Off-line method

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... 

Methods developed for on-line technological control have to be tested for the variation of the product composition due to process variations. However, if rugged analytical procedures are developed these multidimensional methods may only require minimal attention during on-line operation. Multidimensional chromatography for the analysis of complex polymer and industrial samples offers chromatogra-phers high productivity and efficiency and is an excellent alternative to off-line methods. 

By incorporating the entire analytical scheme (enzyme reaction and electrochemical detection) into the flow system a great improvement in precision can be realized. Sample manipulation is minimized because only a single injection into the flow system is required versus sampling of aliquots for the off-line method. Precision is also improved because the timing of the enzyme reaction and detection are much better controlled in the flow system. Finally, less of both enzyme and sample are needed with on-line enzyme reactor methods. 

Many traditional laboratory/off-line methods are now moving in the direction of in-process applications. Online GC had already been introduced in the 1950s. Using chip- and microsystem technology, xGC is now being introduced, which achieves analysis times of 30 seconds and is therefore suitable for quality control. SPME-p,GC is potentially useful for process analysis. 

Comparing on-line and off-line methods of column switching, which of the following advantages and limitations do you think would apply to the on-line technique ... 

On-line techniques are easily automated, but are more expensive as they require additional valves with associated switching equipment. Off-line methods are rather easier to carry out, but, because of the sample collection and re-injection steps, they are slower and tend... 

Warnken et al. [956] have reported an online preconcentration - ultrasonic nebulisation - ICP-MS method that achieved detection limits of 0.26,0.86,1.5, 10, and 0.44 ng/1 for manganese, nickel, copper, zinc, and lead in seawater. This online preconcentration method compares favourably to the state of-the-art off-line methods. 

Step 5 Off-line method or analyzer development and validation This step is simply standard analytical chemistry method development. For an analyzer that is to be used off-line, the method development work is generally done in an R D or analytical lab and then the analyzer is moved to where it will be used (QA/ QC lab, at-line manufacturing lab, etc.). For an analyzer that is to be used on-line, it may be possible to calibrate the analyzer off-line in a lab, or in situ in a lab reactor or a semiworks unit, and then move the analyzer to its on-line process location. Often, however, the on-line analyzer will need to be calibrated (or recalibrated) once it is in place (see Step 7). Off-line method development and validation generally includes method development and optimization, identification of appropriate check samples, method validation, and written documentation. Again, the form of the documentation (often called the method or the procedure ) is company-specific, but it typically includes principles behind the method, equipment needed, safety precautions, procedure steps, and validation results (method accuracy, precision, etc.). It is also useful to document here which approaches did not work, for the benefit of future workers. 

Sulfates are precipitated as BaS04, and then reduced with carbon at 1,000°C to produce CO2 and CO. The CO is either measured directly or converted to CO2 by electrical discharge between platinum electrodes (LonginelU and Craig 1967). Total pyrolysis by continuous flow methods has made the analysis of sulfate oxygen more precise and less time-consuming than the off-line methods. Bao and Thiemens (2000) have used a C02-laser fluorination system to liberate oxygen from barium sulfate. 

This problem is overcome by the Bio View sensor, which offers the possibility to monitor the whole spectral range simultaneously, and by using suitable data analysis and mathematical methods like chemometric regression models 11061. Real-time fluorescence measurement can be used more effectively comparing time-consuming off-line methods. Partial least squares (PLS) calibration models were developed for simultaneous on-line prediction of the cell dry mass concentration (Fig. 5), product concentration (Fig. 6), and metabolite concentrations (e. g., acetic acid, not shown) from 2D spectra. 

There are two basic approaches off-line and on-line. The off-line method, as discussed in the chapters on sample pretreatment, are most often used because they involve either manually or automatically collecting a fraction from a sample cleanup sorbent. The appropriate fraction is transferred and then assayed by a second chromatographic method. The manual steps are time-consuming and potentially introduce significant error to the j precision and accuracy of the method. The on-line method, when fully automated, would have the chromatography system perform sample pre- j treatment by column switching between two or more columns. j... 

The classical way to determine biomass concentration is typically an off-line method, namely to harvest a known aliquot of the culture suspension, separate cells by centrifugation, wash the cells and dry them to constant weight at a few degrees above the boiling point of the solvent (i.e. aqueous medium, usually... 

Infrared spectroscopy is a powerful alternative analytical technology for process monitoring which has found wide application as an off-line method in the chemical and food industries. The additional advantage over other methods is that in many circumstances it is possible to quantify a number of components simultaneously. 

In Figure 8-18, a mixture of acids and bases was analyzed on three types of columns phenyl, polar embedded, and C18 column. Significant differences in selectivity were obtained. The separation could be further optimized by modifying the gradient slope and employing off-line method development tools such as Drylab for further optimization and resolution of the critical pairs. 

In general, tandem systems involving the combination of the liquid chromatograph in-line with the infrared spectrometer have not been very successful and most IR spectra of LC eluents are obtained by what are, in effect, off-line procedures, as in the example given above. The FTIR spectrometer, in its present form, demands too large a sample size and is too insensitive for successful in-line association with modem high-efficiency microbore LC columns. Fortunately, the demand for in-line production of IR spectra from LC eluents is not great and, in most cases, the off-line methods are quite satisfactory for the majority of LC/IR applications. 

The particle beam LC/FT-IR spectrometry interface can also be used for peptide and protein HPLC experiments to provide another degree of structural characterization that is not possible with other detection techniques. Infrared absorption is sensitive to both specific amino acid functionalities and secondary structure. (5, 6) Secondary structure information is contained in the amide I, II, and III absorption bands which arise from delocalized vibrations of the peptide backbone. (7) The amide I band is recognized as the most structurally sensitive of the amide bands. The amide I band in proteins is intrinsically broad as it is composed of multiple underlying absorption bands due to the presence of multiple secondary structure elements. Infrared analysis provides secondary structure details for proteins, while for peptides, residual secondary structure details and amino acid functionalities can be observed. The particle beam (PB) LC/FT-IR spectrometry interface is a low temperature and pressure solvent elimination apparatus which serves to restrict the conformational motions of a protein while in flight. (8,12) The desolvated protein is deposited on an infrared transparent substrate and analyzed with the use of an FT-IR microscope. The PB LC/FT-IR spectrometric technique is an off-line method in that the spectral analysis is conducted after chromatographic analysis. It has been demonstrated that desolvated proteins retain the conformation that they possessed prior to introduction into the PB interface. (8) The ability of the particle beam to determine the conformational state of chromatographically analyzed proteins has recently been demonstrated. (9, 10) As with the ESI interface, the low flow rates required with the use of narrow- or microbore HPLC columns are compatible with the PB interface. 

In linear development of a chromatogram, unidirectional or bidirectional developments of the chromatogram are possible. Similarly, as in liquid column chromatography, there are possible, in this case, either on-line or off-line techniques of sample application, separation, and detection, as well as various modifications (e.g., partly off-line method). Bidirectional development can also be vertical. Using vertical bidimensional development, applying different eluents, components of complex, difficult mixtures can be separated. The separation of such mixtures is also possible by means of this technique using multiple automatic development of chromatogram. 

Separation analysis at the outlet of the detector must respect three major conditions. The first is the cell integrity (i.e., the diagnosis of the particle). This can be operated on line, by means of classical photometric devices operated in the light-scattering mode (opacimetry) at 254 nm. Off-line methods, after fraction collection, are possible and recommended, by microscopy and granulometric analysis (Coulter counting). The second objective is to analyze cell viability. Off-line methods after fraction collection are equally possible. The blue trypan exclusion test, motility measurements, or specific enzymatic activities (esterase) can be performed on an aliquot of the collected fraction. 

Off-line preconcentration systems are less expensive and easier to handle, but often require very large sample volume (500-1000 ml), in some cases hampering the analytical determination due to the low amount of sample available. Furthermore, off-line methods are more contamination-prone, since they require a great number of subsequent steps of sample treatment. In order to reduce sample... 

Off -line control by local equipment also here the analysis is made on a sample, which is taken out from the process, but the response time can be much lesser, which ensure more reliable adjustments on the process. The advantage compared to the distance control is obvious. The analysis methods can be expensive and complicated, but it can be as simple as thin layer chromatography (TLC) [22, 23], Chromatographic methods such as HPLC and GC have been commonly used as off line methods for bioprocess monitoring [24, 25] and HPLC is described in section 2.5. 

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