pounds is greater for XAD-2 than for uptake by the more polar resin, A-26 (Fritz and Tateda, 1968). The capacity of A-26 for benzoic acid and nitrophenol in basic solution is somewhat greater than the resin capacity for chloride, indicating that some sorption of the organics has taken place in addition to the ion-exchange uptake. The sorption by A-26 and XAD-2 resins of 13 neutral organic compounds (n-hexyl ether, benzyl ether, naphthalene, mesitylene, various chlorobenzenes, etc .) added to water at 50 ppb was compared (Grieser et al., 1974). The percentage uptake by a small column of the very polar A-26 anion-exchange resin was only 7% or less in every case. On the other hand, a comparable size column of nonpolar XAD-2 resin resulted in quantitative or nearly quantitative uptake of the same compounds. From basic aqueous solution A-26 sorbs phenols quantitatively (as the phenolate) by ion exchange but still does not sorb the neutral organic compounds. This is being made the basis of an analytical method for isolation and determination of phenols in water. Uptake of Gases by Macroporous Resins Here also the rule seems to be that less polar resins retain nonpolar compounds best, while more polar resins have a preference for more polar substances. For example, XAD-7, which is a polyacrylic resin and more polar than XAD-2 (polystyrene-DVB) retains sulfur dioxide and hydrogen sulfide much longer than the less polar resin XAD-2 (Fritz and Chang, 1974). Gvosdovich et al. (1973) compared the gas chromatographic retention volumes of Chromosorb 101 (polystyrene-DVB) and the more polar resin Chromosorb 104 (polyacrylonitrile-DVB) . They found larger retention volumes for nonpolar alkanes and alkyl benzenes on Chromosorb 101 than on the 104 resin. However, more polar compounds phenols and aniline had a much larger retention
volume on Chromosorb 104; nitrobenzene also had a larger retention volume on the 104 resin. Gas chromatographic retention times on a column packed with XAD-2 resin and another containing A-26 (macroporous anion-exchange resin) under the same conditions showed a retention time of 3.20 min for hexane on XAD-2 but only 0.17 min for hexane on A-26. On the other hand, the more polar compound methanol has a retention time of only 0.85 min on XAD-2 but >14 min on A-26 (Fritz and Kissinger, 1974). Although the hypothesis given earlier is undoubtedly an oversimplification, the experiments cited above do lend support to this viewpoint. Literature Cited Burnham, A. K., Calder, G. V.. Fritz, J . S.,Junk, G. A , , Svec, H. J,, Vick, R., J. Am. Waterworks Assoc.. 65, 722 (1973). Burnham, A. K., Calder, G. V.,Fritz, J . S.,Junk, G. A , , Svec, H. J., Willis, R.,Anal. Chem., 44, 139 (1972). Fritz, J . S.,Chang, R. C., Anal. Chem., 46, 938 (1974). Fritz, J . S.,Kissinger, L. D., "Gas ChromatographyonMacroporous Resins," (unpublished work), Iowa State University, 1974. Fritz, J . S.,Tateda, A., Anal. Chem.. 40, 2115 (1968). Grieser, M . D., Arguello. M. D., Fritz, J. S.,"Sorption by Macroporous Resins." (unpublished work), Iowa State University, 1974, Gvosdovich. T. N., Kiselev, A. V.,Yashin, Ya. I., Chromatographia, 6, 179 (1973). Hollis, 0 . L., Anal. Chem.. 36, 309 (1966). Kirkland, J. J., J. Chromatogr. Sci.. 9, 206 (1971) Junk. G. A., Richard, J. J., Grieser, M. D., Witiak. D., Witiak, J. L., Arguello, M . D . , Vick, R . , Svec, H. J., Fritz, J . S.,Calder,G. V.,J.Chromatogr.. 99, 745 (1974). Witiak, J. L., Junk, G. A., Calder, G. V.. Fritz, J. S.,Svec, H. J., J . Org. Chem., 38, 3066 (1973).
Received for reuieu: October 15, 1974 Accepted February 10,1975
Presented before the Division of Organic Coatings and Plastics, 168th National Meeting of the American Chemical Society, Atlantic City, N.J., Sept 1974.
Disposable Resin Columns in Analytical Chemistry Reiner H. Kopp Brinkmann lnstruments, lnc., Westbury. N e w York 11590
Disposable columns filled with Arnberlite XAD-2 increasingly are used to replace liquid/liquid extraction in clinical laboratories. These columns will double efficiency and, therefore, are particularly helpful in routine analysis when large numbers of samples have to be processed. Presently, the main applications are urine screening for misused drugs and excreted steroids. Procedures are described and critically compared to alternative liquid/liquid extraction techniques. Various parameters affecting recovery rates are discussed.
Amberlite XAD-2 (Rohm and Haas Co., Philadelphia, Pa.) was originally developed for industrial applications such as concentration of water soluble compounds (e.g., enzymes, steroids, and fatty acids) or removal of trace contaminants from surface or waste water. During the past two years, the emphasis has shifted from industrial to analytical applications and two major areas of interest have evolved: (1) high-pressure liquid chromatography, and (2) drug screening and/or steroid 96
Ind. Eng. Chem., Prod. Res. Dev., Vol. 14, No. 2, 1975
screening analysis. Pietrzyk (1973), Fritz (1968) as well as Gustafson (1968) used crushed Amberlite XAD-2 for highpressure liquid chromatography to separate phenol derivatives and amines. They came to the conclusion that because of the large surface area, rigid porous structure, and solvent resistance, this material is a promising adsorbent for HPLC. Our own research and development is centered around its application as an extraction medium for urine or blood
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analysis. i n i s extraction step pnor to tne actual tnin layer chromatography (TLC) and gas-liquid chromatography (GLC) analysis in many cases is more time-wnsuming than any individual step and, therefore, has an essential influence on total cost. In the past, these extractions were done by conventional liquid/liquid techniques with chloroform as the organic phase. Liquid/liquid extraction, particularly if large numbers of samples have to he extracted, is space-consuming and the technique, because of the manual labor involved, is tedious. Furthermore, additional work might he necessary if, during shaking, the two phases emulsify and therefore have to he separated by centrifugation or filtration. Because of the increasing demand for a rapid and efficient drug screening analysis, Brinkmann Instruments developed (in 1972) a disposable column filled with Amberlite, based on a patent by Quam6 (1972) and an analytical hardware system which will optimize efficiency and ease of operation. This extraction techique proved to he superior to conventional extraction and today approximately 10,000-20,000 analyses are performed daily using such technology. With the introduction of four new steroid assay procedures, hopefully many more laboratories will benefit from the advantages this technique has to offer. Procedure Add 20 ml of the buffered sample solution and allow it to pass through the disposable cartridge. For convenience, all cartridges are mounted on a so-called column aspirator which will allow for drainage of the waste. This also serves for the application of vacuum t o speed u p the flow of viscous solutions and to remove excess adsorbed water a t the end of this operation. Gravity flow can he replaced (if reproducibility is needed) by controlled flow, utilizing a peristaltic pump. This special set-up is necessary whenever quantitative recoveries have to he achieved. In order to remove excess pigments or other unwanted material, columns can he washed with either water or buffer solutions. For elution, columns are transferred to an elution rack and connected to a filter cartridge containing phase-separating filter paper. During elution, the filter cartridge will remove residual water and prevent contamination of the organic phase. Approximately 15 ml of dichloroethane-ethyl acetate or dichloroethane-ethyl acetate-isopropyl alcohol are needed to recover the adsorbed drugs. In order to obtain optimum recovery, elution solvents should he added in three steps in order to avoid the solvents rushing through the column without having the proper contact time necessary to elute all the material. The recovered and purified drug solution is then concentrated with the help of a sample concentrator. The sample extract now can he further analyzed either by TLC, GLC, orRIA. In order to optimize operations, a hardware system was developed to handle up to 400 extractions per day. Fortyeight samples are processed simultaneously in three steps: (1) extraction, washing, and removal of excess water; (2) sample elution and filtration; and (3) sample concentration. The middle of the photograph (Figure 1) shows 48 cartridges mounted on the aspirator tray. This unit is used for the initial urine extraction and washing steps. The mounting plate with cartridges is then transferred to the elution unit shown a t the left hand side. T h e second shelf contains phase separating filters and the bottom rack holds the collection tubes. After completing elution, this rack is transferred to a sample concentrator, which will concentrate 48 samples in about 30 min with the help of a vacuum-induced air current and heat. All hardware was
Figure 1. Basic hardware for mass-screening system. The column mounting plates are used in conjunction with an aspirator tray (front) or elution unit (left). The elution unit is provided with phase-separating units and a tube rack which after completing elution is transferred to a vacuum sarn~leconcentrator.
Figure 2. Comparison of amphetamine recovery in liquid/liquid and resin column extraction as a function of pH.
developed to give positive sample identification and the individual units were designed to eliminate accidental side reversal of racks or mounting plates. Some of the advantages of this system are obvious: (1) because of the simplicity of operation, substantial time saving of up to 70% can he obtained; (2) elimination of glassware; (3) only 30% of the space is required than a comparable liquid/liquid extraction unit would need; (4) samples adsorbed in cartridge can he mailed; (5) elimination of emulsification. Some of the advantages are not as obvious: (1) The extraction is not as critically affected by pH as is liquid/liquid extraction (Figure 2) (Kullberg e t al., 1973, 1974). This has two benefits: it allows extraction of multiple compounds with different isoelectric points a t one and the same pH, and, on the other hand, accurate pH adjust. ment is not required. This is particularly important for mass screening procedures where an additional step can he eliminated. (2) Less than 50% of elution solvent is required to obtain the same recoveries as in liquid/liquid extraction. Kullherg and Miller (1973, 1974) reported for morphine that the extraction efficiency procedure using 20 ml of organic solvents could only he matched by liquid/liquid extraction employing 60 ml of a similar solvent mixInd. Eng. Chem., Prod. Res. Dev.. Vol. 14, No. 2, 1975
97
ture. In other words, a savings of 66% can be achieved. (3) Last but not least, the cartridge principle lends itself to automation much easier than liquidlliquid extraction. Sample extraction, various washing steps, oxidation, reduction, and hydrolysis can easily be done with the sample adsorbed on Amberlite prior to elution. Instruments which can be used for automation such as automatic sampling and diluting devices, solenoid valves, and peristaltic pumps are readily available and it is more or less a question of how to optimize their use. In developing a disposable cartridge, the following objectives were considered: (1) the extraction and elution time should be reasonably short; (2) the column should be shipped dry, but ready to use; and ( 3 ) the adsorbent should be free from contaminates which interfere with drug analysis. Amberlite XAD-2 is manufactured with a particle size ranging from 20 to 50 mesh. This particle size gives good results for large columns and relatively slow flow rates; however, it is not suitable for analytical columns with relatively high flow rates. Our requirements to adsorb 75% of the total morphine present in the sample a t flow rates of 4 ml/min were met by using smaller particles. These smaller particles helped in two ways: (1) increased the surface area by 50%, and, what is most important, (b) increased the easily accessible “outside” surface area which is essential whenever fast flow rates are .used. Morphine or alkaloid adsorption was, so to speak, a yardstick since they are present only in low concentrations of 0.1-20 pg/ml and, therefore, most difficult to detect. Columns should be shipped dry but ready to use. Unfortunately, dry Amberlite is water repellent and has to be treated with alcohol before use. Wet columns, on the other hand, present a problem because of weight, leakage, and air entrapment in the column packing. This problem was solved by impregnating Amberlite with water-alcohol mixtures and adjusting the concentration so that the total liquid was fully absorbed and the packing still had its dry appearance. One problem we were not able to solve to our satisfaction was contamination with short-chain hydrocarbons. By washing the raw material several times in nonpolar and alcoholic solvents, the contaminate level can be decreased to a point where it does not represent a major interference with TLC work. However, for trace analysis in GLC, it still represents a problem and it seems that even after removing the major portions by washing, additional material can still be leeched out and it seems impossible to remove it completely.
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During our research with different washing solvents, we noticed that, independent of the final alcohol wash, the nonpolar solvents used before had a strong influence on recovery rates. For instance, a noticeable drop of 5% in the recovery of morphine was found when Amberlite was pretreated with trichloroethane. This drop was even more pronounced for compounds extracted under nonideal pH conditions. For barbiturates a t a pH of 8.5, the recovery drop was from 70% to 37%. A tracer study showed that this washing or pretreatment of solvents affected recovery in two ways. Those which reduce adsorption will, on the other hand, aid desorption and in this way slightly correct the total recovery losses. Since we were able to find elution solvents which would desorb almost 100% of the adsorbed drugs, we concentrated our search on solvents which increased adsorption. We found that for morphine, amphetamine, and barbiturates, pretreatment with nonpolar solvents decreased adsorption and pretreatment with polar solvents increased adsorption. Our findings indicate that the resin matrix will absorb solvent molecules and in this way change its polarity. It is, therefore, possible to influence its adsorption specifically by pretreatment with approporate solvents. The effect of this pretreatment is not diminished by consecutive alcohol washes which are necessary in order to hydrate the resin. In conclusion, analytical Amberlite XAD-2 columns proved to be superior to liquid/liquid extraction techniques for drug and steroid assays. The main advantages besides speed, simplicity, and economy are higher recovery rates and the possibility for automation. Literature Cited Fritz, J. S., Tateda, A , , Anal. Chem., 40,2115 (1968). Grieser, M. D.. Pietrzyk, D. J.. Anal. Chem., 45, 1348 (1973);46, 330
(1974). Gustafson, R. L., Albright, R. L., Heisler, J., Liror. J. A,, Reid, 0. T., Jr., Ind. Eng. Chem., Prod. Res. Dev., 7,107 (1968). Kullberg, M. P., Gorodetzky, W., Clin. Chem., 20, 177 (1974). Kullberg, M. P., Miller, W. L., McGowan, F. J., Doctor, B. P., Blochem. Med., 7,323 (1973). Miller, W. L., Kullberg. M . P., Banning, M. E., Brown, L. D . , Doctor, 8. P., Biochem. Med., 7, 145 (1973). Quame, E.,U.S. Patent No. 3,567,029(1972).
R e c e i v e d for r e v i e w O c t o b e r 15, 1974 Accepted M a r c h 6, 1975 P r e s e n t e d a t t h e D i v i s i o n of O r g a n i c C o a t i n g s a n d P l a s t i c s C h e m i s t r y , 168th N a t i o n a l M e e t i n g of t h e A m e r i c a n C h e m i c a l Society, A t l a n t i c C i t y , N.J., S e p t 1974.
