Reproducibility, automated analysis

For PMMA/additive dissolutions, it was not possible to identify any additive characteristic mass peaks, either by direct laser desorption or with matrix-assistance (dithranol, DHBA or sinapinic acid, 4-hydroxy-3,5-dimethoxy-cinnamic acid). This has again been ascribed to very strong interaction between PMMA and additives, which suppresses desorption of additive molecules. Also, partial depolymerisation of pho-tolytically labile PMMA by laser irradiation may play a role, which leads to saturation of the detector by PMMA fragment-ions and disappearance of additive mass peaks below noise level. Meyer-Dulheuer [55] has also reported MALDI-TOFMS analysis of a coating/2-ethylhexyldiphenylphosphate sample. Quantitative determination of the additives by means of MALDI-ToFMS proved impossible. Possibly the development of reproducible (automated) sample handling procedures or thin films might overcome this problem. 

A fully automated two-dimensional electrophoresis (2DE) system for rapid and reproducible protein analysis is described. 2DE that is a combination of isoelectric focusing (lEE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein expression analysis. Here, all the operations are achieved in a shorter time and all the transferring procedures are performed automatically. The system completed the entire process within 1.5 h. A device configuration, operational procedure, and data analysis are described using this system. 

A method for the automated analysis of volatile flavor compounds in foods is described. Volatile compounds are removed from the sample and concentrated via the dynamic headspace technique, with subsequent separation and detection by capillary column gas chromatography. With this method, detection limits of low ppb levels are obtainable with good reproducibility. This method has experienced rapid growth in recent years, and is now in routine use in a number of laboratories. 

The sample introduction system must be capable of introducing a known and variable volume of sample solution reproducibly into the pressurized mobile phase as a sharp plug without adversely affecting the efficiency of the column. The superiority of valve injection has been adequately demonstrated for this purpose and is now universally used in virtually all modern instruments for both manual and automated sample introduction systems [1,2,7,31,32]. Earlier approaches using septum-equipped injectors have passed into disuse for a several reasons, such as limited pressure capability, poor resealability, contamination of the mobile phase, disruption of the column packing, etc., but mainly because they were awkward and inconvenient to use compared with valves. For dilute sample solutions volume overload restricts the maximum sample volume that can be introduced onto the column without a dramatic loss of performance. On-column or precolumn sample focusing mechanisms can be exploited as a trace enrichment technique to enhance sample detectability. Solid-phase extraction and in-column solid-phase microextraction provide a convenient mechanism for isolation, concentration and matrix simplification that are easily interfaced to a liquid chromatograph for fully or semi-automated analysis of complex samples (section 5.3.2). 

Capillary electrophoresis (CE) is a modern analytical method that is being extensively applied to the characterization of biotechnology-derived products like peptides and proteins [1], Due to its ease of automation and facilitating the development of reproducible routine analysis, CE seems to be well suited for the quality control of biotechnological products, including process monitoring, purity assessments. 

Cool on-column injection is used for trace analysis. Ah. of the sample is introduced without vaporization by inserting the needle of the syringe at a place where the column has been previously stripped of hquid phase. The injection temperature must be at or below the boiling point of the solvent carrying the sample. Injection must be rapid and no more than a very few, usuahy no more than two, microliters may be injected. Cool on-column injection is the most accurate and reproducible injection technique for capihary chromatography, but it is the most difficult to automate. 

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... 

However, many of these tools, while enabling markedly faster and more detailed analysis than paper-based methods, still mimic static, one-by-one paperlike reports with no real-time auditing capability. Moreover, these COTS do not have integrated data analysis and automated data screening capabilities and are not optimized for systematic analyses. Furthermore, the ad hoc analyses that these COTS produce lack interactive, automatic auditing reproducible functions. Thus these tools are often used to produce the same dense, unwieldy paper tables of counts and percentages that were created manually before personal computers became ubiquitous. 

The determination of lead in blood is the most widespread clinical use of ASV The technique is attractive because it is rapid, simple and reproducible A recent advance is to couple ASV to flow injection analysis in order to automate the process so that smaller samples and shorter analysis time can be achieved Lead is also routinely determined in bonemeal meant for human consumption by ASV Both lead and cadmium are determined in agricultural crops by ASV... 

Sample preparation, injection, calibration, and data collection, must be automated for process analysis. Methods used for flow injection analysis (FLA) are also useful for reliable sampling for process LC systems.1 Dynamic dilution is a technique that is used extensively in FIA.13 In this technique, sample from a loop or slot of a valve is diluted as it is transferred to a HPLC injection valve for analysis. As the diluted sample plug passes through the HPLC valve it is switched and the sample is injected onto the HPLC column for separation. The sample transfer time typically is determined with a refractive index detector and valve switching, which can be controlled by an integrator or computer. The transfer time is very reproducible. Calibration is typically done by external standardization using normalization by response factor. Internal standardization has also been used. To detect upsets or for process optimization, absolute numbers are not always needed. An alternative to... 

Generally SFE has proved a greater success than SFC. However, the need for successful automation is a significant restriction in many routine applications. SFE has been promoted as the ideal technique for sample preparation for chromatography. Meanwhile it is clear that this is far too optimistic [77,292]. As shown in Section 3.4.2.7, SFE does not guarantee quantitative analysis. Before any technique can be fully accepted, it should be capable of generating reproducible results. This is clearly not the case in SFE. Also, sample sizes of (on-line) SFE tend to be much smaller than in other methods, such as MAE or ASE (Table 3.4), which raises the risk of nonrepresentative sampling. There is a need for SFE to be carried out on reference materials of known composition determined by an alternative technique. 

Headspace analysis involves examination of the vapours derived from a sample by warming in a pressurized partially filled and sealed container. After equilibration under controlled conditions, the proportions of volatile sample components in the vapours of the headspace are representative of those in the bulk sample. The system, which is usually automated to ensure satisfactory reproducibility, consists of a thermostatically heated compartment in which batches of samples can be equilibrated, and a means of introducing small volumes of the headspace vapours under positive pressure into the carrier-gas stream for injection into the chromatograph (Figure 4.25). The technique is particularly useful for samples that are mixtures of volatile and non-volatile components such as residual monomers in polymers, flavours and perfumes, and solvents or alcohol in blood samples. Sensitivity can be improved by combining headspace analysis with thermal desorption whereby the sample vapours are first passed through an adsorption tube to pre-concentrate them prior to analysis. 

---