Liquid Chromatography of Chlorinated Biphenyls on Pyrolytically-Deposited Carbon Toshihiko Hanal and Harold F. Walton” Department of Chemistty, University of Colorado, Boulder, Colorado 80309
Mlxtures of chlorinated blphenyls were analyzed by liquid chromatography on porous silica coated wlth carbon deposited pyrotyilcaliy from benzene vapor. Moblie phases were ethanol and acetonitrile, each contalnlng 10% of water. Good resolution of lsomerlc compounds was observed In spite of unsymmetrical peaks. Chlorine substltutlon In the 2-posltlon weakened adsorption considerably; In other posltlons it strengthened the adsorption. Elution sequences wlth pyroc a r b o d l c a were compared wtth those for graphfflzed carbon black and nonionic, macroporous polystyrene gel, and enthalpies of adsorption were measured. Graphitized carbon black and pyrocarbon-coatedsilica behaved very differently.
We recently reported the separation of chlorinated biphenyls by chromatography on a cation-exchange resin (1). We compared the elution sequence on the resin with that found on silica bonded with octadecyl groups, and found that whereas the CIs-silica retained the compounds more or less in the order of their chlorine content, the more chlorinated compounds being more strongly retained, the ion-exchange resin showed selective behavior towards biphenyls chlorinated in the 2-position. Such compounds were more weakly retained than the unsubstituted compounds. We ascribed this “2effect” to the twisting of the two phenyl groups around the common carbon-carbon bond, which lessens the overlap and delocalization of the 7r-electron orbitals and decreases the ability to form a-bonds with the aromatic rings of the polystyrene resin. We have now studied elution sequences of chlorinated biphenyls on three other sorbents: a macroporous styrenedivinylbenzene copolymer having no ionic groups, and two materials whose active surface is carbon deposited by pyrolysis of benzene vapor a t 950 “ C . One of these is based on carbon black, the other on silica. The “2-effect” or “twist-effect” was almost absent in the macroporous polystyrene, but very marked in the carbon adsorbents, particularly the pyrocarbon-silica. Silica coated with pyrocarbon showed the greatest selectivity between various isomeric biphenyls and was therefore studied most extensively. Carbon adsorbents have been little used in liquid or gas chromatography, because of the difficulty of preparing them in a suitable physical form. Recently, graphitized carbon spheres and pellets have been offered commercially for use in gas chromatography. Their particle sizes are too large, however (80-100 mesh), for them to be used in liquid chromatography. Ordinary carbon black consists of very fine particles, and pellets made from carbon black powder are too large and too soft for use in liquid chromatography. Kiselev (2,3)stabilized granules of carbon black by heating them in benzene vapor carried in a stream of nitrogen, and showed that the “graphitized carbon black” so produced could be used as a packing in gas chromatography. Guiochon and his coworkers ( 4 , 5 ) improved the technique for coating carbon-black particles with carbon deposited from benzene vapor a t 900 “C and above, and obtained granules in the size range 20 to 1954
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
70 pm that were hard enough to be packed under pressure in liquid chromatography columns. It was difficult to prepare smaller-sized granules because of the aggregation produced as pyrocarbon was deposited. Colin and Guiochon (6) therefore tried to deposit pyrocarbon on particles of porous silica, and showed that if the temperature and the proportion of carbon were carefully controlled, the silica surface could be covered with an adherent layer of carbon without significantly reducing the porosity or the specific surface area. At higher carbon contents than the optimum (about 15%) the carbon layer tended to flake off. The chromatography of alkyl benzenes, other aromatic hydrocarbons, and phenols was reported. Bebris, Kiselev, and others (7)have also prepared silica adsorbents coated with pyrocarbon, and used them for liquid chromatography of hydrocarbons, with 2-propanol as the mobile phase. Turning to the chromatography of polychlorinated biphenyls, the most effective way to analyze these very complex mixtures is by gas chromatography on wall-coated glass capillary columns (8-10). Biphenyl is retained the least, followed by 2-, 3-, and 4-chlorobiphenyl and then more highly chlorinated biphenyls. The commercial product Arochlor 1242, which has an average of 3 chlorine atoms per biphenyl unit, shows 58 peaks. The best resolution by liquid chromatography has been performed on silica gel with dry n-hexane as the mobile phase (11-13). In this system, biphenyl is more strongly retained than chlorinated biphenyls, and the more chorine, the less the retention. Thin-layer chromatography on paraffin oil held on Kieselguhr is also effective; Arochlor 1232 showed 8 zones (12).
EXPERIMENTAL Materials. Samples of carbon black and silica coated with pyrocarbon were given to us by Henri Colin of the Ecole Polytechnique, Palaiseau, France. These materials were made by passing benzene vapor in a stream of nitrogen over particles of carbon black or silica heated to 950 “C. The carbon was batch MT 14-15,particle size 5Cb63 pm. The silica was Spherosil XOB 75, particle size 31.540 pm, coated with 15% of carbon and having a surface area of 90 square meters per gram. We examined both materials by powder x-ray diffraction. The material made from carbon black showed strong lines at spacings of 3.37 and 1.69 A, corresponding to crystalline graphite. The carbon-coated silica showed no trace of the graphite lines, but instead a strong line with spacing 1.99 A and a very weak line at 1.73 A, both identical with the lines obtained from silica gel. We shall therefore call the first material “graphitized carbon black” and the second “pyrocarbonsilica”. Although Colin and Guiochon did not include x-ray diffraction data in their paper (6), they were careful not to use the word “graphite” to describe their carbon-coated silica. A macroporous polystyrene gel, particle size 10 pm, was provided by Hitachi, Ltd., Tokyo, Japan. Acetonitrile was the non-spectro grade of Burdick and Jackson, Inc., Muskegon, Mich.; 95% ethyl alcohol was the normal commercial product. Solvents were mixed with water as desired and degassed before use. Chlorinated biphenyls, biphenyl, naphthalene, and various pesticides were obtained from Analabs, Inc., North Haven, Conn.
E L U T i O N OF C H L O R I N A T E D B I P H E N Y L S
07
1 2.5.4‘TR Ic H LOR o DIPHENYL
/’
p;/
/
,//* \
0 6 1
-1 BIPHENYL
2,4‘ 2,s 3 4
2: 3 , 4 2, 4 , 4 ’
3,3’ GRAPHITIZED CARBON BLACK
ION- EXCHANGE RESIN
(
PYROCARBONSILICA
-
‘IS
BONDED
Figure 1. Elution of chlorinated biphenyls on five sorbents. Solvents: graphitized carbon black, 80 YO CH3CN; ion-exchange resin and C,,-bonded silica, 35% CH3CN; pyrocarbon-silica, 100% CH,CN; polystyrene gel, 70% CH3CN. Abscissas in parentheses are k ‘ for polystyrene gel Equipment. Chromatographic pumps, Model 6000, and a solvent programmer, Model 660, were supplied by Waters Associates, Inc., Milford, Mass. A Chromatronix Model 220 ultraviolet detector (Spectra-Physics,Inc., Santa Clara, Calif.) and sample-injection valve (Glenco Scientific, Inc., Houston, Texas) were used. The columns were of stainless steel, 2.1-mm internal diameter and 50 cm long. Two columns were used in series with pyrocarbon-silica. They were packed by the balanced-slurry method, and were water-jacketed and maintained at controlled temperatures. Void volumes were found by injecting water into flowing streams of alcohol or acetonitrile.
RESULTS AND DISCUSSION Comparison of Different Sorbents. Figure 1 compares the retention volumes of several chlorinated biphenyls on five sorbents. Polystyrene gel and (&-bonded silica show similar selectivity patterns, with the retention increasing as the chlorine content increases. The ion-exchange resin shows a moderately pronounced “2-effect”; that is, chlorine substitution in the 2-position weakens the sorption. We ascribed this effect to a decreased *-electron over1a.p. The difference in behavior between the microporous ion-exchange resin and the macroporous styrene polymer suggests that the solute moiecules can penetrate all parts of the resin and orient themselves freely with respect to the aromatic rings of the polymer network, while in the macroporous material they can only come in contact with randomly-oriented benzene rings on the surface of the small primary aggregates. The greatest “2-effect” was found with pyrocarbon-silica. This material showed greater differences in selectivity than graphitized carbon, and its particle size was smaller than that of our graphitized carbon, promising narrower bands and higher chromatographic resolution. Sample Loading and Elution Volume. Pyrocarbonsilica, unfortunately, gave unsymmetrical peaks with marked tailing, and this effect was worse, the more strongly retained was the solute. The tailing was of thermodynamic origin, not kinetic; if the flow were stopped in the descending part of the “tail” and resumed a few minutes later, the “tail” continued in the same form as it would have done without flow interruption. Because of the tailing, the retention volume a t the peak maximum depended on the amount of solute injected. in 90% acetonitrile at In tests with 3,4,2’-trichlorobiphenyl 70 “C, the corrected elution volume was 6.0 mL with 15 pg injected, 7.7 mL with 0.05 pg. The graph of volume against
’
= f O -- 0 2
2 9
3 0
31
3 2
33
3 4
35
( I / T ) x103
Figure 2. Capacity factors and temperature. Dashed lines, 90% ethanol; solid lines, 90% acetonitrile Table I. Enthalpies of Adsorption ( - A H ) , cal/mol GraphiPyrocarbontized carbon silica black, CH,CN, C,H,OH, CH,CN, Compound 90% 90% 80% 4400 2300 4-C1-biphenyl Biphenyl 4000 2900 1100 3700 600 Naphthalene 2-C1-biphenyl 3000 2700 800 3100 2,2’-CI,-biphenyl 2,5,4‘-Cl3-biphenyl 4000 2700 2300 N o t e : Probable error is +250 cal/mole. the logarithm of the quantity of solute was a straight line. Where retention volumes had to be carefully compared under different conditions, as in studies of the temperature effect (see below), we injected different-sized samples of each solute and compared the volumes that would correspond to equal peak heights a t different temperatures. Graphitized carbon black gave symmetrical peaks with little or no tailing, and the peak retention volume was almost unaffected by the sample size. Temperature Effects. Retention volumes for several solute-solvent combinations were measured over a range of temperatures from 30 to 70 “ C , taking care to compare volumes at approximately equal peak heights as noted above. Retention volumes were consistent to f0.2 mL or better. The capacity factors calculated from the retention volumes are subject to the uncertainty in the void volume; the void volume of 2.80 mL, found by injecting water into the pair of 50-cm columns packed with pyrocarbon-silica, includes the volume of the connections as well as the interstitial volume of the bed itself, and the dead volume of the connections could easily be 0.2 mL. No attempt was made to correct for the dead volume; the measured volume, 2.80 mL, was considered to be the true interstitial volume. Figure 2 shows retention volumes on pyrocarbon-silica as a function of temperature. From the slope of the (log k’ curves, enthalpies of adsorption were calculated. These are presented in Table I, along with similar data for the graphitized carbon column. Generally speaking, the stronger the
l/n
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13,NOVEMBER 1977
1955
Table 11. Capacity Factors Silica-pyrocarbon, 70 "C Acetonitrile, Ethanol, 90% 90% 0.38 0.41 0.58 0.75 0.79 0.72 1.21 1.19 1.42 1.40 1.42 1.41 1.39 1.42 1.88 1.95 1.92 2.20 2.33 1.85 2.67 2.75 2.67 2.75 4.7 4.75 5.5 5.75
Compound 2,2' 2,3,2' ,3' 2 273 294 2,4' 295 2,5,3' 3,4,2' Biphenyl 2,4,4' 2,5,4' 3 4 ?,5 and 3,3' 394 434 Naphtha le n e Aldrin Atrazine DDT
-
-
2.11 0.2 0.38 0.50. 0.70. 1.15
9.0
14.0 15.6 1.65 -
Graphitized carbon, 55 "C Acetonitrile 80%
100%
1.24 1.46 (70") 1.33 1.68 1.79 1.64 1.85
1.03 1.14 1.09 1.19 1.19 1.19 1.23
2.04 1.45 2.63 2.63 2.27 2.71 4.45 5.6 7.5 1.37
1.28 1.14 1.43 1.43 1.43 1.55 2.05
-
-
-
-
2.6
3.0 1.10 -
3 CHLORINATE1 BIPHENYLS
3
BIPHENYL
I
2,4,4' 3,4,2" I '
I
0
1 IO
1
1
1
,
20
in1
Flgure 3. Separation of known compounds on pyrocarbon-silica. Solvent, 9 0 % acetonitrile; temp, 60 O C ; flow rate, 18 mL/h. Ordinates, absorbance at 254 nm, 0.16 unit full scale adsorption the higher is the (negative) enthalpy, and there is no evidence of any large entropy effect that modifies the strength of adsorption: however, one notes that biphenyl is more strongly adsorbed from 90% ethanol than from 90% acetonitrile, even though the enthalpy of adsorption is more negative in acetonitrile. It is very clear that the enthalpies of adsorption on graphitized carbon black are smaller (less negative) than those on pyrocarbon-silica. Selectivity Orders. Table I1 lists capacity factors for a number of chlorinated biphenyls and other substances. It amplifies the information presented in Figure 1. A few measurements were made with pure methanol and pyrocarbon-silica; the capacity factors were roughly 50% greater than those found with 90% ethanol. The "2-effect'' is very marked, especially in the compound 2,3,2',3'-tetrachlorobiphenyl, and it is greater in ethanol than in acetonitrile. Presumably there is some r-bonding between the solutes and acetonitrile, and this would counteract the bonding between 1956
ANALYTICAL CHEMISTRY, VOL. 49. NO. 13, NOVEMBER 1977
12 mi = 3 0 m i n
Flgure 4. Chromatography of Arochlor 1232 on pyrocarbon-silica at 70 O C . First curve, 90% ethanol; second curve, 90% acetonitrile. Ordinates, absorbance at 254 nm, 0.32 unit full scale. See text for numbering of peaks the solutes and the pyrocarbon. The ultraviolet absorption spectrum of 2,3,2',3'-tetrachlorobiphenyl was measured, and it shows that this compound has a large "twist-effect". The spectrum is very like that of 2,2'-bichlorobiphenyl (1). It has two flat maxima a t 280 and 270 nm. The molar absorptivity a t 270 nm is 1140, and it rises rapidly below 250 nm. The pesticides aldrin and atrazine have very low retentions and should not interfere with chromatographic analysis of chlorinated biphenyls. A mixture of DDT isomers gave three well-separated chromatographic peaks, the peak at k' = 0.70 being the highest. Here again, the retention was weaker than that of most of the chlorinated biphenyls. Chromatographic Separations. Figure 3 shows the chromatography of a mixture of known substances on py-
(A)
o l IO
20
30
40
50
60
LINEAR FLOW R A T E , cm/min Flgure 0. Piate height vs. flow rate. Upper cwves for graphitled carbon (80% CH3CN, 55 “C),lower curves for pyrocarbon-silica (90% CH3CN, 70 “C). Curves (a),2,4’-bichlorobiphenyi:(b), 2,5,4’-trichiorobiphenyl; (c), naphthalene; (d), 4-chlorobiphenyl
Figure 5. Chromatography of Arochlor 1232 on pyrocarbon-silica at 70 O C . Curve (A), linear gradlent from 90 % ethanol to 90 % acetonitrile in 30’min;curve (B), linear gradient from (45% ethanol, 45% acetonitrile, 10% water) to 90 % acetonblle In 30 min. Chdlents started immediatety after InJection. Flow rate, ordinates and abscissas same as in Figure 4 rocarbon-silica, and Figure 4 shows the chromatography of a commercial mixture of chorinated biphenyls, Arochlor 1232, which contains 32% chlorine by weight and has an average of two chlorine atoms per biphenyl unit. The contrast between the two solvents, acetonitrile and ethanol, is interesting; ethanol shows a larger 2-effect and gives better resolution between the peaks of 2-chlorobiphenyl and biphenyl, while acetonitrile gives better resolution beyond the peak of biphenyl. This difference suggested that a gradient might combine the advantages of both solvents. Several gradients were tried, using two Waters Model 6000 pumps and a solvent programmer. Two of the best are shown in Figure 5. There is hardly enough improvement in peak separation to warrant using a gradient; however, a gradient combined with multiple-wavelength detection might be effective. T h e identification of peaks by their retention volumes is made difficult by the fact that the retention volume depends on sample size, and two close neighboring peaks seem to affect each other’s retention volumes. We have attempted the following identification of the numbered peaks in Figure 4: Peak No. 1, 2-chlorobiphenyl; peak 2, mixture of 2,4-, 2,4’-, 2,5-; peak 3, biphenyl; peak 4, mixture of 3,4,2‘- and 2,5,3’-; peak 5, mixture of 2,4,4’- and 2,5,4‘-; peak 6, 3-; peak 7, 4-. In addition, four or five minor peaks and shoulders can be seen. By gas chromatography on surface-coated capillary columns, some 15 compounds were identified in Arochlor 1232 (9,14), and a couple of unidentified peaks were seen. The higher Arochlors give many more peaks (10,15). It would seem that with a little improvement, liquid chromatography on pyrocarbon-silica columns might be competitive with capillary gas chromatography. Theoretical-Plate Heights. Figure 6 shows how the plate heights €or the two column packings depend on flow rate. The “plate height” for pyrocarbonsilica was estimated by drawing the steepest tangents to the ascending and descending sides of the peaks and measuring the base intercept, or, alternatively, by measuring the width of the peak a t 0.606 times its height and calling this two standard deviations. Because of
the dissymmetry of the peaks, these numbers are arbitrary, but the effect of flow rate can be clearly seen. For the runs shown in Figures 4 and 5, the flow rate was 0.4 mL/min, or 11.5 cm/min linear flow rate; the corresponding plate height is about 1.2 mm for both materials. Though the plate heights are disappointingly large, it is possible to use long columns, for the pressure drop across a column 1 m by 2.1 mm was only 500 psi (35 bars) for ethyl alcohol at 70 “C flowing a t 0.4 mLJmin. Trace Recovery from Water. T o see if the pyrocarbon-silica column could be used for trace enrichment, we made several tests in which dispersions of Arochlor 1232 in water containing between 25 and 100 ppb &g/L) were pumped through the column, followed by 90% ethanol. The UV absorbance was recorded and chromatograms characteristic of Arochlor 1232 were obtained. Typically, 250-300 mL of the dispersion in water was passed over a period of 3-4 h, then ethanol was passed at 0.4 mL/min with the column at 70 O C . The Arochlor dispersions were made by injecting 100-200 kL of solutions of Arochlor in alcohol into 400 mL freshly-distilled water. Recoveries ranged from 20% to 80%, and depended apparently, on the extent t~ which the chorinated biphenyls had separated from the supersaturated solution before the solution entered the column. These compounds are soluble in water only to the extent of 5 ppb or less. We therefore repeated these tests with naphthalene, whose solubility in water is reported to be about 50 ppm. A solution of naphthalene in water, 0.48 mg/L, was pumped through the column, which was maintained a t 70 O C . Volumes between 50 and 120 mL were passed at 1.0 mL/min, then the column was flushed with pure water and 90% ethanol was passed. The naphthalene peak appeared at the proper retention volume, and from the peak heights, recoveries of 85-95% were measured. Thus it seems that pyrocarbon-silica columns could be used for trace enrichment or preconcentration, and for measuring the very low concentrations of pesticides, aromatic hydrocarbons, and chlorinated biphenyls found in environmental waters. We plan to investigate this possibility.
CONCLUSIONS The two adsorbents, graphitized carbon black and pyrocarbonsilica, show very different chromatographic behavior. Graphitized carbon gives symmetrical elution peaks, showing that its surface is uniform; the same conclusion was reached by Kiselev (2). Pyrocarbon-silica, on the other hand, gives unsymmetrical peaks even at low loadings. Taking the specifk surface of the pyrocarbon-silica to be 90 m2/g, we calculated ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1957
that in our experiments the fraction of surface covered by solute molecules at the column outlet did not usually exceed 0.05%. Even with this small coverage, the adsorption sites were heterogeneous. According to the calculations of Colin and Guiochon (ref. 6, p 52) our adsorbent, with 15% carbon, carried twice as much carbon as was needed to form a monolayer on the surface of the silica base. Electron microscopy indicates that the surface is completely covered with carbon, but Colin and Guiochon point out that it is very likely that some silica is exposed. Probably the carbon is bonded to silica through silicon-carbon bonds, and the surface has some of the character of twodimensional silicon carbide, as well as that of carbon and of silica. The strong 2-effect suggests the presence of two-dimensional basal planes of graphite. No x-ray lines of graphite nor of silicon carbide were seen, so there can be no significant amounts of these materials as bulk crystalline phases. It is easy to understand why the surface of pyrocarbon-silica is heterogeneous. It is not so easy to understand why the 2-effect is so much stronger on this material than on graphitized carbon black. In spite of its heterogeneity, pyrocarbonsilica shows great selectivity among isomeric chlorinated biphenyls and great promise as a chromatographic absorbent. It would be worth testing materials with a somewhat higher proportion of carbon, in hope of finding a more uniform surface, and materials of smaller particle size, which should give narrower bands. With even a modest improvement in column performance, liquid chromatography on pyrocarbon-silica could give resolution equal to capillary gas chromatography for at least the lesshighly chorinated biphenyls. The technique is easier than that of capillary gas chromatography, and its sensitivity could be improved by using lower wavelengths for ultraviolet detection. One could also pass the effluent directly into an electron-
capture detector, as was done by Willmott and Dolphin (16).
ACKNOWLEDGMENT We cordially thank H. Colin and G. Guiochon for the gift of carbon black and pyrocarbon-silica absorbents and for discussions in their laboratory. We also thank K. Fujita of Hitachi, Ltd., for the gift of polystyrene gel, and Curt Haltiwanger of the University of Colorado for the x-ray diffraction study. LITERATURE CITED T. Hanai arid H. F. Waiton, Anal. Chem., 49,764 (1977). T. V. Barmakova, A. V. Kiseiev. and N. V. Kovaleva. Kolbidn. Zh.. 36.
133, 934 (1974). E. V. Kaiashnikova. A. V. Kiseiev. D. Poskus. and K. D. Shcherbakova, J . Chromatoor. 119. 233 (1976). H. Colin. C. Eon, and G . Guiochdn, J . Chromatogr., 119, 41 (1976). H. Colin, C. Eon, and G . Guiochon, J . Chromatogr., 122, 223 (1976). H. Colin and G. Guiochon, J . Chromatogr., 126, 43 (1976). N. K.Bebris, R. G. Vorobieva, A. V. Kiseiev, Yu S.NikRin, L. V. Tarasova. I. I. Frolov. and Ya. I. Yashin, J . Chromatogr., 117, 257 (1976). R. G.Webb and A. C. Mccall, J . Assoc. Off. Anal. Chsrn., 55,746 (1972). R. G. Webb and A. C. McCall, J . Chromatogr. Sci., 11, 366 (1973). J. Krupcik, P. A. Leclercq, A. Simova, P. Suchanek, M. Collak, and J. Hrivnak, J . Chromatogr.. 119, 271 (1976). U. A. Th. Brinkman, J. W. F. L. Seetz, and H. G. M. Reymer, J .
Chrornatoyr., 116, 353 (1976). U. A. Th. Brinkman, A. de Kok, G. de Vries. and H. G. M. Reyrner, J . Chromatogr., 128, 101 (1976). U. A. Th. Brinkman and J. J. de Kok. Fresenius' Z . Anal. Chem.. 283, 205 (1977). D. L. Stalling and J. N. Huckins, J . Assoc. Off. Anal. Chem., 54,801 (1971). D. Sissons and D. Welti, J . Chrornatogr., 60, 15 (1971). F. W. Wiiimotl and R . J. Dolphin, J . Chromatogr. Sci., 12, 295 (1974).
RECEIVED for review May 27,1977. Accepted August 5, 1977. Support is acknowledged from the National Science Foundation, Grant CHE 76-08933. Part of this work was presented at the ACS Award in Chromatography Symposium, New Orleans, La., March 21, 1977.
Dynamic Slurry-Packing Technique for Liquid Chromatography Columns H. P. Keller, F. Erni, H. R. Linder, and R. W. Frei"' Analytical Research and Development, Pharmaceutical Division, Sandoz, Ltd., 4002-Basle, Switzerland
A new packlng apparatus for slmultaneous packing of up to 6 columns Is descrlbed. The principle Is based on a comblnation of stirrlng action and high pressure of up to 500 bar. Data are presented for packlng reversed phase materlals. The choice of slurry solvent Is not critical and nontoxic inert and moblle phase compatible solvents can be used. A column testing procedure whlch takes Into account both plate heights and asymmetry Is discussed wlth a serles of C8 reversed phase columns packed for routine use. The method will provlde columns wHh good separation properties for a column length of up to 25 cm.
It is usually accepted that particles of 5-10 km particle size have to be packed by slurry techniques. Solvent systems with Present address, Department of Analytical Chemistry, The Free University at Amsterdam, De Boelelaan 1083,NL Amsterdam 1011, The Netherlands. 1958
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
suitable equal density or viscosity properties are used to keep the particles in a reasonably suspended state during the packing procedure (1-3). In the very recent literature an alternative method was proposed where silica gel and alumina particles of 3-6 km were packed upward with methanol as a slurry solvent. The pressure was ZOO0 psi using a CE 210 coil pump. The column dimensions varied between 10-100 cm length and 1 5 mm i.d. Because of the few experimental data published, it is difficult to make a comparison with the method presently discussed. In our laboratories a new packing principle has been described (4)which involves a combination of high pressure and stirring action to keep the particles afloat during the column-filling step. The advantages of this principle were primarily the elimination of inconvenient halogenated solvents and the possibility of using slurry solvents similar to the mobile phase which shortens the time of eauilibration for the columns mior to actual use. The disadvant&e was that the quality of coiumn Packing decreased drastically with increasing column len@h, and it has been demonstrated experimentally ( 4 ) that only
