Step-wise Elution

Several methods are applied to reduce the separation time. The best way is the selection of a suitable column and an eluent using isocratic elution. However, much skill and knowledge are required to make such a system. A flow rate gradient, step-wise elution, or eluent composition gradients are commonly applied to reduce separation times. 

Figure 2.6 Step-wise elution of aromatic compounds. Experimental conditions as in Figure 2.2 except the acetonitrile concentration was changed from 55 to 75% in water.

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... 

Fig. 6.28. (A) Preparative gel electrophoresis apparatus with step-wise elution. 1, Gel column 2, dialysis bag 3, 4, capillary tubes 5, 6, platinum electrodes 7, pump 8, monitoring system (UV). (B) Principle of the electrophoretic apparatus with intermittent elution.

Adsorption chromatography usually employs microcrystalline hydroxyapatite columns that adsorb proteins vmder conditions above pH 5. Gradient or step-wise elution with increasing concentration of eluting agent in the mobile phase may be used to release the enzyme required. 

Figure 3. Elution profile of Milfraction from hot-water extraction in Isaria iavonica on a column of DEAE-Sepharose CL-6B column. Mil (100 mg) was eluted with 1/15 Mphosphate buffer (pH 7.2) and the same buffer containing 0.2 or 0.6M NaCl in step wise elution. The sugar content of each fraction was measured at 485.0 nm by the phenol-sufluric acid method.

Elute the antiphosphotyrosine antibodies with 10 mM fynitrophenol phosphate in PBS, pH 7.4. Use a step wise approach. Add a column volume of the elution buffer and wait for 30 min. Apply a second column volume, save the first volume and again incubate for 30 min. Repeat once more, if necessary. The last column volume of eluate is collected by using PBS as the buffer. 

For a step-wise process, consecutive constants are used. In the solvent extraction or extraction by ion exchange resins, the expression for Kj is more complicated than Eq. (25) [211], but is still directly proportional to Pi or Ki. In experiments with single atoms, is defined by the retention time indicated by peaks on elution curves as given by Eq. (1), see Section 1.1. Obtained sequences in the Ki values for a studied series of complexes, e.g. for elements of one group, provide the information about relative stabilities of their complexes, i.e., of Pi. 

The following protocol is an adaptation of the Ciphergen Biosystems, Inc. user manual and of different published research papers. The Ciphergen Biosystems protocol for serum proteins profiling begins with an anion exchange fractionation step that uses step-wise pH elutions. A total of seven fractions (pH... 

As efforts expanded, the need for more practical and efficient systems took on a greater importance. This led to the emergence of elution of polymer supported on packed columns in preference to approaches involving step-wise precipitation. The advantages of the use of columns was noted in a review by Schneider [3]. These include ... 

Flow programming involves initially using a low flow rate to better resolve the early peaks and then increasing the flow rate to elute well-retained components. It can be carried out step-wise or continuously if desired. Basically, improvement of front-end resolution at the expense of back-end resolution is the result. Decreasing the flow rate by 75% will yield a resolution improvement of about a factor of 1.4. The primary advantages of flow programming are its ease of implementation and no need for column reequilibration between samples. 

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