In this work, we have investigated the predictive properties of mixed-mode retention model and oligomeric mixed-mode model, taking into account the contribution of monomeric units to the retention, in hydrophilic interaction liquid chromatography. The gradient retention times of native maltooligosaccharides and their fluorescent derivatives were predicted in the oligomeric series with number of monomeric glucose units in the range from two to seven. The maltooligosaccharides were separated on a packed column with carbamoyl-bonded silica stationary phase and 15 gradient profiles with different initial and final mobile phase composition were used with the gradient times 5; 7.5 and 10min. The predicted gradient retention times were compared for calculations based on isocratic retention data and gradient retention data, which provided better accuracy of the results. By comparing two different mobile phase additives, the more accurate retention times were predicted in mobile phases containing ammonium acetate. The acidic derivatives, prepared by reaction of an oligosaccharide with 2-aminobenzoic acid or 8-aminonaphthalene-1,3,6-trisulfonic acid, provided more accurate predictions of the retention data in comparison to native oligosaccharides or their neutral derivatives. The oligomeric mixed-mode model allowed prediction of gradient retention times using only one gradient profile, which significantly speeded-up the method development.
In this work, we have investigated retention of maltooligosaccharides and their fluorescent derivatives in hydrophilic interaction liquid chromatography using four different stationary phases. The non-derivatized maltooligosaccharides (maltose to maltoheptaose) and their derivatives with 2-aminobenzoic acid, 2-aminobenzamide, 2-aminopyridine and 8-aminonaphthalene-1,3,6-trisulfonic acid were analyzed on silica gel, aminopropyl silica, amide (carbamoyl-bonded silica) and ZIC-HILIC zwitterionic sulfobetain bonded phase. The partitioning of the analytes between the bulk mobile phase and adsorbed water-rich layer, polar and ionic interactions of analytes with stationary phase have been evaluated and compared. The effects of the mobile phase additives (0.1% (v/v) of acetic acid and ammonium acetate in concentration range 5-30 mmol L(-1)) on retention were described. The suitability of different models for prediction of retention was tested including linear solvent strength model, quadratic model, mixed-mode model, and empirical Neue-Kuss model. The mixed-mode model was extended to the parameter describing the contribution of monomeric glucose unit to the retention of non-derivatized and derivatized maltooligosaccharides, which was used for evaluation of contribution of both, oligosaccharide backbone and end-group to retention.
- MeSH
- 1-naftylamin chemie MeSH
- adsorpce MeSH
- aminopyridiny chemie MeSH
- chemické modely * MeSH
- chemické techniky analytické * MeSH
- chromatografie kapalinová * MeSH
- hydrofobní a hydrofilní interakce MeSH
- oligosacharidy chemie izolace a purifikace MeSH
- ortoaminobenzoáty chemie MeSH
- oxid křemičitý chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The applicability and predictive properties of the linear solvent strength model and two nonlinear retention-time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention-time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution-strength combinations. The quadratic model provided the most accurate retention-factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention-time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (<2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention-time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).
- MeSH
- časové faktory MeSH
- chromatografie s reverzní fází * MeSH
- fenoly chemie izolace a purifikace MeSH
- molekulární modely MeSH
- molekulová hmotnost MeSH
- peptidy chemie izolace a purifikace MeSH
- proteiny chemie izolace a purifikace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effect of gradient steepness on the kinetic performance limits and peak compression effects has been assessed in gradient mode for the separation of phenol derivatives using columns packed with 2.6μm core-shell particles. The effect of mobile-phase velocity on peak capacity was measured on a column with fixed length while maintaining the retention factor at the moment of elution and the peak-compression factor constant. Next, the performance limits were determined at the maximum system pressure of 100MPa while varying the gradient steepness. For the separation of small molecules applying a linear gradient with a broad span, the best performance limits in terms of peak capacity and analysis time were obtained applying a gradient-time-to-column-dead-time (tG/t0) ratio of 12. The magnitude of the peak-compression factor was assessed by comparing the isocratic performance with that in gradient mode applying different gradient times. Therefore, the retention factors for different analytes were determined in gradient mode and the mobile-phase composition in isocratic mode was tuned such that the difference in retention factor was smaller than 2%. Peak-compression factors were quantitatively determined between 0.95 and 0.65 depending on gradient steepness and the gradient retention factor.
