Solid phase analytical derivatization as a sample preparation method
Introduction
Sample preparation (SPrep) is a critical step in analytical chemistry. It is certainly required in most standard analytical equipment and even the methods based on the advanced instruments benefit by the separation of analyte from matrix components [1], [2]. The techniques for this separation generally involve fractionation between an aqueous phase and a water immiscible extracting phase. The extracting phase could be a volatile organic solvent or a pseudophase sorbed on a solid phase. Once partition is complete, the extracting phase is removed and analytes are isolated/concentrated by a number of techniques. A considerable body of research was and remains focused on simplifying and automating sample preparation.
LLE is the classical SPrep for biological and environmental samples [2], [3]. Developed in the 1950s and used into the mid-70s this technique led to the first analytical methods for measuring drugs and their metabolites in biofluids. While leading to the classical bioanalytical and environmental chemistry LLE, is nevertheless time consuming, labor intensive and requires large amounts of organic solvents. It is also relatively difficult to automate [4], [5].
Investigators addressed these problems of this classical with a variety of innovative techniques that are discussed below. Throughout these studies the focus was on high throughput techniques, simplification, automation and miniaturization.
Development of SPE overcame some of these disadvantages and reduced solvent consumption, labor requirements and advanced automation [3], [5]. In SPE, the analyte is partitioned between the biological or environmental aqueous liquid phase and a reverse phase column typically C18 linked to a silica support or an organic polymer [5]. The solid phase retains the analyte during removal of the aqueous phase and subsequent washing to remove various components. Analyte can then be desorbed by solvent or thermal desorption.
Although an improvement over LLE, use of SPE presented other problems [3]. It required careful control of the flow rate through the column both for sorbing analyte from the aqueous phase and eluting the analyte with organic solvent. Although SPE requires less eluting solvent, than the corresponding LLE technique the solvent burden is still large. This would be particularly true for laboratories with high throughput.
Matrix solid-phase dispersion extraction (MSDE), “quick, easy, cheap, effective, rugged and safe” (QuEChERS), purge-and-trap extraction and static headspace extraction are some of the recently reported alternatives for SPE.
Elimination of the eluting solvent became possible with the creation of SPME [6], [7], [8]. This is a completely solvent-less technique for separating analytes from their matrix and is fully automated [8]. SPME is thoroughly investigated for determination of analytes from numerous matrices and is one of the most cited techniques in the analytical chemistry literature. SPME involves the use of a fiber coated with the extracting phase which extracts analytes. After extraction analytes are thermally desorbed from the SPME fiber by transferring into an injection port of a chromatographic instrument. Although widely used, SPME cost of equipment and the fibers themselves can be an issue.
A variant of SPME is stir bar sorptive micro extraction (SBSME) in which typically a 0.5–1 mm thick polydimethylsiloxane coating of stir bars is the extraction phase. Stir bars are contacted in aqueous medium to extract analytes [9], [10]. After extraction, either thermal desorption or liquid desorption can be used before analysis.
This LLE technique was developed as an inexpensive alternative to SPME. Jeannot and Cantwell reported the first SDME method for chromatographic analysis [11]. In their first report few microliters of octane was immersed in aqueous sample aid of a Teflon rod. The sample solution was stirred to accelerate extraction. After extraction the rod was removed and a portion of the octane solvent was injected to a GC/FID for analysis. It found a variety of applications but exhibited some deficits such as a small interfacial area, droplet instability when the aqueous phase was stirred at high speed to provide efficient extraction.
The HFLLLE addressed the issue of droplet instability by using a porous hollow fiber with the impregnated organic solvent acts as an interface between the acceptor and donor phases. Pedersen-Bjergaard and Rasmussen reported the first LLLME method based on the use of porous hollow fibers made out of polypropylene [12]. In HFLLLE methods the analytes of interest are extracted from the aqueous donor phase to the thin layer organic solvent impregnated in the pores of the hollow fiber and then to the acceptor phase inside the lumen of the hollow fiber [12], [13]. In the two phase mode the acceptor solution is an organic solvent and therefore more compatible with GC analysis. The acceptor solution is aqueous in the three phase mode which is more compatible with LC analysis. The major advantage of the HFLLLE method is very clean extracts resulting from the small pore size of the hollow fiber which prevents interfering substance particles present in the donor phase entering the acceptor phase and due to the low solubility of organic phase present in the pores. HFLLLE methods have been successfully applied to analyze biological and environmental sample with complex matrices.
This is a three solvent component system consisting an aqueous phase, extracting solvent (nonpolar water immiscible solvent), and disperser solvent (polar water miscible solvent) [14], [15]. A mixture of disperser and extracting solvents are injected to the aqueous phase to form a cloudy solution. The cloudiness consists of microdroplets of water immiscible solvent and the high surface area speeds the extraction process. Extracting solvent containing the target analytes are then separated from the aqueous phase by centrifugation.
These ionic solvents possess low melting points, low vapor pressures, high thermal stability, nonflammability, and good solubility for inorganic and organic compounds [16]. Many physical properties of ILs can be varied by varying the structure according to analyte extraction selectivity, efficiency, and sensitivity needs. These features make ILs an excellent extraction media for many liquid phase microextraction techniques such as DLLME, HFLLLME, and SDME.
Section snippets
Analytical derivatization
Current requirements of analytical methods are high sensitivity, high throughput, ease of use, precision, accuracy and automation. Although instrumentation meets some of these requirements, sample preparation that takes place prior to instrumental analysis is still under active research and development. Despite extra steps reagents, analytical derivatization (AD) of the analyte during SPrep can substantially improve sensitivity of detection, chromatographic separation, and selectivity [2]. Even
Polar phenols
Analytical methods for phenols include GC-MS, high-performance liquid chromatography (HPLC) equipped with ultraviolet and mass spectrometry detectors. However the most common method reported is GC–MS using wall-coated open-tubular (WCOT) columns due to its high sensitivity and separation power [27], [29], [38], [52], [63], [64], [65], [66]. Many of these phenols and alcohols can interact with accessible silanol groups on the fused-silica surface and at low concentrations require analytical
Organic acids by SPAD
Analysis of analytes with the carboxyl functional group is very important in environmental chemistry and biomedical applications due its wide presence. It is a common practice in both GC and LC analysis to derivatize acids [83]. In GC analysis carboxyl group is derivatized in order to improve volatility and to avoid interaction of accessible silanol groups in WCOT columns [67], [68]. In LC analysis carboxylic acids are derivatized to add a chromophore to improve sensitivity and selectivity.
Many
Carbonyl compounds by SPAD
A wide range of carbonyl compounds are found in environmental and biological samples. Analysis of carbonyl compounds at trace level concentrations has become an important topic in research due to demanding applications in environmental monitoring and medical diagnosis. The analysis of trace levels of some of these compounds challenges existing analytical methods because their concentrations are lower than most instrument limits of detection. Research groups in recent times have focused on new
Amine compounds by SPAD
The most common separation technique in amine analysis is HPLC. The molar absorptivity coefficients for amines are low especially due to lack of chromophores except aromatic amines. These compounds show little or no electrochemical or fluorescence activity. The hydrogen atom attached to the nitrogen in amine group has a partial positive charge due to the electro-negativity difference between the two atoms. Analysis of amine compounds by GC using WCOT columns is a difficult task at low
Conclusions
Solid phase analytical derivatization (SPAD) is an attractive tool for determination of wide range of analytes in complicated biological and environmental sample matrices. Low organic solvent consumption, ease of automation, low cost and efficiency in SPAD has helped immensely gain its popularity over the years. More focus on automation can be expected in SPAD method development as time factor is becoming critical in sample preparation methods. Analytical derivatization (AD) increases
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