Offline Technique

LC-MS/MS techniques moved the bottleneck for analyzing NCEs from sample analysis to sample preparation. Several methodologies have been developed to reduce sample preparation time. Online extraction methods are becoming more popular and have many advantages over offline techniques. For example, online sample preparation is easier to automate and minimizes recovery issues. 

In offline techniques, the desorption solvent most often used for SPE with C18 sorbent is ethyl acetate, sometimes associated with isooctane.MTBE is also used. In the case of polymeric phases, methanol, acetonitrile, and acetone are used. ... 

The mRNA level of a bioprocess-relevant gene can be used as an early measure of its expression status. A simple but labor-intensive offline technique for the mRNA analysis of one or a limited number of genes is Northern blotting. Following the separation of the RNA sample in a polyacrylamide gel, the RNA is blotted on a membrane and detected by the hybridization of labeled sequence-specific probes. This technique does not only allow a quantitative analysis of the mRNA level but also gives information about the length and potential degradation products of the RNA. 

As an offline technique the TLC process consists of several individual steps. Traditionally, instruments for each step are controlled by separate more or less sophisticated software. winCATS-Planar Chromatography Manager (CAMAG, Switzerland) is the first integrated software that can communicate with all instruments involved in the TLC process. A win-CATS method file can include information about the stationary phase, samples and their components, standards and their preparation, application parameters, prechromatographic derivatization, development conditions, postchromatographic derivatization, all parameters of densitometric and spectral evaluation, and details of electronic documentation using a... 

Spectroscopic techniques can be applied for monitoring conversion. Nuclear-magnetic resonance (NMR) and Fourier-transform infrared (FTIR) can be applied as offline techniques. However, spectroscopic techniques are arguably most attractive if they can be applied online. For example, attenuated total reflectance (ATR) FTIR has been applied to monitor the copolymerization of butyl acrylate and vinylacetate (Jovanovic Dube, 2003). Raman spectroscopy, in combination with a self-modelling curve-resolution technique, has... 

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... 

TLC plates are of particular interest as substrates for spectroscopy (i) as a storage device for offline spectroscopic analysis (ii) for efficient in situ detection and identification and (iii) for exploitation of spectroscopic techniques that cannot be used in HPLC. Thin-layer chromatography combined with HR MAS (NMR) can be used for compound identification without the need for elution from the stationary phase [413]. Recently also TLC-XRF was found suitable for in situ TLC imaging of elements [414]. The combination... 

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. 

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. 

Off-line measurements differ significantly from on-line techniques. Offline measurements include safety testing in a laboratory, which has the major advantage of providing available time to redesign the process and/or equipment if a risk is identified. Of course, immediate process response is not possible. 

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... 

Table 1.5 Differences between the offline and online techniques...

One important area of stable isotope ratio measurements (SIRMS) is the analysis of gases using offline or online techniques (see also the discussion in Section 8.3.). 

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. 

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