temperature as a variable in pharmaceutical...

18TEMPERATURE AS A VARIABLE INPHARMACEUTICAL APPLICATIONS

Roger M. Smith

18.1 THE INFLUENCE OF TEMPERATURE ONCHROMATOGRAPHY

For much of the early development of liquid chromatography, separationswere carried out at ambient temperature and many laboratories did notattempt to regulate or control the temperature of the column. Frequently, thecolumn would be mounted on the side of the pump or detector and thus wouldbe subjected to changes in the room temperature or changes due to externalfactors, such as sunlight. However, the influence of temperature on the reten-tion times of analytes was well known and had been studied by a number ofgroups—in particular, Melander et al. [1]. They demonstrated that for mostanalytes there was a linear relationship between the retention factor of ananalyte and the inverse of the absolute column temperature (see Chapter 1).However, for a few samples there has been an increase in retention withincreasing temperature usually attributed to entropy effects. In the case ofpolyethylene glycol oligomers, the optimum separation was achieved with anegative temperature gradient [2]. The retention of leucine-phenylalanine at low pH and high % acetonitrile also increased with increasing tempera-ture [3].

As a result, temperature can play an important role in pharmaceuticalanalysis. The precise and accurate control of temperature can improve repro-ducibility and method transferability (Section 18.2). In recent years, the use of

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HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBruttoCopyright © 2007 by John Wiley & Sons, Inc.

elevated or unconventional temperatures have been examined as methods toalter selectivity and column efficiency, either with conventional mobile phases(Section 18.3) or with solvent free systems, such as superheated water (Section18.4). Although normally the interest has been in elevated temperatures, sub-ambient chromatography has provided a number of interesting separations(Section 18.5).

18.2 EFFECTS ON METHOD TRANSFERABILITY AND REPRODUCIBILITY

As pharmaceutical analysis developed and the need for long-term repro-ducibility became more important, instrument manufacturers recognized theneed for temperature stability and by the early 1990s started to include columnovens as an integral part of their instruments. In most cases the temperatureswere controlled near or just above ambient because the aim was to ensure areproducible result rather than to employ temperature as a method variable.However, even now, many chromatographers carry out separations at ambienttemperature, partly on the assumption that the conditions in a heated or air-conditioned building are constant. The reality is often different and the tem-perature around a column can alter quite markedly. Sunlight can shine on acolumn, draughts can blow on the column, or the air-conditioning can be pro-grammed to lower the laboratory temperature overnight or at weekends as acost-saving exercise. The result is that the retention of analyte compounds canmove outside predefined retention windows and the system can show daily orlong-term variations and poor reproducibility.

Of particular concern is that methods that have been developed and testedin one laboratory are often transferred to another laboratory in the same ora different company as the drug product moves from discovery through toxi-cology, stability studies, formulation, scale-up, and eventually to manufactur-ing quality control. Frequently, it is found that at each transfer, a new methodoptimization and a revalidation are required, each taking time and money. Sur-prisingly, relatively little research had addressed this problem. There are onlyfew reports of interlaboratory collaborative studies where the target has beento assess the transferability of retention or resolution. In contrast, the trans-fer of quantitation has been repeatedly examined, but this is based on relativepeaks areas to an internal or external standard measured under the same con-ditions. This usually compensates for differences in retention time. Typically,interlaboratory studies produce retention time reproducibility, which is muchworse than intralaboratory measurements. A comparison of the analysis offorensic drugs in different UK labs [4] and in an international study [5] showedwide variation in relative and absolute retention times even through themobile phases were closely specified and all the columns were from a singlebatch of packing material.

Within a single laboratory, the situation could be improved markedly byemploying temperature control of the column, with an oven or water bath [6].

812 TEMPERATURE AS A VARIABLE IN PHARMACEUTICAL APPLICATIONS

The values were then quite usable for quality control and identification espe-cially when the system was calibrated with standards at frequent intervals. Themain residual source of variation was then batch-to-batch differences betweencolumns, although these differences have been reduced in recent years, anduncertainties in the preparation of the mobile phase, which can be reduced byclose control of the protocols used.

As part of a project to develop a certified reference material for high-performance liquid chromatography [7], it was necessary to demonstrate thatthe proposed method would yield identical results in different laboratories andon different equipment. However, initial results using a specified temperatureand columns from a single batch of packing material gave poor interlabora-tory results, and temperature variations were suspected as a cause [8]. It wasfound that although the ovens in each laboratory were set to the same nominaltemperature, different oven types, air, fan air, convection/conduction, andwater bath gave significantly different results, the worst results coming fromheaters where the column rested against a heated block. The effective tem-perature could be up to 6°C lower than the set value, and this could be mon-itored by using the changes in shape selectivity and hydrophobicity of a testmixture [9]. Similar observations of oven variability were made by Paesen andHoogmartens [10]. The protocol for the CRM was then tightened so all thelaboratories used a water bath or circulating water jacket and specified lengthsof eluent preheating tubing. This gave interlaboratory and intralaboratoryvariations that were comparable and within the acceptable range [11]. Thus toachieve good transferability of a method, not only the obvious factors, such ascolumn make and mobile phase, need to be defined, but also the method ofmaintaining a constant temperature needs to be specified.

Part of the cause of the problem is attributable to differences in the dissi-pation of the fictional heating generated by the movement of the mobile phasethrough the stationary phase. In a liquid bath, this heat is readily lost to thebath as the external temperature of the column is constant along its length,whereas in a noncontrolled or static air system the mobile phase elutes fromthe column at a higher temperature (2–3°C) than the inlet [12]. There is alsoan axial temperature gradient in each case. The effect of different tempera-ture control was also examined by Welsch et al. [13], who found differencesbetween air oven and water baths on normal-phase separations and alsostudied the effect of inlet temperatures. These effects were later studied forreversed-phase separations by Wolcott et al. [14], who suggested a number oftemperature-related reasons for poor method transferability and suggestedhow different effects changed the temperature profile within the column.

18.3 ELEVATED TEMPERATURE AND PHARMACEUTICAL SEPARATIONS

Although temperature has been used for many years to alter separation prop-erties, especially selectivity and efficiency, the operating range has usually been

ELEVATED TEMPERATURE AND PHARMACEUTICAL SEPARATIONS 813

modest, typically up to 40–60°C. The principal aim has been to establish a con-trolled system sufficiently above ambient temperature so that day-to-daychanges in the laboratory conditions have no effect on the separation.Although the use of non-ambient temperatures might offer advantages in bio-analytical methods, it has been noted that the selection often seems arbitraryand without specific justification [15]. However, Brinkman et al. [16] com-mented that temperature was a variable that should be considered in methoddevelopment. This comment was echoed in a recent review of the use of mod-erate temperature changes for drug assays by Zhu et al. [17], who noted that“temperature should be considered as a useful variable to control resolutiononly when components in a mixture are of different types.”

For many years, analysts have been deterred from applying significantly ele-vated temperatures because of concern about the volatility of mobile phasesand the stability of stationary phases and analytes. More recently as a spin-offfrom work with supercritical fluid chromatography, many laboratories learnedhow to handle separations in pressurized columns (up to 300 bar), and hard-ware with pressure-resistant detector flow cells became available. As a con-sequence, the expertise and equipment were commercially available, whichcould control mobile phases above their boiling point. This has enabled theexamination of separations under conditions up to 250°C based on either con-ventional mobile phases or less common solvents, such as superheated water[18]. Temperatures above 80°C, where pressure has to be applied to preventthe mobile phase from boiling, are usually termed either pressurized, super-heated, or subcritical conditions, the latter two terms being more frequentlyapplied to separations with just water as the mobile phase. Either the separa-tions can be isothermal or a temperature gradient can be employed, whichgenerates an effect similar to gradient elution, speeding up the later compo-nents [19, 20]. However, concern is often expressed that the mass of a packedHPLC column might cause the internal temperature to lag behind the ovensetting but as long as the internal temperature is reproducible, a valid methodcan be developed. A number of early studies employed packed capillarycolumns with a low thermal mass [19].

Three main aims have driven these studies: the use of temperature as a variable to optimize separations, an interest in improved efficiency, and the potential for “green” separations methods, such as superheated water chromatography, which can eliminate the organic solvent from the mobilephase.

18.3.1 Effect of Temperature on Selectivity

Although temperature has been proposed as a variable in altering selectivity,it has not been widely used, because the majority of analytes show very similarchanges on changing temperature (especially over the limited conventionaltemperature range). Significant differences may be observed if temperaturecan cause ionization changes or if analytes with very different functional


groups are present. However, care must be taken in these situations becauserelative retention changes with column temperature could result in a lack ofmethod robustness, especially if caused by ionization changes.

A few application and studies have examined temperature effects, such asthe selectivity dependence of the carotenoids [21] on different columns from25–45°C. Studies of the prediction of the influence of temperature and solventstrength on the separation of 47 basic acidic and neutral drugs compoundswere reported by Zhu et al. [22] in an interlaboratory collaborative study.More recently the influence on temperature on selectivity has been reviewedby Dolan [23, 24].

The changes in retention and selectivity can also be exploited in the thermally tuned tandem column concept by Mao and Carr [25], in which thetemperatures of two sequentially linked columns containing different sta-tionary phases can be altered to provide the optimum separation. The tech-nique was applied to the separation of barbiturates, phenylthiohydantoinamino acids [26], and selected basic pharmaceuticals, such as antihistamines(Figure 18-1) [27].

Berthod et al. [28] examined the effect of temperature on chiral separationsbetween 5°C and 45°C using four macrocyclic glycopeptides phases; andalthough the efficiencies increased with temperature, in 83% of cases the chiralselectivity decreased.

18.3.2 Effect of Temperature on Separation Efficiency

One of the reported advantages of raising the temperature of a chromato-graphic separation is an increase in peak efficiency. This is usually attributed


Figure 18-1. Separation of antihistamines on linked columns with different tempera-tures. ODS at 40°C and PBD-ZrO2 at 35°C. Mobile phase 40 : 60 methanol/pH 7 buffer.Solutes: 1, pheniramine; 2, chlorpheniramine; 3, thenyldiamine; 4, bromopheniramine;5, cyclizane; 6, pyrrobutamine; 7, chlorcyclizane; 8, thonylamine; 9, meclizane.(Reprinted from reference 28, with permission. Copyright 2001, American ChemicalSociety.)

to a reduction in the viscosity of the eluent and an increase in the diffusionrate of the analyte as the temperature is increased. A higher diffusion rateshould reduces the mass transfer term effect (the C term) in the van Deemterequation but can also worsen the influence of longitudinal molecular diffusionin the column (the B term) [29]. The improvement in efficiency is normallyregarded as most significant for larger analytes, such as biological and synthetic macromolecules, whose size reduces their mobility [30]. For smallermolecules the effects are relatively small, and often an increase in efficiencycan be attributed to a reduction in the retention factor on raising the temperature.

A second factor, which influences peak shape and apparent efficiency, is thetemperature of the incoming mobile phase relative to the column tempera-ture. The presence of this underestimated factor may have obscured or con-fused previous studies of efficiency. Frequently, it has been claimed that themobile phase in a high-temperature separation must be heated to the sametemperature as the column; otherwise, peaks distortion and broadening areobserved [18, 31]. This was demonstrated by Thompson and coworkers [32, 33], who reported the band-broadening effect of a thermal mismatch andadvocated the use of narrow bore columns to reduce the effects. Guillarme et al. [34] also demonstrated the need for some preheating of the mobile phaseand considered the length of tubing required for effective preheating (within5°C of the oven) particularly with high flow rates.

However, even early studies including those by Cooke et al. [35] and byPoppe and Kraak [36] demonstrated that using a mobile phase slightly coolerthan the column temperature can improve column efficiency. Usually, differ-ences of 10–20°C gave the highest efficiency. For example, Mayr and Welsch[12], who found the highest efficiency for the separation of five hormonesteroids was obtained with the incoming mobile phase at 10°C and a columnat 30°C (Figure 18-2). Spearman et al. [9] reported that in one case reducingthe inlet to 37°C below the column temperature optimized the results.

The effect is thought to have two origins. First, a cooler mobile phase andby implication cooler sample causes an initial sample focusing at the head ofthe column. Second, the cooler eluent flow reduces the analyte mobility at thecenter of the column, thus balancing the enhanced temperature and hencemobility in the center of the column caused by friction heating (Figure 18-3)[14]. At its most serious, not only efficiency but also peak distortion has beenobserved caused apparently by a temperature in-balance. The selection of theoptimum column inlet temperature is not totally clear, and this is an area ofongoing research.

In such a situation the internal diameter of the column might also effectthe equilibration process but Molander et al. [37] found that even using a tem-perature gradient, the differences were minimal for columns narrower than4.6mm internal diameter. A recent study has found that elevated tempera-tures, up to 70°C, markedly improved the efficiency and peak shapes of baseswith intermediate pH eluents [38].


18.3.3 Other Temperature Effects

A side effect of a lack of temperature control is that changes can alter therefractive index of the mobile phase, causing baseline disturbances and reduc-ing sensitivity The problem is principally with refractive index detection [39],but it can also influence spectroscopic detectors and their light path can bedistorted. Temperature has also been reported to alter the nature of some sta-tionary phases. For example, it caused a change in the chiral selectivity of theresolution of dihydropyrimidone acid and its methyl ester on amylose and cel-lulose stationary phases [40].

18.3.4 Applications of Elevated Temperatures

Almost all the high-temperature work on pharmaceutical compounds hasemployed reversed-phase separations. In a series of studies since the 1990s,Greibrokk and co-workers [41, 42] have examined the role of elevated tem-perature and temperature gradients. Many were devoted to polymer separa-tions where the use of a temperature gradient speeded up the larger oligomersand provided clear advantages because of the complexity of the sample.As part of the optimization of the conditions for a separation on a packed


Figure 18-2. Separation of hormones on ChromSpher UOP column at 30°C at differ-ent eluent inlet temperatures: a, 5°C; b, 10°C; c, 22°C; d, 30°C. Compounds: 1, thiourea;2, hydrocortisone; 3, nortestosterone; 4, dehydro-17a-methyltestosterone; 5, testos-terone; 6, 17a-methyltestosterone. (Reprinted from reference 12, with permission fromElsevier.)

capillary, Tran et al. [3] included the effect of temperature on a range of compounds, including naphthalene, acenaphthene, ibuprofen, butylparaben,diethyl phthalate, monoethyl phthalate, amitriptyline, propranolol, ampheta-mine, all-trans-retinol, 13-cis-retinol, and dl-leucine-dl-phenylalanine.

A few applications have employed conventional packed columns, althoughrecent developments in new thermally stable stationary-phase materials havegenerated a renewed interest and the temperature stability of the differentstationary-phase materials has been reviewed by Claessens and van Straten[43]. The new materials have included stable metal oxide materials, based onzirconia (Figures 18-4 and 18-5) and titania [44, 45] and hybrid phases com-bining silica and methylene or ethyl bridges [46]. These have been applied ina number of applications to pharmaceutical compounds (Table 18-1).

One of the most interesting thermally stable groups of stationary phasematerials has been the polybutadiene, carbon and phenyl-coated zirconia


Figure 18-3. Band-broadening due to thermal effects. (a) Ideal case, no thermal effects;(b) effect of incoming mobile phase that is at a lower temperature than the column;(c) effect of frictional heating; (d) combined effect of cold incoming mobile phase andfrictional heating. An oven temperature of 70°C is assumed. (Reproduced from refer-ence 14, by permission from Elsevier.)

phases developed by Carr and colleagues [48, 49]. They reported that at hightemperatures, the zirconia material offered a much higher stability than silica-based columns [50]. Under these conditions the reduced solvent viscosity gaveadvantages as flow rates as high at 5ml/min were feasible [51]. The PBD zir-conia column has been used for the separation of tricyclic antidepressants [50] and lidocaine, quinidine, norephrine, tryptamine, amitriptyline, and nor-triptyline. Some selectivity changes with temperature were noted. The low vis-cosity at 100°C also enabled very small 1-μm particles to be used for theseparation of benzdiazepines (Figure 18-6) [52]. Guillarme applied these techniques to the separation of a series of caffeine derivatives, including


Figure 18-4. The separation of barbiturates on (A) ODS at 30°C, (B) C-ZrO2 at 30°C,(C) ODS at 60°C, (D) C-ZrO2 at 60°C. Mobile-phase 20/80 acetonitrile. (Reproducedfrom reference 27, with permission. Copyright 2001, American Chemical Society.)

theobromine, theophylline, and caffeine when separation on a PBD zirconiacolumn at 150°C took place in less than 1 minute compared to 7 minutes at40°C on a Hypercarb column (Figure 18-7) [35].

In a recent study, Marin et al. [53] used a set of test compounds includingamitriptyline, salicylic acid, and ibuprofen to compare the temperature stabil-ity of six stationary phases at temperatures up to 150°C.


Figure 18-5. The separation of therapeutic tricyclic antidepressants on PBD-coated zirconia at different temperatures. Solutes: 1, lidnocaine; 2, quinidine; 3, norephedrine;4, tryptamine; 5, amitriptyline; 6, nortriptyline. (Reproduced from reference 52, withpermission. Copyright 1997, American Chemical Society.)

18.4 SUPERHEATED WATER CHROMATOGRAPHY

With an increased interest and awareness of the impact of society and indus-try on the environment, there has been a significant attempt in recent years toreduce or replace the usage of organic solvents. Much early work in this areaconcentrated on the application of supercritical and subcritical carbon dioxide,but in recent years superheated (or subcritical/pressurized hot) water (SHW)has become of interest for both chromatography and extraction [43, 54]. Theearliest work was reported by Guillemin et al. [55], who used the term thermalaqueous liquid chromatography. As well as using SHW for the separation of

SUPERHEATED WATER CHROMATOGRAPHY 821

TABLE 18-1. Pharmaceuticals Separated at Elevated Temperature UsingConventional Mobile Phases

StationaryAnalyte Mobile Phases Phase Temperature Reference

Tricyclic — PBD zirconia 40–100°C 49antidepressants

Benzodiazepines Acetonitrile– PBD zirconia 100°C 53water (nonporous)

Barbiturates Acetonitrile– ODS + silica + 30–80°C 27water PDB zirconia

Basic 28pharmaceuticals

Vitamin A and Acetonitrile– Suplex pKb-100 25–60°C 47retinoids water

Figure 18-6. UPLC chromatography of benzodiazepines on a 14.5-cm × 50-μm columnpacked with 1-μm polybutadiene-encapsulated non-porous zirconia particles. EluentpH 7 buffer–acetonitrile 68 : 22 at 100°C. Peaks: 1, uracil; 2, clorazepate; 3, fluni-trozepam; 4, clonazepam; 5, chlordiazepoxide; 6, oxazepam; 7, clorazepate; 8, diazepam.(Reproduced from reference 53, with permission from Elsevier.)

alcohols, carbohydrates, and phenol, they also looked at iprodine and used anon-line FID detector for analysis.

Water has interesting and unusual thermal properties, which have onlyrecently been significantly exploited by chromatographers [56–58].As the tem-perature is increased, thermal motion weakens the hydrogen-bonding so thatthe polarity of water is reduced (Figure 18-8). At 200°C, water has a polaritysimilar to that of methanol; in addition, the viscosity also drops markedly withtemperature and the diffusion rate increases. However, the vapor pressureremains low and by 250°C has only reached 30 bar, well within the normal


Figure 18-7. Effect of temperature on the separation of caffeine derivatives on aHypercarb column (1 mm × 100 mm). (a) Column at 100°C, mobile phase: acetonitrile;(b) Column at 180°C, mobile phase: water/acetonitrile 70/30. Samples: 1, hypoxantine;2, theobromine; 3. theophylline; 4, caffeine 5, β-hydroxyethyltheophylline. (Reproducedfrom reference 35, with permission.)

capabilities of HPLC systems and markedly below the 300–350 bar usuallyneeded in SFC. However, the density is largely unaltered so that the water ineffectively incompressible. Hence the pressure applied to the system has aminimal effect, as long as it is sufficiently high to prevent gasification, it doesnot influence separations.

The principal advantages in the use of superheated water are that it is rel-atively easy to attain and the back-pressures required on the column are small.Thus even a modest length of narrow bore tubing can be employed to providesufficient resistance to prevent boiling in the column and at these pressuresmany conventional spectroscopic flow cells can be used. Because of the hightemperatures, there have been concerns about the thermal stability of the ana-lytes, but of the numerous examples, there have been few reports of instabil-ity or a tendency for accelerated hydrolysis or oxidation, of the reportedexamples, only aspirin has hydrolyzed. Compounds which might be expectedto be labile to oxidation or hydrolysis, such as the paraben antioxidants, havechromatographed without problems even up to 200°C [59].

Because of its solvent properties, SHW up to 250°C has also been used forextractions mainly of environmental samples [59]. At higher temperatures>350°C, the critical point of water can be achieved, but by that point the con-ditions are severe and will probably cause analyte degradation.

18.4.1 Columns for Superheated Water Chromatography

The principal limitation of the use of superheated water has been the ther-mal instability of conventional ODS-silica-based stationary phases, which areunstable above 70°C or 80°C. Early work concentrated on PS-DVB columns,which were stable up to 220°C. Then zirconia-based PBD and ODS bonded


Figure 18-8. Effect of temperature on the relative permittivity of water. (Reproducedfrom reference 59, with permission.)

phases became available with stabilities up to 140°C or 180°C, respectively(Section 18.3.4). In addition, PGC columns can be used up to 200°C, and manyof these materials have been compared to conventional column materials [60, 61]. Hybrid phases, such as ODS-X-Terra, can also be employed up to150°C.

18.4.2 Detectors in Superheated Water Chromatography

Superheated water also offers some novel advantages in detection because theabsence of an organic solvent reduces low-wavelength spectroscopic absorp-tion, eliminates the solvent peaks from NMR spectra, and eliminates thesolvent signal from flame detectors. This has enabled a wide range of uniqueHPLC detection methods to be employed.

UV and fluorescent spectroscopy can be employed down to 190nm becausethere is no solvent interference. Mass spectrometry is easy because the waterprovides good ionization. Flame ionization detection (FID) is of particularinterest because potentially it offers a sensitive and universal detector. Anumber of different interfaces have been used, including heated capillaries,which have been examined by Miller and Hawthorne [62], Ingelse et al. [63],and others [64, 65], who separated a range of analytes including alcohols,amino acids, and phenols. An alternative method employing a cold nebuliza-tion of the eluent has been introduced by Bone et al. [66]. They were able to detect both aliphatic and aromatic alcohols, polymers, carbohydrates,parabens, and steroids.

By using heavy water (deuterium oxide) as the eluent, on-line NMR spec-troscopic detection is simplified as negligible solvent signals are detected tointerfere with the sample signals. This method can be used for drug analysis(Figures 18-9 and 18-10) [67]. By stopping the mobile-phase flow, the peak canbe held in the NMR spectrometer cell, thereby increasing sensitivity orenabling more complex data analysis, such as COSY. This method was alsocombined with on-line mass spectroscopy for a number of model drugs [68]and was used to understand the mechanism of an unexpected selective deu-terium exchange that occurred during the separation of some sulfonamides[69]. The combination of detectors using SHW as the eluent has beenextended, and a train of four on-line detectors (UV spectroscopy, FT-IR, 1H-NMR, and MS) were applied to model pharmaceuticals [70] and ecdysteroidsin plant extracts [71].

18.4.3. Pharmaceutical Applications of Superheated Water Chromatography

One attraction of SHW is that it can be used for reversed-phase separationsand is therefore readily applicable to a wide range of pharmaceutical compounds including barbiturates, sulfonamides, analgesics and steroids(Table 18-2), and anticancer drugs, including 5-fluorouracil, methotrexate, and



Figure 18-9. Separation of barbiturates on PS-DVB column at 200°C with water as theeluent. Samples; 1, barbitone; 2, phenobarbitone; 3, talbarbitone; 4, amylobarbitone;5, heptabarbitone. (Reproduced from reference 59, with permission.)

Figure 18-10. Stop flow LC-NMR of heptabarbitone after separation as in Figure 18-9with D2O as the eluent at 200°C. (Reproduced from reference 59, with permission.)

etoposide (Figure 18-11) [75]. The method could be applied to even relativelynonpolar pharmaceutical compounds, such as the steroids [62]. In a relatedstudy, Tajuddin and Smith demonstrated the on-line coupling of SHW extrac-tion with SHW chromatography for the separation of a series of pharmaceu-ticals [76]. The drugs could be sequentially released from the extraction bystepwise temperature increases (Figure 18-12).

SHW has also been applied to the separation of nutraceuticals, naturalproducts, and biochemicals, including the water-soluble vitamins, thiamine,riboflavin, and pyidoxine (Table 18-3) without significant thermal degradation.

18.6 SUBAMBIENT SEPARATIONS

As well as selectivity changes at low temperatures, such as those reported bySander and Wise [78, 79] for the separation of PAHs (Figure 18-13), subam-bient column temperatures can also alter chromatographic separations, byreducing the rate of the racemization of enantiomers and structural isomeri-


TABLE 18-2. Pharmaceutical Compounds Separated Using Hot and SuperheatedWater

Analyte Mobile Phase Column Temperature Reference

Barbiturates Deuterium oxide PS-DVB 200°C 69Sulfonamide Buffered water PS-DVB 70–190°C 72

pH 3–12Sulfonamide Deuterium oxide PS-DVB 160–200°C 71Steroids Water Zirconia PDB 170–200°C 62Analgesics Deuterium Novapak 80–130°C 70

paracetamol, oxide C18caffeine, andphenacetin

Anticancer drugs Water pH 11.5 PS-DVB Up to 160°C 73and 3.6

Caffeine, Deuterium Oasis HLB 185°C 72paracetamol, oxideamitriptyline, Xterra 85°Cand phenacetin

Paracetamol, Water Hypercarb, Up to 225°C 61antipyrine, and PS-DVB caffeine and zirconia

PBDParacetamol, Water PS-DVB 75–185°C 74

salicylamide,methyl paraben,phenacetinethylparaben.

sation, such as cis–trans interconversions. However, as backpressure rises,eluent viscosity increases and diffusion decreases.

A classic example is the anomerization of the carbohydrates: Sucrose will givetwo broad peaks at room temperature but a single sharp peak by 50°C. At 10°Cit will give two resolved peaks. Lower temperature separation will also result inthe separation of the xylose-mannose and rhammnose-arabinose pairs on anionexchange chromatography [80]. Early work on the Pirkle chiral column withpropanolol gave improved peak shapes and chiral resolution as the temperaturewas reduced from 21°C to −24°C [81]. Improved low-temperature chiral selec-tivity was also reported by Kersten [82] in the separation of beta-amino-3-pyridylpropanoic acid at subambient temperatures. Reducing the temperatureto close to the freezing point of the eluent enabled isomeric dipeptides con-taining proline at the C-terminus to be resolved [83, 84]. Potential anti-arthriticprotein kinase inhibitors also showed enhanced chiral resolution at subambienttemperatures in a study on Chiralcel OD by Whatley [85].

Normal-phase separations at −30°C enabled the determination of the impu-rity profile of a mesylated ester, which underwent in-column cyclization atroom temperature, to be determined [86]. Subambient temperatures down to−10°C were used by LoBrutto et al. [87] to generate a single product from theon-column derivatization of an acetylenic aldehyde with diethylamine.

“Supercritical” fluid chromatography using carbon dioxide as the eluent isoften carried out subcritically at 20°C or 25°C, because the more dense eluent

SUBAMBIENT SEPARATIONS 827

Figure 18-11. Separation of the anticancer drugs: 5-fluorouracil (5-FU), methotrexate(MTX), 7-hydroxymethotrexate (7-OH-MTX), and etopoxide (VP-16) using super-heated water and a PS-DVB column at 80°C. (Reproduced from reference 75, with per-mission. Copyright 2001, American Chemical Society.)


Figure 18-12. Separation of test mixture and fractions after extraction and trappingand sequential elution at increasing temperatures. Separation on PS-DVB column at75–185°C at 15°C/min. Analytes: 1, paracetamol; 2, salicylamide; 3, caffeine; 4, methylparaben; 5, phenacetin; 6, ethyl paraben. Separations: a, direct injection of originalmixture of 1–6 without trapping; b, fraction untrapped at ambient temperature; c, frac-tion released from trap at 70°C; d, released at 90°C; e, released at 110°C. (Reproducedfrom reference 76, with permission from Royal Society of Chemistry.)

TABLE 18-3. Superheated Water Chromatography of Nutraceuticals and NaturalProducts

Analyte Mobile Phase Stationary Phases Temperature Reference

Ginger Deuterium oxide Xterra RP 18 50–130°C 75Ecdysteroids Deuterium oxide C8- XTerra or 160°C 73

C18 X-TerraWater-soluble Deuterium oxide PS-DVB Up to 200°C 76

vitaminsKava Deuterium oxide zirconia PBD 80–160°C 77

enables a greater sample loading and more column interactions, especiallyuseful in chiral separations [88]. These conditions can also change the selec-tivity in “entropically driven” chiral separations, resulting in a reversal of theelution order of some pharmaceuticals [89].

The use of carbon dioxide as the mobile phase also means that it is pos-sible to carry out assays considerably below subambient temperatures. At −50°C, Gasparrini et al. used a DACH-DNB column to resolve the enan-tiomers of the thermally enantiolabile 2-methyl-1-(2,2-dimethylpropanoyl)naphthalene, which can undergo rotation around the CO–CAr bond [54].Reducing the temperature resulted in negligible degradation in column performance.

SUBAMBIENT SEPARATIONS 829

Figure 18-13. Separation of phase selectivity test mixture of phenanthro[3,4-c]phenan-threne (PhPh), 1,2:3,4:5,5:7,8-tetrabenzonaphthalene (TBN), and benzo[a]pyrene(BaP) on polymeric C18 phase (Vydac) at subambient temperatures. (Reproducedfrom reference 81, with permission. Copyright 1989, American Chemical Society.)

18.7 CONCLUSION

Temperature is an important and often ignored parameter in method opti-mization. A lack of temperature control can result in poor inter- and intra-laboratory reproducibility. Increased temperatures can speed up and alterseparations and may improve efficiency and throughput, especially of macro-molecules. High-temperature work using superheated water can eliminateorganic solvents from the mobile phase, simplifying detection and solventinterferences in detection. At lower temperature the reduction in molecularmotion can resolve interconverting chiral and structural analytes.

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