Depending on the amount of racemate available, budgetary constraints, as well as the availability of analytical setup, you can decide which and how many kits you should use for screening. There are 4 kits available per racemate. For exhaustive screening and to ensure that you have not missed an optimal resolving agent/solvent combination, we recommend that you use all 4 kits during the experiment. For primary screening kits, 3 mmol per kit is needed during the experiment. If the amount of racemate available is less, and if you have chiral HPLC available for screening, you may prefer to use the kits with glass vials. If the racemate is acidic, you should use the base kits. On the other hand, if the racemate is of type base, you should use acidic kits. For neutral racemates of type alcohols, amino acids, aldehydes and ketones derivativization to acidic or basic state is required before using the kits.
The experiment involves the following steps:
Choose the right set of kits (A1, A2, A3, A4 or B1, B2, B3, B4) depending on whether the unknown racemate is basic or acidic respectively.
If the racemate is of type alcohol, amino acid, aldehyde or ketone, do the pre-processing as described below.
Add 0.03 mmol of your racemate (1:1 ratio with resolving agent) to each of the 96 vials.
If the racemate is liquid, and you are using the kit for liquid racemate, simply dispense 0.03 mmol of the liquid in to each of the 96 vial by piercing through the seal/capmat.
If the racemate is solid, and you are using the kit for solid racemate, dissolve the needed amount of racemate into a most volatile solvent that it can dissolve in using the minimum amount of solvent necessary. Remove the cap-mat or seal from the kit. Add the right amount of racemate solution so that when the solvent evaporates there will be 0.03 mmol of the racemate in each of the tubes. Evaporate the solvent that the racemate was dissolved in completely. Now take the solvent plate; remove its seal; or pierce through each column using 8 channel pipetter (each column has same set of solvents in exact same order; so there won�t be any contamination) and dispense into the vials of the kit in the exact same position vial.
Heat the rack along with its vials to 80� C (the optimum temperature for most of these experiments) or until the mixture becomes homogeneous.
Allow the kit to cool to ambient temperature. Then, if required, further cool it to 4� C and finally to 0� C and observe any crystallization. Vials with crystals are considered positive tests and need further investigation.
Using crystal initiation techniques, encourage more crystal formation. Vials with no crystals even after this effort are considered negative tests.
Select the vials with crystals (positive tests) and note down the kit�s bar-code along with the vial identification (located at the bottom of the vial).
For the selected vials in 7; separate out the crystals from the filtrate
If chiral HPLC, IR or CE are available, use the filtrate; or the crystals to do further analysis
If instruments in 9 above are not available, analyze each of the crystals separately after liberating enantiomers from it�s' diastereomeric salts for specific rotation using polarimeter Note: Typically, during step 8, only 4 to 8 vials per kit will have crystals and will need to be analyzed further. Further the kits identify separation process for both dextro (+) and laevo (-) rotatory enentiomers. After analysis, we recommend that you choose the best 2 or 3 vials with desired enantiomer; and investigate further along with its' mother liquor for scale-up condition optimization.
Alcohol is neutral in functionality and it is usually resolved by conversion into the mono-ester of succinic or phthalic acid. This hydrogen succinate or phthalate then gets converted into a diastereomeric salt with optically active bases. Steps:
Treat the racemic alcohol with 1X1 molar ratio of phthalic anhydride and greater than 1X1 molar ratio of pyridine. Alternately, 1X1 molar ratio of succinic anhydride may be used instead of the phthalic anhydride
Heat the mixture to 80 to 100� C for 2 hours
Cool the mixture to ambient temperature
Quench the mixture with ice-water containing enough sulfuric acid to make the whole mixture acidic. This mixture will be either hydrogen succinate or hydrogen phthalate. It will be either in the form of an oil or a crystalline solid. If the mixture is oil, treat it with acetone and/or use crystal initiation techniques if necessary to crystallize it
Filter, wash and then dry the mixture. The result is hydrogen phthalate with free carboxyl functions
Use the kits B1, B2, B3 and B4 as described here
Amino acids exist in Zwitter ionic structure. A synthetic amino acid is primarily resolved using one of two types of methods:
a) Protection of Carboxylic Group using Esterification The carboxyl end of the molecule can be protected by esterification followed by diastereomeric salt formation of free amine function and needs screening kits of type A1, A2, A3, A4. Many racemic alpha-amino acids have been successfully resolved by preparing isobutyl or benzyl esters.
Steps:Add a sufficient amount of dilute HCl to the racemate to dissolve it and bring the pH to 3
Cool the mixture to 0 to 2 � C
Esterify by adding (1:1.2 ratio) of isobutyl or benzyl ester
Heat the mixture to 100 � C and then cool it to 0 to 5 � C
Decrease the acidity to ph 7 by adding NaOH.
Use the kits A1, A2, A3, A4 as described here to get the diastereomeric salt
After having identified the ideal candidate vial, remove the ester group introduced in step 3 under mild hydrolysis conditions and verify that no racemization occurs
b) Protection of Amino Group using Formylation The carboxylic group can be screened with the ChiroSolv� kits B1, B2, B3, B4. After having identified the ideal candidate vial, remove the formyl group under mild hydrolysis conditions and verify that no racemization occurs.
Steps:
Add a sufficient amount of NaOH solution to the racemate to dissolve it and bring the pH to 10
Cool the mixture to 0 to 2 � C
Formylate by adding (1:1.2 ratio) of Trithyl Orthoformate
Heat the mixture to 100 � C and then cool it to 0 to 5 � C
Increase the acidity to ph 4 by adding Hydrochloric or sulfuric acid
Use the kits B1, B2, B3, B4 as described here.
After having identified the ideal candidate vial, remove the formyl group introduced in step 3 under mild hydrolysis conditions and verify that no racemization occurs
In order to be resolved by salt formation, aldehydes and ketones must be converted into either acidic or basic derivatives.
a) Acidic derivatives Reagents like 4-sulfonylphenylhydrazine, 4-(4-carboxyphenyl) semicarbazide, 4-hydrazinobenzoic acids (para/meta), or Oxalic acids monohydrazide can be used. These salts can then be resolved by chiral bases.
Steps:
Treat the racemic aldehyde or ketone in minimum amount of methanol
Cool the mixture to 0 to 5 � C
Add one of the above mentioned reagents. The result is a crystalline protected amino acid
Isolate the protected amino acid using filtration or centrifusion
Use the kits B1, B2, B3,B4 as described here.
b) Basic derivatives Carbonyl can be converted into enamine using tertiary amines, which can then be resolved by chiral acids. Alternatively, carbonyl can be treated with bisulphite salts of chiral amines, and the resulting diastereomers can be separated by crystallization
Steps:
Treat the racemic aldehyde or ketone in minimum amount of methanol
Cool the mixture to 0 to 5 � C
Add a tertiary amine like pyrrolidene or piperidene. Alternatively, Sodium Bisulphite may be added. The result is a crystalline protected amino acid
Isolate the protected amino acid using filtration or centrifusion
Use the kits B1, B2, B3, B4 as described here