Packing materials, micro HPLC

In certain cases, affinity columns can be used to fractionate within classes of bound materials for instance, protein A antibody columns have been used to separate the various subtypes of IgG. In this case, the packings are micro-porous, heavily cross-linked polymers and benefit from HPLC operating conditions. Eluting conditions are usually step gradients of buffers with different pHs. The last step of a protein G column is at a very acidic pH and the sample is eluted into a buffer solution that quickly raises the pH to prevent protein denaturation. 

Micro-injections in micro-flow and nano-flow systems are done with injectors in which the external sample loop is replaced with the internal fixed volume within the injector body. HPLC-on-a-chip systems also build the column into the injector body. The internal path within the injector body is abladed with a laser, packed with micro-packing material, and this serves as the separating media. The injector inlet is connected to the pumping system and the outlet to the detector. Sample is loaded into an internal loop in the load position, then injected onto the chip HPLC by turning the injector. Obviously, in a system like this sample size is very limited and the detector is usually a highly sensitive mass spectrometer. 

Micro- and nano-HPLC systems (Fig. 15.11) rely on small-diameter and capillary columns packed with high-efficiency packing materials and very slow flow rates to produce concentrated solutions and sharp chromatography peaks to feed electrospray interfaces for mass spectrometers. 

MEPS Micro-LLE Microextraction in a packed syringe Micro-liquid-liquid extraction Sample enrichment on a small amount (1-2 mg) of solid material inserted into a syringe (100-250 iL) as a plug. Elution with a suitable solvent As LPME Equally applicable to HPLC and GC analysis... 

The buffer system is a combination of buffer for electrophoresis and eluent for the particular chromatographic mode being employed. Figure 5.14 shows the separation of a group of neutral molecules using a capillary packed with a reversed-phase material.38 The buffer was a mixture of 4 mM sodium tetraborate (pH 9.1) and acetonitrile (20 80, v/v). The separation was compared with a micro-HPLC separation in which the same capillary was used but the eluent was pressure driven. As can be seen in Figure 5.14, sharper peaks were obtained with the EOF-driven system. 

More recently, columns have been developed where the stationary phase is formed of a porous polymer network inside the capillary. These are called monolithic phases, and have emerged as an alternative to traditional packed bed columns for use in micro-HPLC. They hold many advantages over traditional packed bed columns, being easy to manufacture since the monolith is formed in situ, often via a one-step reaction process, and its properties such as porosity, surface area, and functionality can be tailored. Another major advantage is that they eliminate the need for retaining frits. These columns can be manufactured from a variety of materials, but the most common include sol-gel, methacrylate-based, acrylamide-based, and styrene-based polymeric structures. 

A variety of micropellicular packing materials has been developed for the analysis of both small and large molecules by various HPLC modes, including ion exchange (lEC), metal interaction (MIC), reversed-phase (RPC), and affinity chromatography (AC). Besides analytical applications, other possible utilization of micro-pelhcular stationary phases includes fundamental kinetic and thermodynamic studies of the retention mechanisms on a well-defined surface. Nevertheless, a relatively limited variety of micropellicular columns are commercially available. They are mainly restricted to ion-exchange and reversed-phase stationary phases. This may reflect certain practical disadvantages of micropelhcular sorbents. 

Micronization in the field of column hardware and packing materials led to the development of microbore and short columns the latter allow separation times sometimes to be shortened by a factor of 10 (83, 385). The advent of microbore columns created a whole new methodology in HPLC with detection limits lowered into the femto-mole range (224, 260, 291). Special micro-metering pumps give pulseless flow rates down to pl/min, and allow direct interfacing of the LC apparatus to a mass or infrared spectrometer. They also open the field of fused silica columns for HPLC use, and it might be not too optimistic to expect theoretical plate numbers of >500000 per LC column for the near future. 

Chip HPLC—Nano-flow, micro-sample HPLC system in which the packed column resides within the body of the injector. Originally touted as the HPLC of the future for nano-level LC/MS, but its potential has been slow to materialize. 

SPME is a fast, simple, and green sample preparation technique that can easily combine the process of sample preconcentration and GC or HPLC determination. However, in some cases, it still has difficulty in the fabrication of SPME fibers. In 2006, Lee s group reported a novel extraction and preconcentration technique termed micro-solid-phase extraction (p-SPE), based on the packing of sorbent material in a sealed porous polypropylene membrane envelope. Recently, Lee s group reported... 

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