Reversal of elution order

Temperature can also be used to optimize enantioselectivity in SFC. The selectivity of most CSPs increases as temperature decreases. For this reason, most chiral separations in SFC are performed at ambient or subambient temperatures [50, 74]. Subambient temperatures are particularly useful for compounds having low conformational stability [75]. Stringham and Blackwell explored the concept of entropically driven separations [76]. As temperature increased, enantioselectivity decreased until the enantiomers co-eluted at the isoelution temperature. Further increases in temperature resulted in reversal of elution order of the enantiomers. The temperature limitations of the CSP should be considered before working at elevated temperatures. 

This reversal of elution order is due to the changed CIP priorities, but not a result of an altered binding and chiral recognition mechanism. Moreover, in yet another study, BOC and DNP-protected a-substituted proline derivatives have been resolved into enantiomers and elution orders were determined [48], The method allowed the sensitive and accurate analysis of samples with regards to their enantiomeric purities. 

Macaudiere, E., Lienne, M., Caude, M., Rosset, R., and Tambute, A. 1989. Resolution of Jt-acid racemates on Jt-acid chiral stationary phases in normal-phase liquid and subcriti-cal fluid chromatographic modes A unique reversal of elution order on changing the nature of the achiral modifier. Journal of Chromatography, 467 357-72. 

Conversion of summarized values (e.g. mean ER cr to the equivalent EF tr) can lead to substantial discrepancies, and should be avoided [109]. In addition, the conventions used in describing ERs and EFs may differ between studies and analytical methods. For example, enantioselective separations on different stationary phases may result in reversal of elution order and lead to different values if elution orders are not known. In this discussion, EFs are used to the extent possible, and both EF and ER are defined using using Equations (4.1) and (4.3), respectively, for those analytes for which the elution order is known unless otherwise indicated. Otherwise, these metrics are defined using Equations (4.2) and (4.4) on the specified column. 

R. W. Stringham and J. A. Blackwell, Entropically driven chiral separations in supercritical fluid chromatography. Confirmation of isoelution temperature and reversal of elution order, AnaZ. Chem. 68 (1996), 2179-2185. 

Drawbacks of the macrocyclic antibiotic type CSPs may be (i) the complexity of rationalizing and/or predicting enantiomer affinity, and accordingly the inability to predict the elution order so that chromatographic assignment of absolute configurations is not possible, and (ii) the total absence of the enantiomeric CSP which would facilitate the reversal of elution order of the SA enantiomers. 

Dipolar compounds such as those with chloro, nitro, and nitrile substituents are more strongly retained on cyano columns, compared to amino or diol [14]. Also, cyanopropyl silica can exhibit acidic or basic character, depending on the mobile phase used. It was shown that a complete reversal of elution order was obtained for phenol and anihne when MTBE and chloroform were used as the mobile phases, because phenol eluted first in the MTBE solvent and second in the chloroform solvent [9],... 

Okamoto, M., Nalcazawa, H., Reversal of elution order during direct enantiomeric separation of pyriproxyfen on a cellulose-based chiral stationary phase,/. Chromatogr., 1991, 588, 177-180. 

A reversal of elution order for the ions Pb2+, Co2+, Zn2+, and Ni2+ is obtained with the use of oxalic acid as the sole complexing agent. Such a separation is displayed in Fig. 3-155. Under these conditions, the metal separation is controlled by anion and cation exchange processes. The degree to which both mechanisms contribute to the separation process is different for each metal ion. The anion exchange mechanism predominates where stable anionic oxalate complexes are formed. In metal ions which do not form stable oxalate complexes, such as Cu2+, the cation exchange mechanism is... 

Gyllenhaal, O., Stefansson, M. Reversal of elution order for profen acid enantiomers in packed-column SFC on chiralpak AD, Chirality, 2005,17, 257-265. 

Stringham, R. W., Blackwell, J.A. Entropically Driven Chiral Separations in Supercritical Fluid Chromatography. Confirmation of Isoelution Temperature and Reversal of Elution Order, Anal. Chem., 1996, 68, 2179-2185. 

Figure 22.2. A plot of log V, for two dissimilar solutes A and B on the same stationary phase as a function of IjT, illustrating the possibility of resolution, no resolution, and reversal of elution order as the column temperature is varied.

For dissimilar species, enthalpies of vaporization are often quite different, so a can depend strongly on temperature. Figure 22.2 shows that at low temperatures, A will elute before B (log Vg < log Vf) but as the temperature is increased, the difference in retention time decreases until at temperature T, log Vg = log Kf. At temperatures higher than T, B elutes before A, since now log Vg > log Vg. Such a reversal of elution order is not uncommon for dissimilar molecular species. 

Okamoto M (2002) Reversal of elution order during the chiral separation in high-performance liquid chromatography Review. Journal of Pharmaceutical and Biomedical Analysis 27 401—407. 

In this example four protic solutes are considered benzoic acid (BA), phenyl-acetic acid (PAA), phenylsuccinic acid (PSA), and 4-l droxybenzoic acid (HBA). For these optimizations, knowing the pA, value for each solute is he fiil. Here the values are [68] 4.20 for BA, 4.58 for HBA, 4.31 for PAA, and 3.78/5.55 for PSA (diprotic). The pH of the mobile phase (at least the aqueous component) is made to cover the range of at least 1 pH unit of the pAj extremes (here 1.7 and 6.9). Table 2.2 lists the k values generated at each pH and then lists all the ratios calculated therefrom. Note that the ratio is always set up so that the first calculated value is > 1 (i.e., solute 1 is better retained than solute 2). That does not mean that the ratio will remain > 1 throughout the experimental pH range. In fact, looking at the k ratio values in Table 2.2 shows two instances where the ratio goes to < 1. Where this happens, a reversal of elution order has occurred. 

Smith and Cooper [601] studied the retention of three nonpolar solutes (phenan-threne, chrysene, perylene) and four polar solutes (nitrobenzene, 1,2-dinitrobenzene, phenol, aniline) in hexane and hexane/x mobile phases (where x = chloroform, methyl r-butyl ether [MtBE], and dichloromethane at the 5%, 10%, 15%, and 20% levels) on cyanopropyl, aminopropyl, and diol columns. From this work, the solvent strength of each mixture was determined for use in predicting chromatographic retention. More importantly, complex solvent/solute/adsorbed solvent/stationary phase interactions were described highlighting important and unique selectivities offered by these combinations. For example, altering the mobile phase composition from 3% MtBE in hexane to 12% MtBE in hexane (on a cyanopropyl support) leads to a decrease in the retoition of phenol and aniline. What is imexpected is the concomitant reversal of the elution order (phenol/aniline to aniline/phenol). This type of reversal of elution order is rare in leversed-phase separations (ion-pair systems notably excluded) but may be a considerable advantage in normal-phase separations. 

A further evaluation of the CB-DNP was reported by Gahm of Amgen using supercritical fluid chromatography (SFC) [27]. He concluded that inclusion com-plexation was the primary factor in retention and enantioselectivity. Better enantioseparation, as well as peak shape together with the reversal of elution order, makes CD-based CSPs unique and complimentary to the more commonly used jt-basic amylose and cellulose columns. 

Figure 7 shows the separation of terbutaline on the V and T CSPs with the same mobile phase. The retention, selectivity, and resolution factors are very different for the same solute eluted on the two CSPs. The key point of the example is the reversal of elution order that is observed on the polarimeter trace (Fig. 7). Such reversal signs a different selector-selectand separation mechanism with the same mobile phase at least with one of the interactions needed for enantiorecognition [29],...

Ma S, Shen S, Lee H, Eriksson M, Zeng X, Xu J, Eandrick K, Yee N, Senanayake C, Grinberg N. Mechanistic studies on the chiral recognition of polysaccharide-based chiral stationary phases using liquid chromatography and vibrational circular dichroism. Reversal of elution order of iV-substituted alpha-methyl phenylalanine esters. J. Chromatogr. A 2009 1216 3784-3793. 

Confirmation of isoelution temperature and reversal of elution order. A/m/. Chem. 1996 68 2179-2185. 

Roussel C, Vanthuyne N, Serradeil-Albalat M, Vallejos J-C. True or apparent reversal of elution order during chiral high-performance liquid chromatography monitored by a polari-metric detector under different mobile phase conditions. J. Chromatogr. A 2003 995 79-85. 

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