Review pubs.acs.org/ac
Capillary Electrophoresis Matthew Geiger, Amy L. Hogerton, and Michael T. Bowser*
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University of Minnesota, Department of Chemistry, 207 Pleasant Street South East, Minneapolis, Minnesota 55455, United States
CONTENTS
Techniques Fundamentals and Separation Modes Sampling and Preconcentration Buffer Additives Capillary Coatings and Surface Modifications Detection Multiple Dimension Separations Applications Nucleic Acids Proteins and Peptides Carbohydrates Metabolites Pharmaceuticals Bioaffinity Cells and Organelles Environmental Materials Author Information Corresponding Author Biographies Acknowledgments References
Lovsky et al. combined CE with atomic force microscopy (AFM) to achieve nanoprinting of proteins with a high degree of control.2 Atomic force controlled capillary electrophoretic printing (ACCEP) demonstrated the ability to perform electrophoretic separation with high resolution both in time and space. The authors suggest the impact this technology could have in the ability for nanoprinting and nanoinjecting proteins on substrates with both positional and timing accuracy. Garcia-Perez et al. developed a statistical integration of nuclear magnetic resonance (NMR) spectroscopy and CE data to describe a pathological state caused by infection in a mouse.3 Two sets of metabolic profiles were independently processed and analyzed prior to integration by statistical correlation. The strategy uses the structural elucidation power of one analytical method to validate and identify metabolites profiled using another analytical platform, demonstrating the utility for characterizing diseases across different analytical platforms for a range of physiological conditions. Joule heating is an inherent limitation of electrophoretic separations. Temperature increases are known to have adverse effects on the quality of separation and detection, so efforts to actively cool the capillary are employed. This leads to temperature differences at various parts of the capillary including the inlet, outlet, and detection window. Musheev et al. described for the first time an experimental determination of temperature differences across the length of the capillary.4 They found that under typical CE conditions, the temperature at the inlet exceeded the temperature in the cooled regions by more than 15 degrees. The authors also developed a universal method for determining electrolyte temperatures and in both efficiently and inefficiently cooled parts of the capillary.5 In light of the limitations caused by Joule heating, this temperature determination tool will allow operators to circumvent problems with hot spots by knowingly moving samples past the inefficiently cooled sections of the capillary prior to application of the separation voltage. To limit the effects of band broadening caused by Joule heating, separations are often carried out in narrow bore capillaries. However, the decreased size results in poorer detection sensitivity, smaller loading capacities, and increased backpressure. Rogers et al. bundled microstructured fibers, of similar dimensions to CE capillaries, for use in capillary zone electrophoresis (CZE).6 Efficiencies approaching 900 000 plates/m were achieved in separating six Cy5 dye-labeled peptides with a 10 cm length of fiber in under 3 min. Sampling and Preconcentration. Sampling. Thi et al. combined microelectrodialysis (μED) with CE for pretreatment
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apillary electrophoresis (CE) is a field that continues to grow. All areas of CE including theory, separation modes, instrumentation, and applications remain highly active areas of research. This review includes a cross section of references from all areas of the field published in the 2 year period between January 2010 and November 2011. Web of Science reports over 4 000 articles, including 396 reviews, published with CE in the title, abstract, or keywords during this time period. Of these we have chosen 218 papers. We have attempted to choose papers that showcase some of the newest and most exciting developments in the field. It should be noted that papers describing electrophoresis in microfabricated devices were excluded since another review in this issue exclusively covers this topic.
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TECHNIQUES Fundamentals and Separation Modes. Anderson et al. reported the first use of capillary electrophoresis (CE) for Western blot analysis.1 A gel-filled capillary was interfaced with a blotting membrane such that proteins were captured as they eluted, thereby eliminating the need for electro-blotting. Mass detection limits of 10 pg were obtained with little optimization, demonstrating improved mass sensitivity over traditional slab gel Western blots. The technique holds promise for sample limited analysis and situations where high throughput, speed, and automation are desired. © 2011 American Chemical Society
Special Issue: Fundamental and Applied Reviews in Analytical Chemistry Published: December 12, 2011 577
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and determination of inorganic cations in biological matrixes.7 The process of dialysis excludes high molecular weight compounds from CE analysis and is therefore an advantageous sampling tool. Optimized electrodialysis conditions yielded recoveries of 96.3− 110%. Membrane lifetime for pretreatment of biological samples was approximately 100 analyses. Tang et al. developed an approach for sampling and analyzing single aerosolized droplets of solution for CE−laser induced fluorescence (LIF) separation and detection.8 Sample particles were accelerated through a nozzle into a small drop of buffered solution, and upon dissolution a portion of the sample was injected. The approach was used to analyze droplets, smaller than 10 nL in volume, containing fluorescein isothiocyanate (FITC) labeled glycine and glutamic acid. Affinity-Based Preconcentration. Dispersive liquid−liquid microextraction (DLLME) was combined with nonaqueous capillary electrophoresis (NACE) for the first time by HerreraHerrera et al.9 The method was used to determine levels of fluoroquinolone antibiotics in mineral and runoff water. Detection limits in the low μg/L range were achieved. Electro-membrane extraction (EME) was employed as a microextraction method by Nojavan and Fakhari.10 The technique facilitated extraction and concentration of enantiomers from biological samples, demonstrating utility for combination with chiral separations of pharmaceuticals in complex biological matrixes. Detection was reported at the ng/ mL level without any other treatment. Wang et al. demonstrated the successful use of in-capillary deposited gold nanoparticles (AuNPs) as a preconcentration tool. The method was applied to the analysis of monohydroxypolycyclic aromatic hydrocarbons in synthetic urine samples. Enhancement factors of 87−100 were reported along with detection limits ranging from 9 to 14 ng/mL. Park et al. employed single drop microextraction (SDME) to selectively preconcentrate zwitterionic amino acids and peptides prior to electrophoretic separation.11 A 350-fold sensitivity enhancement after a 5 min extraction period was reported. The coupling of hollow fiber-liquid phase microextraction (HF-LPME) with microemulsion electrokinetic chromatography (MEEKC) was described by Lin et al. for the separation of six aromatic amines.12 A 30 s extraction time resulted in enrichment factors ranging from 70 to 157. Detection limits were in the single-digit ng/L range. A microemulsion buffer allowed baseline resolution. Ye et al. described the use of a dynamic pH junction coupled online with CE−electrospray ionization-mass spectrometry (ESI-MS) for the analysis of small peptides with similar pI values.13 They reported baseline resolution of four peptides in an 18.3 min separation window with detection limits in the range of 0.2−2.0 nM. Breadmore et al. investigated the use of an ionic liquid-based liquid phase microextraction as a preconcentration and cleanup method for basic compounds prior to direct injection for CE.14 Capabilities of the technique were demonstrated with the basic dye chryisoidine. Up to 1000-fold enhancements in sensitivity were reported, although 200-fold enhancements were more common. To enhance the sensitivity of a developed micellar electrokinetic chromatography (MEKC)-UV method, Wu et al. investigated the use of a coal cinders microcolumn as a solid phase extractant.15 The phenylenediamine isomer analytes were quantitatively adsorbed by the column, followed by desorption with NaOH. An enrichment factor of 33.3 was reported.
A recently developed concentration technique, micelle to solvent stacking (MSS), was employed by Guidote and Quirino to achieve online sample stacking of charged analytes in CZE.16 Cationic micelles were added to the sample solution to cause stacking of anions loaded in the background solution (see Figure 1). Concentration detection sensitivity was enhanced by an order of magnitude. Liu et al. described MSS in an acidic buffer for MEKC.17 Sodium dodecyl sulfate (SDS) micelles were used as carriers for stacking tetrandrine and fangchinoline mixtures. In total, 113to 123-fold improvements in detection sensitivity were realized over traditional MEKC. Electrophoretic Preconcentration. Bahga et al. employed bidirectional isotachophoresis (ITP) to trigger a transformation from ITP preconcentration to electrophoretic separation.18 The method provided a faster and less dispersive transition than traditional (unidirectional) ITP and eliminated the need for intermediate steps between focusing and separation. Botello et al. applied three different setups of ITP to analyze nonsteroidal anti-inflammatory drug compounds by electrokinetic supercharging in CZE.19 The methodology provided an approximately 2000-fold sensitivity enhancement when compared with results obtained without preconcentration. Dawod et al. reported the first hyphenation of electrokinetic supercharging with electrospray ionization-mass spectrometry (ESI-MS) for the online concentration and separation of five drug compounds.20 Supercharging led to a 1000-fold improvement in sensitivity over conventional injection under fieldamplified sample stacking conditions. Detection limits of 180 ng/L were reported. Busnel et al. integrated transient ITP (t-ITP) preconcentration into a CE−ESI-MS sheathless interface to bring detection limits from the nanomolar range into the sub nM range.21 The platform provided good sensitivity and enabled detection of small amounts of materials with very high resolving power. Marak et al. used preparative ITP (pITP) for sample pretreatment followed by direct infusion mass spectrometry with nanoelectrospray ionization (DI-nESI-MS).22 The method has utility for complex samples due to the significant simplification of the matrix afforded by pITP. Additionally, pITP decreased the concentration limit of detection. Lu and Breadmore used electrokinetic supercharging (EKS) preconcentration in NACE to enhance the sensitivity of phenolic acids.23 Sensitivity enhancement factors between 1333 and 3440 were observed over a normal hydrodynamic injection under the same separation parameters. EKS was found to be 36−79 times more sensitive than large-volume sample stacking (LVSS) and anion selective exhaustive injection (ASEI) for the same phenolic acids. Improvements to EKS were investigated by Xu et al. with the intention of enhancing the sensitivity of conventional CZE to single-digit part per trillion (ppt) levels.24 The first successful demonstration of an ITP generated state in an EKS system without the use of an external terminator was reported. Furthermore, the strategy realized an enrichment factor of 80 000 and detection limits in the ppt level. In another paper, the authors demonstrated even greater sensitivity enhancements by modifying the configuration of sample introduction.25 By increasing the sample vial volume, replacing the wire electrode with a ring electrode, and making other minor adjustments, an enrichment factor of 500 000 was achieved and detection limits dropped to less than 1 ng/L. 578
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Figure 1. Schematic of online concentration of organic anions by MSS in CZE with anodic electroosmotic flow (EOF). The background electrolyte (BGE) solution contained an organic modifier, and the sample (S) solution contained two anionic analytes. The capillary was conditioned with BGE, and S was injected (A). Micelles cross the MSS boundary (B). The analytes focus and accumulate as more micelles cross the MSS boundary (C). Analytes were separated by CZE (D and E). Adapted with permission from ref 16. Copyright 2010 Elsevier.
Hou et al. described two-end field amplified sample injection (TE-FASI), a technique to extend the application of FASI preconcentration.26 TE-FASI accomplished simultaneous stacking of cationic and anionic compounds in CZE. After separation, stacked anions and cations were detected by a common detector. Compared with nonstacking conditions, sensitivities for cationic and anionic compounds were enhanced 1000- and 1380-fold, respectively. Bernad et al. separately evaluated two preconcentration procedures, LVSS and FASI, in the analysis of haloacetic acids in water using CZE.27 A 310-fold sensitivity enhancement was achieved with FASI-CZE, higher than the 25-fold enhancement afforded using LVSS. For optimal application, solid phase extraction (SPE) was employed to remove sample salinity. Detection limits of 0.05−0.8 μg/L were obtained with SPEFASI-CZE. See et al. also tested LVSS and FASI preconcentration procedures for the determination of glyphosate, glufosinate, and aminophosphonic acid in drinking water.28 LVSS achieved detection limits of 1.7−11.1 μg/L and 48- to 53-fold sensitivity enhancements. FASI achieved detection limits as low as 0.1− 2.2 μg/L and realized sensitivity enhancements of up to 245- to 1000-fold. Despite the apparent sensitivity enhancements afforded by field-amplified sample stacking (FASS) preconcentration techniques, Huhn and Pyell have investigated limitations to sample loading caused by decreases in analyte migration
velocity produced by injecting a sample plug of lower electric conductivity.29 They concluded that diffusional band broadening limits the obtainable enrichment efficiency of this technique. Hybrid Preconcentration Techniques. Kitagawa et al. employed a combination of sweeping and stacking mechanisms to accomplish large-volume preconcentration of peptides in NACE.30 Sample solutions were injected as a large-volume plug of up to 80% of the effective capillary length. Limits of detection and quantitation in the sub μM range were reported. Zhu et al. described the online combination of single-drop liquid−liquid−liquid microextraction (SD-LLLME) with CE used in conjunction with LVSS combined sweeping.31 The method was highly reproducible, demonstrated by low relative standard deviations, and achieved a 1030-fold enrichment factor. The technique has utility for commercial CE instruments by offering a method to directly handle complex matrixes. A high-sensitivity offline coupled with an online preconcentration method was described by Luo et al.32 Cloud-point extraction (CPE)/cation-selective exhaustive injection (CSEI) and sweeping-MEKC were integrated for the analysis of malachite green. A sensitivity enhancement of 19 000 was achieved relative to CZE. Cheng et al. developed a triple-stacking CE method with solid-phase extraction for the analysis of methotrexate (MTX) and its metabolites in cerebrospinal fluid.33 Stacking 579
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moiety. Furthermore, it was observed that the hydrophobic environment afforded by the microemulsion strengthened interactions between the chiral selectors and enantiomeric targets. Chen et al. evaluated the capabilities of clindamycin phosphate, a new chiral selector, in the enantioseparation of several racemic drugs using MEKC.43 Clindamycin phosphate belongs to a group of lincosamide antibiotics which have only recently undergone analysis for utility in chiral separations. This work demonstrated good separation of enantiomeric compounds including nefopam, citalopram, tryptophan, chlorphenamine, proporanolol, and metoprolol. SDS enhanced the quality of the separation. Achiral Pseudostationary Phases. The use of surfacemodified silica nanoparticles has increased across a variety of applications. Hui et al. compared the effectiveness of diamineand amine-modified silica nanoparticles as pseudostationary phases in CE.44 When added to the running buffer, the nanoparticles coated the inner wall of the capillary and induced a reversal of the electroosmotic flow (EOF). This was found to significantly enhance enantioseparation both in terms of resolution and selectivity. Behavior of the diamine-modified silica nanoparticles was superior to those modified with amines. Luo et al. used phospholipids as additives to the background electrolyte to alter the selectivity for glycans.45 The additive was shown to enhance the resolution. Different injection techniques were employed to maximize the effect of the phospholipid additive (see Figure 2). Concanavalin A and α 1-2,3 mannose were also incorporated to provide additional selectivity.
was performed by sweeping, and an MEKC separation mode was employed. Detection limits were in the sub μM range. Wei et al. combined poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction with field-enhanced sample injection onto a coated capillary electrochromatography (CEC) column for the analysis of antidepressants in human plasma and urine.34 The method was reproducible over a large linear range and afforded detection limits as low as 3.7− 51.5 μg/L. Li and Hu developed a three-phase liquid phase microextraction procedure, hollow fiber based liquid−liquid−liquid microextraction (HF-LLLME), to determine phenylarsenic compounds.35 Additionally, ASEI was applied for oncolumn stacking. This dual preconcentration platform achieved enhancement factors ranging from 155- to 1780-fold and produced detection limits in the range of 0.68−6.9 μg/L. Buffer Additives. Chiral Selectors. Elbashir and Suliman demonstrated that three chiral primary amine compounds exhibited only partial or no separation when β-CD was employed as a chiral selector but found enhanced separation upon the addition of 18-crown-6 (18C6).36 To explain this phenomenon, the authors performed a molecular modeling study based on molecular mechanics and semi-empirical PM6 calculations. In the presence of 18C6, the authors suggest that a sandwich compound is formed between 18C6, the amine, and β-CD. This significantly increases the binding energy and induces strong hydrophobic and van der Waals interactions, resulting in enhanced enantio-differentiation. Jeon et al. synthesized a chiral selector, carboxymethylated cyclosophoraose (CM-Cys), for the enantioseparation of flavonoids including catechin, hesperidin, hesperetin, and eriodictyol among others.37 Xiao et al. synthesized a series of single-isomer cationic β-cyclodextrins to be employed as chiral selectors for the enantioseparation of carboxylic and hydroxycarboxylic acids and dansyl amino acids.38 The effective mobilities of analytes decreased with increasing selector concentration. Yang et al. described the use of cyclodextrin-modified gold nanoparticles (AuNPs) as an enantioselector for the separation of dinitrophenyl-labeled amino acid enantiomers and drug enantiomers by pseudostationary phase-CEC (PSP-CEC).39 The scheme was successful, such that even low amounts of GNPs afforded good resolution and efficiencies as high as 240 000 theoretical plates per meter. Qi et al. developed a new enantioselective method for determining the enzyme kinetic constant of L-amino acid oxidase.40 The method is based on the principle of ligand exchange CE and used Zn(II)-L-valine as a chiral selecting complex in addition to β-CD. The method was successful in determining the enzymatic rate constant and was further applied for analysis of 20 amino acid enantiomer pairs. Linear polysaccharides have demonstrated powerful enantioselective properties. Chen et al. report the use of branched polysaccharides as chiral selectors in CE for the first time.41 Glycogen, an electrically neutral branched polysaccharide, was employed for the enantioseparation of 18 chiral compounds. Enantiomers of ibuprofen, citalopram, cetirizine, and nefopam were baseline resolved in several Tris-buffered systems. Hu et al. employed chiral oils, L-tartrates and an D-tartrate, with various alcohol moieties to make chiral microemulsion buffer additives for the separation of enantiomeric β-blockers with MEEKC.42 The efficiency of a chiral selector in terms of enantioselectivity and resolution was reported to increase with the number of carbon atoms in the alkyl group of the alcohol
Figure 2. Demonstration of how the injection procedure used in part B improves separation performance of phospholipid media. Sample (solid gray) is introduced either directly into phospholipid buffer (wavy lines) or with aqueous buffer plugs (dotted) before and after the sample injection. Adapted from ref 45. Copyright 2010 American Chemical Society.
Nilsson et al. described the use of porous lipid-based liquid crystalline nanoparticles for CE analysis of proteins in their physiological state using unmodified cyclic olefin copolymer capillaries.46 Without the use of the nanoparticles, proteins irreversibly adsorbed to the inner surface of the capillary. The nanoparticle-based pseudostationary phase both prevented this and enhanced the separation. Closely related variants of native 580
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C(12)-MIM-BF(4) led to 59- and 110-fold enhancements in peak heights and corrected peak areas, respectively. The authors attribute this effect to the capabilities of hydrophobic and π−π interactions afforded by the ionic liquid micelles, allowing for better analyte transport. Capillary Coatings and Surface Modifications. Noncovalent Coatings. Liu et al. developed a dynamic coating method using carboxymethyl chitosan (CMC) to improve the reproducibility and sensitivity of CE analysis.57 The coating was applied by rinsing the capillary with CMC solution for 1 min between each run. The coating demonstrated good stability and reduced adsorption for basic proteins. An average efficiency of 90 000 plates/m was achieved for each protein. A new strategy for coating fused silica capillaries through the adsorption of gold nanoparticles (AuNPs) was described by Qu et al.58 The capillary wall was modified with a polyelectrolyte multilayer (PEM) onto which the AuNPs were adsorbed. The new capillary column was amenable to further surface modifications which were applied for separating neutral analytes and both acidic and basic proteins by open tubular-capillary electrochromatography (OT-CEC). Elhamili et al. described the use of N-methylpolyvinylpyridium as a silica surface modifier for the analysis of peptides and protein digests using CE−ESI-MS.59 The surface modifier was immobilized onto the capillary surface by electrostatic interaction with the ionized silanol groups. The resulting cationic surface reversed the EOF. Peak efficiencies of 430 000 plates/m were reported. Two cationic capillary coatings were employed by Sebastiano et al. for the separation of a suspected impurity in preparations of S-adenosylmethionine (SAM).60 N-Methylpolyvinylpyridinum and divalent barium were both used to coat the capillary, causing a reversal or reduction of EOF, respectively. Both methods were successful in separating the impurity (dcSAM), and divalent barium was able to quantify the amount dcSAM in a commercial sample of SAM. Cao et al. employed a graft copolymer, hydroxyethylcellulose-graf t-poly(2-(dimethylamino) ethyl methacrylate) (HECg-PDMAEMA), to suppress the EOF during CE separation.61 When the polymer was physically adsorbed on the previously bare fused-silica surface, more efficient separations of basic proteins were achieved. An added benefit of this scheme was the ease with which different ratios of PDMAEMA could be grafted, thereby changing the surface charge of the coated capillary. This allowed the analysis of acidic and basic proteins in a single capillary. Zhou et al. described a brush-like copolymer comprised of poly(ethylene glycol) methyl ether methacrylate and N,Ndimethylacrylamide (PEGMA-DMA) used as a physically adsorbed coating for protein separation by CE.62 The coatings were shown to suppress and stabilize the EOF over a broad pH range (2.2−7.8). A series of hyperbranched polycarbosilanes were synthesized and used to coat the inner wall of fused silica capillaries by Xu et al. The terminal groups of the hyperbranched polycarbosilanes were modified with the enantioselective 2-O-(2hydroxypropyl)-β-CD. Capillary performance in chiral separations was demonstrated by separating two chiral isomers, ofloxacin and chlorpheniramine. Shen et al. developed a new amphipathic block copolymer, poly(tert-butyl acrylate)(127)-block-poly(glycidyl methacrylate)(86), for the coating in OT-CEC.63 This coating acted both as a surfactant and enhanced the separation of steroids.
green fluorescent protein were separated with efficiencies of 400 000 plates/m within a 2.5 min separation window. With the increasing use of nanoparticles as pseudostationary phases, it is important to understand how nanoparticle chemistry, composition, and concentration affect the separation. Subramaniam et al. reported how varying the length of a nanoparticle pseudostationary phase plug affected the CE separation.47 11-Mercaptoundecanoic acid functionalized gold (Au@MUA) nanoparticles were employed for the separation of Parkinson’s disease biomarkers including dopamine, epinephrine, and glutathione among others. The migration times and peak areas of the biomarkers changed when the plug length was varied at a fixed separation voltage. Liu et al. investigated the influence of cationic poly(amidoamine) (PAMAM) dendrimers on the separation of proteins in acidic buffers using CE−UV.48 PAMAMs adsorbed to the inner wall of the capillary and both suppressed adsorption of proteins and enhanced buffer selectivity toward the proteins. Cao et al. reported the combined use of anionic and cationic surfactants to act as a modified pseudostationary phase in MEEKC for the separation of flavonoids.49 The mixed use of surfactants demonstrated significantly enhanced separation efficiency, and 185- to 508-fold sensitivity enhancements were reported. Cao et al. also investigated the use of single-walled carbon nanotubes (SWNTs) with a different dispersion as additives in MEEKC.50 They compared the effectiveness of surfactantcoated SWNTs (SC-SWNTs) with carboxylic SWNTs for the separation of flavonoids. The SC-SWNT microemulsion was found to be more efficient for the separation. Ionic Liquids. Bwambok et al. synthesized and characterized a new fluorescent chiral ionic liquid (FCIL), L-phenylalanine ethyl ester bis(trifluoromethane) sulfonamide (L-PheC(2)NTf(2)), which has utility as a solvent, chiral selector, and fluorescent reporter.51 This strategy simultaneously offered both chemo- and enantioselectivity toward a variety of analytes. The influence of 1-dodecyl-3-methyl-imidazolium (C(12)MIM)based ionic liquid on the separation of zwitterionic fluoroquinolones with CE was investigated by Liu et al.52 It was reported that sub mM amounts of C(12)MIM-based ionic liquid was effective causing significant separation enhancements. Li et al. employed a polymeric ionic liquid (PIL), poly(1vinyl-3-butylimidazolium) bromide, as a dynamic coating additive for the separation of four basic proteins.53 When added to the BGE solution, the PIL both reversed the EOF by establishing a cationic coating on the inner capillary wall and prevented adsorption of the protein targets. Peak efficiencies ranging from 247 000 to 540 000 plates/m were achieved within an 11 min separation window. The authors also used this PIL in the coelectroosmotic CE separation of aromatic acids.54 Separation efficiencies of 355 000−943 000 plates/m were achieved. Su et al. used SDS and 1-butyl-3-methylimidazolium-based ionic liquids as buffer additives to improve the separation of benzodiazepines. 5 5 1-Butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([BMIM][NTf(2)]) was the most efficient additive due to the interaction of its anionic moiety with the benzodiazepines. Quirino et al. described the use of the long chain ionic liquidtype cationic surfactant, 1-dodecyl-3-methylimidazolium tetrafluoroborate (C(12)-MIM-BF(4)), to enhance the performance of MSS preconcentration in CZE.56 The inclusion of 581
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Figure 3. Schematic representation illustrating the inner wall of (A) fused silica, (B) fullerenol, and (C) latex diol capillaries. Adapted with permission from ref 67. Copyright 2010 Wiley.
allowed separation of the five target compounds in 10 min with small (0.52%) relative standard deviations in migration times. Lin et al. employed a hyperbranched polyether coated capillary to analyze organophosphorous pesticides with MEKC.69 The hyperbranched polyether coatings reduced EOF which subsequently enhanced the detection sensitivity. The coated columns demonstrated good stability. Hu et al. described the covalent attachment of chitosan and silica to the inner wall of an open-tubular (OT) column using a chitosan and silane cross-linking agent (γ-glycidoxy-propyltrimethoxysilane) for CEC.70 The column demonstrated good selectivity for nucleotides, aromatic acids, and inorganic anions. J. L. Chen prepared untreated multiwall carbon nanotube materials (MWNTs), HNO(3)-treated MWNTs, and HNO(3)HCl-treated MWNTs for the covalent attachment inside a silicahydride modified CEC capillary.71 The treated MWNTs resulted in higher EOF values. Luces et al. described a mixed mode separation employing a combination of MEKC and polyelectrolyte multilayer (PEM) coatings for achiral and chiral separations.72 Partial separation of analytes was achieved using each mode individually, but baseline resolution was observed in mixed mode. Stationary Phases for CEC. Li et al. reported the development of several chiral stationary phases (CSPs) with chiral recognition based on the immobilization of β-CD derivatives on an epoxy-activated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith.73 The CSPs were applied to the CEC separation of amino acids and chiral drugs. Jiminez-Soto et al. investigated the use of single-walled carbon nanohorns (SWNHs) as pseudostationary and stationary phases for electrokinetic chromatography and CEC, respectively.74 To determine the efficacy of SWNHs as a
Cheng et al. investigated the use of a molecular film assembled on a capillary surface for the chiral separation of sertraline by MEEKC.64 The assembled film consisted of poly(diallyldimethylammonium-chloride) and β-CD. Baseline resolution was achieved for the four sertraline isomers and enantiomers. Separation efficiency was further enhanced with a borate buffer microemulsion containing ACN, SDS, n-butanol, and n-hexane. Moore et al. synthesized a zwitterionic molecular micelle, poly(ε-sodium-undecanoyl lysinate) (poly ε-SUK), to be employed as a coating in OT-CEC.65 The zwitterionic nature of the micelle, containing both carboxylic acid and amine moieties, allows the coating to have either an overall positive or negative charge. The utility of this coating was shown by separating proteins in human sera under both acidic and basic conditions. Covalent Coatings. He et al. covalently attached δ-gluconolactone to an aminopropyl-derivatized capillary to enhance the separation of biopolymers by CE.66 The resulting hydrophilic coating suppressed EOF and minimized protein absorption, allowing for separations of basic proteins and DNA with efficiencies upward of 450 000 plates/m. Bachmann et al. described the covalent attachment of derivatized polystyrene nanoparticles and derivatized fullerenes to the inner capillary surface for the analysis of peptides and protein digests by CE (see Figure 3).67 Low relative standard deviations in measurements indicated good run-to-run and batch-to-batch reproducibility. The applicability of this procedure was realized by combining CE with MALDI-MS for complex sample analysis. Iwamuro et al. employed a chemically modified capillary with amino groups in the analysis of phosphorus-containing amino acid-type herbicides with CE−MS.68 The amino capillary 582
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however, because the salts and surfactants employed in separation buffer suppress ionization efficiencies and can lead to contamination. Barbula et al. have developed a new interfacing scheme for CE−MS using desorption ESI (DESI) to minimize this effect.84 Two separations were performed with this scheme, and in both cases the effects of salts and detergents on MS analysis were reduced. Lu et al. described coupling SDS-capillary gel electrophoresis (CGE) with matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF-MS) by a poly(tetrafluoroethylene) (PTFE) membrane.85 The PTFE membrane trapped SDS-capillary gel electrophoresis (CGE) separated proteins; SDS is then easily washed away, allowing for subsequent analysis of on-membrane proteins with MALDITOF-MS (see Figure 4). The ability to immunoblot and Coomassiestain the collected proteins was also demonstrated.
pseudostationary phase, their dispersion in surfactants was explored. For CEC, SWNHs were carboxylated and subsequently immobilized in the capillary to produce a stable stationary phase. When compared with the performance of SWNTs for the separation of water-soluble vitamins, it was found that the SWNHs were less useful in EKS but enhanced the separation in CEC. Sun et al. developed a new CSP by bonding isopropylcarbamate functionalized cyclofrucan6 (IP-CF6) to silica gel.75 The new capillary was used for the enantioseparation of 119 racemic compounds containing a primary amine group. Separation of 93% of the compounds was reported, demonstrating good enantioselectivity of the new CSP. An enantioselective OT-CEC column was made with thiolated β-CD modified gold nanoparticles (CD-GNPs) as a stationary phase by Li et al.76 Separation capabilities were demonstrated by analysis of three drug enantiomers. Karenga et al. developed segmented monolithic columns (SMCs) made from two adjoining segments filled with different monoliths.77 One segment was filled with naphthyl methacrylate monolith (NMM) for hydrophobic and π-interactions; the other segment was filled with octadecyl acrylate monolith (ODM) for hydrophobic interactions. The differences in retention mechanisms between the two monoliths allowed for manipulation of the EOF. Zhou et al. described the modification of an open tubular (OT) column with a newly developed stationary phase, hydrophilic polysaccharide CMC, for the separation of basic proteins and opium alkaloids using CEC.78 The column demonstrated stability and high tolerance against acids and bases (0.1 M HCl, 0.1 M NaOH) and organic solvents. Column efficiencies ranging from 92 000 to 132 000 plates/m were reported for the separation of four opium alkaloids in phosphate buffer. Chen et al. designed a porous polymethacrylate ester-based monolithic column for separation of flavonoids by CEC.79 Lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA), and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) were copolymerized in situ to establish the monolith. The column separated four flavonoids in less than 10 min. Miscellaneous. Mei et al. employed three preparation methods for coating capillaries with liposomes: physical adsorption, avidin−biotin binding, and covalent coupling.80 The coating methods were compared based on EOF characterization and performance in neutral compound separations. Detection. Mass Spectrometry (MS). Borges-Alvarez et al. reported on factors influencing the analysis of and identification of peptides using CE coupled online to orthogonal accelerated TOF-MS (CE-oa-TOF-MS) through a sheath-flow ESI interface.81 CE-oa-TOF-MS demonstrated a 10-fold improvement in sensitivity over other CE−MS techniques. The application of MS detection for MEEKC is limited due to ion suppression by the commonly used surfactant SDS. The first example of coupling MEEKC to inductively coupled plasma mass spectrometry (ICPMS) for the separation Pt(II) and Pt(IV) anticancer drugs was presented by Bytzek et al.82 A sheath flow interface was used for coupling. Himmelsbach et al. described a method for the analysis of methylated melamines by CZE with Q-TOF-MS.83 Detection limits of 0.01 mg/L were reported with mass errors lower than 2.3 ppm. The coupling of CE-based separations with MS is usually done with ESI. The capabilities of this mechanism are limited,
Figure 4. Example of results obtained by coupling SDS-CGE with MALDI-TOF-MS. The SDS-CGE trace was monitored with UV absorbance detection (A). PTFE collected proteins were collected and subsequently analyzed by MS (B). Inset I presents an image of a PTFE membrane with collected proteins stained on it. Inset II is an expanded MALDI-TOF-MS spectrum. Adapted from ref 85. Copyright 2011 American Chemical Society.
The first online coupling of chiral micellar electrokinetic chromatography (CMEKC) to atmospheric pressure photoionization MS (APPI-MS) was described by He et al.86 Simultaneous enantioseparation and online detection of four photoinitiators was reported. Maxwell et al. described an interface for CE−ESI-MS which decouples the electrical and solution flow rate requirements of the separation and ionization processes.87 The terminating end of the separation capillary was inserted into a stainless steel 583
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properties of the CNT electrodes allowed for detection limits in the low μM range to be achieved. Despite the potential for fouling, the electrode demonstrated good stability. Fang et al. employed fast-scan cyclic voltammetry (FSCV) detection for a CE separation of neurotransmitters.97 FSCV is advantageous over other amperometric detection schemes because peak identification could be made both by migration time and the cyclic voltammogram. Detection limits as low as 1 nM were reported. Multiple Dimension Separations. Anouti et al. employed a transient moving chemical reaction boundary (tMCRB) to improve the sensitivity of heart-cutting twodimensional (2D)-CE.98 tMCRB was achieved using an ammonium formate sample matrix and acetic acid as a BGE in the first dimension. A stacked fraction from the first dimension was selected, isolated, and separated further in the second dimension using a chiral selector, ((+)-(18-Crown-6)2,3,11,12-tetra-carboxylic acid). The method was used for the enantioseparation of D,L-phenylalanine, and D,L-threonine in with a mixture of 22 native amino acids. An integrated 2D system for both preconcentration and separation was described by Zhang et al.99 Sweeping with an electrokinetic preconcentration was used to inject a large volume of sample solution into the MEKC-CZE 2D scheme. Analyte focusing by micelle collapse (AFMC) was introduced between the two separation dimensions to release analytes from the micelle interior into a liquid zone (see Figure 5). The use of
needle. Here, the inside of the electrode acts as the CE outlet vial, and the outside acts as the ESI emitter. The detection limits for amino acids were typically 5-fold greater when compared with commercial, sheath-flow based CE−MS interfaces. Spectroscopy. Chen et al. developed a method for the detection of organophosphorus pesticides using quantum dot (QD)-enhanced LIF detection for CE (CE−QD/LIF). 88 Quantum dots were immobilized onto the inner wall of the capillary, where they were able to selectively enhance analyte detection. Detection limits ranging from 50 to 180 μg/kg were reported. Diekmann et al. reported a portable microcoil NMR detection scheme for coupling to CE.89 Previous CE−NMR systems employed large NMR magnets and spectrometers which subsequently required long CE separation capillaries. The microcoils offered a briefcase-sized NMR system with improved mass sensitivity. An LOD of 31.8 nmol for perfluorotributylamine was reported with a resolution of 4 ppm. Dada et al. reported a dynamic range for CE−LIF spanning 9 orders of magnitude, approaching the fundamental limits of the system, by employing cascaded avalanche photodiode photon counters.90 The LIF detector used a cascade of four fiber-optic beam splitters connected in series to generate four attenuated signals along with the primary signal. Each line was subsequently monitored by a single-photon counting avalanche photodiode. Scaling allowed the production of a linear optical calibration curve for 5-carboxyl-tetramethylrhodamine from the concentration detection limit of 1 pM up to 1 mM. A mass detection limit of 120 yoctomol was observed by injection into the instrument. A rotary cell for improving the capabilities of CE− chemiluminescence (CE−CL) coupled systems was developed by Wang et al.91 The rotary reaction cell reduced problems with bubble formation and flow blockage. Detection limits were reported as low as 0.91 nM for horseradish-peroxidase in proofof-concept experiments. Another approach for improving the coupling of CE−CL was described by Xu et al.92 Here, an oncolumn fracture is used to decouple the electric field from the CL detection. A detection limit of 0.1 nM was reported for acridinium ester. Ryvolova et al. combined contactless conductometric, photometric, and fluorimetric on-capillary detection methods for single point detection.93 The utility of a single point detection scheme using various detection modes was demonstrated in the analysis of sample mixtures containing fluorescent and nonfluorescent dyes, common ions, underivatized amino acids, and a fluorescently labeled digest of bovine serum albumin (BSA). Electrochemical. A graphene/poly(urea-formaldehyde) composite modified electrode was fabricated and employed as an amperometric detector for CE separations by Chen et al.94 The electrode demonstrated low detection potentials, enhanced S/N characteristics, and high resistance to surface fouling. Li et al. coupled CE with dual electrochemical (EC) and electrochemiluminescence (ECL) detection for the analysis of cardiovascular drugs.95 A central composite design was used for optimizing the operating conditions, and the system was employed in urine sample analysis. A method for analyzing polyphenols in wine by CZE with carbon nanotube (CNT)-modified electrodes for amperometric detection was described by Moreno et al.96 The electrochemical
Figure 5. Results collected from the 2D-MEKC-AFMC/ZCE separation of flavonoids. MEKC is employed as the first dimension separation mode. AFMC preconcentrates fractions from the first dimension prior to CZE separation in the second dimension. Adapted from ref 99. Copyright 2011 American Chemical Society.
dual concentration methods increased detection factors 6000fold when compared to conventional pressure injections. Limits of detection were in the ng/L range, and theoretical plate numbers were on the order of 10 000. Wojcik et al. described a form of 2D-CE, diagonal CE, which employs identical separation modes in each dimension.100 The utility of the platform in studying enzymes was demonstrated with phosphopeptide characterization. The assay facilitates detection of post-translational modifications assuming availability of an immobilized enzyme which reacts under electrophoretic conditions. Li et al. describe the combination of wide-bore electrophoresis (WBE) with CE.101 Three electrodes were used to separate and transfer samples between WBE and CE in a 584
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continuous manner. This avoids problems often associated with precise transfer timing of sample zones from the first dimension into the second. A new LC−CE interface was developed by Skinner et al.102 The interface introduced two second dimension CE capillaries to sample effluent from the chromatographic first dimension. The use of multiple capillaries demonstrated higher first dimension sampling rates and has utility for multiple second dimension separation modes. Dickerson et al. described a method for coupling capillary isoelectric focusing (CIEF) with CZE and LIF detection.103 A focusing step was performed in the first dimension on fluorescently labeled proteins (pIs were preserved). Through a buffer-filled interface, fractions of the first dimension were transferred to the second dimension for further separation. A spot capacity of 125 was reported.
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APPLICATIONS Nucleic Acids. Separation and Sequencing. CE is the primary tool for separation and analysis of DNA. Gao et al. utilized star-shaped poly(N,N-dimethylacrylamide) (PDMA) polymers in capillary gel electrophoresis (CGE) to reduce the viscosity of the separation matrixes commonly used.104 High resolution was achieved between 271 bp and 281 bp, and the starlike polymers were shown to offer higher separation performance than linear polymers. A pulsed field CE method using hydroxyethylcellulose polymer was optimized for the separation of DNA fragments by Li et al.105 Polymer concentration, pulse field strength, pulse frequency, and modulation depth were all varied. It was found that small DNA fragments were better separated with low polymer concentration and modulation depth, whereas larger fragments were better separated with high polymer concentration and modulation depth. Albrecht et al. presented a free-solution conjugate electrophoresis (FSCE) method using drag-tags as long as 516 amino acids for the rapid sequencing of DNA.106 Using a four-color laser induced fluorescence (LIF) detector, 256 bases were read in 30 min using a drag tag of 267 amino acids (see Figure 6). This sequencing was shown to be comparable to the average DNA reading capabilities of current next-generation sequencing systems. Detection of Mutations. The γ-glutamyl hydrolase (GGH) gene plays a role in the metabolism of methotrexate, a chemotherapeutic for the treatment of arthritis and leukemia. A single-strand conformation polymorphism (SSCP) CE method was developed by Chen et al. for the screening of SNP 452 C > T in the GGH gene.107 Genotyping was accomplished in a simple, rapid, and reproducible manner, with results coinciding with DNA sequencing. Methylated bases are only found in a small fraction of the genome; however, they can display significant biological effects. Krais et al. reported a MEKC separation with LIF detection based on fluorescence labeling with BODIPY FL EDA to detect 2′-deoxy-N6-methyladenosine (N6mdA).108 The LOD for N6mdA was 1.4 amol, which was equivalent to 0.01% abundance. The assay was further applied to the analysis of methylation levels of cytosine 5-hydroxymethyl2′-deoxycytidine (5 hmdC). Krais et al. also used CE−LIF to determine 5 hmdC levels in DNA.109 A dsDNA fragment containing a known amount of 5 hmdC was used to optimize separation conditions. The method
Figure 6. Four-color sequencing electropherogram using 267-aa drag tag. Adapted from ref 106. Copyright 2011 American Chemical Society.
demonstrated a LOD of 0.45 amol and was used to show the presence of 5 hmdC in human tissue and cancer cells. Benesova et al. developed a CE method for the assay of the L858R mutation in exon 21 of the epidermal growth factor receptor gene.110 Separation of heteroduplexes was optimized under partially denaturing conditions using 8, 16, and 96 capillary arrays on a conventional CE platform. Detection and Quantification. Hassel et al. developed a MEKC-LIF method for the total nucleotide analysis of both DNA and RNA in freshwater polyp Hydra magnipapillata.111 Two versions of the assay were developed using either deoxyribonucleoside-5′-monophosphates or deoxyribonucleoside-3′-monophosephates for digestion. Both revealed high A+T compositions of 78 and 71% while total DNA methylation was 2.6 and 3.1%. Results were shown to be in good agreement with those found by an established 32P-postlabeling method. Turner et al. used a CE−LIF based approach to monitor the cellular uptake of a 12-mer peptide nucleic acid which was disulfide bridged with other peptides both with and without cell-penetrating features.112 By monitoring the down regulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes, it was determined uptake occurred independent of the cell-penetrating properties of the peptides. Fujii et al. coupled CE with inductively coupled plasma mass spectrometry (ICPMS) for the determination of phosphorus in enzymatically digested DNA as an alternative method of DNA quantification.113 The method was also compared to conventional fluorescence detection, showing similar quantification numbers of DNA and equivalent LODs of 3.1−26 ng/mL. A novel assay for the simultaneous detection of multiple mRNAs was developed by Jiang et al.114 Using denaturing CGE−LIF combined with a tandem adenosine-tailed DNA bridge-assisted splinted ligation, a single capillary was used to detect five mRNAs of Epstein−Barr virus, in addition to mRNA isomers. The linear range of the assay covered 3 orders of magnitude and detection limits were as low as 2.5 zmol. 585
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Figure 7. Urinary peptide patterns displaying differences between chronic kidney disease and human controls. Adapted with permission from ref 116. Copyright 2010 American Society for Biochemistry and Molecular Biology.
́ CGE−LIF was used by Garcia-Cañ as for the monitoring of ligation reactions in the production of long DNA probes.115 The method provided insight into the nature of impurities and allowed for the optimization of reaction conditions. As a result, high-quality DNA probes were obtained and shown to sensitively detect percentages of transgenic maize lower than 1%. Proteins and Peptides. Proteomics. Urine is an attractive source for clinical proteomics due to its ease of collection and correlation with physiology. Good et al. established a CE−MS method for peptidome analysis of urinary peptides and proteins ranging from 800 to 17 000 Da.116 Using defined biomarkers, chronic kidney disease diagnosis was achieved with 85.5% sensitivity and 100% specificity (see Figure 7). Proteomic analysis as a tool for diagnosis of disease is rapidly advancing. Alkhalaf et al. used CE−MS for the diagnosis of diabetic nephropathy (DN).117 Urinary proteome analysis was shown to have 93.8% sensitivity and 91.4% specificity in identifying patients with DN. Significant differences were shown in 60 peptide biomarkers in cases versus controls. Delles et al. used CE with time-of-flight (TOF) MS to diagnose coronary artery disease (CAD) through urinary proteomics.118A pattern of 238 CAD-specific polypeptides were found and used to predict CAD with high confidence as well as monitor the effects of treatment with irbesartan. CE−MS was used by Drube et al. for urinary proteomic analysis in the detection of ureteropelvic junction obstruction (UPJO).119 Results allowed for the prediction of UPJO with 83% sensitivity and 92% specificity for children
