Column procedures

As mentioned in the historical synopsis (Section 5.1), Levine121 perfected the compendial partition column procedure in which aspirin in chloroform is first trapped in an immobile phase of sodium bicarbonate on a column of siliceous earth (celite) then eluted with a solution of acetic acid in chloroform and measured spectrophotometrically. This has been also used for separation in combination products.80 For the determination of salicylic acid in presence of aspirin by this method, see Section 5.61. Ion exchange columns filled with strongly or weakly basic anion exchange resin in the acetate or chloride cycle have also been used for separation of aspirin in combination products. 122 123/l2lf This has also been adapted for a student experiment.125 A Sepha-dex-G25 column has been used for the separation of aspirin from salicylic acid.126... 

Owing to the short path length, the sensitivity of photometric detection in CE is limited. For this reason, procedures are necessary to enhance the sensitivity. This can be achieved by sample pre-concentration, improvement of the optical design and/or alternative capillary geometries [66]. Sample pre-concentration can be done off- or on-column. The off-column procedures are well described in the literature and have been applied extensively in chromatography [67]. The on-column procedures are more specific to CE and are therefore briefly discussed. 

The performance of HPLC separations can be increased not only by the application of two- or multidimensional techniques but also by the use of simulated moving bed (SMB) [100-102] or true moving bed (TMB) techniques [103,104], SMB is a multicolumn separation technique allowing the continuous separation of analyses with higher productivity and smaller eluent consumption than the traditional single-column procedures. TMB... 

Exchange Column Procedure. J. Amer. chem. Soc. 70, 3520 (1948). 125- Tompkins, P. C., L. Wish and J. X. Khym Some Unit Operations for Preparation of Carrier-free Strontium. U. S. Nat. Nuclear Energy Ser., Vol. 9B, Paper 239- New York McGraw-Hill 1951. 

Penefsky introduced the centrifuged-column procedure (see diagram below) that can be used for small aliquots of enzymes and other proteins (a) to exchange buffers and/or other solutes, (b) to measure ligand binding, and (c) to concentrate protein by as much as 10 times. His two-step protocol for rapid buffer exchange can be summarized as follows ... 

Penefsky centrifuged-column procedure, RAPID BUFFER EXCHANGE PENICILLOPEPSIN... 

Column Procedures Are the Most Versatile and Productive Purification Methods Electrophoresis Is Used for Resolving Mixtures Sedimentation and Diffusion Are Used for Size and Shape Determination Protein Purification Procedures... 

Column Procedures Are the Most Versatile and Productive Purification Methods... 

Column procedures are the most effective and most varied of purification methods. Common to all col- umn procedures is the use of a glass cylinder with an... 

Column procedures are useful in fractionating proteins with different affinity properties and sizes. By the use of different column materials in conjunction with specific eluting solutions, highly purified protein preparations can be obtained. 

The rates of the reactions are not usually limiting for in vitro reactions, whereas slow reactions can cause serious mixing and reaction-volume problems in post-column procedures. This effect is not very serious in TLC. 

The advantages of post-column procedures are summarized below ... 

Horwitz et al. have discussed a number of applications for Sr-Resin, including the analysis of 90Sr in high-level nuclear wastes.117"119 In conventional practice, isolation of 90Sr with Sr-Resin is a manual open-column procedure with quantification of the eluted 90Sr carried out as a separate counting step. 

Berggren, D. (1989) Speciation of aluminium, cadmium, copper, and lead in humic soil solutions - a comparison of the ion exchange column procedure and equilibrium dialysis. Int.J. Environ. Anal. Chem., 35, 1-15. 

The liner may also be inverted, reversing the location of its radial restrictions. If the narrower restriction is placed at the top and a wide-bore column into the bottom restriction, the column will enter into the expansion chamber and butt to the narrower end. In this mode, a 26-gauge needle can be used to perform wide-bore on-column injections. The advantage of this mode of sample introduction is that exposure to active sites of the glass liner is eliminated. The disadvantage is that small samples must be used relative to the direct flash. The effect of depositing particles directly on the column is more severe for the on-column procedure, along with wider solvent peaks. 

Coupled column (multidimensional) systems in chromatography have also been developed to improve resolution. The coupled column procedure, as noted in Section 6.4, requires two or more columns of different kinds having different retention mechanisms. 

The classical column procedures for the preparation of lysozyme are difficult to use with high viscosity solutions such as serum. A batch method was developed based on affinity chromatography using deami-nated chitin [82]. The method was found to give a one-step purification of nearly theoretical amounts in tissue homogenates tested. These included tissue homogenates from humans, primates, avian egg white and plants. The use of a chitin-coated cellulose affinity column for purification of lysozyme has also been described [83]. 

When beginning the development of a TE column procedure, an excellent source of solid-phase extraction (SPE) information may be found in SPE device manufacturers literature (or on their websites). Most manufacturers offer at least 25 years of literature references on their products. Their procedures will often provide useful information about compound types and the SPE chemistries used and solvents for cleanup and elution. The same packing material may be used in the TE column, either the same size (usually 40 - 63 /tm for SPE tubes) or it can be a 5- or 10-/um version of it. Buying the TE packing material from the same company that makes the SPE tubes will allow the solvent information to be used with the on-line work. 

The preparation of this analytical reagent is described by Sawicki et al., who developed a sensitive test for formaldehyde and other water-soluble aliphatic aldehydes. Treatment of a drop of an aqueous solution of formaldehyde with excess basified reagent effects conversion to the azine (2). Ferric chloride then oxidizes (1) to (3). which condenses with the azine to form the brilliant blue cation (4. one of the resonance structures).. Spot plate, paper, silica gel. and column procedures described fur the detection and determination of aldehydes are particularly useful fur determination of formaldehyde In auto exhaust fbmes and polluted air. 

M15. Miller, E. J., and Piez, K. A., An accelerated single-column procedure for the automatic analysis of amino acids in collagen and elastin hydrolyzates. Anal. Biochem. 16, 320-326 (1966). 

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