The demand for increased efficiency and speed in pharmaceutical analysis extends from drug discovery and development to clinical screening and trials. The need to meet these throughput requirements without compromising the data required in quality assurance–quality control (QA–QC) applications has highlighted the important technical challenge of providing faster separation in high-performance liquid chromatography (HPLC). This article will focus on the challenges in serial chromatography (i.e., one run at a time) on a single HPLC column. Alternatives such as parallel chromatography and advanced routines using multiple columns with switching valves are best addressed separately. 

The direct approach to increasing throughput is to raise the mobile-phase flow rate (linear velocity) to drive the peaks from the separation column faster. Increasing the mobile-phase flow rate in the widely used 5-μm particle-packed column, however, diminishes column efficiency. To counteract this loss in performance, one can consider two approaches: 					

Using smaller particle-size packing material 

Performing separations under elevated temperature.

The common goal of the two approaches is to reduce the height equivalent to theoretical plate (HETP) and, consequently, to increase the column performance per a given column length in an extended flow-rate region. One must consider several factors in both approaches to achieving ultrafast liquid chromatography (UFLC).  

Generally, columns using smaller particles provide good separation performance, are shorter, and also help to maintain column efficiency as the flow rate is increased. The columns' efficiency is highest at a particular mobile-phase flow rate, but at greater and lesser flow rates, efficiency decreases (HETP increases). This relationship is expressed using the following van Deemter equation: 

 is the HETP (column length required to obtain one theoretical plate—the smaller the 

 is the mobile-phase linear velocity, and 

 is coefficient of mass transfer. The usefulness of smaller packing material with a microsphere-packed column for performing high-speed separation is attributed to the column's efficiency at high flow rates. This effect is demonstrated by the van Deemter curves for a compound that is run on a series of HPLC columns packed with particles of various sizes. As shown in Figure 1, the HETP remains small over a wider flow-rate range if smaller particle-size packing material is used. 

Figure 1: Van Deemter plots of different particle-size packing materials. 			

The plots indicate that smaller particle-size packing material makes it possible to achieve great column efficiency at a high flow rate, thus enabling good, quick separations. One can increase the flow rate of a method developed to meet certain resolution criteria without losing resolution, thereby decreasing the run time. 

It is worth making a few observations about the van Deemter plot. First, the plot only applies to isocratic separations because no term in the equation takes changing mobile-phase strength into consideration. Second, the size, shape, and polarity of the analyte influence the observed van Deemter plot. Small, nonpolar molecules behave as shown in Figure 1. As more complexity and polarity are added to the molecule, however, a reduced region of minimized HETP may be observed. This effect results from the behavior of an analyte on the column, which depends on many different interactions that the van Deemter equation does not consider. 

The downside to using a sub-2-μm-particle column is increased system pressure, which requires a limited column length to remain within the capabilities of the available hardware in the laboratory. This restriction occurs because the pressure arising from the column is in an inverse-square relationship to the particle diameter. Reducing the particle diameter by half results in a fourfold increase in observed pressure. This pressure relationship makes it difficult to use small-particle columns under a high (i.e., optimum) flow-rate range or choose a column long enough to achieve the desired resolution. 

In response to this difficulty, some companies have developed specialized instruments with improved pressure tolerances. These instruments, however, have their own limitations when it comes to the accepted levels of performance expected from HPLC analysis with respect to injection reproducibility and gradient accuracy. In addition, these systems may have a higher cost of ownership because of the extreme conditions under which they operate and the resulting wear and tear on consumable components such as pump seals and injection-valve rotors. The question then arises: Is it really necessary or beneficial to use a sub 2-μm particle to increase the speed when the back pressure increases so much? 

The authors suggest that using a well-packed column with uniform silica gel-based materials of a mid-size diameter is the proper solution (1). Mid-size particles are between 2 and 3 μm. Sub 2-μm particles are defined as small. This may seem like an academic difference, but recall the inverse-square relationship of particle size to pressure. Mid-size particles allow users to achieve high speed and high resolution simultaneously on existing equipment. Consider that the performance difference between a 2.2-μm column, for instance, and a 1.8-μm column is minimal, but the expected back pressure with the 2.2-μm particle column is less than two-thirds that of the sub-2-μm particle, meaning the highest performance level is achieved without sacrificing operability. 

Furthermore, in some cases, small-particle columns, owing to their shorter length, may not be suitable for separating closely eluting peaks. If resolution is critical, the natural strategy is to use a longer column. With a small-particle column, it may be impossible to use a long column because the back pressure exceeds the allowed operating pressure, regardless of the system capability because every system has a limit. As a result, it is necessary to choose either a lower flow rate (thus eliminating the advantage of going to a smaller particle) or to switch to a larger particle size and longer column to reduce the back pressure. 

The time needed to perform the separation is longer, so a balance must be struck between time and resolution. This problem demonstrates that one cannot necessarily have an ideal separation. The separation must be optimized for each individual's needs—the highest speed and the ultimate resolution may not always be possible in the same run. Columns with mid-size particles have proven to be optimal for balancing the time reduction and high resolution required at the highest levels. It is more useful if the same or better separation with a shorter run time can be achieved with lower back pressure.  

Interest in elevated-temperature liquid chromatography (LC) is increasing because better and faster separation can be obtained simultaneously by using higher temperatures. Separation performed at increased temperature has an effect similar to that of reducing particle size. Both techniques accelerate the mass diffusion of the analyte between the stationary phase and mobile phase, thus resulting in a "flatter" van Deemter curve. 

 of the van Deemter equation decrease as temperature increases and result in a lower HETP. High-temperature HPLC also reduces the viscosity of the mobile phase, thus lowering the resistance to flow through the column and allowing greater flow rates compared with ambient conditions. The resistance to column flow is inversely proportional to temperature, such that the column pressure at 80 °C is about 40% lower than that at 40 °C. This relationship highlights the importance of temperature as a factor in accelerating separation. Of course, increasing the temperature of the column raises questions of itself. 

One must consider not only the stability of the analyte, but that of the packing material as well. Much progress on this front has been made in recent years, resulting in longer column life and more selectivity. As long as the packing material is stable under the elevated temperature, this technique is quite effective for getting higher resolution and higher speed without making many changes to existing equipment. 

In successful elevated-temperature HPLC, the column environment is critical because a temperature gradient in the column can distort the peak shape. A forced-air column oven is required to support this application. These ovens are powerful enough to eliminate the need for a column preheater, which would increase the delay volume of the system and negate many advantages gained from elevated temperatures. These preheaters or loops of tubing are necessary for typical block-heating-type ovens. 

Demands for faster analysis times will multiply as researchers attempt to increase sample throughput and maintain data quality. Once the separation is optimized for the highest possible speed and resolution, it is necessary to consider the rest of the HPLC system and its contribution to the total analysis time. Engineers are developing HPLC equipment to work in conjunction with high-speed columns with the goal of faster analysis in mind. 

 The first part of this article dealt with columns, and many examples of fast analyses are available in the literature. Figure 2 shows a comparison between a typical isocratic separation performed on a 5-B5m particle and a mid-size (2.2-μm) particle. Figure 2 shows that the run time was shortened by a factor of 10 with no loss in resolution. Mid-size silica gel-based material reduces column-flow resistance compared with that of a sub-2 μm particle column, thereby allowing high-speed analysis with conventional HPLC systems. Since these HPLC columns achieve a good balance between separation efficiency and pressure, resolution performance is maintained as in a general-purpose column (4.6 mmi.d. × 150 mm, 5 μm), and analysis time is greatly shortened. 

Figure 2: Comparison of Shim-pack VP-ODS with conventional 5-Î¼m particle column and the Shim-pack XR-ODS with 2.2-Î¼m particles. Chromatographic conditions: Mobile phase: waterâacetonitrile (40/60, v/v); XR-ODS: Flow rate: 2.0 mL/min, Temperature: 60 Â°C; VP-ODS: 1.0 mL/min, 50 Â°C; Detection: absorbance at 210 nm; Peaks: fluorene (1), phenanthrene (2), anthracene (3), fluoranthene (4), pyrene (5), chrysene (6), benzo(b)fluoranthene (7), benzo(k)fluoranthene (8), benzo(g,h,i)perylene (9). A Shimadzu Prominence high-performance liquid chromatography system was used. 			

 High-throughput analysis requires reducing the total cycle time, which is defined as the separation, injection interval, and column equilibration. New autosamplers provide some of the fastest injection cycles (10 s per 10-μL injection) and allow users to successfully suppress carryover by careful evaluation of all contact materials in the flow path. When seeking short run times for an assay, the system speed becomes even more critical. Figure 3 shows a separation of different xanthine derivatives completed in 1.2 min using a conventional Shimadzu Prominence HPLC unit optimized for UFLC by reducing the tubing diameter and eliminating as much system-delay volume as possible. At such run times, autosampler speed begins to play an important role. 

Figure 3: Ultrafast liquid chromatography (UFLC) chromatogram of xanthine derivatives (10 Î¼g/mL): xanthine (1), theobromine (2), theophylline (3), caffeine (4). Instrument: Shimadzu Prominence UFLC. Column: Shim-pack XR-ODS (3.0 mmI.D. Ã 50 mmL, 2.2 Î¼m); Mobile Phase: 20 mM (sodium) phosphate buffer (pH 2.6) for A and acetonitrile for B (70/30), B concentration 10% initial to 85% at 1.2 min. 			

 Although speed is important, one also must consider data integrity. Many discussions about UFLC focus on data reproducibility. These discussions arise because the specialized systems designed to tolerate the high pressures that sub-2-μm particle columns endure may exhibit decreased system reproducibility, whether resulting from premature consumable wear or concessions made in the design to accommodate the required pressure tolerance. A few possible causes of poor reproducibility merit examination. 

 The peak widths seen in fast HPLC are extremely narrow. Narrow peak widths require a detector capable of sampling at a speed that ensures enough points to achieve proper integration. If the sampling rate is too slow, one may miss the apex of the peak or get sporadic noise in the baseline that contributes to poor peak integration. Many available systems are capable of this sort of sampling if the user sets them up properly. 

 Carryover has gained a great deal of attention in recent years with the widespread adoption and increased sensitivity of current detectors, especially mass spectrometers. As run times get faster, many people try to accelerate them by using advanced injection routines. Taking advantage of these routines is acceptable as long as cross contamination is reduced. For example, with a flow-through injection mechanism (needle-in-the-flow path), one may take the sample loop off line to begin preparing the next sample. On the other hand, if the loop is not sufficiently flushed or cleaned before it is removed, carryover and poor injection relative standard deviations (RSDs) will result.  

Reduced carryover is an important factor for UFLC, but combating sample carryover may increase analysis cycle time if additional autosampler needle rinsing is required. Rinsing, cleaning, and flushing takes time, so it is important to choose a design that also minimizes the effects of carryover in other ways. 

 Gradient accuracy and precision also influence system reproducibility. If the gradient controller is not sufficiently precise, the gradient curve varies slightly from run to run. It also may cause distorted peak shapes or resolution fluctuation with closely eluting peak pairs, resulting in poor integration. 

No HPLC–UFLC system compensates for poor technique. As users push the limits of their systems, they must bear chromatography basics in mind. Cutting back on postgradient column equilibration time to shorten the analysis cycle affects system performance. Using a small or inefficient mixer for mobile-phase mixing can result in poor data quality. Small-particle-size columns and tubing entail the need for rigor in mobile phase and sample preparation to prevent clogging. Filtering both the mobile phase and samples is no longer an option, but a requirement. A good quality sample sealing mechanism (e.g., vial septa, microtiter plate covers) is critical to achieving optimum autosampler performance for injection accuracy and carryover prevention. A little time spent reviewing the basics goes a long way toward ensuring the collection of reliable data and results in an HPLC–UFLC system that performs well and reliably. The following examples illustrate these concepts. 

Table I shows the system reproducibility of the xanthine separation. The retention-time RSD performance demonstrates that the system's pumps deliver a consistent and reproducible gradient. This feature allows the user to create a specific retention-time window in which to look for this particular peak, thus aiding in identification. The peak-area RSD shows that the autosampler provides a reproducible injection and also suggests that carryover is not a problem. 

Table I: Retention time and peak-area reproducibility of xanthine derivatives.			

Figure 4 and Table II show a paraben separation completed in less than 1 min. The gradient in this separation is from 5% to 95% 

 in just over 20 s. Note that it is possible to maintain data integrity while achieving this kind of performance. 

Table II: Retention time and peak-area reproducibility of paraben mixture.			

 Today's HPLC systems are used for many applications, including UFLC, traditional HPLC, QA–QC work, mass-spectrometry front end, multidimensional chromatography, on-line sample cleanup, and setup as column-switching systems for method development. Some of these techniques may require a special system to achieve the optimum performance for the method (e.g., a bioinert system). 

Figure 4: Paraben separation completed in ,

Articles

The authors review methods for reducing analysis time and increasing throughput that are reliable and maintain data integrity.

Achieving Ultrafast Liquid Chromatography

Although the first enantiomeric separations were carried out during the early past century, it was not until 1973 that the first generally useful chiral stationary phase (CSP), cellulose triacetate, was developed. This phase was used primarily for preparative separations until the work of Professor Pirkle in the United States and Professor Okamoto in Japan made analytical CSPs readily available. Although the Pirkle phases provided good separations, the polysaccharide-based CSPs developed by Okamoto had very wide applicability.  

It was subsequently found that the majority of enantiomeric separations could be achieved using just four different derivatives—two based on cellulose and two based on amylose. These polysaccharide phases were prepared by first derivatizing native amylose or cellulose and then coating these chiral polymers on a silica-gel base. This preparation method led to phase instability to certain organic solvents in which the polymers are partially or fully soluble. 

Many methods have been developed to allow the immobilization or chemical bonding of the polysaccharide-based polymers to the silica support (1). Methods include cross-linking the polymer by use of diisocyanates (2), unsaturated groups that are subsequently polymerized (3, 4), silyl derivatives (5), direct bonding of the polysaccharide to silica by enzymatic or chemical routes before derivatization (6), and cross-linking of the chiral polymer by photochemical or free-radical reactions (7). All of these methods produce somewhat different products with different chromatographic properties. 

Immobilization of the chiral polymer to the silica support has several benefits. The CSP is not destroyed on contact with a solvent that swells or dissolves (even partially) the polymer. The sensitivity of the first-generation CSPs to even small concentrations of such solvents often required the use of dedicated equipment to eliminate all chances of accidental introduction of a "forbidden" solvent.  

Immobilization of the chiral polymer thus brings a new generation of polysaccharide-based CSPs into line with the stability of other high-performance liquid chromatography (HPLC) phases (such as C

 and CN), which means a significant extension of the column lifetime and the elimination of sudden column failure because of an accident. Others benefits of the immobilization process are that otherwise difficult separations can be achieved by using solvents that previously could not be used with the coated phases and that new selectivity can be developed by using previously unusable chiral selectors. 

The 3,5-dimethylphenylcarbamates of amylose and cellulose are the most useful polysaccharide-based CSPs. They are commercialized as "Chiralpak IA" and "Chiralpak IB," respectively (Daicel Chemical Industries, Osaka, Japan) (see Figure 1). Other polysaccharide-based chiral selectors include the 3,5-dichlorophenylcarbamate of cellulose, which was first introduced by Okamoto as a coated CSP. Although it had limited utility because of its solubility in hexane-based mobile phases, it demonstrated sufficiently interesting selectivity when used with pure alcohol mobile phases to be of interest as an immobilized phase. Daicel introduced a CSP of similar structure, immobilized by a proprietary technology, as "Chiralpak IC" (see Figure 1). 

 Figure 1: Structures of the immobilized chiral stationary phases.			

Immobilizing otherwise unstable chiral selectors has great advantages, including the ability to separate enantiomers that cannot be resolved on existing CSPs. The unique structure of Chiralpak IC often allows separations of different selectivity from other polysaccharide-based CSPs. A combination of Chiralpak IC with Chiralpak  IA and IB is replacing the older generation coated phases. The three immobilized columns are complementary, and a separation can sometimes be found on only one column. Figure 2 shows the example of prilocaine where the enantiomers are poorly separated with Chiralpak IA and IB but are well resolved with Chiralpak IC (8). 

Figure 2: Separation of the enantiomers of prilocaine. Green is Chiralpak IA, blue is Chiralpak IB, and red is Chiralpak IC. Mobile phase is hexaneâ2-propanol 8:2; flow rate = 1 mL/min; column dimensions = 250 Ã 4.6 mm.			

Immobilization of the chiral polymer increases solvent stability, which allows modification of the selectivity of chiral separations by the modulation of the solvent composition and its components. Figure 3 shows the exploitation of the mobile-phase constituents. Neither of the conventional mobile phases (hexane–2-propanol or hexane–ethanol) gave good resolution of the enantiomers of indoprofen using Chiralpak IC (8). Dichloromethane-based mobile phase improved separation in a significantly shorter time. 

Figure 3: Separation of indoprofen enantiomers. Column was Chiralpak IC, 250 Ã 4.6 mm. All mobile phases contained 0.1% trifluoroacetic acid. Flow rate = 1 mL/min. Green is 30% ethanol in hexane; blue is 35% 2-propanol in hexane; and red is 2% methanol in dichloromethane.			

The restrictions on solvents that applied to the early generation of coated polysaccharide CSPs made the development of separation methods using them relatively straightforward. One screened with four mobile phase mixtures—hexane:ethanol, hexane:2-propanol, acetonitrile, and 1:1 ethanol:methanol. Following this, the optimization also was simple because there were either "hits" or not, and the opportunity for modifying the separations achieved were limited. This limitation is an important advantage of these CSPs because the vast majority of separations can be found using these four solvents and only four CSPs—the tris-(3,5-dimethylphenylcarbamate) of cellulose and of amylose, cellulose tris(4-methylbenzoate), and amylose tris(

Using immobilized CSPs removes the restrictions of solvents to the extent where any organic solvent with a reasonable viscosity is now accessible. This is a tremendous advantage in that it allows optimization of separations using solvent selectivity effects not available for methods using the coated CSPs. Nonetheless, this also may complicate the development of separation methods simply because of the bewildering array of solvents from which to choose.  

To simplify the task, statistical studies have been conducted to assist in solvent selection and reduce the number that should be tried to a reasonable few while retaining maximum resolution and selectivity. Of course, such statistical guidelines must be based upon a wide range of applications to ensure their relevance, and there will always be some exceptions.  

Two main sets of data, each based on the separation of a different set of 70 compounds, have been developed. Each set was screened using a wide range of solvents, and the success rates arising from each were determined. 

Two measurements of success were used. The first was the number of separations with resolution >1.5 (i.e., baseline separations). The second was the number of separations for which at least a partial separation was attained. Although neither metric is perfect, they can be used to estimate the relative success of separation approaches. Table I shows the results from the first set of data using the three immobilized CSPs available (8). Solvents were the conventional hexane–ethanol and hexane–2-propanol mixtures together with mixtures based on dichloromethane (DCM), methyl 

-butyl ether (MTBE) and tetrahydrofuran (THF). These latter solvents were used either pure or with an admixture of hexane and/or alcohols to adjust the solvent strength to allow elution in a reasonable time. 

Although no one solvent system showed selectivity for all compounds, it is clear that a suitable combination of them can maximize the success rate (see Table I). Based on these data, one can make recommendations on a set of initial screening solvents that would give a high probability of finding separation conditions (see Table II). 

Table I: Success rates using various screening mobile phases.*			

In the second study, the separations were not optimized but were simply screened with a range of solvents. In this study, again with 70 solutes, 61.4% were baseline separated, and 94.3% of all chromatograms showed a selectivity greater than 1. Fourteen percent of the baseline separations required use of the extended range solvents (MTBE, DCM, THF etc.).  

Table II: Proposed screening solvents*			

Chiral separations are generally simple, involving the separation of a pair of enantiomers. For this reason, elution gradients are rarely used for enantioselective chromatography. This approach is somewhat different from achiral separations where elution gradients are frequently used.  

There are, however, some solvent strength issues, especially for some extended range solvents. Although for many compounds the solvent composition of Table II will suffice, some solutes will either not be retained or will not elute under these basic screening conditions. Thus, before starting the screening, one should establish a suitable solvent composition using several isocratic experiments involving various mixtures of solvents or with a gradient. 

Figure 4: Overlay of gradient runs. Column:  Chiralpak IC, 250 Ã 4.6 mm. Mobile phase: A = 30% dichloromethane in hexane; B = 10% methanol in dichloromethane. Both A and B contained 0.1 % diethylamine. Gradient: 0â100% B in 30 min; hold at 100% B 10 min. Flow rate = 1.5 mL/min. UV detection at 250 nm. Blue = Mianserin, Red = Methocarbamol, Green = Sulpiride.			

Figure 4 shows an overlay of three gradient runs with three compounds, and Figure 5 shows the final isocratic separations. The gradient was chosen to run from the least-polar solvent mixture (hexane containing dichloro-methane) to the most polar (dichloro-methane containing methanol). A clear correlation exists between the elution time in the gradient and the solvent composition that allows elution in a reasonable time. Of course, this is not only applicable to methylene chloride as a mobile phase but also can be used with the other solvent systems. Such gradients are made possible only by the immobilization of the CSP. 

Figure 5: Isocratic separations arising from the separations in Figure 3. Top is mianserin, mobile  phase is 20% dichloromethane in hexane. Middle is methocarbamol, mobile phase is 1:1 hexane:dichloromethane containing 2% methanol. Bottom is sulpiride, mobile phase = 10% methanol in dichloromethane. Other conditions are the same as those for Figure 3.			

Supercritical fluid chromatography (SFC) is important in enantioselective separations, especially for the preparative purification and isolation of individual enantiomers. Mobile phases based on alcohols (methanol, 2-propanol) or acetonitrile have been used. Of these, the alcohol-based solvents have been the most useful. Acetonitrile is a much weaker solvent in SFC than it is in HPLC and is often used with added alcohol to improve its elution power. The major problem with existing solvents in preparative separations is that many compounds have limited solubility in alcohols and are better soluble in solvents of intermediate polarity such as chloroform or THF. The sample in this case may precipitate when it is mixed with the mobile phase, thus blocking the inlet frit of the column. Under certain conditions, some coated polysaccharide stationary phases may be stable to small amounts of, for example, chlorinated solvents in CO

. But problems arise if the column depressurizes during operation. The immobilized chiral selectors are not affected by these considerations and allow any organic solvent as SFC cosolvent, thus eliminating solubility problems.  

Selectivity in SFC follows much the same trends as in HPLC, although as noted, the lower solvent strength of aprotic solvents requires the addition of alcohols for the more-polar solutes, not only to ensure rapid elution but also obtain good peak shapes. Figure 6 illustrates the effect on peak shape and retention of 1,2,3,4-tetrahydro-1-naphthol by the addition of 5% methanol to the THF cosolvent. Compounds containing polar groups appear to tail in aprotic solvents. Addition of methanol (or other alcohol) removes this effect. 

Figure 6: Influence of methanol additive in SFC separation of 1,2,3,4-tetrahydro-1-naphthol enantiomers. Column is Chiralpak IA, 250 Ã 4.6 mm. Mobile phase for (a) is 25% THF in CO2 and for (b) is 25% of 5% methanol in THF in CO2. Flow rate = 4 mL/min, back pressure = 150 bar, and temperature = 35Â°C.			

Separation method development in SFC usually is carried out by investigating the selectivity derived from alcohol–CO

 mixtures. This task may be modified by using suitable additives such as ethanesulphonic acid (9), trifluoroacetic acid, or diethylamine (see Figure 7). Sulfinpyrazone is not eluted from the Chiralpak IC column with CO

–2-propanol as a mobile phase. Addition of 0.5% ethanesulfonic acid to the cosolvent allows elution. 

Figure 7: SFC separation of sulfinpyrazone enantiomers. Column is Chiralpak IC, 250 Ã 4.6 mm. Mobile phase is CO2 containing 20% of  0.5% ethanesulfonic acid in 2-propanol. Flow rate = 5 mL/min. Back pressure = 100 bar, Temperature = 35Â°C.			

Once the alcohols have been investigated, other solvents may be tried. The most useful to date are THF and methyl acetate, both with an admixture of methanol to adjust the solvent strength. This approach gave at least partial separations for 92% of a 38 sample set. This work continues with the study of additional cosolvents, particularly MTBE and the chlorinated solvents. 

An important goal in the industry is to bring products quickly to clinical trial. Enantioselective chromatography is used extensively in this activity because it is usually much faster to develop a chromatographic separation that can be applicable to the quantities required for discovery, toxicology, and early Phase 1 testing than it is to develop a crystallization, an enzymatic resolution or, especially, an enantiospecific chemical route. The most accepted stationary phases for this activity are the polysaccharide-based CSPs because they combine the widest range of selectivity with good loading characteristics (10). 

The biggest challenge in the development of a preparative separation is to find suitable solubility. Many compounds show very little solubility in hexane-containing mobile phases and even where polar solvents such as methanol or acetonitrile can be used, the solubility of many products remains low. Thus, a major need for preparative chromatography is access to the solvents in which most samples are well soluble. Several separations involved adding chloroform or methylene chloride to the sample, and sometimes the mobile phase, to enhance sample solubility. Such operations are fraught with peril because the coated polysaccharide CSPs can be damaged very easily by such mobile phases. With immobilized CSPs, this problem disappears, and in a number of separations with HPLC and SFC, chlorocarbon-based mobile phases were used with great success. 

Studying the effects of solubility, however, we found other influences of the mobile phase on preparative separations. The effects of the nature of the mobile phase on the separation of α-methyl-α-phenylsuccinimide enantiomers were studied with a view to establishing the highest production rate for HPLC and SMB separations. Screening against a number of possible mobile phases gave the solubility, retention, and selectivity shown in Table III, which shows considerable variations in the values among the solvents.  

Table III: Solubity, retention, selectivity, and productivity			

Loading studies, which involve the injection of a range of sample loads to evaluate the isotherms of adsorption of the solutes, demonstrated major differences among the solvents. The adsorption behavior of the α-methyl-α-phenylsuccinimide enantiomers depended considerably on the nature of the mobile phase. In some cases (chloroform and acetonitrile/2-propanol), the separation collapsed rapidly on loading with the second peak merging with the first at relatively low load. In the case of MTBE, the second component peak moved to longer retention time at moderate load and only began to merge with the first eluting component at high loadings. The high loading capacity in this system led to high productivity for the separations, both in HPLC and SMB mode. The productivity of the separations (in kilograms of enantiomer produced per kilogram of CSP per day) is shown in Table III for each of the mobile phases.  

Separation in ethyl acetate also had a high loading capacity, which, combined with the high solubility in this solvent, resulted in the highest production rate for both HPLC and SMB. Thus, solubility is only part of the answer for preparative separations. The effect of the solvent on the loading capacity, presumably through its interactions with the stationary phase, is of great importance in preparative separations. This result has been observed only by virtue of the solvent stability of the immobilized polysaccharide-based CSPs. 

The new generation of CSPs, based on immobilized polysaccharides, has proven to be versatile in development of enantioselective separation methods, requiring relatively few stationary and mobile phases for success. The range of selectivity of the CSPs is extended by using solvents hitherto forbidden for the older generation of coated polysaccharide stationary phases not only for HPLC separations but also for SFC methods. Furthermore, the nature of the solvents in preparative separations may have a profound influence on both the solubility of the samples and on the loading capacity of the preparative columns. The extended range of solvents now accessible with the availability of immobilized CSPs extends the potential of preparative enantioselective chromatography to new and larger-scale separations. 

is vice-president of technology at Chiral Technologies, 800 N. Five Points Rd., West Chester, PA 19380, tel. 610.594.2100, fax 610.594.2325, 

 1. T. Zhang and P. Franco, "Analytical and Preparative Potential of Immobilized Polysaccharide-Derived Chiral Stationary Phases" in 

 G. Subramanian, Ed. (Wiley-VCH, Weinheim, 2007). 

 2. Y. Okamoto et al., ""Chiral Stationary Phases for HPLC: Cellulose Tris (3,5-dimethyl-phenylcarbamate) and Tris (3,5-dichlorophenylcarbamate) Chemically Bonded to Silica Gel," 

Polysaccharide-based chiral stationary phases have been developed that comprise chiral selectors immobilized on their support rather than being physically coated. These materials are completely solvent stable, thereby increasing selectivity and and enabling the development of new chiral selectors that have been too unstable in a coated form for general use.

Polysaccharide-Based Chiral Chromatography Media in HPLC and SFC

By itself, supercritical fluid chromatography (SFC) is a well-known technique in pharmaceutical laboratories. Combined with chiral separation media, however, the technique can prove to be a powerful tool for reducing early-phase development costs and time. Although SFC chiral chromatography can be used at a process scale, its most typical application involves separating 50–100 g of materials for toxicology studies during the discovery and preclinical stages (see Figure 1).  

Regis Technologies manufactures chiral stationary phases and performs SFC custom separation work using chiral stationary phases under good manufacturing practices (GMP). 

Figure 1: Preparative separation using supercritical fluid chromatography.			

"We work with a lot of companies that are just starting in their own process and looking for drugs to market," says Francis Mannerino, director of chromatography at Regis Technologies (Morton Grove, IL). "We see compounds in their earliest stage and a lot of them have difficulties in their purification. If they have a chiral separation built in, we try to marry the custom synthesis with the separation work or the purification work."  

The method development for SFC chiral chromatography is similar to that for a typical liquid chromatography (LC) technique. SFC chiral chromatography uses the same columns that are used in LC and the same, readily available stationary phases (see Figure 2). Only the carrier is different. LC requires the use of 100% solvent, such as hexane–isopropyl alcohol (IPA) mobile phase. In SFC chiral chromatography, CO

 liquid is the main carrier with a 5–10% cosolvent added with it. Most cosolvents are hexane, IPA, methanol, and ethanol. About 95% of the separations by LC also can be separated with the SFC chiral method and, in some cases, the resolution can be improved over traditional LC results.   

Figure 2: Scientist using an SFC preparative instrument in the Regis Separations laboratory.			

According to Mannerino, there are two main business advantages of SFC chiral chromatography: separations can be completed at least five times faster than separations using traditional LC and the technique uses about one-tenth of the amount of solvent, which reduces process costs and produces less hazardous waste.  

"What it comes down to is speed. Getting your drug to toxicology testing faster. If you're going to fail, fail fast. Take your resources and put them into another project," says Mannerino. 

As the first company performing SFC chiral separations under GMP, Regis faced several challenges. "For any new technique, qualifying for GMP is very trying,"says Mannerino. Fortunately, the company manufactures active pharmaceutical ingredients and finished-drug products under GMP conditions, so there were quality systems already in place. "We just needed to make the capability GMP, so it was about 40 or 50 new standard operating procedures, and we customized them for SFC equipment and personnel." 

After about 10 months and several dry runs, the company gained GMP qualification. "Our challenge right now on the research side is improving productivity. There are no precedents, so we are developing different methods such as putting columns in series and using different phases in series," says Mannerino. 

Increasing productivity is crucial for shortening cycle time. SFC separation is a continuous process. Once automated systems are developed and the technique is optimized, machines can run all day. "If you want a technique for commercial use, you have to optimize it as much as you can. That's the biggest challenge now:increasing productivity by doing the research," says Mannerino. 

Productivity will play a big role in future advances in SFC chiral methods. Much of the problem in progressing the technique stems from the fact that a lot of SCF work so far has been conducted with proprietary compounds and information is not available.  

Regis scientists are conducting studies involving different chiral phases from two manufacturers, with one column in a series being used for mixing. "I think the biggest improvements will come in the machinery. Advances in the machinery and feedback from companies will move the technology forward," says Mannerino.  "When a manufacturer does research, it a completely different story than when it is done in other settings. We are in constant communication with equipment manufacturers." 

As with any chromatographic technique, continued compliance with US Food and Drug Administration regulations is an issue. For this reason, the approval of SFC chiral methods is another important factor in its success. "A validated synthesis will always be favorable over a chromatographic technique, even when it doesn't make sense financially or scientifically,"says Mannerino. Perhaps the greatest obstacle, however, is convincing high-level personnel. "It's just fighting old-school thinking,"he says. have a lot of upper management in companies that didn't grow up with advanced chromatography techniques, and they didn't go to school or internships with it. There are so many variables that you can't explain in chromatography, and so in their eyes it's not a reliable technique. Many organic chemists won't buy into it. It's changing slowly." 

This article is based on an interview with 

director of chromatography at Regis Technologies (Morton Grove, IL).

The combination of supercritical fluid chromatography with chiral separation media offers several analytival advantages over traditional liquid chromatography techniques.

Supercritical Fluid Chiral Separations

Ultrahigh pressure liquid chromatography (UHPLC) has altered liquid separation procedures, but it has not changed the fundamental theory. Although UHPLC enables the use of sub-2-μm high-performance liquid chromatography packing materials, it is beneficial for a practicing chromatographer to consider the overall benefits and limitations of this technology. The author discusses the principles of chromatographic separations and explains how specific parameters such as stationary-phase selectivity and efficiency affect chromatographic resolution.  

Since the late 1960s, when modern  HPLC became a viable tool for practicing chemists, continual advancements have been made in the technology. Considering analytical-scale HPLC columns in particular, over time there has been a movement from irregular to spherical particles, as well as an overall decrease in the size of the particles used in the packing material. For spherical silica particles, this shift has been from 10 μm to 5 μm, then to 3 μm, and most recently to sub-2-μm particles. When using a smaller particle-size packing material, it is possible to increase the efficiency, or number of theoretical plates, and expand the range of usable flow rates.  

However, this technique results in proportionately higher back pressure as liquid mobile phase is forced through a much more tightly packed bed. Conventional HPLC systems are capable of handling pressures as high as 5000 psi, which until recently limited the usable particle size to approximately 3 μm, depending on flow rate, mobile phase composition, and column temperature. The advent of UHPLC, which uses instrumentation capable of handling back pressures greater than 14,000 psi, makes using columns packed with sub-2-μm packings possible. With UHPLC, extremely fast and efficient liquid separations are possible.  

Figure 1: The resolution equation indicates that selectivity has the greatest influence on resolution.			

The overall goal in HPLC, and now UHPLC, is chromatographic resolution, whether between target analytes or between an analyte and the sample matrix. Considering, as a guideline, the fundamental relationships given in the resolution equation (see Figure 1), one can better understand the process of resolving mixtures. The resolution equation comprises three terms:  selectivity (α – 1),

) Each of these terms is affected by the specific conditions chosen when developing an analytical method. How well one resolves the analytes and how quickly it is done depend upon the analyst's ability to control these three factors. Selectivity, which is governed predominantly by analyte interactions with both the stationary and mobile phases, is arguably the driving force behind separations because it affects resolution to the greatest degree. The smaller particles used in UHPLC primarily affect the efficiency, 

, of the resolution equation. Although small particles can improve a separation, they are only one contributor toward the goal of resolution. 

Figure 2: Efficiency, N, is inversely proportional to the particle diameter dp; therefore, as particle size decreases, efficiency increases. 			

Columns packed with sub-2-μm particle sizes can be used to increase resolution and shorten analysis times. How is this done? To better understand the effect of particle size, consider two more concepts in separation science—the theoretical plate and the van Deemter equation. 

Figure 3: Efficiency, N, is directly proportional to the column length L; therefore, efficiency increases as column length increases.			

As previously mentioned, particle size affects the efficiency term of the resolution equation. Efficiency is ultimately derived from the theoretical plate model of chromatography. Conceptually, a plate refers to one complete equilibrated transfer, or partition, of a solute between the mobile and stationary phases. 

, therefore, is a qualitative term used to measure the number of theoretical plates in a given column or the extent to which a solute partitions between the mobile and stationary phases. In relation to particle size, 

 is inversely proportional to the particle diameter (see Figure 2). Put simply, as particle size decreases, efficiency increases, and more resolution is possible with the same length of column.

 Figure 4: The van Deemter equation, an empirical equation describing the relationship between efficiency, as measured by theoretical plate height H, and linear velocity is governed by three cumulative terms: A eddy diffusion, B longitudinal diffusion, and C mass transfer.			

Practically, this means that shorter analysis times through the use of smaller particle-size packings can be achieved by two means. First, 

 is directly proportional to the column length 

 (see Figure 3). Therefore, an analyst can decrease the length of the column as the particle size decreases, still maintaining resolution and shortening the analysis time. Second, and probably more significant, higher flow rates can be used without a substantial loss in resolution. The van Deemter equation is an empirical formula describing the relationship between plate height 

 (the height of one theoretical plate) and linear velocity μ (see Figure 4). This equation explains why higher flow rates can be used with small-particle columns without compromising resolution. Smaller plate-height values correspond to greater peak efficiencies because more plates or analyte partitions can occur over a fixed length of column. The van Deemter equation is governed by three cumulative terms: eddy diffusion 

. Eddy diffusion is caused by disturbances in the solute flow path and is largely unaffected by flow rate. Smaller particles give rise to less interstitial space and therefore, through a decreased 

 term, have higher overall efficiencies. Mass transfer is the movement of the analyte in and out of the stationary phase. Through this term, higher flow rates typically result in poorer efficiencies. 

Figure 5: An empirically determined van Deemter plot for a biphenyl test probe demonstrates that sub-2-Î¼m packings are minimally affected by flow rate, thus allowing higher flow rates to be used and resulting in faster analysis times.			

If we look at an empirically determined van Deemter plot of efficiency versus flow rate, when using a 1.9-μm particle size (see Figure 5), we can see that, in practice, smaller particle sizes are minimally affected by higher flow rates. Column efficiency does not diminish when flow rate increases, as indicated by the relatively flat slope of the curve. Peak efficiency is comparable even when the flow rate increases to 1 mL/min on a 2.1-mm internal diameter column. This illustrates the most significant influence that small particles have on chromatographic separations—a much wider range of usable flow rates, which translates into significantly faster analysis times. This benefit, and the shorter column lengths needed for a given resolution, result in higher sample throughput without loss of separation quality. 

Figure 6: A biphenyl stationary phase is more selective for aromatic or fused-ring compounds than a conventional C18 phase, providing improved resolution.			

UHPLC does maximize efficiency (e.g., theoretical plates), but, as defined by the resolution equation, stationary-phase selectivity is still a crucial consideration when attempting to resolve mixtures of compounds. By choosing a stationary phase that produces optimum selectivity for the specific compounds of interest, one can maximize the benefit of UHPLC. For example, the use of a biphenyl stationary phase can greatly enhance a separation. A biphenyl stationary phase differs from that of an alkyl (e.g., C18) phase in that the phenyl rings present can employ pi–pi interactions. This creates a separation mechanism with improved selectivity for aromatic or fused-ring compounds (see Figure 6). When using a biphenyl stationary phase with a highly efficient 1.9-μm particle-size column, one can produce fast, highly selective separations of steroids (see Figure 7). A 1.9-μm biphenyl column can separate a test mix of seven hormones in less than 2 min, a feat not possible through C18 selectivity (1, 2).  

Figure 7: A biphenyl stationary phase, combined with a 1.9-Î¼m packing, makes possible a fast and highly selective analysis of steroids.			

Another example of the advantage of a carefully chosen stationary phase for UHPLC is a separation using a pentafluorophenyl (PFP) propyl stationary phase. This fluorinated phase is extremely retentive and selective for organohalogens or other compounds containing basic or electronegative functionalities. The analysis of fluoroquinolones such as ciprofloxacin, norfloxacin, and enrofloxacin demonstrates this characteristic. When compared with C18 and cyanopropyl stationary phases, the PFP propyl phase is more selective and more retentive (see Figure 8). The US Food and Drug Administration recently announced import controls on seafood from China, where the use of fluoroquinolones in food animals is permitted. All incoming shipments are now detained at the border until proven to be free of drug residues. In this specific example, the use of LC–MS–MS is preferred. Using a PFP propyl stationary phase to heighten the retention of fluoroquinolones is advantageous in LC–MS analysis because it requires higher organic percentages to elute the compounds. With electrospray ionization (ESI), higher amounts of organic in the mobile phases help in desolvation, thereby lowering the detection limits. In addition, unwanted adduct formation can be reduced, and charge competition from matrix interferences are lowered as the fluoroquinolones are better resolved from the matrix components (3).  

Figure 8: A pentafluorphenyl propyl stationary phase offers greater retention and selectivity for fluoroquinolones than either C18 or cyano phases, making it well suited for LCâMS analyses.			

In a final example, using the same premise of heightened selectivity of a fluorinated phase toward halogenated drug compounds, benzodiazepines and two metabolites, a mix commonly assayed on a C18 column, can be resolved in nearly 4 min on a PFP propyl column (see Figure 9). To get the same level of selectivity from a C18 column, a shallower gradient would be needed, thus prolonging the analysis time. Because the selectivity of a PFP propyl stationary phase elutes the benzodiazepines in quick succession, a simple gradient allows for the earlier elution of the more polar metabolites, while maintaining a fast overall run time.  

Figure 9: Fast, selective analysis of benzodiazepines is made possible by combining the speed of UHPLC with the enhanced selectivity of a pentafluorophenyl propyl stationary phase.			

Although the sub-2-μm particle sizes used in ultrahigh pressure liquid chromatography make fast, highly efficient assays possible, selectivity is still a critical parameter to consider. The examples reviewed here demonstrate the impact of selectivity on overall chromatographic resolution and speed of analysis. Proper stationary-phase selection is the most important factor in optimizing resolution and, with sub-2-μm particles, allows the benefits of UHPLC to be fully realized. 

is a pharmaceutical innovations chemist at Restek Corporation, 110 Benner Pike, Bellefonte, PA 16823, tel. 814.353.1300, fax 814.353.1309, 

 1. R. Freeman, R. Lake, and L. Nolan, "Enhanced Resolution of Endocrine Disrupting Hormones," 

 2. R. Lake, "Optimize Selectivity and Efficiency in UHPLC Separations," 

 3. R. Lake, "Simplified LC/MS/MS Analysis of Fluoroquinolones," 

Ultrahigh pressure liquid chromatography maximizes efficiency, but, as defined by the resolution equation, the stationary phase is still a crucial consideration when attempting to resolve mixtures of compounds.

Using Selectivity to Optimize UHPLC Separations

When it comes to controlling the quality of a final active pharmaceutical ingredient, high-performance liquid chromatography (HPLC) is one of the techniques used by quality control departments to analyze the final product. HPLC has gained popularity because of its efficient separation performance. Unfortunately, this separating power is rarely considered as a potential purification process at the commercial scale. The reason is that chromatography is always perceived as an expensive technique because of poor throughput and high solvent consumption. Although production-scale HPLC may have been expensive in the past because of glass columns, it is not today, thanks to major improvements in the technology. 

Highly efficient packing materials, also called 

, with narrow particle-size distribution are available and can be used at high pressure. As a result, high throughput can be achieved with high purity and high recovery compared with traditional "column" chromatography. Furthermore, additional improvements to the technique allow for counter-current continuous operation that further reduces the need for solvent and significantly improves process throughput. This technique, called 

 (SMB), is currently in use in many industrial applications at large scales (see Figure 1). For example, the purification of sugar is conducted using large diameter SMB units with a throughput of several tons of purified fructose or glucose per day. 

Figure 1: Commercial-scale simulated moving bed unit, 5 columns of 1 m in diameter. This unit can process several hundred tons of racemic solution per year. (PHOTO: AMPAC FINE CHEMICALS)			

SMB has been presented in many different ways in the scientific literature. Using a merry-go-round of 5–8 columns and a set of valves per column, a simple batch separation process can be converted into a continuous operation. This is achieved by changing the relative velocities of each compound in the columns. By "moving" the columns at a constant speed against the eluent flow, the most-retained component appears to move with the columns while the less-retained component flows with the eluent. As a result, a gap between the two fractions is created and can be filled continuously. To avoid accumulation, outlet streams must be collected on either side of the point of entry of the feed. This is truly a counter-current process and the packing material is used more efficiently. The only limitations are that SMB separations are conducted in an isocratic mode and that only two product streams can be isolated. This makes SMB an excellent candidate for binary separations such as the separation of enantiomers.  

Method development and chiral applications

The number of drugs entering the market that have one or more chiral centers has grown significantly in the past decade. Knowing that one enantiomer can potentially carry side effects has brought regulatory authorities such as the US Food and Drug Administration to favor the single enantiomer version of a drug whenever possible. Specific crystallization, enzymatic or chemical resolution, or chromatography can be added as a unit operation to the existing synthesis route to obtain the single enantiomer. Or a completely new asymmetric route can be designed to obtain the desired single enantiomer. As a result, pharmaceutical companies have invested substantial resources in trying to find the right chiral catalyst or the right enzyme that will provide the single enantiomer at a satisfactory enantiomeric purity.   

The development of a chromatographic chiral separation is a straightforward process that requires a few steps, described as follows.  

 There are a handful of chiral stationary phases (CSP) that provide greater than 80% probabilities of finding a good separation. If these phases are not working, there are a few more that may be suitable. The selection of solvents for the separation traditionally has been limited to acetonitrile, alcohols, and mixtures of alcohols with heptane. However, with the development of more robust CSPs that can handle solvents such as acetone, MTBE, toluene, and ethers, more possibilities exist. These solvents allow for high solubility and low viscosities, which are key elements to achieve high throughput. The screening process typically takes 1–2 weeks. From this study a handful of conditions can be identified as potential candidates.  

Determine the amount that can be separated.

 The "loading study" step is aimed at determining the maximum loading capacity of the CSP. The study consists of injecting on an analytical column (4.6-mm diameter X 250-mm long) packed with preparative CSP (typically 16 or 20 μm) an increasing volume of a concentrated solution of the racemic mixture until the separation is lost (see Figure 2). From these data valuable information about the behavior of the compound as a function of the concentration is collected. This information is entered into a computer model which in turn calculates the parameters required to operate an SMB unit. At this point an estimation of the production rate is obtained and the size of the SMB unit required to produce the desired amount of the single enantiomer can be calculated. 

Figure 2: Example of a loading study. By increasing the volume injected, the peaks change shape according to their equilibrium isotherm. This information is used to model the SMB process and calculate the throughput. (FIGURE: AMPAC FINE CHEMICALS)			

 Simulations are not always perfect, and it is usually a good practice to demonstrate the separation on a small-scale unit. On such a unit, equipped with columns of 4.6 or 10 mm in diameter, 20–200 g of material can be processed (see Figure 3). This amount is enough to achieve steady state and obtain a representative sample of the product. The columns can be packed in house or purchased from the packing manufacturer. 

Figure 3: Bench-top simulated moving bed unit, 8 columns of 4.6 mm or 1 cm in diameter. This unit is used for proof of concept. (PHOTO: AMPAC FINE CHEMICALS)			

This step can take 2–4 weeks, which is long enough to evaluate the productivity as well as the robustness and the stability of the separation. At this stage, chemists can start developing the isolation process post-SMB separation (e.g, crystallization, solvent exchange, drying).  

 The scale-up from the demonstration run is straightforward. Chromatography processes are scaled up on the basis of the linear velocity at the particle level (i.e., the flow rate is multiplied by the ratio of the column diameters squared.) For example, if 50 g of racemic feed can be processed per day on a 10-mm diameter column, then a 50-mm SMB unit can process 1250 g per day (25X). This is a very easy process to scale-up. As a matter of fact, performances often improve with the diameter of the columns because of the favorable ratio of column to piping volume.   

Following the demonstration step, it is frequent that 5–25 kg are separated using the 8 X 50-mm unit to supply material for Phase I. This material can be made according to current good manufacturing practices and purified in a matter of weeks. This work is still conducted on lab-scale equipment, and the overall cost is very reasonable. 

 Typically, a campaign involving the separation of 200–500 kg of racemic feed follows the scale-up phase. Normally, this is to supply material for Phase II or Phase III clinical trials. At this stage, the separation process is well characterized. The effort is more concentrated in the handling of solvent (recycling, composition adjustment etc.) and the product recovery (solvent exchange, crystallization, drying, etc.). All these steps are conducted in equipment that is similar to commercial-scale units. This provides a complete and accurate picture of the final process at commercial scale.  

 Provided that the feed material is available, a process can be developed from method screening to commercial quantities in a mater of months. SMB as a unit operation to produce an API has been approved by FDA. For example, Escitalopram, an antidepressant developed by Lundbeck (Copenhagen, Denmark), was one of the first drugs to be approved using an SMB step. Since then, additional drugs have been approved, and a lot more are in the pipeline.  

At this stage, this process, like any other process, must be well defined and ready for validation. The definition of the batch for a continuous process is based on time in the unit. This time can be the time required to process enough material to fill a dryer or fill the reactor for the next chemistry step. This must be defined clearly so that the pedigree of the product can be traced. 

The stability and robustness as well as the cost of the chiral stationary phase are always a concern when a new SMB process is discussed. AMPAC Fine Chemicals (AFC) has been producing several hundred metric tons of an API with SMB as the penultimate step of manufacturing using the same packing material for the past eight years. Unpacking the columns every 12 or 18 months is sometimes required to remove the fines that accumulate as a result of slow attrition of the packing. Thus, for a commercial-scale project involving the separation of hundreds of metric tons per year over several years, the cost of the packing material amortized during three years is in the range of $5–10 per kilogram of product manufactured. 

The cost of operating an SMB unit is not as expensive as it is commonly believed. Most of the solvent used for the process is recycled at three levels. The first level is internal to the SMB. The second level is at the falling films used on each outlet to preconcentrate the product. At this point 75–85% of the solvent is recycled. Finally, the solvent removed from the drying step can be recycled to achieve a total greater than 99%. AFC received an award from the Department of Toxic Substance Control (DTSC) and the Chemical Industry Council of California (CICC) for the implementation of the solvent recycling on the large-scale manufacturing using SMB. Several thousands of gallons of solvent are recycled instead of being wasted, thereby making SMB a green technology. Currently, the overall solvent usage at AFC for commercial-scale units (6 X 800 mm and 5 X 1000 mm) is less than a few drums of solvents per month.  

The labor required for the operation of an SMB unit is also relatively low. A single operator can monitor the unit, and from time to time, a second operator is required for preparing the feed or for recovering the product from the dryer. 

The cost of equipment installation is still relatively high mostly because of the cost of the unit and the amount of ancillary tanks required to operate the system. Nonetheless, this cost will eventually decrease as more units are sold around the world. 

It is quite possible that for a separation with a decent productivity (2 kg of feed/day/kg of CSP or better) and relatively high annual volumes, the price of the product falls into the range of $75–150/kg — all included.  

SMB is not limited to chiral separations. Any separation that can be turned into a binary separation is potentially an excellent candidate for SMB. Removal of troublesome impurities by SMB is a good example. Troublesome impurities are closely related to the main product or are dimers or trimers of the product and are very difficult to remove. Sometimes multiple crystallizations are required to meet the final product specifications.  

As an example, at AFC we purify a natural product by SMB. There are numerous impurities in the natural product, but one in particular that is very difficult to remove by traditional purification processes. Removal of this impurity by crystallization would result in a substantial loss of the expensive natural product to the mother liquor, which in turn makes crystallization a very expensive technique. Work was undertaken at AFC to develop a commercially viable SMB process to achieve high purity and high recovery of the natural product. The chromatogram of the feed analyzed under the SMB conditions shows only two peaks, but each peak represents the combination of several species. The SMB not only removes the troublesome impurity but it also removes a significant portion of the other impurities, thereby bringing the product total purity from ~75% to >96%.  

Because it provides high purity, high yields, and potentially high throughput, chromatography is the ultimate purification technique. With recent improvements in technology and stationary phases, the cost of using chromatography has significantly decreased, making this unit operation a very cost effective and commercially viable technology. 

					 see In the Loop:   Continuous Chromatography for Chiral (and Other) Separations

 is director of chromatography business development at AMPAC Fine Chemicals, PO Box 1718, MS 1007, Rancho Cordova, CA 95741-1718, tel. 916.357.6242, fax 916.353.3523, 

High performance liquid chromatography has become an efficient technique at the production scale, and simulated moving bed chromatography provides several benefits during processing.

Pharmaceutical Technology-10-01-2007

Achieving Ultrafast Liquid Chromatography

Polysaccharide-Based Chiral Chromatography Media in HPLC and SFC

Supercritical Fluid Chiral Separations

Using Selectivity to Optimize UHPLC Separations

Simulated Moving Bed Chromatography: A Powerful Unit Operation