Simultaneous chromatography and electrophoresis: two-dimensional planar separations
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- Stevenson, P.R., Dunlap, B.E., Powell, P.S. et al. Anal Bioanal Chem (2013) 405: 3085. doi:10.1007/s00216-013-6737-0
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Single-dimension separations are routinely coupled in series to achieve two-dimensional separations, yet little has been done to simultaneously exploit multiple dimensions during separation. In this work, simultaneous chromatography and electrophoresis is introduced and evaluated for its potential to achieve two-dimensional separations. In simultaneous chromatography and electrophoresis, chromatography occurs via capillary action while an orthogonal electric field concurrently promotes electrophoresis in a second dimension. A novel apparatus with a dual solvent reservoir was designed to apply the concurrent electric field. Various compounds were used to characterize the apparatus and technique, i.e., vitamins, amino acids, and dyes. Improved separation is reported with equivalent analysis times in comparison to planar chromatography alone. The feasibility of simultaneously employing chromatography and electrophoresis in two dimensions is discussed.
KeywordsPlanar chromatographyAmino acidsSeparations/instrumentationElectrophoresis
Simultaneous chromatography and electrophoresis
Chromatography in a single dimension may be insufficient for separation of complex mixtures. Conventional techniques of altering mobile and stationary phase compositions and/or adjusting separation conditions (e.g., temperature, flow rate, stationary phase film thickness, etc.) are often tedious, time consuming, and occasionally fruitless. In addition, the separation conditions required may result in excessively long analysis times. To overcome these challenges, faster higher-resolution separation techniques have been developed and optimized (e.g., ultra performance liquid chromatography and capillary electrochromatography). In addition, comprehensive two-dimensional separation techniques occurring in series have emerged enhancing separation through improved effective peak capacity [1, 2]. This approach has been widely successful allowing for the analysis and characterization of many complex mixtures: i.e., biological compounds [3–6], pharmaceuticals [7–9], polymers [10, 11], hydrocarbons [12, 13], fungicides , and pesticides . Yet, these serial procedures have several disadvantages. The second dimension typically requires further analysis time and/or must be relatively fast in comparison to the first; thus, a slow first dimension and/or a limited-efficiency second dimension is/are required . Consequently, to obtain the advantages offered by multidimensional separations, analysis times are commonly increased in comparison to single-dimension separations.
Simultaneously accommodating separation in multiple dimensions poses significant challenges, and the use of conventional columns is impractical. Two or more independent separation mechanisms must be chosen that can occur simultaneously in orthogonal dimensions without introducing antagonistic effects. When simultaneously coupling chromatography with electrophoresis, the solvent must serve both as an electrolytic solution to promote electrophoresis and as a mobile phase to promote chromatography. Ideally, both separations occur independent of each other, and the altering of conditions for one dimension does not affect separation in the second dimension. Then, if separation is possible, detection subsequently presents unique challenges.
Reports of simultaneous multidimensional separations are antiquated and scarce. In 1948, planar chromatography was performed using a conventional planar chromatography apparatus with voltage applied orthogonal to the chromatographic separation . However, with no modifications to the planar chromatography apparatus, the electric current was assumed to pass mostly through the solvent reservoir (rather than the chromatographic plate) resulting in analysis times of over 16 h! In 1949, a technique for carrying out a continuous or discontinuous separation of compounds by passing a background electrolyte under gravity flow through filter paper at right angles to an electric field was introduced [18–20]. Shortly after its introduction, work was done to improve the technique by designing the separation apparatus with [19, 21] and without [20, 22] the filter paper sandwiched between glass plates. This work, and other similar work with the electric field applied parallel rather than orthogonal to the direction of compound migration [23–27], is called electrochromatography.
In 1973, van Ooij reported the construction of the first apparatus truly designed for the simultaneous operation of electrophoresis and chromatography . Separation occurred horizontal to the bench top with the apparatus developed to suit the orientation. The apparatus used graphite electrodes, several mobile phase reservoirs, and was optimized for the separation of aquochloro complexes of iridium. The term “chromatophoresis” was proposed.
More recently, Nurok published several articles describing pressurized planar electrochromatography where approximately 1 kv/cm is applied across the thin-layer chromatography plate to drive the mobile phase via electroosmotic force. This single-dimension technique has greatly improved separation efficiency in significantly reduced analysis times compared to traditional planar chromatography .
Despite limited work and potential challenges, simultaneous multidimensional separations offer many advantages, i.e., onetime (bulk) sample analysis, reduced analysis times, improved effective peak capacity, and enhanced compound identification. Considering the advantages, simultaneous multidimensional separations merit further development and characterization. It is the purpose of this work to present and evaluate a novel, simple, and versatile apparatus that allows for simultaneous chromatography and electrophoresis in orthogonal dimensions and to contribute to the further development of such techniques. Unique from previous work, the apparatus presented accommodates vertical capillary action for a chromatographic separation, greatly simplifying the design, and increased voltage is permitted by preventing the movement of electric current through the solvent reservoir, dramatically decreasing separation time . Throughout this paper, we call the approach simultaneous chromatography and electrophoresis (SCE).
Ammonia and 1-propanol (ACS certified) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ethanol (200 proof) was purchased from Heath Link (Jacksonville, FL, USA) and used with ninhydrin (Aldrich Chemical Co., Milwaukee, WI, USA) for the detection of amino acids. Glycine (electrophoresis grade) was acquired from Sigma-Aldrich (St. Louis, MO, USA).
The copper anode and cathode were submerged in the solvent of the large and small glass vessels, respectively. Only after the solvent from both vessels migrated above the height of the notch could current flow orthogonal to capillary action; this typically coincided with the initial spotting height of the compounds on the plate approximately 6 mm from the bottom of the plate. Current would generally begin at 0.0 mA, jump to 0.1 mA when the solvent from each vessel made contact, and then slowly increase to approximately 0.4 mA. The larger glass vessel, containing the entire plate and smaller vessel, was enclosed to minimize solvent evaporation. Voltage was applied using either a Spellman Bertan series 225 (amino acids) or a Spellman SL60 (dyes and vitamins) power supply (Hauppauge, NY, USA).
Hydrophilic vitamins were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aqueous solutions of thiamin, riboflavin, ascorbic acid, folic acid, and pyridoxine were separately prepared at 1 mg/mL. Complete dissolution of riboflavin and folic acid was not possible at these concentrations. Separation occurred employing 1-propanol/1 mM aqueous ammonia buffered to pH 9.3 (2:1) in 16 min at 500 V. Ultraviolet light (254 nm for thiamin, ascorbic acid, and pyridoxine, and 365 nm for folic acid and riboflavin) was used for detection.
Amino acids were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aqueous solutions of glycine, glutamic acid, proline, leucine, and lysine were separately prepared at 1 mg/mL. Separation occurred employing 1-propanol/1.0 mM aqueous ammonia buffered to pH 9.3 (2:1) in 30 min at 500 V. Ninhydrin, 0.1 % (m/v), in ethanol was used for detection.
Aqueous solutions of tartrazine, allura red, erythrosine, and erioglaucine were supplied by McCormick & Co., Inc. (Hunt Valley, MD, USA). Standard solutions were prepared by diluting 0.25 mL with 3.75 mL water. Crystal violet, methylene blue, bromocresol green, and methyl red were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aqueous solutions of bromothymol blue and methyl red were separately prepared at 1 mg/mL; crystal violet and methylene blue were prepared at 0.25 mg/mL. Separation occurred employing 1-propanol/1.0 mM aqueous glycine buffered to pH 2.4 (2:1) in 15 min at 500 V. All dyes were visible after separation.
Conditions for all separations were chosen through experimentation. Only conditions resulting in maximized chromatographic and electrophoretic separations are reported. Due to the electric current limit of the power supplies, 500 V was the maximum voltage achievable employing the chosen separation solutions.
Results and discussion
Feasibility of technique
Figure 2a shows four spots for the planar chromatography of five vitamins, with ascorbic acid and pyridoxine comprising one spot. All five vitamins show improved separation in Fig. 2b in the same analysis time when employing SCE. Occasional vertical streaking was seen with folic acid. The more acidic, negatively charged vitamins (i.e., folic acid and ascorbic acid) migrate toward the positively charged anode, while the more basic, positively charged vitamins (i.e., thiamin and pyridoxine) migrate toward the cathode. Chromatographic separation also appears to be consistent with log P values; riboflavin and thiamine have the largest (0.1) and smallest (−4.0) log P values and also have the largest (0.75) and smallest (0.13) Rf values.
Figure 3a shows four spots for the planar chromatography of five amino acids with glutamic acid and glycine comprising one spot. All five amino acids show improved separation in Fig. 3b in the same analysis time when employing SCE. Lysine, having the most basic substituent (pKa = 10.8) and being positively charged, moves toward the cathode, while glutamic acid (having the most acidic substituent, pKa = 4.3) moves toward the anode. The other amino acids show little lateral migration. Chromatographic separation is consistent with log P values; leucine, having the largest log P value (−1.29), also has the largest Rf value (0.72).
Figure 4a shows four moderately separated regions for the planar chromatography of eight dyes. All eight dyes are separated in Fig. 4b in the same analysis time employing SCE. The negatively charged dyes found as sodium salts (i.e., tartrazine, allura red, erythrosine, and erioglaucine) and the positively charged dyes found as chloride salts (i.e., crystal violet and methylene blue) migrate toward the anode and cathode, respectively. The relatively neutral methyl red displays little lateral migration.
Compound charge likely affects both its chromatography and electrophoresis. Thus, changing pH may alter separation in both dimensions and the techniques are not completely orthogonal. In addition, it is recognized that electrolysis of water creates a more acidic region near the anode and a more basic region near the cathode potentially introducing a pH gradient parallel to the electric field .
Characterization of horizontal migration
Rf and μa for vitamins, amino acids, and dyes
μa (cm2/Vs) e-6
0.66 ± 0.04
5.7 ± 0.5
0.52 ± 0.08
4.8 ± 0.6
0.73 ± 0.03
−1.7 ± 0.4
0.75 ± 0.02
1.5 ± 0.4
0.13 ± 0.02
−1.7 ± 0.4
0.51 ± 0.05
4.0 ± 0.4
0.53 ± 0.03
−0.60 ± 0.04
0.71 ± 0.02
−0.08 ± 0.04
0.26 ± 0.02
−9.4 ± 0.6
0.45 ± 0.03
−0.76 ± 0.05
0.69 ± 0.02
7.3 ± 0.8
0.91 ± 0.03
4.3 ± 0.9
0.80 ± 0.06
−8.0 ± 2.0
0.79 ± 0.03
1.1 ± 0.4
0.28 ± 0.02
−3.2 ± 0.9
0.91 ± 0.02
5.9 ± 0.9
0.78 ± 0.02
8.5 ± 0.8
0.73 ± 0.03
11.4 ± 0.9
This work introduces a dual reservoir apparatus allowing for simultaneous chromatography and electrophoresis in two orthogonal dimensions. The apparatus is novel in design, simple in assembly, and easily applied to simple mixtures. The dual reservoir design promotes the passage of electric current through the TLC plate allowing for separation in as few as 15 min. Vitamins, amino acids, and dyes were used to characterize the technique. Each sample mixture showed improved separation employing SCE over traditional planar chromatography. Separations were consistent with known log P values and compound charges. Further evaluation is imminent, yet the merits of this technique and its potential are notable. The feasibility of simultaneously employing electrophoresis and chromatography in two separate dimensions has been demonstrated.
The authors gratefully acknowledge the financial support provided of the BYU—Idaho Chemistry Department. We also offer sincere appreciation to Milton L. Lee of Brigham Young University for the donation of the power supplies used in this work.