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

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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 0.1 M HCl) and buffer Barcelona, Spain (see http://www.ub.es/gesq/mcr/mcr.htm). (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 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 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 programs for Principal R2 Component Analysis, Principal Component Regression and www.lcgceurope.com 3 Saurina

The most significant parameters affecting the CFD system were temperature, pH, reagent concentration and residence The most significant parameters affecting time. The experimental ranges under study were 40–90 °C for the CFD system were temperature, pH, temperature, 8–11 for pH and 1–10 mM NQS. The influence of residence time was evaluated for a combination of total flow- reagent concentration and residence time. rates (from 0.7–2.4 mL/min) and reaction coil dimensions (reaction coil length from 1–6 m and i.d. 1.1 mm). The results indicated that stronger reaction conditions were required for An electrokinetic injection mode was chosen because of the the derivatization of acidic amino acids compared with non- design of the flow interface. Hydrodynamic injection was not acidic amino acids which could be derivatized under milder recommended because the pressure applied for the injection conditions. Conditions finally selected (see experimental could damage the interface. Furthermore, electrokinetic section) were those that led to a quantitative formation of injection provided enhanced sensitivity as the analyte derivatives. In our opinion, complete formation of derivatives preconcentration was based on stacking processes (e.g., field- would not strictly be required in the continuous-flow amplified injection). derivatization (note that this is a crucial point in off-line For the optimization of separation conditions the following precapillary derivatization). This assertion is supported by the parameters were considered: injection volume, composition of fact that the degree of development of the derivatization the running buffer, voltage and temperature. Despite much reaction of a given analyte in the flow system is the same in any effort spent achieving a suitable separation of all amino acid run, and it is reproducible from sample to sample. These results derivatives, problems dealing with poorly resolved peaks may are a direct consequence of the timing control that is still arise (Figure 6). In this instance, overlapping peaks could characteristic of flow systems. be attributed to the high structural similarities of amino acid derivatives. However, this is where the application of chemometric methods could be of great importance. Figure 3: Detail of the continuous flow interface. A scheme showing the general guidelines proposed for the evaluation of CE data is shown in Figure 7. In the classic Pt electrode CE capillary approach, quantification is commonly based on univariate calibration with linear regression, using the peak areas (or the peak heights) as analytical responses. This strategy is valid when Out (for emptying the interface) the electrophoretic peaks are completely resolved. However, with unresolved peaks selectivity of the analyte signal is not

Waste achieved so that the determination is interfered with. For this In Out reason, mathematical methods for checking the purity or From the continuous flow derivatization system homogeneity of CE peaks are useful. Peak purity studies are crucial for investigating the presence of interferences hidden in the CE peak of a given analyte. An experimental strategy for Level of liquid solving this drawback is based on re-optimizing the separation conditions to avoid co-migrations. However, depending on the complexity of the sample, a full separation of all components might not be achievable.

Figure 4: Example of a 3D electropherogram recorded with a CE–DAD system and chemometric strategies for the CE–DAD data analysis. Types of data: (a) peak area (or peak height) at a given wavelength; (b) spectral profile at a given migration time; (c) time profile at a given wavelength; (d) full spectra taken over migration time.

15 1 (a) t Univariate 1 10 calibration 4 1 n 2 3 (b) 1 Multivariate 5 t t calibration 1 n 5 (c) (PLS, ANN,...)

0 (d) 1 n

Absorbance (mAU) t 1 600 Peak purity -5 Characterization Peak resolution 40 400 t 39 n 38 37 Time (min) Wavelength (nm)

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Derivatization and sample with which it was impossible to quantify the amino acids in this preconcentration procedures deserve particular system. special attention in CE because low Conclusions sensitivity and poor detection limits are the Derivatization and sample preconcentration procedures deserve main weaknesses of this technique. special attention in CE because low sensitivity and poor detection limits are the main weaknesses of this technique. Here, derivatization is performed in a continuous-flow system An alternative approach lies in chemometric methods for coupled to CE. The preconcentration in CE can be • deconvoluting each component present in overlapping peaks accomplished especially via stacking processes. Although in the to obtain pure peak profiles present study field-amplified injection was applied as a • improving the quantification in overlapping peaks. preconcentration technique, more efficient procedures such as Both spectral and electrophoretic profiles can be used as large-volume sample stacking could be implemented. multivariate data for further analysis using methods such as Furthermore, depending on the analytical problem and the partial least square regression (PLS) or artificial neural characteristics of the sample, additional preliminary operations networks (ANN). Furthermore, more complex 3D data can be may be required. For instance, solid-phase extraction, dialysis analysed with factor analysis techniques (e.g., evolving factor or gas diffusion could be sequentially combined with the analysis or multivariate curve resolution). derivatization plus preconcentration procedure according to Thus, a reasonably accurate quantification could be analytical needs. Another aspect of the proposed hyphenated accomplished using multivariate calibration methods, even system is the application of chemometric techniques for data when the analytes were present in poorly resolved peaks. As an analysis. The combination of CE with mathematical treatments example, strongly overlapping components such as histidine takes advantage of synergistic effects on the analyte resolution and isoleucine derivatives were determined in sample mixtures which cannot be reached separately; that is, via the analytical with prediction errors ranging from 5–10%. These results power of the electrophoretic and mathematical separation of represent a tremendous improvement with respect to univariate compounds. Finally, in contrast to expensive systems calibration (with prediction errors of approximately 50–100%), combining series of instrumental techniques, the present approach is readily available in most laboratories and can be easily adapted to the study of other analytical problems. Figure 5: Scheme of the manual off-line precapillary derivatization procedure of amino acids with NQS. Figure 7: Flow diagram of steps involved in the analysis of Injection multidimensional CE data generated from hyphenated systems. (a) classic approach when having resolved peaks; (b) alternative chemometric approach when having overlapping peaks.

CE–DAD data NQS Sample Buffer 65°C, 5 min Acidification Filtration

Time 20 min Peak purity methods (a) Classic (b) Alternative approach approach Figure 6: Electropherogram of a standard amino acid solution Checking Yes No under the experimental conditions selected. for resolution

Chemometric data analysis Asp Fully resolved Poorly resolved 10 His lle NQS peaks peaks

Gln 8 Val Selectivity! Lack of Selectivity! Hyp Asn Trp Leu Phe Pro Glu 6 Ser Lys Me Thr Orn Univariate Analysis of Mathematical 4 Peak calibration spectral or resolution of areas Gly methods peak profiles peak profiles

Absorbance (mAU) 2

Multivariate Deconvolution 0 calibration methods 30 35 40 45 50 55 60 methods Migration time (min) Results Results

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References 1. M. Valcárcel, A. Ríos and L. Arce, Crit. Rev. Anal. Chem., 28, 63–81 (1998). 2. C. Mardones, A. Ríos and M. Valcárcel, Electrophoresis, 22, 484–490 (2001). 3. P. Kuban and B. Karlberg, Trends Anal. Chem., 17, 34 (1998). 4. P. Kuban et al., J. Chromatogr. A, 912, 163–170 (2001). 5. Z-L. Fang, Z-S. Liu and Q. Shen, Anal. Chim. Acta, 346, 135–134 (1997). 6. R.M. Latorre, J. Saurina and S. Hernández-Cassou, J. Chromatogr. A., 871, 331–340 (2000). 7. R.M. Latorre, J. Saurina and S. Hernández-Cassou, Electrophoresis, 21, 563–572 (2000). 8. R.M. Latorre, S. Hernández-Cassou and J. Saurina, J. Sep. Sci., 24, 427–434 (2001). 9. S. Sentellas et al., J. Chromatogr. A, 909, 259–269 (2001). 10. S. Sentellas et al., Electrophoresis, 22, 71–76 (2001). 11. B.G.M. Vandeginst et al., Handbook of Chemometrics and Qualimetrics: Part B, (Elsevier, Amsterdam, 1998). 12. “Matlab for Windows”, The MathWorks Inc., Natick, Massachusetts, USA. 13. B.M. Wise and N.B. Gallagher, PLS Toolbox 2.0 for use with MATLAB, Eigenvector Research Inc., Manson, West Virginia, USA (1997). 14. R.M. Latorre, J. Saurina and S. Hernández-Cassou, J. Chromatogr. Sci., 37, 353–359 (1999).

Javier Saurina is associate professor at the Department of Analytical Chemistry, University of Barcelona. He obtained his degree in Chemistry in 1988, degree in Pharmacy in 1996, MSc in Chemistry in 1990 and PhD in Chemistry in 1997. His main research fields include the development of analytical methods for environmental, pharmaceutical and food analysis based on capillary electrophoresis, liquid chromatography and flow injection analysis, and the development of sensors and biosensors. Another area of interest concerns the application of chemometric techniques to the analysis of data generated in the above-mentioned studies. In the last fifteen years, he has published around fifty papers in scientific journals and presented more than fifty communications to international symposia.

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