Achiral columns

Chromatographic Method. Progress in the development of chromatographic techniques (55), especially, in high performance Hquid chromatography, or hplc, is remarkable (56). Today, chiral separations are mainly carried out by three hplc methods chiral hplc columns, achiral hplc columns together with chiral mobile phases, and derivatization with optical reagents and separation on achiral columns. All three methods are usehil but none provides universal appHcation. 

Achiral Columns Together with Chiral Mobile Phases. Ligand-exchange chromatography for chiral separation has been introduced (59), and has been appHed to the resolution of several a-amino acids. Prior derivatization is sometimes necessary. Preparative resolutions are possible, but the method is sensitive to small variations in the mobile phase and sometimes gives poor reproducibiUty. 

Derivatization with Optically Active Reagents and Separation on Achiral Columns. This method has been reviewed (65) a great number of homochiral derivatizing agents (HD A) are described together with many appHcations. An important group is the chloroformate HD As. The reaction of chloroformate HD As with racemic, amino-containing compounds yields carbamates, which are easily separated on conventional hplc columns, eg (66),... 

The enantioselective determination of 2,2, 3,3, 4,6 -hexachlorobiphenyl in milk was performed by Glausch et al. (21). These authors used an achiral column for an initial separation, followed by separation of the eluent fraction on a chiral column. Fat was separated from the milk by centrifugation, mixed with sodium sulfate, washed with petroleum ether and filtered. The solvent was evaporated and the sample was purified by gel permeation chromatography (GPC) and silica gel adsorption chromatography. Achiral GC was performed on DB-5 and OV-1701 columns, while the chiral GC was performed on immobilized Chirasil-Dex. 

Let us assume that a given compound has a purity of 98 % ee, and that this compound is reacted with a derivatizing agent which has also a purity of 98 % ee. The two major compounds plus the minor impurities in the compound to be analyzed and the derivatizing agent will create a set of four diastereomers. Two pairs of diastereomers (-i-)-A(-i-)B and (-)-A(-)-B as well as (- )-A(-i-)-B and (-i-)-A(-)-B are enantiomeric pairs, and thus elute together on an achiral column. Therefore, a peak area of 98.011 % will be detected for (-i-)-A(-i-)-B, which leads to a purity of 96.03 % ee for (-i-)-A. This is a quite significant deviation from the true value for (-i-)-A. 

Achiral-chiral LC-LC is most often used to separate the desired analyte from interfering components, such as matrix components, metabolites, excess derivatiza-tion reagent, or other impurities. Separating such interferents from the analyte allows for better analyte quantification or enantiomeric ratio determination. Also, achiral columns are seen as a way to protect the more expensive chiral columns from matrix components that might become irreversibly retained and deteriorate column performance. Short achiral columns (trap columns) are sometimes used to reconcentrate the chiral analyte after a previous separation (either chiral or achiral) as a type of online enrichment. Configurations that combine an achiral column for increased selectivity and trap column(s) for online enrichment are relatively common, though this type of configuration requires more columns and increases complexity. 

In achiral-chiral LC-LC, the mobile phases used with the achiral and chiral columns must be miscible with one another. Since the enantiomeric separation is usually the most difficult to optimize, it is usually the separation that dictates the mode of operation of the total analysis. Thus, it makes sense that a chiral column that operates in the normal phase mode would require an achiral column that also works in the normal phase mode. Polar organic mode chiral separations are universal in that they can be paired with an achiral column that operates in either the reverse phase or normal phase mode. The choice of the achiral column is always determined after selecting the chiral column and the mode of operation. As with traditional liquid chromatography, different achiral columns will give different selectivity. 

Additionally, the inj ected matrix must also be miscible with the solvents used in the separations. For normal phase mode separations, all water must be removed from the injected matrix. Since many of the complex matrixes, such as plasma, urine, and other biological fluids contain a large amount of water, this requires more time consuming sample preparation. However, water can be injected into a polar organic or reverse phase mode separation. Even within the same mode, mobile phases that are very different can cause large disturbances in the baseline. Oda et al., (1991) solved this problem by inserting a dilution tube followed by a trap column in order to dilute the mobile phase used on the achiral column. Following the dilution tube, a trap column was used to reconcentrate the analyte of interest before the enantiomeric separation. 

Compound Mode0 Achiral column(s) Chiral column References... 

If it suspected that the enantiomers of interest are coeluting, a correction to the areas of the affected peaks may be applied. The correction can be determined from a knowledge of the peak areas on a nonpolar and polar achiral column. If the shape of the peak and the areas are the same on both columns, then no coelution is occurring. If the areas are different, then a correction factor can be applied by comparing retention times of the separated enantiomers, and subtracting the areas of those peaks that are coeluting, which were determined from the chromatograms obtained from the achiral columns. It should be emphasized that this is not an appropriate correction to make if accurate quantitative information is required. 

New columns should be conditioned following the manufacturer s instructions. Most are conditioned at lower temperatures than for achiral columns. The temperatures used for separation should be as low as possible, which leads to better separation and less deterioration of the sample. 

Etherfication in DMF using Ba(OH)2 as base usually gave syn-syn-triethers.122 The racemic 61a thus obtained was converted in a pair of diastereomers by introduction of the chiral (-)-menthoxyacetyl group. These diastereomers 61b could be separated by HPLC using an achiral column (Zorbax ODS). Finally, the... 

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