric
substances. Where will GPC go from
here?-We will see it applied, with ever
increasing frequency, t o the solutiop of
more and diverent separation problems as
x general $separation tool, not only for high
moleeul~rweight substances, but in every-
day applications involving materials cov-
ering t,he entire molecular weight range
from 10 t o lo'! Certainly, it will find an
immediate niche where thermally unstable
low moleeuler weight mixtures are in-
volved (where gas chromatography is in-
applicable) or where siee separation offers
an insight, into the qualitative nature of
unknown substances (cj. above discussion
of the correlation of GPC retention vol-
umes with molar volumes).
The recent and future development of
low cost instrumentation will put GPC, as
well as other liquid chromatographic tools,
in the quality control laboratory and i t
will, no doubt, find applications in plant
monitoring and control. When low cost,
high efficiency column substrates become
available, GPC will find an increased num-
ber of applications in the chemical process
industries. Some molecular siae-based
sepamtions artre being used, on an industrial
scale, in the pharmeeeutical industry
where s. relatively high conversion cost can
be ju~tified. ltecycle operation will im-
prove the economies, but only a drastic de-
crease in the cost and efficiency of the sub-
strate will make sny real impression in this
area.
The wide variety of applications and in-
genuity exhibited by workers in the solu-
tion of 'ieal" problems with GPC support
the present writer's feeling that when con-
sidering GPC as an avenue of approach
one should not ask, "Am I doing it right?"
but rather he should ask, W i l l i t do the
job?"
References
17. Punooa.J. R.,J n . . m n MATE,R.D.. 3.P.11)-
me, Sei.. A l , 6 , 243 (1968).
18. B A L ~ ES., T..et al., Ind. &no. Clem.. Plod.
Rcs. Dcuel., 8 (1). 54 (1969).
19. CASES.J.. AND G*RKILL,D. R..Sep'n. 5ci.. 2 ,
421 (1967); ibid.. 4, 15 (1969).
20. Gnusrs~c,Z., d el., J . P o l y m r Sci., B5, 753
(1967).
(Calinued a page A614)

Volume 47, Number 8, August 1970 / A51 3