CHIRAL SEPARATIONS. CHIRAL DYNAMIC CHROMATOGRAPHY
6. ULTRA-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
chromatography (Wolf, 2005; Trapp and Schurig, 2000). It was therefore found out that the DC methods, in the whole of their different forms, can ensure the coverage of a very wide range of activation energies, from the very small value of about 15 kcal/mol (Gasparrini et al., 2000) to the much greater value of 38 kcal/mol (Trapp et al., 2002). A schematic representation of the optimal range of applicability of DC [including gas chromatography, high-performance liquid chromatography (HPLC), and UHPLC] and DNMR techniques in the determination of activation barriers for isom- erization processes is given inFig. 3.7.
InFig. 3.6some representative examples of isomerizations are collected whose activation barriers determined by DC methods range between the extreme values of 14.7 and 38.0 kcal/mol. Both temperature and residence time of the interconverting species inside the column are parameters that can be tuned to approach the upper or lower limits of the above range. The residence time may be effectively modulated by changing the flow rate, but also by drastically improving the chromato- graphic efficiency. Very high efficiencies, in fact, allow better resolutions in shorter times, and this strongly reduces the residence time of stereolabile species having extremely low activation barriers to interconversion. A second strategy is to reduce the column temperature, and several examples of cryo- chromatography on chiral stationary phases (CSPs) at temperatures ranging from 50C down to80C are listed inFig. 3.6. The chemical and stereochemical diversity of the solutes investigated, together with the values of energy barriers spanning from 14.8 to 24.8 kcal/mol, indicate that the DC approach is well suited to study a broad range of intriguing chiral molecules with labile stereogenic elements. The implementation of novel and more efficient chromatographic materials, columns, and improved hardware, such as in the UHPLC approach, may introduce additional resolving power and speed of analysis, thus extending the application range of the dynamic technique.
to the ever-increasing complexity of samples that must be analyzed. To perform fast separations while maintaining acceptable efficiency, resolution, and overall chromatographic performance, a compro- mise is necessary between eluent flow rate, column length, and back pressure.
According to theoretical treatments of the LC chromatographic process, one potential approach to increase column efficiency is to decrease the average size,dp, of the packing particles in the column (Giddings, 1965; Knox, 1977; Poppe, 1997). Thus, during the last three decades, HPLC has witnessed a smooth evolution of the packing material size from the original 10 to 5mm, and later to 3mm. A parallel shortening of the standard column length has been observed, moving from the classical 30 or 25 cm to the 10 or 5 cm format, or even to very short 2e3 cm column lengths for fast (but lower efficiency) separations in the subminute range. To retain high efficiency together with reduced analysis time, sub- 2mm spherical porous particles have been proposed as new packing materials in the last years (Wu et al., 2001; Jerkovich et al., 2003). Columns packed with sub-2mm particles have very low perme- abilities (permeability is proportional tod2p), whereas their optimum flow velocity for maximum effi- ciency is higher compared to columns packed with larger particles. The combination of reduced particle size with high eluent flow rates results in a drastic increase in the column inlet pressure, which is proportional to the inverse ofdp2(Desmet et al., 2006; Neue and Kele, 2007; Gritti and Guiochon, 2008).
Columns packed with sub-2mm particles very quickly move outside the pressure range of classical HPLC (roughly up to 40 MPa) when they are operated at their optimum flow rates or above, and this poses two problems in terms of mechanical stability of the SP particles and in terms of dedicated instruments, both of which are required to function under operating pressures reaching 100 MPa or higher.
Today, several manufacturers produce analytical LC instruments that are able to deal with pressures higher than 40 MPa, such as UPLC for ultra performance liquid chromatography or other variants of the technique (RRLC for rapid resolution liquid chromatography, UHPLC, VHPLC for very high-pressure liquid chromatography). Columns and SPs compatible with extreme pressures are available as well.
However, the large repertoire of SP chemistries presented by HPLC columns is not found for the UHPLC counterpart (Guillarme et al., 2007; Wales et al., 2008; Carr et al., 2009).
Enantioselective LC systems can, in principle, benefit from a substantial increase in sample throughput by employing smaller particles packed in short columns and using high linear velocities of the eluent. Unfortunately, if the number and types of achiral conventional SPs for UHPLC applications are scarce compared to HPLC, the situation is even worse in chiral LC, where no UHPLC-dedicated CSP is commercially available so far.
Recently, brush-type CSPs for UHPLC applications have been developed as the result of transition from the 5mm to the sub-2mm format of the underlying silica particles (Cancelliere et al., 2010; Kotoni et al., 2012b; D’Acquarica et al., 2014; Cavazzini et al., 2014). These UHPLC brush-type CSPs combine the use of reduced particle size with established chiral selectors for the generation of advanced materials with high throughput and/or high resolution capabilities. Two well-known selectors have been selected for this transition.
The first one is the DACH-DNB CSP (Cancelliere et al., 2006). It was prepared starting from 1.9mm spherical silica particles using a synthetic strategy that generates the intermediate DACH-CSP in a single step, starting from a slurry of bare silica, the chiral 1,2-diamine, and glycidoxypropyl- trimethoxysilane. Subsequent treatment of the intermediate silica with dinitrobenzoyl chloride gave the final CSP wherep-acidic aromatic fragments are fixed on the diamine framework through amide linkages (Fig. 3.8).
When the efficiency of a stainless-steel (1004.1 mm I.D.) column packed with 1.9mm DACH- DNB CSP was monitored as a function of the eluent flow rate (van Deemter analysis), a value of Hmin¼5.2mm was found at the optimum linear velocity,mopt¼4.00 mm/s, using methyl benzoate as a test solute and 10% chloroform inn-hexane as the eluent. The van Deemter plot of the column packed with 1.9mm DACH-DNB CSP showed a flat portion at high linear velocities of the eluent, suggesting a potential high efficiency use of the column in the subminute separation regime (Fig. 3.9).
Indeed, several very fast chiral separations on the 1.9mm DACH-DNB CSP have been reported for a range of compounds including alkylearyl sulfoxides, secondary phosphine oxides, and acylated amines. The combination of high flow rates, short column length (504.1 mm I.D.), and large enantioselectivity (a¼1.87) resulted in a complete separation of the enantiomers of a chiral 1-naphthamide in less than 15 s (Fig. 3.10).
The second selector used for the transition from the 5mm to a sub-2mm format is the Whelk-O1 (Kotoni et al., 2012a,b; Cavazzini et al., 2014). The Whelk-O1 selector was successfully covalently immobilized onto 1.7mm large surface area totally porous spherical silica particles. Columns packed with the 1.7mm Whelk-O1 CSP showed excellent kinetic performance that was then fully exploited to reduce analysis time to 10e45 s for a number of racemates and/or to enhance resolution of more difficult enantiomeric pairs. The resolution of a broad set of compounds, including alcohols, polar sulfoxides and phosphine oxides, and acidic drugs, was achieved on these columns. Comparison with commercial columns packed with 5mm particles clearly illustrated the advantages in terms of speed gain, peak shape, resolution, and solvent consumption (seeFig. 3.11).
The chromatographic separation of stereolabile chiral compounds is one particular case in which enantioselective UHPLC columns and hardware find immediate practical applications. Two extreme scenarios can be envisaged when stereolabile chiral compounds, featuring energy barriers to enan- tiomer interconversion lower than 18 kcal/mol, must be resolved by chromatography. In one situation, the column temperature can be lowered down to cryogenic temperatures to a point where the half-life times of the interconverting enantiomers are commensurate with the analysis time. With high-viscosity eluents, the column inlet pressure rises rapidly with decreasing temperature, and only UHPLC systems
silica
NHCO N
NO2
O2N O
XO
C
NO2 NO2 O
FIGURE 3.8
Chemical structure of the DACH-DNB CSP.
FIGURE 3.9
Representation of the van Deemter plot of the column packed with 1.9mm DACH-DNB CSP.
0 6 12 18
= 4.00 ml/min P = 256 bar
2 Rs = 3.7
seconds FIGURE 3.10
Ultra-fast enantioresolution ofN-(1-(naphthalen-5-yl)ethyl)-1-naphthamide on the 1.9 mm DACH-DNB CSP packed into a stainless-steel (504.1 mm I.D.) column.
can be used under these extreme experimental conditions. In the other situation, the chromatographic time scale is shifted into the seconds regime by the combined use of short columns packed with sub 2-mm particles and high eluent flow rates. Under these conditions, the overall analysis time can be cut by a factor of 10, compared to a conventional column, and the time scale of the separation can approach the time scale of the enantiomer interconversion at a given temperature.
With the aim of comparing the UHPLC technique advantages with the well-consolidated HPLC, atropoisomeric chiral species have been resolved on a chiral column based on the DACH-DNB selector, and the chromatographic time scale was shifted from the minutes into the seconds range. This is the case FIGURE 3.11
Van Deemter plot for chiral and achiral compounds on Whelk-O1 chiral stationary phases. (A) 2504.6 mm I.D.
column packed with fully-porous particles of 5-mm average size. (B) 1004.6 mm I.D. column packed with fully-porous particles of 1.7-mm average size. Eluent: hexane/dichloromethane 8:2 (v/v)þ3% methanol.
of a chiral bis-ketone with two stereogenic axes (Fig. 3.12), which was effectively resolved into its three stereoisomers (a couple of conformational enantiomers and an achiralmeso-form). In chromatographic runs performed at 10C by resorting to both HPLC (Gasparrini et al., 1995) and UHPLC methods (Cancelliere et al., 2010), the very different residence times that the stereoisomers spent inside the column appeared evident. Accordingly, a marked difference in the extent of isomerization is clearly visible in the resulting dynamic chromatograms, the plateau almost lacking in the case of UHPLC (Fig. 3.12). Schematically, if we state that the residence time decreases by a factor of 10 when changing from HPLC to UHPLC (a quite low value, relative to optimized conditions), it may be generalized that, to assure the same half-life time (t1/2) of the process, the operating temperature has to be increased by a DTamount predictable by the following linear equation:DT¼0.8611DGsþ1.0676.
This means that to measure an activation energy of 15 kcal/mol, an operating temperature of 49C will be required by UHPLC, for a run time of about 1 min, whereas HPLC will require63C for a run time of about 10 min. From a different point of view, the same transition of technique (i.e., from UHPLC to HPLC) would allow one to measureDGsvalues lower than about 1 kcal/mol at any established temperature in the range100 to þ30C, under the same conditions (Fig. 3.7). In general, a much more marked modulation of plateau zones may be obtained by suitable modest column temperature changes, compared to huge changes of the eluent flow rate (Cancelliere et al., 2010). Thus, very high activation barriers, not far from 40 kcal/mol, can be estimated by heating the column up to 200C, for at1/2 of about 30 min. Such temperature values are typical of HRGC, which is suitable only for thermally stable and volatile compounds. A quite great number of stereo- isomerizations (commonly enantiomerizations) have been studied by dynamic HRGC (DHRGC), featuringDGsvalues almost close to 30 kcal/mol. Selected examples from the literature are collected inFig. 3.6. In the case of thalidomide, an extreme enantiomerization barrier of 38.0 kcal/mol at 220C was reported (Trapp et al., 2002).
0 2 4 6
HPLC
8 10 12
min 0 20
UHPLC
40 60
sec O
O
FIGURE 3.12
Comparison of the ultra-high performance liquid chromatography (UHPLC) technique advantages with the well- consolidated high-performance liquid chromatography (HPLC).