The most important difference between the CHIRALPAK IA, IB, and IC columns and the traditional polysaccharide columns is their robustness and stability to mobile phase composition. In the CHIRALPAK IA, IB, and IC columns, the stationary phase is immobilized on the packing material instead of the coating process used in Daicel's other chiral columns.

This immobilization confers two major advantages. One is that the CSP can no longer be changed or destroyed by the use of a “forbidden” solvent — there are no forbidden organic solvents with the new columns. The other advantage is that with this total freedom of choice of solvent it is possible to develop new separations not previously possible, thus further extending the range of polysaccharide-based columns to chiral separations. See Question 7 for information on how the extended range of solvents can be used to attain high levels of success for method development with these new columns.

The similarities between CHIRALPAK IA and IB and the other columns lie in the chiral selector. Both columns are based on the same chiral selectors as CHIRALPAK AD-H and CHIRALCEL OD-H®: the 3,5-dimethylphenylcarbamate derivative of amylose and cellulose, respectively. CHIRALPAK IC, on the other hand, has a totally new chiral selector — the 3,5-dichlorophenylcarbamate derivative of cellulose — which is too soluble in common organic solvents to be available as a coated phase.

The difference between CHIRALPAK IA / CHIRALPAK AD-H and between CHIRALPAK IB / CHIRALCEL OD-H lies in the immobilization of the stationary phase. Because the conformation of the polymer may be slightly influenced by the fact of the immobilization, there may be some small selectivity differences between the immobilized and coated support. These effects may be positive or negative, but are unimportant when compared to the far greater stability of the CSP to solvents, and to the possible improvement of separations through the exploitation of the wide range of solvents that may be used with these columns. As noted, CHIRALPAK IC has a unique chiral selector which is not available as a coated phase and therefore has the potential to develop unique separations not available with other Daicel CSPs.

There are currently no known organic solvents which will damage the CHIRALPAK IA, IB, or IC columns in any way.

Extensive testing has been carried out with many common organic solvents, including hexane or heptane/alcohols, methanol, isopropanol, ethanol, acetonitrile, dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, acetone, methyl acetate, MTBE, dimethylformamide, dimethylacetamide, etc.

Where the columns have been extensively used and have perhaps become fouled with impurities or non-eluted compounds they can readily be cleaned by flushing with dimethylformamide, THF, or ethyl acetate.

When used in reversed-phase chromatography, the columns should not be operated below pH 2 or above pH 7. The upper range of the CHIRALPAK IA column only can be extended to pH 9, provided that borate buffer is employed, and that a guard cartridge is used and is changed at least once every 200 injections at this pH.

Generally, the best procedure is to dissolve a sample in mobile phase, whenever possible. Care must still be taken when injecting sample dissolved in a solvent that has greater solvating power than mobile phase. This is a general chromatographic problem in that this may cause distortion of the chromatographic peaks, thus affecting the analytical results. In preparative chromatography there is a risk that sample from a concentrated injection in a good solvent will precipitate on the column, once the sample comes in contact with a mobile phase in which it has poorer solubility. An additional problem could occur if the column has greater attraction for sample diluent than for mobile phase. In such cases, sample diluent may stick to the column and affect the selectivity or efficiency of future injections.

For some applications, samples are presented in dimethylsulfoxide (DMSO). This solvent will not permanently harm these chiral stationary phases but is retained on the column under certain mobile phase conditions. Repeated injections of samples diluted in DMSO may produce a gradual decrease in column efficiency on a CHIRALPAK IA column which can be corrected by occasional flushing with dimethylformamide. Injections of samples diluted in DMSO are not recommended on CHIRALPAK IB columns, as such injections cause a more severe and immediate decline in column efficiency due to retention of this solvent on the column. This too is reversible following flushing of the column to remove the DMSO.

Of course, since the stationary phase is immobilized, there are no issues with stability of the column under any of the above circumstances.

Unless there are special circumstances, it is strongly recommended that immobilized columns be used for any application. This is because of their far greater stability in operation than other Daicel chiral columns. Another advantage of immobilized columns is their stability to strong solvents like THF, ethyl acetate, and the chlorinated solvents. This allows development of separations in these solvents which often give different selectivity in comparison with the usual solvent set used for chiral chromatography. Further, in preparative chromatographic applications, the use of such solvents can greatly enhance the sample solubility and thus the potential production rate for the separation.

In circumstances where a conventional, coated column is specified (in a validated procedure where an immobilized column is not yet approved, or when an existing method meets the specs of the separation) it may be appropriate to use coated columns rather than immobilized columns.

During the introduction of the full range of immobilized columns, there may be separations requiring a selectivity for which there is not yet an immobilized column. In such cases, other Daicel columns remain available for use.

The same additives — diethylamine for basic compounds and trifluoroacetic acid for acidic compounds — used for other chiral columns may be used with CHIRALPAK IA, IB, and IC. Studies conducted on the CHIRALPAK IB column have shown that ethylenediamine (EDA), ethanolamine (EtNA), and butylamine (BuA) may enhance the resolution and peak shape of basic compounds separated on that column compared to the resolution obtained with DEA additive.

A good rule of thumb is to use a basic additive with an amine functionality similar to that of the compound being resolved

CHIRAL TECHNOLOGIES has introduced the Analytical Method Development service. For primary screening, our experienced technical team will test your material on four Chiral Stationary Phases, including both immobilized and coated phases that are deemed most suited to your compound. Up to seven mobile phases will be used in this screening process.

Compounds with more than one chiral center are expected to require significantly more effort, and therefore a more extensive screening and optimization regimen will be employed. Please call our technical services group at 1-800-6CHIRAL to discuss these Advanced Method Development procedures and to obtain a quote prior to sending your sample.

Where no information is available, the compound must be screened against a variety of columns in order to find one with the appropriate selectivity. Because of their robustness and versatility, we strongly recommend that you first try the separation on CHIRALPAK IA or CHIRALPAK IB. Experience in our laboratories shows that most separations can be achieved using these columns in combination with the wide range of possible mobile phases. If the separation cannot be achieved using these columns, other Daicel polysaccharide columns should be tested, starting with CHIRALPAK AS-H and CHIRALCEL OJ-H.

If you prefer, you can ask CHIRAL TECHNOLOGIES to develop a separation, using our analytical method development service. Please contact your local account manager for more details.

If you would like technical assistance in performing a literature search for your application, send an e-mail to questions@chiraltech.com. Please provide structures of the compounds you wish to separate. You can also send names of well known compounds with similar structural features or specific derivatives of these compounds, so that a structure-based search can be performed. In many cases, a separation of your compound using one or more Daicel chiral columns, or one closely related to it, will already have been reported in the literature. Based upon this search, we can usually recommend the column most likely to separate your compound.

In addition, a general column selection guideline is available in our brochure, Technical Support, Products and Services for Chiral Analysis and Separation. Click here to view our brochure.

More specific guidelines can be found in the DAICEL Application Guide for Chiral Column Selection, at http://www.daicelchiral.com/appguide. It lists over 350 non-proprietary racemic compounds that have been successfully separated. These guidelines are based on empirical observations and may not correctly predict the separation of your compound.

Always consult the Instruction Sheet shipped with your column, before exposing your column to any mobile phase. The immobilized columns, CHIRALPAK IA and CHIRALPAK IB may be used with any solvent. This is not true for traditional Daicel columns, but by taking a few simple precautions, you can greatly enhance their lifetimes. The Instruction Sheets refers to solvents that can be used with that specific column. You should carefully heed the caution statements found at the top of each Instruction Sheet for solvents to avoid. Some solvents listed may be acceptable as mixtures. Mixtures of three solvents should be avoided for all but immobilized columns as the solubility of the polysaccharide polymer is unknown and may be increased.

In contrast to immobilized columns, traditional Daicel coated columns should never be used with solvents such as methylene chloride, chloroform, THF and DMSO. Such solvents solubilize the polysaccharide polymer at the head of the column which then reprecipitates as the solvent is diluted, resulting in a plugged column. Please be aware that even small quantities of incompatible solvents introduced in sample dilutions, or left in transfer lines (including autosampler lines) can rapidly degrade or destroy a column. Even residual amounts of forbidden solvents in samples may shorten the life of the column. For these reasons we recommend using the immobilized columns, CHIRALPAK IA and CHIRALPAK IB.

If you are unsure whether or not a particular solvent can be used with your column, assume that it is incompatible and avoid using it until you have contacted us at questions@chiraltech.com. Replacement Instruction Sheets can be rapidly sent to you by FAX or e-mail; contact CHIRAL TECHNOLOGIES at 610-594-2100 or e-mail us at questions@chiraltech.com if you need a replacement.

Click here to view a table of compatible solvents for our most popular columns.

There are six columns available for reversed phase applications: two immobilized columns, CHIRALPAK IA and CHIRALPAK IB, and four coated columns CHIRALPAK AD-RH, CHIRALCEL OD-RH, CHIRALCEL OJ-RH and CHIRALPAK AS-RH. All columns are compatible with mobile phases conventionally used in reversed phase chromatography, although only immobilized columns may be used with THF as the organic mobile phase component. The columns are stable to much the same pH and ionic strengths as are conventional reversed phase columns. It is the silica support, rather than the chiral phase, which limits stability. The amylose-based columns can be used to pH 9 if borate buffers are used, although this approach does not seem to be useful for the cellulose-based media.

A detailed discussion of reversed phase chromatography on Daicel phases can be found in the paper by Tachibana et al on our Technical Papers page.

R-H series columns are stable in the pH range from 2 to 7, and can be used in the extended pH range of 7 to 9 with borate buffer. Aqueous 0.2 M phosphate buffer at pH 2, and aqueous 0.2M borate buffer at pH 9 are the recommended starting points for acidic and basic solutes respectively, that are likely to require additives. Note that these are recommended starting conditions for developing a method. In actual practice a lower concentration of buffer or a pH between 2 and 9 may yield the best separation. Mobile phases made from pH 1 perchloric acid, are the recommended starting point for use with the CROWNPAK CR(+) and CROWNPAK CR(-) columns only.

Column performance can be measured by many parameters. These include column efficiency, selectivity and resolution, peak symmetry and column pressure drop. There are many possible reasons for change of column performance while in operation. Some are the normal chromatographic problems which can occur with any column. Some, especially for coated Daicel columns, relate to specific properties of the columns and of the stationary phases.

In general, if a column problem is suspected, it should first be thoroughly flushed (see its operating instructions) and then tested under the QC conditions used when it was originally packed. The results of this test can usually help diagnose the problem.

Since the solution of such problems is simpler when immobilized columns—CHIRALPAK IA and CHIRALPAK IB—are used, the resolution of problems with these columns will be addressed first. Later we will note differences between the traditional coated columns and the immobilized columns.

Operating Pressure: With immobilized columns, the source of an increase in operating pressure is usually the inlet frit. This can be blocked either by solids in the sample or entrained in the mobile phase. They can also be blocked by the introduction of a sample which was dissolved in a solvent stronger than the mobile phase; as it mixes with the mobile phase, material can be precipitated from solution and is filtered out by the frit. This can be corrected by changing or cleaning the inlet frit. It is sometimes difficult to remove the inlet frit and such removal always comes with the danger of disturbing the packed bed of the column. One easy experiment is to reverse the flow direction through the column in the hope that the foreign matter will be washed from the frit. It is, of course, always better to prevent such problems by the use of (and regular replacement of) a guard cartridge.

Sudden increases in operating pressure with traditional coated columns can be due to the effects of solvents on the chiral stationary phase. If the pressure increase is due to the introduction of a solvent which can damage the stationary phase, it is usually too late. To prevent such an occurrence, it is vital to ensure that the entire HPLC system is flushed of potentially harmful solvents before the column is connected to the system. Proper sample clean-up and preparation are also vital. Small amounts of non-allowed solvent in a sample preparation may seem insignificant, but these low level residues often dissolve the chiral polymer, which leads to a rapid decay in column performance. In these cases, the test chromatogram will almost certainly show a marked drop in column efficiency and selectivity. Although in some cases a prolonged flushing with 2-propanol may improve the situation, usually the column is most likely dead and will need to be replaced.

Column Efficiency: In most cases, changes in column efficiency are accompanied by changes in peak symmetry or peak shape. In rare cases, a reduction in efficiency accompanied by the appearance of shoulders on the peak trailing edge may be due to void formation at the head of the column. This could be due to dissolution of the silica support by the mobile phase conditions (usually in reversed phase mode), over-pressurizing of the column, collapsing the silica particles or a poor initial packing of the column. While we take great care in the packing and QC process, occasionally a poorly packed column can slip through the net. To eliminate this possibility, we strongly recommend that all columns be tested on receipt with the QC test that accompanies each column. Most loss in efficiency problems are due either to partial blockage of the inlet frit (see above) or are due to the adsorption of material at the head of the column.

Adsorption of material at the head of the column can be seen where the samples are not pure and contain components which are strongly adsorbed on the stationary phase. This can often be resolved when using immobilized columns by flushing with a strong solvent such as DMF. This approach cannot be realized with the coated columns and the best that can be done is to flush them with the strongest compatible solvent, often 2-propanol. For those cases in which recommended washing fails to restore performance, more drastic washing may be needed. Such washing procedures carry a significant risk of column damage, so they are best used as a measure of last resort. For more information on such procedures, contact questions@chiraltech.com. This is usually not so successful a process and represents another reason why we strongly recommend using the immobilized columns wherever possible.

Sometimes an established separation cannot be duplicated on a new column. While there can be some lot-to-lot variation in column performance, this situation more often results because the established separation is dependent on some type of column conditioning that the new column has yet to be subjected to. The older column may have a “memory effect” in which additives used in the past history of the column have become adsorbed on the stationary phase, and are crucial to the current separation. With immobilized columns, a simple flush with DMF may be all that is necessary to “reset” the stationary phase. In many cases, the problem can be resolved by conditioning the new column for a few hours with mobile phase that contains the pertinent additive. In those cases in which the separation is still not restored, the method may need to be redeveloped with a different mobile phase, column, or temperature. For this reason, we recommended developing new separations on a new column, or one for which the mobile phase and sample history are documented.

E-mail questions concerning columns which are not working properly are always welcome at questions@chiraltech.com. To avoid delay in receiving an accurate response to your question, describe your problem as completely as possible.

Many neutral and weakly basic or acidic compounds do not require any additives, and most method development procedures are best initially carried out without additives. Compounds that are strongly basic or strongly acidic will tend to adsorb on Daicel polysaccharide columns, with the result that broad or tailing peaks will be observed. The adsorption occurs at the most active sites on the silica support in the column. To overcome this problem, compounds are added to the mobile phase that will preferentially adsorb on the most active sites, displacing solute molecules, and making these sites unavailable for solute adsorption. The most common additives are trifluoroacetic acid for acidic solutes and diethylamine or triethylamine for basic solutes. These additives are quite soluble even in non-polar solvents, and they can be used equally well in either normal phase or polar organic phase modes. A second advantage of these additives is that they can greatly enhance the solubility of compounds that would otherwise have low solubility in a given mobile phase. Additives such as DEA and TFA can be added to the mobile phase at concentrations up to 0.5%. Longer column life may result if the concentration of additive can be held to 0.2% or less. And even better concentration is 0.1% or less.

Some users develop methods using additives from the outset. One popular system is to use both DEA and TFA in the mobile phase. In all cases where additives are used in development, care must be taken to ensure that when the method is transferred to another column that the additive is necessary for the separation. Often it can be eliminated. You should also ensure that the new column does not need to be conditioned by the additive before the separation will work reproducibly.

Our Chiral Separation Service is an important part of the comprehensive offering of products and services we provide. This service is available to clients who need to quickly obtain pure enantiomers, but who don’t have the time, equipment, or facility to scale-up an analytical separation to isolate them. To initiate a Separation Service project, contact your CHIRAL TECHNOLOGIES sales representative, or contact us by telephone at 1-800-6CHIRAL or by e-mail at chiral@chiraltech.com. Starting, if necessary, with a confidentiality agreement, clients then provide us with 100 to 500 mg samples for evaluation. Our expert staff then uses a wide range of commercial and proprietary CSPs to identify the optimum CSP, and conditions for a preparative separation of the compound. If a promising separation is identified, you will then be provided with a quotation for the separation to be performed. Enantiomers are typically returned with an optical purity of >98% e.e. (chemical purity = purity of the racemate and yield >85% for each isomer).

For analytical purposes, high sample concentrations are usually not necessary. A sample preparation of 1 mg/ml, or even less, in mobile phase is usually sufficient. If your sample is an acid salt of a base, then addition of 0.1% DEA to the sample solvent may help solubility by converting the material to the free base, which usually is more soluble. Conversely, If your sample is a salt of an acid, then addition of 0.1% trifluoroacetic acid may improve solubility.

If you are using one of the immobilized phases, try to dissolve your sample in some other solvent in which it has good solubility. When the application is analytical, there should be no issues with sample solvent-generated effects on the results, providing the sample injection volume is small. This is especially true if the chosen solvent has a similar solvent strength to that of the mobile phase. For preparative applications, this can be more problematic since the sample can precipitate from solution, once the injection mixes with the mobile phase. In such cases it is best to use the sample solvent as one of the mobile phase components to enhance the mobile phase solubility.

Where the column used is a traditional, coated column, then try to dissolve the sample in 100% methanol, ethanol, isopropanol, or acetonitrile. These polar solvents can be used with nearly all of our most popular chiral stationary phases. In general, organic solvents such as toluene, chloroform, methylene chloride, MTBE, tetrahydrofuran, acetone, MEK, ethyl acetate, dimethylformamide, dimethyl sulfoxide, and pyridine must be avoided when dissolving samples to be injected into the traditional coated columns. Even small amounts of these solvents, not removed during sample clean-up, can dissolve the chiral stationary phase and lead to dramatically shortened column life.

If your sample will only dissolve in aqueous solvents, then it can be used in reverse phase mode with IA, IB, AD-RH, AS-RH, OD-RH and OJ-RH columns. Mobile phases and dilution solvents of methanol/H2O ethanol/H2O, isopropanol/H2O, or acetonitrile/H2O can be used in reverse phase mode. Please consult the Mobile Phase Solvents Chart for reverse phase composition limitations associated with specific CSPs. Care should be taken to control the pH between 2 and 9 in order to avoid dissolving the silica support of the chiral stationary phase.

The pressure limit specified in the User’s Guide for a Daicel chiral column applies only to the pressure drop across the column itself and not to the rest of the chromatographic system. For example, if the total pressure drop measurement in your system, at normal operating conditions, is 1000 psi, but the system pressure (without column) is 300 psi, then the pressure drop across the column would be 700 psi, which would be acceptable for most columns. A high system pressure probably indicates that there is a partial blockage, possibly in connecting tubing, an in-line filter, or a valve channel. Whenever possible, this blockage should be systematically located and the problem component replaced.

High operating pressures often result from material blocking the frits of the column. In some cases, this material may be removed through the use of recommended washing procedures. To prevent such problems, it is always wise to use a replaceable in-line filter or guard column before the analytical or semi-preparative column.

Flow rates for semi-preparative columns can generally be scaled up from the flow rate developed on an analytical column, by a factor proportional to the volume comparison of the two columns. When operating at the higher semi-preparative flow rate, you may need to increase the diameter of connecting tubing or the volume of the detector flow cell in your system. Alternatively, you may need to slightly reduce the flow rate on the semi-preparative column to stay under the recommended maximum operating pressure.

High back-pressures may result if a column is eluted at normal flow rates with solvents such as pure ethanol or isopropanol, due to the high viscosity of these solvents. If you are experiencing high back-pressure when using these solvents, reduce the flow rate to a level that brings the back-pressure under the recommended maximum operating pressure. Since these solvents are often associated with column-cleaning procedures, you should be able to operate at higher flow rates once you return to your normal operating mobile phase.

Generally, lower flow rates are not necessary when using H-series columns, with the exception of -RH columns. These reverse phase columns generally have higher pressure drops due to small particle size and the increased viscosity of aqueous mobile phases. As a general rule, 4.6 mm i.d. -RH analytical columns should be operated at a flow rate of 0.5 ml/min or less.

A rapid build-up in pressure with the traditional coated columns can be a symptom of a serious column problem. Introduction of incompatible materials (from sample or mobile phase) can be rapidly destructive to a column. When such a problem occurs, chiral stationary phase will dissolve or be lifted into the mobile phase, only to drop out of solution downstream, plugging the flow path, and causing a rapid build-up of column pressure.

Analytical separations can be predictably scaled up to the semi-preparative level using some fairly simple calculations. When resolution of a given quantity of racemate is the desired objective, it is best to first optimize using an analytical column to yield the maximum possible loading. If you are fortunate to have more than one possible separation method, each method can be tested separately to determine which one gives the best overall loading. For each attempt to achieve maximum loading, start by making injections of a solution of the target racemate, as concentrated as possible, in the mobile phase. Using a detection wavelength selected to keep the peaks on scale, increase the injection volume until the valley between the enantiomers begins to rise. This should give you an experimental loading weight WE = maximum concentration of racemate (Cmax) x largest analytical injection volume before overload (VAmax). For ballpark estimation purposes, typical WE values for an analytical column are 1-10 mg/injection.

The relative loading capacity (LCR) on the 0.46 x 25 cm analytical column is assumed to be "1." For various-size semi-preparative and preparative columns, the relative loading capacity and associated flow rates can be determined from the following table.

The answer to the question: How much can I load?, is (WE) x (LCR). Thus, if the typical load on an analytical column is 1-10 mg, then a typical load on a 2 x 25 cm semi-preparative column is 19 –190 mg/injection.

What size column do I need? is a typical preparative question that is inter-related to the question: How many injections (N) are you willing to make? To determine the size column that you need, the following equation can be used:

Where is the loading capacity of the column needed to achieve your objectives.

EXAMPLE: A researcher needs to isolate 1 gram each of two enantiomers from a racemic mixture. It has been determined that 48 half-hour runs made in a 24-hour period would be a reasonable number of preparative injections. The maximum analytical loading weight is 2 mg = .002 g (1 mg of each isomer). What relative loading capacity is required if 48 injections are to be made?

Consulting the table, it can be seen that a 2 x 25 cm column with a relative loading capacity of 19 would probably best meet the researcher’s needs.

Another typical question is: How much sample can I load on the semi-preparative column that I already have? Determine the maximum load for an analytical column, and multiply this value by the relative loading capacity of your column to determine the preparative loading; i.e. WPREP = WE x LCR. Use a preparative injection size that is equal to your maximum analytical injection volume multiplied by the relative loading capacity; i.e. VPREP = VAmax x LCR.

The older CHIRALCEL and CHIRALPAK columns are based on 10-micron particles, whereas H-series CHIRALPAK and CHIRALCEL columns are based on 5-micron particles. The H-series columns have chiral stationary phases identical to their non-H counterparts (i.e., an AD® and AD-H column have the same chiral stationary phase), which provide similar selectivity to the standard columns while providing much better chromatographic efficiency. The smaller 5-micron particles thus give better overall resolution. This means better performance for the most difficult separations, or for separations in which impurities would otherwise interfere with the main components. For those situations in which speed of analysis is most important, a 15 cm H-series column may give an equivalent separation with a shorter analysis time, than the same separation on a 25 cm non-H column. -RH series (reverse phase) columns are also based upon 5-micron particles.

In this context it can be noted that the immobilized columns, CHIRALPAK IA and CHIRALPAK IB are available only in a 5-micron particle size for analytical separations.

Part A – Things To Do when Using Daicel Chiral Columns

1. Read carefully the User's Guide that was shipped with the column. There are major differences in stability between the immobilized 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 CTI. Please note, however, that not all of the alternative solvents have been evaluated over all 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 Sheets that are provided with each column for solvent limitations). HPLC-grade EtOH is used for methods developed at CTI. HPLC-grade EtOH is denatured with 5% IPA and 5% MeOH. 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 CTI 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 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. Don’t operate your Daicel column above the recommended maximum pressure limit.
2. Don’t 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. Don’t 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. Don’t discard the Instruction Sheet or the test chromatogram that comes with each column. If you lose or misplace the Instruction Sheet for your column, it can be replaced by contacting us at chiral@chiraltech.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.

SFC works very well with Daicel chiral columns. Carbon dioxide as a mobile phase bulk fluid has solvent properties similar to hexane with lower viscosity and flammability. Carbon dioxide can be used with all the modifiers used in HPLC (alcohols, acetonitrile) plus others such as methanol that are immiscible in hexane. Chiral selectivity is normally comparable in SFC and HPLC but better resolution is observed in SFC due to its higher efficiency at typical flow rates. Higher flow rates may be used in SFC because of the low viscosity of CO2 resulting in faster separations. We have discovered that carboxylic acids requiring acidic mobile phase additives in HPLC can be eluted in SFC without such additives.

A common concern is the effect of the high pressures used in SFC on column stability. The pressure drop across the column is the important factor in column stability. This pressure drop is lower in SFC than HPLC and Daicel columns have proved very stable to SFC conditions. We recommend that when the column is not in use, it be removed from the SFC, flushed briefly with isopropanol to displace CO2 (that would evaporate leaving a dry column), and capped. When using a Daicel column in SFC that had been used in HPLC, it is necessary to first flush the hexane with isopropanol, as CO2 will not efficiently flush hexane, and a noisy baseline will result.

SFC can offer several advantages in preparative applications. Separations are faster and isolation of the product from the mobile phase is also faster as the bulk of the mobile phase evaporates as part of the collection process. With the lower pressure drop experienced in SFC, the use of higher efficiency 5-micron particle H-series columns for preparative application is feasible. CHIRAL TECHNOLOGIES offers 1-, 2-, 3- and 5-cm ID columns packed with ADH, ASH, ODH or OJH in SFC column hardware.

In many cases, installing a guard column upstream of your analytical or semi-preparative column is a cost effective strategy. The purpose of a guard column is to protect the analytical or semi-preparative column from materials that would either adsorb on the column, or which would dissolve some of the column packing. Guard columns thus serve a sacrificial function; when a guard column is nearing the failure or breakthrough point, it can be discarded and replaced at a fraction of the cost of a new column. Knowing when to replace a guard column can be determined from observations about your chromatography. Loss in separation between peak maxima, increased peak broadening or tailing, or increased pressure drop in your system are all signals that a guard column may need to be replaced.

A guard column should contain the same packing as the analytical or semi-preparative column, and specific guard columns are available for all Daicel chiral columns. A guard column containing a different chiral stationary phase may actually diminish the separation. A non-specific guard column might absorb some sample or mobile phase impurities; however, it would degrade the separation by adding more volume to the sample flow path without increasing the separation.

The end fittings for a guard column are exactly the same as the main column. Therefore a short piece of narrow i.d. connecting tubing is needed between the guard column and main column. Guard columns for all H-series and R- or RH-series columns are cartridge type. A universal cartridge holder can be used for any 5-micron column, together with a specific disposable cartridge containing the same 5-micron packing as the main column.

In most cases, running a gradient with a Daicel chiral column is counterproductive. The time required to re-equilibrate the column back to initial mobile phase conditions is typically longer than any time-savings realized from the use of a gradient.

There have been reports from some users that they find gradients useful in method development, although in general the gradients are best run over relatively limited range to avoid the long equilibration times. This avoids the situation where either the solutes elute immediately or not at all. This may be an application-dependent phenomenon where a particular class of compounds lends itself better to gradient elution than others.

Preparation of Aqueous Mobile Phases
Preparation of pH 2 buffer: Weigh 6.80 g of monobasic potassium phosphate (KH2PO4: FW 136.09) into a 500 volumetric flask (100 mM). Purified water is added to the mark. The pH of this resulting solution is approximately 4.5. In a second 500 ml volumetric flask 5.76 g of phosphoric acid (H3PO4--85% by weight) is added and diluted to the mark with purified water (100 mM). The potassium phosphate solution is transferred to a 1L flask. Using a pH meter, the phosphoric acid solution is added until the pH is adjusted to 2.

Preparation of pH 9 buffer: Weigh 1.24 g of boric acid into a liter volumetric flask and dilute to the mark with purified water (20 mM); (H3BO3: FW 61.83). Weigh 7.63 g of sodium borate decahydrate (Na2B4O7•10H20 --FW = 381.37) into a second 1-liter volumetric flask and dilute to volume with purified water. Take 500 ml of the sodium borate buffer and transfer to a 1-liter flask and, using a pH meter, adjust the pH to 9 by adding the boric acid solution.

Preparation of pH 1 Perchloric acid: Weigh 16.3 g of commercially available 70% perchloric acid into a 1-liter volumetric flask and dilute to the mark with distilled water. Other pH values can be obtained as follows:

1. pH 2.0 – 100 ml of pH 1 perchloric acid is diluted to 1 liter.
2. pH 1.5 – 316 ml of pH 1 perchloric acid is diluted to 1 liter.
3. pH 1.3 – 500 ml of pH 1 perchloric acid is diluted to 1 liter.

Important Safety Message Regarding Perchloric Acid Mobile Phases: Perchloric acid and perchlorates are extremely hazardous materials. If perchloric acid or perchlorates are used in the mobile phase, their use should be limited to analytical purposes only. You should never use perchlorate buffers for preparative or semipreparative purposes. Do not attempt to evaporate an aqueous solution containing perchloric acid (or sodium perchlorate) as a method to isolate an enantiomer. This solution can explode if heated to evaporation. If you are not already using perchloric buffers in your laboratory, we strongly recommend that you read carefully the MSDS for perchloric acid and sodium perchlorate. Additional information can be found on Web sites such as: http://www.ab.ust.hk/sepo/tips/ls/ls011.htm or http://www.auburn.edu/administration/safety/crcperchloric.html.