Anal. Chem. 1995,67,3456-3463
Comprehensive Three-Dimensional Separation of Peptides Using Size Exclusion Chromatography/ Reversed Phase Liquid Chromatography/Optically Gated Capillary Zone Electrophoresis Alvin W. Moore, Jr., and James W. Jorgenson* CB 3290, Department of Chemistty, Univetsi@of North Carolins-Chapel Hill, Chapel Hill, North Carolina 27599
Multidimensional separation methods are attractive because of their potentially high peak capacities. Coupledcolumn systems in particular offer the advantages of online detection and automation. With the development of rapid two-dimensional(2D) analysis, it becomes possible to consider three-dimensional (3D) separation systems. In such a 3D system, effluent from a slow first dimension is repetitively sampled into a rapid 2D system. In the resultant data, each sample component has been subjected to three separative displacements, and the overall peak capacity is the product of that of each of the three dimensions. This paper demonstrates a comprehensive coupled-column3D analysis of peptides. Size exclusion chromatography (SEC) is used as the first dimension to separate sample components by molecular weight, over an analysis time of several hours. The SEC effluent is repetitively sampled on-line into a rapid 2D reversed phase liquid chromatography/capillary zone electrophoresis (CZE) system with an analysis time of 7 min. Detection of sample zones is done only after the final CZE separation, by laser-inducedfluorescence detecton. Analysis data from this system consist of a series of 2D “slices” of the SEC effluent, which when stacked together give the 3D separation “volume”. In the previous paper, we described a method of doing rapid comprehensive two-dimensional (2D) analysis of peptides. This system used a coupled-column approach to link reversed phase liquid chromatography (RPLC) with optically gated capillary zone electrophoresis (CZE). In a coupledcolumn 2D system, effluent from the first column is repetitively sampled into the second, with detection usually only after the second dimension. If it is a comprehensive system,’ the entire effluent from the first column is sampled into the second. This is in contrast to a heart-cutting technique, in which only small regions of interest from the first separation are sampled into the second. For a comprehensive coupled-column system, each entire analysis in the second dimension yields only a single “point” in the first. Thus, if possible, it is advantageous to have the analysis time of the second method be much shorter than the kst, so that the separation in the first dimension can be well sampled. This restriction usually results in overall analysis times of several hours for a 2D method. However, optically gated CZE makes possible rapid 2D analyses, with total analysis times of 5 min or (1) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62,978-984.
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less. Because these 2D analyses can be done in only a few minutes, it becomes possible to consider comprehensive threedimensional (3D) separations. A coupled-column 3D system could be constructed by repetitively sampling effluent from a first dimension column into a subsequent two-dimensional system. If the entire effluent of the first column is sampled into the 2D method, the 3D method is likewise termed comprehensive. In a sequential 3D system of this kind, the separation data will consist of a series of 2D analyses of samples of the first column effluent. These 2D “slices” can then be “stacked together to give a 3D separation “volume”in which each of the three axes represents a different separation method. The motivation for development of 2D systems is the large gain in peak capacity of the 2D method over its component onedimensional (1D) methods. Giddings has shown that the peak capacity of the 2D method is roughly the product of the peak capacities of the component 1D methodsS2In the same work, he showed that this applies for a multidimensional system of any number of dimensions, n. Thus a 3D system is of interest because the total peak capacity will be roughly the product of all three component dimensions. Since the peak capacity of a 2D method is inherently high, even a modest peak capacity in the first dimension will make a significant difference in the peak capacity of the overall 3D method. Giddings has reported a personal communication from J. F. K. Huber of a three-dimensional ~eparation.~ This method relied on coupling three chromatographic columns in a 3D analysis requiring 102 h with over 6000 peaks. From the description, the method involved some heart-cutting of particular peaks and was not a comprehensive approach. In this paper, we demonstrate a comprehensive coupled-column 3D system. A size exclusion chromatography (SEC) column is used for the first dimension, to give a rough separation of peptides based on their molecular size. Effluent from this column is then sampled into a 2D RPK/ CZE system described in the previous paper. This system further separates the components based on their hydrophobicity and electrophoretic mobility. Detection of sample zones is done only at the end of the CZE analysis, after they have passed through all three separation modes. (2) Giddings, J . C. HRC CC,j.High Resolut. Chromatogr. Chromafogr.Commun. 1987.10,319-323. (3) Giddings, J. C. In Multidimensional Chromatography; Cortes, H. J., Ed.; Marcel Dekker, Inc.: New York, 1990; pp 1-28.
0003-270019510367-3456$9.00/0 0 1995 American Chemical Society
Fast-CZE I (2SeC I analysla I (LaM Po.ltlo")
Flgun 1. Schematic diagram of 3D SEC/RPLC/fast-CZE instrument. See text for explanation
EXPERIMENTAL SECTION
Samples and Reagents. Only samples and reagents specific to this experiment are listed here. All components in common with the previous paper are as described therein. Hen ovalbumin was purchased from S i a . Ammonium acetate and boric acid were purchase from EM Science (Gibbstown, NJ). Methanol (Fisher Optima grade) was purchased from Fisher Scientific pair Lawn,NJ). Digest Conditions. A 10 mg/mL solution of hen ovalbumin was prepared in 100 mM borate buffer, pH 8.5. This solution was heated in a boiling water bath for 5 min to denature the protein. A 37 mg/mL solution of trypsin was prepared in the same borate buffer, and 5 pL of this solution was added to 0.75 mL of the albumin solution. The mixture was allowed to react for 24 h at 37 "C, after which it was stored in the freezer. The ratio of ovalbumin to trypsin (w/w) in this solution was 411. Thirty-five peptide fr'agments of ovalbumin were expected in the digest if digestion was complete.' Tagging Conditions. Fluorescein isothiocyanate (FITC) was dissolved in 955 (v/v) acetone/pyridine solution. A 65pL sample of this solution was added to 300 pL of digest solution, and the mixture was allowed to react in darkness for 24 h at room temperature. The concentration of the FTK solution was adjusted to give a Sfold molar excess of tryptic peptides over FITC in the sample. The final concentration of FITC-tagged components in tbis sample was 2.2 mM. The tagged digest sample was diluted 150 into CZE buffer before analysis by 2D RPLC/fast-CZE. The tagged digest sample was diluted 1:lO in SEC mobile phase before analysis by SEC alone (with W absorbance detection, for method development) or by 3D SEC/WLC/fast-CZE. Instrumentation. The 3D instrument is diagrammed in F i r e 1. Reading from the left, the sample is injected onto the SEC column and separated by size,passes through into an SEC/ RPLC interface in the center of the F i r e , and then into the 2D RPLC/fast-CZE system on the right. The components of the individual separation systems will be described first, followed by a description of the interface between the SEC and RPLC systems. SEC. The SEC column was slurry packed in-house. A 4.6 mm i.d. x 25 cm column blank with O.5pm Kel-f encased frits (4) Nisbet, A D.; Saundry, R H.:Moir, A J. G.; Fotherdl. L A Fothergill,J. E. Biockem. J 1981, 115. 335-345.
(Alltech Associates Inc.. Deerfeld, IL) was packed5 with HEMA stationatyphase (5pm particles, lWA pores) (Alltech Associates). HEMA is a hydrophilic macroporous copolymer of Zhydroxyethyl methacrylate and ethylene dimethaaylate. For the SEC analysis, a 3WmL stainless steel solvent reservoir (Whitey Co., Highland Heights, OH) connected to a helinm gas cylinder was used as an isocratic solvent pump. Solvent flow rate through the column was controlled by varying the applied gas pressure. The SEC solvent was 85%methanol/water (v/v), with 100 mM h a 1 concentration of ammoninm acetate. This solvent was chosen on the basis of earlier work in our group6 to maximize ideal size exclusion behavior of the peptides and reduce other nonideal chemical interactions with the stationary phase. The solvent was filtered with O.Zpm syringe filters (Gelman, Inc.) before use, and a 0.5 pm precolumn in-line filter was inserted between the sample injection valve and the head of the SEC column. For the data shown here, the SEC solvent flow rate was 11pL/min, driven by helium pressure of 3.1 bar (45 psi). Sample was injected onto the SEC column with a six-port Rheodyne valve fitted with a 20. pL sample loop. Initial method development work was done with the SEC column outlet connected directly to a Hewlett-Packard (HP) 1050 wiablewavelength W absorbance detector. The absorbance was monitored at 488 nm to detect only the FlTGtagged components, with 2.0 absorbance units full scale (AUFS). RPLC. The RPLC column was a 5 cm x 2.1 mm id.. 5 p m particle diameter, 300b-CS column (RocklandTechnologies,Inc.). The elution gradient and the RPLC injection valve were the only other changes in the LC system from that described in the previous 2D paper. The LC pump flow rate was 250 pL/min. Solvent A was 10 mM sodinm phosphate buffer, pH 6.85. Solvent B was 40% acetonitrile/60% solvent A The gradient was from initial conditions of 15%B to 75%B over 3.5 min and then a return to initial conditions from 3.5 to 3.6 min. This gave a change in acetonitrile from 6 to 30%. The gradient time was shorter than that of the 5min gradient described in the previous paper to allow a faster overall LC cycle time without substantial loss of LC resolution. This allowed for more frequent sampling of the SEC effluent. The manual Rheodyne valve used for RPLC injections in the previous paper was used here for sample injection onto the SEC (5) Kennedy, R T.:lorpenson, 1.W. A n d Ckem. 1989, 61. 1128-1135. (6) Larmann, I. P.. Jr. Ph.D. University of North Carolina-Chapel Hill. 1993.
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column. Injections onto the RPLC column were done with an electrically actuated six-port valve (Valco Instrument Co. Inc., Houston, TX) fitted with a 2@pL sample loop. In Figure 1, the automated valve is shown in the load position, in which the sample loop was slowly filled with a diluted fraction of the SEC effluent over the course of a 2D analysis. The valve was controlled by a digital output line of the data acquisition board, through an interface constructed in-house. Fast-CZE. The fast-CZE system is described in detail in the previous paper and The CZE capillary was 8 cm x 6 pm i.d.,
[email protected]. with 1.7 cm between the gating and probe beams of the laser. The applied voltage was 20 kV to give an applied field of 2.5 kV/cm. Because the gating beam is the actual point of sample injection, and the probe beam is the point of sample detection, the voltage actually available for the separation is only the voltage between the beams or 4.25 kV. The gating and probe beams were generated by a 1-W Coherent Innova 70 argon ion laser (Coherent Inc., Palo Alto, CA) operated at 400 mW at 488 nm. The fast-CZE injection time was 15 ms. SEC/LC Interface. (a) SEC Flow Redudion. Under ideal circumstanceswith a coupledcolumn system, the geometry and flow rates of the SEC and RPLC columns would be matched. That is, the amount of effluent generated by the SEC system during the time of a RPLC analysis would be equal to the injection volume of the RPLC column. In this way, all of the SEC effluent would be sampled into the LC system. In practice, this can be done by using a smaller internal diameter first dimension column and a larger internal diameter second dimension column. Then the optimum flow rate for the first is significantly smaller than for the second, and the ideal described above can be realized. For example, a SEC column with a flow rate of 1pL/min might be connected to a RPLC column which could receive a D p L injection. Then RPLC analyses done every 10 min would inject the full effluent flow to the SEC column. Smaller bore SEC columns, both 1- and 2.1-mm i.d., were packed in our laboratory, but the measured separation efficiencies of these smaller columns were far less than that obtained with the 4.6"-i.d. column. Thus, the 4.h"mi.d. column was used. Because the SEC column used in this system was of larger i.d. than the RPLC column, there was a flow incompatibility. Even with low SEC flow rates, the amount of effluent generated during the course of a RPLC analysis was more than could be stored in a sample loop and injected into the RPLC column. Thus, a discrete sampling approach was used, in which the repetitive RPLC analyses sampled some fraction of the SEC effluent and in which some fraction of the SEC effluent is lost between RPLC analyses. Consider the case, for example, if the SEC flow rate was 10 pL/min, the RPLC injection volume was 20 pL, and the RPLC analysis time was 5 min. In 5 min, the SEC system generates 50 p L of effluent, but the LC system can only inject 20 pL. Thus 60%of the SEC effluent is lost. This is not a concern if the RPLC system can sample the SEC effluent rapidly relative to the SEC peak width, so that no SEC peaks will be missed in the SEC effluent lost between WLC samplings. A better arrangementuses a flow splitter in which a fraction of the SEC effluent is diverted to waste before the LC system. In the example above, a 4060 flow splitter between the SEC column and the WLC column would divert 6 pL/min to waste. Then only 4 pL/min would go to the (7) Monnig, C. A; Jorgenson, J. W. Anal. Chem. 1991, 63, 802-807. (8)Moore, A W., Jr.; Jorgenson. J. W. Anal. Chem. 1993,65, 3550-3560.
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LC sampling loop, or 20 pL in the 5-min RPLC analysis time. This method has the advantage that the entire SEC effluent is analyzed by RPLC (there are no "losf? fractions of the SEC effluent), but some of the sample is lost in the flow splitter. A flow-splitter arrangementsimilar to that described above was used to split off only a fraction of the SEC effluent for injection into the RPLC. The splitter itself was a in. stainless steel tee (Swagelok), with an extension 2 cm long brazed onto one side of the tee. The splitter capillaries were referred to as the LC split (33 cm x 50 pm id.) and the waste split (37 cm x 100 pm id.). As shown in Figure 1, the LC split capillary passes through the tee and extension and is positioned very near the inlet of the SEC effluent. Effluent from the SEC column flows into the tee and across the end of the capillary. A fraction of the SEC effluent flows into the LC split, while the remainder flows out the waste split. The measured flow rates were 10 pL/min for the waste split and 1pL/min for the LC split. (b) SEC Mobile Phase Dilution. The high methanol content of the SEC mobile phase made it difficult to directly inject a fraction of the SEC effluent into the RPLC system. The initial RPLC solvent contained only 6%acetonitrile,while the SEC solvent was 85%methanol. Injection of as little as 5 pL of sample in SEC solvent resulted in significant breakthrough of early-eluting peaks in the solvent front of the RPLC analysis. To circumvent this problem, the SEC mobile phase was diluted with CZE buffer before injection into the RPLC system. Tests were done with varying dilution factors and injection volumes to find the best compromise between them. The SEC effluent was diluted 1:l (v/v) with CZE buffer so that the organic concentration was 42.5% (with 10 pL injected) and 1:3 (v/v) so that the organic concentrationwas 21.25% (with 20 pL injected). It was found that the 1:l dilution gave the most breakthrough, and the 1:3 dilution the least. To dilute the SEC effluent on-line, a 25bpm-i.d. tee (Valco Instruments Co. Inc.) was connected to the LC split line. A syringe pump (SAGE Instruments, Orion Research Inc, Boston, MA) with a 5mL syringe was used to deliver 4 pL/min CZE buffer to the tee to dilute the SEC effluent. This 1:4 dilution gave an approximately 17%methanol concentration in the solution entering the sample loop, and 20 pL of this was injected onto the RPLC column. This gives a total flow into the RPLC sample loop of 5 pL/min. With a RPLC analysis time of 6.35 min, this is 31.75 pL. Because only 20 pL is injected in each RPLC analysis, the remaining 11.75pL is lost. Again this is not a significant problem if the RPLC analyses are rapid relative to the peak width of the SEC peaks. Though some of the SEC effluent is missed, it is does not result in SEC peaks totally missed by the RPLC sampling. Flow Rate Measurement. Flow rates through the splitter capillaries were measured using a piece of
[email protected] with two windows burned into the polyimide coating. The measured distance between the ends of the windows and the known capillary internal diameter give a known volume. The meniscus of the front of the flow of liquid through this capillary can be seen by the unaided eye, and the time taken to fill the region between the windows can be measured with a stopwatch. This time can be used to calculate a volumetric flow rate. The measurement capillary can be temporarily connected to the end of the splitter capillary with a Direct-ConnectCapillary Connector union (Alltech Associates, Inc), After each measurement the measuring capillary was blown out with a flow of gas.
RPLC/CZE Interface. The unique RPLC/fast-CZE interface is described in detail in the previous paper. RPLC effluent flows into a tee and across the end of the CZE capillary. A small fraction of the LC effluent is continuously electromigrated into the CZE capillary. No valve is needed, because the actual fast-CZE injections are done optically with the gating beam. Data Acquisition, Timing, and Instrument Control. The detection optics and electronics for fast-CZE are described in detail in the previous paper and elsewhere.7*8 Data acquisition and instrument control software was written in-house using LabVIEW 2 software (National Instruments, Inc., Austin, TX). Data acquisition was done with a multifunction A/D interface board (Model NB-MIO-16X, National Instruments) in a Macintosh I1 computer. Three-dimensional separations place unique demands on the data acquisition system and the instrument control mechanism. To acquire the 3D data set, a number of repetitive 2D “slices”of the SEC effluent were taken. As described in the previous paper, 2D data acquisition is done in double-buffered mode. Data are acquired in the foreground and written to disk in the background. While this method handles the large 2D data sets very well, it leaves the computer free to do nothing else. Thus, all instrument control functions must be accomplished between successive 2D data acquisitions. To do repetitive RPLC gradients and synchronize the gradients with the data acquisition, the start of each gradient was triggered by the computer. To control the HP1050 pump with the computer, two digital input/output (I/O) lines on the data acquisition board were connected through a custom-made cable to the pump’s ninepin Analytical Products Group (APG) interface. This is the same interface used for local communication between individual HewlettPackard HPLC system components bumps, detectors, autosamplers) to signal analysis start and stop and error conditions. A 16@mspulse from high to low on one line started the gradient, while a similar pulse on the other line stopped the gradient. The actual gradient elution parameters were programmed into the pump through the pump front panel, not through the computer. The actual gradient covered only 3.6 min. After this time, the pump had returned to initial conditions and the column was reequilibrating. The gradient end time in the pump was set to 11 min, but the 2D analysis time was only 6.35 min. Thus, the pump waited at the initial conditions of the gradient for each start signal from the computer. The computer initially started the gradient in the pump and then stopped and restarted the gradient at the beginning of each new 2D data acquisition. The automated injection valve was controlled in a simiiar manner through another digital output line of the A/D board. High and low on the digital line corresponded to the inject and load positions on the valve, with timing controlled by the software. Control of the instrument for a 3D analysis was as follows. The sample was injected onto the SEC column. After the appropriate amount of “dead time”, 3D data acquisition was begun. The LabVIEW program would start the gradient, switch the valve to the inject position, and then return the valve to the load position after 10 s. A “slice” of 2D data was acquired over 6 min and written to disk. Immediately afterward, the pump gradient was stopped and restarted and the valve switched to the inject position to inject the next sample and repeat the entire process. The 3D data acquisition program ran the 2D data acquisition as a subroutine,repeated for the number of “slices”desired. The time
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Figure 2. SEC analysis of FITC-tagged tryptic digest of hen ovalbumin. UV absorbance detection at 488 nm, 2.0 AUFS. Digest conditions and analysis conditions as described in text. Vertical lines indicate points of 2D sampling of the SEC effluent in a 3D analysis.
of each sample injection was written in the lilename of each stored 2D data set. Data Processing. All 2D slices were treated in exactly the same manner. First, all were moving median filtered with a moving median of rank 32 (see previous paper) with another LabVIEW program. Similarly,the data were smoothed with a five point linear smooth to reduce baseline noise. SpyGlassTransform, Format, and Dicer (SpyGlass Inc, Chicago, Illinois) were used to view the 2D and 3D data sets as color or gray-scale images. The LabVIEW data files were imported into Transform to generate 2D images and Format was used to annotate those images for publication. To examine the entire 3D data set, the individual 2D slices were combined into a 3D Dicer file. Dicer allows the display and manipulation of a 3D “volume” of data. RESULTS AND DISCUSSION
Figure 2 shows the results of a 1D SEC analysis of the FITG tagged ovalbumin digest sample. UV absorbance was monitored at 488 nm, so that only the FITC-tagged components were detected. The original tagged digest mixture was diluted 1:lO in SEC mobile phase and the injection volume was 20 pL. The measured flow rate through the column and detector was 11pL/ min. At this flow rate, with column dimensions of 4.6 mm x 25 cm, a totally included peak eluted at 260 min. Only the last 5 h of a total 8 h analysis time is shown. Obviously, this is much slower than we would normally run a 1D SEC analysis. However, as the first dimension of a 3D separation, this slow SEC has two advantages. First, the SEC separation generally improves as the flow rate decreases. The extended time in the column will lead to increased diffusional broadening of sample zones, but there is also more time for equilibration of sample components with the pores of the stationary phase. During the SEC method develop ment, resolution of sample components in successive analyses continued to improve as flow rate was decreased to 25 pL/min. Below this point, increased diffusional band-broadening effects due to the greater analysis time began to counteract the improved SEC resolution. Second, the long SEC analysis time allows more frequent sampling of the SEC peaks by the 2D system because the SEC peaks are broader in time. The peaks in Figure 2 are 5-15min wide. With a 2D RPLC/fast-CZE analysis time of 6.35 Analytical Chemistty, Vol. 67, No. 19, October 1, 1995
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min, the broad SEC peaks ensure that each peak is sampled at least once into the 2D system. The vertical bars on Figure 2 show where individual 2D samples were taken of the SEC effluent. Notice that most of the visible peaks are intersected by one or more 2D slices. 2D slices were also taken from 305 to 370 min but with no detectable peaks. Though some components are visible with the W absorption detector, after dilution and injection into the RPLC (with further dilution in the LC column), the sample zones are apparently too dilute to be seen in fastCZE. The latest peak at 430 min probably would have been detectable, but 2D data were not taken so late in the SEC run. It should be noted that an injection of 0.1%formamide solution under the same conditions (with detection at 230 nm) resulted in a peak at 260 min. Since formamide should be totally included in the pores of the SEC stationary phase, all peaks after this point are separated by some mixed-mode mechanism. The multiple components visible early in the largest peak are probably larger peptides, separated by size exclusion. The components late in the down slope of the same peak, and the later eluting components, are resoived by some combination of SEC and other retention modes such as adsorption or ion exchange. figure 3 shows gray-scaleplots of some of the actual 2D slices (samples) of the SEC analysis. The LC analysis time is 6 min, but the gradient formed in the pump requires 3 min to pass through the proportioningvalve, pump, LC column, and conned 3460 Analytical Chemistry, Vol. 67, No. 19, October 1, 1995
ing tubing to reach the top of the fastCZE capillary. Thus, there are no peaks in the LC dimension at times earlier than 3 min. The CZE analysis time is 2 s, but most of the CZE peaks occur in the last half of the CZE analysis. The slices in Figure 3 therefore focus on 3-6 min LC time (on the vertical axis) and 1.4-2.0 s CZE time (on the holizontal axis). The slices shown cover most of the major multicomponent SEC peak centered at about 230 min. The best signal to noise (S/M ratio and the majority of the peaks are seen in the central slices at 220-240 min, corresponding to the maximum of the SEC peak. While in the earlier slices the SEC time reveals some information on the relative size of components seen in that slice, in the later slices we can only say that molecular size was one of the factors controlling its position. In the slices taken after 255 min, the SEC separation is mixedmode. Figure 4 is a 3D representation of a region of the 3D data set. The intensity of peaks is shown in inverted gray scale, so that lighter areas indicate peak maxima or larger peaks. Of the 20 2D slices taken, only 14 are included in this image. The peaks are shown as "stac!d of disks of varying gray-scale intensity. Because there are many more LC points than SEC points (14 slices = 14 points), and even more CZE points, Dicer is used to scale the values to give the 3D volume shown. In the 3D image, values between the actual data points are linearly interpolated to fill the desired volume. In this way, more than 14 horizontal surfaces can be drawn through the volume to better show the shape of
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min. Data for the entire SEC analysis are shown on the vertical axis. Only a portion of the CZE analysis data is shown on the horizontal axis. the peaks in space. To see only the peaks, Dicer can be used to set some of the inverted gray-scale levels to be transparent. That is, the background gray level seen in the slices shown in F i r e s 3 and 4 can be set to be transparent, so that only the peaks are visible in the data volume. Without this feature, visualization of the entire data volume would result in a gray cube, with the peaks hidden behind baseline levels of gray above or in front of them. Only peaks intersecting the faces of the cube would be visible. Though the 3D "volume" of data was collected as a series of horizontal RPLC/CZE "slices", other 2D "slices" of the data can be extracted in any combination of two dimensions to focus on distinct parts of the data set. F i r e 5 is a SEC/CZE "slice" taken at a single WLC retention time. All of the points shown in F i e 5 correspond to an RPLC retention time of 4.76 min. The entire range of the SEC analysis time sampled is given on the vertical axis. Remember that this is actually only 20 points. A region of the CZE analysis similar to that shown in Figure 3 is given on the horizontal axis. As in the other gray-scale plots, peaks are represented as dark spots on the gray background. There are
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Figure 6. Individual SEC analyses taken from Figure 5 at palticular CZE migration times. Three individual columns of data from Figure 5 are shown. These plots are individual SEC analyses of compounds whose RPLC retention time is 4.76 min and whose CZE migration times are 1.44, 1.49, and 1.85 s. respectively. As mentioned in the text. the SEC analysis is seriously undersampled.
three darker spots (larger peaks) near the center as well as two lighter bands (smaller peaks) above and to the right of the these bands. As would be expected, these spots correspond to those seen in F i r e 3 at the appropriate R P E and SEC retention times and CZE migration times. Notice that the three larger peaks near the center are well resolved in the CZE dimension but only partially resolved in the SEC dimension. If the data in this plot were summed horizontally, across the rows, it would give the equivalent SEC analysis with no CZE separation. Obviously, any peaks with the same SEC retention time would be unresolved by SEC alone. The three large peaks in the center would have very similar SEC retention times and would be largely unresolved. Similarly, summing vertically down the columns would give the equivalent CZE separation with no SEC separation. Part of the power of the multidimensional approach can be realized by examining the data in a single column or row. A single column of data from Figure 5 gives the SEC analysis for all compounds of a particular RPLC retention time and a particular Analytical Chemistry, Vol. 67, No. 19, October 1, 1995
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CZE migration time. Similarly, a single row of data gives the CZE analysis for all compounds of particular SEC and RPLC migration times. Figure 6 shows tluee SEC analyses taken at the CZE times of the three large peaks in Figure 5. Obviously the peaks are extremely nndersampled. The RPLC/CZE analysis time of 6.35 min limits how often the SEC separation can be sampled. However, even the smallest peak is sampled by at least two points. Though the undersampling limits the accurate determination of peak heights and retention times, some information can still be obtained. These peaks would probably be unresolved by SEC alone, and it would thns be dficult to estimate the number of components present Also, rough estimates of the SEC peak width can be obtained from this figure. The smallest peak is roughly 20 min wide at the base, and the largest is roughly 30 min wide. The 3D system relies on a flow splitter and a dilution tee after the SEC column to interface the SEC to the LC. Because a signiicant amount of sample is "lost" in the interface, some lower concentration components might not be detected in the 3D analysis. Also, we did not acquire data over the entire SEC analysis. To see small peaks possibly too dilute to detect in the 3D system, and to see which peaks might have been omitted because of their late SEC elution, we compared the 3D analysis to a 2D analysis of the same sample. The original tagged ovalbumin digest was diluted 1:50 in CZE buffer, and 2 0 ~ was L injected onto the RPLC column. The same 2D analysis conditions were used as in the 3D analysis. To compare this to the 3D separation, the 20 2D slices taken of the SEC were summed. A comparison of the direct 2D analysis and the summed slices is shown in Figure 7. Comparing panels A and B. the results of the 2D analysis and the summed data are largely what would be expected. Most of the larger peaks are present in both (A) and (B). Noticeably, a large peak in the 2D analysis at 1.82 s CZE time, 4.6 min LC time is missing from the summed data. Also two peaks from a group of three seen at 3.253.75 min LC time in the 2D analysis are not present in the summed data. Most of the peaks in the summed data are shifted to slightly later LC times than in the 2D analysis alone. There are a number of very faint spots (small peaks) seen in the 2D analysis which are not seen in the summed data. This is to be expected because of the greater sample dilution in the 3D analysis, as was mentioned earlier. Notice that for most of the components, the pattem seen in the summed data is very similar to that of the 2D analysis alone, both in peak position and intensity. Peak Capacity. To estimate the peak capacity of the overall 3D method, we calculate the peak capacity of each dimension separately and take the product of the three values so obtainetl. The peak capacity of a separation with unit resolution is defined as
n, = L/4a where L is the total distance (or time) over which the sample zones (peaks) are distributed and u is the average standard deviation of the peaks separated! Calculation of the peak capacity for the RPLC and CZE is relatively straightfomd, as shown below, but is more diflicult for the SEC. In the SEC analysis shown in Figure 2, in the part of the run over which 3D data was (9) Giddings, 1. C. Unlfld Seoorofion Science; John W i l q & Sons: New York. 1991.
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5'01 5.1
"i
1
4.0
. 1.1
, 1.5
.
, 1.6
4 1.7
1.8
1.9
CZE (*-I
Figure 7. Comparison of summation of 2D slices taken in 3D analysis and same sample analyzed by 2D RPLC/fast-CZE alone: (A) Point-to-point summation of all 20 2D data slices, including those shown in Figure 3. (6)2D RPLC/fast-CZE analysis of FITC-tagged
ovalbumin digest. collected, there are no baselineresolved peaks from which an estimate of a can be easily obtained. To estimate the peak capacity of the SEC separation, we use the data from Figure 6. Because peaks widths there are 20-30 min, and data were acquired over about 125 min, a rough estimate of the SEC peak capacity is nspc % 4-6. In the calculations below we use the middle value of nyc = 5. In Figure 7B, the RPLC peaks elute over a range of 2.3 min, from 3.2 to 5.5 min. The average peak width in the RPLC dimension is approximately 0.10 min (4 standard deviations). Based on eq 1,this gives a RPLC peak capacity of nmc = 23. Similarly for CZE, the peaks span 0.60 s, from 1.4 to 2.0 s. An average peak width is approximately 0.025 s (4 standard deviations), and again with eq 1 this gives a CZE peak capacity with unit resolution of nCz = 24. The peak capacity of the RPLC/CZE system alone is 23 x 24 % 550. When the SEC is added, the overall peak capacity of the 3D method is 5 x 550 % 2800. Though the peak capacity of the SEC dimension is small compared to the RPLC and SEC, it results in a substantial increase in the peak capacity of the overall method when multiplied by the RPLUCZE peak Capacity. CONCLUSIONS A threedimensional separation identifies sample components
by three different characteristics. Here, molecules are located in
a 3D space by their size, hydrophobicity, and electrophoretic mobility. If there is sufficient resolution in each of the three dimensions, 3D “location”becomes a powerful means of component identification. As the number of separation methods increases, the possibility of two different molecules having the same location in each dimension decreases. However, this identificationis not without cost. In our present system, very concentrated samples are used to overcome dilution factors inherent in the design of the SEC/RPLC interface. This would be a problem in a sample-limited situation. Also, for elutiodmigration times in three dimensions to be strictly comparable, rigorous control of temperature in all three dimensions would be necessary. Thermostatic control of all three separation systems would avoid retentiadmigration time drift caused by changes in temperature over the course of the analysis. It would also be desirable to use a faster computer which would be able to acquire data and control the instrument simultaneously. This would enable not only more accurate timing but also better use of the 3D system. For example, the SEC analysis could be done more rapidly or be better characterized because LC analyses could be overlapped (see previous paper) to better sample the SEC. The major advantage of a 3D method is of course the gain in peak capacity. As with a 2D system, the increase in peak capacity
is multiplicative. Because the peak capacity of a 2D system is already high, the addition of a thiid dimension with only a modest peak capacity can significantly increase the peak capacity of the overall method. As shown in the estimate of peak capacity above, addition of the SEC as a thiid dimension increases the peak capacity of the overall method by a factor of 5. ACKNOWLEDGMENT
This research was supported by grants from the National Science Foundation (CHE 9215320) and the National Institute of Health (GM 39515) and by a gift from Hewlett-Packard. The HP1050 pump and W absorption detector were donated by Hewlett-Packard, The HEMA stationary phase was donated by Alltech Associates. The RPLC column was donated by Rockland Technologies. Received for review October 25, 1993. Resubmitted July
7,1995. Accepted July 14, 1995.@ AC9506802
@
Abstract published in Advance ACS Abstracts, September 1, 1995.
Analytical Chemistry, Vol. 67, No. 19, October 1, 1995
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