Overview of Conventional Chromatography

How much protein must be purified How pure must the protein be These two questions establish the criteria for selecting specific chromatographic techniques and for judging the efficacy of any individual purification step or overall purification procedure. The answers to these simple questions depend upon the nature of the source material from which the target protein will be purified, the intended use for the purified protein, and the methods available to assess the purity of the target protein. 

The amount of protein required from a purification process depends primarily on the intended use for the target protein. However, the amount of protein that will be used during the purification process to develop methods and assess purity must also be considered. It is necessary only to estimate whether analytical ( 1 mg), preparative ( 1 g), pilot ( 1 kg), or process ( 1 kg) amounts of target protein will be needed. This is often referred to as the scale of operation. When the source material is very 

The yield from any purification step may be reported as total protein, percentage of total activity, or change in specific activity. The yield information from progressive purification steps is used to construct a purification table (e.g., see Table B4.2.1). 

In Table B4.2.1, the yield is reported as percent of total activity remaining from the 

Methods for assessing protein purity must quantitate the amount of target protein relative to the amount of contaminant(s) in the sample. This requires two separate analytical methods one for the taiget protein and one for the total protein including contaminants. For example, with enzymatic proteins, assessment of functional purity typically relies on kinetic assays in which the amount of substrate consumed or product produced per unit time is proportional to the amount of enzyme present. The total activity of the sample is calculated and compared to the total amount of protein to give the specific activity of the sample. Methods for determining total protein are discussed in Chapter Bl. An increase in specific activity at a purification step reflects a loss of contaminant proteins. 

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B4.1 Overview of Protein Purification and Characterization B4.2 Overview of Conventional Chromatography... 

The use of essential oils is increasing because of the increase in the number of their apphcations and in the framework of natural and environmentally friendly materials. Many times the analysis of their components is quite complex due to the high number and the diversity of compounds in their composition. In this entry a general overview of the extraction methods is given by comparing conventional hquid-liquid and sohd-hquid methods with new alternative ones, such as supercritical fluid extraction and microwave-assisted extraction. Gas chromatography methods and examples are treated and important issues such as detection systems, modem hbraries for compounds identification, as well as multidimensional or hyphenated techniques are discussed. The use of these modem techniques and methods has improved resolution and sensitivity in essential oils determination and could open the possibihty of future work in this area of chromatography. 

An overview and discussion is given of literature methods published after 1989 devoted to the ion-interaction chromatographic determination of inorganic anions. Seventy references are quoted. Ion-interaction chromatography makes use of commercial reversed-phase stationary phase and conventional high-performance liquid chromatography instrumentation. The basis of the technique, the modification of the stationary phase surface, the choice of the ion-interaction reagent as well as the dependence of retention on the different variables involved are discussed. Examples of application in the fields of environmental, clinical and food chemistry are presented. The experimental conditions of stationary phase, of mobile phase composition as well as detection mode, detection limit and application are also summarized in tables. 1997 Elsevier Science B.V. 

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