In the Laboratory
Micellar Electrokinetic Capillary Chromatography in the Undergraduate Curriculum: Separation and Identification of the Amino Acid Residues in an Unknown Dipeptide Using FMOC Derivatization Timothy G. Strein,* James L. Poechmann, and Mark Prudenti Department of Chemistry, Bucknell University, Lewisburg, PA 17837; *
[email protected] The use of capillary electrophoresis (CE) in analytical and biochemical research has rapidly increased in the last decade. Since it is clear that CE techniques have become widely accepted by the scientific community, it is important to include CE methodology and technology in the undergraduate laboratory sequence. In this manuscript we briefly describe the operational principles behind micellar electrokinetic capillary chromatography (MEKC), a particularly useful mode of CE that employs micelles in the operational buffer. We also present an example of a simple undergraduate laboratory experiment involving MEKC to determine the amino acid content of a dipeptide. Overview of Micellar Electrokinetic Capillary Chromatography Capillary electrophoretic separations, like slab gel electrophoretic techniques widely used by biologists, involve separation schemes in which chemical species are separated via differential migration of charged species in an applied potential field. Unlike slab gel methods, CE separations occur in a narrow-bore capillary tube, usually with a 25–100-µm inner diameter. Under most operational conditions in an unmodified fused silica capillary, there is a strong flow of solution from the anodic to the cathodic end of the capillary. This flow, which is referred to as an electroosmotic flow (EOF), typically sweeps all components through the capillary tube so that all analytes injected at one end will exit the column at the other end. Consequently, the data output, which is called an electropherogram, is similar in appearance to an HPLC chromatogram. The superb resolving power of electrophoretic separations with high potentials, coupled with a single online detection scheme, has made CE an increasingly popular choice for the separation of samples from biological, chemical, and agricultural sources (1). A schematic of a typical CE setup is shown in Figure 1. We recently reported an undergraduate laboratory experiment involving capillary zone electrophoresis (CZE), the simplest mode of operation in CE (2). In CZE, the operational buffer that fills the capillary and the reservoirs at either end of the capillary is simply an aqueous buffer solution. When the separation potential (10–30 kV) is applied, EOF sweeps the solution inside the capillary toward the cathodic end. When a sample for analysis is then injected at the anodic end, a separation of the charged chemical species occurs within the flowing stream. Cations will migrate ahead of the EOF with their characteristic electrophoretic mobilities, neutral and zwitterionic species will move along with the EOF, and anions will lag behind the EOF by their respective mobilities. In 820
CZE, neutral and zwitterionic components are unresolved from one another. MEKC, on the other hand, is capable of separating neutral species as well as charged analytes. The MEKC system, like the CZE system, consists of a fused silica capillary column 20–100 cm in length and a 50–100 µm inner diameter, which is filled with an aqueous buffer solution and is immersed in that same buffer at each end (Fig. 1). The buffer solution used in MEKC, however, contains a surfactant at a concentration in excess of its critical micelle concentration (cmc). In this experiment, sodium dodecyl sulfate (SDS), one of the most prevalent surfactants for MEKC work, is used as the micelleforming species. SDS has a cmc of approximately 8 mM; thus, at a concentration above 8 mM, the SDS monomers aggregate into spherical micelles (Fig. 2). Since the micelles have a hydrophobic interior, they serve as a nonpolar pseudostationary phase within the capillary column. After the capillary is filled with the desired MEKC buffer solution and the buffer has reached equilibrium with the inner walls of the capillary, the “front” (anodic) end of the capillary is placed into the solution of interest and a small volume (typically 1–50 nL) of the analysis solution is injected into the anodic end of the capillary. Injection of the analysis solution is accomplished either electrokinetically (by applying a potential to generate electroosmotic flow) or hydrodynamically (by exerting pressure on the analysis solution at the front end of the capillary, generating gravity flow) for a given amount of time. The injection end of the capillary is then placed back into the front-end buffer reservoir and a large separation potential (between 10 and 30 kV) is applied across the capillary via platinum electrodes immersed in the buffer reservoirs at each end of the system. EOF moves the entire solution toward the cathodic end. The SDS micelles carry a large negative charge (Fig. 2); thus, the micelles migrate “upstream”, opposite the direction of the EOF. The hydrophobic interior of the micelles allows partitioning of neutral analytes on the basis of their hydrophobic character. A schematic view of the process within the capillary is shown in Figure 3. The separation of analyte species in MEKC results from a combination of (i) differential migration (electrophoretic movement) of the “free” ions in the potential field and (ii) differential interaction (partitioning or electrostatic) of analytes with the negatively charged, migrating micelles. The migration times of anionic analytes tend to be unaffected by the use of SDS micelles, since anionic analytes have little affinity for either the hydrophobic interior or the anionic exterior of the micelles. An individual neutral analyte will elute at a time determined by its affinity for the micelle, which is migrating “upstream”, relative to its affinity for the aqueous moving phase,
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In the Laboratory
which is moving at the rate of the EOF. This separation process, governed by polarity, is completely analogous to HPLC; however, with SDS micelles in solution, the “stationary” phase is actually moving. Cationic analytes can be retained by an interaction with the exterior of the micelle, apparently via ion-pairing equilibria. It should be noted that micelles can be formed using anionic (as described above for SDS), cationic, zwitterionic, or nonionic surfactants; thus, in principle, MEKC separations can be tailored to fit any application. There are also more advanced modes of CE designed to separate, for example, optical isomers by using buffer systems with chiral additives, or biopolymers by using gel-filled capillaries. Isoelectric focusing can also be accomplished quite efficiently in capillaries (3– 6 ). The following discussion is confined to the MEKC separation scheme using SDS as the micelle-forming species. Figure 1. Schematic of general capillary electrophoresis (CE) setup. Before attempting to build or use such a setup, please see the note concerning safety at the end of this paper.
MEKC Theory The initial pioneering work with MEKC was performed by Terabe et al. (7–9). The equations that describe the elution behavior of neutral analytes in MEKC are similar to the familiar chromatographic equations. The primary difference in MEKC is that all neutral solutes must elute in the “window” between the dead time (teo), the time it takes the EOF to sweep an unretained solute past the detector, and tmc, the time it takes a solute that is irreversibly solubilized in the micelle to pass the detector (Fig. 3). The capacity factor (k′), also called the retention factor, for MEKC analytes can be expressed as k′ =
t i – t eo t eo 1 –
Figure 2. Schematic 2-D representation of the structure of an SDS micelle. The center of the micelle is hydrophobic; the outer perimeter is hydrophilic. The micelles are negatively charged and migrate “upstream” in the MEKC capillary.
Figure 3. Schematic of the MEKC separation processes within the capillary. Neutral solutes (analytes) will be retained on the basis of their affinity for the nonpolar interior of the micellar pseudostationary phase.
ti t mc
In this equation, ti is the time for an individual neutral analyte to reach the detector, teo is the time for an unretained neutral species (MeOH, acetone) to reach the detector, and tmc is the time it takes a micelle to be swept, against its migration, through the column to the detector (measured using quinine). Note that as tmc goes to infinity, the equation becomes the more familiar equation used to calculate a capacity factor in HPLC. This equation can also be expressed in terms of mobilities (7–9). The theory for charged analytes becomes more complex owing to the fact that the unbound analytes have an electrophoretic mobility of their own; that is, unlike neutral analytes, when charged species are “free” in solution, they migrate toward the electrode of opposite charge. A rigorous treatment of this situation accounts for the effects of both separation mechanisms independently and requires knowledge of how the analytes migrate in the absence of micelles. Although the above equation for retention factor can be used for qualitative identification of analytes that possess a net charge, qualifying terminology must be used to define the calculated value. For this manuscript, we will refer to this calculated value as the apparent capacity factor (k′app). The word “apparent” is used to signify that the measured ti (and therefore the calculated value of k′app) is not a true measure of only the hydrophobic/ hydrophilic interactions of the analyte with the micelle. For a more comprehensive discussion of the theory, the reader is referred to the original work (7–9) or to one of the many fine texts now available on this subject (3–6 ).
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Figure 4. FMOC derivatization reaction for an amino acid.
This manuscript presents a simple laboratory experiment designed to illustrate the principles of MEKC to undergraduate instrumental analysis and biochemistry students. The determination of the amino acid content of a dipeptide by cleavage, followed by derivatization with 2-(9-anthrylethyl) chloroformate (FMOC) (illustrated in Fig. 4) and separation with HPLC, is a common laboratory experiment and is performed in Bucknell’s biochemistry laboratories (10). A fine manuscript detailing MEKC separations of NDA-derivatized amino acids has recently appeared in this Journal (12). We present here an experiment with FMOC-derivatized amino acids designed to illustrate both the separation power and the complementary utility of MEKC and HPLC in a single integrated laboratory experiment suitable for use in the undergraduate laboratory. Experimental Procedures
Overview An in-house lab manual describing the fundamentals of CE and HPLC (consisting primarily of the text from the introduction of this manuscript and from the introductions in refs 2 and 10), with an outline of the instrument-specific experimental steps to be taken, is sold to the students at the beginning of the semester. Each student or pair of students prepares a series of standard solutions of FMOC–amino acid derivatives to determine the electrophoretic and chromatographic characteristics of each derivative. Each student is then given an unknown dipeptide and is instructed to determine the two amino acids in the dipeptide by cleaving the peptide bond, derivatizing the amino acids, and performing MEKC and HPLC separations on the resultant products. In a separate part of the same laboratory exercise, the students will also determine the N-terminus of the dipeptide by derivatizing the free amine functionality using dansyl chloride, cleaving the peptide bond, and identifying the derivatized residue with thin layer chromatography (TLC). Chemicals All amino acids, FMOC, FMOC–amino acid derivatives, and dipeptides were obtained from Sigma Chemical Co. (Milwaukee, WI). Boric acid was obtained from Baker Chemical Co. (Phillipsburg, NJ), SDS was obtained from ICN Biochemicals Inc. (Irving, CA), and quinine sulfate, HPLC-grade acetone, acetonitrile, and methanol were obtained from Fisher Scientific (Fair Lawn, NJ). All chemicals were used as received without further purification. Solutions A 20 mM borate buffer pH 9.2/25 mM SDS solution was prepared for MEKC by first dissolving the appropriate mass of boric acid in deionized water and adjusting the pH
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with ca. 1 M NaOH. The appropriate mass of SDS was then added before dilution to the final volume.1 Stock solutions of 1.00 × 10 {3 M of each standard FMOC–amino acid derivative were prepared in 25-mL volumetric flasks using the MEKC buffer as the solvent. A stock solution of 1.00 × 10 {3 M quinine sulfate was prepared in a 100-mL volumetric flask by dissolving the appropriate mass of quinine sulfate in ca. 75 mL of the MEKC buffer. Ten milliliters of methanol (MeOH) was added before dilution to the final volume with the MEKC buffer. From these stock solutions, standard solutions containing 1.00 × 10 {4 M of the FMOC–amino acid derivative and 1.00 × 10 {4 M of quinine (tmc marker) were prepared for analysis using the MEKC buffer as the solvent. One standard solution containing 1.00 × 10 {4 M of 15 different FMOC-amino acid derivatives as well as quinine was also prepared. All solutions to be analyzed in the MEKC system were filtered through a 0.2-µm disposable syringe filter obtained from Whatman Inc. (Clifton, NJ). All solutions were refrigerated when not in use.2
Preparation of FMOC–Amino Acid Derivatives Most amino acids do not possess a suitably strong chromophore for use with separation techniques that employ UV detection. In order to employ UV detection, the amino acids must be derivatized with a chromophoric group such as 2-(9-anthrylethyl) chloroformate, as shown in Figure 4. Because only nine FMOC–amino acid derivatives are commercially available, some derivatives for use as standards need to be prepared in house. An earlier publication from this department provides the details of our method (10), which was adapted from that first reported by Einarsson et al. (11). Briefly, standard amino acid solutions of 0.5 mM are prepared in 0.2 M borate buffer, pH 7.7. Five hundred microliters of 15 mM FMOC in HPLC-grade acetone is added to an equal volume of the amino acid solution. Two milliliters of hexanes is added after about 40 s. The reaction vessel is stoppered with a plastic cap and shaken. The hexane layer is removed by pipet and the aqueous layer is extracted with an additional 2 mL of hexanes. The aqueous layer, which is the lower layer, is transferred by pipet to a 10-mL volumetric flask, where a standard solution is prepared for analysis by adding 1 mL of the stock quinine solution before dilution to the final volume with the MEKC buffer.3 Preparation of the Unknown The peptide bond must first be cleaved in order to determine the amino acid content of the dipeptide. This process, which is termed peptide hydrolysis, has been documented in an earlier publication (10). Briefly, 0.2 mL of 6 M HCl is used to dissolve 2 mg of the dipeptide. This solution is sealed in Pyrex tubing (6 mm o.d.) and heated in an oven at 110–120 °C for 18–24 h. A 50-µL aliquot of the cooled solution is transferred to a 1.5-mL Eppendorf tube and concentrated to dryness in a Savant Speed Vac Concentrator equipped with a vacuum pump. The resulting solid is dissolved in 0.2 mL of 50 mM borate buffer, pH 9.0, and 2.0 mL of 0.2 M borate buffer, pH 7.7, is added immediately. The pH of this solution is then adjusted with HCl or NaOH to between 7.5 and 8.0. The procedure for preparing the FMOC derivatives is then followed, substituting a 0.5-mL aliquot of the resulting solution for 0.5 mL of the amino acid solution.
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In the Laboratory
A
B
Figure 5. Representative MEKC separations of two FMOC-amino acid derivatives. Teo is the time it takes an unretained neutral species to reach the detector, Ti is the time it takes an individual analyte to reach the detector, and T mc is the time it takes a micelle to be swept, against its migration, through the column to the detector. Tmc is marked with quinine. A: Methionine (M); T eo is marked with methanol. B: Threonine (T); Teo is marked with acetone.4
Table 1. Calculated k ′app Values for FMOC-Derivatized Amino Acids under MEKC Separation Conditions One-Letter Designation
k ′app ± SD a
Serine
S
1.195 ± 0.007
Asparagine
N
1.232 ± 0.008
Threonine
T
1.262 ± 0.009
Alanine
A
1.320 ± 0.009
Amino Acid
Glycine
G
1.437 ± 0.010
Proline
P
1.631 ± 0.012
Valine
V
1.743 ± 0.013
Tyrosine
Y
1.90 ± 0.02
M
2.26 ± 0.02
I
2.66 ± 0.03
Glutamic acid
E
2.82 ± 0.03
Aspartic acid
D
3.23 ± 0.04
Leucine
L
3.33 ± 0.03
Phenylalanine
F
5.28 ± 0.07
Arginine
R
112.9 ± 1.1
Methionine Isolucine
Values for k′app are the average of values calculated from three individual injections of a mixture of the FMOC derivatives. a
Micellar Electrokinetic Capillary Chromatography Students perform the MEKC experiments using either an HP3D CE (Hewlett Packard, Wilmington, DE) or a locally constructed CE unit. The home-built CE unit employs a Spellman (Plainview, NY) high-voltage dc power supply, platinum electrodes, and a Spectra-Physics variable-wavelength UV detector fitted with a capillary flow cell (Spectra-Physics, Fremont, CA). The high-voltage end of the of the home-built system is housed in a Plexiglas box equipped with two interlock switches for operator safety (please see the note on safety at the end of the manuscript). Both instruments are equipped with a PC workstation and Hewlett Packard Chemstation software. Unmodified fused-silica capillary (75 µ m i.d., 360 µ m o.d.) was obtained from Polymicro Technologies (Phoenix, AZ). The following parameters were used for the CE analyses illustrated in this paper: (i) total column length (Lt), 64 cm; (ii) effective column length, injection to detection (Ld), 49 cm; (iii) voltage, 18 kV, with a resulting current across the capillary of 28–34 mA; (iv) injection by application of the separation voltage, 18 kV, for approximately 2 s; (v) column temperature, ambient; and (vi) detection, UV at 248 nm. High Performance Liquid Chromatography A Hewlett Packard 1100 HPLC system equipped with a UV detector operating at 254 nm, a ternary pumping system, and a data acquisition workstation was used for all HPLC separations. A 250 × 4.6-mm Alltech Adsorbosphere 5-µm C18 column (catalog no. 287062) was used to separate the FMOC– amino acid derivatives. Mobile phase A was a 10:40:50 mixture of acetonitrile, methanol, and acetate buffer. Mobile phase B was a 50:50 mixture of acetonitrile and acetate buffer. The acetate buffer was prepared by adjusting the pH of a mixture containing 1.00 mL of trimethylamine, 3.00 mL of glacial acetic acid, and 900 mL of deionized water to 4.2 with sodium hydroxide solution. The mixture is then diluted to 1 L in a volumetric flask. The following parameters were used for the HPLC elution program: (i) a 3-min isocratic elution with 100% A, (ii) a 9-min linear gradient from 100% A to 100% B, (iii) an 11-min isocratic elution with 100% B, and (iv) a flow rate of 1.3 mL/min. The injection volume was 10 µ L and the column temperature was ambient. Results and Discussion (with Typical Student Results) Representative electropherograms of two FMOC–amino acid derivatives (methionine and threonine) are shown in Figure 5. The electroosmotic flow is marked by either a dip in the baseline (Fig. 5A) caused by a local absence of absorbing species in the injected plug of sample, or by a positive peak (Fig. 5B) in samples containing acetone, which absorbs UV radiation at 248 nm. In both cases, the end of the elution window is marked by quinine, which is known to bind strongly with SDS micelles. The FMOC–amino acid derivatives will elute between these two boundaries, and k′app can be calculated for each individual standard as described in the experimental section of this manuscript. Values for k′app for each of the 15 FMOC–amino acids considered in this work are given in Table 1. It should be noted that small variations in the pH and ionic strength of the buffer system, the inner surface of the capillary (capillary history), and the ionic strength of the sample injected can cause deviations in the rate of electroosmotic
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A A MEKC
B
B HPLC
C
Figure 6. Separation of 15 FMOC-amino acid derivatives using (A) MEKC and (B) HPLC. Key: serine, S; asparagine, N; threonine, T; alanine, A; glycine, G; proline, P; valine, V; tyrosine, Y; methionine, M; isoleucine, I; glutamic acid, E; aspartic acid, D; leucine, L; phenylalanine, F; and arginine, R.
flow. Consequently, it is unwise to depend solely on the migration time that is observed as a particular analyte passes the detector as the means of peak identification; it is advisable to calculate k′app for best results. An electropherogram illustrating the separation of 15 derivatized amino acids is given in Figure 6A. The same mixture separated by HPLC is shown in Figure 6B. The MEKC separation is shorter, the column does not require a re-equilibration time between runs, and a higher efficiency of separation is evident. Nonetheless, the complementary MEKC and HPLC results are needed to conclusively identify a few of the amino acids. For example, the MEKC separation shows adequate, but not complete, baseline resolution of serine (S), asparagine (N), threonine (T), and alanine (A). If one of these amino acids were in an unknown sample, an HPLC separation of the same mixture might further substantiate the tentative peak identification assignment based on MEKC alone. However, the HPLC method results in coelution of glycine (G) and threonine (T) as well as of alanine (A) and tyrosine (Y), making the HPLC separation inadequate on its own for the determination of these amino acids. In addition, the HPLC procedure cannot determine methionine (M) because this FMOC-derivatized amino acid coelutes with an absorbing by-product of the derivatization, “FMOH”, the alcohol formed when FMOC reacts with water. Clearly, the complementary nature of the data provided by the HPLC and MEKC systems is desirable for full characterization of the amino acids an unknown dipeptide. Representative separations of a few cleaved dipeptides using MEKC and HPLC are given in Figures 7 and 8, respectively. Many of the individual amino acids can be conclusively
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Figure 7. Representative MEKC separations of the FMOC-amino acids from several cleaved dipeptides. A: serine–glutamic acid; B: glycine–threonine; C: arginine–aspartic acid.
identified with either of the two separation techniques. Both HPLC and MEKC can resolve and identify serine (S) and glutamic acid (E) as shown in Figures 7A and 8A. However, a few of the derivatized amino acids require the use of both techniques to conclusively identify the constituent amino acids. The examples shown in Figures 7B,C and 8B,C illustrate some of the strengths and weaknesses of each technique. Using the data obtained via MEKC or HPLC separations coupled with a bit of deductive reasoning, any of the 15 amino acids considered in this experiment can be conclusively identified. By choosing the unknowns prudently, the instructor can ensure that students will need to use both techniques to conclusively determine the amino acid content of the unknown dipeptide. In this laboratory exercise, students become familiar with the complementary utility of MEKC and HPLC to identify amino acids. They learn the practical advantages of MEKC, such as a run time, including a total run time less than onehalf the length associated with HPLC and a greatly reduced volume of solvent required for the analysis. Students also gain an appreciation for the strengths and weaknesses of these two methods for separating this particular class of compounds. Perhaps most importantly, however, they actively participate in a problem-solving, research-oriented laboratory exercise. A Note on Safety When working with home-built CE instrumentation, it is very important to ensure that all students are protected from the high potentials employed with this technology. The high-potential end of our home-built system is housed in a Plexiglas box with a hinged lid that opens from the front.
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A
Acknowledgments Supported by a Cottrell College Science Award from Research Corporation (CC4333) and by The Camille and Henry Dreyfus Foundation (#SU-94-011). Notes
B
C
1. SDS forms bubbles very easily when dissolved in aqueous solutions. These bubbles must be allowed to settle overnight before dilution to the final volume. 2. SDS dissolved in water becomes “cloudy” when refrigerated. The SDS must be redissolved by gently warming the solution to room temperature before use. 3. Quantification becomes difficult because the dilution ratio is only approximate, since the concentration of the FMOC–amino acid derivative in the aqueous–acetone layer is unknown. 4. Although MeOH is still in solution, the absorbance due to acetone dominates the signal seen at teo .
Literature Cited
Figure 8. Representative HPLC separations of the FMOC-amino acids from several cleaved dipeptides. A: serine–glutamic acid; B: glycine– threonine; C: arginine–aspartic acid.
The top lid has two interlock switches to ensure that the highpotential lead is not “live” when the lid is open. The first switch kills the power supply when the lid is opened, and the second switch, which controls a vacuum relay, bleeds the residual voltage to ground through a bank of resistors. We highly recommend the use of safety devices such as interlock switches on the lid of a Plexiglas box be used to avoid unintentional contact with the live high-potential lead. Upon request, we will be glad to provide further details concerning the construction and safe operation of a home-built CE system.
1. Monnig, C. A.; Kennedy, R. T. Anal. Chem. 1994, 66, 280R– 314R. 2. Thompson, L.; Veening, H.; Strein, T. G. J. Chem. Educ. 1997, 74, 1117–1121. 3. Capillary Electrophoresis: Theory and Practice; Grossman, P. D.; Colburn, J., Eds.; Academic: San Diego, 1992. 4. Capillary Electrophoresis Technology; Guzman, N. A., Ed.; Dekker: New York, 1993. 5. Baker, D. R. Capillary Electrophoresis; Wiley: New York, 1995. 6. Kuhn, R.; Hoffstetter-Kuhn, S. Capillary Electrophoresis: Principles and Practice; Springer: New York, 1993. 7. Terabe, S.; Otsuka, K.; Ichikama, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111–113. 8. Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834– 841. 9. Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 348, 39– 47. 10. Clapp, C. H.; Swan, J. S.; Poechmann, J. L. J. Chem. Educ. 1992, 69, A122–A126. 11. Einarsson, S.; Josefsson, B.; Lagerkvist, S. J. Chromatogr. 1983, 282, 609–618. 12. Weber, P. L.; Buck, D. R. J. Chem. Educ. 1994, 71, 609–612.
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