Off-Line Techniques

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is carried out in a test tube under controlled conditions with aliquots being taken at timed intervals. These aliquots are then analyzed for the electroactive product and the enzyme activity in the sample calculated from the generated kinetic information. 

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol 

In addition to enzyme activity, the concentration of an nonelectroactive substrate can be determined electrochemically by this technique. By keeping the substrate (analyte) the limiting reagent, the amount of product produced is directly related to the initial concentration of substrate. Either kinetic or equilibrium measurements can be used. Typically an enzyme which produces NADH is used because NADH is readily detected electrochemically. Lactate has been detected using lactate dehydrogenase, and ethanol and methanol detected using alcohol dehydrogenase 

Lequea et al. used the activity of tyrosine apodecarboxylase to determine the concentration of the enzyme cofactor pyridoxal 5 -phosphate (vitamin B6). The inactive apoenzyme is converted to the active enzyme by pyridoxal 5 -phosphate. By keeping the cofactor the limiting reagent in the reaction by adding excess apoenzyme and substrate, the enzyme activity is a direct measure of cofactor concentration. The enzymatic reaction was followed by detecting tyramine formation by LCEC. The authors used this method to determine vitamin B6 concentrations in plasma samples. 

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. 

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The category of algebraic equation models is quite general and it encompasses many types of engineering models. For example, any discrete dsmamic model described by a set of difference equations falls in this category for parameter estimation purposes. These models could be either deterministic or stochastic in nature or even combinations of the two. Although on-line techniques are available for the estimation of parameters in sampled data systems, off-line techniques... 

FTIR in multiply hyphenated systems may be either off-line (with on-line collection of peaks) [666,667] or directly on-line [668,669]. Off-line techniques may be essential for minor components in a mixture, where long analysis times are required for FT-based techniques (NMR, IR), or where careful optimisation of the response is needed. In an early study a prototype configuration comprised SEC, a triple quadrupole mass spectrometer, off-line evaporative FTIR with splitting after UV detection see Scheme 7.12c [667]. Off-line IR spectroscopy (LC Transform ) provides good-quality spectra with no interferences from the mobile phase and the potential for very high sensitivity. Advanced approaches consist of an HPLC system incorporating a UV diode array, FTIR (using an ATR flow-cell to obtain on-flow IR spectra), NMR and ToF-MS. 

Coupled LC-LC can separate high-boiling petroleum residues into groups of saturates, olefins, aromatics and polar compounds. However, the lack of a suitable mass-sensitive, universal detector in LC makes quantitation difficult SFC-SFC is more suitable for this purpose. Applications of multidimensional HPLC in food analysis are dominated by off-line techniques. MDHPLC has been exploited in trace component analysis (e.g. vitamin assays), in which an adequate separation for quantitation cannot be achieved on a single column [972]. LC-LC-GC-FID was used for the selective isolation of some key components among the irradiation-induced olefinic degradation products in food, e.g. dienes and trienes [946],... 

Tobias et al. [665] have described a method in which the GC effluent is passed into a combustion furnace to convert the organic hydrogen content into water, which is then selectively reduced to hydrogen in a reduction furnace containing Ni metal. The final stream is transmitted to the IRMS via a heated Pd filter, which passes only hydrogen isotopes to the ion source. For a benzene sample a precision of < 5 %o was obtained for <52HSMOw> which approaches the performance of off-line techniques and the requirements for studies of natural variability. This already meets requirements for analysis of D-labeled compounds used in tracer studies [666,667]. 

The speed of analysis provided by LC-NMR can potentially give significant efficiency gains over off-line techniques. However, there are several factors that need to be considered before deciding which approach to take ... 

In Other words, the assurance of quality by measurement of process impurities in the end product has been replaced by assurance of quality by the removal of variance in the process (by continuous monitoring of a continuous process). Naturally, whether online process analysis is being used as a surrogate for an alternative off-line technique to measure specific analytes or as a monitor to reduce process variance it needs calibration and validation. These stages require measurement of process analytes by a reference off-line technique, usually HPLC, and subsequent demonstration that the resulting calibration model has reliable predictive power. 

Nanospray is a miniaturized version of electrospray. In the original setup of Wilm and Mann (8) it is utilized as an off-line technique using disposable, finely drawn (1 -gm tip), metallized glass capillaries to infuse samples at 10-30 nL/min flow rates. This allows more than 50 min analysis time with just a 1-pT sample. Due to the formation of much smaller droplets and the more effective ionization, there is often no need for LC separation, since the separation is accomplished in m/z or by MS/MS. However, limited reproducibility with respect to quantification and a more complex sample preparation can be seen as drawbacks. An on-line version for hyphenation with capillary and nano-LC as well as CE (slightly modified) is now commercially available. 

The morphology of particles is an important characteristic that can seriously affect powder handling and end-use properties. Off-line techniques are often not suitable for monitoring industrial processes since extracting the particles from the process can alter their shape, e.g. particles taken from a crystallizer may fracture or aggregate. 

The analysis of such systems is often complex. One solution to this problem is the use of preparative SEC, to provide fractions for subsequent analysis by off-line techniques such as mass spectrometry, Fourier transform infrared and nuclear magnetic resonance. This technique can also be used to isolate pure polymer fractions for subsequent use in SEC calibration. 

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. 

The temperature governs the calorimetric signature of this process and the analysis of the heat released, in combination with other off-line techniques, allows to discern the sequence of the events involved and to quantify the heat released in each event. 

Mass spectrometry (MS) techniques are based on measurement of the charge/mass (m/z) ratio of molecules ionized by electron impact, laser irradiation, or by interaction with electric fields. Several MS methods have been applied in the study of grape and wine proteins initially as off-line techniques, then coupled to LC and CE techniques. 

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. 

There are a variety of other spectroscopy techniques available for investigating reaction kinetics. However, MALDI-TOF-MS allows for the determination of rate constants regardless of the incorporation of a chromophore, and at a wide variety of buffer concentrations. Both of these advantages are useful if one wishes to study enzymatic reactions under naturally occurring conditions. The advantages of MALDI-TOF-MS can be compared to spectrophotometric methods, which require a chromophore, and electrospray ionization mass spectrometry, which is sensitive to buffer concentrations. As MALDI-TOF-MS is an off-line technique requiring quenching for the analysis we performed, a rapid mixer must be incorporated into the system. 

Heat transfer surface cleaning techniques can be applied either online or off-line. Online techniques (usually used for tubeside applications) include various mechanical techniques (flow-driven or power-driven rotating brushes, acoustic/mechanical vibration, chemical feeds, flow reversal, etc.). Off-line techniques include chemical cleaning, mechanical cleaning by circulating particulate slurry, and thermal baking to melt frost/ice deposits. 

Once the sampling protocol has been decided, the next step is analysis of the sample(s). Of course, sampling and analysis may also be integrated. In the past 50 years, the type of assays that were done in-process exploited the nonselective properties of the process stream, such as density, viscosity and conductivity. This monitoring has been achieved by both automatic and automated instruments. Selective properties of the process stream, such as chemical composition, were usually measured by taking grab samples and examining them in a laboratory by off-line techniques as they are more difficult to adapt for process stream analysis. 

The type of ionization method is determined by the method of sample introduction. The most useful sources with GC are El and Cl, and with LC, ESI (or another API method, e.g., APCI or APPI). MALDI is a stand-alone ionization method that is best suited to peptides and proteins. Because MALDI is an off-line technique, samples can be investigated multiple times as opposed to LC, where peaks cannot be revisited without reinjecting the sample. Although it is highly desirable to have multiple ionization sources, the budgets for dedicated instruments may not allow such luxuries. 

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