Anal. Chem. 1981, 53,
pholipid composition results usually in a large neutral lipid response close to the solvent front if the UV detection or the flame ionization detection is applied (1,3,18). This response is avoided by the use of the phosphorus analyzer as the detector. A lipid sample containing approximately equal amounts of neutral lipids and phospholipids was chromatographed and only a minor interference due to the neutral lipids was found (Figure 4). This interference is apparently due to the high overloading of the ashing unit with neutral lipids which all are eluted as one sharp band. In this particular case the total phospholipid load was about 500 nmol and the measured absorption maximum at the top of the PC peak was approximately 1.6. Both the load and the maximum absorption are close to the upper limits of the method and are not as high in routine analysis where the neutral lipid interference is hardly visible if phospholipids predominate in the total lipid sample. In this study we used solvent systems which can be applied also for UV detection. This was done only to obtain a better comparison between the published HPLC data on the phospholipid analysis using UV detection and our studies on the application of the automatic phosphorus analyzer as the detector. It should be emphasized that common organic solvents do not interfere with the phosphorus determination and thus regular and less expensive solvents can be used for the HPLC separation if the automatic phosphorus analyzer is used for detection and quantitation.
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1235-1238
LITERATURE CITED (1) Jungalwala, Firoze 6.; Evans, James E.; McCluer, Robert H. Blochem. J . 1976, 155, 55-60. (2) Van Kessel, W. S.M. Geurts; Hax, W. M. A,; Demel, R. A,; DeGier, J. Biochim. Blophys. Acta 1977, 486, 524-430. (3) Kluchi, Kan; Ohta, Teruo; Ebine, Hideo J. Chromatogr. 1977, 133, 226-230. (4) Kaltaranta, J. K.; Nicolaides, N. J. Chromatcgr. 1981, 205, 339-347. (5) Cooper, M. J.; Anders, M. W. J. Chromatogr. Sci. 1975, 73, An7-44 i _“I I. 7
,
(6) Z. Z. Physiol. . . Schiefer, Hans-Gerd; Neuhoff, Volker Hoppe-Seyler’s .. Chem. 1971, 352, 913-926. (7) Fiske, Cyrus H.; Subbarow, Yellapragada J. Biol. Chem. 1925, 66, 375-400. - . - . - -. (8) Bessman, Samuel P. Anal. Biochem. 1974, 59, 524-532. (9) Geiger, Paul J.; Roberts, Carmel M. Biochem. Siophys. Res. Commun. 1979, 88, 508-514. (10) Warden, C. H.;Friedkin, M.; Geiger, P. J. Biochem. Blophys. Res. Commun. 1980, 94, 690-696. (11) Kaltaranta, J. K.; Gelger, P. J.; Bessman, S. P. J. Chromatogr. 1981, 206, 327-332. (12) 9 Bligh, i1-91E.7. G.; Dyer, W. J. Can. J. Blochem. Physiol. 1959, 37, - . . - .. . (13) Bessman, Samuel P.; Geiger, Paul J.; Lu, TsungCho; McCabe, Edward R. B. Anal. Biochem. 1974, 59, 533-546. (14) Geiger, Paul J.; Ahn, Shunwoo; Bessman, Samuel P. Methods Carbohydr. Chem. 1980, 8 , 21-32. (15) Hurst, W. J.; Martin, R. A. J. Am. Oil Chem. SOC. 1980, 57, 307-310. (16) Slpos, J. C.; Ackman, R. G. J. Chromatogr. Sci. 1978, 16, 443-447. (17) Radln, Norman S. J. LlpM Res. 1978, 70, 922-924. (18) Gross, Richard W.; Sobel, Burton E. J. Chromatogr. 1980, 707. 79-85.
RECEIVED for review December 1,1980. Accepted March 25, 1981.
Characterization of Microporous Polystyrene-Divinylbenzene Copolymer Gels by Inverse Gel Permeation Chromatography David H. Freeman* and Steven B. Schram’ Department of Chemistry, University of Maryland, College Park, Maryland 20742
The pore propertles of microporous polystyrene-divlnylbenzene copolymer (PSDVB) gels are lnvestlgated wlth Inverse gel permeatlon chromatography (GPC). The results Indicate a monomodal pore size distribution. The measured pore size ranges from 77 A for 1 % dlvlnylbenzene (DVB) to 13 A for 16% DVB. The GPC technique, when callbrated wlth solvent regaln measurements, gives the size of the tetrahydrofuran carrler molecule as 4.9 f 0.5 A. This compares to 4.5 A based on reported solvation effects. The gel surface area, near 2000 m2/g, shows no major dependence on cross-linking for 1-8% DVB. The method appears to offer reasonable accuracy, and It gives new insight to the pore structure of gels.
Copolymers of styrene and divinylbenzene (PSDVB) are used for gel permeation and gel partition and as synthetic intermediates for ion or ligand exchange resins. These materials become swollen when they absorb solvents of similar polarity. The swollen state consists of a microporous gel phase which may or may not be mixed with structural voids (macropores) depending on the diluents present during polymerization (1-3). While the micropores are the likely result of Present address: Sid E. Williams Research Center, Life Chiropractic College, Mariett,a,GA 30010. 0003-2700/81/0353-1235$01.25/0
interconnecting chain and link cages (4),their actual structures are not easily elucidated. We have chosen in the present study to apply the inverse gel permeation chromatography technique (5-1 0) to a series of microporous PSDVB samples. Our purpose is to explore the relationship between cross-linking and pore properties in the swollen gel state. Previously, we showed (6) that the Giddings theory (11) of gel permeation provides a way to obtain the average pore size from the chromatographically measured surface area. The empirical adaptation of Giddings’ theory involves experimental measurement of the distribution coefficient, D, using -In D = go g,L (1) where L is the “mean external length” of probe solutes, gois an empirical parameter that refers to a hypothetical solute of zero length, and g, is the specific surface area, g , = 4/d, with the pore diameter, dwre,derived from a simple capillary model. Molecular size, L, calculations are referred here to data on aliphatic hydrocarbons; for normal CnH2n+2alkanes L = Lo kN (2) where N is the number of alkane carbon atoms and the terms LO= 3.32 and K = 0.45 are derived (6) from alkane viscosity data. The fractional pore volume in a microporous gel is obtained as follows. The measured value of D is the equilibrium concentration ratio
+
+
0 1981 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
D = hS/Vs)/hrn/Vrn)
(3)
where n is the amount of solute, V is the volume, and the subscripts s and m refer, respectively, to stationary (gel) and mobile (carrier) phases. The gel phase porosity Pggis defined (6) as the fractional volume, Vg,available for permeation by the solvent.
pgg =
vg/vs
(4)
By defining the distribution coefficient for the solvent, and substitution of eq 4 in (3), one obtains
(5) If we choose D to refer to the distribution coefficient of the carrier liquid, the concentration ratio in parentheses in eq 3 should be equal to unity so that Dsolvent becomes
According to eq 6 the porosity of a gel (or of any porous material) may be determined when go, gl,and Lsolvent are known. Since go and gl are the calibration coefficients based on solutes of known size, pore volume determination depends upon the reference size L of the solvent. In the present study we have measured this by using independent regain and volume swelling measurements in order to obtain Pa directly. We used GPC to give the go, g, coefficients. The value of Lsolventwas then obtained. This reasoning leads to an interesting test of the ability of the GPC technique to measure the effective molecular size of the carrier molecule. EXPERIMENTAL SECTION Samples of spherical particles of microporous PSDVB copolymers were identical with those provided by the Dow Chemical Co. for an earlier study where microscopy was used t o measure their swelling properties (12). We used samples with 1, 2, 4, 8, and 16% DVB cross-linking. Solvent and Solutes. Solvents used were tetrahydrofuran (THF) and reagent grade toluene. The THF was distilled from freshly cut sodium under a nitrogen atmosphere and stored at 5 OC until used. THF has a solubility parameter close to that for PSDVB. This is needed to help assure the nonenthalpic conditions required for size exclusion chromatography in the absence of other partitioning or sorptive effects. Solute standards include n-alkanes and a polystyrene sample of narrow molecular weight range at 10000 Daltons from Pressure Chemical Co. (Pittsburgh, PA). The alkane standards were tested by both gas and gel permeation liquid chromatography and were found t o contain no significant impurities. Apparatus, The equipment used included a solvent pump (Waters Associates, Model 6000A), a six-port injection valve (Rheodyne Model 7120 with a 100-pL injection loop), and a refractive index (RI) detector (Laboratory Data Control). The data collection system utilized a Wang 2200 microcomputer (13)which allowed data to be collected at 1 point/s with 8-bit resolution of the detector output, Le., to 1 part in 256. Columns and Packing Procedures. A thin slurry of each gel material in THF was packed into glass columns (0.635 X 12.9 cm) by pouring the slurry into the column through a larger reservoir column placed on top. In order t o avoid compression of the soft gels, we maintained solvent flow rates of under 0.5 mL/min and pressures of less than 300 psi during packing. Column packing was followed by carrier recirculation with the flow continuing until bed settling was complete. The following calibration procedure was then carried out. Calibration Procedure. The calibration of each column involved triplicate measurement of the retention volumes of each of the calibrating solutes. The empty column volume, V,, was measured before or after the calibrating measurements were carried out. This was obtained from the weight difference after filling the empty column with water.
THF carrier was pumped at room temperature (22-25 OC) at 0.5 mL/min through the column until a stable base line was
observed with the RI detector. At this point, each of the alkane solutes,0.5% (w/v) in THF, was sequentially injected in triplicate series. The void volume, Vo,was taken from the retention volume of the excluded 10K polystyrene peak. Regain Measurements. The internal pore volume of each gel was measured for toluene and THF regain by centrifugal filtration. The procedure, as reported earlier (14),was adapted as follows. To ensure complete solvent permeation, each sample was gently boiled in the regain fluid for 30 min prior to measurement. The slurry was transferred to a filter tube for 15 min of centrifugal filtration at lOOOg measured at the mid-sample position. The centrifuge temperature was kept at 0 "C to reduce evaporation. Samples were weighed in closed containers immediately following centrifugation and again after vacuum drying at 100 "C. The mass of fluid in the pores was taken from the weight difference after correction for a 5% volumetric retention error (14,15). Pore volumes for each gel were determined by this method for both THF and toluene. Regain results for internal porosity were compared to corresponding values found from the previous microscopy analysis (12). A combined average THF reference porosity value was calculated for each gel in the following manner. The THF/toluene weight regain ratio was used to multiply the microscopy values for toluene swelling in order to obtain values expected if the measurements were done in THF. We averaged this value with the THF regain value and obtained the final reference values. GPC Calibration, The GPC results were analyzed as follows. For each solute injected, symmetrically shaped peaks were recorded. The retention volume (Vd of each solute was taken from the difference between the injection time and that for the location of the chromatographic peak maximum, multiplied by the flow rate. Automated data analysis yielded retention volumes precise to within 1%. From the retention volumes, the distribution coefficients were calculated by using D = ( VR - Vo)/ V, where Vo was the retention volume of the excluded solute and V , was calculated from V , - Vp The -In D and L values were treated by linear regression analysis to obtain values for the slope, gl,and intercept, go, for each data set. RESULTS AND DISCUSSION The n-alkane hydrocarbon distribution coefficient determinations for the PSDVB gels in T H F are plotted in Figure 1. The observation of a linear relationship (-ln D vs. L) provides further support that GPC results tend to be precisely consistent with Giddings' theory (11)which we have adapted for the correlation and interpretation of GPC calibration data. The observation of a simple linear relationship between -In D and L is interpreted to indicate the presence of a gel phase having a single pore size distribution. This contrasts markedly with findings of adjacent linear regions with different slopes which were observed earlier and attributed to a bimodal size distribution of micropores and macropores (6). Each of the measurement sets shown in Figure 1 were treated by least-squares analysis to obtain the go and g1 coefficients. (The data set containing only two points was treated more simply.) The pore size is obtained directly as 4 times the reciprocal slope. The effective size of the alkane standards has been reported (6) in angstrom units, P\ = lo-'' m, and the pore size is directly obtained in the same units. No independent basis is available for measuring the size of the randomly oriented polymeric cages. However, the basic tetrafunctional network topology of the cage structure is largely predetermined (4)due to the use of the 4-connective DVB cross-linker. The cross-link density in the copolymer is known from the monomer ratio and polymer density. The corresponding distance between cross-links, d,, in the swollen gel can be calculated. This is obtained by using the following expression
(7)
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
3wl / 1691 D V B
.In
D
/
I
,
/
1.32
0.66
0.OC
Figure 1. Gel permeation chromatographic behavior of microporous PSDVB gels (% DVB = 1, 2, 4, 8, and 16, bottom to top) in tetra-
hydrofuran for n-alkane solutes. ~~
~~
Table I. Results of GPC Calibration ( 2 3 "C) of Cross-Linked PSDVB Network Gels in Tetrahydrofuran
GPC calibration coeff
crosslinking X
1 2 4 8 16
go
g1
-0.018 0.01 0 0.008 -0.636 -3.346
0.052 0.074 0.109 0.283 0.301
intercrosspore link dist, size, A A , d, dpore= from 4k, eq 7
I1 54 31 14 13
42 30 22 16 12
where E, is the network equivalent weight of the copolymer (I6),q is the swelling ratio which will be discussed shortly, pc is the measured unswollen copolymer density of 1.06 g/cm3, JV is Avogadro's number, and X is the mole fraction of cross-links. As given in Table I, the more lightly cross-linked gels (X = 1, 2, 4) show a 2-fold ratio of dPle to d,, while the ratio is closer to unity for the 8 and 16% cross-linked gels. Thus, the more heavily cross-linked gels show a lesser dependence of dPre upon cross-linking. This shows that the divinylbenzene becomes less effective in reducing the pore size
as its proportion is increased above 4 mol % . The results in Table I are consistent with the idea that two types of cage structure may be involved. The flexible and randomly oriented polystyrene chain segments should control the pore size when the DVB concentration is low. In highly cross-linked PSDVB there may be a fixed cage size contribution to the pore structure due to self-reaction of the divinylbenzene or to other steric factors. The present determinations of gel porosity include independently memured regain (centrifugal filtration) and volume swelling (microscopy). These results are summarized in Table 11. Precise agreement is found between these two independent techniques. Each pair of fitted coefficients to eq 6 were then used to find corresponding L values which make PgB= D. (LTHF). This equates the chromatographic and nonchromatographic values of porosity. The resulting L T H F values are given in Table 11. The values of L T H F among the different PSDVB samples were combined to obtain the average value of 4.9 f 0.5 A where the tolerance is the standard deviation which, for the present result, is the same as the SDM multiplied by the Student t for n = 5 a t a 95% confidence level. The value for L m found here can be compared to results reported by Hendrickson and Moore ( I 7)who found N = 2.54 (eq 2) as the size increase due to alcohol or alkyl halide monosolvation in tetrahydrofuran solvent. Substituting this in eq 1 gives 4.5 A for the size of the T H F molecule. The agreement between the two sets of results is within experimental error. Given the average value for & determined by the present GPC techniques, the determination of gel porosity by GPC can now be based on the measured go,gl coefficients using eq 6. The results are given in Table 11. The individual ratios of P ~ p to c the more accurate values obtained by regain have a standard deviation of f0.06. This serves to estimate the relative uncertainty in porosity determination by GPC using the techniques we have described here. Our final task is to obtain the surface area for the PSDVB gels. The conventional expression for specific surface area in the swollen network per unit weight of the polymeric matrix ( 6 ) is
The results, given in Table 11,show that the surface area does not undergo any discernible trend with cross-linking or with pore size. This set of microporous PSDVB gels displays an average surface area of 1780 f 370 m2/g (SD). This corresponds to 21% relative standard deviation (RSD) among the different samples. This is significantly more precise than a worst case test of the preciseness of the method where 12 deliberately varied column packing conditions for the same material gave 28% RSD in the surface area and 11%RSD in the pore size (8).
Table 11' Porosity and Pore Surface Area from GPC Derived from Regain Calibration and from Average Size of Solvent (THF) Molecule pore vol fraction cross-linking
regain
microscopy
X
PA
PB
av p,,
LTHF,A a
0.799 0.734 0.633 0.458 0.294
0.788 0.717 0.614 0.456 0.302
0.794 0.726 0.624 0.457 0.298
6.0 4.2 4.3 5.0 5.0 4.9
1
2 4 8 16
av
1237
solvent size,
* 0.5
porosity from surface area, GPC,b PGPC S, m 2 k 0.84 0.69 0.58 0.47 0.31
1890 1850 1710 2240 1230
a Obtained from eq 5 using reference value of average pore volume fraction (PA + PB )/2 with g,,g , coefficients from Table I for each gel. Obtained from eq 5 with g o ,g, values for each gel using the averaged value of L T H =~ 4.9 A .
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Anal. Chem. 1981, 53, 1238-1240
The surface area determination can also be examined in
Halasz. I. Ber. Bunsenges. Phys. Chem. 1975, 79, 731-732. Freeman, D. H.; Poinescu, I. C. Anal. Chem. 1977, 49. 1183-1188. Halasz, I.; Martin, K. Angew. Chem., Int. Ed. Engl. 1076, 77, 901. Schram, S. B.; Freeman, D. H. J . Llq. Chromafogr. 1980, 3(3), 403-41 7. (9) Nikolov, R.; Werner, W.; Halasz, I. J . Chromafogr. Sci. 1980, 76, 207-216. (IO) Werner, W.; Halasz, I. J. Chromafogr. Scl. 1980, 78, 277-283. (11) Glddings, J. C.; Kucera, E.; Russell, C. P.; Meyers, M. N. J . Phys. Chem. 1966, 72, 4397-4408. (12) Freeman, D. H.; Patel, V. C.; Smith, M. E. J . Polym. Scl. 1965, 3, 2893-2902. (13) Schram, S. B. Ph.D. Dissertation,University of Maryland, College Park, MD. .---, 1978. (14) Scatchard, G.; Anderson, N. J . Phys. Chem. 1961, 65, 1536-1539. (15) Freeman, D. H.; Angeles, R. M.; Enagonlo, D. P.; May, W. E. Anal. Chem. 1973. 45. 788-774. (16) Freeman, D. H.; Currle, L. A.; Kuehner, E. C.; Dixon, H. D.; Paulson, R. A. Anal. Chem. 1970, 42, 203-209. (17) Hendrlckson, J. G.; Moore, J. C. J . Polym. Sci. 1966, 4 , 167-188. (5) (6) (7) (8)
terms of a hypothetical cylindrical chain model of polystyrene. The repeat distance for polystyrene is 2.5 A so that the overall chain length, I , corresponding to 1 g (0.0096 mol) can be calculated. The measured surface area, A = 1780 m2, can be equated to that of a cylinder, rD1,and this gives a chain diameter, D, of 3.9 A. This value can be compared to a value of 5.2 A based on the model compound, ethylbenzene, whose apparent size (N= 4.8) was reported by Hendrickson and Moore (17). This suggests that the apparent surface area in a PSDVB gel structure is closely related to the molecular threads that constitute the network structure. LITERATURE CITED (1) Seidl, J.; Malinsky, J.; Dusek, K.; Heltz, W. Adv. Polym. Scl. 1967, 5 , 113-213. (2) Mlllar, J. R.; Smith, D. G.; Kressman, T. R. E. J . Chem. Soc. 1965, 304. (3) Dusek, K. J . Polym. Scl., PartCl967, 76, 1289-1299. (4) Zlablcki, A. Po/ymr 1979, 20, 1373-1381.
RECEIVED for review January 12,1981. Accepted March 25, 1981. This research was supported by the National Science Foundation, Grant No. CHE-77-11313.
Spectrophotometric Determination of Aniline by the Diazotization-Coupling Method with N-( I-Naphthy1)ethylenediamine as the Coupling Agent George Norwitr and Peter N. Kellher" Chemistry Department, Villanova Universlty, Vlllanova, Pennsylvania
79085
The factors affecting the spectrophotometric determination of aniline by the diazotization-coupling technique using N-( 1naphthyi)ethylenediamine (also called N-( 1-naphthaieny1)1,2-ethanedlamine) (N-na) as the coupling agent in an acidic medium are investigated. The effect of nitrite concentration, effect of time for diazotization, effect of temperature for diazotization, and necessity for destroying excess nitrite (by the use of suifamic acid) are the same as for the coupling method using H-acid (8-amino-l-hydroxynaphthaiene-3,6-disuifonic acid) as the coupling agent. Acidity in the N-na method has a dual effect In that it affects the diazotization and coupling. There Is a plateau for maximum absorbance over the range of 0.5-5.0 mL of 1 N hydrochloric or sulfuric acid. The concentration of N-na has a pronounced effect on the intensity of the color and time required for development of the color. Ethanol, methanol, and acetone can cause low results.
N-(1-Naphthy1)ethylenediamine (N-(l-naphthalenyl)-l,2ethanediamine; N-na; Cl&17NHCH2CH2NH2),next to H-acid (8-amino-l-hydroxynaphthalene-3,6-disulfonic acid), is the most widely used coupling agent for the spectrophotometric determination of aniline by the diazotization-coupling technique. The conditions used previously for the N-na method vary considerably (1) and no comprehensive study has been made of the factors involved. It is the purpose of the present paper to make such a study, particularly since the N-na method has certain advantages over the recently reported (I) H-acid method. EXPERIMENTAL SECTION Apparatus and Reagents. Bausch and Lomb Model 70 spectrophotometer (1-cm cell).
All chemicals were reagent grade. Standard aniline solution no. 1 (1mL = 10.00 mg of aniline). Dissolve 1.000 g of aniline in ethanol and dilute to 100 mL in a volumetric flask with ethanol. Prepare fresh weekly. Standard aniline solution no. 2 (1mL = 1.00 mg of aniline). Dilute a 10-mL aliquot of standard aniline solution no. 2 to 100 mL in a volumetric flask with water. Prepare fresh every 3 days. Standard aniline solution no. 3 (1mL = 0,010 mg of aniline). Dilute a 5-mL aliquot of standard aniline solution no. 2 to 500 mL in a volumetric flask with water. Prepare fresh daily. Sodium nitrite solution (1%)and sulfamic acid solution (3%). Prepare fresh every 3 weeks. N-na reagent (0.75%). Add 0.375 g of N-naphthylethylenediamine dihydrochloride to about 45 mL of water while stirring and dilute to 50 mL. Prepare fresh every 4 days. Preparation of Calibration Curve. Transfer 0.00,2.00,4.00, 5.00, and 6.00 mL of standard aniline solution no. 3 (1mL = 0,010 mg of aniline) to 50-mL volumetric flasks and dilute to about 35 mL with water. Add 2.0 mL of 1N hydrochloric or sulfuric acid. Add 1.0 mL of sodium nitrite solution (l%), swirl, and allow to stand 5 min. Add 1.0 mL of sulfamic acid solution (3%))swirl, wash down the sides of the flask, and allow to stand for 10 min. Add 2.5 mL of N-na reagent (0.75%),swirl, and dilute to the mark. Mix, remove the stopper (to permit the escape of nitrogen gas), and allow to stand 75 min or more. Measure the absorbance at 555 nm against distilled water, deduct the blank, and plot absorbance against mg of aniline per 50 mL. Procedure. Transfer an aliquot of the sample containing preferably 0.03-0.05 mg of aniline to a 50-mL volumetric flask, dilute to about 35 mL, and proceed as described in the preparation of the calibration curve. RESULTS AND DISCUSSION Effect of Amount of Nitrite, Effect of Time for Diazotization, Effect of Temperature for Diazotization, and Necessity for Destroying the Excess Nitrite. The conditions previously established for the determination of aniline
0003-2700/81/0353-1238$01.25/00 1981 American Chemical Society
