Eluent viscosity

Assuming an eluent viscosity of 1 cP, K can be read from Table 2.1 and the theoretical linear velocity of an eluent at any given pressure can be calculated. For the less rigid Sephadex G types, the maximum operating pressures at which the relation between superficial velocity and applied pressure is still linear are given in Table 2.1. Exceeding the pressures listed will result in bead compression, a reduction in pore volume, and a decreased flow rate. 

Using other methods for the calculation of plate count can result in different numbers, depending on peak shape. It should also be kept in mind that many other operational parameters, such as eluent viscosity, column temperature, flow rate, and injection volume, will influence the results of the plate count determination. 

The dominating working mode of polymer HPLC is the straight elution of small volume of sample solution along the column. There were attempts to introduce the differential and vacancy procedures in SEC. In both cases, the eluent was a diluted solution of the polymer while either a proper sample or a reference polymer [314] or even pure eluent [315] was injected. A broader application of these proposals was hindered by both the high eluent viscosity and the large sample consumption. Moreover, the dependences of log M vs. Vr (Equation 16.12) obtained by the conventional and the differential/vacancy methods are mutually shifted. The deviation between both dependences was attributed to the nonequilibrium situation in the vacancy polymer HPLC [316],... 

Frequently, IEC separations are carried out at elevated temperatures. This is because the kinetics of the ion-exchange process may be improved dramatically. The efficiency of the column may be increased by a factor of three if the temperature is increased from 30 to 70 °C [371], An additional advantage of an increased column temperature is a decrease in the eluent viscosity and hence a reduced pressure drop over the column. 

Results of efficiency enhancement studies have been controversial. Increasing the temperature lowers eluent viscosity and system back pressure, leading to the nse of (1) higher flow rates (shorter cycle times) [9], (2) longer columns, and (3) smaller particles that enhance efficiency in their own right. However, efficiency is also expected to increase because high column temperatures involve (1) faster adsorption-desorption kinetics, (2) enhanced diffusivity, (3) lower mass transfer resistance (C in the van Deemter Equation 6.4), and (4) flatter van Deemter curves. 

Another thing to consider with gradient elution is changes in the eluent viscosity. When gradient elution with a hydro-organic mobile phase is used (e.g., methanol-water), systematic variations in the flow rate are expected under conditions of constant-pressure operation, and systematic variations in the operating pressure will be found when a constant flow rate is used [1]. The compressibility of the solvent is species-specific. 

Fourth dimensionless parameter, dimensionless separation impedance, E (also known as efficiency ), which embraces retention time, pressure drop, plate number, eluent viscosity and retention factor, is taken as a measure of quality ... 

Higher temperatures are often used in ion-exchange chromatography (60-80 °C) in order to obtain a lower eluent viscosity, an increase in plate number and shorter retention times. 

Viscous Fingering effect This phenomenon is observed when the viscosity of the injected sample is significantly larger than the eluent viscosity. A hydro-dynamic instability appears within the column, leading to a fingering elution of the viscous solution in the bed. This disturbance of the flow will strongly impact the obtained efficiency. A reduction of the injected concentration is efficient to correct this effect, which can appear during a process scale-up. 

In order to design the appropriate liquid chromatography separation system, it is necessary to nnderstand on molecular level some basic principles and tendencies of the processes taking place in the chromatographic column. Above processes resnlt in differences in retention of sample constituents to allow their mutual separation. Extent of retention of macromolecules within colutim reflects the volume of mobile phase needed for their elution, their abovementioned retention volume, V. For the sake of simplicity, let us consider constant overall experimental conditions that is the elnent flow rate, temperature and pressure drop. The latter two parameters are dictated not only by the inherent hydrodynamic resistance of colunm that is inflnenced by the eluent viscosity, size and shape of packing particles but also by the sample viscosity, which may be rather high in polymer HPLC. Further, only one variable molecular characteristic of separated macromolecules will be... 

The plate number is affected by several variables, including the particle diameter, eluent viscosity and flow rate, the internal volume of the apparatus and the rate of equilibration. The internal volume of the equipment is obviously important and should be minimized by using small-bore tubing ( 0.01" i.d.), keeping the lengths as short as possible. 

Kinetic improvement is solely about the time needed to generate a certain number of theoretical plates. Plates per time depend on molecular diffusivity, which can directly be influenced by temperature, mainly through reduction of eluent viscosity. From this, we can conclude a maximum practical speedup potential by temperature by factor of 2, as this is the accessible range to influence the diffusion coefficient in practice. 

As noted before, the effect of temperature on the retention of peptides and proteins is more complex than found for small molecules in RPC and HIC because both the molecular diffusivities and the conformational status change with temperature. For example, for small molecules in RPC, column efficiencies typically increase while retention decreases as the temperature is increased [397], With peptides and proteins, on the other hand, retention often increases with temperature in the RPC or HIC modes under conditions that promote unfolding, although decreased retention times may arise at some specific temperature values for some proteins. Similarly, peak shape may also become impaired due to these conformational effects, despite the reduction in eluent viscosity and concomitant increase in the molecular diffusivity of the peptide or protein with temperature. 

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