19. What are some do’s and don’ts when using Daicel chiral columns?

Part A – Things To Do when Using Daicel Chiral Columns

1. Carefully read the Instruction Manual. There are major differences in stability between the immobilised columns and the traditional coated Daicel columns, as well as between individual coated columns.
2. Flush the entire HPLC system with the appropriate solvent (including the sample loop, autosampler rinse solvent [if used], and detector), before attaching the column to the instrument.
3. Use only recommended solvents to ensure maximum column life. A list of alternative solvents is available from Chiral Technologies. Please note, however, that not all of the alternative solvents have been evaluated overall mixture ranges and have not been evaluated over extended periods of use. Consequently, prolonged use of these alternative solvents could shorten column life considerably.
4. Use simple mobile phases. Chromatographic separations on normal-phase columns are usually achieved with simple mobile phases such as heptane/isopropyl alcohol (IPA), 95/5 to 50/50 v/v, or heptane/ethanol (EtOH), 95/5 to 50/50 v/v. Note: several of the older polysaccharide columns are not stable to alcohol percentages over 15% (see individual column Instruction Manuals that are provided with each column for solvent limitations). HPLC-grade EtOH is used for methods developed at Chiral Technologies, but you should be aware that sometimes HPLC-grade EtOH is denatured with 5% IPA and 5% MeOH. This can obviously have an influence in the resolution. DO NOT USE EtOH DENATURED WITH BENZENE OR OTHER NON-ALCOHOL DENATURANTS.
5. Equilibrate the system to a stable baseline after attaching the column and starting the solvent flow. Equilibration usually requires a minimum of thirty minutes at a flow rate of 1 ml/min. At lower flow rates or lower detector sensitivity, longer equilibration times may be required.
6. Samples should be free of insoluble particulates. It is recommended that a guard column always be used to prevent contamination of the main column. Note: Guard columns are available for all the polysaccharide chiral phases. It has been our experience that a significant number of column problems arise due to the plugging of column frits. This problem can be completely eliminated by using a guard column or an inline filter (2 micron or less).
7. To distinguish enantiomers from achiral impurities, try running at multiple wavelengths (chiral peaks will have the same relative proportion to each other at all wavelengths), or use different types of detectors such as a chiral detector and/or a refractive index detector. For racemic compounds with multiple stereogenic centers (chiral centers), each pair of enantiomers will retain the same signal ratio at all wavelengths.
8. Dissolve the sample in mobile phase constituents only, to avoid possible on-column precipitation and/or injected solvent effects. If the mobile phase will not dissolve the sample, contact Chiral Technologies for assistance.
9. Flush the column with the appropriate storage solvent when the analysis is completed. Aqueous buffers are commonly used as a mobile phase component when using columns in the reversed-phase mode. When a buffer solution has been used, it is imperative that the column be flushed with the identical mobile phase, without the buffering salt present, before the column is converted to the recommended storage solvent. In addition, when mobile phase modifiers (i.e., acids or bases) are used, the columns should be flushed thoroughly with the same mobile phase, without the modifier present, before flushing the column with storage solvent. When acidic or basic modifiers, such as trifluoroacetic acid (TFA) or N,N-diethyamine (DEA), are used as mobile phase modifiers, it is satisfactory to leave this mobile phase in the column overnight. However, if the column will not be used for several days it is recommended that the system be flushed with a mobile phase that does not contain modifiers so that the column is not damaged. Note: When the column is no longer being used, it should be removed from the HPLC system and capped tightly at both ends to avoid evaporation of the solvent. When these polysaccharide columns are used in the normal-phase mode without the modifiers, the column can remain attached to the HPLC system for up to a week without being flushed. Polysaccharide columns last for years under proper care but can degrade quickly if the storage instructions are not followed.

Part B – Things Not To Do When Using Daicel Chiral Columns

1. Do not operate your Daicel column above the recommended maximum pressure limit.
2. Do not use dilution solvents and mobile phases that are not listed on the Instruction Sheet for your column. Not all columns are compatible with the same solvents; therefore, don’t assume that a special solvent that worked fine on a different column previously will be OK on your current column.
3. Do not leave buffers and additives in your column if you are planning to store it for a long time. Do follow the recommended storage instructions that are found on the Instruction Sheet that comes with each column.
4. Do not discard the test chromatogram that comes with each column.  The Instruction Manual for your column can be found under Quick Links on any page of the website. Or contact us: Chiral Technologies Europe (CTE), support@cte.daicel.com, Chiral Technologies, Inc. (CTI), questions@cti.daicel.com, or Daicel Chiral Technologies India (DCTI), chiral@chiral.daicel.com

If your column develops a performance problem, it may be necessary to test it to determine whether or not it has the same selectivity and efficiency as it had when new. Having the original test chromatogram is a good way to compare current performance to when it was new.