Anal. Chem. 1995,67,1191-1196
Representing lsocratic Multicomponent Soiid-Phase Extraction Data by an Extension of Liquid-Liquid Extraction Theory David Emlyn Hughes* and Keith E. Gunton Bristol-Myers Squibb Company, Pharmaceutical Research Institute, P,0. Box 4755, Syracuse, New Yo& 13221-4755
The theory and a model of representation for isocratic multicomponent solid-phase extraction (SPE) data are presented. The model requires the determination of extraction recovery as a function of extractantvolume. The recovery/extractant volume data are then analyzed to determine whether further wash optimization studies might be promising or if another SPE stationary phase should be chosen. Similarly extracted species are represented in a consistent manner, allowing systematic variation of the wash solvent composition to optimize selectivity. Extraction of a mixture of alkyldimethylbenzylammonium chloride is performed, and analysis of the resulting data is seen to be consistent with extraction of a homologous series. A complex mixture of epipodophyllotoxin-related compounds is analyzed by the model presented, and selective wash conditions are developed. The effect of mass and volume on SPE column loading is demonstrated by extraction of a series of p-hydroxybenzoic acid ester compounds. The model allows inference of the selectivity, extent of irreversibly bound ana&, loading, and type of elution profile. Solid-phaseextraction (SPE) as a sample preparation technique has become very popular in recent years, and several reviews are This technique often has several advantages over liquid-liquid extraction in many sample preparatory schemes; oftencited reasons include ~electivity,~ high recovery! fast analysis time,9-13low cost, and ease of a ~ t o m a t i o n . ~ ~When - ’ ~ SPE and liquid-liquid extractions are compared as sample preparation (1) Lim, C. K Trends Anal. Chem. (Pen. Ed.) 1988,7 (9), 340. (2) Wu, R; Huang, W. Fenri Shiyanshi 1988,7 (9), 34. (3) Wintersteiger, R GIT Fachz. Lab. 1988,32 (4), 345. (4) Christie, W. W. Adv. Lipid Methodol. 1992,1. (5) Scheurer, J.; Moore, C. M. J. Anal. Toricol. 1992,16 (4), 264. (6)Clarke, G. S.; Robinson, M. L. Anal. Proc. (London) 1985,22 (5), 137. (7) Verhaeghe, B. J.; Lefevere, M. F.; De Leenheer, A P. Clin. Chem. 1988, 34 (6), 1077. (8) Salari, H. J. Chromatogr., Biomed. Appl. 1987,63 (l), 103. (9) Kames, H. T.; Rajasekharaiah, K; Small, R E.; Farthing, D. J. Liq. Chromatogr. 1988,11 (2), 489. (10) Richard, J. J.; Junk, G. A.Mikrochim. Acta 1986,1 (5-6), 387. (11) Wachob, G. D. LC Mag. 1983,1 (2), 110. (12) Wright, D. S.; Pachla, L. A.; Gibson, D. M.; Jordan, R A. j. Chromatogr., Biomed. Appl. 1987,61 ( l ) , 223. (13) Brooks, M. W.; Jenkins, J.; Jimenez, M.; Quinn, T.; Clark, J. M. Analyst (London) 1989,114 (3), 405. (14) Ni, P.; Guyon, F.; Caude, M.; Rossef R]. Liq. Chromatogr. 1988,11 (5), 1087. (15) Ni, P.; Guyon, F.; Caude, M.; Rosset, Rj. Chromatogr,,Biomed. Appl. 1988, 68 (2), 255. (16) McMurrough, I.; Byme, J.; Collins, E.; Smyth, M. R; Cooney, J.; James, P. J Am. SOC.Brew. Chem. 1988,46 (2), 51. 0003-2700/9510367-1191$9.00/0 0 1995 American Chemical Society
techniques, SPE usually is referr red;'^-^^ however, at least one studyz2found liquid-liquid extraction to be more precise. A lesscited but important advantage of SPE is the possibility of routinely obtaining enrichment factors of 100-100023-25and even recovering and concentrating species from a liquid chromatographic eluate.z6 Finally, SPE columns that have been loaded with analyte often have a rather long storage life,27328allowing analysis to be performed at a later time. The SPE protocol has been somewhat standardized. A study of 25 randomly selected SPE papers in the literature indicates that 88%used the technique of washing the impurities from the SPE column with a weak solvent followed by elution of the species of interest with a strong solvent. In this conventional approach, the SPE attempts to take advantage of the selectivity of the c0lumn.~3~O In fact, if a suitable separation is not achieved, a column with a different stationary phase is selected. Hence, the current SPE papers reflect the earlier prejudice31that selectivity changes are best achieved by choice of column type rather than by varying the composition of the eluting solvent. This paper will attempt to present a simple model to represent multicomponent SPE data. Homologous compound series will be shown to exhibit similar extraction profiles. Selectivity as a function of the extractant (wash) composition is investigated rather than the more conventional SPE column choice, trial-and-error protocol. Development of the Model. Approaching SPE sample preparation in the traditional weak wash, strong elution solvent manner is appealing since this “digital”method development does not require extensive data interpretation. The choice of wash (17) Kames, H. T.; Opong-Mensah, K; Farthing, D.; Beightol, L. A J. Chromatogr., Biomed. Appl. 1987,422,165. (18) Li,F.; Lim, C. K; Peters, T. J. Chromatographia 1987,24,637. (19) Sarkar, M. A.; Kames, H. T.]. Chromatogr., Biomed. Appl. 1988,422,329. (20) Bellar, T.A; Budde, W. L. Anal. Chem. 1988,60 (19), 2076. (21) Jemal, M.; Ivashkiv, E.; Valatin, P.; Cohen, A I. J. Chromatogr., Biomed. Appl. 1986,381,424. (22) Prasad, V. IC; Ho, B.; Haneke, C.j. Chromatogr., Biomed. Appl. 1986,378, 305. (23) Steinheimer. T.R; Brooks, M. G. Int. J. Enuiron. Anal. Chem. 1984,17 ( a , 97. (24) Vastano, S. E.; Mihe, P. J.; Stahovec, W. L.; Mopper, K Anal. Chim. Acta 1987,201, 127. (25) Junk, G. A; Richard, J. J.J Res.Nut. Bur. Stand. (US.)1988,93 (3), 274. (26)Wilcox, C. D.; Phelan, R M. J Chromatogr. Sci. 1986,24 (4), 130. (27) Marble, L. IC; Delfino, J. J. Int. Lab. 1989,19 (3), 16. (28) Green, D. R; Le P a p , D. Anal. Chem. 1987,59 (5), 699. (29) Hartley, R;Lucock, M.; Becker, M. ]. Chromatogr., Biomed. Appl. 1987, 415,357. (30) Salari, H.; Steffenmd, S. J. Chromatogr., Biomed. Appl. 1986,378,35. (31) McDowall, R D.; Pearce, J. C.; Murkitt, G. S.j. Phatm. Biomed. Anal. 1986, 4 (I), 3.
Analytical Chemistry, Vol. 67, No. 7, April 1, 1995 1191
solvent components and composition cannot be inferred by the current model; traditional conditions may often be available in the liquid chromatographic and thin-layer chromatographic literature for similar systems. Some a~thors32,~~ offer recovery versus volume of elution representations to aid in interpretation of extraction data. While the recovery versus volume graphical procedure is helpful, it suffers from a lack of theoretical basis which would allow the analyst to differentiate between useful and inefficient extraction schemes. Solid-phase extraction is often used when a complex matrix is involved, and hence the extraction profiles of several, and sometimes many,34species must be characterized. The limitations of recovery versus volume of extractant representations are quite serious for simple systems and even more limiting, it is suspected, for multicomponent systems. A theoretically based, simple representation for SPE data is currently available. Classical liquid-liquid extraction theory may be used as a starting point to develop a suitable SPE graphical representation. Some reasons why liquid-liquid theory, in a modified form, might be applicable are the following. (1) The solid phase is usually in contact with the compositionally same extractant throughout an extraction step, Le., the separation is usually an isocratic as opposed to a gradient process. Even step gradient procedures are relatively uncommon in the l i t e r a t ~ r e . ~(2)~ .A~ very ~ small amount of the stationary phase is associated with the analyte (on the order of 1 ppm, Le., 1 pg of analyte/g of stationary phase) during the extraction. Small variations in the analyte/stationary phase ratio would not be expected to significantly affect the extraction efficiency, since essentially all the stationary-phase sites are always available. This assumption becomes less realistic as the complexity of the matrix and concentration of the analyte are increased. The ideal relationship for liquid-liquid extraction relating the total (cumulative) recovery to the extraction efficiency is
where Res is the total cumulative recovery, Ro is the extraction efficiency, and n is the number of extractions. When applying liquid-liquid theory to liquid-solid extractions, allowance must be made for irreversible loss of analyte to the stationary phase in the loading step. This modification is unnecessary in ideal liquid-liquid extraction theory, since all of the solute is available in one of the liquid phases. In liquid-solid extraction, however, it is anticipated that at least small amounts of analyte may be bound to the SPE stationary phase and not available for extraction. Although little theoretical work is available on liquid-solid extraction applicable to SPE, we have previously di~cussed3~ some general guidelines that are useful here. In the simple isothermal model presented here, it is assumed that the extractant simply removes species retained by the SPE stationary phase (there are no nonideal considerations, e.g., associative or dissociative reactions, mutual dissolution of (32) Johnson, E. L.; Reynolds, D. L.: Wright, D. S.; Pachla, L. A J, Chromatogr, Sci. 1988,26,372. (33) Veals, J. W.: Lin, C. C. Am. Lab. 1988 (April), 42. (34) Bagnati, R: Benfenati, E.; Davoli, E.; Fanelli, R. Chemosphere 1988,17 (1). 59-65. (35) Hudgins. W. R.; Stromberg, K. J. Liq. Chromatogr. 1987,10 (15), 3329. (36) Oleszek. W. J. Sci. Food Agn’c. 1988,44 ( l ) , 43. (37) Hughes, D. E. Anal. Chem. 1983,55,78.
1192 Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
the matrix and extracting solvent, side reactions, or salt effects/ salting-in, s a l t i n g - o ~ t ~ ~To - ~ the ~ ) . extent that the system is ideal by the previous definition, it is expected37that the total extraction recovery RT will be defined by
RT = RretRext
(2)
which includes the multiplicative factor Rret, which may be identitied as the limiting recovery due to irreversible solute retention by the SPE stationary phase. Combining eqs 1 and 2 yields RT = R r e t [ l -
(1- RJ”]
(3)
It is clear that Rretrepresents a limiting recovery: an exhaustive extraction scheme would approach a total recovery equal to Rret. It may also be clear that the concept of “irreversibly retained solute” is relative since increasing an extractant solvent strength might allow analyte to be eluted that was unavailable under milder conditions. Further developing the model by taking the natural log of both sides and rearranging,
ln(Rre, - RT) = n ln(1 - R,)
+ ln(Rret)
(4)
To the extent that ln(Rrer- RT) h(1- RT),
ln(1 - RT) = n ln(1 - RJ
+ ln(Rret)
(5)
A plot of ln(1 - RT), or more conveniently, -ln(l - RT),versus n will therefore yield a straight line in this ideal model. Either the number of extractions n or the directly proportional quantity V, the total volume of extraction, may be plotted on the abscissa. The extraction efficiency and R,,, factor may be calculated from the slope and intercept, respectively. If quantitative use of the representation is required (e.g., slope or intercept), then the unitless quantity n must be employed. Most applications, however, rely on the relative shape and position of elution profiles and not on the absolute values, and for these examples, the total extractant volume V will be used as the independent variable. In the latter case, V will be expressed in milliliters. Extraction Profiles in the Current Model. A favorable extraction scheme in the current model is one in which the extraction efficiency, or slope, is much larger for the impurities than for the analyte in the wash step. In the elution step, the most important requirement is that the analyte is efficiently eluted independent of the extraction profile for the impurities. Gaussian and Langmuir Extraction Profiles. A linear relationship between -ln(l - RT) and Vimplies that the zone of interest was eluted from the SPE column in a manner consistent with a modified ideal liquid-liquid extraction model. Since many elution profiles (or distributions) have been postulated for similar systems, including exponentially modiiled b i G a u s ~ i a nPoisson,46 ,~~ L a n g m ~ i r , ~omb ~ < bin ~ 8at ion^^,^^ functions, and others,j1-j5 it would be surprising if all SPE data were to (38) Berg, Eugene W. Physical and Chemical Methods of Separation: McGrawHill: New York, 1963: pp 7-15. (39) Morison, G. H. Anal. Chem. 1950.22,1388. (40) Masterson, W. L.: Lee, T. P. j. Phys. Chem. 1970,74,1776. (41) Ringbom, A Complexationin Analytical Chemisty; Interscience: New York, 1963; pp 10-50. (42) Groenewald, T. Anal. Chem. 1971,43,1678. (43) Giddings, J. C. Dynamics of Chromatograflhy,Part I; Marcel Dekker: New York. 1965; p 20. (44) Foley, J. P. Anal. Chem. 1987,59, 1984. (45) Grusha, E.: Myers, M. N.; Giddings, J. C. Anal. Chem. 1970,42,21.
1 2
4
0
--
-
Gaussian I Current Model Langmuir, L-0.10
1
2
3
4
5
6
7
Volume (mL) Figure 1. Various peak profiles in the modified liquid-liquid representation.
conform to the simple model presented here. For liquid chromatography in general, a Gaussian57or modified Gaussian band profile is often assumed when the column is not overloaded either by concentration or by volume of analyte. Practical method development using SPE would not routinely require determination of whether the system is in either the “mass overload” 51.56 or the analytical range, and hence a model appropriate to overload will also be included, i.e., the Langmuir solution of the ideal model of ~hromatography.~~ As Figure 1indicates, a Gaussian elution profile in the current model results in rather dramatic curvature if we require that the extraction be camed out to a high (ca. 98%) recovery. Gaussian 1 represents a more efficient extraction than Gaussian 2. Independent of efficiency, the Gaussian band profile provides a representation characterized by dramatic curvature. In a similar fashion, the ideal model for chromatographic analysis under column overloading conditions is the Langmuir i~otherm,4~ which yields similar profiles over a relatively large (0.03-0.25) loading factor range. EXPERIMENTAL SECTION Apparatus. The HPLC system was a Waters 600E gradient chromatograph, a Waters 717 autoinjector, and a Perkin-Elmer LC 290 variable wavelength detector set at 254 or 210 nm. The data were collected, stored, and analyzed using a PE Nelson Series 900 interface, an IBM PS/2 Model 60 computer, and Nelson Analybcal2600 chromatography software. The analytical columns were Jones Chromatography Apex ODS 5 for the separation of ~
(46) Kuceva, E. /. Chromatogr. 1965,19,235. (47)Golshaw-Shirazi, S.; Guiochon, G. Anal. Chem. 1988,60, 2364. (48) Golshaw-Shirazi, S.; Guiochon, G. Anal. Chem. 1989,61,462. (49) Chesler, S. P.; Cram, S. P. Anal. Chem. 1971,43,1922. (50) Fraser, R D.B.; Suzuki, E.Anal. Chem. 1966,38,1770. (51) Gmbner, 0. Anal. Chem. 1971,43,1934. (52) McWilliams, I. G.; Bolton, H. C. Anal. Chem. 1969,41,1762. (53) Gmsha, E.: Myers, M. N.;Schettler, P. D.; Giddings, J. C.Anol. Chem. 1969, 41,889. (54) Chesler, S. N.; Cram, S. P. Anal. Chem. 1973,45,1354. (55) Eble, J. E.: Grob, R L.; Antle, P. E.: Snyder, L. R]. Chromafor. 1987, 384,25. (56) Barber, W.E.: Carr, P. W. Anal. Chem. 1981,53, 1939. .Chromatogr. 1986,363,1. (57) Knox, J. R;Py-per, H.M. 1
phydroxybenzoic acid ester, a Waters-Bondapak irregular 10 cyano (300 x 3.9 mm) for the separation of alkyldimethylbenzylammonium chlorides, and a Jones Chromatography Apex phenyl RP 5 for the separation of the epipodophyllotoxin series. Solidphase extraction studies were performed on J. T. Baker 3 mL (50 mg sorbent weight) disposable SPE columns; CIS(capacity 2-5% by weight) for phydroxybenzoic acid ester and epipodophyllotoxins, and cyano (capacity 2-5% by weight) for alkyldimethylbenzylammonium chloride. Reagents and Chemicals. Standards of phydroxybenzoic acid esters (methyl, ethyl, propyl, and butyl) were purchased from Sigma Chemical Co. Alkyldimethylbenzylammonium chloride standards were purchased from Aldrich Chemical Co. Buffers and reagents were analytical grade, and the solvents used were HPLC grade. The epipodophyllotoxins were obtained from Bristol-Myers Squibb Co. The compounds and designations were as follows: [ (5R)-[5a,5@,8aa@*)I 1-5[3,5dimethoxy-4-(phosphonooxy)phenyl]-9-[ (4,60-ethylidene-~-~-glucopyranosyl) oxy]-5,8,8a,%tetrahydrofuro[3’:6,7lnaphtho[2,W-l,Sdioxol-6(5~+ne(etoposide phosphate, pptoxin I); (5R)-[5a,5aB$aa,9B(r*) I l-%~-~-glucopyranosyloxy)-5(4hydroxy-3,5dimethoqq1henyl)-5,8,8a,%tetrahydrofuro{3’:6,7lnaphtho [2,3$1-1,3-dioxol-6(5aH)+ne(hgnan P, pptoxin ID; [ (5R)-[5a,5@,8aa,gB(R*)1I-%[ (4BOethylidene;B-~-glucopyran~ syl)oxy]-5(4hydroxy-3,5dimethoxyphenyl) -5,8,8a,%tetrahydrofurc[3’:6,7]naphtho[2,3-d]-1,3-dioxol-6(5aH)-one(etoposide, pptoxin III) ; and [ (5R)-[ 5a,5aa,8aa,9B(R*)] I-%[ (4,60ethylidene;B-~-glucopyranosyl)oxy1-5(4hydroxy-3,5-dimethoxyphenyl)-5,8,8a,%tetrahydrofuro[3’:6,7lnaphtho[2,3-dl-1,3-dioxol-6(5aH)+ne (picro ethylidene lignan P, pptoxin N). Methods and Procedure. (a) Chromatography. PHydroxybenzoic acid esters were separated on a Jones Chromatography Apex 5 cl8 column using a mobile phase of acetonitrile/ water (30/70) at a 1.4 mL/min flow rate. A mixture of alkyldimethylbenzylammonium chlorides was separated on a Waters cyano column using a mobile phase of 0.1 M sodium acetate (PH = 4.8) buffer/acetonitrile (50/50) at 2.5 mL/min flow rate. The epipodophyllotoxins were separated on a Jones Chromatography Apex phenyl 5 column using a mobile-phase gradient initially consisting of acetonitrile/0.02 M ammonium phosphate monobasic buffer (pH = 2.5) (10/90) at 1.5 mL/min flow rate for 3 min, followed by a linear gradient to acetonitrile/0.02 M ammonium phosphate monobasic buffer (PH = 2.5) (27/73) in 1 min. (b) Solid-Phase Extraction. In the course of the extraction protocol, care was taken during column conditioning, sample addition, and sample elution to ensure that the SPE stationary phase remained wet. (c) p-Hydroxybenzoic Acid Esters. A cl8 cartridge was conditioned by washing the column with 2 volumes of methanol, followed with 2 column volumes of water using positive pressure. A solution of phydroxybenzoic acid esters was prepared in methanol at 8 mg/mL and serially diluted in water. A 5.0 mL aliquot of a 0.24 mg/mL phydroxybenzoic acid esters solution was added to the preconditioned column and expressed. pHydroxybenzoic acid esters were eluted from the SPE cartridge by successive 0.5 mL aliquots of methanol/water (70/30) and collected separately in 0.5 mL volumes. These aliquots after dilution to 3 mL each were injected into an HPLC to study the extraction efficiency for each phydroxybenzoic acid ester. SimiAnalytical Chemistty, Vol. 67,No. 7, April 1, 1995
1193
larly, a C18 cartridge was conditioned by washing the column with 2 volumes of methanol, followed with 2 column volumes of water using positive pressure. A 0.5 mL aliquot of the 8 mg/mL p-hydroxybenzoic acid ester solution was added to the preconditioned column and expressed. p-Hydroxybenzoic acid esters were eluted from the SPE cartridge by successive 0.5 mL aliquots of methanol/water (70/30) and collected separately in 0.5 mL volumes. These aliquots after dilution to 3 mL each were injected into an HPLC to study the extraction efficiency for each phydroxybenzoic acid ester. (d) Epipodophyllotoxins. A cartridge was conditioned by washing the column with 2 volumes of acetonitrile, followed by 2 column volumes of water using positive pressure. A solution of epipodophyllotoxins was prepared in acetonitrile/water (50/ 50) at 300 pg/mL. A 0.5 mL aliquot of the diluted sample was added to the preconditioned column and expressed. Epipodophyllotoxins were eluted from the SPE cartridge by successive 0.5 mL aliquots of acetonitrile/0.02 M ammonium phosphate monobasic (PH 4.8) (70/30) and collected separately in 0.5 mL volumes. These aliquots were injected into the HPLC to study the extraction efficiency for each epipodophyllotoxin. A second experiment consisted of conditioning a c18 cartridge by washing the column with 2 volumes of acetonitrile, followed by 2 column volumes of water using positive pressure. A solution of epipodophyllotoxins was prepared in acetonitrile/water (50/ 50). A 0.5 mL aliquot of the diluted sample was added to the preconditioned column and expressed. Epipodophyllotoxins were eluted from the SPE cartridge by successive 0.5 mL aliquots of acetonitrile/0.02 M ammonium phosphate monobasic (PH = 4.8) (30/70) and collected separately in 0.5 mL volumes. These aliquots were injected into the HPLC to study the extraction efficiency for each epipodophyllotoxin. (e) Alkyldimethylbenzylammonium Chlorides. A cyano cartridge was preconditioned by aspirating 2 column volumes of acetonitrile followed by 2 column volumes of water through the column using vacuum. A solution of alkyldimethylbenzylammonium chlorides (c8, Clz, C14, and C16 isomers) was prepared in water at 5 pg/mL. A 1mL aliquot of the diluted sample was added to the preconditioned cartridge column and allowed to aspirate. Alkyldimethylbenzylammonium chlorides were eluted from the SPE cartridge by successive 1 mL aliquots of 1 M hydrochloric acid/methanol (50/50) and collected separately in 1mL volumes. The aliquots after dilution to 2 mL each were analyzed by HPLC to study the extraction efficiency for each alkyldimethylbenzylammonium chloride. RESULTS AND DISCUSSION
Interpreting Extraction Profle Data. The slope, shape, and intercept of the current representation allow analysis of the solidphase extraction with respect to selectivity, extent of irreversibly bound analyte, loading, and type of elution profile. Although the discussion is directed toward the elution step for a single column, it could also be applied to columns in tandem58,59and the very important column preconditioning step, which allows for more selectivem,61and sensitive@procedures. With respect to selectivity, the shape and slope (more generally, the first derivative) may be classified as favorable or unfavorable as a function of which (58) Wells, M. J. M.; Michael. J. L. J Chromatogr. Sci. 1987,25 (8), 345. (59) Bemdge, J. C.; Broad, L. A. J. Pharm. Biomed. Anal. 1987,5 (5). 523 1194 Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
step, wash or elution, and what species, analyte or impurity, are being studied. In the popular analyte-retained mode of SPE, a favorable extraction contour for the analyte would have a low slope for the wash cycle but a high slope (extraction efficiency) for the elution cycle. Correspondingly, it would be preferred that impurities be eluted rapidly in the wash step and gradually in the elution step. However, since the elution step usually involves a solvent which more readily elutes all species, it is unlikely that the ideal impurity elution profile would often be achieved. The rapid elution of the impurities is not a concern in most cases, since the SPE column has ideally been depleted of these species in the wash step, as mentioned previously. In the less popular impurityretained a similar but more straightforward situation exists for the elution profile. In this mode, the wash and elution steps are usually combined into a single step which allows elution of the analyte but retention of the impurities. A more complex situation involves changing from reverse-phase to normal-phase wash or elution (often preceded by air-drying of the SPE column)ffi or use of an ion-pairing agent.66 It is tempting to define what value of the slope of the elution contour (or average slope if the contour is not a straight line) d e h e s “gradual” or “rapid”elution. Although some SPE methods pass very large volumes of extractant through the column,67most literature methods confine themselves to 1-10 mL. In a similar manner, most reported methods recover 75-100% of the species of interest, with a majority of the methods recovering in excess of 90%. Within these guidelines, gradual elution might be defined as having a slope of the representation of less than 0.1 mL-l, and rapid might be defined as having a slope in excess of 0.5 mL-’. It is clear that the satisfactory recovery and elution of the analyte and impurities must be decided in each analytical case and that the definition presented here of gradual and rapid elution is illustrative and quite arbitrary. In a similar manner, the main requirements for successful SPE schemes involve the efficient retention and elution of appropriate species and are less dependent on the exact shape of the elution profiles. Although it is expected that many of the empirical profiles will be linear, the general guidelines presented here are useful for most other profiles as well. In the case of a linear relationship, analysis of the data is particularly straightforward. A final caveat concerns adequate batch-to-batch reproducibility of the SPE columns, which although assumed for this discussion, is not always the case.68 Even a simple model for SPE would be expected to represent extraction data such that similar extraction patterns could be discerned. For the homologous set of alkyldimethylbenzylammonium chlorides (benzalkonium chlorides) with alkyl chain lengths of 8, 12, and 14, for example, a reverse-phase extraction (60) Lopez-Ada, V.; Milanes, J.; Dodhiwala, N. S.; Beckert, W. J. J. Chromatogr. Sci. 1989,27 (5), 209. (61) Junk, G. A; Avery, M. J.; Richard, J. J. Anal. Chem. 1988,60,1347. (62) Mamyama, H.; Ide, M. J. Anal. Toricol. 1988,12 ( l ) , 33. (63) Hartley, R; Lucock, M.; Cookman, J. R; Becker. M.; Smith, I. J.; Smithells, R. W.; Forsythe, W. I. J. Chromatogr., Biomed. Appl. 1986,380, 347. (64) Nomura, N. S.; Kuhnle, J. A: Hilton, H.W. Int. Sugar J. 1984,86 (1029). 244. (65) Kohler, P. W.; Su, S.Y. Chromatographio 1986,21 (9), 531. (66) Nelis. H. J.; Merchie, G.; Lavens, P.; Sorgeloos, P.; DeLeenher, A. P. Anal. Chem. 1994,66,1330. (67) Bardalaye, P. C.; Wheeler, W. B. J. Enoiron. Anal. Chem. 1986,25(1-3), 105. (68) Vendrig, D. E. M. M.; Holthuis, J. J. M.; Erdelyi-Toth, V.; Hulshoff, A. J. Chromatogr., Biomed. Appl. 1987,414. 91.
ii 0.7
)r L
0.6
a,
> 0.5 0 0
a tT
0.4 0.3
0.2 0.1
0.0 -0.1
0
1
2
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5
6
7
0
1
2
3
Volume (mL)
4
6
5
7
8
9
10
Volume (mL)
Figure 2. Total recovery from SPE as a function of extractant volume for alkyldimethylbenzylammoniumchlorides. Extractionfrom a cyano SPE column with 1 M HCVmethanol (50/50).A, c8; 0,C12; and 0, c14.
Figure 4. Extractionof epipodophyllotoxins(pptoxins I-IV) from a SPE column with 0.02 M ammonium phosphate (pH = 4.8)/ acetonitrile (30RO). c 1 8
7
A
2.5
5
n
1.5
Y
Y 7
c I
0.5
-0.5
0
1
2
3
4
5
6
7
Volume (mL)
0
1
2
3
4
5
6
7
Volume (mL)
Figure 3. Current model representation of SPE data for alkyldimethylbenzylammonium chlorides. A, c8; 0,(212; and 0, c14.
Figure 5. Extraction of phydroxybenzoic acid esters (parabens) from a c18 SPE column with methanoVwater (70/30), 1.2 mg total loading.
with the higher-chain compounds retained longer but with similar extraction profiles might be expected. In the recovery versus volume of extractant representation mentioned earlier, which appears on Figure 2, the expected similarity in extraction profiles is not obvious. In the current model, which appears in Figure 3, the order of retention and similarity of extraction profiles is clear: a similar retention mechanism (as expected) appears to be operating for the three species with the alkyllstationary-phase interaction increasing as the analyte chain length increases. In terms of selectivity, the 8 and 12 carbon analogs can be efficiently separated from the 14membered chain analog beyond a volume of ca. 4 mL. None of the species appears to present a problem with regard to irreversible binding to the stationary phase, since there does not appear to be a signficant intercept, and the extraction profile is consistent with either the Langmuir or the Gaussian representation. There is no indication of column overload, as will be discussed below. The extraction profile for four epipodophyllotoxin-related compounds eluted from a C18 SPE column with 0.02 M ammonium phosphate (PH= 4.8)/acetonitrile (30/70) appears in Figure 4.
As long as two species have the same slope (for straight-line or ideal representations) or parallel tangents (for curved representations) for some extractantcomposition, the efficiency of extraction is equal to that point, and hence no selectivity, or enrichment, is possible. When the four species are eluted from a C18 column with 0.02 M ammonium phosphate (PH= 4.8)/acetonitrile (30/ 70), all four extraction profiles are linear and parallel. To overcome these difficulties, we might be tempted to change SPE stationary phase to attempt to gain selectivity that the extraction system described lacks. If attention is now turned instead to the eluting solvent, some selectivity can be achieved. In Figure 4, an aqueous acetonitrile solution was used to elute the same substituted epipodophyllotoxins. As is often the case, a weaker elution solvent results in a more spec@c separation. Epipodophyllotoxins designated pptoxin I11 and pptoxin IV may now be separated from the other species by prior elution of pptoxin I and pptoxin 11. Loading. An important parameter for solid-phase extractions is the loading factor, and more importantly, how the SPE separation is affected by sample loading mass. Analytical or lowAnalytical Chemisfry, Vol. 67, No. 7, April 1, 1995
1195
7
6
4
P P
-E+Z (Methyl ester)
(Ethyl ester)
+Z
0
I (Propyl ester)
1 1
0
4-Z (Butyl ester) 1
1
1
1
1
2
1
1
3
1
1
,
4
1
5
,
1
6
1
7
Volume (mL) Figure 6. Extraction of phydroxybenzoic acid esters from a c18 SPE column with methanol/water (70/30), 4.0 mg total loading.
level loading is characterized by relatively high specificity and may facilitate column-to-column reproducibility. As an SPE system approaches overload for a multicomponent system, the dif€erent zones begin to coalesce, and chromatographic specificity decreases. At the same time, the elution contour (-ln(l - RT) vs v) changes from the quite curvilinear Gaussian (analytical) shape to the less curved Langmuir (overload) contour. Hence, we would expect for real systems that the current
1196 Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
model would represent an extraction near overload as a set of contours with less curvature and smaller differences in slopes than the corresponding low-level analytical separations. Figure 5 is the representation for the SPE extraction of four phydroxybenzoic acid esters (parabens) from a C18 column. For 1.25 mg loading of the SPE column, the elution profiles are similar, as would be expected for a homologous series. In a similar manner, the order of retention is also expected to increase from the methyl to the butyl ester, which is observed experimentally. When 4.0 mg is loaded on the column (Figure 6), signs of overload in the form of loss of specificity between the methyl and ethyl esters appear concomitantly with a decreased range in slope differences across the homologous series due to a leveling of extraction efficiency. In summary, the model presented here provides a consistent representation of multicomponent SPE extraction data, which allows optimization of extraction selectivity by judicious choice of wash solvents rather than the SPE stationary phase. Graphic representation and interpretation of multicomponent data are straightforward and allow analysis of SPE extraction conditions with respect to selectivity, type of extraction profile, and column loading. Received for review September 8, 1994. January 18, 1995.@
Accepted
AC9408991 @
Abstract published in Advance ACS Abstracts, February 15,1995.
