ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
important that regulatory officials seeking to establish effluent standards for PCBs in discharge media such as paper mill effluent take the findings of this and other related studies into consideration, so that the standards may be enforced rationally in light of analytical variability.
ACKNOWLEDGMENT The efforts of the participating analysts in this study are greatly appreciated, as is the cooperation of the paper manufacturer that assisted in the collection of the effluent used for the comparison study. S. Kleinert and T. B. Sheffy of the Wisconsin Department of Natural Resources (DNR) and D. J. Dube of the Wisconsin Laboratory of Hygiene assisted in the establishment and coordination of the program.
(2) Easty, D. B., Wabers, B. A. Tappi1978, 61(10), 71-74. (3) Ball, J.; Gibson, T.; Priznar, F.; Delfino, J.; Dube, D.; Peterman, P. "Investigation of Chlorinatedand NonchlorinatedCompounds in the Lower
(4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
LITERATURE CITED (1) Ayer, F. A,, Ed.; "Proceedings, National Conference on Polychlorinated Biphenyls, November 1975, Chicago". EPA-560/8-75-004; Envkonmental Protection Agency: Washington, D.C.. 1976.
2239
Fox River Watershed". EPA-905/3-78-004; Environmental Protection Agency: Chicago, Ill., 1978. Fed. Regisf. 1973, 38(199), Part 11, 28758. Young, S. J. V.; Finsterwalder, C. E.; Burke, J. A. J . Assoc. Off. Anal. Chem. 1973. 56(4). 957-961. Finsterwak; C. E.'). Assoc. Off. Anal. Chem. 1974, 57(3), 518-521. Webb, R . G.; McCall, A. C. J . Chromafogr. Sci. 1973, 1 1 , 386-373. Sawyer, L. D. J. Assoc. Off. Anal. Chem. 1978, 61(2), 272-281. Sawyer, L. D. J. Assoc. Off. Anal. Chem. 1978, 67(2), 282-291. "Standard Methods for the Examinationof Water and Wastewater", 14th ed.; American Public Health Assocktion: washington, D.C., 1975; p 94. Easty, D. B.; Wabers, B. A. Anal. Lett. 1977, 10(11), 857-867. Holden, A. V. Pestic. Monit. J . 1973, 7, 37. Harvey, G. R.; Miklas, H. P.; Bowen, V. T.; Stelnhauer, W. G. J . Mar. Res. 1974, 32, 103. Pavlou, S. P.; Hom, W. Mar. Chem. 1978, 4 , 155. van Hove Holdrinet, M. "Preliminary Results of an Interlaboratory PCB Check Sample Program". In "Pesticides, Environmental Quality and Safety", Suppl. Vol. 111, Coulston, F., Korte, F., Eds.; Georg Thleme Publ.: Stuttgart, 1975; pp 51-56.
RECEIVED for review May 31, 1979. Accepted August 9,1979.
Quaternized Porous Beads for Exclusion Chromatography of Water-Soluble Polymers C. P. Talley and L. M. Bowman* Research and Development, Calgon Corporation, Post Office Box 1346, Pittsburgh, Pennsylvania 15230
The novel approach of using Ion-exchange chromatographic supports for size exclusion chromatography of neutral and cationic water-soluble polymers is presented. A quaternary ammonium group, a strong anion exchanger, has been bonded to the surface of porous silica glass in the pore sire range from 40 to 25 000 A. Chromatograms are shown of poly(2-vinylpyridine) and dextran molecules using acidic salt solutions as eluents. Molecular weight calibration graphs, theoretical plate height plots, and concentration effects are discussed in detail.
ammonium ion is an excellent choice since it creates a positively-charged surface to repel the positively-charged polyelectrolyte. The support was prepared by reacting 3aminopropyltriethoxysilane with the glass followed by reaction of the terminal NH2 group with 3-chloro-2-hydroxytrimethylammonium chloride. The procedure was similar to those described in the literature for the silanization of glasses (2, 8, 9) and is illustrated in the equations below: (1)
Glass) -Si-OH
Gel permeation chromatography (GPC), originated in 1965 by J. Moore ( I ) , has primarily focused on nonaqueous applications and many advances have been achieved. However, progress in the area of aqueous exclusion has not been as rapid owing to the many experimental difficulties encountered ( 2 ) . Aqueous GPC has been confined to neutral and anionic polymers since commercially available supports work favorably for a number of these materials ( 3 ) . Cationic polymers have not been chromatographed because porous silica glasses and cross-linked copolymer resins tend to be anionic in nature leading to solute-support interactions. G. B. Butler ( 4 ) , in 1976, chromatographed cationic polyelectrolytes on quaternized styrene/divinylbenzene supports with limited success. Some of the disadvantages observed were: (1) the swelling of the resin which was a function of ionic strength; (2) the support compression as the flow rate increased; (3) the limited porosity of these supports. The porous silica glass supports were felt to serve as the ideal substrate if adsorption could be eliminated. They have a fixed pore and particle size and can be used at the high flow rates needed for microparticulate high resolution chromatography. Attempts to deactivate the surface by silanization ( 3 ) and by the addition of cationic surfactants to the eluent (5, 6) have been unsuccessful. In this paper, we are introducing the concept of using ion-exchange supports on glass to reduce solute-glass interactions (7). The ion-exchange support chosen to chromatograph cationic polymers must be a strong anion exchanger. The quaternary
+
(CH3CH20j3Si(CH2)3 NH?
>-.
HZO
OH
I
Glass)-Si-O-Si(CH2)3 NH2 OH Si
I
OH
(2)
I
Glass)-Si-O-Si(CH,j3 NH?
I
-
OH
a)
I
(;lass)-Si-O-Si(Cli?)3
I 0
I Si
Si
I
?
(3)
I
Glass)-Si-C-Si-(CH2)3 NH?
I
+
CICHZ hHCH2
'\
(CH3)3 'IC
P
Si
>-pH
7 4
Si I 0
H
OH
I
I
1
)-Si-O-Si-(CH2)3-N-CH2 CHCH,@(CH3)3CP
I
0
I
Si
0003-2700/79/0351-2239$01.00/00 1979 American Chemical Society
VI,
2240
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
Table I. Quaternized Supports nominal sample pore size, A 40 porous silica
CPG-75 CPG-170 CPG-350 CPG-1400 BIO-2500
EM-GEL 5,000 EM-GEL 10,000 EM-GEL 25,000 CPG-1500
40 75 170 350 1400 2 500 5 000 10 000 25 000 1500
Table 11. Molecular Weights of Poly(2-vinylpyridine) particle
size, gm 39-75 39-75 39-75 39-75 39-75 39-75 79-125 79-125 79-125 5-10
sample PVP-2 PVP-3 PVP-4 PVP-5 PVP-6 PVP-7
-
% x io4
Q X
io4
3.1 9.2
2.9 8.9
46.0
44.0
23.0 2.2
20.0
60.0
5.2
EXPERIMENTAL Apparatus. The gel permeation chromatograph consisted of a Waters Model 301 Refractive Index Detector, a Laboratory Data Control Constametric I liquid pump, and a DuPont Model 834 Automatic Liquid Sampler. All columns were made using 91-cm lengths of 0.952-cm 0.d. stainless steel tubing. All connecting fittings were 0.952 cm to 0.158 cm low dead volume Swageloks with 5-pm porous frits. The aqueous GPC eluent was 0.1 N in HN03and 0.1 N in NaN03. Elution volumes were measured with a Waters Liquid Volume Indicator whose count volume was 3.6 mL. A Hewlett-Packard 3354A Lab Data System was used in data acquisition and reduction. All samples were made up at 0.2 wt % based on active polymer, and the injection volume was varied from 0.25 to 2.0 mL. Materials. The Na3P04,K3P04,NaN03, HC1, and HNO, were ACS grade purchased from Fisher Scientific Company; the 3aminopropyltriethoxysilane from PCR, Inc.; and the 3-chloro2-hydroxytrimethylammonium chloride from Story Chemical Company. All porous silica glasses described were purchased from either Electro-Nucleonics or Sigma Chemical Company and the EM-Gel supports from Scientific Products Company. All support data are listed in Table I. Dextran standards were purchased from Pharmacia and poly(4-vinylpyridine)from Polysciences,Inc. The poly(2-vinylpyridine)standards were synthesized by Pressure Chemical Company. Poly(N,N-diallyldimethylammonium chloride) is a standard product of Calgon Corporation. The quaternized poly(4-vinylpyridine) and Nfl-dimethylpiperidinium chloride were prepared at the Calgon Research Laboratories. Procedure. The bare glass supports were activated at room temperature for 24 h in 6.0 N HC1, washed with deionized water, filtered, and dried (9). To 125 cm3of activated support was added 150 cm3 of a 10% aqueous solution of 3-aminopropyltriethoxysilane. The solution was evacuated until the cessation of bubbling and placed in an oven at 75 "C for 2 h. After cooling, the silanized support was washed exhaustively with water on a Buchner funnel and dried in an oven for 12 h at 100 "C. The dried support was added to 400 cm3 of a 5 wt % solution of 3chloro-2-hydroxytrimethylammoniumchloride and buffered at pH 7.4 by a standard phosphate buffer. This solution was held at 85 "C for 12 h and the quaternized support was filtered, washed with HzO, and dried. The columns were prepared by standard dry packing techniques (8). Characterization of the glass after Step I of the reaction by elemental analysis showed that 95% of the surface had been covered. Upon completion of Step 111, a quaternary ammonium titration indicated that SO% of the surface had been quaternized. POLYMER CHARACTERIZATION The anionically polymerized, isotactic poly(2-vinylpyridine) samples synthesized by Pressure Chemical Company were
%/%in 1.07 1.04
1.05 1.15 1.10 1.15
2.0
Table 111. Plate Counts flow
Supports were prepared with Electro-Nucleonics Porous Silica Glass of pore size 40 A, Corning Controlled Pore Glass (CPG) of pore size 75 A to 3000 A, Bio-Rad Bio-Glas of pore size 2500 A, and E. Merck EM-Gel of pore size 5000 to 25 000 A. The actual pore sizes of the quaternized supports are dependent on the percentage of the surface covered and the charge repulsion between polymer and surface. Furthermore, these effects should be more pronounced in the smaller pores than in the larger ones.
-
column 700 A CPG 5000 A EM-GEL 1500 A CPG
particle rate, size, pm mL/min 39-75 75-125 5-10
1.12
1.04 1.10
initial count, plates/ meter 1900 1150 5180
characterized in our laboratory. A Brice Phoenix Model 3200 Light Scattering Photometer was used to determine weight average molecular weights a t 546 nm. The weight average molecular weights were determined in benzene a t 25 "C from a Zimm plot using the angular dependence from 35' to 135". The dnldc value, measured with a Brice Phoenix Model 5000 refractometer a t 546 nm, was found to be 0.075 mL/g. The number average molecular weights were determined in benzene a t 25 OC with a Hewlett-Packard Model 502 membrane osmometer equipped with a nonaqueous membrane (18527A) and a nonaqueous capillary (18551A). All molecular weight data are listed in Table 11.
RESULTS AND DISCUSSION Quaternized supports were prepared from each of the porous substrates described in the Experimental section. The coating procedure appeared to work equally well for all of the supports as based on initial theoretical plate counts using N,N-dimethylpiperidiniumchloride in the aqueous 0.1 N H N 0 3 + 0.1 N NaN03 eluent. Examples of initial column plate counts are shown in Table 111. The different values of plate counts listed in Table I11 can be attributed to the different particle sizes (IO). In all samples chromatographed on these columns, no peaks were noted after total penetration with the exception of an anion-exchange peak resulting from the polymer counterion. In the first 3 months of usage, all columns showed a decreased plate count followed by a leveling off value. For example, a column packed with quaternized CPG-75 and run a t a flow rate of 1.08 mL/min exhibited plate counts/meter of 1680 initially, 790 after 3 months, and 780 after 9 months. The reason(s) for this decrease is currently under investigation. The aqueous eluent chosen for use in this system was 0.1 N HN03 0.1 N NaN03. The acid was necessary to protonate the secondary amine functionality resulting from Reaction 3 and/or the residual primary amine group remaining due to any failure of Reaction 3 t o go t o completion. The highly acidic medium also tended to suppress the ionization of free silicon hydroxyl groups on the support surface which may have escaped silanization. A salt concentration of the order of 0.1 N or greater was necessary to reduce the polyelectrolyte effects (12, 14) exhibited by the highly-charged cationic polymers chromatographed on this system. The salt concentration was held a t a minimum to prevent salting out of the polymeric solute (15) during chromatography. Figures 1, 2, and 3 are chromatograms of a poly(2-vinylpyridine), a dextran, and a quaternized poly(44nylpyridine), showing some polymers which can be chromatographed on these supports. T o determine whether these quaternized
+
2241
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
il
3'
L
?E
1
2 L'l
C
Figure 1. Chromatogram of a 460 000 mol wt poiy(2-vinylpyridine)in 0 1 N HNO, 0.1 N NaNO, on an &column set of quaternized supports varying in porosity from 75 to 25000 A Flow rate = 2 mL/min
+
7 5 0 JEXTRAN Ir'w = 250
CC?
TOTAL "ENETQATIOW PEAK
i:E
22'
238
249
260
271
252
293
304
315
326
3:-
348
RETENTION VOLLME (mi)
Figure 4. Calibration curves for poly(2-vinylpyridine)and dextran in 0.1 0.1 N NaNO, on a 10-column set of quaternized supports N HNO, varying in porosity from 40 to 25000 A. Flow rate = 2 mL/min
+
Figure 2. Chromatogram of a 250 000 mol wt dextran in 0.1 N HNO, 0.1 N NaNO, on an 8-column set of quaternized supports varying in porosity from 170 to 25000 A. Flow rate = 2 mL/min
+
7I 13'
216
1
1 3
"
RE'EhT
.
I L..
I i
I L
I
,
C N $0 l t 4 E V I
, 22'
]
/
]
I
1
~
l
,
228 224 2617 2 7 : 252 293 304 315 RETENTION VOLUME iml)
,
l
326
33i
5
Figure 3. Chromatogram of a quaternized poly(4-vinylpyridine)in 0 1 N HNO, 0.1 N NaNO, on a 10-column set of quaternized supports varying in porosity from 40 to 25000 A. Flow rate = 2 mL/min
Figure 5. Log (hydrodynamic volume) vs. retention volume for poly0.1 N NaNO, on a (2-vinylpyridine) and dextran in 0.1 N HNO, IO-column set of quaternized supports varying in porosity from 40 to 25000 A. Flow rate = 2 mL/min
supports were operating by the exclusion mechanism, the series of narrow molecular weight distribution poly(2vinylpyridine) samples and a series of dextran standards from Pharmacia (16) were chromatographed. Figure 4 is the plot of log (weight average molecular weight) vs. retention volume for these polymers. Both series gave a linear relationship with retention volume which was an indication that separation by size was occurring. As expected. the two calibration curves are parallel rather than superpositioned. Poly(2-vinylpyridine) is charged, leading to a more extended structure (11,17-19) in solution, whereas dextran would conform to a more compact structure. In other words, a particular molecular weight poly(2-vinylpyridine) eluted before the same molecular weight dextran. Furthermore, a plot of the log (hydrodynamic volume) vs. retention volume for poly(2-vinylpyridine) and dextran does not superimpose as shown in Figure 5. If the universal
calibration (20) is to hold for these polymers in this chromatographic system, the two lines should coincide. Since the lines are parallel, it can be concluded that the universal calibration does not hold for comparison of neutral to charged polymers. However, the validity of the concept has not been established when comparing polyelectrolyte to polyelectrolyte or neutral polymer to neutral polymer in this system. The effectiveness of the coating procedure was determined by measuring theoretical plate height as a function of eluent velocity for a cationic polymer, poly(N,N-diallyldimethylammonium chloride), and monomer, N,N-dimethylpiperidinium chloride. Figure 6 is a plot of plate height vs. velocity for these species. In both cases, the plate height decreased with velocity obeying a typical plate height vs. velocity plot for polymer and monomer (21). Furthermore, the elution volumes of the polymer and monomer did not vary with velocity.
+
+
2242
I
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
L 0
I
N N DIMET~YLPIPERIDINIUMCHLORIDE
001
002
003
001
005
006
007
0C8
039
010
011
012
VELOCITY (CM/SEC)
Figue 8. Plate height vs. velocity for poly(N,Ndial~imethyiammonium chloride) and N,Ndimethylpiperidiniumchloride in 0.1 N HNO, 0.1 N NaNO, on a 1Ocolumn set of quaternized supports varying in porosity from 40 to 25 000 A
POLY ( N N DIALLY3'WETHYLAMMOUIUM CHLORIDE
+
Table IV. Concentration vs. Retention Volume Concn., wt % 0.1 0.2 0.3 0.4 0.5
retention volume, mL 270 272 27 1 27 0 27 1
The effect of polymer concentration on chromatographic behavior was determined by measuring the elution volumes and relative areas of a poly(N,N-diallyldimethylammonium chloride) sample over a range of concentrations. Figure 7 is a plot of relative area as a function of concentration and Table IV lists retention volume as a function of concentration. These data indicate that retention volume is independent of concentration up to 0.5 wt %. In addition, the detector response is linear with concentration up to -0.3 wt %. Therefore, in a normal operating range, the chromatographic system is essentially independent of concentration. The reproducibility of the system was assessed by injecting a sample of poly(N,N-diallyldimethylammonium chloride) 5 times at 0.25 wt %. The total area and retention volume varied *6% a t the 95% confidence level. The initial data obtained with these quaternized supports indicate that a variety of cationic and neutral polymers can be chromatographed on silica glass deactivated by converting i t to an ion-exchange support. The size exclusion mechanism appears to work with minimal solute-substrate interaction. A number of authors (11-13) have stated that, since polyelectrolyte size is dependent upon salt concentration, the true molecular weight and molecular weight distribution must be carefully evaluated if determined from aqueous size exclusion chromatography. The ionic quaternary ammonium groups lining the pore surface may serve to significantly increase the ionic strength of the environment felt by a solute molecule as it enters a pore, particularly if it can come into intimate contact with the pore surface. The effect becomes more pronounced as the pore size decreases. The effect of ionic strength on elution, the chromatography of other cationic polyelectrolytes, and the range of application of the universal calibration need further investigation. It should also be
0
0
I C 1
I I I 04 02 03 CONCENTRATION lV/T )
I
05
I
6
Figure 7. Relative area vs. concentration for poiy(N,Ndiallyidimethylammonium chloride) in 0.1 N HNO, + 0.1 N NaNO, on a 10column set of quaternbed supports varying in porosity from 40 to 25 000 A. Flow rate = 2 mL/min mentioned that although a broad range of pore sizes was used here, other authors (22) have shown this not to be the optimum case.
CONCULSIONS In this paper, we have found that silica-based ion-exchange supports can be used for the size exclusion chromatography of cationic polyelectrolytes. After initial equilibration, these supports appear to be stable for several months in an acidic medium. For poly(2-vinylpyridine) and dextran molecules, the log molecular weight vs. retention volume relation appears to hold. This novel approach should be applicable to anionic polymers using a suitable cation-exchange support such as a sulfonic acid. LITERATURE CITED (1) J.
(2) (3) (4) (5) (6) (7) (8) (9)
(IO) (11)
(12) (13)
(14) (15) (16) (17) (18) (19) (20) (21) (22)
C. Moore, J. Polym. Sci., Part A ,
2. 835 (1964). F. E. Regnier and R. Noel, J. Chromatogr. Sci., 14, 316 (1976). A. R. Cooper and D. P. Matzinger, Am. Lab., 9, 13 (1977). G. B. Butler, US. Patent 3,962,206, June 8, 1976. A. L. Spatorico, J. Appl. folym. Sci., 19, 1601 (1975). A. L. Spatorico and G. L. Beyer, J. Appl. Polym. Sci., 19, 2933 (1975). C. P. Talley et ai., U.S.Patent 4,118,316, October 3, 1978. M. Lynn and A. M. Filbert, in "Bonded Stationary Phases In Chromatography", E. Grushka, Ed., Ann Arbor Science Publishers, Ann Arbor, Mich., 1974, p 1. S. H. Chang and F. E. Regnier, U.S. Patent 4,029,583, June 14, 1977. J. C. Glddlngs and K. L. Mallik, Anal. Chem., 38, 997 (1966). T. K. Yamashita, Polym. J., 4, 262 (1973). B. Stenlund, Adv. Chromatogr. 14, 37 (1976), K. G. Forss and B. G. Stenlund, J. Po&m. Sci., Po&m. Symp., 42, 951 (1973). J. C. Giddings, G. Lin, and M. N. Myers, J. LiquM Chromatogr., 87 1, 1 (1978). S. Fundano and K. Konlshi, J. Chromatogr., 87, 117 (1973). Pharmacia Fine Chemicals, Product Bulletin, August 1971, p 14. A. J. Hyde and R. 8. Taylor, Polymer, 4, 1 (1963). K. Matsuzaki, T. Matsubara,and T. Kanal, J. P@m. Sci., Polym. Chem. Ed., 15, 1573 (1977). S. Arichi, Bull. Chem. SOC. Jpn., 41, 548 (1968). H. Benoit, Z. Grubisic, P. Rempp, D. Decker, and J. G. Zilliox, J. Chirn. Phys., 63, 1507 (1966). J. C. Giddings, L. M. Bowman, and M. N. Myers, Anal. Chem., 49, 243 (1977). W. M. Yau, C. R. Glnnard, and J. J. Kirkland, J. Chromatogr., 149, 465 (1978).
RECEIVED for review February 5, 1979. Accepted August 15, 1979.
