Modern HPLC for Practicing Scientists

Second- and higher-order data generation and calibration: A tutorial

“…In chromatography, the retention factor (k), which is the degree of retention of the 621 sample constituent in the column, plays an important role in most analysis: in practice 622 analytes elute with retention factors between 1 and 20, with a peak with k equal to 0 623 indicating a constituent which does not interact with the stationary phase [67]. 624…”

Peptide fractionation in proteomics approaches

“…The separation is based on the analytes partition coefficient between the polar mobile phase and the hydrophobic (nonpolar) stationary phase. In the case of peptides, more polar peptides elute first while less polar peptides interact more strongly with the hydrophobic groups that form a 'liquid-like' layer around the solid silica support [12]. RPLC has been extensively applied in peptide separation for its ease of use with gradient elution, compatibility with aqueous samples and versatility of the retention mechanism, allowing changes in the separation brought by changes in the pH, organic modifier or additives [13].…”

“…Hydrophilic interaction LC (HILIC) is somewhat similar to normal-phase chromatography and it is most commonly used to separate polar analytes and hydrophilic peptides [12]. This separation is characterized by the use of a hydrophilic stationary phase, that is, cyano-, diol-, amino-and other bonded phases [18], and a hydrophobic organic mobile phase [19].…”

“…Polymer-based matrices were introduced in the late 1990s allowing a wider pH range (2-12) and introduced the ana lysis of basic peptides not properly resolved in acidic conditions [26]. The ana lysis of basic compounds using RP-HPLC has been extensively studied [4,[11][12][13] since the presence of isolated silanol groups in the stationary reversed phase leads to strong interactions with the basic compounds, resulting in a nefarious peak tailing [27]. The use of polymeric stationary phases in the first dimension was suggested as a suitable alternative, owing to their lack of silanol groups, making them more stable at a high pH, and using mobile phases without any salts [14].…”

Chemometric strategies for enhancing the chromatographic methodologies with second-order data analysis of compounds when peaks are overlapped

“…In most chromatographic analysis, analytes elute with retention factors between 1 and 20 allowing their complete separation. A peak with k equal to 0 is a component that does not interact with the stationary phase and elutes in the void volume [2]. Chromatographic separations can become a difficult task when complex samples have to be analyzed.…”