Online detection

Online detection using 4H nuclear magnetic resonance (NMR) is a detection mode that has become increasingly practical. In a recent application, cell culture supernatant was monitored on-line with 1-dimensional NMR for trehalose, P-D-pyranose, P-D-furanose, succinate, acetate and uridine.33 In stopped-flow mode, column fractions can also be analyzed by 2-D NMR. Reaction products of the preparation of the neuromuscular blocking compound atracurium besylate were separated on chiral HPLC and detected by 4H NMR.34 Ten isomeric peaks were separated on a cellulose-based phase and identified by online NMR in stopped-flow mode. 

A schematic representation of a CE system is presented in Figure 9.1. In this diagram, the CE components have obvious counterparts to those found in slab gel electrophoresis. Instead of buffer tanks there are two small buffer reservoirs, and the capillary takes the place of the gel (or more accurately, a gel lane). The capillary is immersed in the electrolyte-filled reservoirs, which also make contact with the electrodes connected to a high-voltage power supply. A new feature to the conventional gel electrophoresis format is the presence of an online detection system. 

A special mention in the field of enantioselective HPLC separations must be made of chiro-optical detection systems, such as circular dichroism (CD) and optical rotation (OR), which can be also used to circumvent the low UV detectability of chromophore-lacking samples [40, 61]. While sensitivity of chiro-optical detection is not always sufficient to perform enantiomeric trace analysis, the stereochemical information contained in the bisignate spectropolarimetric response is useful in establishing elution order for those compounds not available as single enantiomers of known configuration. An example of application of different online detection systems (UV and CD at 254 nm) in the enantioselective separation of a racemic sulfoxide on a commercially available TAG CSP is reported in Figure 2.12, under NP conditions. 

Quantitative RT PCR (qRT PCR) can be used to accurately determine the levels of messages within given preparations of RNA. qRT PCR thermocyclers provide rapid online detection and quantification of mRNA, however, the initial purchase cost and the cost of reagents may be prohibitive for some laboratories. Methods of semiquantitative RT PCR have been used and good descriptions of these techniques are available (Samhrook and Russell, 2001). However, the same cDNA populations should not be used for differential display reactions and verification that a potential differential display band represents a differentially expressed gene. For this reason, independent cDNA samples should be prepared if both the screening and verification methods rely on PCR. qRT PCR, therefore, should be used in conjunction with other methods to verify that a differential display band represents a differentially expressed gene. 

Figure 3. Generalized process for lithium-ion separator manufacturing. Each step of the separator manufacturing process has online detection systems to monitor the quality of the separator.

Fig. 5. Online detection of a pollutant in the influent stream of a municipal sewage plant with the nitrification biosensor installed in the testing plant in Fig. 4 (from [82])...

Health World Online. Detection of Opiates in Urine. Available online at http //www.healthy.net/clinic/lab/labtest/007.asp. Accessed June 2, 2006. 

The activity of dihydropyrimidine dehydrogenase can be measured in peripheral blood mononuclear cells, fibroblasts, and liver using radiolabeled thymine followed by separation of radiolabeled thymine and the radiolabeled reaction products with reverse-phase HPLC combined with online detection of the radioactivity [12]. 

In recent years, those methods that do accept original samples (e.g., from soils or water) without major treatment and concentration have gained importance due to the small degree of denaturing and of added pollutants. This section will focus on the methods that are suited to characterize the organic matter in its dissolved state. Powerful methods are also in use for online detection of chromatographic or otherwise obtained fractions. 

Yoshida et al. reported that generation and online detection of highly reactive carbocations from carbamates were accomplished by integrating an electrochemical microreactor with an FTIR spectrometer [57]. They also demonstrated that both the carbocations and nucleophiles could be generated using the paired electrochemical flow system to give the coupling products in reasonable yields (Scheme 4.42) [58]. 

Liquid chromatography mass spectrometry (LC-MS) is now routinely used in analytical laboratories. Traditional IPRs are non-volatile salts that are not compatible with MS techniques because they play a major role in source pollution that is responsible for reduced signals. Moreover the final number of charged ions that reach the detector is impaired by ion-pair formation actually IPRs added to the mobile phase to improve analytes retention exert a profound effect on analyte ionization. Chromatographers who perform IPC-MS must optimize the eluent composition based on both chromatographic separation and compatibility with online detection requirements. 

IPC-MS combined methods must be optimized with respect to separation and compatibility with online detection involving the constraints detailed in Table 12.2 regarding the composition and volatility of the mobile phase. The major concern of chromatographers who deal with this combined technique is the reduced signal caused by source pollution of non-volatile IPRs. Moreover, the efficiency of droplet development, which in turn affects the number of charged ions that ultimately reach... 

Finding an online detection method is an important step in carrying out any analytical or preparative chromatography operation. In a preparative operation, a reliable online detector can be used to determine at which time the desired product is eluting from a chromatography column. 

Flow-through methods with online detection 508... 

Figure 12.2. Microcolumn flow-through manifolds for fractionation analysis in environmental solids (ti) continuous-flow system with either fraction collection or online detection (Jj) flow-injection setup with discrete injection of extractant (c) sequential injection system as assembled for fully automated sequential or single extractions. MC microcolumn, C coil, SP syringe pump, PP peristaltic pump, R reagent, D detector, W waste, SV selection valve, IV injection valve. (Adapted from Mir6 et al., 2005a.)...

Figure 12.5. Comparison of EDTA extractrograms for Cu as obtamed via off-line and online measurements. Subfraction volume 1 mL and 175 iL for off-line and online detection, respectively. (Reproduced from Chomchoei et ah, 2005a copyright American Chemical Society.)...

It is worth noting that a single sample with a broad MWD is enough to make the calibration curve if the number of end-groups per chain is well-defined. Online detection by H NMR offers not only an absolute calibration curve but also information on the molecular weight dependency of some polymer properties such as tacticity or copolymer composition at the same time. Thus, efforts... 

---