CALIBRATION OF GPC SYSTEMS

Gel permeation chromatography separates sample molecules by differences in effective molecular size in solution.
Separation is accomplished as a result of the pore size distribution in the packing material. Molecules too large to
penetrate any of the pores are unretained by the column packing ( totally excluded ) and elute first . Slightly
smaller molecules penetrate some of the pores, are retained on the column, and elute somewhat later. Molecules
small enough to penetrate all of the pores are retained on the column longest and elute last. The consistency of
this technique allows determinations of weight- average molecular weight ( M y ), number-average molecular
weight ( Mj ), and molecular weight distribution, and dispersity for polymeric materials. ^
^
In many instances , it is not necessary to obtain If it is deemed necessary to obtain numerical data ,
numerical values for these parameters. Differences attention must be paid to :
in molecular weight distributions are obvious if
GPC curves are compared. Curve overlays are suffi¬ Sample preparation ,
cient for most quality control uses.
Sample injection ,

Column selection ,
ALKYD COMPARISON ON p. STY RAGEL
Calibration method ,
PACKING: /i S T Y R A G E L 103 A, 5 0 0 A , 5 0 0 A, 1 0 0 A, 1 0 0 A
SOLVENT : THF
FLOWRATE : 4 . 0 m l/ m i n Baseline determination , and
DETECTORS : R 1 4X
Computation .

H E A V Y - BO D1 1 D A L K V D
-
LIGHT BODIED A L K Y D
— * 5 \ M!’I i PHEPAH VI' ION

Sample preparation for GPC , as for any mode of

0
TIME ( min )
If obtaining numerical values for the molecular
weight averages and dispersity parameters is impor
tant , GPC can generate the necessary data . With
proper calibration procedures, statistical data very
close to that obtained by classical techniques are
generated . Usually molecular weights obtained by
GPC are relative to the calibration method employed .
The procedures given here are general approaches ;
sometimes more in -depth work is required . Refer
ences to more sophisticated calibration techniques
are given on the last two pages.
liquid chromatography , is usually quite easy. The
polymer to be analyzed is dissolved in the mobile
phase and chromatographed within the next 24
hours. Agitation ( with a magnetic stirrer or lab ¬
oratory shaker ) aids in dissolving the sample.
¬
ULTRASONIC DEVICES MUST NOT BE USED
BECAUSE THEY MAY CAUSE POLYMER DEG¬
RADATION .
After the sample is dissolved , it should be filtered
to remove any material likely to clog the columns.
Filters with pore sizes between 0.4 /mi and 0.5 /im
¬ should be used . Waters Associates’ Sample Clarifica ¬
tion Kit for organic solutions provides a convenient
means of filtering samples for the ALC /GPC 200 series

W A H R S' \ ^ Í H ¡ * i t , m Í KM ID CHROMATOGRAPHY PEOPLE

of instruments. For filtering aqueous solutions for use
with the water-wettable GPC packing materials use
Waters’ Sample Clarification Kit for aqueous solutions.
Both kits are described in DS-043.
The presence of a cross-linked microgel in a sample
that appears clear to the eye may be revealed by
filtration . If a microgel is present , it can clog the
filter and prevent the passage of smaller molecules. If
the filter clogs, excessive and increasing pressure will
be needed for filtration. Should this occur, decrease
the sample concentration and / or use a prefilter.
For most polymers a sample concentration of
0.25% will minimize the possibility of viscous finger ¬
ing caused by high concentrations of polymer , par¬
ticularly high molecular weight polymers. Viscous
Fingering can result in changes in peak retention
volumes and band shapes.
Along with sample concentration , mobile phase
consistency is an important consideration in sample
preparation . All solvents require agitation. This
may be supplied by a magnetic stirrer or by a large
mixing chamber , depending upon the GPC instrument
used . A list of the most commonly used GPC sol¬
vents is given in the table below.
S AM P I T. ] INJECTION
Samples can be injected through a fixed volume
valve and loop as in traditional GPC or the Model
U6K Universal Injector may be used . For high¬
speed / high-resolution GPC , on /aSTYRAGEL? the
Model U 6K must be used . For a complete descrip¬
tion of this injector , refer to DS 036.
Before injecting the sample , however , a stable
baseline must be obtained .

An unknown sample should be chromatographed COLUMN SELECTION

at successively decreasing concentrations until con ¬
stant retention volume and chromatographic band
shape are observed . However , a balance must be
made between a concentration that avoids viscous
fingering and one that provides adequate detector
response. A response that approximates that obtain
ed with the calibration standard is ideal .
Use of the proper columns is essential in obtain ¬
ing optimum separations. Columns should be chosen
so that their calibration curves are linear over the
polymer distribution range. Samples may have to be
¬ chromatographed initially on a very broad range
column set in order to estimate the range of interest .

MOST COMMONLY USED GPC SOLVENTS

BOILING REFRACTIVE
SOLVENT POINT DENSITY VISCOSITY INDEX
(°C)

TETRAHYDROFURAN 66 0.889220 0.51 @ 25° 1.407020

1, 2,4-TRICHLOROBENZENE 213 1.462325 0.50 @ 135° 1.552425
TOLUENE 110.6 0.86620 0.52 @ 25° 1.489324
mera — CRESOL 202 1.03420 16.9 @ 20° 1.534820
/V, A/- DIM ETHYL FORMAMIDE * 153 0.944525 0.90 @ 25° 1.4280325
WATER t 100 0.999920 1.0 @ 20° 1.3330020
CHLOROFORM 61.2 1.48920 1.447620
1 , 1 , 2, 2 TETRACHLOROETHANE 146.5 1.5865825 1.4941920
TRIFLUOROETHANOL 73.6 1.382325 0.9 @ 38° 1.290720
*DMF should not be used with packings with pore sizes less than 103 A
°.

^ Water should not be used across STYRAGEL or /xSTYRAGEL .

 Such a broad range column set is fashioned by join ¬ TYPICAL GPC CALIBRATION CURVE
ing several columns of different exclusion limits and
different linear ranges. However , for optimum resolu ¬ n5
tion , a column set should be chosen so that each col¬
umn plays an important part in the separation . The
range of the column set should only be broad enough
to cover the range of interest . n *

GALIIlKATlOiN MKTHOl) Ñ „3 &

to
cc
EXCLUSION SELECTIVE PERMEATION
Both direct and indirect methods of calibrating
GPC columns have been used to obtain molecular a y
weight averages. With polymers for which narrow § n2
K

molecular weight distribution standards are commer¬

TOTAL
cially available ( e. g. , polystyrene ) , a direct calibration PERMEATION
on the basis of molecular weight can be made . If
standards are not commercially available , they must id \

either be prepared or an indirect method used .

V¡ -
Vo ELUTION VOLUME ( Ve) VT
Hired Calibration

Prepare fresh calibration standards in the same man ¬ ANALYSIS CONDITIONS

ner as samples, but use 0.1 % concentrations. (Stand¬ SOLVENT : THF

ards available from Waters Associates are listed in the COLUMNS: 106, 105. 104, 103
table on the following page.) As many standards as
TEMP: °
30 C
FLOW: 1 ml / min. 910 p /ft
possible should be run in the range of interest. If the
sample is totally unknown, it may be necessary to chro¬ 1.8x10
®

1.8 x 10® 48,000 A

matograph it first to determine the estimated range. 867 K 20, 200 A
867 K 9, 800 A
173 K 4,160 A
Be sure each injection point is clearly marked . LiJ 104 2.360 K
N
With the Model U 6 K Universal Injector this is done CO
1, 220 A
20 K 480 A
automatically .
< 98 K
10 K 244 A
117 A

Measure the retention volume or time of each 3

LU 51 K
p - DCB 7.7A

standard from the start of injection to the maximum o 103

of the chromatographic peak . Plot this value (on the 20 K

linear x-axis) on semilog paper vs. the corresponding 10 K

value (on the logarithmic y-axis) of the peak molecular

5K
weight , peak M (listed on the standard ), for each stan¬ 102
dard. Draw a smooth curve through the points plotted
to obtain the calibration curve.

26 28 30 34 36 38 £) o- DCB
COUNTS
Indited Calibration

If narrow molecular weight distribution standards

of the polymer of interest do not exist , such standards
can be prepared by GPC fractionation of a broad
molecular weight distribution sample or an indirect
method of calibration can be applied.
Stylized and actual GPC calibration curves are shown
above. It should be emphasized that each calibration curve
is related to a unique set of columns. If even one column of
that set is replaced — even by another with the same exclusion
limit -the whole set must be recalibrated .
 sample
POLYSTYRENE is then calculated for the polymer according to the
equations given in the section on computation. These
Nominal molecular size averages are then converted to mole¬
Catalog Weight Average
Number Molecular Weight
Suggested cular weight averages by a Q factor, /. e. , a conver¬
Concentration
Mw % sion factor determined for the polymer of interest.
26971 2,000
^ ^ = QAj^
25169 4,000
0.25 - QA y , M
0.25
25171 10,000 0.25
25168 20,000 0.25
25170 80,000 0.10 Q is experimentally determined by obtaining GPC
41995 100,000 0.10 curves for a number of samples of the polymer of in ¬
41984 200.000 0.10
25166 400,000 0.05 terest whose molecular weight averages are known
25167 700,000 0.05
61970 2,500,000
( via light scattering , osmometry , viscometry , etc.)
0.05
41746 4,000,000 0.05 Q, the number of molecular weight units per angstrom
of molecular size, can then be calculated by the follow¬
POLYPROPYLENE ( GLYCOL ) ing equations:
Nominal
Catalog
Number
Weight Average
Molecular Weight
Suggested
Concentration
Q= Mw / Aw = MN / AN
Mw %
Similar Q values should be obtained from both
41993 800 0.25
41994 1.200 0.25 equations. If not , the Q-factor method is not
41985 2,000 0.25 applicable , and another calibration method must
41983 4,000 0.25
be chosen.

A GPC Calibration Kit ( request DS 045 ) is also available
from Waters Associates. The kit contains standards , syringes,
and semilog graph paper .
One of these indirect methods which can be applied
successfully is the “ Q- Factor Method ” . Briefly, it
consists of estimating the molecular size averages of
the polymer from its GPC curve and a molecular size
calibration curve for polystyrene. The polystyrene
calibration curve is prepared as described in the
section on direct calibration except that the MOLE¬
CULAR SIZE (extended chain length in angstroms)
is plotted instead of molecular weight — molecular
size is given on the standard container. Molecular size
When using the Q - factor method, the analyst
should realize that wide deviations in results will
result if his samples are of copolymers which vary in
composition , blends of two polymers where the blend
ratios differ , or polymers where the degree of branch¬
ing or cross-linking varies from sample to sample.
BASELINE DETERMINATION
On the chromatogram , mark the retention volumes
Vt
for the start ( Vj) and finish ( ) of the polymer chro¬
matogram . Draw a linear baseline from before Vj to
after Vj.. If the baseline is drawn from Vj to Vj., small
rises may be overlooked .

33
POLYSTYRENE |/\34
polystyrene sample.
/ \ PACKING : STYRAGEL
/ l SOLVENT: THF
/ \ FLOWRATE : 2.0 ml / min
32 /1 l DETECTORS: Rl : 8x
135
HEIGHT 82 mm
\ 36
31/ \\37l 38
1 HT > 42
VI
-uiTi 11 I. I 11 ixi
28 2930 '
MINIMJ \Jr
Vi BASELINE Vt
FIG. 3
COM P i l i ATI O PS Once the molecular size averages have been deter¬
mined , multiply them by the previously determined
Once the baseline has been determined , measure Q-factor to obtain the corresponding molecular
peak heights to three significant figures for about 15 weight averages. Dispersity then is the ratio ,
equally spaced points along the GPC curve . Tabulate MW/ MN.
these data under the headings shown below.

1 2 3 4 5
RETENTION HEIGHT CHAIN LENGTH
VOLUME OR ( mm ) OR MOL WT Col 2 /Col 3 Col 2 X Col 3
COUNTS

If calibrating directly , list molecular weights in EXAMPLE

column three and determine molecular weight av¬
erages by the following equations:
The chromatogram on the preceding page is of a
polystyrene sample. Since narrow molecular weight
Mjyj = 2 Column 2/ 2 Column 4 distribution standards are available for this material,
a direct calibration method was chosen. Eight poly¬
= 2 Column 5 / 2 Column 2 styrene standards were run and the calibration curve
shown below obtained.
These equations are based on the theoretical
equation :
(masa total de la población) POLYSTYRENE MOLECULAR
(Fig. 4)
2 NjM:
b WEIGHT CALIBRATION CURVE
M* o 1 1

2 NjMj b 1 '
(generación de sistema polidisperso discreto)
Q 867 K

where b is a constant , any whole integer , and M* = 411 K

MN when b = 1 , when b = 2, and Mz when

Mw
b = 3. 173K
98 K
The elution volume is directly relatable to Mj, the
51 K
molecular weight , and the amplitude of the curve is
directly relatable to Nj, the number of molecules
20 K
present at that molecular weight.
C3 104 10 K
If using the Q-factor for indirect calibration , list £ 5K
chain length in angstroms in column three and deter¬ DC
<
mine molecular size averages by replacing M in the
preceding equations with A.
8
LU

° 103 SOLVENT:
COLUMNS:
THF
5 x 10®, 7 x 105,
Ajy[ = 2 Column 2 / 2 Column 4 1.5 x 10* 103,
700 A
Ayy - 2 Column 5 / 2 Column 2 TEMPERATURE : 22°
UCC
FLOW RATE : 2 ml / m in o- DCB
102 STANDARDS USED: 867 K 20 K
i b 411 K 10 K
2 N: A; 173K 5K
0
A* _ _
98 K o- DCB
¿ TT71 51 K
N:1 A1:
0

44
then A * = AN when b = 1, Aw when b = 2 , and COUNTS
A when b = 3.
^
 _
Values obtained from the chromatogram and from the calibration curve were then entered on the sheet
shown here . was calculated to be 13,000 and Myy 2 ,068 ; D = 2.5 .
Mn=2/4
(Fig. 3/Fig. 4)

ft 1
WATERS ASSOCIATES INC.

CALCULATION SHEET
Ni*Mi
2
interpolado
Mi
3
Ni
4
Mw=5/2

Ni*Mi^2
5
RETENTION HEIGHT CHAIN LENGTH
VOLUME OR ( mm ) OR MOL WT Col 2/Col 3 Col 2 X Col 3
COUNTS
30 1.0 340 K 0.000029 340,000
31 17 162 K 0.000105 2,754,000
32 82 77 K 0.001065 6,314,000
33 194 35K 0.005543 6,790,000

34 180 19 K 0.009474 3,420,000

35 90 12 K 0.007500 1,080,000
36 41 7.8 K 0.005256 319,800
37 26 5.2 K 0.00500 135,200
38 13.5 3.6 K 0.003750 48,600
39 8.5 2.0 K 0.004250 17,000
40 6 1.3K 0.004615 7,800
41 2.5 820 0.003049 2,050
42 0.5 510 0.000980 255

662 0.050616 21 ,228 ,705

MN = 2 Col 2/ 2Col 4 Mw = 2 Col 5/ 2 Col 2

MN = 662/0.05062 = 13,000 Mw = 21 ,228,705/ 662 = 32,067.53

The calibration techniques described here are general approaches. The following list of references will be use¬
ful if more sophisticated calibration techniques are required .
 REFERENCES
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Educ
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¬
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matography , Monaco , 1969.
¬

ments on Linear and Branched Polystyrenes of Known

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Structures ” , /. Chim. Phys., 63, 1507 (1966 ).

lecular Weights by Gel Permeation Chromatography. Un ¬
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¬
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.
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¬
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