Micro-separation techniques

Rozing, G., Dittmann, M.M., Rozing, G.P. (1996). Capillary electrochromatography —a high-efficiency micro-separation technique. J. Chromatogr. A 744, 63-74. 

Ruchel, R. (1977). Two-dimensional micro-separation technique for proteins and peptides, combining isoelectric focusing and gel gradient electrophoresis. J. Chromatogr. 132, 451 168. 

Vastly reduced solvent consumption for micro-separation techniques has advantages in that it gives superior solvent suppression when protonated solvents are used [88]. Reduced solvent volumes also make the use of fully deuterated solvents more attractive, eliminating the need for solvent suppression [87]. A low-volume capillary probe with a 7 pi cell volume (1.5 pi active) is commercially available and its application to metabolite identification has been reported [89]. 

Boemsen, K. O. (2000). Using the TopCount microplate scintillation and luminescence counter and deep-well lumaplate microplates in combination with micro-separation techniques for metabolic studies, in Application note, AN004-TC. Packard Instrument Co. Available at http //www.perkinelmer.com. 

Fig. 7.1. Schematic that summarizes the relationship between the various micro-separation techniques. Line 1 lists the various micro-separation techniques. Below each method in line 2 is the type of flow or combination of flow (=>) that exists for each technique while in line 3 is the relative contribution of solute/bonded phase interactions and electrophoretic mobility to the separation mechanism.

Due to its high sensitivity and specificity, mass spectrometry (MS) is a detector of choice in micro-separation techniques such as CE. This selective detector provides additional advantages by allowing high speed analysis, giving information about the mass, and potentially, fhe structure of fhe separated compounds. This... 

The small synthetic scale used for production of many labeled compounds creates special challenges for product purification. Eirst, because of the need for use of micro or semimicro synthetic procedures, the yield of many labeled products such as high specific activity tritiated compounds is often low. In addition, under such conditions, side reactions can generate the buildup of impurities, many of which have chemical and physical properties similar to the product of interest. Also, losses are often encountered in simply handling the small amounts of materials in a synthetic mixture. As a consequence of these considerations, along with the variety of tracer chemicals of interest, numerous separation techniques are used in purifying labeled compounds. 

The spectrum of new analytical techniques includes superior separation techniques and sophisticated detection methods. Most of the novel instruments are hyphenated, where the separation and detection elements are combined, allowing efficient use of materials sometimes available only in minute quantities. The hyphenated techniques also significantly increase the information content of the analysis. Recent developments in separation sciences are directed towards micro-analytical techniques, including capillary gas chromatography, microbore high performance liquid chromatography, and capillary electrophoresis. 

The ionspray (ISP, or pneumatically assisted electrospray) LC-MS interface offers all the benefits of electrospray ionisation with the additional advantages of accommodating a wide liquid flow range (up to 1 rnl.rnin ) and improved ion current stability [536]. In most LC-MS applications, one aims at introducing the highest possible flow-rate to the interface. While early ESI interfaces show best performance at 5-l() iLrnin, ion-spray interfaces are optimised for flow-rates between 50 and 200 xLmin 1. A gradient capillary HPLC system (320 xm i.d., 3-5 xLmin 1) is ideally suited for direct coupling to an electrospray mass spectrometer [537]. In sample-limited cases, nano-ISP interfaces are applied which can efficiently be operated at sub-p,Lmin 1 flow-rates [538,539]. These flow-rates are directly compatible with micro- and capillary HPLC systems, and with other separation techniques (CE, CEC). 

High-performance liquid chromatography (HPLC) has become a standard separation technique used in both academic and commercial analytical laboratories. However, there are several drawbacks to standard HPLC, including high solvent consumption, large sample quantity, and decreased detection sensitivity. Micro-HPLC (pHPLC) is a term that encompasses a broad range of sample volumes and column sizes (as shown in Table 3.1), but Saito and coworkers provided narrower definitions in their review based on the size of the columns. ... 

In 1990, Bushey and Jorgenson developed the first automated system that coupled HPLC with CZE (19). This orthogonal separation technique used differences in hydrophobicity in the first dimension and molecular charge in the second dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 (xL/min volumetric flow rate, with a column of 1.0 mm ID. The effluent from the chromatographic column filled a 10 pU loop on a computer-controlled, six-port micro valve. At fixed intervals, the loop material was flushed over the anode end of the CZE capillary, allowing electrokinetic injections to be made into the second dimension from the first. 

The advancement of modern biochemistry and developments in micro- and macromolecular separations have been intimately linked. Capillary electrophoresis offers major advantages over other separation techniques, including speed and resolving power. The potential of capillary electrophoresis seems so vast that it will significantly complement the technology of high-performance liquid chromatography. However, because of unique characteristics of capillary electrophoresis, it will also... 

Genf, L. Demirel, M. Giiler, E. Hegazy, N. Micro-encapsulation of ketorolac tromethamine by means of a coacervation-phase separation technique induced by addition of non-solvent. J. Microencapsulation 1998, 15 (1), 45-53. 

Separated by Gas Chromatography, using a Potassium Bromide Micro-pellet Technique, Z. Anal, Chem. (1969) 246, 294-297. 

---