Gradient separations mobile volume

Developing a gradient separation involves varying the percent change in mobile phase composition per unit volume of mobile phase delivered. This rate of change (ROC) in solvent composition per unit volume is graphically represented in Figure 7-7. As the slope of the line is decreased, the resolution of the separation will improve until the separation is limited by the efficiency of the column. The ROC value can be calculated by ... 

Fig. 2 Separation of hypothetical solutes A-K with a wide range of polarity (a) five-step simple gradient of mobile phase (concentration of modifier Cmod iu the range 0.05-0.7), volume of solvent expressed in void volume units (dimensionless) (b) two-step development (solid lines). In the first step, polar solutes E-H are separated the less polar solutes A-D, poorly separated during the first step, are separated during the second step.

Schramm et al. [43] used a simplified classic cell model to describe gradient elution chromatography. This model was used to optimize the duration of a linear gradient and the volume of the injected feed, using a simplex algorithm, in the case of the chromatographic separation of a ternary mixture, using two different objective fimctions (Pr x Y and the specific solvent consumption, CS,- = Pyr/vii, where F-p is the volumetric flow rate of the mobile phase, t is the period between two successive injections and nii is the mass flow of component i). 

Method transfer from one laboratory to another one (from development to routine, from manufacturer to customer and so on) can be difficult because HPLC separations are influenced by many parameters. At the new place the resolution of a critical peak parr can be worse than required or the whole chromatogram looks different. In order to prevent such surprises, whenever possible, it is necessary to describe every detail of the method column dimensions, stationary phase (maybe even the batch number), preparation of the mobile phase (the order the individual components are mixed can be critical), temperature, volume flow rate, extra-column volumes of the instrument, the dwell volume in the case of gradient separations (see Section 4.3) as well as detection and integration parameters. It can be useful to designate alternative stationary phases, i.e. materials which are located close to each other in representations such as Figure 10.9. The true temperature in a column oven must be verified because it can differ from the requested one Method transfer also includes the detailed description of sampling, storage and sample preparation. 

A clean-up process-scale RP-HPLC step has been introduced into production of human insulin prb. The C8 or C18 RP-HPLC column used displays an internal volume of 80 1 or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 11.4). Separation is achieved using an acidic (often acetic-acid-based) mobile phase (i.e. set at a pH value sufficiently below the insulin pi value of 5.3 in order to keep it fully in solution). The insulin is usually loaded in the water-rich acidic mobile phase, followed by gradient elution using acetonitrile (insulin typically elutes at 15-30 per cent acetonitrile). 

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