Offline analytics

In order to achieve results with close-to-conventional testing conditions, the parallel reactor setup for liquid-phase reaction must mimic the real process conditions of the later process as nicely as possible. The main efforts to be realized lie in the miniaturization and integrated construction of the parallel testing setup and the automation of process control combined with suitable online and offline analytical methodologies. 

If product moisture is measured offline, analytical results can be used to adjust K and 7), manually. If an online analyzer is used, the analyzer controller would be most effective in adjusting the bias 7] as is done in the figure. 

The potential offline analytical methods include UV, FTIR, proton NMR, carbon-13 NMR, gas chromatography, and capillary electrophoresis. With the exception of capillary electrophoresis and UV absorbance, none of these methods can... 

An offline analytical HPLC method with small injection volumes can be used to confirm the composition of cuts collected from a preparative run. Ideally, an analytical HPLC method provides an acceptable separation of the mixture components, displays peaks that can be assigned to known components, and gives a detector response that is a linear function of analyte concentration. Using isolated standards and calibrating the analytical detector for the concentration of each component is a good way to know the mass of each component eluting from a chromatography column, when such standards are available. 

The term online implies that analytical methods gain information directly from the process. This is in contrast to offline analytics, where a sample is taken from the process and transferred to a central analytical laboratory with sophisticated instrumentation. Formally, online analytical techniques can be further classified into atline, online, inline, and noninvasive techniques [6, 7]. 

Offline analytical techniques such as gas chromatography and high-pressure liquid chromatography are utilized to determine the minuscule amounts of reaction products produced. In cases where the products were not separated with these techniques, the percentage yields are sometimes reported in terms of the substrate used or remaining instead of the product produced. Measurement of the peak area or height is the most commonly utilized method for estimation of the substrate used before and after the reaction. 

Figure 3 SFE system configured for offline analyte collection using a standard 6 ml SPE column and inline using a packed sorbent bed. Reproduced with permission from O Keefe, Residue Analysis in Food, p. 96 (2000) Taylor and Francis.

The major attribute that distinguishes planar techniques from column chromatography is that in the former separation and detection are discontinuous ( offline )- In column chromatography analytes are carried through the entire column and monitored at the end, usually by flow-through detectors measuring changes in some physical characteristics of the effluent (optical... 

Off-line SFE is inherently simpler for the novice to perform, since only the SFE (and analyte collection) step needs to be understood. In off-line SFE further cleanup or a pretreatment step can be employed to eliminate interferences. With off-line SFE, sensitivities are limited by the fact that only about 1 p,L of the collection solvent is generally injected into the GC. The daily sample throughput can be higher using offline SFE, since SFE-GC requires that the GC be used for a sample collection device (rather than performing chromatographic separations) during the SFE extraction, whereas several off-line extracts can be loaded into an autosampler for unattended GC analysis. 

There are two general types of multidimensional chromatography separation schemes those in which the effluent from one column flows directly on to a second column at some time during the experiment, and those in which some type of trap exists between the two columns to decouple them (off-line mode). The purpose of a trap is often to allow collection of a fixed eluate volume to reconcentrate the analyte zone prior to the second separation step, or to allow a changeover from one solvent system to another. The use of offline multidimensional techniques (conventional sample cleanup) with incompatible mobile phases, is common in the literature, and replacing these procedures with automated on-line multidimensional separations will require continuous development efforts. 

Extracts of significant results from the calculations are stored in the analysis database. These results are adequate for a quick glance at the analytical results. The raw data can be downloaded to an offline computer if further reduction is desired. 

FIGURE 5.14 Software to read individual files from an offline GC instrument into the 2D software. Figure courtesy of Kroungold Analytical (2007). 

A variety of online solid extraction devices and applications have been developed for bioanalysis. Many are easy to build in laboratories or commercially available. Unlike offline methods, minimal operator intervention is needed for daily sample analysis after online applications are set up, so the approach is both labor- and cost-effective. The technique can also minimize errors arising from manual operations, eliminate potential inconsistencies caused by different operators, and provide accessibility of LC/MS/MS applications to laboratories that have minimal analytical expertise. 

Hopfgartner et al. (2002) compared ternary column online SPE LC/MS and TFC with offline 96-well plate SPE LC/MS to quantitate three drug candidates in human plasma. A protein precipitation step was performed before the SPE LC/MS. Dual trapping columns (YMS AQ, 10 x 2.0 mm, 5 /tm) were used with an analytical column (Intertsil Phenyl, 50 x 2.1 mm, 5 /tm). The run cycle was 3 min calibration range was 0.2 to 250 ng/mL. The run cycle was 2 min with a calibration range of 5 to 1000 ng/mL for TFC. Offline SPE LC/MS achieved the same calibration range with a run time of 2 min. 

Because the instability of the N-oxide metabolite, which was subjected to decomposition during sample preparation (solvent evaporation during offline SPE), online SPE LC/MS became the method of choice for the application. Hsieh et al. (2004) built a system with two TFC cartridges and one analytical column, and another system with two TFC cartridges and two analytical columns for GLP quantitative bioanalysis of drug candidates. A Turbo C18 (50 x 1.0 mm, 5 /.mi, Cohesive Technologies), an Xterra MS C18 (30 x 2.0 mm, 2.5 /mi), and a guard column were used. Protein precipitation preceded injection. The cycle times for the two systems were 0.8 and 0.4 min. 

Online SPE LC/MS/MS is commonly used for bioanalytical applications in the pharmaceutical industry. Column switching systems and TFC systems are easy to build and control. Sophisticated commercial systems and SPE cartridges are readily available. Compared to offline sample preparation, the online approach can save time and labor. However, the development of online SPE bioanalytical assays remains analyte-dependent. Generic methods can be applied to many analytes. For extremely hydrophobic, hydrophilic, and ionic analytes at normal pH range and analytes with a variety of hydrophobicity and pKa values, analyte-specific methods must be developed. An understanding of the chemistry of the analytes and SPE is critical. 

Molybdenum isotope ratio measurements by MC-ICP-MS (Plasma 54) have been carried out using Zr or Ru elemental spikes to study the mass discrimination during the whole analytical procedure including sample preparation.146 A laboratory fractionation of Mo isotopes of about 0.15 % is observed during ion exchange by offline Mo separation. Using this analytical technique, possible natural isotope variation of Mo can be determined with a precision of 0.02 %. 

Long-lived radionuclides occur at extremely low concentrations, especially in environmental samples, therefore several authors have proposed matrix separation and enrichment of the analytes before analysis.21,24,26,3 39 Radiochemical methods often require very careful and time consuming separation and enrichment processes and measurement procedures of a-, (3- and -emitting radioactive species at the trace and ultratrace level using conventional radioanalytical techniques 40-43 Trace/matrix separation, which is performed offline or online in order to avoid possible isobaric interferences, matrix effects and to reduce the detection limits for the determination of long-lived radionuclides, is also advantageous before ICP-MS measurements as the most widely applied mass spectrometric technique. 

Of more immediate interest are approaches that permit offline TLC-MS in which the spots are scraped out from the layer and the analytes are either extracted from the sorbent to be transferred to the mass spectrometer as discrete samples or are introduced without sorbent removal into the spectrometer on a direct insertion probe (51). TLC-MS quantification and confirmation efficiency can be further enhanced by submitting the TLC extract to an additional chromatographic separation using a different technique prior to the final MS analysis. Advantages of this approach over direct TLC-MS include extra cleanup through the additional chromatographic separation (52). This has been realized in the TLC-GC-MS analysis of eggs and meat for chloramphenicol residues (49). 

Liquid chromatography cleanup on a LiChrosorb Diol column has been further proposed for the offline purification of chloramphenicol residues from bovine muscle and eggs (32). An online approach based on reversed-phase principles has also been described for isolation of chloramphenicol residues from swine kidney by an automated column switching system (63). Use of a protein exclusion column (Hisep) has been also suggested in an online trace-enrichment method for the determination of chloramphenicol in animal tissues (52). By employing a column-switching system, all chloramphenicol that eluted from the protein exclusion column was trapped at the entry of a 5 m Supelcosil LC-18 preconcentration column, to be subsequently back-flashed into the analytical column. 

There are cases where HPLC separation is performed not in order to quantify the alcohols but as a technique for the purification of the analytes to be subjected to further instrumental analysis. This is the case, for example, with the identification and determination of the structure of an abscisic acid in starfruit extract (Averrhoa carambola L.). The separation and purification of the analytes was carried out also with HPLC using a mobile phase of diethyl ether, whereas the structure was elucidated by H and UC-NMR (6). In a similar way, to separate the sterols and alkanols from the unsaponifiable matter from olive oils on a silica column, a gradient composed of hexane/diethyl ether was chosen in an offline system (7), whereas an online HPLC-HRGC system uses as its mobile phase hexane/isopropanol (8). 

The fraction of column effluent containing the analyte and internal standard can be either collected manually for subsequent reinjection onto the second (analytical) column (offline operation) or diverted directly onto the second column via a high-pressure switching valve (online operation). For manual collection, a drop-counter-fraction collecting system rather than a volume collection system has been recommended (117). The fraction is collected in a small tapered tube, and the solvent is carefully evaporated off under a stream of nitrogen. The residue is then dissolved in a small volume of a suitable solvent for the analytical separation. Because the sample is reconstituted in offline operation, the potential problem of mobile phase incompatibility between the two HPLC systems is avoided, and hence any semipreparative/analytical combination can be used. 

The column-switching system was applied to the determination of STR and DIHS in pork and bovine muscle and kidney. Perchloric acid was used to precipitate proteins and extract analytes from the tissue. The clear supernatant was further cleaned up by an offline SPE on a cation-exchange SPE column. After the washing of the cartridge with water, the analytes were eluted with phosphate buffer (pH 8.0) and diluted with HSA, perchloric acid, and water. The enrichment was achieved in the online mode. After loading the sample, the enrichment precolumn was flushed with HSA at pH 3.3 for 5 min. Using the ratio of MeCN to aqueous component of 83 17... 

An automated system for offline SPE is ASPEC XL. The main advantage of using offline SPE is that the analytes can be stored in disposable preconcentration cartridges. Polymeric sorbent Lichrolut EN or ENV was used for groundwater preconcentration (42,43). 

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