Off-line Sample Preparation Techniques

Membrane separation techniques, which are used mainly in industrial processes, include dialysis, electrodialysis, reverse osmosis, ultrafiltration, 

Chapter 3 Instrumentation for High-Performance Liquid Chromatography 

Dialysis has been applied to the preparation of a wide range of sample types, ranging from foodstuffs to physiological fluids. Membrane-based sample preparations for chromatography have been reviewed by Van de Merbel et al.60 In ordinary dialysis, solutes are transferred from a concentrated to a more dilute solution as a consequence of the concentration gradient. 

In electrodialysis, an applied electric field rather than a concentration gradient is used to draw ions across the membrane. Because it is faster than ordinary dialysis, electrodialysis is often used in biochemical analyses for purposes of fractionation, concentration, and desalting. Reverse osmosis (RO) is a process that uses semipermeable membranes to allow water permeation however, the membranes act as a barrier to the passage of dissolved and suspended particles. Typically, RO membranes are used to extract pure water from aqueous solutions of dissolved salts, such as seawater. The particle size cutoff is typically 0.0001 yum with driving pressures of 200 to 800 psi (1.4 to 5.5 MPa).61 

Separation process Driving force Separation mechanism Species passed Membrane structure Application 

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In order to reduce or eliminate off-line sample preparation, multidimensional chromatographic techniques have been employed in these difficult analyses. LC-GC has been employed in numerous applications that involve the analysis of poisonous compounds or metabolites from biological matrices such as fats and tissues, while GC-GC has been employed for complex samples, such as arson propellants and for samples in which special selectivity, such as chiral recognition, is required. Other techniques include on-line sample preparation methods, such as supercritical fluid extraction (SFE)-GC and LC-GC-GC. In many of these applications, the chromatographic method is coupled to mass spectrometry or another spectrometiic detector for final confirmation of the analyte identity, as required by many courts of law. 

The three main formats for sample preparation used in drug-discovery are protein precipitation (PPT), SPE, and LLE. Several examples of off-line sample preparation have been reported and involve SPE [37,38,46,47], LLE [38,48], and PPT [39,49]. In each of the examples cited, semi- or fully automated strategies for liquid handling were incorporated to enhance throughput. Even with the recent popularity of on-line methods, off-line techniques continue to be widely employed. The key advantage to off-line methods is that sample preparation may be independently optimized from the mass spectrometer and does not contribute overhead to the LC-MS injection duty cycle. 

Many different sample preparation techniques are available to the drug discovery scientist. Off-line sample preparation procedures include protein precipitation, filtration, dilution followed by injection, liquid-liquid extraction (LLE), and solid-phase extraction (SPE). Typically, these procedures are performed in an automated, high-throughput mode that features a 96-well plate format. Online sample preparation procedures include SPE and turbulent flow chromatography (TFC) with conventional chromatographic media or restricted access media (RAM). These online approaches are often simple and easy to automate. 

There has also been a move from slow manual sample preparation techniques to faster automated techniques. Automated sample preparation can be carried out on-line (with sample preparation connected directly to the analysis system) or off-line (sample preparation is automated, but the sample has to be manually transferred to the analysis system). Automated sample preparation offers the potential of performing sample clean-up, concentration, and analyte separation in a closed system. This reduces the sample preparation time, and the whole sample becomes available for analysis, leading to improved limits of detection. It also removes some of the human element from a procedure, thereby improving precision and reproducibility. Eurther-more, automated sample preparation reduces cost by using... 

The sensitivity of the method is evaluated by the limit of detection and quantification. In general, there are no specific criteria for the limit of detection, but for bioanalysis purposes, the limit of quantification is established for the concentration with precision and accuracy lower than 20%. Limits of detection and quantification obtained in CE with UV detection are generally higher than in HPLC, owing to the small optical path in the detection window and injected volumes. As discussed previously, by using preconcentration procedures (off-line sample preparation and/or stacking sample injection techniques) and a more sensitive detection system, particularly LIE and MS detection, suitable detection or quantification limits could be obtained for the apphcation of the methods to the analysis of real samples. 

Until this point, the sample preparation techniques under discussion have relied upon differences in polarity to separate the analyte and the sample matrix in contrast, ultraflltration and on-line dialysis rely upon differences in molecular size between the analyte and matrix components to effect a separation. In ultrafiltration, a centrifugal force is applied across a membrane filter which has a molecular weight cut-off intended to isolate the analyte from larger matrix components. Furusawa incorporated an ultrafiltration step into his separation of sulfadimethoxine from chicken tissue extracts. Some cleanup of the sample extract may be necessary prior to ultrafiltration, or the ultrafiltration membranes can become clogged and ineffective. Also, one must ensure that the choice of membrane filter for ultrafiltration is appropriate in terms of both the molecular weight cut-off and compatibility with the extraction solvent used. 

There are many sample preparation techniques listed in texts, from a simple filtration or centrifugation to many other kinds of extraction procedures, including both liquid-liquid and solid-phase extraction. When any type of sample preparation is used, it often is done manually if only a few samples are involved. If a large number of samples are to be analyzed, the entire procedure should lend itself to automation. Regardless of the number of samples, most sample preparation is done off-line that is, the samples are prepared first with one of the methods listed, then placed into an automated sample injection system for sequential analysis of all samples. 

On-line methods refer to techniques where sample cleanup occurs on-Une en route to the mass spectrometer. The common thread to all on-line techniques is column-switching (CS). CS refers to the use of HPLC valves to couple multiple LC columns in configurations that permit sample cleanup and analysis to occur on-line. Such techniques eliminate the manual wash and transfer steps associated with off-Une sample preparation and are readily automated. 

Two very comprehensive reviews of current trends in sample preparation have been published by Kinsella et al. and by No kav and Vlckov. This chapter includes topics discussed in these specific reviews. The different off- and on-line sample preparation procedures mentioned above are described. General items regarding extraction procedures are discussed, followed by a discussion of the current sample preparation techniques, with some examples of applications. 

Determining the N-isotopic composition of sedimentary OM requires that N2 gas be prepared from the sample. The gas is then fed into a mass spectrometer for analysis. An excellent, comprehensive review of mass spectrometry, and the various sample preparation techniques that may be used for N-isotope analysis, is given in Owens (1987). Attention here will be confined to some of the more pertinent practical aspects of isotopic analysis with particular reference to the system used at UB. As indicated in the Introduction, the adoption of nitrogen isotopes as a standard technique in the earth and biological sciences was hindered by the time-consuming, off-line preparation techniques that most studies required. A few labs have stuck to the original, wet-chemistry sample preparation, feeling... 

Many of the sample preparation techniques for ion chromatography can also be performed using online instrumentation, which can be easily automated and is less time-consuming than off-line techniques. ... 

Due to the complexity of environmental real samples and the low relative abundance of pollutants, preparative cleanup and preconcentration steps are usually required. Although sample preparation techniques are widely developed in conventional formats and off-line, it is important to point out that from the different steps related to the analytical process, sample preparation is notably the least developed in miniaturized devices. However, very exciting unique opportunities could be expected because of... 

Application of rotating coiled columns has become attractive for preparative-scale separations of various substances from different samples (natural products, food and environmental samples) due to advantages over traditional liquid-liquid extraction methods and other chromatographic techniques. The studies mainly made during the last fifteen years have shown that using rotating coiled columns is also promising for analytical chemistry, particularly for the extraction, separation and pre-concentration of substances to be determined (analytes) before their on-line or off-line analysis by different determination techniques. 

Although SFE and SFC share several common features, including the use of a superaitical fluid as the solvent and similar instrumentation, their goals are quite distinct. While SFE is used mainly for the sample preparation step (extraction), SFC is employed to isolate (chr-omatography) individual compounds present in complex samples (11 -15). Both techniques can be used in two different approaches off-line, in which the analytes and the solvent are either vented after analysis (SFC) or collected (SFE), or on-line coupled with a second technique, thus providing a multidimensional approach. Off-line methods are slow and susceptible to solute losses and contamination the on-line coupled system makes possible a deaease in the detection limits, with an improvement in quantification, while the use of valves for automation results in faster and more reproducible analyses (16). The off-line... 

Principles and Characteristics Although early published methods using SPE for sample preparation avoided use of GC because of the reported lack of cleanliness of the extraction device, SPE-GC is now a mature technique. Off-line SPE-GC is well documented [62,63] but less attractive, mainly in terms of analyte detectability (only an aliquot of the extract is injected into the chromatograph), precision, miniaturisation and automation, and solvent consumption. The interface of SPE with GC consists of a transfer capillary introduced into a retention gap via an on-column injector. Automated SPE may be interfaced to GC-MS using a PTV injector for large-volume injection [64]. LVI actually is the basic and critical step in any SPE-to-GC transfer of analytes. Suitable solvents for LVI-GC include pentane, hexane, methyl- and ethylacetate, and diethyl or methyl-f-butyl ether. Large-volume PTV permits injection of some 100 iL of sample extract, a 100-fold increase compared to conventional GC injection. Consequently, detection limits can be improved by a factor of 100, without... 

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