Anal. Chem. 1981, 5 3 , 1822-1828
1822
Characterization and Applications of Amberlite XAD-4 in Preparative Liquid Chromatography D. J. Pletrzyk" and J. D. Stodola' Chemistry Department, The University of
Iowa, Iowa City, Iowa
52242
Amberlite XAD-4, a nonpolar adsorbent which can be employed in reversed-phase chromatography, was evaluated as the stationary phase for preparative liquid chromatography. The cdumn parameters studied include XAD-4 particle size, packing of particles, column diameter, flow rate, mass and volume overload, and eluting conditions. Columns that were 8.0 mm i.d. (3/e in. 0.d.) X 25 cm packed with 37-44 pm XAD-4 particles provided efficiencies of 500-1500 piates/m. Similar efficiencies were also obtained on a 20.5 mm i.d. (1 In. 0.d.) X 32 cm column packed with 75-105 gm XAD-4 particles. Flow rates over the range studied did not have a significant effect on capactty factor or efficiency for the two columns. For a capactty factor of about 2 the mass overload limit, defined as a 10% decrease in efficiency, was about 0.23 % wt/wt load on the 8 mm i.d. cdwnn and 0.25 % wVwt load on the 20.5 mm i.d. column. Mass overloading is possible, and for &'less than 10 for the sample, the two columns can handle quantities of a few hundred miillgrams and several grams, respectively. I n general, these trends are similar to those observed for silica and aikyi-modtfied silica stationary phases. Eluting conditions studied Include pH control under conditions in which a k a type columns cannot be used and/or in the presence of mixed solvents. Mixtures of benzenesulfonic acids, chiorophenoxyacetlc acids, phenols, amino acids, and dipeptides were separated.
High-performance liquid chromatography (LC) has become a dominant force in the separation of complex mixtures. Since many of the major features of analytical LC (ALC) can be used advantageously in preparative liquid chromatography (PLC), this latter technique has also undergone major growth in recent years (1-3). In general, a PLC separation is one where the sought q t e r material, which may range from milligram to multigram quantities, is separated in a sufficient quantity of acceptable purity in a reasonable time period in order to continue into the next step in the application of the material. The two most widely used stationary phases in PLC are silica gel and alkyl-modified silica. Both are commercially available in micre and macroparticle bulk form and prepacked columns of varying diameters. One major limitation is that their applications are restricted to a mobile phase pH of 2-8. It has also been suggested (2) that silica columns in some PLC applications can introduce metal ions, act as catalysts, and irreversibly retain components; the latter two can contribute to significant sample loss. Amberlite XAD copolymers have been widely used as sorbents for stripping and concentrating organic compounds from air (4),water (5,6),biological fluids (7),and food samples (8) and as a stationary phase in ALC (9-11). The XAD properties which are important to these stripping and LC applications are the following: (1)They sorb weak (as charged Present address: The Upjohn Company, Control 7824-41-1,
Kalamazm, MI 49081.
0003-2700/81/0353-1822$01.25/0
and uncharged species) and strong organic electrolytes and nonelectrolytes. (2) Analyte retention can be altered by variations in composition, type of organic solvent, pH, and type and concentration of salts or counterions in the mobile phase. (3) Analytical columns packed with microparticle XAD or similar copolymers will provide efficiencies similar to alkyl-modified silica (I1,12).(4) XAD copolymers (20-50 mesh) are inexpensive. ( 5 ) The XADs are available with different pore sizes and surface areas, and have large loading capacities. (6) The XADs are stable throughout the pH range of 1-13. These properties are also very useful in PLC applications, particularly the extension of the mobile phase pH to a range not attainable when using silica type stationary phases. The purpose of this study was to evaluate the characteristics and applications of Amberlite XAD-4 columns in PLC in order to determine whether these columns are useful alternates to silica and allcyl-modified silica columns. XAD-4 is a copolymer of styrene and divinylbenzene with a surface area of 750 m2/g and an average pore diameter of 50 A. Chromatographically, it functions as an adsorbent (exhibiting retention orders similar to that found on alkyl-modified silica). Some reports of preparative separations on the XADs have appeared (13-15) but, in general, these have dealt with low column loadings and did not focus on general column or elution variables.
EXPERIMENTAL SECTION Reagents. The organic compounds were obtained from Eastman Organic Chemicals and Matheson, Coleman and Bell (MCB). Amino acids and dipeptides were obtained from Sigma. Benzene, methanol, 95% ethanol, and LC grade acetonitrile were obtained from MCB. Amberlite XAD-4 was purchased as 20-50 mesh beads from Mallinckrodt. Column Preparation. Bulk XAD-4 was batch cleaned by successive overnight Soxhlet extractionswith methanol, benzene, and three separate portions of methanol. The cleaned beads were filtered and dried in a vacuum oven at 45 O C for at least 4 h. They were then wetted with 1:l EtOH/H20 and the resulting slurry was ground in an Osterizer blender. The pulverized slurry was wet sieved by hand on a stack of nine U.S. Standard sieves ranging from 80 to 400 mesh with 1:9 EtOH/H20 as the wash solution. Particles which did not pass through the 80 mesh sieve were reground. Each fraction was rinsed into a beaker containing 1:l EtOH/H20. The slurry was stirred vigorously and placed in an ultrasonic bath for several minutes to disperse aggregates followed by settling for 3-5 min. Fines, which did not settle during this period, were removed by suction with a disposable pipet attached to a water aspirator. The settled material was reslurried by addition of water (about one-fourth the volume of the slurry), stirring, and ultrasonication. The slurry was allowed to settle as before and fines were removed. This process was repeated until each fraction was shown to be free of f i e s by examination with a 100 X American Optical microscope. Stainless steel tubing (3/8 in. 0.d. x 8.0 mm i.d. and 1.0 in. 0.d. X 20.5 mm i.d.) was cut to the appropriate length and cleaned with soap and water, acetone, chloroform, and acetone. End fittings were obtained from Parker H d i (3/8 in. X in. with 5-pm frits) and Waters (1 in. with a 10-pm frit). The inlet was a Swagelok cap fitted with a silver soldered 1/18 in. 0.d. tube. The 8.0 mm i.d. X 25.0 cm columns were slurry packed in an upward direction using a custom-built high-preasure slurry packer. Slurry concentrations of XAD-4 were 10-15% (wt/v) in degassed 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER -__-
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Table I. Comparison of Packing Consistency of XAD-4 Columns VO
(void a vol
column dimensions i.d., o.d., length, weight of mm in. cm XAD-4, g 2.36' 8.00
20.5
318
30.0 25.0
1
32.0
118
0.46 4.4 40
Ve
XAD-4 particle size,Nm 45-65 37-44 75-105
(empty packing tube vol), density, mL g/mL 1.3 12.5 108
0.35 0.35
0.37
packed col% filled umn), 100 ( V emL Vo)/Ve 0.76 8.3
41 34
70
35
specific permeability, c m ~
obsdb
predicted
1.5 X
1.6 X e 1.5 X 1 0 - I 0 f
1.6 X 10.' 8.1 X
lo-'
*
See ref 11 for calculation method. All values calculated for 1:4 a Measured using NaNO, or lycine peak. 5.6 mL/min, P = 20 atm. e 28.0 mL/min, P = 94 atm. 11.0 mL/min, P = 51 atm. EtOH/H,O. From ref 10. 1:l to 1:9 EtOH/H20 and the initial slurry flow rate during packing was 5 hL/min. When the tube was beginning to pack the flow rate was increased to 22 mL/min. At least 20 tube volumes (- 250 mL) were pumped during the packing process. If dead space appeared at the column inlet, it was filled with small portions of wetted particles. The 20.5 mm i.d. X 32 cm column was dry packed by filling it to about 95% capacity and then slowly wetting it by pumping 95% EtOH at 0.5 mL/min into the column. The bed was allowed to settle overnight and slurried XAD-4 was added to fill any remaining dead space. The XAD-4 bed at the column inlet end was covered with a piece of Whatman No. 35 filter paper cut to fit into the Swagelok inlet cap. Column conditioning was done by pumping 500 mL of 1:l EtOH/H20 followed by 500 mL of 1:4 EtOH/H20 through the column. If there was still a void space, it was filed with XAD-4 slurry. Because of the possibility of swelling, columns were always equilibrated with at least 10 column volumes of mobile phase before they were used. If the peak for NaN03 or glycine (unretained solutes) showed unusual tailing, the bed at the inlet was examined for a void space and, if present, it was filled with XAD-4 particles. Instrumentation. Modular LC instrumentation was used. Components consisted of a 4-L glass solvent reservoir with a magnetic stirrer, an Altex Model 100 dual piston pump with preparative heads and pistons (28 mL/min max.), a Rheodyne 905-19 injector fitted with either an Altex 1.1-mL sample loop or a 10.2-mL loop constructed from 25 ft of 0.05 in. i.d. X in. 0.d. stainless steel tubing, a Beckman DBG dual-beam spectrophotomer with a 1-cm pathlength 0.5-mL flow cell, and an Altex Model 153 detector with either a 1-cm,8pL flow cell or a 0.5-mm, 2-pl flow cell. Analytical HPLC was performed on a Waters Model 202 instrument with a 30 cm X 4.6 mm i.d. Waters C18p-Bondapak 10-pm column. Hamilton syringes were used to inject samples. Column dead volume was determined by using samples (NaN03 or glycine) that were not retained at the mobile phase conditions being used.
RESULTS AND DISCUSSION A number of factors must be evaluated in designing an LC system for the preparative separation of milligram to multigram mixtures. For example, the operating conditions, such as choice of stationary phase, particle size, flow rate, and mobile phase composition, which are routinely of concern in ALC, must be adjusted appropriately. In addition, the sizes of the column (diameter and length) and the sample load (mass and volume) are increased to permit a maximum sample throughput that yields an acceptable recovery and purity. An understanding of how each of these variables affects the performance of a PLC system should in principle allow optimization of the variables for a given separation. Samples a n d Stationary Phase. The samples studied were chosen as test compounds because they exhibit a wide range of solubilities, they were readily available at high purity, their retention on XAD-4 or related copolymers had previously been characterized (9-12), they are easily detected, and their retention properties were such that the chromatographic variable being studied could be widely altered. Several different copolymers of styrene and divinylbenzene are available
?L24L-A // 1 I
40
80
I20
160
dp (urn)
Flgure 1. Effect of XAD-4 particle size on plate height for an 8 mm i.d. X 250 mm column.
and potentially useful as stationary phases in PLC. XAD-4 was chosen for the study because it has the largest surface area and porosity. Thus, it should provide the largest sample loading and maximum retention at a given condition. Column Design. Table I lists the specific permeability and packing density for the preparative level XAD-4 columns used in this study and for one XAD-4 analytical column. The packing density and specific permeability of these columns are similar despite the large differences in column diameter. This indicates that the packing arrangements within the preparative columns are reasonably reproducible and favorable for preparative applications. Baum et al. (11)has suggested that the observed permeabilities for XAD columns are lower than predicted values because the smallest of the irregularly shaped particles f i the channels between the larger particles. It should be noted that the flow rates and operating pressures needed to use the XAD-4 preparative columns in Table I are readily attained with standard LC pumps. Figure 1illustrates how XAD-4 particle size influences plate height for a 8.0 mm i.d. column. The particle size is a median value representative of the fraction collected by sieves. The upper and lower curves indicate the plate heights found for dry-packed and slurry-packed 8.0 mm i.d. columns, respectively. The sample was 3.5 mg of p-methylbenzenesulfonic acid injected in 10-pL volumes using a 1:95 95% EtOH/H20 mobile phase. Linear velocities were 0.23 f 0.04 cm s-l (a flow rate range of 5.6-8 mL/min) for all experiments except for the 40-pm particle size where the maximum flow rate available (28 mL/min) provided a linear velocity of 0.96 cm s-l. Inlet pressures never exceed 800 psi. Although not shown, similar results were found for other benzenesulfonic acid derivatives as samples. Slurry packed columns with the smallest particles (37-44 pm), lower curve in Figure 1,provided the best performance as expected and were similar to that provided by analytical columns (2.4 mm id.) of 45-65pm XAD-4 particles (IO). Plate heights of 0.084.2 cm were typically observed for the 8.0 mm i.d. column which corresponds to about 500-1200 plates/m.
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
An improvement in column performance would be realized by using smaller XAD particles with a narrow size range, but the 37-44-pm (325-400 mesh) particles represent a particle size that can be easily isolated in the laboratory. Analytical columns packed with 3.6-8.4-pm XAD-2 (11) and 10-pm spherical poly(styrene-diviny1benzene) (12) have been shown to provide plate numbers in excess of 15-20000 plates/m. The upper curve in Figure 1, which indicates a poorer column performance, was obtained by using a dry packed column. Efficiencies were about half that found for a slurry-packed column. However, they are still high enough so that the column, and the convenience of dry packing, can be used in many applications. The effect of XAD-4 particle size was not studied in detail with the 20.5 mm i.d. column. For 75-105-pm particles and linear velocities of 0.38 cm s-l, efficiencies on the 20.5-mm column were nearly identical with the 8.0 mm column. Furthermore, efficiencies for the 20.5 mm i.d. column could by reasonably predicted by infinite diameter considerations (16). Thus, separations, which are successfully achieved on the 8.0 mm i.d. column, may be directly scaled up for higher loadings to the 20.5 mm i.d. column. Since the efficiencies are nearly the same for the two columns, the scale up factor for equivalent performance is given by the ratio of the two column packing weights or 8.4. Flow Rate. Preparative scale separations are designed to provide maximum throughput (mass/time) with acceptable recovery and purity of the collected material. Optimization of throughput requires the use of the highest flow rate which yields a level of efficiency sufficient to satisfy the recovery and purity requirements. The relationship between efficiency or capacity factor and flow rate (linear velocity for silica and alkyl-modified silica columns) has been reviewed in detail (2). For the 8.0 mm i.d. XAD-4 column it was shown that flow rate did not significantly influence capacity factor or efficiency when using a mass overload over the flow rate range, 1.68-11.2 mL/min (0.0834.56 cm s-l linear velocity), studied. In these experiments p-chlorobenzenesulfonic acid was the sample and was used at a mass overload condition of 2.3% wt/wt (a 1-mL injection of a 102 mg/mL sample) with a 1:9 95% EtOH/H20 mobile phase. At an analytical loading a typical Van Deemter plot was observed. Similar results were found for other benzenesulfonic acids as samples at concentration at and below mass overload conditions. The higher the linear velocity the greater the throughput is through a given column. For the p-chlorobenzenesulfonic acid sample used in the above study at 0.42 cm s-l (8.4 mL/min and a void volume of 8.4 mL), it was shown that on the average throughput corresponded to about 24 mg/min for collection of the sulfonic acid from the beginning of the peak to 10% of the peak maximum on the tail. At the experimental conditions (It' = 1.2) the peak geometry was defined by a bandwidth of 0.6 min at half-height, 2.7 min width a t 10% height, and skewed with a tailing factor of about 3 due to the mass overload. Although throughput can be increased by further increase in linear velocity, it also results in an increase in peak broadening. Column Overload. Increasing the sample load delivered to the column will also improve throughput. However, it is necessary to overload only to the point where isolated sample purity and recovery requirements are met. Column overloading may occur in two ways: (1) dilute solutions are injected onto the column in large volumes (volume overload) or (2) small volumes of concentrated samples are injected (mass overload). A special w e of mass overloading can occur when the sample is injected as a solution in which the sample solvent has a much weaker eluting strength than the mobile phase (17). In this case the stationary phase at the head of the
1'
io 2
4
6
mL
8
1
0
Injected
Flgure 2. Effect of injection volume (volume overload) on half-height peak width and apparent capacity factor: (A) 8.0 mm i.d. X 25 cm, 63-74 p m X A D 4 column, a 1:9 9 5 % EtOH/H20, pH 7.0, 0.02 M phosphate buffer mcbk phase, a flow rate of 5.6 mUmin, and injection of 5.5 mg/mL oc-leu; (B) 20.5 mm i.d. X 32 cm, 75-105 p m XAD-4 column, an aqueous pH 9.1, 0.02 M NaHfl, buffer mobile phase, a flow rate of 22 mL/min, and injection of 50.6 mg/mL &Val.
column becomes locally overloaded as the plug of sample solvent passes through the column. Volume Overload. The effect of volume overload on peak shape has been widely studied by using silica-based preparative columns (2,3,17,18). In general, an evaluation of the effect of injection volume on both the 8.0 mm and 20.5 mm i.d. XAD-4 columns indicated trends that are in agreement with the silica column results. Several different samples were used to evaluate this. In all cases sample concentrations were kept low to prevent mass overloading and the mobile phase conditions were purposely chosen to keep retention times short. The injection volume range studies was from 1 to 10 mL. It is clear that the peak front from the two XAD-4 columns (experimental conditions are listed in Figure 2) remains in the same position on the chromatogram regardless of the volume injected and that the tail of the peak has the same shape but is displaced a t its base further down the chromatogram as injection volume is increased. Figure 2 compares how the half-height peak widths (Wl/J and observed capacity factor for the peak changes as the injection volume is increased on the 8 and 20.5 mm i.d. column. It should be noted that the former column is affected to a greater extent than for the latter column over the range of injection volumes studied. For the 20.5 mm i.d. column, neither W1,, or peak maximum is significantly affected until the larger injection volumes are reached or a t the range where the injection volume is about 10% of the column dead volume (see Table I for typical column dead volumes). For the 8.0 mm i.d. column peak maximum increases markedly while Wl12 increases modestly above a 4-mL injection. For this column 10% of the column dead volume occurs a t about 0.8 mL. It has been suggested that injection volumes on silica preparative columns should not exceed 10% of the column dead volume if volume broadening effects are to be prevented (19); this general guideline also applies to the XAD-4 columns. Mass Overload. Introduction of a concentrated sample produces a localized saturation of the stationary phase at the head of the column. As sample concentration is increased, mass overloading occurs and efficiency is lost. Also, peak geometry changes in that peak tailing increases and, usually, peak maximum shifts toward the origin. Figure 3 depicts the decrease in peak maximum and in efficiency that occurs on an 8.0 mm i.d. XAD-4 column as sample concentration was increased for a fixed injection volume maintained a t the volume overload limit. Defining the mass overload quantitatively for a given column and eluting condition is very complicated since the operating conditions are such that retention isotherms are no longer linear and a large number of sorption sites are deactivated. Thus, operational definitions (2, 3,20) have generally been
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
Flgure 4.
Effect of sample loading (mass overload) on plate number and capacity factor. Conditions: 8.0 rhm i.d. X 25 cm, 37-44 pm XAD4 column, a 1:9 95% EKH-(IH,O mobile phase, a flow rate of 5.6 mL/min, and 1.O-mL injections of p-chlorobenzenesulfonic acid. Figure 3.
Table 11. Effect ofGading on a 20.5 mm i.d. XAD-4 Column”
sample, mg/mL
k’
No.5
% wt/wt load
30 50 100 200 300 400 500
1.04 0.93 0.87 0.79 0.76 0.69 0.69
290 285 267 246 193 170 177
0.08 0.13 0.25 0.50 0.75 1.00 1.25
Illustration of a combined mass/volume overload. Condi-
tions: 8.0 mm i.d. X 25 cm, 37-44 pm XAD-4 column, an aqueous moMle phase, a flow rate of 5.6 M m i n , and lnjectkm of three Merent
benzenesulfonlc acM concentrations. ~~~~~
~
Table 111. Results of Combined Mass/Volume Study on a 20.5 mm i.d. XAD-4 Column’ mL sample, injected mg/mL k’ Noesb Noelc A,.,C 0.5 1.0 3.0 6.0 10.2 0.5 1.0 3.0 6.0 10.2
20.5 mm i.d. x 320 mm, 75-105 I.rm XAD-4, 1-mL injections, 1:9 95% EtOH/H,O, and 22 mL/min flow rate for the sample p-toluenesulfonic acid.
used to define mass overload. Thus, the overload has been suggested to occur when the plate number, peak width, or capacity factor decreases by a factor of 10%. In Figure 3, the mass overload corresponds to a p-chlorobenzenesulfonic acid concentration of 15 mg/mL or 0.34% wt/wt column loading using a 10% decrease in plate number as the definition of mass overload. However, it is still important to note that even at 80 mg/mL, or at about 1.8% wt/wt loading, the column still provides 100 plates. Thus, prep-level separations on the XAD-4 are possible at even higher overloads where selectivity is large and many plates are not needed for the separation. The 20.5-mm column exhibits the same general trends in performance when mass overloading occurs. Data illwtrating this are listed in Table I1 and show that the mass overload occurs at approximately 100 mg/mL or a 0.25% (wt/wt) load. However, many plates are still available at even 5 times this mass overload limit and preparative separations in excess of 100 mg are possible on the 20.5 mm i.d. column. For example, two- and three-component mixtures of benzenesulfonic acids at 2.5-3 g total mass have been successfully separated in a single pass with only modest crossover. The theoretical maximum loading for the 8.0 mm i.d. column was estimated by considering the weight and surface area of the stationary phase and approximating the number and area of the adsorption sites. For the compound used in Figure 3, where the k’is 1.6 and only one-tenth of the column dead volume is used for sample injeetion, the calculated maximum loading was found to be about 21 mg/mL vs. about 15 mg/mL by experiment. A similar favorable comparison, considering the approximations used, was found for the 20.5 mm i.d. column. The mass overload limit on the XAD-4 occurs at about the same level on a weight-to-weight basis for compounds with approximately the same k’. Also, the overload limit in many cases will decrease as retention increases while in others the lower capacity is characteristic of the sample. This has been observed on alkyl-modified silica coluinns (21). In general the eluting conditions should be adjusted so k’ < 10 in order to obtain a reasonable column overload. For example, benzoic
1825
52.8 52.8 52.8 52.8 52.8 528 528 528 528 528
137 110 120 110 90
161 145 148 148 111
0.8 0.9 1.0 1.0 0.8
1.45 1.34 0.85 0.61 0.34
85 77 44 33 23
114 105 40 24 16
0.8 0.8 0.4 0.3 0.2
32 cm column packed with 75-105 at 22 mL/min for the sample p-toluenesulfonic acid. Calculated from half-height. C Calculated at 10%of peak height by A. = peak taillpeak front. a
20.5 mm i.d.
1.37 1.37 1.52 1.52 1.52
X
pm XAD-4 1:9 95% EtOH/H,O
acid has a k’ of 8.5 in 1:l 95% EtOH/H20 on the 8.0-mm XAD-4 column and mass overloads at above 4 mg/mL. A combined mass and volume overload effect is shown in Figure 4 for the 8.0 mm i.d. column. The volume overload effect begins to be visible at about 10% of the dead volume or a t 0.83 mL. Thus, for the 4.0 mg/mL sample the mass overload limit is not reached and only volume overloading occurs producing a shift in peak maximum to longer apparent retention times when the injection volume exceeds 1 mL. When the sample exceeds the mass overload limit, for example, at small volumes of the 40 and 410 mg/mL samples, the peak maximum shifts markedly toward the origin as injection volume increases up to about 1mL. Larger injections of these two samples give peak maxima at about the same position because the mass and volume overload effects cancel. The very large shift in k’ with even small increases in injection volume for the 410 mg/mL sample indicates that a grossly overloaded column becomes less useful since the k’value drops sharply. It should be noted, however, that the sharp decrease in k‘ occurs when the overload is about 40 times the mass overload. Similar behavior was observed on the 20.5 mm i.d. XAD-4 column. Since the mass loading limit is larger, weight levels 10 times that for the 8.0 mm i.d. column were used. Table I11 lists the apparent k’values, efficiencies calculated by two methods, and the tailing factor for two series of injections at two concehtrations. As expected this column is less sensitive to mass/volume overload than found for the 8.0 mm i.d. column. For the 52.8 mg/mL sample, which is just at the m a s overload, retention and peak shape undergo little change over the volume range injected. For the 528 mg/mL sample, which represents a mass overload, retention and plate number decrease rapidly at a gross overload corresponding to about 5-10 times the mass overload and is accompanied by an increase
1826
ANALYTICAL CHEMISTRY, VOL. 53,NO. 12, OCTOBER 1981
I
iI
li n
b-20min4
I
Figure 5. Separation of p-toluenesulfonlcacid at three concentration ratios. Condttions: 20.5 mm i.d. X 32 cm, 75-105 pm XAD-4 XAD-4 column, a 1:4 95% EtOH/H,O mobile phase, a 16.8 mL/mln flow rate, a 1mL Injection of a solution where the total mass Is 500 mg/mL, and a V , = 70.5 mL.
in peak tailing. The effect on peak shape is particularly evident when comparing plate number calculated at 10% peak height, this has been previously shown to be a more sensitive indicator of column performance (22). Purification. Figure 5 shows the separation (R * 1)of p-toluenesulfonic acid (k’ = 1.0) from 2,5-dichlorobenzenesulfonic acid ( k ’ = 2.5) at weight ratios of 40:1, 1:1, and 1:40. This demonstrates that purifications on the XAD-4 are possible a t a significant overload and that both minor and major components can be isolated from the mixture. The total mass, 500 mg or a 1.25% wt/wt load, and sample volume were constant for the three separations. Thus, the major component in the 4 0 1 and 1:40 mixtures and both components in the 1:l mixture were mass overloaded. The percent recovery and purity were established by collecting fractions at 1-min intervals (16.8 mL) and determining their composition by ALC; these results are shown in Figure 6. In the 40:l mixture only one peak is observed due to detector saturation. However, the peak front is p-toluenesulfonic acid and 95% of it may be collected at greater than 99% purity. The trailing edge contains the dichloro compound and was recovered at about 70% purity because mass overloading of the p-toluenesulfonic acid causes it to tail and overlap into the dichloro peak. The 1/40 mixture was separated with a recovery of about 90% and 95% of the p-toluenesulfonic acid and dichloro acid, respectively, at a greater than 99% purity. A gross mass overloading of the more highly retained compound apparently causes the column to approach displacement chromatographic conditions and facilitates the elution of the first compound. There is considerably more overlap in the 1/1 chromatogram due to both components being at a mass overload. About 80% of the two compounds were collected at greater than 99% purity. The overlap region which contains about 20% of the total mass injected, although not done, could be concentrated and recycled. Mobile Phase Variables. The use of XAD-4 and related copolymers as stationary phases in ALC has been studied extensively and these data provide a basis for suggesting the eluting conditions for the PLC of many different types of mixtures (9-12). Parameters that can be varied are pH, organic solvent-water ratio, type of organic solvent, ionic strength, and type of organic counterions (pairing ions). Although adding inorganic buffers and salts to the mobile phase can complicate recovery of the separated components, often the salts can be easily removed by passing the collected fraction through a XAD-4 column using a mobile phase solvent mixture where the sample has retention. In contrast, removal
Mole percent of p-toluenesulfonic acid and 2,5dichlorobenzenesulfonic acid for 16.8-mL fractions collected from the separations in Figure 7. Analytical column conditions: A 4.6 mm i.d. X 30 cm, 10 pm, Waters C,, p-Bondapak column, a 1:4 EtOH/H20, pH 5.8, 0.1 M NaCI, 0.02 M phosphate buffer mobile phase, a flow rate of 1.0 mL, and a 10-25-~Linjection. Figure 6.
of organic counterions is more difficult and these would be used in PLC only when other suitable eluting conditions are not readily obtained. The eluting power of organic solvents on XAD-4 is similar to that observed on other reversed stationary phases such as alkyl-modified silica. However, the stability of XAD-4 toward the entire pH range, unlike silica-based stationary phases which are generally useful only from pH 2 to 8, offers several advantages in PLC. First, the selectivity (a)for a given pair of organic acids, bases, or ampholytes changes with pH. Thus, adjustment of the mobile phase pH to make a large will also permit a higher loading of the column. Second, elution orders for many ampholytes are reversed when comparing retention between an acidic and basic mobile phase. Third, the addition of buffer salts will often sharpen chromatographic peak shapes (9,10,23). Fourth, the option is available to use a basic mobile phase to separate weak organic acids in their ionized form or modestly strong organic bases in their neutral form or to use an acidic mobile phase to separate weak organic bases in their ionized form and modestly strong organic acids in their neutral form. Ampholytes can be separated as cations, neutral forms (or zwitterions), or anions. Adding inorganic salts to the mobile phase reduces bandwidth and increases retention on analytical XAD-4 columns (9, 10, 23). In PLC it was found that as column loading increased retention decreased despite the presence of added salt (0.1 M NaC1). Thus, a t large mass overloads retention was virtually the same for mobile phases with or without salt. In contrast, at the mass overload or below it the added salt increased retention significantly. Several different mixtures were separated on the 8.0 mm and 20.5 mm i.d. XAD-4 column to illustrate the versatility of these columns and eluting conditions, In most cases the columns were not mass or volume overloaded and often larger quantities could be separated. A large mass overload, however, is not possible in all cases because of limited sample solubility in the typical mobile phases used in reversed-phase LC. This has also been observed on alkyl-modified silica columns (Y-3). Thus, the limiting factor determining column loading is often sample solubility and not the column or mobile phase conditions. Mixed Solvent. The mixture listed in Figure 5 was separated at a 2:1 p-methykdichloro ratio where the total mass was 0.86 g (2.2% wt/wt load) and 2.40 g (6.0% wt/wt load) on the 20.5 mm i.d. XAD-4 column using a 1:9 95% EtOH/H20 mobile phase a t 22 mL/min. This is a weaker
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981 amino acids
a AI0mg/mL 21 b Ile 7 3 c T y r 01
%T
8
'
'J
'
1827
A
' I C
B J 1' l5min
2 rnL injection
l5mm 0 05 mL injection
Figure 7. PLC separation of polynuclear aromatics. The column in Figure 3 was used with a 2:'l 95% EtOH/CH3CN mobile phase for A and a 5 1 9 5 % EtOH/CH,CN mobile phase for B with both at a flow rate of 5.6 mL/min.
Figure 8. PLC separation of a mixture of three amino adds at two different loadings. The column in Figure 3 was used with a 0.02 M H3P04 at two different loadings.
eluent than used in Figure 5 and, thus, a greater mass loading is possible. The Fz'values at analytical concentrations are 1.2 and 2.8, respectively, and a base line separation was obtained even at higher total mas8 loadings. Both components were grossly mass overloaded even for the lower weight sample. Other three- and four-component benzenesulfonic acid mixtures were separated on the 8.0 and 20.5 mm i.d. column at total mass loadings corresponding to 2-3% wt/wt. The XAD-4 columns are readily used with mobile phases containing large amounts of organic solvent. For best column performance the transition from a mobile phase that is largely H 2 0 to one that is largely organic solvent or vice versa should be done gradually. The retention order for chlorinated phenoxyacetic acids in EtOH/H20 mixtures is 2,4,5-trichloro- > 2,4-dichloro- > p-chloro- > o-chlarophenoxyacetic acid (9).In scaling up the analytical separations to a preparative level on the 8.0 mm i.d. column, only the ortho/para mixture was difficult to resolve; all other combinations were readily resolved at column loadings below and in small excess of the mass overload conditions. For the ortho/para mixture crossover occurs but significant enrichment for each is achieved in a single pass through the column even at 50 mg total load or about 5 times the mass overload. Also, collection of the leading edge of the ortho peak and trailing edge of the para peak provides each in good purity. A mobile phase of 1:195% EtOH/H20 at a flow rate of 5.6 mL/min was used for this separation (126 = 5.0 and k k = 6.7). A 3:2 95% EtOH/H20 mobile phase at 5.6 mL/min was used to separate a mixture of aniline (21 mg, k'= 1.6), o-chloro(82 mg, k'= 3.6), and 2,4-dichloroaniline (57 mg, k'= 7.8) on the 8.0-mm column. The first is just above the mass overload while the latter two are grossly mass overloaded (3.6% wt/wt for the total sample). Resolution corresponded to R = 0.95 for adjacent peaks. Figure 7 illustrates the preparative separation of two different polynuclear aromatic (PNA) mixtures on the 8.0 mm i.d. column using a mobile phase that is almost free of H20. The introduction of large quantities of PNAs into the column is difficult because of their limited solubility in common reversed-phase eluants. This was overcome by using benzene or indane as the sample solvent. Since both of these are strong eluting agents relative to the EtOH-CH&N mixtures used as the mobile phases, the PNA peaks are broadened by the movement of the benzene or indane through the column. Thus, the extent of the broadening will be dependent on the sample volume injected and the mismatch between the eluting power of the mobile phase and sample solvent. The broadening effect was minimized by dissolving the PNA sample in 5 mL of benzene (Figure 7A) or indane (Figure 7B) and diluting it to 50 mL with 95% EtOH. An aliquot of each was
then injected into the column and the weight of benzene or indane corresponds to the amount present in the samplle solution. In Figure 7 benzene, indane, and naphthalene are mass overloaded while the rest are below a mass overload. The total mass loadings in Figure 7 are 4.5% and 1.4% wt/wt, respectively. pH Control, Several separations were performed to illustrate the advantage of pH control to improve resolution and the use of the XAD-4 column at mobile phase pH conditions where silica or alkyl-modified silica cannot be used. In these experiments no column deterioration was found 81s the result of using either an acidic or basic mobile phase. In general, the optirnum eluting condition for the separation of amino acids and peptides on reversed stationary phases is to use either an acidic or basic mobile phase (10,12);alkylmodified silica, however, is limited to a mobile pH range of 2-8. Figure 8 shows the preparative level separation of two Ala-Ile-Tyr (k' = 2.4,4.6, and 8.2, respectively) mixtures a t a ratio of 210731 on n 8.0-mm column using an acidic mobile phase. If a basic mobile phase (pH 12) is used, Ala and Ile are readily separated. But, because of the additional acidic site on the side chain of Tyr, its retention is very low and the Tyr peak will appear with the Ala peak (k'w< kil, at pII 12). Thus, by using pH control the elution order of Ile-Tyr can be reversed; in acid Ile elutes first while in base Tyr elutes first (10, 12). In the mixtures in Figure 8 only Ala approaches the mass overload. Resolution for the 7.1 mg (0.18% wt/wt load) sample is R b . n e = 1.0 and R g l e .=~0.95, while for thle 14.2 mg (0.36% wt/wt load) sample, R was 0.94 and 0.71, respectively. Figure 8 also illustrates an example of the separation of a minor component since Tyr is only 0.35% by weight of the total sample. The fact that its peak has the largest area is due to ik3 comparatively large molar absorptivit:y at the detection wavelength. Dipeptides are similar to amino acids in their retention behavior on XAD-4 as a function of pH. However, the effect of side chain structure on retention is more significant; ALC data for dipeptides and other peptides on XAD columns are provided elsewhere and serve as a basis for predicting many PLC separations (10, 12). Figure 9 illustrates a special case of dipeptide separation; namely, the separation of dipeptide diasteromers. The D,D-L,L mixture is less retained (k' = 2.0:) than the D,L-L,D (k' = 3.3) mixture; this elution order can be explained by considering the orientation of the side chain within the diastereomers (12).Either an acidic or basic eluting condition can be used; the former provides a more favorable resolution for this sample. If a lower load (5.5 mg) and flow rate (2.8 mL/min) are used, the separation is close to being: a base line separation. Because retention is very low a t the eluting condition used.,larger mass loadings are possible, but at lower resolution (far example, R changes from about 1.3
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
1
dipeptide dimtereomers
DL-bla-DL-Ala 22 m g h L a
also found for the separation of phenoxyacetic acid and aniline mixtures from basic and acidic mobile phases where they are anions and cations, respectively.
SUMMARY
b
a D(L)-AIa-LXL)-4a b D(L)-Alo-L(D)-Ala %T
JLJb 1.0 m i injected
2.2 rnL injec
Bmdn
Figure 9. PLC separatlon of a diastereomeric dipeptide sample. Conditions were the same as in Figure 8.
b o-isopropyl00488g
c o,o-diisopropyl0 0473 g
*d
PLC columns packed with 37-44 pm XAD-4 particles generate efficiencies of 500-1500 plates/m depending on the sample and mobile phase and in general provide good column performance over a wide range of flow rates and mobile phase conditions. If better efficiencies (similar to alkyl-modified silica) are required microparticle (
