Peaks, shape peak tailing, measurement

The methods of quantitative analysis are essentially tiiose inherited from gas chromatography. Peak hei t or pe area can be measured, either manually or with electronic devices. Peak height measiu-ements have the advantage of simplicity but are sensitive to changes in peak shape peak area measurements should always be used where peaks are broad and tailing. 

When the column is ready to be used, the chromatogram of a suitable test mixture should be obtained. The plate number and retention times of the test solutes should be noted, and the peaks should have a satisfactory shape (minimal tailing). For measurement of the plate number, the recorder should be used at a high chart speed. Fig. 5.1b(i) and (ii) show test chromatograms for a C-18 column prepared by the above method, and Fig. 5.1c and 5.Id show the data that you should report with the chromatogram. The retention for an unretained peak is taken as the small baseline disturbance just before the first peak. 

Spectroscopic measurements, such as electron energy loss spectroscopy (EELS), infrared (IR) and Raman spectroscopy, can probe the very small energy excitations across the Fermi level (Ef) or band gap by changes in the peak shape such as via the Drude tail in photoemission. However, such lines-shape changes can also be caused by a number of experimental effects making these measurements difficult. 

The simplest form of an HPLC SST involves comparison of the chromatogram with a standard one, allowing comparison of the peak shape and the peak width baseline resolution. Additional parameters that can be experimentally calculated to provide quantitative SST report include the number of theoretical plates, separation factor, resolution, tailing or peak asymmetry factor, accuracy, and precision (RSD of six measurements). Resolution may also be combined with a selectivity test to check the resolution of the analytes from components present in the sample matrix. If matrix components interfere with a method, a matrix blank may be included in the SST. Peak shape and asymmetry, or tailing factor, can... 

In the analytical procedure, an accurately measured aliquot of the product is diluted Avith a diluent (normally the mobile phase) and the resulting sample solution is injected into the HPLC. Because the majority of injectable pharmaceuticals are clear solutions, typically a simple dilution step is all that is needed for sample preparation. However, if the parenteral product is an emulsion or a suspension, appropriate steps must be taken to dissolve the product to achieve a clear solution (ultrasonication, filtration, etc.). For the assay procedure, the sample concentration chosen should be such that the peak areas obtained from multiple injections from the same sample are reproducible with minimum variance (<2% relative standard deviation). Peak shape and retention time also play important roles in the precision of the assay. A tailing factor less than 1.5 and a capacity factor less than 10 for the active peak are generally required for a good analytical method. A reference standard solution having the same concentration and using the same diluent as the sample solution is prepared. 

Under ideal conditions, chromatographic peaks should have Gaussian peak shapes with perfect symmetry. In reality, most peaks are not perfectly symmetrical and can be either fronting or tailing (Figure 2.8). The asymmetry factor (As) is used to measure the degree of peak symmetry and is defined at peak width of 10% of peak height (W0.i). Note that Tf is used here instead of T, as in the USP, because T often stands for temperature. 

At lesser sensitivity requirements, or if a measurement of the peak shape is desired, a continuous scan of mass to record full peaks may be preferred. In modern instruments, control is really a digital process, so that the continuous scan mode is essentially peak hopping with a small mass interval—0.1 amu or less per step. Very high dynamic range instruments, in which the peak tails are to be measured, might use 50 or more steps per amu. 

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