On-line Sample Preparation Techniques

Autosamplers are used to inject samples automatically. They tend to be used for routine analyses, after a chromatographic method has been developed and validated. Some autosamplers can be programmed to perform dilutions and standard additions prior to autoinjection, and these 

Chapter 3 Instrumentation for High-Performance Liquid Chromatography 

The other form of on-line solid-phase extraction procedures involves column-switching techniques. Column switching employs valves that can be switched manually or automatically between a number of columns at predetermined times.67-69 For sample cleanup the analyte of interest is retained on the primary or precolumn while the interfering matrix components are eluted to waste. The analytes are then diverted to a second or analytical column where they are separated for identification and quantification. 

When column switching is used for sample cleanup, the technique is known as zone cutting. If the fraction of effluent to be transferred from the precolumn to the analytical column is at the front of the precolumn chromatogram, the technique is known as front cutting. Heart-cut technique 

Most analysts will be required to troubleshoot the chromatography system at some stage. Although chromatographic instruments are considerably more rugged now than they once were, components still have limited lifetimes and need to be replaced. As operator error can be the cause of a problem, it is important to understand the operation of the system in order to minimize downtime. The best approach to troubleshooting, however, is preventive maintenance the most important points to remember are filter, degas, and flush.71 

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Although on-line sample preparation cannot be regarded as being traditional multidimensional chromatography, the principles of the latter have been employed in the development of many on-line sample preparation techniques, including supercritical fluid extraction (SFE)-GC, SPME, thermal desorption and other on-line extraction methods. As with multidimensional chromatography, the principle is to obtain a portion of the required selectivity by using an additional separation device prior to the main analytical column. 

Dialysis can also be used as an on-line sample preparation technique for the deproteinization of biological samples prior to HPLC. Selecting the appropriate semipermeable membrane for the dialyzer can prevent interference from large macromolecules. Samples are introduced into the feed (or donor) chamber, and solvent is pumped through the lower acceptor (or recipient) chamber. The smaller molecules diffuse through the membrane to the acceptor chamber and are directed to an HPLC valve for injection. In case of low concentrations of compounds of interest, a trace enrichment step may be required this is accomplished with a column placed downstream of the dialyzer that will retain the analyte until the concentration is sufficient for detection. After this step, the analyte can be backflushed into the HPLC system. The technique is useful for blood studies as sampling can be achieved continuously without blood withdrawal. A commercial on-line system, such as Asted from Gilson, used for both cleanup and enrichment by a combination of dialysis with SPE, is shown in Fig. 7. 

On-line sample preparation techniques for ion chromatography,/. Chromatogr. A, 804, 55, 1998. 

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. 

Before compounds in biological matrices can be analyzed by LC/MS/MS, the samples must undergo a preparation procedure. There are a variety of techniques available for sample preparation including offline sample preparation techniques (liquid-liquid extraction, protein precipitation, and solid phase extraction) and on-line sample preparation... 

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. 

One way of subdividing on line sample preparation-GG techniques is into solvent-free techniques and techniques requiring the introduction of some 10 to 100 p.1 of an organic solvent into the GG. As regards the latter group, 10 years ago, such amounts of solvent were called very large. Today, LVI has become... 

As an alternative approach towards the above requirement, Somorjai introduced the method of electron lithography [119] which represents an advanced HIGHTECH sample preparation technique. The method ensures uniform particle size and spacing e.g. Pt particles of 25 nm size could be placed with 50 nm separation. This array showed a uniform activity similar to those measured on single crystal in ethylene hydrogenation. The only difficulty with the method is that the particle size is so far not small enough. Comprehensive reviews have been lined up for the effect of dispersion and its role in heterogeneous catalysis [23,124,125]. 

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. 

In some manufacturing process analysis applications the analyte requires sample preparation (dilution, derivatization, etc.) to afford a suitable analytical method. Derivatization, emission enhancement, and other extrinsic fluorescent approaches described previously are examples of such methods. On-line methods, in particular those requiring chemical reaction, are often reserved for unique cases where other PAT techniques (e.g., UV-vis, NIR, etc.) are insufficient (e.g., very low concentrations) and real-time process control is imperative. That is, there are several complexities to address with these types of on-line solutions to realize a robust process analysis method such as post reaction cleanup, filtering of reaction byproducts, etc. Nevertheless, real-time sample preparation is achieved via an on-line sample conditioning system. These systems can also address harsh process stream conditions (flow, pressure, temperature, etc.) that are either not appropriate for the desired measurement accuracy or precision or the mechanical limitations of the inline insertion probe or flow cell. This section summarizes some of the common LIF monitoring applications across various sectors. 

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