Mobile phase peak broadening

Errors in the molecular weight data from HPSEC are usually due to improperly prepared samples, column dispersity, or flow rate variations. The sample to be analyzed should be completely dissolved in the mobile phase and filtered prior to injection onto the column. A plugged column inlet frit will invalidate results. In addition, do not load the column with excess sample. Column overloading affects the accuracy of data by broadening peaks, reducing resolution, and increasing elution volume. For best results, the concentration of the injected sample should be as low as possible while still providing adequate... 

The characteristics of the sorbent in the precolumn may lead to problems when coupling the two systems. Therefore, when the analytes are more retained in the precolumn than in the analytical column, peak broadening may appear, even when the analytes are eluted in the backflush mode (40). This has been solved with a special design in which the analytes retained in the precolumn are eluted with only the organic solvent of the mobile phase and the corresponding mobile phase is subsequently formed (40, 41). 

The matrix, which in most reported applications appears to be glycerol, may either be incorporated directly into the mobile phase pre-column or added postcolumn. If added to the mobile phase, its effect on the separation must be considered, while if added post-column, significant peak broadening may be observed. 

Also, peak broadening appeared to increase with increased concentration of n-heptane. It is quite possible that mobile phase composition and/or polymer type affects axial dispersion. 

M tris-HCl buffer solution (pH 7.94) was used as mobile phase. The peak separation obtained with a three column set (G 3000 PW + 2 G5000 PW) is comparable to the peak separation obtained in the present study for the molecular weight range, 120,000 to 3.6 X 10. Peak broadening appeared to be appreciable although no calculations of single-species variance were done. 

Mass transfer in either the stationary or mobile phase is not instantaneous and, consequently, complete equilibrltui is not established tinder normal separation conditions. The result is that the solute concentration profile in the stationary phase is always displaced slightly behind the equilibrluM position and the mobile I se profile is similarly slightly in advance of the equilibrium position. The combined peak observed at the column outlet is broadened about its band center, which is located where it would have been for instantaneous equilibrium, provided the degree of nonequllibrluM is small. The stationary phase contribution to Mass transfer is given by equation (1.25)... 

Based on the previous analysis of the different transport phenomena, which determine the overall mass transport rate, the structure of the solid phase matrix is of extreme importance. In the case of any chromatographic process, the different diffusion restrictions increase the time required for separation, since any increase of the flow rate of the mobile phase leads to an increase of the peak broadening [12]. Thus, the improvement of the existing chromatographic separation media (column packing of porous particles) and hence the speed of the separation should enable the following tasks ... 

Traditionally, most pharmaceutical assays are isocratic analysis employing the same mobile phase throughout the elution of the sample. Isocratic analyses are particnlarly common in quality control applications since they nse simpler HPLC eqnipment and premixed mobile phases. Notable disadvantages of isocratic analysis are limited peak capacity (the maximnm nnmber of peaks that can be accommodated in the chromatogram), and problems with samples containing analytes of diverse polarities. Also, late eluters (such as dimers) are particularly difficult to quantitate in isocratic analysis due to excessive band broadening with long retention times. 

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