5


essentially amirror-image of the structure discussed above, providing compelling evidence that pseudoenantiomericmolecular recognition characteristics of Cinchona alkaloids is also fully preserved at the molecular level [23]


(Figure 2). 2.2. RetentionMechanisms


The chromatographic retention behavior of cinchona carbamate-type selectors for acidic analytes shows, independent fromthemobile phasemedia, classical ion-exchange characteristics. Retention can be conveniently modeled on the basis of the well-established stoichiometric displacementmodel [24]


,


predicting an inverse linear relationship between binding affinity and the concentration of a given counterion present as displacing species in themobile phase. Provided that constant pH ismaintained, the level of chiral discrimination of a given analyte generally remains uncompromised by changes of the counterion concentration in themobile phase, allowing a convenient manipulation of analysis time without sacrificing enantioselectivty.


The nature of the employed counterion, however, was found to affect both retention and enantioselectivity [2]


.Generally, the elution


strength of the counterions in the RPmode decreases roughly in the order citrate > phosphate > formate > acetate. The impact of the nature of the counterion on enantioselectivity is difficult to generalize and appears to involve specific competitive interactions at the binding site of the selectors.


The impact of themobile phase pH on the chromatographic performance characteristics of cinchona carbamate-type selectors can be readily rationalized considering that the quinuclidine unit serving as the active anion exchange functionality is a relatively weak base, rendering these selectors as weak anion exchanger systems [11]


. Consequently,


variations of themobile phase pH value will affect the degree of protonation for both the basic quinuclidine integrated within the selectors and the acidic function in the analyte. Highest levels of chiral recognition and affinity are generally obtained at pH values ensuring the co-existence ofmaximumconcentrations of protonated selector and deprotonated analyte species.With hydro-organicmobile phases and simple carboxylic acids this ideal pH value is in the range of pH 4 to 6. For acids with lower pKa values, such as phosphoric acids or sulfonic acids, the optimal pH values may be shifted to lower values.


An attractive feature of Cinchona carbamate anion exchange-type selectors is the fact that chiral recognition is not restricted to certain mobile phase environments [2]


; successful


enantiomer separation can be achieved in essentially allmajormobile phasemodes,


Figure 3. Separation of stereoisomers of an acidic drug intermediate on a CHIRALPAK QN-AX column under supercritical fluid conditions employing carbon dioxide methanol as mobile phase. Reproduced from [27] with permission.


including reversed phase (RP), polar organic (PO), nonpolar (NP) and even in supercritical fluidmobile phases.


In hydro-organicmobile phase environments cinchona alkaloid-based CSPs exhibitmixed- mode RP/weak anion exchange retention characteristics [11]


. The relatively hydrophobic


nature of cinchona carbamates along with the linker functionalities employed for immobilization renders the surface of these phases lipophilic, and expresses in water-rich mobile phase systems RP retention increments that contribute significantly to the global retention characteristics. At constant ionic strength and pH the global retention behavior follows trends consistent with the linear solvent strength theory, predicting an inverse relationship between log k and the volume fraction of organicmodifier in the mobile phase. This hydrophobic retention increment can be effectively attenuated via an increase of the content of the organic modifier in themobile phase. Formost applications strong hydrophobic interactions are unfavorable as they add retention and generally compromise enantioselectivity. Enforcing hydrophobic interactions by using water-rich hydro-organicmobile phase systems, however,might be favorable for applications requiring enhanced levels of chemoselectivity, e.g., for the separation of complex (diastereomeric)mixtures [25]


.


Formost applications, POmobile phases, consisting ofmethanol and/or acetonitrile and a low concentration of acetic acid and ammoniumacetate as displacing ionic species, are preferable over other RPmobile phasemodes [2]


. Specifically for highly


lipophilic acidic analytes, POmobile phases provide superior levels of enantioselectivity and fast elution through efficient suppression of non-specific interactions. Also, the lower viscosity of POsolvents as compared to water- rich RPmobile phases favorsmass transfer and reduces column pressure drop, resulting in higher efficiencies and improved longevity of the column packing. Polar organicmobile phases composed of acetonitrile and/or


methanol and volatile carboxylic acids, devoid of difficult to remove salt additives, are good options for preparative applications, facilitating product recovery.


Certain classes of cinchona-type selectors were found to operate successfully even with NPmobile phases systems. For example, a hybrid urea-linked epiquinine-calixarene-type CSP operated with amobile phase composed of chloroformand acidic acid as an acidic displacer resolved the enantiomers of N-tert-butoxycarbonylproline with high levels of selectivity [26]


. No elution was


observed in the absence of acetic acid. Upon increasing the acetic acid concentration, linear ln k vs. ln[CH3COOH] dependencies were observed with this specific separation system, suggesting that anion-exchange processesmay still operative even in relatively NPmobile phase environments.


Retentionmechanisms related to those seen in NPmobile phase systems alsomay underlie the successful chiral separation of acidic analytes with cinchona carbamate-type CSPs inmobile phases composed of supercritical carbon dioxide and polar alcoholicmodifiers. The separation of a mixture of the four diastereomers of a hydrophobic acidic drug intermediate could be achieved using a commercial quinine tert.- butylcarbamate CSPs in combination with methanolmodified carbon dioxide [26]


(Figure


3). It is interesting to note the elution of the acidic analyte could be effected without additives. Thismay be seen as evidence that carbon dioxide is sufficiently acidic to function as an efficient displacer for anion exchange applications.


2.3. Analytical Applications 2.3.1. HPLC Applications


A chiral stationary phase based quinine 09- tert-butylcarbamate has been successfully applied for the enantiomer separation of thyroid hormone (T4) thyroxin and itsmono- deiodine analog triiodothyronine (T3) [25] Sensitive and robust assays for the enantiomer


.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60