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Hyphenation in Capillary Electrophoresis: from Sample Pretreatment to Data Analysis

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Hyphenation in Capillary Electrophoresis: from Sample Pretreatment to Data Analysis Hyphenation in Capillary Electrophoresis: From Sample Pretreatment to Data Analysis Javier Saurina, Department of Analytical Chemistry, University of Barcelona, Spain. This article describes a hyphenated system composed of a continuous- flow manifold for sample pretreatment coupled on-line to a capillary electrophoresis (CE)–diode array system. In combination with chemometric analysis the possibilities of extracting more information from CE data are explored. Introduction • Robotic systems: The use of a robotic arm to introduce a vial Hyphenations in liquid and gas chromatography are mature containing a discrete volume of pretreated sample into the disciplines and systems such as liquid chromatography–mass CE system.1,2 spectrometry (LC–MS) and gas chromatography–mass • Flow methods: Sample treatment is performed in a flow spectrometry (GC–MS) are well established techniques system. The versatility and feasibility of flow systems commonly used in analytical laboratories. In comparison with (including flow-injection analysis and continuous-flow chromatography, hyphenation in capillary electrophoresis (CE) analysis) make these procedures especially powerful for is still in its infancy, but is receiving increasingly more implementing certain preliminary operations of an analytical attention. Critical aspects of CE hyphenation include the process.3–5 minute volumes of sample injected (typically a few nL) and On-line procedures open up the possibility of automation, small flow-rates (in the order of nL/min). To solve these simplification and miniaturization of a wide variety of sample technical limitations interfaces have been developed either by pretreatments for routine applications. These procedures may adapting existing high performance liquid chromatography also lead to improved precision, higher sample throughput, (HPLC) ones or through the design of completely new ones. reduced sample and reagent consumption, and more cost- However, the aim of this article is not to discuss new effective operations. Consequently, the hyphenation of developments in the coupling of detectors to CE, but instead preliminary sample treatments with separation techniques to focus on hyphenation from a different perspective; that is, seems to be an attractive way to enhance the analytical the integration of experimental steps that comprise the potential, providing more robust and reliable methods. analytical procedure. Of course, on-line procedures combined with CE also have From this perspective, a CE method should not be thought some disadvantages. The main problem in these couplings of merely as a separation of analytes, but as a series of steps arises in the design of a suitable interface to make system flow- leading to the resolution of an analytical problem (Figure 1). rates compatible with the low injection volumes required in Among these steps are sample pretreatments prior to sample CE. In fact, the interfaces in question are not yet commercially injection and data analysis following chromatographic available. separation. Another important feature of hyphenated systems is the huge Solid-phase extraction, liquid–liquid extraction, dialysis, gas amount of data they are capable of generating. Typically, a CE diffusion and derivatization are among the typical sample system coupled to a fast-scanning spectrometric detector (e.g., treatments required prior to sample injection into the CE diode array devices or mass spectrometers) records full spectra system. The main objectives of these preliminary steps include at regular intervals over the entire electropherogram. The analyte preconcentration, removal of sample matrix and obtained three-dimensional (3D) data provide a rich source of improvement of analyte detection. In most instances, these information that can be interpreted or analysed by appropriate sample preparation techniques are implemented through chemometric techniques.6–11 Thus, the analytical potential of manual, off-line procedures. However, the shortcomings of hyphenated systems is greatly enhanced in combination with off-line manipulations include reduced precision, increased mathematical tools for extracting information from a 3D data time consumption and manual handling of toxic reagents and analysis. organic solvents. This article describes a hyphenated system comprising of a Various strategies have, therefore, been proposed for continuous-flow derivatization (CFD) system coupled to developing on-line hyphenated CE systems, including CE–diode array detection (DAD), followed by chemometric 2 LC•GC Europe November 2002 Saurina data analysis (CDA). The potential of this approach is A CE method should not be thought of illustrated by the CE determination of amino acids. merely as a separation of analytes, but as a series of steps leading to the resolution Experimental Hyphenated system: Figure 2 shows a scheme of the of an analytical problem. CFD–CE–DAD–CDA system. Here, amino acids were labelled with 1,2-naphthoquinone-4-sulfonate (NQS) to yield Partial Least Square Regression. More specific programs for derivatives that could be detected spectrophotometrically (see checking peak purities and deconvoluting overlapping peaks the reaction scheme in Figure 2). were obtained from the Chemometrics Group within the The continuous-flow set-up was composed of channels for Analytical Chemistry Department of the University of sample, reagent (0.06 M NQS ϩ 0.1 M HCl) and buffer Barcelona, Spain (see http://www.ub.es/gesq/mcr/mcr.htm). ϩ (0.05 M Na2B4O7 0.1 M NaOH) solutions. Solutions were pumped by a peristaltic pump using standard Tygon tubing. Results and Discussion The derivatization reaction was developed in a PTFE reaction A comparison of manual off-line and on-line derivatization coil (3 m ϫ 1.1 mm i.d.) heated to 70 °C using a water bath. methods for amino acid analysis with NQS showed that the The resulting derivatized sample solution was continuously continuous-flow procedure was much faster and more introduced into the flow interface (Figure 3). The level of straightforward. When the process was performed manually liquid in the interface was kept constant by means of a waste additional steps, such as acidification and filtration, were channel while derivatization was performed. After sample necessary (Figure 5).14 Conversely, these steps were injection, the interface was quickly emptied through an unnecessary in on-line derivatization because the formation of additional channel (see Figure 3). relatively insoluble components, from side reactions, was A P/ACE capillary electropherograph (Beckman Coulter, negligible in the flow system. Minimizing side reactions also Fullerton, California, USA), with a diode array helped to avoid ghost peaks appearing in the electropherogram. spectrophotometric detector was used. Electrophoretic runs This fact was attributed to the continuous renewal of sample were acquired in the spectral range 190 to 800 nm at regular solution in the interface of the CFD system so that freshly 1 s intervals. The corresponding data were processed with derivatized samples were always injected. Beckman P/ACE station (v 1.0) software. Injections were performed electrokinetically by applying 10 kV for 25 s. A fused-silica capillary (Teknocroma, Sant Figure 1: General guidelines for the evaluation of CE data. Cugat, Barcelona, Spain) of 75 µm i.d. (375 µm o.d.), with an Resolution effective length of 58.7 cm and a total length of 67 cm was Sample Separation Detection Data of the used. Amino acid derivatives were separated at 20 kV and treatment techniques techniques treatment analytical 25 °C using a running buffer consisting of 40 mM sodium problem Integration of the analytical process tetraborate aqueous solution (pH 9.2) plus isopropanol (30%, v/v). A forward pressure of 0.5 psi was applied 35 min after injection to accelerate migration of the acidic amino acids. Data generation and analysis: Figure 4 shows a 3D plot obtained from a CE–DAD system in which the absorbance Figure 2: (a) Schematic of the CFD–CE–DAD–CDA and values were taken as a function of wavelength and migration (b) the derivatization reaction. time. From such data, peak areas, spectral and electrophoretic profiles, and even more complex arrangements (e.g., full (a) Capillary electrophoresis spectra over the migration time) can be used as analytical Pumps responses in further studies. Thermostatic Diode array detector In general, the analysis of CE data is mainly aimed at Sample bath Mixing quantifying the analytes of interest. For this purpose, univariate Buffer coil calibration methods are commonly used, although multivariate NQS Reaction methods could be applied to multidimensional data, such as coil spectral or electrophoretic profiles. Other relevant tasks PC (e.g., checking for peak purity, sample characterization and Interface Waste channel classification) may be performed with a mathematical treatment of CE data. Additional channel A description of the wide variety of chemometric tools for the analysis of CE–DAD data is beyond the scope of this article, but more information can be found in references 6–11. Software: The P/ACE software was used to generate ASCII (b) O O files suitable for analysis with MATLAB (The MathWorks Inc., O O Natick, Massachusetts, USA).12 MATLAB includes some R1 ϩ ϭ software packages for statistics and artificial neural networks NH pH 9 Time 2 min R2 etc., for characterization and quantification. Multivariate R 13 1 calibration methods were available using PLS Toolbox. This SO3 N toolbox includes mathematical
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