r
Liquid Chromatography of Aromatic Hydrocarbons on lonExchange Resins David M. Ordemann and Harold F. Waiton” Department of Chemistry, University of Colorado, Boulder, Colo. 80309
Polycyclic aromatic hydrocarbons and halogenated hydrocarbons, including chlorinated biphenyls, are separated chromatographically on columns of low-cross-linked polystyrene-type cation-exchange resins. The counterions affect retention, doubly- and triply-charged Ions being more effective than singly-charged. Best results were obtalned with Ca and Fe( Ill), using acetonitrile-water or methanol-water solvent mixtures.
Ion-exchangeresins based on polystyrene absorb uncharged organic compounds, especially those having aromatic character. The charge type of the resin is of secondary importance. In 1957, Sherma and Rieman (1)described the “solubilization chromatography” of alcohols, esters, ketones, and aromatic hydrocarbons on columns of anion- and cation-exchange resins, using as eluents mixtures of water with methanol or acetic acid. The proportion of nonaqueous solvent was increased as elution proceeded. Recently Funasaka et al. ( 2 ) studied the chromatography of several substituted benzenes and naphthalenes on six different ion-exchange resingwith six different solvents. Anion-exchange resin columns were used by Scott and others ( 3 , 4 )to analyze the polar organic constituents of body fluids and contaminated waters. Polycyclic aromatic hydrocarbons and their chlorinated derivatives are of current interest because of their environmental importance. Chromatography on bonded packings ( 5 , 6) and on silica gel (7,8) has been used to analyze mixtures of these compounds. A combination of gas and liquid chromatography was used by May et al. (9) to analyze aromatic hydrocarbons in sea water. Gas chromatography on a liquid crystal stationary phase (10) is very effective for distinguishing isomers. In our laboratory, we have studied the chromatography of various polar aromatic compounds on anion- and cationexchange resins (11,12) and noted, among other effects, the effect of the resin counterions. We have now extended these studies to polycyclic aromatic hydrocarbons and a few of their chlorinated derivatives, using both anion- and cation-exchange resins of the polystyrene type. Experiments with cation-exchange resins are described in this paper.
EXPERIMENTAL Materials. Most of our work was done with the sulfonated polystyrene cation-exchange resins, Aminex 50W-X4,20-30 p, and Aminex A-7,8% cross-linked, 7-11 p , both supplied by Bio-Rad Laboratories, Richmond, Calif. A custom-made copolymer of 2-methyl-5vinylpyridine with 9% divinylbenzene was supplied by the same company. The nonionic, macroporous styrene-divinylbenzene copolymer, Amberlite XAD-2 (Rohm and Haas co., Philadelphia) was also tested. Pure phenanthrene, biphenyl, and chlorinated biphenyls were supplied by Analabs, Inc., North Haven, Conn., and other hydrocarbons by Aldrich Chemical Co., Milwaukee, Wis. High-quality solvents were used. Columns, Chromatographic Equipment, Procedure. Waterjacketed glass columns, 6.3-mm i.d., fitted with adjustable Teflon (PTFE) plungers and tolerating pressures of 500-1000 psi (35-70 bars) were supplied by Glenco Scientific, Inc., Houston, Texas, and by Laboratory Data Control, Riviera Beach, Fla. Injections were made by septum or by sample-injectionvalves. Pumps were made by Waters 1728
Associates, Milford, Mass., and by Laboratory Data Control. Ultraviolet detectors were supplied by Spectra-Physics, Inc., Santa Clara, Calif., and were used at 254 nm. The columns were normally kept a t 55-65 “C by circulating water fr0m.a constant-temperature bath. The resin beds were 18-35 cm long. Mixed solvents were used, one component being water and the other either methanol, acetonitrile, or isopropyl alcohol. Solvent gradients were sometimes used. Flow rates were between 12 and 30 m l h , with pressures up to 300 psi (20 bars). With the soft, 4% cross-linked resin, it was important not to use excessive pressure, or the resin bed collapsed, sometimes irreversibly. At moderate pressures, the columns could be operated continuously.
RESULTS Comparison of Ionic and Nonionic Resins. The ionic functional groups of ion-exchange resins play no direct part in the absorption of uncharged organic compounds. Their main function is to become hydrated and cause the resins to swell and become permeable. Comparing the retention of biphenyl on a column of Aminex 50W-X4 ion-exchange resin with that on a column of nonionic, macroporous Amberlite XAD-2, we found much stronger retention on XAD-2. The capacity factor, k’, was 2.5 at 65 “C with 80%acetonitrile as the solvent, compared to 1.6 with 33% acetonitrile for the ion-exchange resin (see Table 111). The bands were much broader with XAD-2, because this resin was used in ita normal commercial form, as porous granules 0.5 mm in diameter. Counterion Effects. The absorption of hydrocarbons by cation-exchange resins depends on the counterions. Divalent ions give considerably stronger retention than univalent, and trivalent ions give somewhat stronger retention than divalent ions. This effect was first noticed in batch equilibration tests with 8% cross-linked resin in its sodium and calcium forms. The distribution ratio of biphenyl in isopropyl alcohol-water mixtures was almost twice as great for the calcium form of the resin than it was for the sodium form. Column elution tests confirmed this result. A selection from a large number of elution data with different counterions is given in Tables I and 11. Cross-Linking. Table I1 shows that the higher-cross-linked resin retains hydrocarbons more strongly. The bands were significantly broader with this resin, however. The effective plate height for fluoranthene, for example, was more than 1.5 times as great for the 8% cross-linked resin than for the 4% cross-linked resin with Mg as counterion. ‘Forthis reason, the 4% cross-linked resin was chosen for more detailed study. Comparison of Different Resins. The anion-exchange resin, Bio-Rad AGl-X4 37-75 microns, was tested. It retained hydrocarbons roughly twice as strongly as calcium-loaded Aminex 50W-X4, and the chloride form gave slightly stronger retention than the sulfate form. However, the plate height of AGl-X4 was about five times as great as that of 50W-X4. Cross-linked 2-methyl-5-vinylpyridineresin retained hydrocarbons some four times as strongly as Aminex 50WX4-Ca, but it, too, gave broad bands with marked reverse tailing. Solvents. Mixtures containing water and up to 60% of an organic solvent were used. Acetonitrile and methanol gave the narrowest bands, corresponding to their low viscosities. Rel-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1976
l
Table I. Elution of Phenanthrene from Aminex 50W-X4a 2-Propanol 25%
Counterion
Na Ag 4"
Ace tonitrile, 37.5%
37.5% H
k'
H
k'
H
4.8
... ...
3.8
1.0
...
4.4 7.9 7.4 8.2
0.7 0.7 0.8 0.5
...
... 0.3 ... 0.3 ...
8.4 9.1
0.3 1.3
...
...
...
5.2 8.6 9.1 11.1
0.2 Ca 0.3 A1 Fe(II1) 0.4 Th ... ... ... a Flow rate, 24 ml/h; temp. 60 H = effective plate height, mm.
...
6.3
...
7.9
Oowex-SOx4-Fe, 35cm x 0.63cm
BIPHENYL
SOLVENT:
CHsCN, 37.5%
IDENEl II
-J
h' = capacity factor;
O?;
-
+
pH 1.9; 60°C
FLOW RATE:
24mi/hour
W
k'
...
-
COLUMN:
NAPHTHALENE
a
FLUORENE
rn
m V _I -J
LL 3
*0
PHENANTHRENE
-d w
11 '0
1/11 (1
0
z a
m
FLUORANTHENE
A
LT 0 m
A
PYRENE
m
a
Table 11. Comparison of Counterions and Cross-Linkinga Resin crosslinking
Counterion
8%
Mg
Anthracene
k' for Fluoranthene
3 -4
1.9 2.4 0.9 1.15 1.3
Zn
...
Pyrene
Flgure 2. lsocratic elution on Fe-loaded resin
4.5 5.1 2.3 2.9 3.2
Na 1.8 Mg 2.3 Ca 2.6 a Solvent, 50% CH,CN by volume; flow rate, 24 ml/h, temp. 55 OC;k' = capacity factor. 4%
Resin, Aminex 50W-X4-Fe: solvent, acetonitrile,37.5% (v/v); temp. 60 O C : flow rate, 24 ml/h. Column dimensions, 35 cm X 0.63cm
w:D o w e x - 5 0 x 4 - F e . FLOW R A T E :
3 8 c m x 0.63cm
30ml/hour
SOLVENT.
2504 - - - - - -----50%
(gradient)
W _I
a m V 1 1
3 LL
N
-s w 0
z 4:
m
a
m 0
m 4
30
40
50
60 70 (v/v)
PER CENT C H 3 C N
Flgure 1. Capacity factor (column distribution ratio) and solvent composition (+) fluoranthene; ( 0 )pyrene: (0) phenanthrene. Resin, Aminex 50W-X4-Ca. Temp. 65 O C
ative retention volumes of different hydrocarbons were almost unaffected by the choice of solvent, though we did notice that chlorinated naphthalenes were poorly resolved in isopropyl alcohol-water mixtures, compared to acetonitrile-water mixtures. Chlorinated biphenyls were resolved nearly as well in methanol-water (60%methanol by volume) as in acetonitrile-water (33% acetonitrile). Elution volumes were lower in acetonitrile-water mixtures than in methanol-water mixtures having the same proportion of organic solvent. Elution volumes increased rapidly with decreasing proportion of organic solvent. Figure 1 shows an almost linear correlation between the logarithm of the corrected elution volume and the proportion of acetonitrile by volume ("50% by volume" means that equal volumes of acetonitrile and water were mixed). This is a common relationship, and shows that the energy of transfer of solute from the solution to a standard state is proportional to the volume fraction of the mixed solvents.
0
12
24
36
48
60ml
Figure 3. Gradient elution on Fe-loaded resin Resin, Aminex 50W-X4-Fe; solvent, acetonitrile: gradient, 25% to 50% in 45 min; temp. 60 O C : flow rate, 30 mllh. Column dimensions, 35 cm X 0.63 cm. Quantities injected (pg): indene 10, naphthalene 17,biphenyl 10, fluorene 7, phenanthrene 4, fluoranthene 24, pyrene 24
Elution Sequences and Separations. Two extended series of tests were made with the resin Aminex 50W-X4,one with Fe(II1) counterions, the other with Ca(I1). Iron-Loaded Resin. With Fe(III), the solvents were made 0.01 M in nitric acid to prevent hydrolysis. A very small leakage of iron from the resin was observed, but it was steady, and stable baselines were obtained with the uv detector. Figures 2 and 3 show chromatograms obtained with this column. With 20% acetonitrile as solvent, we separated a number of single-ring and two-ring aromatic hydrocarbons. They were eluted in this order: benzene, toluene, ethyl benzene, orthoxylene, meta- plus para-xylene (not resolved), indene, naphthalene, biphenyl. Naphthalene and biphenyl were well resolved with good baseline separation. Anthracene and phenanthrene were not resolved. Compounds with larger molecules than pyrene were sepa-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1976
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(a)
w
O.O!
E
4
d
m
v)
a
N
0
w
v)
0
m
z a a
a
m 0
ln m a
T I M E A N D VOLUME
Flgure 4. Elution on Ca-loaded resin
Resin, Aminex 50W-X4-Ca; temp. 65 "C; column dimensions, 18 cm X 0.63 cm. Peaks, in order of elution, are: biphenyl, 0.2 pg; phenanthrene, 0.025 pg; fluoranthene,0.3 pg; pyrene, 0.6 pg. Curves (a)and (b),50% acetonitrile (v/v); (a)24 mllh: (b) 12 ml/h. Curve (c),37.5% acetonitrile (v/v), 24 ml/h C
Table 111. Effects of Chlorine Substitutiona Compound
k'
Flgure 5. Absorptivity at 2 5 4 nm
0
5
lOml
Biphenyl and chlorinated biphenyls
Resin, Aminex 50W-X4-Ca; temp. 65 " C column dimensions, 18 cm X 0.63 cm: solvent, 33% acetonltrile (vlv); flow rate, 21.5 ml/h. Peaks, In order of elution, are: 2,2'-bichlorobiphenyl, 3 pg; 2-chlorobiphenyl, 1 pg; biphenyl, 0.2
1.60 16 500 Biphenyl 1.30 5 400 2-Chlorobiphenyl 1.03 1000 2,2'-Bichlorobiphenyl 3 2 000 2.00 4-Chlorobiphenyl 2.85 36 000 4,4' -Bichlorobiphenyl Naphthalene 1.7 2.6 1-Chloronaphthalene 8.0 10 000 Anthracene 15.5 50 000 9 ,lo-Dichloroanthracene a Resin, Aminex 50W-X4-Ca;solvent, 33% CH,CN by volume; temp. 65 O C ; It' = capacity factor. Molar absorptivities are approximate.
duced. Another consequence is reduced absorption of ultraviolet light. Where no steric interference occurs, however, the effect of chlorine substitution is to increase the retention by the resin and also to increase the ultraviolet absorption. These effects are seen in Table 111. The 3- and 3,Y-substituted chlorobiphenyls were retained somewhat more strongly than their 4- and 4,4'-isomers.
rated with a solvent mixture consisting of acetonitrile, tetrahydrofuran, and water, in ratios 41:5 by volume. These elution volumes were found a t 55 "C on the same column used for Figures 1and 2: fluoranthene 17, pyrene 19.5, chrysene 23.5, benzo[a]pyrene 29.5, perylene 34 ml. Effective plate heights were 1.5 mm at 24 m l h , which is to say that the bands for the hydrocarbons beyond pyrene were undesirably broad. A mixture of biphenyl, 4-bromobiphenyl, and 4,4'-dibromobiphenyl was cleanly separated on a column 18cm X 0.63 cm, with 37% CH&N a t 55 "C. Elution volumes were, respectively, 8.0, 10.5, and 14.0 ml. The presence of a halogen atom always increased the elution volume except in the case of 2-substituted biphenyls (see below). Calcium-Loaded Resin. The advantage of calcium as a counterion, compared with iron, is that it is not hydrolyzed by water and there is no need to acidify the solvent. Retentions and bandwidths are comparable. Representative chromatograms of a hydrocarbon mixture on a calcium-loaded 4% cross-linked resin are shown in Figure 4. They show the effects of solvent composition and flow rate. With 50% acetonitrile at 12 ml/h, acceptable resolution of biphenyl, phenanthrene, fluoranthene, and pyrene was achieved in 40 min. For fluoranthene, the plate number of the 18-cm column was 1050, and the effective plate number was 750. A chromatogram of a mixture of chlorinated biphenyls and biphenyl itself appears in Figure 5. It shows that chlorine atoms in the 2 position decrease the retention rather than increasing it. They cause the two phenyl groups to be twisted out of their common plane, and as a result, *-electron overlap is lost and attachment to the aromatic resin polymer is re-
DISCUSSION An interesting feature of these results is the effect of the counterion. The stronger absorption found with divalent counterions, compared with univalent, may simply be due to the fact that one doubly-charged ion takes up less space than two singly-charged ions, and leaves more room for absorbed molecules. A more likely explanation is that electrostatic fields within the resin are stronger with doubly-charged ions. Their position of maximum stability is close to one of the singlycharged fixed ions, not midway between two of them. A strong electrostatic field would induce a dipole moment in an aromatic molecule and strengthen its absorption. Silver ions are known to form a-bonded coordination complexes with aromatic hydrocarbons, but the few tests that we made showed little difference in retention between a silver-loaded and a sodium-loaded resin, probably because water is present and the silver ions are hydrated. The major cause of the binding of aromatic hydrocarbons by polystyrene resins is, almost certainly, a-electron overlap. The behavior of 2-substituted biphenyls supports this idea.On a column packed with Clg-bonded silica, we found that 2chlorobiphenyl was retained more strongly than biphenyl itself, whereas the reverse was true on the cation-exchange resin. As to the practical utility of the 4% cross-linked cationexchange resin for chromatography of these compounds, it would seem that, in most circumstances, a (&-bonded packing would work better. Certainly this is true for molecules having more than four aromatic rings, for the transfer of big molecules in and out of a resin is just too slow. For molecules the size of pyrene and smaller, however, the resin may be a useful alternative. Resolution is as good as on the bonded packing, with
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1976
5000 plates per meter and better. Elution peaks on the resin are very symmetrical, even a t high loadings, which makes it easy to detect small amounts of closely-eluted impurities. We found, for example, that our high-grade phenanthrene had a small trace of an impurity which eluted 0.6 ml after the phenanthrene peak. The higher capacity of the resin makes it better for preparative purposes than the bonded packing. Columns of 4% cross-linked resin are easy to pack and to use. Chromatography is slower than on CIS-bonded packings, but not much slower, and if running times of 30-60 min are acceptable, rosin columns may be of practical value.
(3) C. D. Scott, Sep. Purif. Methods, 3, 263 (1974). (4) W. W. Pitt, R. L. Jolley, andC. D. Scott, Envlron. Sci. Techno!.. 9, 1068 (1975). (5) B. B. Wheals,C. G. Vaughan, and M. J. Whitehouse, J. Chromatogr., 106, 109 (1975). (6) C. H. Lochmuller and C. W. Amoss, J. Chromatogr., 108, 85 (1975). (7) B. Coq, C. Gonnet, and J. L. Rosca. J. Chromatogr., 106, 249 (1975). (8) U. A. Th. Brinkman, J. W. F. L. Seek, and H. G. M. Reymer, J. Chromatogr., 110,353 (1976). (9) W. E. May, S. N. Chesler, S.P. Cram, B. H. Gump. H. S.Hertz, D. P. Enagonio, and S. M. Dyszel, J. Chromatogr. Sci., 13, 535 (1975). (10) 0. M. Janlnl, K. Johnston, and W. L. Zielinskl, Anal. Chem.. 47, 670 (1975). (11) P.Larson, E. Murgia, TongJung Hsu,and H. F. Walton, Anal. Chem., 45, 2306 (1973). (12) H. F. Walton, J. Chromatogr., 102, 57 (1974).
LITERATURE CITED
RECEIVEDfor review April 19,1976. Accepted July 1,1976. This work is taken in part from the M.A. Thesis of D.M.O. (University of Colorado, 1975). Support is acknowledged from the National Science Foundation grant MPS 73-08592.
(1) J. Sherma and W. Rieman, Anal. Chim. Acto, 20, 357 (1957). (2) W. Funasaka, T. Hanai, T. Matsumoto, K. Fujimura, and T. Ando. J. Chromatogr., 88,87 (1974).
Determination of Anhydrotetracyclines in Tetracycline by HighPressure Liquid Chromatography Richard F. Lindauer," David M. Cohen," and Kevin B. Mesnnelly" Pfizer Inc., Quality Control Dlvlslon, Brooklyn, N. Y. 11206
A method has been developed for deteamlnatlon of (4s)anhydrotetracycline (ATC) and (4R)-anhydrotetracycline (EATC) by high-pressure liquid chromatography. The method uses a cation-exchange column and a neutral 0.3 M ethylenediaminetetraacetate bwffer. Response is linear and the estimated limits of detection are 12 ng of ATC and 7 ng of EATC. At 429 nm, the recoveries of ATC and EATC added to tetracycllne at the 0.6% level were quantltatllve wlth relative standard devlatlons of 0.7% and 1.4%, respectively. Paeparation and characterization of a suitable assay standard is described and possible application to other tetracyclines is discussed.
Commercial preparations of the broad-spectrum antibiotic (4s)-tetracycline (TC) may develop varying levels of (48)anhydrotetracycline (ATC) and (4R)-anhydrotetracycline (EATC) if stored under adverse conditions-e.g., high temperature and humidity (I).Limits for EATC in TC bulks and products have been established by the Food and Drug Administration ( 2 ) .Because ATC and EATC are readily interconverted (3),accurate determination of both epimers is desirable. Resolution of ATC and EATC from TC has been achieved by partition chromatography (1, 2, 4, 5 ) , gel permeation chromatography (6),and gas chromatography (7), but these methods were not rapid. High-pressure liquid chromatography (HPLC) with certain reverse-phase systems gave poor resolution of ATC from EATC (8, 9). Shorter analysis times and good resolution of ATC, EATC, TC, (4R)-tetracycline (ETC), and chlortetracycline have recently been reported by isocratic reverse-phase ion-pair HPLC (10) and by gradient-elution reverse-phase HPLC (111; however, no evidence was presented to rule out formation of ATC and EATC by partial dehydration of TC and ETC in the highly acidic mobile phases used. Reproducibility data were not given for the ion-pair system, but in the gradient method the relative standard deviation for EATC was only f6%. Several ion-exchange HPLC systems with short analysis
times and adequate efficiencies for the anhydrotetracyclines nevertheless appeared unsuitable for our purposes sjnce the anhydrotetracyclines eluted on the tail of the main tetracycline peaks (12). However, a preliminary cation-exchange HPLC system (13) in which h' values for the anhydrotetracyclines were low appeared to be more promising. The latter system, which used a dilute phosphate/ethylenediaminetetraacetate (EDTA) buffer at pH 7 as the mobile phase, was investigated and modified in an effort to develop a suitable assay for ATC and EATC in TC bulks and products. This paper describes both the resulting system and methods of preparing and characterizing a suitable quantitative standard for ATC and EATC.
EXPERIMENTAL Apparatus. For determinations a t wavelengths greater than 260 nm, a Du Pont Model 843 pump module was used in conjunction with a Schoeffel Model SF 770 multiwavelength absorbance detector and a Spectrum Scientific Model 1020 electronic filter (operated at a nominal cutoff frequency of 0.1 Hz).At 254 nm, a Du Pont Model 840 liquid chromatograph was used. Injections were made with a (nomi1 injector valve (Du Pont No. 204590), and all connecnal) 1 0 - ~loop tions were made with either stainless steel or Teflon capillary tubing. The temperature of the column was maintained to within 10.2 O C by a Varian water jacket (No. 96-000048-00and No. 37-000324-00)and a Lauda K-2/R circulator. Spectrophotometry was performed on a Cary 14 recording spectrophotometer. Column. A straight 2.1-mmX 1-mstainless steel column (Du Pont No. 820983903)was thoroughly cleaned with hot detergent solution, rinsed with hot water, hot 30% nitric acid, hot deionized water, and methanol, and dried. Stainless steel column plugs (Du Pont: inlet porosity, 10 pm; outlet porosity, 2 pm) were used, and the column was packed with Zipax SCX (Du Pont No. 820960003) using the modified tap-fill procedure (14). A prepacked column of the same dimensions (Du Pont No. 820950002)gave comparable results. Prior to first use, columns were conditioned by elution with 150-200 ml of the mobile phase. Mobile Phase. The nearly saturated mobile phase may be prepared readily in the following manner. Add 229 g of disodium EDTA dihydrate (Fisher Certified) to 1.8 1. of 0.27 M aqueous sodium hydroxide at 70 O C and stir to dissolve. Filter (1.2-pm membrane, Millipore type RAWP) and cool to room temperature under vacuum (100-150 Torr)
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