KiloMentor Stresses the Importance of the Integrity of the Reactor at Scale

Laboratory equipment costs just a minuscule fraction of that of process equipment. For that reason, scientists can perform a reaction that requires strong aqueous alkali in a glass round-bottomed flask even though one knows that at the end of the reaction the flask will be opaque and etched by the dissolution of a portion of the glass itself. One the other hand, precautions must be taken that a large scale reactor, that is expected to have a long useful life should nor e partially dissolved or pitted or weakened by any reactor contents. A process development chemist must never put a large scale reactor at risk. Consideration should be paid early on that reaction conditions are not incompatible with the materials of construction. Engineers are particularly knowledgable in this area and can provide an early warning that particular conditions must be vetted. This is normally done in the laboratory by placing weighed tiles of reactor surface material into the laboratory reactor throughout the process step of concern and at its conclusion, these tiles are fished out and carefully reweighed. Any experimentally significant difference between before and after weighings is suggestive that teethe reaction conditions are eroding the reactor surface material, 

At the same time, the experiment will detect any unexpected effect of the reactor’s material on the course of the process's reaction.

Loss of the surface of the reactor can also be caused by abrasion. The surface is simply rubbed off and probably remains as fine insoluble particles inside the reactor. Very little can be done about this except togged away from the abrasive reagent. Sometimes this problem can be solved by packing the abrasive agent tightly into a special column-shaped reactor tube and rapidly circulating the reaction mixture solution through the column past the insoluble abrasive agent.

Loss of the reactor surface may simply be caused by excessive pH and this cane controlled by an adjustment in the reactor material itself.

Another cause is the use of or the creation of a very strong chelating agent which simply rips metal ions out of the reactor surface. I have encountered such a situation. I was able to overcome the corrosion simply by adding a stoichiometric quantity of an inorganic iron salt into the reactor with the rest of the reagents. As the chelator formed it complexed the iron cations and left the reactor alone!

Identifying Chemical Process Stopping Points for Working in the Kilolab or Pilot Plant

It is not as if there is no planning in the laboratory. If a synthetic lab procedure is so long that the reaction and workup cannot be completed in a single day, chemists can use their experience to extrapolate from similar procedures and guess at what points manipulations can be stopped and under what conditions intermediate solutions or crude solids can be stored without damage. Occasionally there are misjudgments and surprises and a product will be prepared in lower than expected yield or poorer purity. But then even in the worst situation what is lost is no more than a couple of man-days of labor and the price of the starting materials consumed. Also, in the laboratory, because the capacities of refrigerators, freezers, and evaporators are so much greater than the quantities of material being transformed, there are do-able fixes at almost any stage for the situation where a stoppage is forced.

There is no room for such risk-taking on-scale. For advanced intermediates that are themselves the product of a series of sequential steps, one misstep can be economically disastrous. The more points in the process that have been verified as safe-to-stop, by actual test results, the more confidently the process team can be. Moreover, to be a safe stopping point it must be proven safe not just for the quantity and quality of the product but also for the protection of the processing equipment.

As a general rule once a reaction has been initiated, the kinetics must be allowed to run undisturbed to the proper end-point according to the batch sheet. The dynamic transformations cannot be expected to respond to any speed up or slow down without some quantity or quality deviation. After the endpoint condition has been reached and the reaction quenched then the mixture is likely more stable and various stopping points during the work-up can be tested by holding portions of a process mixture for given periods under controlled conditions and examining the mixtures and isolating the product to see whether an unacceptable deviation has occurred or not.

It is more difficult to demonstrate a good stopping point where the mixture in the process equipment is heterogeneous. The difficulty is taking a representative sample for analysis out of a heterogeneous mixture to show that no change affecting quantity, quality, or the protection of the reactor has occurred.  Since one cannot easily take a precise fraction of a heterogeneous mixture, working up that fraction after a pause will not accurately tell you whether the yield would have been different.

Finally, to fairly test the stability of an aliquot at a proposed stopping point the aliquot must be left in contact with a sample of the reactor material. In my experience, this is rarely ever done. At the very least it should be kept in mind where an aliquot might be corrosive to the reactor material.

Getting Free from Dipolar Aprotic Solvents at Scale

Obtaining a Reaction Product Free from Polar Solvents: Efficient extraction of highly polar solvents from reaction mixtures.
following the late Dr. Phillip Hultin

 Dr. Philip Hultin was a professor at the University of Manitoba, Canada who passed away in 2018. Although I did not know this man, the following article, which I present with a few edits, is valuable for process chemists scaling up extractive work-ups. Searching Philip Hultin extraction in Google will provide the original unedited article.

The Problem of the Standard Practice

The most common workup for reactions conducted in DMF or DMSO is to drown out (dilute) with a larger volume of water, extract with a solvent such as ether or dichloromethane, and then wash the organic phases numerous times with water.  The shortcoming with this is that dipolar aprotic solvents also have significant solubility in organic solvents and they can also behave as phase-transfer agents drawing a significant amount of the desired reaction products into the aqueous layer.  The whole process can become very time-consuming with multiple extractions and back-washings.

A more effective way, in the laboratory setting, to remove these solvents is a series of separatory funnel extractions that somewhat mimic liquid-liquid partition chromatography.  Indeed, it is a counter-current extraction but wherein only one phase is moving.  The method retains all the lipophilic materials,  does not create large volumes of waste, and reduces the time required to achieve essentially complete separation of dipolar aprotic solvents such as DMF or DMSO from reaction mixtures.

The procedure uses one larger separatory funnel and three or four smaller ones. For laboratory-scale extractions it is convenient to have a rack that can hold all the separatory funnels in a row.  For “large scale” extractions where the first separatory funnel is larger than 250 mL capacity, stands with rings are more appropriate. 

The Laboratory Procedure

1.  After quenching the reaction and diluting with enough ether to easily dissolve the expected products everything is poured into the large funnel #1 and the reactor flask washed with a little ether. There, it is diluted gradually with a generous amount of water. As the water is added two phases start to separate. Doing the addition of liquids in this order may reduce or eliminate the amount of agitation needed to equilibrate the layers; furthermore, the effect of further small increments of water on the volume of upper and lower phases can be more easily assessed and this correlates with the partitioning of the dipolar aprotic solvent.
  2.  Into each of the remaining funnels #2 through #5 is placed a smaller portion of ether.
3.  Funnel #1 is shaken and allowed to settle. 
4.  The aqueous (lower) layer is run out into funnel #2. 
5.  Funnel #2 is shaken, and while it settles, an additional portion of water is poured into #1 and shaken.

6.  The aqueous layer from #2 is run off into #3, the aqueous from #1 is run into #2, and more water is added to #1. 
7.  All funnels are shaken and allowed to settle.

8.  The process of running the aqueous layers into the next funnel in sequence is repeated until all funnels have been shaken with water and the first aqueous portion resides at the bottom of funnel #5 having passed through each of the ether layers.
9.  It is then run off into the beaker. 
10. The remaining aqueous layers continue to move through the funnels and eventually into the beaker as well.

When this sequence is finished, all the ether solutions have been washed five times with water, and all the water washes have been back-extracted as well. If the remaining ether layers are analyzed by TLC, it will likely be observed that reaction products are in funnels #1 through #3 or maybe #4.  Ether layers that contain products are pooled and can be processed further or dried and evaporated.  In general, NMR analysis of the crude material will not show any sign of residual DMF or DMSO after this treatment.

What is happening here is this: when the quenched mixture is initially extracted in funnel #1, most of the polar solvent goes into the water layer.  Some products and ether are also partitioned into the water, and some polar solvent remains in the first ether layer.  The aqueous layer moves into funnel #2 where it encounters fresh ether.  This extracts products out of the water, and it may also take up some polar solvent.  The process is repeated in each funnel.

But now consider the subsequent water washes.  When the organic layer in funnel #1 is washed with water, the residual polar solvent is extracted.  The actual amount of this polar material is relatively small since most of it went off with the first portion of water.  Thus, much less if any of the desired products are partitioned into the water.  This wash moves through the subsequent ether layers, removing polar solvent from each.  Each successive water wash moves polar solvent forward through the funnels, but as the amount of polar solvent in the earlier funnels drops the ability of each water layer to remove the desired product is reduced too.  The result is essentially an “elution” of the polar solvent with “retention” of the less-polar organic products in the earlier ether layers.

In general no more than five water washes are needed although in certain situations more may be required.

The process can be adapted to the use of solvents that are heavier than water as well.  If the extraction solvent is dichloromethane, the stationary phase becomes the water.  Funnel #1 is charged with the aqueous quenched mixture and dichloromethane, and smaller portions of water are put into funnels #2 through #5.  Dichloromethane portions are passed in sequence through the funnels and collected in the beaker having had the polar solvent washed out.

Modification for Working at Scale

The laboratory procedure described above would be unworkable at scale because too many vessels are required. The goal should be to retain the benefit of multiple extractions and multiple backwashes while reducing the vessels to just two. This could perhaps be modified by using one organic water-immiscible solvent denser than water and one that is less dense with a final combining of these two and recovery of the more volatile by fractional distillation. Using dichloromethane as the more dense liquid and isopropyl acetate as the less dense one the procedure might look as follows:

1. Dilute the organic reaction mixture with 1 part of isopropyl acetate and 3 parts water. 2. Stir and separate the lower water into a stirred tank.
3. Backwash the water with 1 part methylene chloride and return this to the mix with the isopropyl acetate in the reactor vessel.
4. Discard the water plus dipolar aprotic solvent from the holding tank.
5. Remove the methylene chloride by distillation from the reactor.
6. Add 3 parts of water to the residual isopropyl acetate containing the desired organic products and stir and settle.
7. Draw off the water containing the remaining dipolar aprotic solvent.
The products are retained in isopropyl acetate in the reactor.

Choosing the Scale for Laboratory Project Management Directed towards Chemical Process Development

Deciding the scale at which laboratory work should be done is a project management determination not a scientific one. The proper answer depends upon the physical resources of the laboratory, the budget for the particular project, the scale required to meet the final objective and the time available to meet that objective.

Whether the economic unit is a business or an educational institution the most frequent size at which synthesis experiments are performed can be quickly gauged by looking at the most common sizes of reaction glassware in the drawers of people working at the lab bench. For research conducted in schools, small scale work is more typical because the cost of chemicals is a substantial part of the overall expense. There the cost of student labor is low so using more labor is not a hardship for the professor. An addition consideration that reinforces this tendency is that academic research often works on targets that are many steps away from commercial starting materials. These target materials are time consuming to prepare and so the objects upon which publishable experimentation are conducted are precious and need to be hoarded.

On the other hand, in company laboratories, where the object is to produce either processes for manufacturing or families of compounds for property testing and where the wages of the scientists are a substantial part of the overall cost, the cost of the chemicals is a smaller proportion and working at larger scale saves project time and budget. Again, this will be reflected in the size of standard equipment found in the laboratory.

Although it is most convenient to work at the normal average scale set by the laboratory facilities this can be trumped by a particular project’s requirements so long as that is reflected in the project’s budget. Where this might be true, a discussion of the scale at which different parts of the work are to be performed should take place with the project manager to avoid later misunderstandings.

 http://chemjobber.blogspot.com/2011/11/process-wednesday-rb-woodward-on-scale.html

What is more, when the final objective is to produce hundreds or thousands of kilograms, even more risk related to scale up differences would be introduced if one starts working with only milligram quantities. Besides, if the cost of starting material is so high as to limit the scale of experimentation to the milligram range, it is also quite likely to be too high for commercial implementation at all. Another consideration is that developing a process step requires that many test samples be taken during the run to follow the reaction and to assess the qualities of the intermediates. A non-micro scale of operations is required to allow for representative and meaningful sampling.

What Size Steps for a Process Scale Up?

Why not simply jump from the scale at which a process step was developed to the scale at which it is planned to operate commercially?   Risk of catastrophic failure is the answer. The near optimal conditions for operating in laboratory equipment can still be quite different with respect to a number of variables from what must be done in a pilot plant. Just for starters, some parameters such as heating, cooling, stirring and the times for reagent additions cannot be physically matched on scale because of equipment limitations. Also other surprises can occur as one increases the size of operations and these can lead to product of unacceptable properties. Perhaps one ought to ask instead, “How well have I been able to scale-down the pilot   plant environment and reproduce it in my laboratory equipment."

 ‘Scaling down’ is the exercise of selecting the laboratory scale equipment that can best model operating conditions and provide data for mathematical models that successfully simulate pilot or production scale operations. Risk can be reduced by performing appropriate testing on such equipment.. If the experimentation has been conducted using exactly the same quality for solvents, reagents, processing aids and catalysts, the biggest source of deviation in scale up is removed. 

If the processing times including times of addition, times for transfer and times for filtration are approximately the same as will be used in the pilot plant, risk is reduced. 

If the corrosiveness and abrasiveness of the reactants have been tested upon the reactor construction materials this reduces another risk.

 If the procedure is insensitive to the agitation rate over a wide range another sensitivity has been allowed for.

 If the sensitivity to traces of air and moisture is known and taken into consideration life is simpler.

Using Functionalized Polymers at Scale in Process Chemistry

Functionalized polymers can serve as scaffolds for process intermediates, as reagents, as co-reactants, as catalysts, or as a solvent phase; however, using polymers in process chemistry violates “atom economy” in a massive way. Using polymers in any capacity adds to the mass used without incorporating that mass into the product; therefore, using functionalized polymers must provide a large compensating benefit.

The compensating benefit could be:

In safety and regulatory affairs by avoiding

• smelly reagents like sulfides and thiols 
• explosive reagents such as aromatic peracids, sulfonyl azides
• toxic waste by immobilizing Cr, Sn, Se, Ni
• trace heavy metals that are avoided Ag
• reagents that are toxic: crown ethers, HMPA cosolvent, cryptates
• reagents that cause sensitization: carbodiimides

Avoiding normal small-molecule reagents that cause difficulties in work-up

• triphenylphosphine oxide
• ureas from carbodiimides
• emulsifiers
• phase transfer catalysts
• mineral or organic acids by replacement with cation-exchange resin
• mineral bases that introduce water-soluble alkali and alkali earth salts with anion-exchange resins

Avoiding reagent degradation (where the regular reagent is too unstable)

• Lewis acid impregnated microporous resin AlCl₃ impregnated into carbon
• chromic acid impregnated charcoal
• potassium impregnated graphite
• polymeric trityllithium

Polymeric Protection as a Phase Tag

• scavenger resins to remove residual excess reagent
• starting reagent so that an excess can be used
• capture and release purifications
• cosolvent extraction phase (macroreticular polystyrene)

Removal of Trace Components by selective reactivity

• removal of oxygen (example)
• removal of heavy metals (like using EDTA)
• removing singlet oxygen
• removal of water: carboxymethylcellulose sodium, butyrolactone 
• removal of organic solvents: molecular sieves
• removal of carbonyls: semicarbazide on silica; site isolation
• mono protection of symmetrical substrates
• telescoping process steps using two antagonistic reagents immobilized on separate resins such as periodic acid/ borohydride for first cleaving then reducing 1,2-diols

Recovery of Expensive Catalysts

Solvents

• polyethylene glycol as a solvent for sodium hydroxide or potassium hydroxide
• polyethylene glycol as a dispersing agent during solvent switches based on evaporation to dryness
• polyethylene glycol as distillation chaser

Because of the lack of atom economy to be cost-effective reactions using polymers as processing chemicals or reagents should be used in the latter portion of reaction sequences when small improvements in yield can produce overwhelming cost benefits.

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.

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.

Switching Solvent at Scale: Using a Minimal Stirrable Volume Chaser

Working at laboratory scale one can switch from a reaction solvent to a work-up/extracting/crystallizing solvent by evaporating the reacting solvent to dryness on the rotary evaporator adding the second solvent and scrapping and stirring the oily neat solid off the walls of the flask and back into solution. So long as consideration is given to not decompose the solutes there is no problem.

With a few kilograms, using a large rotovap containing polypropylene beads to trap solids, the same thing can be done. The free-flowing beads trap the solutes; then, they can be redissolved in the second solvent and the polypropylene beads filtered.

In the pilot plant removing polymeric beads from the reactor is not possible. Evaporation to dryness is not possible. A possible solution might be to add into the reactor an inert, high boiling fluid in a volume such that the total volume of the non-volatile solutes plus this fluid reached the minimum stirrable volume. Now the first solvent could be distilled out of the reactor completely because at the end there would remain the minimum stirrable amount of non-volatiles containing all the non-volatile reaction pot contents.

What should be the properties of a minimum stirrable volume chaser? Well if we are going to get it separated from the reactor components of interest by extraction it must be immiscible with some standard organic solvents. This suggests that if the reaction products of interest are at least moderately polar the volume chaser should be a high-boiling paraffin. Such a chaser would be immiscible with either methanol or acetonitrile. Polar or semi-polar compounds would easily be extracted out of the paraffin phase. The paraffin chaser could be saved, drummed off, and reused for repeats of the same reaction.

Traces of paraffin can be removed from methanol by forming the insoluble complex with urea. The complex and excess free urea would be filtered off from the methanol solution.

In the event that the desired reaction products are more nearly apolar, the volume chaser should be itself polar. Liquid polypropylene glycol or glycerol can be used. These will work because the desired reaction components as an oil in either of these can be separated by liquid-liquid extraction with any organic solvent that isn’t appreciably miscible with them. Traces of either polypropylene glycol or glycerol can be precipitated as complexes with anhydrous calcium chloride at an appropriate point in the work-up.

A final possibility is to use a chaser fluid that can be removed in some other way. A possibility of this type would be to use acetic anhydride bp. 140 C which would drive over many solvents that are not reactive with it and that can be subsequently hydrolyzed to acetic acid which can be removed in water followed by an aq. bicarbonate extraction.

In a similar way, quinoline could be used as a chaser then washed out as a salt in water and recovered by subsequent basification of the aqueous extract.