Anomalies in HPLC

Influence of a strong sample solvent on analyte dispersion in chromatographic columns

“…2) is observed. The concentration distribution c a,m is thus found to be bimodal, a property which is seen in practice in RPLC [1][2][3][5][6][7]9,48,49]. As time goes by, the analyte spreads to a larger region with peak tailing increasing towards the upstream direction.…”

“…This occurs in isocratic elution [1][2][3][4][5][6][7][8] as well as in gradient elution [9,10], and can also happen in hydrophilic interaction liquid chromatography [11]. This is detrimental to the separation performance.…”

Effects of the gradient profile, sample volume and solvent on the separation in very fast gradients, with special attention to the second-dimension gradient in comprehensive two-dimensional liquid chromatography

“…The sample solvent may very significantly affect the quality of separation [24][25][26][27][28][29][30][31][32][33][34][35][36][37]. It is well known that samples dissolved in a weaker solvent than the mobile phase (such as in water in reversedphase LC) are adsorbed in a narrow zone at the top of the column, so that the bandwidths at the time of elution are suppressed with respect to the injected sample volume.…”

“…This "on-column sample focusing" can be used to increase the detection selectivity by injecting relatively large volumes of diluted samples [35,36]. On the other hand, injection of a sample dissolved in a stronger eluent than the mobile phase may cause band broadening or even distortion or splitting [24][25][26][27][28][29][30], which was attributed to solubility effects [31], or to differences in the viscosity of the sample solvent and that of the mobile phase, giving rise to "viscous fingering", which can affect the dispersion of localized samples in porous media [25,[32][33][34]. Some experimental results suggest that the differences in the elution strength of the sample solvent and the mobile phase are the most important factor affecting this undesirable behavior [25].…”

Distorted Chromatographic Peaks

“…An example of column overload is given in (6), where nadolol in HPLC gives constant peak height above about 0.5 mg/mL while maintaining constant peak area.…”