Detection integrated with separation

The clean-up separation manifolds are quite similar for different detection systems. They can also be integrated with separation manifolds based on other principles such as gas-liquid, or liquid-liquid separation. A good example is that described by Marshall and van Staden [33]. These workers used an on-line ion-exchange column to remove interfering base metals in the determination of selenium and arsenic with a FI hydride generation AAS system, which also incorporated a gas-liquid separator. The system is shown schematically in Fig. 4.4. More recently, Tyson et al.[40] reported on a column separation system which effectively removed the interference of large concentrations of copper in the determination of hydride forming elements. 

In most situations analysts can achieve a rapid reasonable separation of compounds using an appropriate standard CE method with generic operating conditions [877]. This eliminates or reduces dramatically the need for method development. Major instrumental error sources in CE are detection, integration and injection. General guidelines for validation of CE methods are available and similar to those of HPLC [878]. Validated CE methods often perform the same as, or better than, the corresponding HPLC methods. 

The FPI principle can also be used to develop thin-film-coating-based chemical sensors. For example, a thin layer of zeolite film has been coated to a cleaved endface of a single-mode fiber to form a low-finesse FPI sensor for chemical detection. Zeolite presents a group of crystalline aluminosilicate materials with uniform subnanometer or nanometer scale pores. Traditionally, porous zeolite materials have been used as adsorbents, catalysts, and molecular sieves for molecular or ionic separation, electrode modification, and selectivity enhancement for chemical sensors. Recently, it has been revealed that zeolites possess a unique combination of chemical and optical properties. When properly integrated with a photonic device, these unique properties may be fully utilized to develop miniaturized optical chemical sensors with high sensitivity and potentially high selectivity for various in situ monitoring applications. 

Flow-through sensors based on integrated optical detection and a liquid-liquid separation are relatively scant since the analytes are rarely determined by their photometric or luminescence properties. Thus, with few exceptions, these sensors use amperometric detection —as noted earlier, ISEs and ISFETs are dealt with separately in Section 4.6. 

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).

HPLC has transformed quality control in the pharmaceutical and chemical industries, as it provides a rapid means of checking the purity of samples and even allows for the purification of small amounts of samples by preparative HPLC. The majority of such systems use L V to detect and quantify substances as they elute from the separative column. UV detectors are usually variable wavelength and can be used to detect molecules with absorption maxima above 210 nm by measuring the absorbance of the eluent. When a UV-absorbing substance is eluted from the HPLC column, it absorbs UV radiation at the appropriate wavelength for its chromophore. The amount of UV absorbed is proportional to the quantity of substance being eluted, and is converted into a peak on a chart recorder. Integration of each peak allows the relative quantities of the components of the solute to be determined. 

Figure 4.2 A schematic diagram of an integrated polymer monolith NCE with ESI-MS detection, (a) 1, separation channel 2, double-T injector 3, ESI source 4, eluent reservoir 5, sample inlet reservoir 6, sample waste reservoir 7, eluent waste reservoir that houses a porous glass gate 8, side channel for flushing the monolithic channel and 9, ESI emitter, (b) Cross-sectional view of reservoir 7, showing the position of the semipermeable glass gate, (c) Image of on-chip junction between the separation channel and the ESI emitter [25].

The focus of the present chapter is limited to review major accomplishments with partially integrated microcolumn separation systems that have been achieved in the last five years. Partial integration here refers in most cases to the fluidic part of the system which consists for example of a network of interconnected microchannels. The examples chosen have all been developed at least to the level of functional models and demonstrate principal feasibility. Many aspects of great importance in this context, such as chip fabrication, detection issues, higher levels of functional integration, etc., will be discussed in chap. 1 and 2 of this volume. 

By far most of the work discussed in this review has been based on LIF detection, usually with an 488 nm Ar-ion laser as the excitation source. Only very few other examples exist in the literature where other detection principles were investigated. One of these exceptions is an integrated detection cell for chip CE that has been described by Liang et al. [78]. In combination with the U-shaped separation channel, two additional well aligned channels to take up the excitation and collection fibers where micromachined in a glass plate. The U-cell provides a longitudinal path of 120 -140 pm in length parallel to the flow direction and can be used both for absorption and fluorescence measurements. The absorption detection limit was 0.003 AU ( 6 pM of a fluorescein dye) in the fluorescence mode a detection limit of 3 nM fluorescein (20 000 molecules) was achieved. 

For MS detection, the microfluidic chip has been coupled to various ionization interfaces (ESI, MALDI) and to different mass analyzers (by single quadrupole, triple Q, ion-trap, MS/MS, and FTICR). The chip can be single- or multi-channel, and can be integrated with various sample handling processes such as preconcentration, digestion, and separation. The following two sections describe the two ionization interfaces (ESI and MALDI) that precede the MS analysis. 

Challenges for HPLC method development are for combination products where more than one active is present in the same formulation. Since degradation products are of big concern from a safety perspective, HPLC will continue to be utilized as a major separation technique during formulation development to develop the most safe and efficacious formulations to be used for human use. Many different types of bonded phases are currently available for routine HPLC analysis in addition, very selective and sensitive detection techniques can be integrated with HPLC to help an analytical chemist control the final quality of the drug product. Furthermore, the availability of this technique makes it the first and sometimes the only choice for the analysis of degradation products. 

The analytical pervaporator can be used in combination with a flow-injection manifold, either in the upper chamber when the pervaporated species must be derivatized for adaptation to the detector and/or in the lower chamber for the pervaporation of analytes from liquid samples or slurries. Alterations of either the auxiliary dynamic manifold or the pervaporator itself are required when the pervaporation step is assisted by focused microwaves, the separation step assists in the continuous monitoring of an evolving system, untreated solid samples are used or pervaporation is integrated with detection. 

Fig. 4.22. Monitoring of pervaporation kinetics. (A) By integrating separation and detection. (B) By coupling pervaporation with a retention step integrated with detection. For abbreviations, see previous figures. (Reproduced with permission of Wiley Sons.)...

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