Optimization peak resolution

Optimized HPLC separation allows most betaxanthins to be separated on a Cl8 reversed phase stationary phase according to their respective polarities. - Considerable progress was achieved by the introduction of a highly polar silica-based column, which allowed major improvement of peak resolution, especially at early... 

The three case studies outlined here give some indication of the worst difficulties that have been experienced with peak resolution, and have one factor in common the presence of an additional amorphous or paracrystalline peak was suspected in each case. All three optimization programs worked well when the input parameters were well chosen, but constraints were necessary in the more difficult cases and when broad peaks were present. Judicious choice of input parameters always speeds the ultimate solution and the use of BASELINE is most helpful. 

Peak resolution is usually easier if well chosen background parameters are input and if constrained optimization methods are utilised. Misleading results can be obtained if the constraints are too limited and tests with unconstrained optimization are desirable if at all possible. In particular, the possible presence of paracrystalline or intermediate phase peaks must be tested with extreme care in order to avoid ambiguity. It is not sufficient to have a good mathematical resolution alone, all peaks must be significant in crystallographic or structural terms. The incidental measurement of peak-area crystallinity is considered to be of secondary importance to the resolution of overlapping peaks. 

The principal methods of interfacing SFC with ICP-MS have been discussed by Carey and Caruso [94]. Where packed SFC columns are used, the SFC restrictor is connected to a heated cross flow nebulizer and the nebulizer gas flow carries the sample to the plasma. For the more commonly used capillary columns, the SFC restrictor is passed through a heated transfer line that is connected directly to the torch of the ICP-MS. For optimal resolution of peaks, the restrictor should be positioned so that it is level with the injector of the ICP torch. This position may be varied slightly (Fig. 10.15). Heat is applied where the transfer line and torch connect to prevent freezing of the mobile phase when it decompresses after exiting the restrictor. To transport the analyte to the plasma efficiently, a gas flow of approximately 0.8-1.0 mL/min is used. This gas flow may also be heated to improve peak resolution. 

The main parameters to optimize the separation factor and peak resolution, respectively, are as follows ... 

These effects stem from thermodynamic principles. If in a separation with a given purity requirement the peak resolution is not high enough due to these effects there is no way to overcome this problem by increasing column efficiency, which means optimizing the fluid-dynamic term of the resolution equation (Eq. 2.33). If a tag-along effect occurs the use of a smaller particle diameter has no effect, as long as the... 

Table II contains the optimal empirical resolutions calculated from the weighted least squares analysis of counted peak maxima. The empirical r from each set was used to calculate, from an unweighted east squares fit, a component number from each series in that set. The results for each set were averaged, and the mean and standard deviation for each set are reported In Table II.

Several studies have employed chemometric designs in CZE method development. In most cases, central composite designs were selected with background electrolyte pH and concentration as well as buffer additives such as methanol as experimental factors and separation selectivity or peak resolution of one or more critical analyte pairs as responses. For example, method development and optimization employing a three-factor central composite design was performed for the analysis of related compounds of the tetracychne antibiotics doxycycline (17) and metacychne (18). The separation selectivity between three critical pairs of analytes were selected as responses in the case of doxycycline while four critical pairs served as responses in the case of metacychne. In both studies, the data were htted to a partial least square (PLS) model. The factors buffer pH and methanol concentration proved to affect the separation selectivity of the respective critical pairs differently so that the overall optimized methods represented a compromise for each individual response. Both methods were subsequently validated and applied to commercial samples. 

Tyr-D-Arg-Phe- PheNH2 EKC Plackett-Burman (screening) Factorial (optimization) Central composite (optimization) CD concentration, TEA concentration, BGE concentration, % MeOH, % ACN, ionic strength, temperature, voltage CD concentration, TEA concentration, % MeOH, % ACN CD concentration, % ACN Peak resolution, analysis time Peak resolution, analysis time Peak resolution, analysis time (25)... 

Once a reasonably suitable column phase has been identified, the dimensions of the column employing it should be selected, and then the separation is optimized by varying the flow and temperature parameters. The column dimension decisions will depend on the difficulty of achieving the necessary peak resolutions, which is likely to increase with the number of analytes to be measured. The column s analyte capacity depends on these dimensions as well. The column dimensions, carrier gas flow, and column oven temperature parameters thus chosen will determine the time required to perform each GC run. 

All mass spectrometers require some degree of day-to-day tuning of operational parameters to provide optimal peak shapes, resolution, and sensitivity, as well as calibration of the mass scale. These aspects are important for the characterization of molecules and their fragments, the resolution of one compound from another, and... 

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