Anal. Chem. 1993, 85, 2903-2989
Stabilization of Reactive Species within Polystyrene Divinylbenzene Polymer Networks A. J. Bourque and I. S. Krull’ Department of Chemistry, 102 Hurtig Building, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115
B. Feibusht Supelco, Inc., Supelco Park, Bellefonte, Pennsylvania 16823
The effect of cross-linking, surface area, and porous nature of modified polystyrene-divinylbenzene (STY-DVB) reagents has been investigated. The supports were prepared via two techniques and modified to contain various chemical functionalities. These reagents were used in an on-line reactor for automated derivatization of amines in HPLC. The reproducibility of the response vs the physical nature of the porous support and the chemical functionality was determined. The ability to stabilize highly reactive acylating reagents toward high concentrations of aqueousbase was found to be a complexinteraction of pore size distribution, percent cross-linking, surface area, and absoluteloadingof the analytical reagent on the porous support.
INTRODUCTION Immobilized reagents have been used for organic and analytical chemistry for more than two decades.’“ The most important factor in the success of the immobilized methods ia the ability to carefully manipulate the chemical environment in which the reaction occurs. Solution Chemistry, Biphasic Systems. As early as 1884,the ability to use a water-reactive species to derivatize highly water soluble compounds was realized.415 This concept of extractive acylation described the chemical reaction which occurred a t the interface of the two immiscible liquids. An improvement on this method came with the advent of micellar chemistry.6 Bridging the gap between immiscible solvents and micelles are the phase-transfer reagents.7 Ionic surfactants, both cationic and anionic, a t levels below the criticalmicelleconcentration (cmc),can ion pair with a species of the opposite charge to form neutral analytes having enhanced partition ratios in apolar organic solventa.7 Solid-PhaseChemistry, Biphasic Systems. I t is possible to use an insoluble polymer as the nonpolar hydrophobic solvent. This is the basis of reversed-phasechromatographic supports and many solid-phase extraction techniques.8 Impregnation of an analytical reagent, pentafluorobenzyl bromide, in the polymeric porous resin allowed extractive
* Author to whom corrrespondence should be addressed. t Present address:
212 Primrose Lane, Ambler, PA 19002. (1) Merrifield, R. B. Biochemistry 1964,3,1385. (2) Patchornik, A.; Kraus, M. A. Pure Appl. Chem. 1976,46,183. (3) Gao, C.-X.; Krull, I. S. Biochromatography 1989,4, 222. (4) Schotten, C. Ber. Dtsch. Chem. Gee. 1884,17, 2544. (5) Baumann, E. Ber. Dtsch. Chem. Ges. 1886,19,3218. (6) Duynstee, E. F. J.; Grunwald,E. J. Am. Chem. SOC.1959,81,4540. (7) Dehmlow, E. V. Angew. Chem., Znt. Ed. Enggl. 1977,16,493. 0003-2700/93/0365-2983$04.00/0
alkylation of carboxylic acids from various matrices.@These applications have taken advantage of the recoverability and reusability of the immobilized reagents. However, with the exception of the solid-phase extractive alkylation? few of these methods fully utilized the ability to manipulate the microenvironment of the support. Solid-PhaseChemistry, Triphasic Systems. The introduction of polar groups to the solid phase generates regions of localized polarity which are not attainable in the biphasic solutionsystem.loJ1 A polymeric analog of dimethyl sulfoxide was prepared on polystyrene and used to catalyze the nucleophilic reaction of inorganic and organic anions with various alkyl bromides.12 This concept, first reported by Regen, was called ‘triphase catalysis”.13 Solid-Phase Reagents with Reactive Centers. The earliest efforts followed the successes of Merrifield“ and Patchornik,16 who utilized solid supports for the synthesis of peptides. Patchornik’s scheme was an inverse-Merrifield approach. The polymer was used as the amino acid reagent for adding the next amino acid to the growing peptide chain. Replacing the amino acid with a detector-sensitive acylating reagent made these reagents analytically useful for derivatization/detection schemes in conjunction with HPLC analyses.3 The early choices for polymeric supports and analytical labels were based on commercial availability and prior literature. A chloromethylated 1% divinylbenzene-polystyrene (DVB-STY) microporous resin was modified to contain a mixed anhydride.16 Thisreagent did not yield a reproducible response from injection to injection when used in an on-line precolumn reactor under R P conditions. The support and chemical functionality were changed to a highly cross-linked macroporous STY-DVB support which was chemically modified to contain a hydroxybenzotriazole active ester.17 This reagent could not be used on-line under RP conditionsbecause of ita sensitivity to water. The next reagent investigated was a 4% DVB-STY microporous resin which was modified to (8) Nielen, M. W. F.; Frei, R. W.; Brinkman, U. A. Th. In Selectiue Sample Handling and Detection in High-Performance Liquid Chromatography; Frei, R. W., Zech, K., Eds.; Elwvier: Amsterdam, 1988;Vol. A, P 5. (9) Rosenfeld,J.; Mureika-Ruseel,M.;Yeroushalmi,S. J. Chromatogr. 1986,358,137. 1978,100,7779. (10) Regen, S. L.; Nigam,A. J. Am. Chem. SOC. (11) Sun, J. J.; Fritz, J. 5.J. Chromatogr. 1992,690,197. (12) Janout, V.; Hrudkovh, H.; Ceffelln, P. Collect. Czech. Chem. Commun. 1984,49, 2096. (13) Regen, S. L. J. Am. Chem. SOC.1976,97, 6956. (14) Medield, R. B. J. Am. Chem. SOC.1963,85,2149. (15) Fridkm, M.; Patchomik, A.; Katchaleki, E. J. Am. Chem. SOC. 1966,88, 3164. (16) Chou, T.-Y.;Colgan, S. T.; Kao, D. M.; Krull, I. S., Domhel, C.; Bidlingmeyer, B. A. J. Chromatogr. 1986,367,335. (17) Chou, T.-Y.; Gao, C.-X.; Colgan, S. T.; Krull, I. S.; Dorschel, C.; Bidlingmeyer, B. A. J. Chromatogr. 1988,454, 169. Q 1893 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993
contain a 3-nitrobenzophenon-4-yl ester.18 This reagent had the appropriate characteristics of good reactivity and good stability. However, its dinitrobenzoyl ester again made this reagent too reactive to be useful on-line in RP chromatogr a p h ~ An . ~ investigation ~ of cross-linking and surface area effects suggested that resins with low cross-linking and high surface area would yield reagents with superior characteristics, i.e., high reactivity and good longevity.20 This paper investigates the parameters of cross-linking and surface area of DVB-STY supports prepared via two methods. The resins prepared by both techniques were modified to contain a 3-nitrobenzophenon-4-yl 9-fluoreneacetyl group. These acylating reagents were placed in an on-line precolumn reactor, heated to 70 "C, and used to derivatize a homologous series of n-alkylamines dissolved in aqueous acetonitrile at pH 11-12. The analysis was repeated 50-100 times using the same reactor. Using the support which yielded the highest and the most reproducible response, the 9-fluoreneacetyl group was replaced with a 3,5-dinitrobenzoate tag. In a similar experiment, the optimal DVB-STY resin was modified to contain the hydroxybenzotriazole linkage. This was labeled with the 9-fluoreneacetyl tag and the above studies were repeated at room temperature. The reasons for hydrolytic stability of these polymeric reagents are discussed. Application of some of these reagents toward on-line derivatization for enhanced chiral recognition on Pirkle-type stationary phases will be shown.
EXPERIMENTAL SECTION Materials and Reagents. All separations were performed on a 5-pm, 250 X 4.6 mm i.d. Supelcosil LC-M-DB column (Supelco Inc., Bellefonte, PA). Polymers used for preparation of the solid-phase reagents were obtained from Rohm and Haas (Spring House, PA) and Supelco, a division of Rohm and Haas. 9-Fluoreneacetic acid and other reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI). HPLC solvents (Omnisolv grade) were donated by EM Science, Inc. (Gibbstown, NJ). All solvents were filtered through an off-line 0.45-pm filter (Supelco), and a 0.2-pm PTFE membrane (Whatman, Inc., Clifton, NJ) for on-line degassing and filtration, and were blanketed with helium (2 psig) during use. Samples were filtered through 25-mm-diameter, 0.45-pm nylon 66 syringe tip filters (Supelco). Apparatus. On-line derivatizations and HPLC resolution of the derivatives were performed with a Gilson Model 232 autosampler, two Gilson 203 HPLC pumps, a Gilson 115variablewavelength UV detector, and a Gilson 121fluorescence detector with excitationat 254 nm and emission from 305 to 395 nm (Gilson Medical Electronics, Inc., Middleton, WI). The twomobile-phase components were mixed by a Gilson Model 811B dynamic mixer (1.5-mL volume mixing chamber). A silica saturator column (100 X 4.6 mm id., 30-40 pm silica) was prepared and placed on-line between the mixer and the injector. On-line derivatization hardware consisted of a 27 X 2.1 mm i.d. guard column dry packed with ca. 65 mg of polymeric reagent. The reactor was installed on-line in place of the injection loop on the Model 7010 Rheodyne injection valve (Rheodyne, Inc., Cotati, CA). The reactor temperature was varied by incremental changes in the applied voltage to the heating tape (0.5 in. X 24 in., 120 V, Thermolyne Corp., Dubuque, IA) which had beenwrapped around the reactor. A 0.5-pm on-line filter (Rheodyne) was placed between the injector and the guard column. Data were acquired and the system was controlled through a Gilson 621 Data Master attached to an AST Premium 286 computer (AST Research, Inc., Irvine, CA). Centrifugation was performed using 15-mL glass vials in an IEC HN-S centrifuge (DamoniIEC Division, Needham Heights.,MA). Surface areas and pore size distributions were measured on a Quantachrome Autosorb-6 (Quantachrome, Syosset, NY). (18) Gao, C.-X.;Chou, T.-Y.;Krull, I. S. Anal. Chem. 1989, 61, 1538. (19) Bourque, A. J.; Krull, I. S. J. Chromatogr. 1991,537, 123. (20) Bourque, A. J.; Krull, I. S. J. Chromatogr. Sci. 1991, 29, 489.
Table I. Physical and Chemical Character of Polymeric Reagents po1y m er particle cross-linking loading I.D. type size (pm) (7%-DVB) (mequiv/g) 901-027 910-18 91051b 91053" 91035 910-033b 1842 1841 1851 1850 1849
100-Atemplate IOO-A template 1OO-A template 2004 template 100-A template 100-Atemplate suspension suspension suspension suspension suspension
16 16 16 16 16 16 45-75 45-75 10-15 10-15 10-15
6
1.71 1.39
12 12 12 24
1.24 0.64
80
0.34
3 6
1.61 0.40
6
1.62 1.56
8 12
1.48
1.17
" An 80% DVB analog (910-096) had a surface area of 380 m2/g for pore sizes of >20 A and an average pore size of 79 A. b Surface area of 520 m2/g for pores of >20 A and an average pore size of 59
A.
Synthesis of Supports. Some of the STY-DVB resins were prepared by a templating polymerization technique and others by a suspension polymerization technique. The templated resins were all ca. 16-pm irregular particles, prepared from known concentrations of DVB and STY monomers using 100-and 200-A silica. The suspension polymerization resins were prepared from known concentrations of DVB and STY monomers using various surfactants and porogens to create different particle sizes and, perhaps, porosites (Table I). Templated Polymerization: Preparation of the C1-Modified Silica. Silica, 100 g (100 A, IMPAQ RG1020-Si, PQ Corp. Conshohocken, PA) was dried under vacuum at 120 "C for 2 h, suspended in a solution of 47.6 mL of hexamethyldisilazane in 500 mL of dry toluene heated to reflux for 4 h. The suspension was cooled to room temperature, filtered, and washed with 500 mL each of toluene, dichloromethane, and methanol (MeOH). The modified silica was dried at 60 OC under Nz overnight. Templated Polymerization: Preparation of 12%DVB Support. C1-Modified silica, 30 g, was placed under vacuum and 25.9 mL of a mixture containing 85% styrene, 12% divinylbenzene, 3% ethylvinylbenzene, 1%VAZO-67, 1% Lupersol 10M75, and 1% tert-butyl perbenzoate was added. Inhibitors had previously been removed from the monomer mixture by passing it through a bed of 2 mL of tert-butylcatechol remover (Aldrich Chemical, Co., Milwaukee, WI) and 2 mL of hydroquinoneimethyl ester remover (Aldrich). The resulting suspension was shaken until it became a smooth flowing powder and then a nitrogenpurged aqueous solution containing0.7% Elvanol51-05 and 1.4% sodium sulfate was added. The suspension was added to a 600mL Parr reactor (Model 4563, Parr Instrument Co., Moline IL). The suspension was agitated at 400 rmp under 50 psig nitrogen pressure and subjected to the following thermal gradient: hold 5 min at 40 "C, incrase 1'Cimin to 88 OC, hold 100 min, increase 1"Cimin to 120 "C, and hold for 200 min. The flask was allowed to cool to room temperature, and the supernatant was decanted. The resulting solid was resuspended in deionized water, allowed to settle, and decanted to remove fines. This was repeated a total of five times. The solid was then suspended in 500 mL of 3 N KOH in 1:l MeOH/H20 and stirred overnight at room temperature. The suspension was filtered, washed to a neutral pH with deionized water, and washed with 300 mL of MeOH. A 2-gsample of the resulting solid was further washed with 100mL of 1:l acetonitrile (ACN)/H20 (80 "C), 100 mL of ACN (80 "C), and 100 mL of THF (70 "C). The polymer was dried under high vacuum at 50-60 OC for several hours. Suspension Polymerization: Preparation of 12% DVB Support. A solution was prepared containing 3.6 g of acacia gum arabic and 3.6g of Marasperse N-22 in 493 gof degassed, deionized water by heating in a 1-L reaction vessel at 60 OC under nitrogen and agitation until solution was effected. A mixture of 11.0 g of 55% DVB, 39.0 g of styrene, 20 g of methyl isobutyl ketone, and 0.5 g of benzoyl peroxide was purged with nitrogen for 15 min and then charged to the aqueous solution. The resulting dispersion was vortexed with a high-shear homogenizer for 1
ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993 2981 OC7-FA, 910-051b
OC7-FA, 910-18
AC7-FA. 1849-BP-FA
6.5
F
(P-BP)
0'
(P-HoBTA)
Figure 1. Structures of polymerlc reagents used.
min, heated with agitation to 80 "C for 60 min, and held for 16 hat 80 O C . After being cooled to room temperature, the porogen was removed via azeotropic distillation at 80-85 "C and the resulting solid was washed with 3 X 200 mL of deionized water and 3 X 100 mL of MeOH. A 2-g aliquot of this material was further washed, following the protocol for the templated resin. The chemical functionalities attached to the above supports are provided in Figure 1. The two polymeric leashes, an o-nitrobenzophenol (P-BP) and a 1-hydroxybenzotriazole (PHoBTA), were converted to the three active esters of a O-fluorenylacetate (FA), a 3,bdinitrobenzoate (DNB), and a 3,sdinitrophenyl carbamate (DNPC) (Figure 1). The synthesis of these reagents and the mechanism of reaction with nucleophiles have been reported e l s e ~ h e r e . ~ ~ ~ ~ ~ Characterization of Polymeric Reagents. The polymers were characterized for surface area and pore size distribution by N. T. Miller of the PQ Corp. Loading determinations of the modified polymers were performed in-house, using a controlled saponification technique (Table I).2o Reproducibility Studies. The polymers were placed online in the 27 X 2 mm reactor, and 50-pL aliquota were analyzed. The analyte was a homologous series of n-alkylamines prepared in 1:l 100 mM NaOH/ACN at a 1 pM level (n-butyl- through octylamine, 73-129 ppb). The area of n-heptyl-9-fluorenylacetamide vs the number of injections was plotted (Figure2). Areas of the other amine derivatives were also recorded, though not reported here. The exact derivatization protocol used with the automated Gilson apparatus has been reported.22
RESULTS AND DISCUSSION Initial studies of the feasibility of derivatization of nucleophiles in biological fluids using on-line, precolumn derivatization in reversed-phase HPLC indicated that some solid-phase reagents were quite useful for derivatization of strong nucleophiles in the presence of large concentrations of hydroxide.23 A solid-phase reagent with an average pore size of 430 A acta as a restricted access medium (RAM)for larger molecules, while allowing access of small molecules to the reactive, reagent sites within the pores of the resin.23 However, when used for repetitive, on-line derivatizations in a precolumn reactor, only certain solid-phase reagents gave (21) Bourque,A. J.;Krull,I. S.J.Pharm. Biomed.Anul. 1999,11,496. (22) Bourque, A. J.; Krull,I. S.; Feibush, B. Biomed. Chromatogr., in
press. (23) Zhou, F.-X.; Krull, I. S.; Feibush, B. J. Chromutogr. 1992, 609, 103.
5
1'0
1'5
2'0 25 30 NUMBER OF IKIECTDNS
35
40
45
50
Flguro 2. Polymerization conditions vs reproducibiNty of the final support: 910-051B, tempiated resin wlth suspension polymerizetion condltions(l2%DVB);910-18, templatedrdnfoibwlngexpwi"tai conditions (12% DVB); 1849, resin prepared by suspension polymerization (12% DVB). Reactlon conditions: 50 pL of 1 pM rrelkylamims (C4-C8) in 50% ACN containing 50 mM KOH, 35 8 at 60 OC v8 63 mg of P-BP-FA. Reactor dimensbns: 27 X 2 mm, 0.2-pm SS frits. Analytical column: 250 X 4.6 mm, 5-pm Supeicosii LC-DB-18, 4545% ACN over 5 min.
reproducible, injection-*injection responses. Depending on the surface area, pore size distribution, and degree of crosslinking of the polymeric support, percent derivatizationswere seen to increase, decrease, or stay the same with subsequent injections. Definition of Reproducibility. Reproducibility w i l l be the term used to describe the character of the plots of the derivative peak area vs number of injections of the same sample into the reactor. The various factors that affected the reproducibility will be discussed in their perceived order of importance. Cross-Linking. The amount of divinylbenzene used to prepare the solid-phase support appeared to have the largest effect ontherun-brunreproducibility. The abilityto protect the reagent from water and to decrease physical changes (i.e., swelling) is presumed to be the reason for the dependence of reproducibility on cross-linking. Lower cross-linking of the support decreased the steric hindrance toward diffusion through the interconnected polymer chains and enhanced (increased)the amount of the reagent bonded within the walls of the pores. The low tendency of hydrophilic species (e.g., hydroxide) to penetrate into the hydrophobic gel phase (the pore walls) prevented the concentration of the reactive reagent sites from rapidly decreasing due to nonselective reactions (i.e., hydrolysis). The low percentage of cross-linking of the solid-phaee reagent yielded the following two beneficial results: Loading. A low cross-linking of the polystyrene increased the proportion of monosubstituted phenyl rings and their accessibility to modification. This resulted in an increased loading. Higher loadings yielded longer lifetimes and faster rates of on-line reaction. Hydrophobicity. The second benefit of low cross-linking of the polymer support was the generation of larger concentrations of active ester within the hydrophobic regions of the STY-DVB support. The hydrophobic polymer chains allowed hydrophobic, low molecule weight, nucleophilic analytm a preferred access to the active ester sites. Micropolarity a n d Swelling. The degree to which the polymer chains were interlinked (percent cross-linking) defined the ability of the resin to swell. The polystyrene support, prior to the present modification scheme, could be described as a hydrophobic medium. However, incorporation of polar groups into hydrophobic resins produces regions of micropolarity which add considerable heterogeneity to the medium.24p2sThe hydrophilicity of sulfonated polystyrene is (24) Regen, S.
L.J. Am. Chem. soc. 1975, 97, 5956.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993
an excellent example of a polar modification which completely changes the wetting/swelling properties of the modified matrix. In these studies, the ability of the polymeric reagent to swell changed with depletion of the reactive sites. This was due to the formation of the sodium o-nitrophenolate residue each time an active ester reacted with a nucleophile. This was most pronounced for supports prepared with 6 ?Z or less DVB. As the polymer aged with use it became more hydrophilic. The polymer swelled in the aqueous/organic media, effectively increasing the pore size and surface area and, thus, the kinetics of the nucleophilic reaction. The worst cases in this group were the reagents prepared on 60-pm microporous STY-DVB of less than 4 % DVB. These resins initially had very low reactivity due to their nonporous nature in the nonswollen state and increasingly became more reactive due to rapid increases in the relative pore size and surface area as the resin aged. The intermediate range of cross-linking, 8-12 '3 DVB, yielded the most reproducible behavior in the final modified supports. Presumably, at these levels of cross-linkage, the increase in accessibility and kinetics due to swelling and wetting were compensated by the decrease in kinetics due to depletion of the active, reagent sites as the material aged. Influence of Polymerization Conditions. The difference in the polymerization techniques of the templated and suspension supports was considered a source of the unusual stability of the templated resins. T o ensure that differences in support character were not chemical in nature (i.e., different cross-linking or microenvironment), the templated support was synthesized under conditions used in the suspension polymerization technique. The support that resulted was modified and showed no deviation from a support made following the original templating conditions (i.e., concentration of catalyst, pressure, and temperature) (Figure 2). From these studies, it would appear that the unique character of the templated resins was a function of their controlled pore networks and high surface areas, even for the lightly crosslinked support. Surface Area. Templated polymers gave better reproducibility and higher yields than the suspension polymers of the same cross-linking (Figure 2). We attribute the more stable behavior of the templated resins to higher, accessible surface areas than the suspension supports. Reagents with low surface areas would produce poor yields of modified analyte and would not be as useful as those that produced larger conversion yields. However, absolute yield would not affect the reproducibility, only the limit of detection obtained when the reagent was used for trace analysis. Surface area measurements could not be obtained for the most useful polymeric supports, i.e., templated supports with low (8-12% DVB) cross-link levels. The supports presumably suffered pore collapse upon removal of the solvent. However, in the on-line reactor, the support is stored in 100% ACN at 60-70 "C. These conditions presumably allow the resin to swell and regain the porous network and surface area mirror imaging the original silica template. The templating technique is an effective method (perhaps the only existing method) for generating a lightly cross-linked, highly accessible surface area, polystyrene support. Other methods, which produce high-surface-area supports, use extensive levels of divinylbenzene in the monomer mixture. Not only are these supports difficult to functionalize, leading to low concentration of reactive species, but as their gel phases are the least penetrable by external reagents, most of the ~~
~
(25) Balakrishnam, T.; Babu, S. H.; Perumal, A. J. Polym. Sci., Part A: Polym. Chem. 1990,28, 1421.
O C 7 - F A , 910-18 ( 1 O O A )
O C T F A , 910-053 (200A)
5 5
2 5. 2. 1 5. 1. 5.
.
0
o
, 5
.
, '3
.
, '5
.
,
.
,
.
,
20 25 33 NUMBER OF INJECTIOYS
.
, 35
.
, LO
.
, 45
. 5c
Flguro 3. 12% DVB templated supports prepared with 100-200-A silica. Reaction conditions: as in Figure 2.
reactive sites are present on the rigid surface of the pores, prone to hydrolysis.26 Porosity. The templated reagents were prepared using silica (RG1020-Si, PQ Corp.) which had a mean pore diameter of 100 A. To determine the effect of pore size on reproducibility of derivatization, a support was prepared by the same technology, but with 200-A silica (RG-2020-Si, PQ Corp.). This reagent initially gave higher yields than the same reagent based on 100-Asilica, but after 20 derivatizations ( 5 h of use) leveled off to the same analytical response (Figure 3). In the templated polymerization process, the resin produced mirror images of the templating silica, where the silica pores become the resin network and vice versa. The pore size of the silica template may not be reflectedin the final pore size distribution of the resultant polymers, as here. Pore structure and surface area meaurements have been acquired for the 80% DVB materials (910-033 and 910-096) (Table I, Figure 4). The average pore sizes of the dry polymers were ca. 59 A using the 100-8, (910-033) silica template and 79 8, (910-096) using the 200-A template, roughly half of the templating silica particle diameters. Both bare polymers, underivatized with reagents, showed a remarkably narrow pore size distribution (Figure 4). In addition, the supports showed surface areas for mesopores (>20 A) of 520 and 372 m2/g, respectively, and mesopore volumes of 0.77 and 0.72 mL/g, respectively. The determinations of pore diameter and distribution were possible because the support was crosslinked to the extent that it retained its pore structure upon removal of all solvents. It was not possible to measure these physical properties for lower cross-linked materials, because the pores collapsed when solvents were removed. Such materials in the dry state appeared to be nonporous. There is no obvious physical method to measure pore structure and surface area of lightly cross-linked materials in the solvated state. The actual pore diameter of the lightly (6-12 5%) crosslinked resins under the derivatization conditions might be accessed by size exclusion chromatography using appropriate molecular markers.27 Stabilization via Microenvironment Effects. It was initially found that 80 % cross-linked, silica-templated, porous, solid-phase reagents could not be used on-line for derivatization of strongly basic aqueous/organic samples.22 A crosslinked reagent based on templated 24 % DVB was much more stable under these conditions. It was postulated that hydrophobic shielding of the active ester from hydroxide might be occurring. To investigate this further, a templated resin was prepared with a 1 2 % DVB level. The reagent based on this support was stable enough to be used for repetitive, sequential, on-line sample injections in 0.5 N KOH a t elevated (26) Ford, W. T. In Polymeric Reagents and Catalysts;Ford, W. T., Ed.;ACS Symposium Series 308; American Chemical Society: Washington DC. 1986: D 247. (27) W ' h g Q. C.; Hosoya, K.; Svec, F.; Frechet, J. M. Anal. Chem. 1992, 64,1232.
ANALYTICAL CHEMISTRY, VOL. 85, NO. 21,NOVEMBER 1, 1993
2887
1.56
1.41
1.25
1.c9
0.94
0.78
0.63
0.47
0.31
0.16
0.00 1
Flgure 4. Pore size distrlbutlons of polymer supports. Measured from volume differentlal of nitrogen desorption (mL/g) vs pore radius: (a) resin 910-033 (templated from 100-A silica) and (b) resin 910-096 (templated from 200-A silica). Apparatus described in Experimental Section.
temperatures. This was the first real indication that hydrophobic shielding of the STY-DVB gel phase protected the polymer-bound active ester from depletion due to reaction with hydrophilic nucleophiles, such as hydroxide and water. The increased stability of the 12 % DVB templated resin was not due to an increased concentration of analytical label. Although the loading of the 12 % DVB templated resin was higher, the difference could not account for the increased stability (Table I). If increased loading were the only cause for increased on-column reproducibility (stability), then the peaks of hydrolysis products which were present in each chromatogram would also be expected to increase with increased loading. In fact, they decreased. Thus, the extra amount of active ester was present in the support in regions that were not easily accessible to hydroxide ions. Many hydrophobic alkylamines, on the other hand, interacted with these active sites with yields of greater than 90% in 30 8 at 60 "C. Addition of SDS a t basic pH greatly decreased the lifetime of all reagents due to its aqueous wetting of the hydrophobic regions. The 12% DVB templated support was affected the least and thus showed the best reproducibility. It apparently possessed just enough rigidity, due to its crosslinking, to prevent adverse swelling and aqueous exposure to active ester sites. To further investigate the correlation between the crosslinking and the increased stability, a reagent waa prepared on a 6% templated support. However, this reagent tended to increase its derivatization efficiency with consecutive usage. Although the support conferred excellent stability to active ester groups incorporated within the gel-phase regions, its pore size and surface area chagned with time due to the lighter cross-linked structure. Hydrophobic Protection of Highly Water Reactive Reagents. A hydroxybenzotriazole (P-HoBTA) leash was
prepared on the 12% DVB templated support. A 9-fluorenylacetyl (FA) label was attached to the support (P-HoBTAFA), and this was compared to the data obtained for the P-BP-FA, keeping all conditions, but temperature, constant. While the P-BP-FA was run at 60-70 "C, the P-HoBTA-FA was used a t room temperature (ca. 21 "C). Very similar reproducibility data were obtained. The detector-sensitive label on the templated 12% DVB P-BP support was changed to a more reactive moiety, the 3,5-dinitrobenzoyl group (DNB). This reagent gave >90% conversions of n-alkylamines in under 30 s a t room temperature. The increase in kinetics was due to the more inductive nature of the DNB label as compared with the FA label. The ultimate test of the ability to protect water-reactive reagents within polystyrene matrices was to label the PHoBTA leash with the DNB group. This reagent on other supports was able to derivatize several classes of weak nucleophiles, but was not useful for repetitive injections in aqueous so1vents.m By increasing the speed of certain solvent delivery steps with the autosampler, the exposure of the reagent to aqueous conditions was reduced to ca. 20-s derivatization time and 10-s on-line (inject position) before the autosampler was switched back to the load position and flushed with anhydrous solvent. The reproducibility of the reagent was poor (Figure 5). With the P-HoBTA-DNB, the surface sites were so reactive that they hydrolyzed quickly upon interactingwith the basic aqueousjorganicsamples. Sites deeper within the gel-phase regions of the 6 % DVB polymer were more protected, and it was these sites which generated the plateau at much lower conversions (Figure 5). The lower conversion may have been due to a combination of slower diffusion and the overall decreased loading due to hydrolysis of most of the active ester sites.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993
2988
O C 7 , 910-18-BP-DNB
O C 7 , 910-027-HoBTA-DNB
6 4
6
t
L
51 C
5
' 0
15
20
25
35 40 NUMBER OF INECTIONS 30
45
50
55
69
Figure 5. Stabilization of reactive species. Immobilization of the P-BP-DNB on the 12% DVB templated support provided good stability and high reactivity,while the P-HoBTA-DNBon the 6 YO DVB templated support was not stable. Reaction conditions: 50 pL of 70 pM rralkyiamines (C,-C,) in 50% ACN containing 50 mM KOH, 35 s at 21 O C (room temperature)vs 63 mg of reagent. Reactor dimensions: 27 X 2 mm, 0.2-pm SS frlts. Analytical column: 250 X 4.6 mm, 5-pm Supelcosii LC-DB-18, 45-85% ACN over 5 min.
On-Line Derivatization for Chiral Recognition. A further example of increased stability via lowered cross-linking was the ability to stabilize the P-HoBTA-DNPC reagent (Figure 1). This reagent was difficult to work with in the past due to its low loading and water lability.21 Through improved support design, it was possible to increase the lifetime of the reagent and use it on-line for normal-phase derivatization of racemic amines, followed by Pirkle-type, chiral separations. The reagent was much more stable under normal-phase conditions, but under such conditions, derivatization did not occur. No derivatization of alcohols occurred in dichloromethane/hexane (HEX) solvents containing triethylamine (TEA). The polar solvents which were ideal for the derivatization of alcohols, ACN with TEA, were not miscible with the mobile phase (190% HEX). Both the P-BP-DNB and the P-HoBTA-DNB on a 6% DVB templated support could be used effectively for the online derivatization of amines in normal-phase solvents. This provided a convenient method for generating derivatives of racemic amines which could be separated directly on a Pirkletype stationary phase. The ability to perform rapid derivatization of enantiomers a t room temperature significantly reduced diffusional band broadening (Figure 6). The on-line derivatization and resolution of the enantiomers of 2-aminooctane as their 3,5-dinitrobenzamides was easily accomplished with no apparent band broadening. The plot of area counts for both enantiomers vs number of derivatizations showed a gradual diminution in response over 20 injections. The reduced lifetime in the present normal-phase mode vs the same reagent in the reversed-phase mode may be due to the inability of the normal-phase solvent to support addition/ elimination reactions within the solid-phase reagent. Depletion of the active reagent sites by water and/or solvent impurities may occur faster than reactions with the nucleophilic racemic compounds. These additionielimination reactions are promoted and accelerated by polar-type solvents, such as ACN or ACN/HOH. As a final example of the utility of these resins, the P-HoBTA-DNPC was prepared on the 6% DVB templated support and placed on-line for normal-phase, Pirkle-type derivatizations. This reagent also showed very little band broadening, but a peak corresponding to a hydrolysis product was present in each chromatogram (Figure 7). The lifetime of the reagent under on-line conditions was poor, probably a function of the low loading of the reagent. It was found that typical loadings of the dinitrophenyl carbamate on the 6% DVB templated support ranged from 0.15 to 0.40 mmol/ g. Attempts to increase the loading through the use of 2 or more equivalents of dinitrophenyl isocyanate during the
4-
il
--_
_--,
,
.
.
.
-_-
rime (min 1
__I
20
Flgure 6, On-line derivatizationwith PHoBTADNBfor chirai recognition. Racemic 2-aminooctane dissolved in 1:1 DCMIHEX (0.1 % TEA) was
manually injected directly into the on-line reactor, allowed to react for 2 minat room temperature,and switchedon-line. The polymeric reagent was the 6 % DVB support (9 10-027) containing a hydroxybenzotrlazole labeled with a 3,5dinitrobenzoate group (P-HoBTA-DNB). HPLC conditions: 90:5:5 HEX/DCM/EtOH at 2.0 mL/min (600 psi), 250 X 4.6 mm i.d., 5-pm LC-(@-naphthyl urea column.
L'
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PdA. -L L ---i)
Tirnc
imiii)
20
Flgure 7. On-line derivatization with chirai recognition. Sample and derivatization conditions as in Figure 6. The polymeric reagent is the 6 % DVB templated polymer containing a hydroxybenzotriazole labeled with a 3,5dinitrophenyicarbamategorup (P-HoBTA-DNPC). The peak eluting between the enantiomers is due to hydrolysis of the solid-phase reagent. HPLC conditions as in Figure 6. tagging procedure increased the background peaks more than the derivative signal. Similarly, increased derivative yield with the addition of an organic base "catalyst" was offset by increased side products or byproducts.
CONCLUSION Solid-phase extraction has allowed for protection of watersensitive, solid-phase reagents toward aqueous/organic phases which contain large concentrations of hydroxide. The optimal cross-linking level of the porous solid-phase support was 8-12 % DVB. Higher cross-linking levels prevented immobilization of active ester sites within the gel phase of the porous support. These reagents showed rapid hydrolysis when exposed to basic aqueous/organic media. Reagents prepared with less than 8 % DVB exhibited changes in surface area due to swelling with repeated usage. The increase in the exposed surface area changed the kinetics of reaction leading to nonreproducible behavior. The high surface area of the lightly cross-linked, porous supports was only attainable via the
ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, lQQ3 1989
templating technique. It was the high surface area which made the use of mild conditions for derivatization possible. It is quite likely that these reagents have many important future applications, as yet unrealized.
ACKNOWLEDGMENT The automated HPLC equipment was donated to Northeastern University by Gilson Medical Electronics, Inc. (R. Reinhardt and M. Muncey), Middleton, WI. This work was supported, in part, by an unrestricted grant from Pfizer, Inc., Pfizer Central Research, Analytical Research Department (G. Forcier and K. Bratin), Groton, CT, an NIH Biomedical Research Support Grant to Northeastern University (S. Fine), RR07143, Department of Health and Human Resources (DHHS), and a research and development contract from Supelco, Inc. (J. Crissman and B. FJ, division of Rohm &
Haas Corp., Bellefonte,PA. HPLC solvents were donated to Northeastern University from EM Science, Inc. (G. Niessen) Gibbstown, NJ. We extend special thanks to D. Conklin and Q.-T. Lai of Supelco, Inc. for synthesis of the polymeric support and to D. Tracy of Gilson Medical Electronics, Inc., without whose programming expertise this work would not have been possible. We also thank N. T. Miller of PQ Corp. (Conshohocken, PA) for pore characterization of some of the supports used and G. Lein of Rohm and Haas for technical revisions of the manuscript. Last, we thank our colleagues at Northeastern University, F.-X. Zhou and D. H. Fisher, for helpful discussions. RECEIVED for review February 8, 1993. Accepted July 13, 1993.8 Abstract publishedin Advance ACS Abstracts, September 15,1993.
