An ‘In Situ’ Source of Carbon Disulfide Reagent: An Otherwise Unsafe Chemical

Potassium ethyl xanthate

It is often the case that a process step is vetoed because a reagent or processing chemical is regarded as too risky. Occasionally, KiloMentor will try to identify such problem materials and suggest work arounds.

Some chemicals are regarded as too difficult to work with on scale and processes that require them are nearly always automatically rejected for scale-up. Carbon disulfide is in that category. This low boiling liquid has an auto-ignition temperature of 90 C and so broad a flammability envelope (1.3-50 Vol. % in air), that a fire is likely to result if a bit of CS₂ escapes from the process equipment and finds a  source of ignition, which can apparently be as innocuous as a steam leak. This point was made in the article, Holistic Route Selection, by Ronald B. Leng, Mark V.M. Emonds, Christopher T. Hamilton, and James W. Ringer,  Org. Process Res. & Dev. 2012, 16, 415-424. Nevertheless, these authors eventually stuck with advancing a process involving carbon disulfide reagent but only after adopting mitigation systems. To quote from the paper, “Ultimately a well-engineered CS₂ management system using returnable CS₂ containers, all-welded transfer lines, precise liquid metering, nitrogen blanketing, oxygen sensors, fugitive vapour sensors, and an automatic water deluge system were lines of defence that were incorporated into the process design…”
One lesson of this paper is that essentially any chemistry can be handle safely, if one is prepared to pay enough for the engineered system(s) needed to safeguard it. The problem is that unless the volume of the product is very high (their product was an agrochemical) and the profit margin good and stable, the up-front capital costs are likely to sink the project because of an inadequate discounted cash flow. Multipurpose plants simply cannot afford the required safety systems. It is not that the systems don’t exist.
This same paper points out that a large part of the risk using CS₂ occurs as much from when the material is being transported, stored, moved, measured as from the residue that remains in the waste streams.  The Dow Corporation developed a method of generating enough CS₂ to serve as a reagent ‘in situ’ which eliminated the risks from transporting, storing, moving, measuring and transferring reagent into the reactor. They did this by acidification of the solid salt, potassium ethyl xanthate which created carbon disulfide in situ. Using this sourcing, they obtained the same yield in the requisite transformation; however, they found that in order to completely consume their expensive intermediate substrate a slight excess of carbon disulfide was needed and this left some of it in the post-reaction mixture which gave issues in the subsequent centrifugation and gas venting system. This particular problem would not eliminate this approach for other process steps involving carbon disulfide reagent where an excess is unnecessary or when scrubbing of an excess can be simple.

The Particular Advantage of a Solvent More Dense than Water as Reaction Solvent

The KiloMentor Blog has emphasized the usefulness of phase-shifting during the work up as a simple and powerful element in the isolation/purification of process intermediates. Separation into aqueous acid-soluble, aqueous base soluble, and neutral organic soluble fractions is often part of that effort. On scale, there is a cost advantage and also an advantage in simplicity if the phase in which the product is dissolved is retained in the reactor throughout the work up processing. If at any point it is in the phase that is drained off, an extra vessel is needed. If the expected product is to be concentrated and separated from byproducts and co-products by extraction into water at some pH or other, it is desirable that the organic phase that, after the phase shift, comprises the waste products and not the product, should be the lower phase. That way it can be drained off through the bottom port of the reactor, leaving the product dissolved as a salt, in the aqueous phase and still in the same reactor. The salt of this desired product can then be neutralized and taken up in another solvent less dense than water. The wastewater phase can then itself be drained off leaving the desired product still in the original reactor. This way only a single vessel is required since the waste fractions can be drummed off. 

For this to work dependably the first organic solvent must be distinctly denser than water. In the past, carbon tetrachloride, methylene chloride or chloroform were the common solvents that could function in this way; however, all of these are now less desirable for health and safety reasons. Some remaining solvents that are denser than water are trichloroethylene, tetrachloroethylene,  chlorobenzene, 1,2-dichlorobenzene, benzotrifluoride (BTF),  p-chlorobenzotrifluoride, and 3,4-dichlorobenzotrifluoride. It has been reported that   benzotrifluoride (BTF), one of the simplest of these, often does not separate easily into distinct phases.  
p-Chlorobenzotrifluoride has a much larger density difference compared to water and so is probably superior in this regard. Chlorobenzene may work but it might have the same slow separation as benzotrifluoride, since the density is closer to 1.00. 1,2-Dichlorobenzene has a price advantage on a volume basis over other choices. Since its function is to transport away only reaction wastes, the high bp. is not a disadvantage.

Thus, in situations where a reaction product can either be extracted into aqueous acid or aqueous base as part of the work-up, there is a benefit if the reaction solvent can be performed in one of these solvents that are denser than water.

If mixtures of one of these chlorinated solvents combined with some other lower boiling solvent turns out to provide improved yields in a particular transformation, you can still retain this advantage. The more volatile cosolvent used with the chlorinated one can be removed by simple distillation before the phase separation.

Optimizing Complex Organic Chemical Process Schemes

The KiloMentor Blog concerns itself with process development. It assumes that an adequate process scheme has been chosen for the development. A process scheme is the sequence of reaction transformations that take commercially available substances and create from them a desired material. KiloMentor’s concern with choosing the best process scheme to be scaled up is to increase the likelihood that considerations important in scale-up are properly weighted when selecting the most preferred route.

Steps to make this important selection of the process route are amplified below.  

1. Prepare alternative schemes for the synthesis of the desired product from commercial materials. The methods for preparing process schemes will not be discussed here. Write balanced equations for each reaction step if a reagent is known. Prepare preliminary costings for the candidate routes.

2. Consider selecting two different routes: one to meet the demand for timely delivery of the final product to be used in other parts of the project and a second to be developed into the path for a commercially viable product.

3. Rate the alternative schemes prepared in 1 versus the criteria, musts, wants, ruggedness, analogs in the literature, risk of failure, convergence, phase shifting of intermediates, overall cost, throughput, waste, safety, and atom economy. Use some ranking methodology such as in the Kepner-Tregoe method.

4. If you are going to use two schemes: one to deliver material in a timely fashion for other departments and one for the commercial route, the last step should be the same for both. If the last step is not the same, the last purification should be very rugged and capable of removing many impurities. It is a major problem if the final steps are going to be different and the final isolation-purification is not rugged. The reason is that specifications and pharmacological and sometimes clinical data is collected using material from the research synthesis and this work might have to be redone if the products from both syntheses do not have the same impurity profiles.

5. Choice of whether 1 or 2 routes will be worked.

6. If a timely research route will be used to supply material for development run through that route to give the first look at the product. Often the route used to supply early batches to other teams working towards the commercial product is the laboratory procedure that has been modified as needed to increase scale and throughput. Operations such as column chromatography which cannot be adapted for scaling are worked around but changes are as few as possible. The idea is not to optimize just to make it workable for larger batches. 
7. Gather literature about the steps of the planned commercial route. The objective is to obtain as complete an understanding as possible of everything in the literature, which seems pertinent to the transformations you are going to try. This would include substructure searches on all the intermediates to discover the closest analogous structures existing in the literature; the closest analogous reactions including reagents, interfering or non-interfering groups, and solvents; reviews and mechanism studies of the transformations being studied.

8. Route for research products should only be improved as much as necessary to deliver the commitment for the material. If the ingredients are inexpensive work only on the isolations. If the ingredients are expensive only address the big-ticket items and changes that are substantial and quick.

9. A quick run through the proposed reaction sequence in one of its embodiments. In this work, the intermediates are split and one portion is purified before going on but the other portion is taken on without purification to see whether the subsequent reactions will (purge) purify the mixtures and, if there is no purification, indicate to the analytical department the impurities that are likely to most difficult to remove. At the end of the process, analytical and spectroscopic methods are used to evaluate whether the desired substance has been created and in what purity and yield. This is possible because the product is available from the research route. This exercise will provide a level of confidence that the desired product can be produced by the route. There is no point in optimizing a route that cannot be made to work!

10. Do a revised costing of the commercial proposed route to determine priorities for optimization. Remember optimization can go on forever but at some point, time will run out. The object is to have the best process and the lowest cost possible before the time expires!

11. The starting points for optimization are the paper reaction scheme, the best analogous transformations in the literature, your literature file, your costing, and your quick preliminary process run.

12. Make a preliminary assessment of what reactions might be telescoped together. The criteria for considering telescoping in good process work is that no intermediate is isolated unless its isolation contributes significantly to the purification. In multipurpose plants, there is an additional reason for isolating intermediates and that is to provide convenient and stable stopping points so that the flexibility of the plant is maintained. Stopping points are important as patches to protect against problems. Consecutive reactions are only candidates to be telescoped together in the process if there are no phase shifts in the purification of the first of the candidate reactions, which substantially contribute to purification. This is so because two telescoped reactions are normally done consecutively in the same solvent, phase shifts are usually done during the work-up. Since the phase shifts in the first work-up are lost in the process of telescoping, the purification, which occurs in these phase shifts will also be lost.

13. Choose the reactions to begin optimization on. Optimization of the early steps gains better access to material for the later steps. Attack the “must fix” problems first since if these can’t be addressed the route must be abandoned.

14. It is very advisable to fix the last step early on. If you can decide at the outset what the last step will be, you can design it to be as chemically simple as possible and optimize conditions to get the same polymorph every time. If you can’t fix the final step then add a recrystallization step. It may seem redundant or wasteful of material but for highly active compounds whose activity is very dependent on physical form, it may be the best route.

15. Assess the dilution (D) in L/Kg at which each proposed process step is run in the better literature examples and assign a rough numerical value. Assign a time (H) in hours for the completion from beginning to end of each process step. The conversion of starting material X_i  Kg to Y_i Kg of product in step I can be expressed in the expression X_i/Y_i Kg of X_i giving rise to 1.0 Kg of Y_i.

16. Validate the method of “in situ” analysis for the intermediate that is being optimized. Remember that each process transformation is divided into the reaction transformation yield, which measures the quantity of intermediate in the reaction solution as determined by assay divided by the theoretical quantity of material in solution expected AND the isolation yield which is the isolated weight divided by the quantity of intermediate in the reaction solution as determined by assay.

17. Select among the discontinuous variables the most promising reagent and solvent. This is a mouthful and includes some enormous assumptions. One is more likely to make better choices if one performs a reaction-based electronic literature search for the transformation required in the presence of all the non-reacting functional groups that will be present in the particular process substrate. The sample conditions that you unearth must be such that your particular substrate will be soluble. Identify as many solvents as possible that have worked with the preferred reagent. Searching in monographs like Organic Reactions and Fieser and Fieser can also give leads to start conditions. Aim to use a reagent and condition close to a literature condition. There are no special bonuses for being unusual unless the usual fails. Only if there is a very substantial advantage to be gained by using a less tested or untested condition should it be considered. Such advantages are cheap reagents, convenient solvents, high-load solvents, improved stability, safety/lower toxicity, and solvent convergence. If these conditions apply then try a “wish” reaction using the desired conditions or reagent being particularly careful and aiming for a positive result. If you do not immediately get a measurable yield of product, give up. There is a tremendous waste of time possible in 0 yield reaction space!

18. Select the screening parameters (up to 7), which will be used in the optimization. An understanding of the mechanism of the reaction can be very useful in choosing the screening parameters. If you have extra space for screening parameters choose one to be the effect of absolute concentration on the reaction. This parameter is crucial to getting the optimal throughput of mass in the process. Additionally the higher concentration reaction may proceed substantially faster. Certain reaction variables are only important when one of the reactions or associations is fast compared to the time of addition. Direct or inverse addition, mixing method and rate, addition time…. Sampling the reaction mixture at half-addition time and checking for product formation and/or starting material consumption can give a good clue as to whether these variables are likely to be important.

19. Run the eight screening runs collecting samples at staged time intervals. Quench the samples and then analyze them all to determine the reaction transformation yields and the time profile of the product/by-product productions. Half-addition analysis can also be done when just half of a material has been added. The purpose is to see whether the reaction is so quick that a substantial amount of reaction has occurred at the half-addition point. Using statistics determine the significant variables from those tested. In the screening be vigilant for conditions that substantially increase particular impurities. It may be critical later to identify these impurities and it will be much easier to isolate these impurities using reaction conditions, which give much larger yields of them.

20. 20.  Using half-log paper predict the likelihood that the optimization will meet the target of the yield improvement that is desired. If it is unlikely, choose some new screening parameters including the best of the old ones, and rerun the eight experiments and reevaluate.

21. Compare your best result from the screening reactions. If it is already close to the target or if the yield is good enough to practically produce material then switch your attention to the isolation method. If the isolation method has no major problems switch to the next target reaction and do its screening parameters.

22. Optimization is a cost optimization, not a yield optimization. Remember optimization time is wasted time if the route has any irremediable flaws.

23. Suppose we are continuing with the optimization using the identified statistically significant parameters. Simplex optimization is the simplest methodology and it is psychologically most satisfying. It is also the easiest method for dividing up the work so that everyone has a satisfying activity. As the start point in the simplex choose those conditions that had the highest reported reaction yield but also include lower yield conditions which showed a rising trend for product that had not peaked at the final time. Another advantage of Simplex is that if during the work any new significant parameter gets identified it can immediately be included in further optimization. Again in Simplex runs surprising increases in the level of any impurity may provide a method to synthesize and identify that impurity.

24. Work on the isolation is best performed on larger batches of material prepared by the best available reaction conditions. Isolation experiments are evaluated by the weight of clean intermediate obtained. Isolation studies are preferably done using split runs. That is, a reaction mixture is divided into equal portions, and alternative isolations are tested against one another. Besides yields, the purity profiles of the isolated product are examined and compared. Analyses of the fractions during the isolation development are useful because these form the basis for the creation of in-process checks required in the process. During the isolation particular attention must be paid to the maximum volume. The point of maximum volume in the entire reaction step very often occurs during the isolation and this maximum volume limits the throughput of the process step. The volumes, which are typically used in development research and literature preparations are enormously excessive and many of the treatments are precautionary more than necessary. Large washes contribute to the waste, which must be processed and the expense. Remember to include waste disposal in the costing.

25. Developing a scheme for robust isolation is not easy. Although there are reaction databases there are no isolation databases. Each isolation should be considered de-novo and a think tank approach taken to gather a wide range of ideas, which could form the basis for a rugged isolation. The objective should be to achieve what is called “practical purity”. By this I mean that there is no point in removing impurities, which will automatically be removed by the further chemical transformations of the material or by the phase shifts in subsequent steps. Practical purity is a purity that has removed impurities that interfere in further processing. Therefore it may be a good idea to take some of the crude unpurified product directly from the evaporation of the reaction liquors and perform the next reaction (or reactions) to see what impurities must be cleaned up and what impurities are taken care of by the later transformations.

26. Swish TLC on the product of these isolations can also provide concentrated samples of impurities. Multiple recrystallizations of small samples of product reusing the recrystallization solvent can also provide impurity samples. An impurity that does not change its proportion to the main product during screening or/and optimization is probably an impurity coming from a reactant. It is probably an isomer or a homologue. Check the quality of your commercial ingredients. Use materials of a quality available in kilogram quantities.

27. Solvent changes may be required in the work-up. Use azeotropes and other methods to efficiently do solvent switches without waste.

28. Practical concerns such as filter speed, filter media, emulsions, crystal form interfaces number of reactors, and reactor transfers are of concern, particularly in isolation. These and the elimination of chromatography must be solved.

Reagent Selection

Certain reagents are clearly disfavoured based on cost. 

A reagent that has not been shown to function in a solvent, which will dissolve the required substrate is a very poor reagent choice. This is true because functional group transformations are very sensitive to solvent choice and information about a transformation is not generally transferable between different solvents. This is particularly true when the change is between radically different solvent types.

 A reagent that has not been shown to be compatible with all the functional groups, which are supposed to be unreactive during a transformation cannot be accepted uncritically. This is not as serious a problem as the solvent because often a potential interfering reaction turns out to be kinetically uncompetitive with the desired reaction or the reaction conditions can be adjusted to make it uncompetitive. Selectivity in the reaction is better than using extra protection-deprotection steps. 

Most “optimization” done in reaction method publications by academic authors are one-variable-at-a-time studies with a single model substrate and are useless in assessing the scope of a reaction.

Solvent Choice for Optimization

A pure solvent is preferred so that the recovery of solvent for recycling is possible. Very often solvent recycling is not practiced in early development. Solvent recovery is uneconomic in multipurpose plants. If for solubility reasons the dilution using a pure solvent must be high, then a solvent mixture, which greatly enhances throughput could be considered. Solvent mixtures made up of readily separable components are promising. If the more polar component is more volatile than the less polar solvent there is a good likelihood that the product can be crystallized upon removing the more volatile component.  A pure solvent is preferred for crystallization otherwise getting the proper solvent ratio reproducibly can be a problem.

The solubility of the substrate and product should be determined in solvents with the biggest possible differences in principal properties. Are the different solvents all miscible together? Consider using no solvent. This provides the highest throughput but the least temperature control.

Solvents differing in their principal properties are the most likely to optimize differently.

Shortening the Reaction Time of a Process Step At-Scale

Many synthetic reactions are second or higher kinetic order. Once initiated in a particular reactor at a particular concentration (solvent volume), they proceed most rapidly in the initial stage and then slow down as the starting materials are consumed and their concentrations decline. As a consequence, the major portion of reaction time is spent waiting for the last small part of the reacting to finish because the concentrations of agents in these multi-order kinetics have become relatively low.

From these same considerations when a reaction is exothermic, the larger part of the exotherm occurs in the early stage when concentrations are highest. It is for this reason that process chemists religiously avoid mixing the full stoichiometric quantities of all the reactants together first and then initiating the reaction (say by heating). The reason: this is a recipe for a disastrous runaway reaction. Instead, in the preferred approach, one essential reactant is added gradually to a mixture of the other chemicals at the reaction temperature. Operating this way, any unwanted exotherm above what can be balanced by cooling, can be choked off by stopping the addition.

The question considered here is whether, after the faster part of the reaction has passed, anything can be done to accelerate the later slower stage of the reaction so that the overall reaction time can be reduced? If the reaction is being conducted at the reflux temperature of a single pure solvent, the reaction can in principle be accelerated, without changing the steady reaction temperature, by distilling away part of this reaction solvent. In this situation the reaction temperature is the boiling temperature of the solvent and such distillation removes solvent and increases starting material concentrations without changing the reaction temperature. Because removing solvent increases the concentrations of all the solutes including all the starting materials, the rate of their consumption will increase and the point of effective disappearance of starting materials will arrive quicker. For example, if the volume for a bimolecular reaction is reduced in half, the concentrations are doubled and the rate of reaction will be increased by a factor of four.

Of course there is a limit to how low the volume can be taken in a standard reactor. The volume cannot practically be reduced below where the reactor contents can be effectively stirred (the minimum stirrable volume). Also the volume must not be reduced below the level at which the reacting materials begin to precipitate because the reaction’s kinetics are almost certainly dependent upon a homogeneous solution.

Another advantage for the process of concentrating the reaction mixture is that the volume at the point of maximum volume is likely to be lowered. This will result in a higher product throughput; that is, more kilograms can be synthesized in fewer batch repeats. If the volume at the point of maximum volume can be reduced in half (for the sake of simplicity of example) you would only need half as many repeats of that process step to transform the same amount of starting materials. 

A potential difficulty with such a concentrating procedure as I am describing can arise if some important element of the process co-distils with the solvent and is so removed. Again for example a volatile catalyst co-distilled when the solvent was being reduced this would slow down or stop the desired reaction despite the increased concentrating of the co-reactants. Although some reaction ingredients may not be blown out of a reaction mixture when distilled in the lab, distilling in the plant can have substantially different characteristics and one needs to be aware of the possible loss of even quite non-volatile materials via an aerosol. There are physical traps (called impingers) that can capture aerosol droplets and return them to the reactor to overcome this.

Resort to this concentration strategy described above is only needed when an unacceptably long time is required to get complete reaction at an acceptably low temperature. Of course it can only be practiced if a solvent is found that facilitates the desired reaction at the solvent’s boiling point.

Alternately the pressure in the reactor can be controlled so that the solvent that is most desirable for the reaction boils at the desired temperature.

Reactions that are bimolecular but exhibit pseudo-first order kinetics because one reactant is present in large excess can also be accelerated by this strategy.

This strategy could also be applied to a reaction conducted at the azeotropic boiling point of a binary solvent mixture.

Strategies for Characterizing Trace Impurities Important for Regulatory Compliance in Pharmaceutical Synthesis

In setting the specifications for a pharmaceutical substance, its unknown impurities must typically be less than 0.1% area/area with respect to the main signal peak using the standard detector for that particular method. The usual instrumentation is reverse-phase liquid chromatography.  Even so, regulatory agencies prefer that impurities be identified.  Impurities that might structurally resemble genotoxic substances should be absent. If an unequivocal structure has been assigned to a minor component, it is possible a higher concentration of that impurity level can be accepted by regulators providing an extra incentive to discover that structure.

HPLC-Mass Spectroscopy & HPLC-MS-MS

With the correct instrumentation and method development, a skilled analyst can greatly reduce the number of possible structures for an HPLC impurity peak.  Nearly always this requires that the HPLC mobile phase consist of either a volatile salt buffer, such as ammonium acetate, or no buffer at all. When developing new analytical test methods the first choice for buffers should be volatile buffers.  General analytical methods are well described and easily searchable so no more need be said here.

 It is worth discussing what can be done when the above standard approaches fail or are inapplicable for some reason.  For one thing, it is sometimes possible to narrow down a list of suspect structures and devise means to isolate or synthesize a sample of the hypothesized material.

Clues Suggestive of Structure

1. Is the impurity acidic, basic or neutral?  This can be determined by aqueous acidic and basic extractions.

2. Does the quantity of impurity remain fairly constant with respect to the main component even when different methods of purification are tried? If yes, this is suggestive,  either of a homologous structure in which there is some slight difference in a side-chain between the impurity and the active drug, or of a positional isomer relationship between the impurity and the main constituent.  Such impurities usually come from impurities in one of the starting material building blocks.

3. HPLC is the most common present-day method of pharmaceutical analysis and diode array variable UV wavelength detectors are routinely available for such analyses. So how does the UV spectrum of the impurity measured by the diode array detector compare to the UV spectrum of the main product?  Does this narrow down structural possibilities?

4. Is the impurity’s increase most probably a function of the degree of scale-up? 

5. Have you performed a laboratory run in which the duration of the addition times are the same as in the plant?  Even if obtaining very slow rates of addition on the laboratory scale is too technically difficult or requires unavailable equipment the same effect might be obtained by adding a quarter of the dropping funnel charge, then waiting for ¼ of the plant addition time; adding the next quarter of the charge and waiting another quarter of the plant addition time and so on.  Impurities often arise from the wider variation in the stoichiometric ratios that are present during the lengthened addition period on scale.

6. Is the impurity occurring only in the most recent runs and not occurring under somewhat different earlier conditions?  This is a key question that arises out of Kepner-Tregoe problem analysis.  The answer may trigger an insightful guess at the structure of the impurity connected with the possible change that caused it.

7. Do you already know some means to obtain a sample free of this impurity?  Even if this is expensive and impractical it provides information to fashion a separation/identification method.

8. Is the main component (active API) reactive with some easily removed and quantitatively reacting material (such as hydrogen)?

9. If one prepares the sample for analysis differently, does the impurity increase, decrease, or remain the same? That is to say- is it actually an artefact of the analytical method?

Swish Chromatography

Does “swish chromatography” increase the relative concentration of the impurity versus the main peak? 

Could trituration (swish TLC) with an atypical liquid in which the main component is poorly soluble give an enriched composition? Among atypical solvent I would include methylamine, ethylamine, sulphur dioxide, dinitrogen-pentoxide, carbon disulfide, nitromethane, acetonitrile, perfluoromethylcyclohexane, tetrachloroethylene, trifluoroacetic acid,  carbon tetrachloride, dicyclopentadiene and perylene sulfone. These solvents are quite volatile and can be readily removed. The perylene sulfone and dicyclopentadiene both  decompose to volatiles upon heating under vacuum and methylamine, ethylamine, carbon disulfide, sulphur dioxide and dinitrogen pentoxide are gases under normal conditions or are very volatile.

Is extractive crystallization possible to selectively phase switch the impurity?

Switching from HPLC to TLC for Isolation

Is there some means to find the equivalent TLC,  Rf for the impurity, which you are identifying in HPLC by a RRT?

On this question, I do not know what the literature provides but here is a possible method: Suppose one runs a preparative TLC using a solvent system that gives an Rf of 0.05-0.1 for the main component but one performs multiple elutions on the plate and then removes the adsorbent in horizontal strips from that plate and runs these according to one’s HPLC procedure.  Multiple elutions with a poor solvent system provides the best chance for separation of impurities. If the impurity is not co-eluting on TLC, this HPLC analysis of bands from the prep plate will locate the TLC Rf range where the HPLC impurity of interest is located. Even when this Rf region has been located, the impurity is not necessarily one that can be readily visualized on the TLC plate. The HPLC impurity may be undetectable by conventional TLC at the concentration being spotted.  Nevertheless, if it works, you have found a preparative scaleable method for separating the impurity even if you cannot detect the impurity by normal visualization.  If the location of the HPLC impurity is well resolved from the main compound, you will be able to simplify your TLC method by increasing the polarity of the elution solvent trying to get a method that only requires a single development of the plate, but if the separation is difficult, the simplification may not be possible and you will have to live with a multiple elution method.  

Run a preparative scale column, collecting multiple fractions in the Rf region where your qualitative study has shown the impurity to come and analyze these fractions by HPLC to pick out those with the highest concentration of the unknown impurity.

Hypothesizing the Structure of Potential Impurities

Perhaps you can hypothesize a possible identity for the troublesome impurity from the answers to series of questions. 
What modifications of conditions increase the impurity? What modifications decrease it? Does the Rf provide a clue to the polarity and so make some structures more likely and others less likely? Is the Rf consistent with the proposed functional groups? Does the degree that it partitions between different liquid phases provide a clue?

If you have a potential mechanism for formation of the hypothesized impurity, is it going to be easier to simply synthesize this potential impurity and test it instead of trying to isolate and purify the impurity from the product mix?

Is a composition that results from intramolecular self-reaction possible? Think of possible side reactions that could yield such products.  These materials, because they have a molecular weight about double the API itself and similar functional groups can be difficult to crystallize out.  Size exclusion chromatography can be very powerful for distinguishing materials differing by 700 atomic units.  Advantageously the high molecular weight impurity elutes first!

Is reaction with a solvent, solvent impurity, or reagent impurity possible? 

Could the impurity be present in a very small amount but have a very large extinction coefficient compared to the main substance, so that the HPLC detector signal was exaggerated in a molar comparison?  The diode array spectrum may be useful to assess the likelihood of such a situation. Absorptions with very high extinction coefficients usually have extensive conjugation and longer wavelength absorption.

Can color-forming TLC reagents be useful to identify the functionality in the impurity?  If the impurity is separable by TLC one can often perform derivatizations on the TLC plate, which signal some functionality by a coloration of the spot or band. In contrast, this is not readily applicable to HPLC separation. 

Could you obtain a KBr/IR by concentrating a preparative plate sample on a triangle piece of KBr. Could you obtain a mass spectrum or ms/ms from a TLC or HPLC sample?

Does steam distillation help to decrease the impurity level?  If steam distillation reduces an impurity the most frequent conclusion is that the impurity was some solvent-like material.

Combining Yield Optimization with Impurity Identification

Performing simplex or other process step optimization can provide some conditions, which result in a dramatic increase in the impurity level. These conditions are unsuitable for optimizing but such a sample may be an easier mixture from which to purify the impurity.

Using Coloured Derivatizing Agents

Can colored derivatives be useful? Note you will not know whether the impurity of interest formed a derivative unless you know that it contained the prerequisite functional group.  The main component should not form the derivative.

Chromatography of colored derivatives is simpler because the experiment can be followed visually on the chromatographic plate or on a column whichever is used.

4-phenyl-azo-benzenesulfonic acid chloride has been used as a derivatizing agent for primary and secondary amines [R.D. Desai and C.V. Mehta, Indian J. Pharm. 13, 211 (1951).] Hydrolysis in conc. HCl-dioxane E.O. Woolfork, W.E. Reynods and J.L. Mason J. Org. Chem. 24, 1445 (1959).

4-phenyl-azo-benzoyl chloride is a derivatization for alcohols. [E.O. Woolfork, F-E. Beach and P. McPherson, J. Org. Chem. 20, 391 (1955).]

4-(4-nitrophenyl-azo)-benzoylchloride can be used to form derivatives from primary and secondary alcohols, amines and thiols. E. Hecker, Ber. 88, 1666(1955).

E.S. Amin and E. Hecker Ber. 89, 695 (1956).

A. Butenandt, T. Beckmann and E. Hecker, Z. Physiol. Chem.324, 71 (1961).

A. Butenandt, D. Stamm and E. Hecker, Ber. 94, 1931 (1961).

Because an impurity that moves closely with the main product probably has the same polar functionalities as the main compound, if one can make a colored derivative of the main composition, the unidentified impurity will also most likely form the same derivative. Now, however, a TLC chromatographic separation will much more easily show up the impurity and allow sensitive variation of the elution system to separate this small minor band. Swish TLC can also probably be usefully applied to the now colored, highly crystalline derivative of probably decreased solubility, and the impurity itself recovered by breaking apart the derivative.

Liquid-Liquid Extraction using Hydrotropes as an Alternative to Fractional Crystallization for Purification at Scale

How does one purify a mixture of structurally similar neutral compounds that is about 80% one isomer and 20% the other? If you adopt fractional crystallization the most likely outcome is that you do purify the major compound but the recovery is about 60%. The lost material is in the mother liquors in an approximate 50:50 w/w ratio with the minor constituent.

You could try liquid-liquid partition, even trying several of these in series resembling a rough counter-current extraction. The problem is that there aren’t that many liquid phases that are mutually immiscible and more frequently at least one component of any pair that is immiscible will exhibit poor solubility for most of the multifunctional organic compound mixtures that you want to separate. Yes- water and hydrocarbons are immiscible but neither one dissolves most organics well. Yes- acetonitrile and hydrocarbons are immiscible, but most organic mixtures do not partition competitively between them. Yes, hydrocarbons and perfluorocarbons are immiscible but again distribution between them is usually overwhelmingly into one or the other. Then there are less well-known ones such as MIBK/sulfolane which might be promising, but these are few.

What is needed is a way to modify water so that it has an increased capacity to dissolve organic compounds of interest while still remaining substantially immiscible with those common organic solvents which also have a good ability to dissolve a target mixture. This is what hydrotropes can do.

Two important strengths of the methodology: (i) the solubilization capacity of the hydrotrope is a strong function, usually exponential, of the hydrotrope concentration and 
(ii) mere dilution of the hydrotrope with water is enough to recover dissolved materials.

Triethylamine as Reaction Solvent and Workup Extractant

Triethylamine is more often thought of as a reactant or acid trap. It is inexpensive enough, however, to be considered for a role as a reaction solvent.

 Triethylamine has a critical solution temperature with water. Below 18°C they are miscible but immiscible above this temperature. Thus it is thermomorphic and this provides a potential for simplified isolations.

Triethylamine is likely to dissolve neutral or basic substrates which can deliver hydrogen bonds without actually causing proton transfer.  Alcohols, phenols, amides, N-hydroxyl amides, thioamides, primary and secondary amines, meet the criterion. 

As a solvent it could not be used in oxidizing environments because of the ease of forming an N-oxide or the loss of one of its lone pair electrons.

A reducing environment would not cause any problems. Its' use would be problematic in the presence of electrophiles since it would tend to compete to react with them. Halides, epoxides, etc. are incompatible. With acidic substrates it would be inclined to form salts. It should be compatible with organometallic agents and indeed may stabilize these.

Triethylamine has a boiling point of 90°C. It does not form explosive peroxides like diethyl or diisopropyl ethers. It can be expected to be close to diethylether in solution properties. It might be useful as an extraction solvent so long as the substrate being isolated is not electrophilic. In the same way that liquid ammonia can be a reaction solvent so could triethylamine.

Producing the most likely impurities of a Given Product for Use in Developing a Powerful Analytical Method

In order to develop a good purity analysis for an organic substance one needs to have some method to assess different methods. The better the method separates and quantifies more impurities from the product. A better method the more impurities it separates and the greater the degree of separation between the closest impurity and the product without losing some degree of separation for any single impurity. A better method separates distinctly even an impurity designed to have a very minor difference from the product. Is there a way to prepare a product sample with larger amounts of the most likely potential impurities?

There are two classes of impurities. Impurities that are product degradation derived; that is, they come from the desired product and arise from reactions upon the desired product after it has been isolated and purified. A different class of impurities are formed at the time of the synthesis of the desired product and which were not completely removed by the isolation and purification process performed before packaging of final product. These impurities are process characteristic. It is this second type that are considered here. These impurities are produced in greater or lesser amounts by variation in the continuous variables controlling the process.

A third group of impurities are created by changing the discontinuous variables of the process step, such as reagents, reagent purity, solvent, solvent purity, substrate purity, processing chemicals When the discontinuous variables are not altered this set of impurities do not appear and do not need to be further mentioned herein.

Temperature is the most significant continuous variable and it can produce the most substantial changes in chemical reactivity. If we are seeking a complete reaction it is likely that the transformation is self terminating and will essentially stop when the correct time has expired with very little occurring after this required reaction time. Increasing the temperature by 10 C according to a rule of thumb will double the rate of reaction. This will also allow competing reactions which are limited under the more preferred conditions to compete and produce by-products.

Thus an increase in the temperatures of each of the different stages of the reaction by 10 C and a decrease in the time by half in each stage should produce more impurities in the final product and these impurities should reflect realistic possible impurities.   If possible the extent of disappearance of starting material should be kept about the same.