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Anal. Chem. 1991, 63,2888-2891
Detection of Anionic Polymers by Postcolumn Ligand Exchange with Fluorescent Cerous Ions via a Cation-Exchange Membrane Per Olof G. Edlund*.’ Kabi Invent, Novum, Blickagingen 6 D, S-14152Huddinge, Sweden
Sven P. Jacobsson’ Kabi Pharma, Box 1828,S-17126 Solna, Sweden
A fluorescence method has been developed for detectlon of anionic polymers such as heparin, carrageenans, and other sulfated polysaccharldes. The method Is based on addition of the Ce3+ ion, which forms fluorescent complexes with the polymers. The reagent administration is facilitated by use of a catlonaxchange membrane reactor, and the detectlon scheme can be used both in flow InJection analysis or as a postcolumn reactor after chromatographic separation. Fundamental studies were performed in a flow Injection analysis system, where the cerous Ions were added in two different ways: either by mixing the carrler stream with a cerous sulfate solution followed by removal of excess Ce3+ by exchange against sulfurk acid protons in the membrane reactor or by dlrect additlon of Ce9+ions through the membrane reactor. I n both cases, Ce” was exchanged for the cationic counterions, and the polymers were detected indirectly by fluorescence as their cerous complexes. the ion-exchange membrane acted as a capacitor for Ce3+ and only a low concentratbn of Ce3+In the carrier or regenerant solution was needed to saturate the membrane. the detection was selective, wlth hlgh response to poiyanionic polymers but little or no response to aclds wtth low molecular weighl and a small negative response to complex-forming agents llke oxalic acid. The best slgnai to noise ratio was obtained at a low ionic strength of the carrier. The detection limit by flow injection analysis (FIA) for low molecular weight heparin was 5 ng (1 pmoi) with 50 mM ammonium acetate as the carrier and 0.5 ng with pure water as the carrier. The response was linear withln three decades, and a preclslon of 0.6% RSD was obtained during flow InJectionanalysis. The effect of temperature, flow rate, membrane length, structure of analytes and concentration of reagents is described. The appllcatlon as a detector for chromatography Is demonstrated by sire exciusion chromatography of low molecular weight heparin and carrageenan.
INTRODUCTION Sulfated polysaccharides appear commonly in biological systems. Chondroitin 4-sulfate and chondroitin 6-sulfate are the most abundant mucopolysaccharides in the body and occur in skeletal and soft connective tissue. Dermatan sulfate is present in soft connective tissue and skin, arterial walls, and heart valves. Heparin is well-known for it ability to inhibit the coagulation of blood, and low molecular weight (4000-6000 Da) heparin like Fragmin is used to prevent thrombosis after Present address: Kabi Pharmacia AB, S-11287 Stockholm, Sweden. 0003-2700/91/0363-2888$02.50/0
surgery. Carrageenan is isolated from red seaweed and consists of alternating copolymers of a-(1,3)-and @-(l,.Q)-linked Dgalactopyranose units. Carrageenan is commonly used as a gelling or complexation agent in foods and pharmaceuticals. All of the polymers mentioned above are polydisperse in nature and are of high molecular weight. Chromatographic methods for characterization of the intact polymers have been based on size exclusion chromatography (I). Other methods for characterization of heparin have been based on enzymatic or nitrous acid cleavage of the polymer (2-4)followed by separation of the oligomeric hydrolysis products by reversed-phase ion-pair chromatography or ion-exchange chromatography. Heparin oligosaccharides have been detected by UV (2) suppressed conductivity (4)and by UV detection as their per-0-benzoylderivatives (5). Oligogalacturonic acids with up to 50 residues have been determined by anion-exchange chromatography with pulsed amperometric detection (6). Both UV detection at short wavelengths, and suppressed conductivity detection offer low selectivity and limited sensitivity for sulfated polysaccharides, and there is a need for other detection techniques both for flow injection analysis (FIA) and chromatography. A detector that gives a signal proportional to the degree of sulfation would also be valuable for characterization of such polymers. Postcolumn techniques involving suppressor columns or membranes can be used in several ways to facilitate the detection of analytes as reviewed by Dasgupta (7).When the sample anion emerges from the column together with its countercation, it is possible to exchange the countercation with a detectable ion by cation exchange or to exchange the anion for some other anion by anion exchange. Downey and Hieftje (8) have described a system called “replacement ion Chromatography”where a cation-exchange column was used to exchange Li+ for a countercation associated with an eluting anion. The effluent was then diverted to a total consumption burner for measurement of the atomic emission of lithium. Shintani and Dasgupta (9) have described two systems for postsuppression ion exchange. One system was based on cation exchange of the suppressed eluate, consisting essentially of water, where the analyte counterion (H+) was exchanged for Ce3+. The second system was based on the change in pH during elution of acids where the release of anthralinate from a cation exchanger was decreased due to protonation by the eluting acids. A negative response was obtained in this case. This work describes a study of a fluorescence detection method for sulfated polysaccharides based on cation exchange of the counterion for fluorescent Ce3+. The polysaccharides, which all carry multiple charges, can be regarded as solubilized ion exchangers that exhibit a high affinity for cerous ions over monovalent ions. The reagent administration is facilitated by use of a cation-exchange membrane reactor. The use of the detection method is demonstrated both in flow injection 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
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Figure 1. Instrumental setup with pumps for carrier/mobile phase (Pl) and external channel flow (P2).
analysis (FIA) or as a postcolumn reaction following chromatographic separation.
EXPERIMENTAL SECTION Chemicals and Reagents. Heparin, chondroitin sulfate, K-, 1-, and A-carrageenan were obtained from Sigma (St. Louis, MO) and low molecular weight heparin (Fragmin) was obtained from Kabi (Stockholm, Sweden). Cerous sulfate was obtained from GFS chemicals (Columbus, OH). All other chemicals were of analytical grade quality. Water was purified in a Milli-Q systems from Waters (Bedford, MA). Instrumentation. The mobile phase for LC or the carrier for FIA was pumped with an LC pump Model LC-SA from Shimadzu (Kyoto, Japan), and samples were injected manually with a loop injector Model 7125 from Rheodyne (Cotati, CA). Strong cation-exchangetubular membrane reactors constructed essentially as device type 2C (see ref 9),with an active membrane length of 0.5 or 1 m, were obtained from SciTech (UmeA, Sweden). The regenerant solution was pumped on the outside of the membrane (external channel), counter-current to the mobile phase/carrier (internal channel), with aid of a peristaltic pump, Model FIAstar 5103 from Tecator (Stockholm, Sweden). In this study, the membrane reactors were operated in two different modes. In the first mode, cerous sulfate was added to the carrier solution,which was flowing in the internal channel, and in the external channel sulfuric acid (0-50 mM) was flowing. In this mode, the anionic polymers were equilibrated in an aqueous cerous sulfate solution, with the same concentration of cerous sulfate as in the carrier solution, prior the analysis. In the second mode, 50 mM ammonium acetate carrier was pumped at a flow rate of 0.6 mL/min through the 1m membrane reactor, where cerous sulfate (2-4mM) in 50 mM sulfuric acid was flowing in the external channel at a rate of 2.8 mL/min. This carrier was also used as the mobile phase during size exclusion chromatography on a Superdex column with 13-rm particles, 10 X 300 mm (Pharmacia, Uppsala, Sweden). A Biosil SEC guard column with 5-rm particles, 7.8 X 80 mm (Biorad,Richmond,CA) was also used essentially to desalt samples during FIA (low-resolutionSEC). Band-broadeningeffects were studied by measurement of the increase in peak width caused by adding the membrane reactor between the injector and detector. The membrane reactor was immersed in a water bath with regulated heating and cooling for variation of the temperature. The membrane inner flow exit was connected to a fluorescence detector Model RF-550 or RF-530 from Shimadzu, operated at excitation and emission wavelengths of 250 and 350 nm, respectively. The instrumental setup is presented in Figure 1. Peak heights and areas were measured manually. RESULTS AND DISCUSSION A continuous exchange of Ce3+for H+occurs when a solution of cerous ions is pumped through the ion-exchange membrane capillary with sulfuric acid flowing in the external channel. The selectivity of the membrane for Ce3+vs H+is expected to be very high, and the membrane will be essentially in the cerous form. When an anionic analyte with high affinity for Ce3+passes through the membrane, the Ce3+removal by the ion-exchange membrane will be inhibited to some degree depending on the relative affinity of the membrane and the analyte for Ce3+. It was found that the response obtained for carrageenans was much higher than what could be expected for a solution equilibriated with a concentration of cerous sulfate as low as, for example, 8.5 pM. The analyte was apparently capable of extracting Ce3+from the membrane, which seemed to act as a buffer for this ion. This hypothesis
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k-carrageenanconc. (pg/ml) Figure 2. Calibration curves for K-carrageenan by F I A showing the improvement in linearity with increasing cerous ion concentration (blank square = 2.8 pM and filled square = 8.4 pM Ce3+) in the carrier and with 10 mM sulfuric acid flowing in the external channel. was verified in the following manner. The membrane was disconnected and the system was purged with pure water. Then a carrageenan solution was injected every second minute with water as the carrier. The response decreased slowly to about 33% of the initial value after 60 injections. The initial decrease was faster when a solution of sodium sulfate (1.2 pM) or magnesium sulfate (1.6 pM) was used instead of pure water as the carrier. After 60 injections the response was about 20% of the initial value, although the initial decrease was more pronounced for the divalent cation. This suggests that a high response can be maintained as long as the ion-exchange membrane is kept in the cerous form. Furthermore, the cerous ion concentration required in the internal or external flow will depend on the concentration of other cations in the carrier and regenerant solutions, which compete in the ion-exchange process. In a series of experiments conducted in the first operational mode, i.e., cerous sulfate in the carrier flow (internal channel) and sulfuric acid in the regenerant flow (external channel), the effect on various operational variables on response, signal to noise ratio, linearity, ion-exchange effiency for Ce3+,and selectivity was studied. In these experiments the carrageenans and Fragmin were used as test probes. In the first mode, which could be characterized as a typical setup for flow injection analysis, the concentration of the cerous ion (2.8-22 pM) in the carrier flow correlates directly to the response, signal to noise ratio, and linearity obtained; i.e., an increase of the concentration of cerous ions also results in an increase of these parameters. Typical calibration curves and their dependence on the concentration of cerous sulfate are shown in Figure 2. A cerous ion concentration of 17 p M in the aqueous carrier was required to obtain good linearity (three decades) for K-carrageenan,while 4 mM Ce+ in 50 mM sulfuric acid was required in the external flow channel using the second system with 50 mM ammonium acetate as the carrier. Competing cations (0-8 mM Na+) in the carrier flow have a negative effect on the parameters response, signal to noise ratio, and linearity, whereas the effect of the carrier flow rate (0.25-1.75 mL/min) was more complex, yielding a maximum for these parameters (Figure 3). Parenthetically, a similar optimum in carrier flow rate was observed for Fragmin when the 1-m membrane was operated in the second mode with Ce3+ flowing in the external channel. The flow optimum of the response is hard to explain and illustrates the complex dynamics involved in the system. The ion-exchange efficiency was positively affected by a decrease of the flow rate. For example, for K-carrageenan the ion-exchange efficiency decreased from 99% at a flow rate of 0.25 mL/min to 87% at a flow rate of 1.75 mL/min (Figure 3). In this study the ion-exchange efficiency for Ce3+was defined as the decrease
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ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991
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Flgure 3. Response of K-carrageenan (lower curve) and exchange efficiency of ce3+ as a function of carrier fbw rate. fhe carrier was 2.8 pM cerous sulfate in pure water. The external channel flow was 10 mM sulfuric acid pumped at 2.8 mL/min.
in fluorescence response of the carrier c a d by the placement of the membrane reactor between the injector and the detector. Buffer solutions without cerous sulfate were used to nullify the fluorescence detector. The ion-exchange efficiency was also positively affected by the increase of the concentration of sulfuric acid in the regenerant phase (external channel) and going from 0.5 to 1.0 m membrane reactor length. However, a decrease of the membrane reactor length improved the results obtained on response, signal to noise ratio, and linearity. The response of the carrageenansand Fragmin were independent of the temperature in the interval 13-36 "C. An exception was K-carrageenan for which the response decreased by 15% from the lowest to the highest temperature. If the ion-exchange rate was a rate-limiting factor, one would expect an increase in response with increasing temperature. The opposite behavior seen for K-carrageenan may be due to a conformational change in the polymer chain when the temperature is increased. It is anticipated that it will be more difficult for a uncoiled polymer to coordinate the placement of three sulfate groups around a cerous ion. These results suggest that the sulfated polysaccharides studied were saturated with the maximum amount of Ce3+that could be coordinated under steric limitations. The response, i.e., the slope of the calibration curve, for the different carrageenans varied with the degree of sulfation in the order X > L > K. However, in the presence of competing cations such as Na+ and Mg2+,the relative response reduction was in the order X < i < K. The increase of sodium sulfate concentration in the carrier stream from 2 to 4 mM caused a relative response reduction of X-carrageenan by a factor of 2, whereas the relative response reduction for i-carrageenan was equal to 6, and for K-carrageenan 8. The carrier cerous ion concentrationin this case was 11.2 pM, and 10 mM sulfuric acid was pumped at a flow rate of 2.8 mL/min through the external channel. The difference in sensitivity toward competing cations makes it possible to characterize the carrageenan composition of an unknown sample by measuring the response at three different sodium concentrations and comparing the response with those from the standard carrageenans. This idea was tested by preparing mixtures containing the three carrageenans in different proportions. These mixtures were analyzed at three different concentration levels of the competing sodium ion. The resulting proportions of the three carrageenans compared well with the known values. While further validation of this technique is required, the results suggest that useful information about the carrageenan composition can be obtained with this method. The second mode was not studied as extensively as the first mode with regard to the effect of the operational variables on the parameters, but essentially the same effect on response and linearity was obtained by varying the length of the
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Flgura 4. Recorder trace for FIA of Fragmin on the second system. The Ces+ concentration was 4 mM in the 50 mM sulfuric acid extemal channel flow of 2.8 mL/min. Ammonium acetate (50 mM) was used as carrier at a flow rate of 0.6 mL/min.
membrane, the concentration of cerous sulfate, the flow rate of the mobile phase/carrier, and competing ions as that of the first mode. In conclusion, the short membrane length (0.5 m) gave the best sensitivity with a low concentration of cerous ions in the carrier stream, while a higher response was obtained for the 1 m membrane reactor operated in the second mode with a buffer solution as the carrier and cerous ions in the external channel flow. Quantitation by FIA. A buffer (50 mM ammonium acetate) was used as the carrier during quantitation by FIA because most samples contain some kind of buffer and the samples have to be dissolved in the carrier to avoid a blank response from a buffer different from the carrier. A typical application could be samples containing Fragmin recovered from an in vitro dissolution testing of a pharmaceutical preparation. In this case the dissolution buffer and the carrier could be equivalent. Instead of introducing the cerous sulfate via a mixing tee between the injector and the membrane reactor, the cerous sulfate (4 mM Ce3+)was added to the sulfuric acid (50 mM) used for ion exchange of the carrier. The background fluorescence increased when buffers were used in the carrier, as compared with using pure water as carrier. This resulted in an approximately 10-fold higher detection limit, due to a lower signal to noise ratio. Nevertheless, as little as 5 ng of Fragmin could be detected at a signal to noise ratio of 3 from a 20-pL injection with a linear dynamic range of almost three decades. A fast analysis was obtained by FIA (Figure 4) and a precision of 0.6% RSD was obtained for repeated injections of 0.8 pg of Fragmin. The detection limit for carrageenans at low ion strength was 0.2 ng for L- and A-carrageenan and 0.4 ng for K-carrageenan with 11.2 pM cerous ions in the carrier and 10 mM sulfuric acid in the external channel of the membrane reactor. The selectivity of mass detection was examined by analysis of a number of compounds covering polyanionic polymers, low molecular weight acids, salts,and complex-formingagents (see Table I). The detection system gave a 17-fold lower response to citric acid when compared with heparin, while monovalent anions like chloride gave a very weak response. This clearly demonstrates the high selectivity of the proposed system as a detector for polyanionic compounds. The calibration curve for chloride gave an exponentialcurve while citrate gave a response close to linearity within the same concentration range (Figure 5). The relative response of
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991 Table I. Selectivity of Detection
compd
re1 mass responseo
heparin fragmin &-carrageenan citric acid chondroitin sulfate polygalacturonic acid NaCl sodium acetate oxalic acid NaHIPOI
100 87 67 6 5.3 3.7 0.33 0.08 -0.2 -0.4
The relative mass response was determined by FIA on the second system with 50 mM ammonium acetate as the carrier and 2 mM cerous sulfate in 50 mM sulfuric acid in the external channel flow. A negative sign indicates a negative response.
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Flgure 6. Separatlon of 2 pg each of L-carrageenan and Fragmin by slze exclusion chromatography on a Superdex-75 column (chromatogram A). Chromatogram B shows a low-resolution SEC separatlon of Fragmin from chlorkle on a Blosll guard-column where 20 pL of a salhe solution containing 21 pglmL of Fragmln was injected. Detection was carried out in the second mode regime.
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3 4 5 6 m w Flgure 5. Response curves of sodium chlorlde (bwer curve) and cltric acid obtained with 50 mM ammonium acetate as the carrler. Cerous sulfate (2 mM) In 50 mM sulfuric acM was used In the external channel flow.
chloride was estimated from the lower part of the curve where the response was proportional to the concentration. Chromatographic Separations. A Biosil size exclusion guard column was used for analysis for samples dissolved in a matrix different from the carrier composition used during FIA (Figure 6, chromatogram B). The resolution on this column was low but sufficient to separate the Fragmin fraction from the salt fraction. The postcolumn reactor was useful as a detector in other chromatographic applications, as demonstrated in Figure 6, chromatogram A, where Fragmin was separated from carrageenan on a Superdex SEC column with 50 mM ammonium acetate as the mobile phase. The reador's (1 m) contribution to band broadening measured as the peak-volume variance was 3500 pL2 for Fragmin and about the same for the other analytes in Table I. The band broadening was somewhat higher than expected for a 120 pL membrane reactor. The band broadening may be due to reasons other than the hydrodynamic dispersion, e.g. the ion-exchange process. This has, however, not been further investigated.
C0NCLUSION The postcolumn reactor was rugged and easy to use because the response was not very sensitive to the temperature or the flow rate in the external channel (regenerant) and its composition. The reactor proved to be very useful for flow injection analysis of sulfated polysaccharides or as a detector for liquid chromatography with a linearity of three decades. The detection system gave high response to heparin and carrageenans but low response, if any, to monovalent anions. The use of nonsuppressed eluents improved the selectivity of sulfated polysaccharides, and the best sensitivity was obtained with pure water or buffers with low ion strength in the carrier. ACKNOWLEDGMENT The comments on the manuscript by Knut Irgum and Satish Singh are greatly appreciated. We also wish to thank h a Hallden for technical assistance. LITERATURE CITED (1) DeVries. J. X. J . Chromatogr. 1989, 465, 297-304. (2) Linhardt, J.; Rice, K. G.; Kim. Y. S.; Lohse, D. L.; Wang, H. M.; Loganathan, D. J . Blochem. 1988, 254, 781-787. (3) Guo. Y.; Conrad, H. E. Anal. Biochem. 1888, 168, 54-62. (4) Linhardt, R. J.; Gu, K. N.; Loganathen, D.; Carter, S. R. Anal. Blo&em. 1989. 181, 288-296. ( 5 ) Karamanos, N. K.; Hjerpe. A.; Tsegenidis, T.; Engfeldt, B.; Antonopou10s. C. A. Anal. Biochem. 1988. 172. 410-419. (6) Hotchkiss, A. T.; Hicks, K. B. Anal. Blochem. 1990, 184. 200-206. ( 7 ) Dasgupta, P. K. J . Chromatogr. Scl. 1989, 2 7 , 422-448. (8) Downey, S. W.; Hiettje, G. M. Anal. Chlm. Acta 1983, 153, 1-13. (9) Shintani, H.; Dasgupta, P. K. Anal. Chem. 1987, 59, 1963-1969.
RECEIVED for review May 13,1991. Accepted September 23, 1991.
