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Anal. Chem. 1984, 56, 1521-1524
featureless in an optical microscope and had a medium reflectance value typical of vitrane. Of particular interest is the clear appearance of scandium, ytterium, and elements of the lanthanide series. These trace elements, and others, such as vanadium have not been previously detected on a microscopic scale in coals. The extent of molecular ion suppression for these organic compounds is considerably greater than that found earlier in SI-SIMS spectra typical of metals (8)or metal oxides (7). For example, in the SIMS analysis of PTFE the SI-induced suppression of the major molecular fragment CF+ ( m l e 31) has been found to be suppressed by greater than a factor of 1 X lo5 compared to a SIMS spectrum taken with normal mounting. The best suppression factors for FeO+ and SiO+ in spectra of these metals is 1 X lo4 (8). This suggests that the kinetic energy distributions of these typical organic molecular ions is, in general, considerably narrower than those for metal oxide or metal cluster ions. The implication of the above is that SI-SIMS achieves its highest degree of effectiveness in the analysis of organic substrates. With background molecular ions reduced by over 5 orders of magnitude throughout most of the mass range, it is indeed possible to predict detection sensitivities in the ng/g for many elements. The detection of trace inorganic elements within an organic matrix would be much more difficult and time consuming by spectroscopic or activation techniques, both of which lack the microscopic analytical capabilities of SIMS. Work has begun on quantitation of certain elements in a coal matrix using an ion implantation technique (9). The other advantage of the SI-SIMS technique to polymers resides in the complete absence of charging effects on any surface, no matter how insulating. Alternative methods for charge compensation, such as metallic surface coatings and electron flood guns, are of limited use particularly where depth profiling causes a change in charge compensation requirements.
Registry No. PTFE, 9002-84-0; Li, 7439-93-2; B, 7440-42-8; 0, 7782-44-7; Ca, 7440-70-2; Na, 7440-23-5; Mg, 7439-95-4;Al, 7429-90-5; Si, 7440-21-3; S, 7704-34-9; C1,7782-50-5;K, 7440-09-7; Ti, 7440-32-6; Cr, 7440-41-3; V, 7440-62-2; Mn, 7439-96-5; Fe, 7439-89-6; Ag, 7440-22-4; Ba, 7440-39-3; Ce, 7440-45-1;P, 772314-0;Sc, 7440-20-2; Co, 7440-48-4; Ni, 7440-02-0; Cu, 7440-50-8; Sr, 7440-24-6; Zr, 7440-67-7; La, 7439-91-0; Pr, 7440-10-0;Nd, 7440-00-8;Y, 7440-65-5; polystyrene, 9003-53-6. LITERATURE C I T E D Benninghoven, A.; Jaspers, D.; Sichtermann, W. Appl. Apby. 1976, 1 1 , 35-37. Gardeiia, J. A.; Hercules, D. M. Anal. Chem. 1980, 52, 226-232. Campana, J. E.; Decorp., J. J.; Coiton, R. J. Appl. Surf. Sci. 1981, 8 , 337-341. Briggs, D.; Wooton, A. B. S I A , Surf. Interface Anal. 1982, 4 , 109-1 15. McIntyre, N. S.;Chauvin, W. J.; Martin, R. R.; McPhee, d. A. Scanning Electron Microsc. 1883, 1 1 1 , 1115-1127. McIntyre, N. S.; Martin, R. R.; Chauvln, W. J.; Winder, G. C.; Brown, J. R.; McPhee, J. A., submitted to fuel. Metson, J. 6.; Bancroft, G. M.; McIntyre, N. S.; Chauvin, W. J. S I A , Surf. Interface Anal. 1983, 4 , 181-185. McIntyre, N. S.; Fichter, D.; Robinsgn, W. 6.; Metson, J. 6.; Chauvin. W. J., submitted to S I A , Surf. Interface Anal. Ramseyer, G. 0.; Morrison, G. H. Anal. Chem. 1983, 55, 1963-1970.
N. S . McIntyre* W. J. Chauvin Surface Science Western Laboratory Faculty of Science University of Western Ontario London, Ontario N6A 3K7, Canada
R. R. M a r t i n Department of Chemistry University of Western Ontario London, Ontario N6A 5B7, Canada
RECEIVED for review December 8,1983. Accepted March 26, 1984.
AIDS FOR ANALYTICAL CHEMISTS Liquid Chromatography of Cephalosporin C on Substituted Polystyrene Resins Daniel Sacco* and Edith Dellacherie Laboratoire de Chimie-Physique Macromol6culaire. C.N.R.S.-E.R.A. 23, ENSIC-1, rue Grandville, 54042 Nancy Cedex, France The extraction of biological compounds from fermentation liquors usually involves many difficulties due to the presence of related contaminants and various mineral salts. Of the various purification methods, chromatographic techniques have become prominent (I), owing to the great variety of chromatographic media that are commercially available. Thus in the particular case of cephalosporin C, a P-lactam from which many semisynthetic antibiotics are produced, much work has been done to improve its separation by liquid chromatography. Because cephalosporin C is markedly hydrophobic at low pH, it can be purified moderately well by chromatography on activated charcoal columns ( 2 , 3 ) .The use of other nonpolar adsorbents, such as Amberlite XAD type resins (4-6),has made it possible to improve significantly the quality of these separations based on adsorption. Salto et al. (3, proceeding from the work of Pietrzyk et al. (8-11), determined what
phenomena underlie the interactions between this compound, several of its derivatives, and macroporous styrene-divinylbenzene resins (XAD-4). In addition, cephalosporin C has been purified by combined use of anion-exchange and cation-exchange resins, though this method necessitates long and complicated procedures (12-14). More recently, a new analytical-scale separation procedure has been developed only for derivatives of cephalosporin C, using reversed-phase high-performance liquid chromatography (15). Commercial resins bearing CIS (16, 17) chains were chosen. Generally speaking, one can note that the main feature of the various adsorbents used for the separation of cephalosporin C from the contaminants in its biosynthesis medium is their relatively poor selectivity. Here we describe a study of styrene-divinylbenzene resins bearing immobilized ligands with characteristics intended to
0003-2700/84/0356-1521$01.50/00 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
!!able I. Structures of the Resins Useda + I
COOH
-CH2 - N H - ( C H z t 4 - C H
1
L \
"2
COOH 2
0
COOH 3 2 4
\-I
\
"2
COOH
0
*;I II -C H2-( N H -C H -C ) -N H-(C H2 l4 C H
L
4
\
"2
5 kOOH
-cH,-NH
-icn,i,
-COOH
6
7
8
P O a
- C H - N H -CH;CH2-OH
9
-CH;NH
10
-CH2-CH2-CH,
P is polvstwene-divinvlbenzene Polymer.
be complementary with those of cephalosporin C, so they may interact preferentially with it. The effect on the chromatographic behavior of these new resins of the ionic strength of the aqueous eluting solution was determined. These various studies enabled us to identify a binding ligand that interacts strongly and selectively with the antibiotic. The new adsorbent has been used to separate cephalosporin C from a related contaminant, deacetylcephalosporin C, and from various contaminating amino acids usually present in the fermentation medium.
EXPERIMENTAL SECTION Reagents. The 0-amino acids and the organic acids were from Fluka (Switzerland). Glycylglycine and (paminopheny1)alanine were from Bachem (Switzerland). Polystyrene resins cross-linked with divinylbenzene (Bio-Beads S-X1, divinylbenzene content 2%, particle size 200-400 mesh) were from Bio-Rad (Richmond,CA). Synthesis and Properties of the Polyfunctional Resins. The various resins used (Table I) were synthesized as described elsewhere (18). The chemical capacity of these new adsorbents determined both by elementary analysis and by potentiometric
titrations is between 0.6 and 0.9 ligand groups mmol per g of polymer. The quantities of dry resins present in the various chromatographic systems were between 4.3 and 5.3 g. Procedures. Stainless steel tubing (0.95 cm id.) and fritted end fittings were purchased from Waters Associates. Columns were 30 cm long and were packed by the classical slurry-packing technique (19). A Waters Model ALC 200 liquid chromatograph was used. The flow rate was 1mL/min and the pressure was 400 psi. The concentration of the solute solutions was 60 mM and a volume of 20-150 MLwas injected with a Hamilton syringe into the chromatographic system. The capacity factors ( k ? were calculated from the expression k' = ( VR - Vo)/Vo, where VR is the elution volume of the chromatographic peak and V , is the column void volume. The void volume (Vo = 6.3 mL) was determined as the elution volume of poly(ethy1ene glycol) with molecular weights greater than 1500.
RESULTS AND DISCUSSION Search for a Binding Ligand Specific for Cephalosporin C. The extraction of cephalosporin C from its biosynthesis medium usually requires several operations owing to the lack of specificity of the adsorbents used hitherto. First the antibiotic is partially purified by adsorption chromatography or ion-exchange chromatography or by a combination of both; then cephalosporin C is recovered in a pure state by selective precipitation as its zinc salt. In order to simplify this procedure, the characteristics of the classical chromatography phases were improved by attachment of polyfunctional sites. The nature and geometry of the binding ligands immobilized on the stationary phase (Table I) were best adapted to the nature and geometry of cephalosporin C; they were chosen to favor both ionic interactions between complementary ionic functions and hydrophobic interactions between the spacer arms joining the various functions of the binding ligand and the hydrophobic part of the antibiotic. In order to evaluate the effect of the various functions borne by these binding ligands, other resins bearing simpler sites were synthesized (Table I). Interactions in Water. Cephalosporin C and deacetylcephalosporin C were completely retained by all the adsorbents except by the propylamine resin. Glutamic, aspartic, chloroacetic, and propionic acids were also irreversibly adsorbed to these resins. On the contrary, neutral amino acids were partially retarded (Table 11). These results show that the presence of a carboxyl function without an adjacent amine function is very important and that it is responsible for the irreversible adsorption in water. This hypothesis is confirmed by the fact that unlike free glutamic acid, y-methyl glutamate ester was eluted at a volume similar to that of alanine. Effect of the Salt Concentration of the Eluent. Addition of salt (NaCl) to water affected significantly the retention of previously adsorbed solutes (aspartic and glutamic acid, cephalosporin C, deacetylcephalosporin C). Their retention volume decreased when the ionic strength of the eluent increased, as shown in Figures 1 and 2. Furthermore, it can be seen in these figures that the rate of decrease in retention volume for any given compound was not the same on all the supports. Thus, one can see that certain binding ligands exhibit a high affinity toward cephalosporin C, the e-L-lysine
Table 11. Capacity Factors Calculated for Solutes Loaded onto Various Stationary Phases and Eluted with Watera adsorbent no. 7 8 9 5 6 1 2 3 4 solutes ( D and L ) 0 0 0 0 0 0 0 0 0 lysine alanine 0.20 0.30 0.10 0.20 0.25 0.40 0.35 0.20 0.10 0.65 0.55 0.80 0.70 0.60 0.30 methionine 1 0.75 0.60 phenylalanine 3.35 1.45 1.50 1.65 1.40 1.80 1.50 1.30 0.85 a The elution volume of 7-methyl glutamate was identical with that of alanine on all the resins.
________-
10 0 0 0 0
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
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k
k,*
4
'*
7 . 6 5 4 .
3 .
1 .
k'
'c
I .
Flgure 3. Capacity factors of various solutes in 0.25 M aqueous NaCl solution on resins bearing various monofunctional, difunctional, or trifunctional ligands: (m) chloracetic acid; (0)cephalosporin C; (A) glutamic acid; (0)propionic acld; (0)phenylalanine; (V)aspartic acid. For numbers 1 to 9, see Table I . A Is trlmethylarnine resin.
Figure 1. Capacity factors calculated for the resins 1, 2, 3, and 4, as a function of reciprocal of NaCl concentration: (0)cephalosporin C; (A)glutamic acid; (A)deacetylcephalosporinC; (V)aspartic acid; (0)phenylalanine; (V)methionine; (El) alanine.
1
4
4
10
IO
4b
*
CYMjl
7
5
a. 6. 4.
2. 0.
I 4
10
40
'c&y
9
Figure 2. Capacity factors calculated for the resins 5, 6, 7, 8, and cephalosporin 9, as a function of reciprocal of NaCl concentration: (e) C; (A)glutamic acid; (A)deacetylcephalosporinC; (V)aspartic acid; (0)phenylalanine; (V)methionine; (a)alanine.
resin (1)having the greatest affinity. On the other hand, the k' values of neutral amino acids were not decreased by increasing the ionic strength of the eluent, which suggests that their retention is due not to ionic but mainly to hydrophobic
interactions, whose strength depends on the binding ligands used. Role of the Structure and Functions of the Solutes. On comparison of the k'values presented in Figure 3 (0.25 M NaC1) several points are noteworthy: (1) aspartic and glutamic acids were always retained less than propionic acid, (2) the retention of cephalosporin C is always slightly greater than that of propionic acid and much weaker than that of chloroacetic acid on all the resins except on the €+lysine resin (I),on which cephalosporin C is much more adsorbed than all the other solutes. It appears clearly from these figures that the adsorption on the polyfunctional resins is not solely due to interactions between the carboxylate group of acidic solutes and the binding ligand since, in this case, their order of elution should be strictly related to the pK, values of their carboxylic groups. These results demonstrate well that the hydrophobic moieties of few solutes greatly contribute to their retention. On the contrary, it seems that the presence of an a-aminocarboxylic group in the solute is not a favoring factor for their adsorption (see glutamic and aspartic acids, alanine). Role of the Structure and Chemical Functions of the Binding Ligands. As shown in Table I (resins 1,2, 3 , 4 , 5 ) , the presence on the ligand of functions complementary to those of the antibiotic does not necessarily result in a strong association. In fact, the spatial distribution of the same ionic functions affects the strength of the interactions. Likewise, the nature of the spacer arms linking these various functions is an important parameter. Moreover, the presence of a C5 carbon chain (resins 1, 6, 7) results in a substantially increased affinity to the binding ligand for cephalosporin C, whereas a decrease in the length of this aliphatic chain (resins 6,8) produces the opposite effect. In addition, it is noteworthy that commercial resins containing classic binding sites (ethanolamine or trimethylamine) do not interact strongly and specifically with cephalosporin
C. In summary, though a search for a binding ligand capable of interacting effectively with a given solute must be based on complementary ionic characteristics between the two molecules, it is necessary to optimize the characteristics of the spacer arms linking these functions with those of the compound being purified. Thus for the extraction of cephalosporin C, the best arrangement of ionic groups and hydrophobic moieties in the binding ligand was obtained by immobilizing the +amino group of lysine on a classical chromatographic support.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
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Table 111. Chromatographic Characteristics of the Mixture of Cephalosporin C and Deacetylcephalosporin C on the Resins with the Greatest Affinity, Isocratic Elution with Aqueous NaCl Solutions concn of resolution selectivity pure resin the eluent, factora factora cephalosporin C no. M R 01 recovered, % 1 0.25 0.78 2.90 93 2 0.1 0.65 1.55 75 3 0.1 0.65 2.10 73 4 0.1 0.65 2.10 72 5 0.1 0.70 2.50 74 6 0.25 0.72 2.10 87 7 0.25 0.75 2.10 86 a Resolution and selectivity factors defined between cephalosporin C and deacetylcephalosporin C. CY = (Ve, - V,)/(Ve, - V,) where Ve, is elution volume of cephalosporin C, Ve, is elution volume of deacetylcephalosporin C, and V,, is void volume of the column. R = 2(Ve, - Ve,)/(W, + w,) where w I is width of cephalosporin C peak and w ,is width of deacetylcephalosporin C peak.
0
20
40
80
80
ve
mi
Flgure 4. Chromatogram of a mixture of cephalosporin C and various
Contaminants on the e-L-lysine resin, isocratlc elution: refractometric detection; flow rate, 1 mL/min; r w m temperature; eluent, 0.25 M NaCI; CC, cephalosporin C; DACC, deacetylcephalosporin C. Separations. Mixtures of cephalosporin C and various contaminants were then chromatographed isocratically in the optimal saline solutions. The values for the resolution and selectivity factors between cephalosporin C and deacetylcephalosporin C make it possible to classify the chromatographic materials according to their effectiveness (Table 111). Best separations in analytical scale were obtained with the e-L-lysine resin (1)in a 250 mM NaCl solution (Figure 4), which confirms the high specificity of the immobilized ligand e-L-lysine for the antibiotic. T o extract cephalosporin C satisfactorily from its biosynthesis medium, two conditions are nevertheless necessary: it must be separated as completely as possible from its contaminants, and it must be recovered in the smallest possible elution volume to avoid the necessity of concentrating. T o satisfy the second requirement, a second purification procedure was developed, in which the ionic strength of the eluent solution wm varied during separation (Figure 5) in order
I
1
0
L.&
20
40
80
Ve
Flgure 5. Chromatogram of a mixture of cephalosporin C and various
contaminants on the e-L-lysine resin, gradient elution: refractometric detection; flow rate, 1 mL/min; room temperature; eluent 0.25 M and 1 M NaCI; CC, cephalosporin C; DACC, deacetylcephalosporin C.
to collect the cephalosporin C in a volume about half that with isocratic elution. The second method, which makes it possible to recover cephalosporin C more rapidly and in the least-dilute solution possible, nevertheless has the disadvantage that the chromatographic column must be reequilibrated with the 250 mM NaCl starting buffer between uses. These two separation techniques will therefore have complementary uses. The isocratic technique will be preferable when it is desired to inject mixtures continuously, as in studies of the composition of a biosynthesis medium throughout a fermentation. On the other hand, the stepwise elution method is better for recovering cephalosporin C from its biosynthesis medium.
ACKNOWLEDGMENT The authors thank J. Nee1 for many helpful discussions and T. Geoffroy for her excellent technical assistance. Registry No. CC, 61-24-5; DACC, 1476-46-6. LITERATURE CITED Hussain, Y. M.; Cantwell, F. F. Anal. Chem. 1978, 5 0 , 491-496. Nara, K.; Ohta, K.; Katamoto. K.; Mizokami, N.; Fukuda, H. German Patent 2 309 899, 1973. Nara, K.; Katamoto, K.; Ohta, K. Japanese Patent 8040615, 1980. Voser, W. German Patent 2021 696, 1970. Ciba. S.A. French Patent 750 292, 1970. Kawamura, K.; Tsukazoshi, S. Japanese Patent 76 32 791, 1976. Salto, F.; Prieto, J. G. J . Pharm. Sci. 1981, 7 0 , 994-998. Pletrzyk, D. J.; Kroeff, E. P.; Rotsch, T. D. Anal. Chem. 1978, 5 0 ,
4g7
Pietrzyk, D. J.; Chi-Hung, Chu a / . Chem. 1978, 5 0 , 502-511. Chem. 1977, 49, 757-764. Pietrzyk, D. J.; Chi-Hung, Chu An Pietrzyk, O'Connor,D.S.J.;C.Chi-Hung, Methods Chu Enzymol. Anal. 197e m , 4. 31977, , 296-299. 4 9 , 860-867.
Abraham, E. P.; Newton, G. F.; Trown, P. W. French Patent 1 353 113, 1964. McCormick, M. H. US. Patent 3 467 654, 1969. Marian, G. Y. J . Chromatogr. 1978, 150, 221-224. Salto, F. J . Chromatogr. 1978, 161, 379-385. Grombez, E.;Van Den Bossche, W.; De Moerloose J . Chromatogr. 1979, 169, 343-350. Sacco, D.; Dellacherie, E. Makromol. Chem. 1982, 182, 763-771. Kirkland, J. J. "Chromatographie en Phase Liquide"; Gauthier-Viiiars: Paris, 1972.
RECEIVED for review July 20, 1983. Accepted February 10, 1984.
