Sequential elution methods

The development of the sequential elution methods makes it possible not only to cleanly fractionate the three cellulase components, but to do the fractionation with very little loss of enzyme. The total recovery of major enzyme components, summarized in Table III, is considerably higher than those reported previously by other researchers (8,9). Table III also gives the molecular weights of the three enzyme components. 

Ultrasonic nebulizers have also been employed in continuous flow systems as interfaces between sample preparation steps in the analytical process and detection by virtue of their suitability for operating in a continuous mode. Thus, preconcentration devices have commonly been coupled to atomic spectrometers in order to increase the sensitivity of some analytical methods. An enhancement factor of 100 (10 due to USNn and 10 due to preconcentration) was obtained in the determination of platinum in water using a column packed with polyurethane foam loaded with thiocyanate to form a platinum-thiocyanate complex [51]. An enhancement factor of 216 (12 with USNn and 18 with preconcentration) was obtained in the determination of low cadmium concentrations in wine by sorption of metallic complexes with pyridylazo reagents on the inner walls of a PTFE knotted reactor [52]. One special example is the sequential determination of As(lll) and As(V) in water by coupling a preconcentration system to an ICP-AES instrument equipped with a USN. For this purpose, two columns packed with two different resins selective for each arsenic species were connected via a 16-port valve in order to concentrate them for their subsequent sequential elution to the spectrometer [53]. 

Since this review was completed, a number of interesting developments have been obtained in this laboratory and elsewhere in various protein fractionation techniques at subzero temperatures. Let us mention a work on isoelectric focusing and electrophoresis by M. Per-rella, A. Heyda, A. Mosca and L. Rossi-Bemardi (Anal. Biochem., in press) a work by C. Le Peuch and C. Balny on the sequential elution of proteins bound by hydrophobic interaction chromatography [FEBS Lett. 87,232(1978)] a work by K. Andersson, Y. Benyamin, P. Douzou, and C. Balny about organic solvents and temperature effects on desorption from immunoadsorbents (J. Immunol. Methods, in press, 1978). 

Another concept to consider in reversed-phase elution is selective elution. Selective elution consists of using sequential elution solvents to selectively remove several classes of solutes, or using wash solvents to remove impurities that will interfere with the analysis. An example of selective elution is the separation and isolation of herbicides and their metabolites by a reversed-phase C-18 mechanism. Figure 3.5 shows the separation of alachlor, a herbicide, and its sulfonic-acid metabolite. In this method, both compounds are sorbed to the C-18 resin by a reversed-phase mechanism. Even the ionic sulfonic acid is bound to the C-18 bonded phase. The metabolite, whose structure is shown in Figure 3.5, is a surface-active compound and is bound by reversed phase with its ionic functional group solvated by the aqueous phase. The parent compound is eluted with ethyl acetate while the ionic metabolite stays bound to the C-18 resin. Apparently the solubility of the ionic metabolite in ethyl acetate is too low for dissolution. When methanol is applied to the column, the sulfonic acid metabolite elutes from the column. Thus, a fractionation is obtained by selective elution (Aga et al., 1994). 

Brown, D.H., Wells, J.M., 1988. Sequential elution technique for determining the cellular location of cations. In dime, J.M. (Ed.), Methods in Bryology. Proc. Bryol. Meth. Workshop, Mainz. Hattori Bot. Lab., Nichinan, pp. 227-233. 

Details on the preparation of pyrolysis oils at SERI in the entrained-flow, fast ablative pyrolysis reactor can be found in a report by Diebold and Scahill (2). The oils in Figure 1 were obtained from two runs, 40 (c and d) and 41 (a and b), the oils being collected from a packed scrubber (a and c) and a cyclone scrubber (b and d). The oil obtained from the packed scrubber in run 41 was subjected to sequential elution by solvents chromatography (SESC) according to the method of Davis et al (7). The HPSEC of fractions 1 through 6 appear in Figure 3. Fraction 1 was eluted with 15% toluene in hexane (yield 0.4%), Fraction 2 was eluted with chloroform (yield 1.5%), Fraction 3 was eluted with 7.5% ether in chloroform (yield 7.5%), Fraction 4 was eluted with 5% ethanol in ether (yield 19.5%), Fraction 5 was eluted with methanol (yield 38.1%), and Fraction 6 was eluted with 4% ethanol in THF (yield 3.1%). The oils displayed in Figures 4 and 5 were produced from run 66. 

The method was used successfully in the isolation of descobaltocobalamin from a crude extract of Chromatium by Koppenhagen et al. (92). An aqueous ethanol/acetic acid extract from 500 g wet cells was applied on an Amberlite XAD-2 column (4.0 X 7.0 cm) and sequentially eluted with 0%, 2%, 10%, 20%, and 50% /er/-butanol (v/v) in water. Hydrogenobyric acid eluted at 10%, desco-baltcobalamin at 20%, and uroporphyrins at 50% butanol. After insertion of the... 

In environmental analytical applications where analyte concentrations, e.g. surfactants or their metabolites, are quite low, extraction and concentration steps become essential. Solid phase extraction (SPE) with cartridges, disks or SPME fibres (solid phase micro extraction) because of its good variety of SP materials available has become the method of choice for the analysis of surfactants in water samples in combination with FIA as well as LC—MS analysis. SPE followed by sequential selective elution provides far-reaching pre-separations if eluents with different polarities and their mixtures are applied. The compounds under these conditions are separated in the MS spectrometer by their m/z ratios providing an overview of the ionisable compounds contained in a sample. Identification in the sense it has been mentioned before, however, requires the generation of fragments. 

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