Gradient elution principles

Significant number of modern HPLC separation methods are based on gradient elution or continuous variation of the eluent composition during the analytical experiment. Theoretical description of the gradient separation is very complex and, to large extent, nonexistent. It is possible to use basic HPLC theory for phenomenological explanation of gradient retention dependencies. 

Linear velocity of chromatographic zone as a function of the eluent composition u(c) [as derived in equation (2-38)] in the most simple model of the retention process (partitioning) is 

For a linear gradient, eluent composition is a linear function of time  

Analyte retention time is the time that an analyte travels through the column, so the integral of the analyte linear velocity by time should be equal to the column length  

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Ceruloplasmin (from human blood plasma) [9031-37-2] Mr 134,000. This principle Cu transporter (90-90% of circulating Cu) is purified by precipitation with polyethylene glycol 4000, balchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin... 

The introduction of the commercially available mass detector meant an expansion of the possible conditions for TG separations, including that of gradient elution. The mass detector showed very narrow and symmetrical peaks, which indicated its usefulness for this application. The goal was to find a system that could be used to separate any natural TG mixture regardless of its carbon number distribution. The basic principle of this detector, i.e., the light scattering, originally was presented by Charlesworth (104). 

He et al. (1998) developed a gradient elution reversed-phase HPLC technique for separation of pungent principles. HPLC-UV-electrospray MS was used successfully to identify the individual pungent constituents in the chromatogram of ginger extract. Seven compounds were identified positively as the major pungent constituents of... 

Isocratic versus Gradient Elution. The principles of chromatography just described hold true for isocratic elution schemes, in which the nature of the mobile phase remains constant throughout the de-... 

Theoretical principles, approaches, applications, and advantages of gradient elution are described in detail in a large variety of books and review articles. We recommend reading some of those issued in the last 2 years and the papers referenced therein (see Ref. 2 for some examples). In this entry, we will briefly discuss various types of gradient elution and compare different ways of gradient generation. 

The other two principles that detect changes in the solvent properties are conductivity and density measurements. Conductivity as a detection principle can only be used for ionic substances. The detection range is quite high, but the detector is sensitive to changes in solvent composition and shows a baseline shift if gradient elution is applied. 

There are six modes of HPLC currently in use for secondary metabolite analysis, namely, HP-RPC, HP-NPC, HP-IEX, HP-HILIC, HP-SEC, and HP-AC. The principles of these different modes are explained below. All of these various chromatographic modes can be operated under isocratic (i.e., fixed eluent composition), step gradient, or gradient elution conditions (variable step or continuous changes in eluent composition), except for SEC, which is usually performed under isocratic conditions. All modes can be used in analytical, semipreparative,8 or preparative9-14 situations. 

A second method utilizes the Fresnel principle (Figure 19-44), which relates the transmittance of a dielectric interface to the refractive indices of the interface materials. Such an interface may be formed between a glass prism of selected optical properties and the liquid whose refractive index is to be measured. These detectors are difficult to use with gradient elution systems, and temperature control of the solvents is critical. 

A detailed discussion of the theoretical principles underlying gradient elution and the design of such separations is outwith the scope of this text. The interested reader is directed towards an excellent expose of the topic by Snyder et al. [95]. 

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