2414
Anal. Chem. 1983, 55, 2414-2416
Calibration of Size Exclusion Chromatography Columns for Molecular Weight Determination of Poly(acrylonitri1e) and Poly(vinylpyrro1idone) in N,N-Dimethylformamide Sadao Mori
Department of Industrial Chemistry, Faculty of Engineering, Mie University, Tsu, Mie 514, Japan Calibration curves for poly( acrylonitrile) (PAN) and poly(v1nyipyrroiidone) (PVP) were constructed by using polyethylene oxide (PEO) as primary standards. Molecular weight of PEO at retentlon volume I , (MA),,was converted to molecular weight of PAN or PVP, (M&, by the equation M, = 8(MA);. Constants 8 and f were calculated by using two polymer samples of PAN or PVP of known molecular weights and the PEO Calibration curve. Constants 8 and t for PAN were 12.76 and 0.866, and those for PVP were 3.15 and 0.959. The Mark-Houwink parameters for PAN and PVP were estimated by the SEC method Independently of those for PEO. The relationships between [VIMand retention volume for those polymers suggest that PVP elutes under the mechanism of size excluslon, while PAN suffers the secondary effects even if LlBr Is added to dhnethylformamlde (DMF). Elution behavior of PAN and PVP In DMF and the effect of adding LlBr to DMF are discussed.
Poly(acrylonitri1e)(PAN) and poly(vinylpyrro1idone) (PVP) are important polymers as synthetic fibers and as capsular material for medical use, respectively, and accurate determination of molecular weight for these polymers is of primary importance for process and quality controls. Size exclusion chromatography (SEC) is a widely used analytical technique for the characterization of synthetic polymers. Most commonly employed solvents for SEC are tetrahydrofuran (THF) and chloroform, both of which, however, do not dissolve these two polymers. N,N-Dimethylformamide (DMF) has found widespread application in SEC analysis, but it has also been noted that peculiarities of elution behavior for such polar polymers are directly related to the use of DMF. The addition of lithium bromide (LiBr) to DMF has been attempted to eliminate these peculiarities (1-3). In SEC, the construction of a calibration curve for the column system is required at first and a series of polystyrenes (PS)of relatively monodisperse character are mostly employed for this purpose. Because of the poor solubility of PS in DMF, the retardation of retention volume (V,) for PS was observed (3), and as the result of the retardation, the divergence between the universal calibration curves for PS and PAN obtained by plotting [VIMand V , ( 4 ) is a matter of course. The abnormality of elution of PS in DMF may be explained by partitioning or adsorption of PS on the gel. Polar polymers such as poly(ethy1ene oxide) (PEO)must be employed as standards. Comparison of the PEO calibration curve in DMF with one obtained on the same column set in THF revealed no difference between them (5). To calculate the molecular weight averages for any polymers other than PEO by a PEO calibration curve, it is necessary to transform molecular weight units of PEO in the curve to those for the polymer specified. One of the techniques used to transform the molecular weight units is so-called ”universal calibration” (6),and this method has been shown to have wide applicability. In order to aply the universal calibration method, however, it must be clear that the elution of both the 0003-2700/83/0355-2414$01.50/0
sample and the standard depends strictly on their hydrodynamic volumes ([VIM)in solution without any secondary effects, in addition to the knowledge of the Mark-Houwink parameters for both polymers. It must be examined before use whether the abnormality of elution for PAN and PVP in DMF can be completely suppressed by adding LiBr into DMF. In the previous paper (7), we proposed a method for preparing a calibration curve for a polymer where only broad molecular weight distribution samples were available. The proposed method requires neither prior knowledge of the Mark-Houwink parameters nor the assumption of any formula for the calibration curve. Retardation of elution due to secondary effects (5) will not disturb the application of this method. This paper presents the calibration of SEC columns for PAN and PVP in LiBr-DMF using PEO as the primary standard. Adaptability of the Universal calibration method to PAN and PVP is also discussed.
THEORY Let us first assume that the molecular weight (MB);of a species of polymer B is related to the molecular weight (MJi of a species of polymer A eluting at the same retention volume i by the expression (MB)j =
S(M.4);
(1)
where s and t represent constants (7). Let polymer A be a PEO standard experimentally used to construct a primary calibration curve and polymer B be a polymer requiring analysis (PAN or PVP in this paper). Suppose that two weight average molecular weights, A7r~l and measured by an absolute method, such as light scattering, of two samples of polymer B with a broad molecular weight distribution are given (secondary standards). Let the chromatograms of the polymer samples, B1 and B2, be given by height hBl,iand hB2,; as a function of retention volume i. The weight average molecular weights of two polymers, B1 and B2, may be computed from the chromatograms hBl,i and hB2,iof the polymers and a calibration curve for PEO using eq 1and may be related to the true values measured by an absolute method by the formulas
c(hBl,is(MA)i?/ ChBl,i
(2)
MI,W= C(hBZ,iS(MA)it)/ChBZ,i
(3)
=
Combining eq 2 and 3 and rearranging we obtain
The goal of the calculation is to find a value t where both sides of eq 4 are equal and to calculate the value s by substituting the value of t into eq 2 or 3.
EXPERIMENTAL SECTION SEC measurementswere performed on a Jasco TRIROTAR high-performanceliquid chromatograph (Japan SpectroscopicCo., Ltd., Hachioji, Tokyo 192, Japan) with a Model SE-11differential refractometer. Columns were two Shodex AD-SOM/S high-per0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983 2415
i
2&
7
9
8
10 11 12
13 14 15 16 17 18 19 20
i
Retention Volume (mL)
Figure 1. SEC chromatograms of PAN obtained in DMF (a)and in 0.01 M LiBr-DMF (b). firof PAN = 485000.
t
.i , ,
, , , , , , , , , , , , , ,
10'1
$
, 7
8
9
10 11 12 13 14 15 16 17 18 19 20
11
12
13 14 15 16 Retention Volume (mL)
18
17
Figure 3. Calibration curves of molecular weight (Ad) vs. V , experi-
mentally determined for PEO and calculated for PAN and PVP samples.
J 15 , 7
8
9
10 11 12 13 14
16 17 18 19 20
Table I. Molecular Weight Averages and Intrinsic Viscosity Data of Sample Polymers sample PAN-1 -2 PVP-1 -2
Retention Volume (mL)
Figure 2. SEC chromatograms of PVP obtained In DMF (a)and in 0.01 M LIBr-DMF (b). M, of PVP = 1310 000.
formance SEC columns (25 cm X 8 mm i.d.) (distributed by Shoko Co., L a . , Minato-ku, Tokyo, 105,Japan) packed with a mixture of polystyrene gels of nominal exclusion limits of io3, io4, IO5, and lo6 A. This column is used exclusively for DMF. Columns were thermostated at 60 "C in an air oven, Model TU-100. The primary calibration curve was constructed with PEO standards (obtained Toyo Soda Co., Ltd.,) and cpmmercial poly(ethy1eneglycols) (PEG). The ratios of M,and M,, for these PEO and PEG were between 1.02 and 1.10. Polymer samples used as polymer B in this work were two PAN and four PVP. The molecular weight averages of these polymers were determined by light-scattering in our laboratory. Intrinsic viscosities of PAN, PVP, and PEO were measured using an Ubbelohde type capillary viscometer (viscometer constant was 0.00463 cSt/s and viscosity ranged from 1.7 to 5 cSt) at 60 OC in 0.01 M LiBr-DMF. The mobile phase was 0.01 M LiBr-DMF and the flow rate was adjusted to 1.0 mL/min. Sample concentrations were 0.1% (w/v) for calibration and 0.2% (w/v) for PAN and PVP. A 0.1-mL loop was used to inject these sample solutions. The number of theoretical plates was 15000 when 0.033 mL of a 1%benzene solution was injected.
RESULTS AND DISCUSSION Effect of LiBr. The elution behavior for PAN and PVP in DMF and in a 0.01 M LiBr-DMF eluent is shown in Figures 1and 2. The upper chromatograms (a) were obtained in DMF and the lower ones (b) in a 0.01 M LiBr-DMF solution. The retention volume at the exclusion limit of this column system is estimated from the data for the Shodex A80M/THF system to be about 10 mL (see also Figure 3). Portions that eluted before 10 mL may be attributed to the formation of a supermolecular structure such as aggregation or association of molecules (8),which cannot pass through some of the interstitial volume around the gel particles in addition to being unable to penetrate all of the gel pores. The addition of LiBr in DMF eliminated the multiple peaks and increased retention volume. The shapes of chromatograms obtained by adding LiBr improved as expected. No difference in retention volume and in elution behavior for PEO was observed when LiBr was added to the DMF.
Mr 485 000 333 000 1310 000 1200 000 60 000 11 000
-3 -4
1
[r)
1.726 1.254 0.652 1.350 0.207 0.0586
Table 11. The Values o f t and s and the Recalculated Molecular Weight Averages for PAN and PVP PAN t = 0.866; s = 12.76 PVP t = 0.959; s = 3.15
.
sample PAN-1 -2 PVP-1 -2 -3 -4
-
Mr 484700 332500 1336800 1199600 60 000 9 500
-
Mn 122100 121800 214000 243500 27000 4200
ii?,/M, 3.97 2.73 6.25 4.93 2.22 2.29
Calculation of t and s. The primary calibration curve constructed with PEO is shown in Figure 3. The specifications of characterized sample polymers used in this work are listed in Table I, PAN-1 and -2 and PVP-2 and -3 being used to calculate t and s. The values of MA at each retention volume i were obtained from Figure 3 for PEO (polymer A) and those of hgl and hg2 from chromatograms of PAN-1 (polymer Bl) and PAN-2 (polymer B2) or PVP-2 and PVP-3. The constants t and s in eq 1,calculated from eq 4 and 2, are shown in Table I1 with the recalculated weight average molecular weights for PAN and PVP in addition to number average molecular weights as reference. The agreement was reasonable for PVP-1 and -4.The calibration curves for PAN and PVP, calculated by using the constants t and s in Table I1 and eq 1,are shown in Figure 3 along with the experimental PEO calibration curve. Mark-Houwink Parameters. The Mark-Houwink parameters for PEO in 0.01 M LiBr-DMF at 60 "C were obtained by measuring intrinsic viscosities of several PEO standards followed by relating them with molecular weights
[771PE0
= (5.00 x 10-4)~0~353
(5)
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
Because of the lack of narrow fractions of known molecular weights, another procedure was attempted to obtain the Mark-Houwink parameters for PAN and PVP. Several SEC procedures to get the Mark-Houwink parameters are proposed (9).They are based on the assumption that the product of molecular weight and intrinsic viscosity is proportional to the hydrodynamic volume of polymer in solution and that solutes which have the same hydrodynamic volume elute at the same retention volume. However, as mentioned in the introduction, though the abnormality of elution for PAN and PVP was improved by addition of LiBr into DMF, the product of molecular weight and intrinsic viscosity should not be used as the universal parameter responsible for separation unless it is guaranteed that steric exclusion is the only separation mechanism. Therefore, Mark-Houwink parameters cannot be calculated without this guarantee by using eq 8 and 9 in the literature (7) or by the method proposed by Braun (9). If a calibration curve of molecular -weiught vs. retention volume for a polymer is known, the following equation can be derived (7)
[??I = K(Ch,Mi”/ChJ
(6)
where “a” and “K”are the Mark-Houwink parameters and [q] is the intrinsic viscosity of the polymer. The height of the chromatogram is expressed as hi and Mi is the molecular weight at the retention volume i. If there are two polymer samples whose intrinsic viscosity values are known, one parameter “a” can be calculated by
-[all- - Ch.l,i~ia/Chl,L [VI2
Ch2,iMia/Ch2,i
(7)
A trial value of “a“ is assumed and the calculation of eq 7 is repeated until the value of “a” minimizes the difference between both sides of eq 7. Subsequently, a value of “K”can then be derived from eq 6. We already constructed calibration curves of molecular weight vs. retention volume for PAN and PVP (Figure 3). At this step of calculation, the knowledge of the universal calibration curve of [VIMvs. retention volume of PEO was not required, so that Mark-Houwink parameters cdculated from eq 6 and 7 are effective regardless of the secondary effects in elution. Samples used in this calculation were PAN-1 and -2 and PVP-2 and -3 in Table I and the values of Mi were taken from Figure 3. The Mark-Houwink equations for PAN and PVP thus calculated are
PAN
= (1.46x 10-6)~0397
(8)
PVP
= (1.85 x 10-4)~0.646
(9)
The relationships between [TIM and retention volumes obtained by using these equations and Figure 3 are shown in Figure 4. The curve for PVP is nearly on the same curve for PEO, suggesting the effectiveness of hydrodynamic theory, while PAN still has secondary effects to polystyrene gels. Therefore, the universal calibration method cannot be applied to the calibration of SEC system for PAN. Fortunately, our method can be applied to such systems since the effectiveness of universal calibration is not required. There are several methods to obtain the Mark-Houwink parameters by SEC. Most of them assume the effectiveness of the hydrodynamic concept. One attempt is the calculation of the parameters from eq 5 and the respective s and t values for PAN and PVP and gives PAN
K = 3.87
X
lo*
a = 0.9088
PVP K = 6.910 X a = 0.7236 These values are obviously different from those of eq 8 and 9. However, the universal calibration curves of [TIM and
y\
I
\,
‘. ,
1o2
11
12
13 14 15 16 Retention Volume (mL)
17
18
Figure 4. Universal calibration curves of [q]Mvs. V , for PEO, PAN,
and PVP samples. retention volumes obtained by using these values and Figure 3 piled up on the curve of PEO. This observation is a matter of course, because these values of Mark-Houwink parameters are completely dependent on the PEO calibration curve and eq 5. The calculation of the Mark-Houwink parameters independently on the parameters of PEO is useful to discuss the polymer-gel interaction in addition to obtaining the precise Mark-Houwink parameters. Stability of PEO, PVP, and PAN in DMF. PVP and PAN in 0.01 M LiBr-DMF were stable at least for 3 months (the experimentalperiod in our laboratory) without any visual change in chromatograms as well as molecular weights, PEO below 100OOO molecular weight being stable, too. On the other hand, chromatograms of PEO over 150000molecular weight degraded g r a d d y with standing time without change of peak position: the intensity of the peak decreased in contrast to the peak of ethylene glycol which increased with standing time. After the PEO solution was allowed to stand for 3 months, most of the peaks of PEO disappeared. These observations may suggest that the degradation of PEO is not random cleavage but successive decomposition from the end of linkage.
ACKNOWLEDGMENT The author wishes to express his appreciation to T. Kat0 for the measurements of light scattering of the polymers and to T. Suzuki and N. Fujita for technical assistance. Special thanks go to M. Suzuki for encouraging this work. Registry No. Poly(acrylonitri1e)(homopolymer),25014-41-9; poly(vinylpyrro1idone) (homopolymer), 9003-39-8. LITERATURE CITED (1) Cha, C. Y. J. folym. Sci., fati B 1969, 7, 343-348. (2) Hann, N. D. J . Polym. Sci. W77, 15, 1331-3339. (3) Dubin, P. L.; Koontz, S.; Wright K. L., I11 J. folym. Sci. 1977, 15, 2047-2057. (4) Kranz, D.; Pohl, H. U.;Baumann, H. Angew. Makfomol. Chem. 1972, 26, 67-84. ( 5 ) Matsuzaki, T.; Inoue, Y.; Ookubo. T.; Tomita, B.; Mori, S. J. Liq. Chrmtogr. 1880, 3 , 353-365. (6) Grublslc, 2.; Rempp, P.;Benoit, H. J . folym. Sci., f a f f6 1967, 5 , 753-759. (7) Mori, S. Anal. Chem. 1961, 53, 1813-1818. (8) Coppola, G.; Fabbri, P.; Pailesi, B.; Bianchi, U. J. Appl. folym. Sci. 1972, 16, 2829-2834. (9) Braun, G. J. Appl. folym. Sci. 1971, 15, 2321-2333.
RECEIVED for review June 20, 1983. Accepted September 6, 1983.
