Step elution procedures

Expose the column to several cycles of the low pH elution procedure (see step 10 below) to remove any noncovalently adsorbed antibody. 

Step elution is similar to gradient elution in that the composition of the mobile phase changes during the separation process. However, in step elution the change is not continuous. It involves a sudden change in the composition of mobile phase followed by a period where the mobile phase is held constant. This procedure results in a mobile phase profile that resembles a series of steps. 

A gradient former is not required for step-wise elution procedures, but most oligonucleotide fractions need gradient elution to achieve the required resolution. That is, the ionic strength, or other property, of the eluting buffer is varied continuously as the elution proceeds. The simplest type of gradient former is made from two open tall... 

Further improvement of throughput in LC-MS analysis may be achieved by step-gradient elution. This elution format is essentially an on-line solid-phase extraction (SPE) process, where the samples are loaded onto the column, washed with aqueous mobile phase to remove water soluble impurities, and compounds are eluted with a mobile phase of high organic content. The technique combines the simplicity of FIA with the benefit of the removal of impurities and buffer components before mass spectrometry detection. In this case, selectivity is achieved by mass spectrometry alone without chromatographic separation. The technique has been used for compound purity assessment and quantitation. An on-line back-flush SPE-MS technique has been used by Marshall for quality assessment of the combinatorial libraries [112]. This back-flush elution procedure provides a very effective in-line removal method... 

By this technique of combining affinity partitioning with affinity chromatographic elution, the advantages of the two procedures were combined. The rapid and effective removal of cell debris in the extraction procedure, and finally, the efficient elution procedure of the affinity chromatographic step was achieved. 

The isooctane solution is eluted through a long, narrow (6 mm X 90 cm) column of 2% partially deactivated alumina. The solvent elution procedure is as follows 5 mL prewash of cyclohexane, 10 mL sample, 30 mL 5% methylene chloride in cyclohexane, 30 mL 10% methylene chloride in cyclohexane, 30 mL 50% methylene chloride in cyclohexane, and 30 mL of methylene chloride. Ten-milliliter cuts are collected, counted, and combined according to the radioassay data. The combined cuts are reduced to approximately 0.05 mL on a steam bath under nitrogen after adding 5 fxL of n-hexadecane to prevent going to dryness. An aliquot (10 fxL) of this concentrate is used for the GC-UV finishing step. 

Isolation procedures for many biochemicals are based on chromatography. Practically any substance can be selected from a crude mixture and eluted at relatively high purity from a chromatographic column with the right combination of adsorbent, conditions, and eluant. For bench scale or for a small pilot plant, such chromatography has rendered alternate procedures such as electrophoresis nearly obsolete. Unfortunately, as size increases, dispersion in the column ruins resolution. To produce small amounts or up to tens of kilograms per year, chromatography is an excellent choice. When the scale-up problem is solved, these procedures should displace some of the conventional steps in the chemical process industries. 

Neomycin has been produced by growing the organism, Streptomyces No. 3535, in a suitable nutrient medium under appropriate stationary or submerged aerobic (viz shaken) conditions, and then isolating and purifying the substance, e.g., by procedure of the sort described in the figure including various steps of adsorption, recovery by elution, separation from impurities, and precipitation. 

An affinity sorbent based on WPA-PG carrying immobilized human IgG was applied to the isolation of the first component of the complement (Cl) from human serum and for its separation into subcomponents Clr, Cls and Clq by the one-step procedure [126,127]. Cl was quantitatively bound to the sorbent at 0 °C. The activities of subcomponents Clq and Clr2r2 in the unbound part of the serum were found to be 0.8% and 3.3% of the initial activities in serum. This fraction, therefore, could be used as a R1 reagent for determining the hemolytic activity of Cl. Apparently, the neighboring macromolecules of immobilized IgG resemble to some extent an immune complex, whereas Cl formation is facilitated due to the mobility of polymer chains with the attached IgG macromolecules (Cl is usually dissociated in serum by 30%). After activation of bound Cl by heating (30 °C, 40 min) the activated subcomponent Clr is eluted from the sorbent. Stepwise elution with 0.05 mol/1 EDTA at pH 7.4 or with 0.05 mol/1 EDTA + 1 mol/1 NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent Cls and subcomponent Clq. 

To determine secondary alkanesulfonates in sewage wastewaters, solid phase extraction (SPE) and a single-step procedure which combines elution and injection port derivatization for analysis with GC-MS were developed [36]. Again a tetrabutylammonium ion pair reagent was employed both to elute the secondary alkanesulfonates as their ion pairs from CI8-bonded silica disks and to derivatize sulfonate ion pairs under GC injection port conditions. Secondary alkanesulfonates were effectively recovered from samples of raw sewage (>92%) and from primary (>98%) and secondary (>85%) effluents. No... 

In summary, the selection procedure consists of three steps (1) compare each spectrum in X with all spectra already selected by applying eq. (34.14). Initially, when no spectrum has been selected, the spectra are compared with the average spectrum of matrix X (2) plot of the dissimilarity values as a function of the retention time (dissimilarity plot) and (3) select the spectrum with the highest dissimilarity value by including it as a reference in matrix Y,-. The selection of the spectra is finished when the dissimilarity plot shows a random pattern. It is considered that there are as many compounds as there are spectra. Once the purest spectra are available, the data matrix X can be resolved into its spectra and elution profiles by Alternating Regression explained in Section 34.3.1. 

Note each lot of Florisil must be checked for acceptable recovery of flumioxazin prior to initiating the column cleanup procedure. Adjust elution volumes and/or the solvent mixture as necessary to achieve >90% recovery for this step. Sample cleanup with Florisil may not be required for some water samples. 

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