Integrated detection-separation-reaction

BI SP The flow cell is filled by injecting in the flow system a homogeneous bead suspension of an appropriate solid support previously loaded with the chromogen ic reagent. The solid beads work as a flow-through chemical sensing microzone integrating online separation/reaction/ detection 0.02-0.14 13-16 4.02-4.19 Fruit juices, pharmaceuticals 2003 282... 

Figure 2.1 — Variants of integrated reaction, separation and detection in continuous-flow analytical systems. (1) Reaction/separation. (2) Reaction/detec-tion. (3) Separation/detection. (4) Reaction/separation/detection.

The equipment required to develop this type of sensor is very simple and resembles closely that used to implement ordinary liquid-solid separations in FI manifolds. The only difference lies in the replacement of the packed reactor located in the transport-reaction zone with a packed (usually photometric or fluorimetric) flow-cell accommodated in the detector. Whether the packing material is inert or active, it should meet the following requirements (a) its particle diameter should be large enough (< 80-100 fim) to avoid overpressure (b) it should be made of a material compatible with the nature of the integrated detection system e.g. almost transparent for absorbance measurements) and, (c) the retention/elution process should be fast enough to avoid kinetic problems. 

Gottschlich et al. [134] developed a microfluidic system that integrated enzymatic reactions, electrophoretic separation of the reactants from the products, and postseparation labeling of the proteins and peptides prior to fluorescence detection (see Fig. 12). Tryptic digestion of oxidized insulin p-chain was performed in 15 min under stopped flow conditions in a heated channel serving as the reactor, and the separation was completed in 60 s. Localized thermal control of the reaction channel was achieved using a resistive heating element. The separated reaction products were then labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and detected by fluorescence detection. 

FIGURE 16.20 Integrated cycle sequencing reaction, sample cleanup, separation, and detection system. 

Bio)chemical reactions may take place prior to or after the continuous separation module and are intended to enhance or facilitate mass transfer, detection or both. The earliest and simplest approach to integrated analytical steps in continuous-flow systems involves a combination of chemical reactions and continuous separations [4,5]. Such is the case with the formation of soluble organic chelates of metal ions in liquid-liquid extractions with the ligand initially dissolved in the organic stream [6], the formation and dissolution of precipitates [7], the formation of volatile reaction products in gas difiusion [8] and that of volatile hydrides in atomic absorption spectro-... 

The above exceptions leave relatively few sensors based on integrated separation and detection, particularly of the types involving gas-liquid and liquid-liquid interfaces, which require the detector to be responsive to the gas or ion (molecule) transferred across the membrane. The scope of liquid-solid interactions is somewhat broader as it enables not only retention of the analyte and monitoring of some intrinsic property, but also to retain a product of a previous reaction, thereby substantially expanding the possibilities. 

Flow-through sensors based on integrated reaction, separation and detection... 

Figure 5.1 — Classification of (bio)chemical flow-through sensors based on integrated reaction, separation and detection according to whether the three processes take place sequentially (A,B) or simultaneously (C) at the sensing microzone. S sample R reagent. (Reproduced from [1] with permission of the Royal Society of Chemistry).

Figure 5.2 — Classification of (bio)chemical flow-through sensors based on integrated reaction, separation and detection according to the type of separation technique involved.

Figure 5.3 shows the different possible ways in which the ingredients of the (bio)chemical reaction can take part in the sensing process. For example, the analyte can be retained temporarily and take part in the separation process. The reagent can be present in the solution used to immerse the sensor or immobilized in a permanent fashion on a suitable support. Also, the catalyst can be introduced directly across a membrane or be permanently immobilized. Finally, the reaction product can be the species transferred in the separation process or also be temporarily immobilized. These and other, more specific alternatives that are described below are all possible in (bio)chemical flow-through sensors integrating reaction, separation and detection. 

The fact that the species transferred across the sensor membrane (the analyte or reaction product) must be a gas limits application of this type of flowthrough sensor, which, however, is still more versatile than are the sensors based on integrated separation (gas diffusion) and detection [4] described in Section 4.2 in fact, while these latter can only exploit physico-chemical properties of the analytes transferred, sensors based on triple integration allow the implementation of a (bio)chemical reaction and formation of a reaction product, so they are applicable to a much wider variety of systems with adequate sensitivity and selectivity. 

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