Effect of column temperature on enantioseparation of dihydropyrimidinones using alcohol solvated Chiralpak AS and AS-H columns

In this study we report the application of column temperature programs to compare the chromatographic behaviors of four commercially available dihydropyrimidinone (DHP) compounds on Chiralpak AS and AS-H phases under different alcohol solvation conditions such as 1-propanol (1-PrOH), 2-methyl-1-PrOH (2-Me-1-PrOH), 1-butanol (1-BuOH), 2-BuOH, and tert-BuOH (t-BuOH) in n-hexane (n-Hex). It was observed that the apparent retention factors of the DHP compounds on the AS phase in t-BuOH/n-Hex mobile phase had the largest reduction (>27%) among the mobile phases studied after a heating and cooling temperature cycle (10 to 50 back to 10 degrees C). However, the average reduction of the apparent retention factors of the compounds on t-BuOH solvated AS-H was less than 4%. No clear trend of changes in the apparent retention factors on AS and AS-H columns was observed in other alcohol modified mobile phases. Step-temperature programs showed that alcohol solvated AS phase had larger changes in the apparent retention factors with time than those of AS-H phase at the elevated temperature step (50 degrees C). Both t-BuOH solvated AS and AS-H phases showed kinetic behaviors in chromatographic processes at the elevated temperature.     

Unusual effect of column temperature on chromatographic enantioseparation of dihydropyrimidinone acid and methyl ester on amylose chiral stationary phase

This paper reports an unusual effect of column temperature on the separation of the enantiomers of dihydropyrimidinone (DHP) acid and its methyl ester on a derivatized amylose stationary phase by normal-phase liquid chromatography. The separation of the DHP acid enantiomers was investigated using both carbamate-derivatized amylose and cellulose stationary phases (Chiralpak AD and Chiralcel OD) with an ethanol-n-hexane (EtOH-n-Hex) mobile phase. On the amylose phase, the van 't Hoff plot of the retention factor of the S-(+)-DHP acid was observed to be non-linear while that of R-(-)-DHP acid was linear. Likewise, the van 't Hoff plot for DHP acid enantioselectivity was non-linear with a transition occurring at approximately 30 degrees C. Furthermore, the van 't Hoff plot for the DHP acid enantioselectivity factor for data taken when heating the column from 5 to 50 degrees C was not superimposable with the same plot prepared with data from the cooling process from 50 to 5 degrees C. This observation suggested that the stationary phase was undergoing a thermally induced irreversible conformational change that altered the separation mechanism between the heating and cooling cycles. Similar phenomena were observed for the separation of the enantiomers of the DHP ester probe compound. The conformational change of the AD phase was shown to depend on the polar component of the mobile phase. When 2-propanol (2-PrOH) was used as the modifier instead of EtOH, the van 't Hoff plots for DHP acid were linear and thermally reversible, suggesting that no such irreversible conformational change occurs with this modifier. Conversely, when the AD phase was pre-conditioned with a more polar methanol (MeOH) or water containing mobile phase, thermal irreversibility of DHP acid enantioselectivity was once again observed. Interestingly, when the stationary phase was changed to its cellulose analogue, the Chiralcel OD, all van 't Hoff plots for the retention and selectivity of DHP acid were thermally reversible for both EtOH-n-Hex and 2-PrOH-n-Hex mobile phases.     

Retention mechanisms in super/subcritical fluid chromatography on packed columns

Whereas the retention rules of achiral compounds are well defined in high-performance liquid chromatography, on the basis of the nature of the stationary phase, some difficulties appear in super/subcritical fluid chromatography on packed columns. This is mainly due to the supposed effect of volatility on retention behaviours in supercritical fluid chromatography (SFC) and to the nature of carbon dioxide, which is not polar, thus SFC is classified as a normal-phase separation technique. Moreover, additional effects are not well known and described. They are mainly related to density changes of the mobile phase or to adsorption of fluid on the stationary phase causing a modification of its surface. It is admitted that pressure or temperature modifications induce variation in the eluotropic strength of the mobile phase, but effects of flow rate or column length on retention factor changes are more surprising. Nevertheless, the retention behaviour in SFC first depends on the stationary phase nature. Working with polar stationary phases induces normal-phase retention behaviour, whereas using non-polar bonded phases induces reversed-phase retention behaviour. These rules are verified for most carbon dioxide-based mobile phases in common use (CO(2)/MeOH, CO(2)/acetonitrile or CO(2)/EtOH). Moreover, the absence of water in the mobile phase favours the interactions between the compounds and the stationary phase, compared to what occurs in hydro-organic liquids. Other stationary phases such as aromatic phases and polymers display intermediate behaviours. In this paper, all these behaviours are discussed, mainly by using log k-log k plots, which allow a simple comparison of stationary phase properties. Some examples are presented to illustrate these retention properties.     

Comparative Chiral Separation of Thalidomide Class of Drugs Using Polysaccharide-Type Stationary Phases with Emphasis on Elution Order and Hysteresis in Polar Organic Mode

The enantioseparation of four phthalimide derivatives (thalidomide, pomalidomide, lenalidomide and apremilast) was investigated on five different polysaccharide-type stationary phases (Chiralpak AD, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) using neat methanol (MeOH), ethanol (EtOH), 1-propanol (PROP), 2-propanol (IPA) and acetonitrile (ACN) as polar organic mobile phases and also in combination. Along with the separation capacity of the applied systems, our study also focuses on the elution sequences, the effect of mobile phase mixtures and the hysteresis of retention and selectivity. Although on several cases extremely high resolutions (R_s > 10) were observed for certain compounds, among the tested conditions only Chiralcel OJ-H column with MeOH was successful for baseline-separation of all investigated drugs. Chiral selector- and mobile-phase-dependent reversals of elution order were observed. Reversal of elution order and hysteresis of retention and enantioselectivity were further investigated using different eluent mixtures on Chiralpak AD, Chiralcel OD and Lux Amylose-2 column. In an IPA/MeOH mixture, enantiomer elution-order reversal was observed depending on the eluent composition. Furthermore, in eluent mixtures, enantioselectivity depends on the direction from which the composition of the eluent is approached, regardless of the eluent pair used on amylose-based columns. Using a mixture of polar alcohols not only the selectivities but the enantiomer elution order can also be fine-tuned on Chiralpak AD column, which opens up the possibility of a new type of chiral screening strategy.     

Analysis of polymer additives in high-temperature liquid chromatography

A high-temperature liquid chromatographic technique is employed for the separation of commercially available polymer additives to enhance the resolution and speed. Separation efficiencies and elution behaviors for seven phthalate plasticizers and five antioxidants are evaluated at elevated column temperatures and with a thermal gradient. Diamondbond C18 (octadecylsilica), Zirchrom PS (zirconia-based polystyrene), and Zirchrom PBD (zirconia-based polybutadiene) columns are selected for the study because of their thermal stability. The temperature programming is controlled with a column oven in conjunction with an independent mobile phase preheater and a post-column effluent cooling assembly. Van't Hoff plots show that the reverse-phase liquid chromatography mechanism is maintained over a wide range of column temperatures. A 1% increase of acetonitrile in the mobile phase is estimated to have a comparable effect as a 7-7.5 degrees C column temperature increase on the retention time changes.     

Effects of stationary-phase polarity on retention in reversed bonded phase HPLC columns

Summary Variations in retention and selectivity have been studied in cyano, phenyl and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns was correlated with polar group selectivities of mobile phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers.     

Comparison of Various Columns for the High-Speed HPLC Analysis of Drugs of Forensic Interest

Abstract

A comparison of the use of various commercially available columns for the high-speed reverse-phase ion-pair high performance liquid chromatographic separation of drugs of forensic interest is discussed. The columns include a Partisil 5 ODS-3 RAC, a Partisil 5 C8 RAC, a Radial Pak microBondapak C18 cartridge, a Perkin-Elmer HS/5 C18 and a Perkin-Elmer HS/3 C18. The mobile phases employed contain water, acetonitrile, phosphoric acid, and sodium hydroxide, with or without hexylamine. When a mobile phase without an amine modifier is employed, retention times were at least halved, except with a HS/3 C18 column, over those obtained with conventional columns. Basic drugs did not elute when the above mobile phase is used with a HS/3 C18 column. In addition, the selectivities of the other high speed columns were similar. Further reductions in retention times and different selectivities were obtained when an amine modifier is utilized. Column performance parameters such as n, V and v are presented for the colupns examined. A new column performance parameter S which is (n/V)^1/2 is introduced and discussed.     

Effect of high-temperature on high-performance liquid chromatography column stability and performance under temperature-programmed conditions

Six commercially available analytical (4.1 or 4.6 mm i.d.) columns were evaluated under temperature-programmed high-temperature liquid chromatography (HTLC) conditions to access their stability and performance at extreme temperatures. Seven components consisting of acidic, basic and neutral compounds were analyzed under temperature-programmed conditions and solvent gradient conditions using three different mobile phase compositions (acidic, basic and neutral). Each column was checked with a two-component test mix at various stages of the evaluation to look for signs of stationary phase collapse. Three zirconia based stationary phases studied exhibited column bleed under temperature-programmed conditions. The other three columns, a polydentate silica column, a polystyrene-divinylbenzene (PS-DVB) polymeric column, and a graphitic carbon column performed well with no evidence of stationary phase degradation. The R.S.D. for the retention times and efficiencies were less than 10% for most conditions, and not more than 15% during the course of the evaluation for each column. The polydentate silica stationary phase was temperature programmed to 100 degrees C, the PS-DVB stationary phase was temperature programmed up to 150 degrees C, and the graphitic carbon column was used with temperature programming up to 200 degrees C. Comparable peak capacities and similar retention behaviors were observed under solvent gradient and temperature-programmed conditions. Temperature programming with dynamic mobile phase preheating can replace solvent gradient analysis without a loss of peak capacity when used with 4.1 or 4.6 mm columns.     

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography has been investigated in the present study. Columns with inner diameters of 0.32, 0.53, 3.2 and 4.6 mm were compared with respect to retention and efficiency characteristics using temperature gradients from 30 to 90 degrees C with temperature ramps of 1, 5, 10 and 20 degrees C min(-1). The columns were all of 15 cm length and were packed with 3 microm Hypersil ODS particles. Alkylbenzenes served as model compounds, and the mobile phase consisted of acetonitrile-water (50:50, v/v). The study revealed that the column ID is not a critical limiting factor when performing temperature programming in LC, at least for columns narrower than 4.6 mm inner diameter in the temperature interval 30-90 degrees C. The retention times for all components on all columns were highly comparable, with similar peak profiles without any signs of peak splitting. The use of mobile phase pre-heating when using the larger bore columns was avoided by starting the temperature gradients close to ambient. However, the relative apparent efficiency was inversely proportional to column inner diameter, making the capillary columns generally more functional towards temperature gradients than the larger bore columns with respect to chromatographic efficiency. In addition, the capillary columns possessed higher robustness towards temperature programming than the conventional columns.     

Insights into the Retention Mechanisms on Perfluorohexylpropylsiloxane-Bonded (Fluophase-RP) and Octadecylsiloxane-Bonded (Betasil C18) Stationary Phases Based on the Same Silica Substrate in RP-LC

The solvation parameter model is used to elucidate the retention mechanism on a perfluorohexylpropylsiloxane-bonded (Fluophase RP) and octadecylsiloxane-bonded (Betasil C18) stationary phases based on the same silica substrate with acetonitrile–water and methanol–water mobile phase compositions. Dewetting affects the retention properties of Fluophase RP at mobile phase compositions containing less than 20% (v/v) acetonitrile or 40% (v/v) methanol. It results in a loss of retention due to an unfavorable change in the phase ratio as well as changes in specific intermolecular interactions. Steric repulsion reduces retention of bulky solutes on fully solvated Betasil C18 with methanol–water (but not acetonitrile–water) mobile phase compositions but is not important for Fluophase RP. The retention of weak bases is affected by ion-exchange interactions on Fluophase RP with acetonitrile–water, and to a lesser extent, methanol-water mobile phases but these are weak at best for Betasil C18. The system constants of the solvation parameter model and retention factor scatter plots are used to compare selectivity differences for Fluophase RP, Betasil C18 and a perfluorophenylpropylsiloxane-bonded silica stationary phase Discovery HS F5 for conditions where incomplete solvation, steric repulsion and ion-exchange do not significantly contribute to the retention mechanism. Lower retention on Fluophase RP results from weaker dispersion and/or higher cohesion moderated to different extents by polar interactions since solvated Fluophase RP is a stronger hydrogen-bond acid and more dipolar/polarizable than Betasil C18. Retention factors for acetonitrile–water mobile phases are highly correlated for Fluophase RP and Betasil C18 except for compounds with a large excess molar refraction and weak hydrogen-bonding capability. Selectivity differences are more significant for methanol–water mobile phases. Retention factors on Fluophase RP are strongly correlated with those on Discovery HSF5 for acetonitrile–water mobile phases while methanol–water mobile phases retention on Fluophase RP is a poor predictor of the retention order on Discovery HS F5.     

Selectivity comparisons of monolithic silica capillary columns modified with poly(octadecyl methacrylate) and octadecyl moieties for halogenated compounds in reversed-phase liquid chromatography

Stationary phase selectivities for halogenated compounds in reversed-phase HPLC were compared using C18 monolithic silica capillary columns modified with poly(octadecyl methacrylate) (ODM) and octadecyl moieties (ODS). The preferential retention of halogenated benzenes on ODM was observed in methanol/water and acetonitrile/water mobile phases. In selectivity comparison of selected analytes on ODM and ODS, greater selectivities for halogenated compounds were obtained with respect to alkylbenzenes on an ODM column, while similar selectivities were observed with a homologous series of alkylbenzenes on ODM and ODS columns. These data can be explained by greater dispersive interactions by more densely packed octadecyl groups on the ODM polymer coated column together with the contribution of carbonyl groups in ODM side chains. For the positional isomeric separation of dihalogenated benzenes (ortho-, meta-, para-), the ODM column also provided better separation of these isomers for the adjacently eluted isomers that cannot be completely separated on an ODS column in the same mobile phase. These results imply that the ODM column can be used as a better alternative to the ODS column for the separation of other halogenated compounds.     

Analytical and semi-preparative HPLC enantioseparation of novel pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety using immobilized polysaccharide chiral stationary phases

The direct HPLC enantioseparation of a novel series of chiral pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety was performed on two immobilized polysaccharide chiral stationary phases (Chiralpak IA, Chiralpak IC) using n-hexane (n-Hex)/dichloromethane (DCM) mobile phase with 5% alcohol additive. Good baseline separation of the enantiomers was achieved using amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA) on analytical scale. The analytical method was further scaled up to semi-preparative loading to obtain small amounts of both the enantiomers of pyridazin-3(2H)-one derivative. The semi-preparative resolution of all compounds was successfully achieved with n-hexane/dichloromethane/ethanol (EtOH) as mobile phase using a semi-preparative Chiralpak IA column. The first fractions were isolated with purities of >99.9% (enantiomeric excess (e.e.), and the second fractions were obtained with purities of >98.2% (enantiomeric excess). The assignment of the absolute configuration was established for the F1 fraction of compound a-2 by single-crystal X-ray diffraction method.     

Comparison of liquid chromatographic separations of geometrical isomers of substituted phenols with β- and γ-cyclodextrin bonded-phase columns

The capacity factors (k′) of several substituted phenols were measured by using β- and γ-cyclodextrin bonded-phase columns with mobile phases varying from the classical normal-phase conditions (e.g., heptane/2-propanol) to the reversedl-phase conditions (e.g. water/2-propanol). The cyclodextrin columns have unusual selectivities in both normal- and reversed-phase separations because of their large number of hydroxyl functional groups and their ability to form inclusion complexes with substrates. The occurrences of minima in plots of log k′ vs. percent organic modifier for various substituted phenols are considered to result from solute/solvent competition for interaction with the stationary phase and from the relative hydrophobicity of the stationary and mobile phases.     

Chromatographic studies of unusual on-column degradations of aniline compounds on XBridge Shield RP18 column in high pH aqueous mobile phase

This paper reports unusual on-column degradations of aniline compounds on Waters XBridge Shield RP18 column when ammonium hydroxide in water and acetonitrile were used as mobile phases in liquid chromatography. The change of the level of on-column degradation of a model compound (Compound 1) with time was observed in the first fifteen injections when started at 60 °C. During a subsequent cooling program from 60 °C to 10 °C with a 10 °C interval, the levels of the degradation products of Compound 1 changed with the change of temperature and reached a maximum at 40 °C. The on-column degradation of Compound 1 was observed when started at 10 °C in the first injection, however, the magnitude of the change of the level of on-column degradation of Compound 1 with time in the first fifteen injections was much smaller than that at 60 °C. During a subsequent heating program from 10 to 60 °C with a 10 °C interval, the levels of the degradation products of Compound 1 increased with the increase in temperature but without a maximum. The change of the degradation product levels of this model compound in the heating process is not super-imposable with that in the cooling process, which demonstrates the degree of the degradation also depends on the heating or cooling process. Column history studies demonstrated that the on-column degradation of Compound 1 changed dramatically on the used columns at both starting temperatures while the dependency of heating and cooling processes on on-column degradation still existed. The unusual on-column degradation of Compound 1 on the used columns can be regenerated in a very similar fashion with an acetic acid column-wash procedure, but is not identical to that on the new column. Similar degradations of other commercially available aniline compounds were also observed with this high pH aqueous mobile phase system.     

Joule heating in packed capillaries used in capillary electrochromatography

Effective heat dissipation is critical for reproducible and efficient separations in electrically driven separation systems. Flow rate, retention kinetics, and analyte diffusion rates are some of the characteristics that are affected by variation in the temperature of the mobile phase inside the column. In this study, we examine the issue of Joule heating in packed capillary columns used in capillary electrochromatography (CEC). As almost all commonly used CEC packings are poor thermal conductors, it is assumed that the packing particles do not conduct heat and heat transfer is solely through the mobile phase flowing through the system. The electrical conductivity of various mobile phases was measured at different temperatures by a conductivity meter and the temperature coefficient for each mobile phase was calculated. This was followed by measurement of the electrical current at several applied voltages to calculate the conductivity of the solution within the column as a function of the applied voltage. An overall increase in the conductivity is attributed to Joule heating within the column, while a constant conductivity means good heat dissipation. A plot of conductivity versus applied voltage was used as the indicator of poor heat dissipation. Using theories that have been proposed earlier for modeling of Joule heating effects in capillary electrophoresis (CE), we estimated the temperature within CEC columns. Under mobile and stationary phase conditions typically used in CEC, heat dissipation was found to be not always efficient. Elevated temperatures within the columns in excess of 23 degrees C above ambient temperature were calculated for packed columns, and about 35 degrees C for an open column, under a given set of conditions. The results agree with recently published experimental findings with nuclear magnetic resonance (NMR) thermometry, and Raman spectroscopic measurements.     

Influence of stationary phase solvation on shape selectivity and retention in reversed-phase liquid chromatography

In the past few decades, shape selectivity has drawn a great deal of attention from chromatographers. The chemistry and characteristics of bonded stationary phases such as phase type, length of bonded phase, surface coverage, and silica surface material have an effect on the shape selectivity of the columns. Although the effects of bonded phase shape selectivity are relatively well understood, one remaining question is the effect of intercalated solvent on shape selectivity. The intercalation of organic modifier and water molecules into the stationary phase is believed to introduce more rigidity into bonded alkyl chains in RPLC. The use of gas chromatography (GC) opens a new dimension to approach this question. C18 columns 4 cm in length were prepared in our laboratory and used in both LC and GC experiments. Shape selectivity and thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase have been determined as a function of monomeric octadecyl stationary phase bonding densities over the range of 1.44-3.43 micromol/m2 and a polymeric phase (nominal surface coverage 4.77 micromol/m2). Comparing LC and GC experiments, we observed: (a) similar relationships between shape and phenyl selectivities with monomerically bonded C18 phase densities; (b) different correlation of thermodynamic quantities (DeltaH degrees , DeltaS degrees , and DeltaG degrees ) versus bonded phase densities. The effects of high temperature and residual silanol groups are sources of difficulty in elucidation of the intercalated mobile phase role in selectivity and retention for GC measurements.     

朊病毒蛋白113-120序列多肽的反相液相色谱保留行为与构象稳定性的关系

朊病毒（Prion)蛋白是人和动物慢性中枢神经系统退化病的传染源,该蛋白的113－120序列被认为在其致病和传染机理中起着重要作用。以反相高效液相色谱为分析手段,研究了Prion蛋白113－120序列多肽的色谱保留行为。通过比较不同温度、不同流动相条件下该多肽色谱保留行为的变化,发现在乙腈溶液为上时,1nKw随温度的变化关系和Van′t Hoff曲线均比简单,说明该多肽在乙腈溶液中所采取的构象均较稳定,不易受温度的影响。以甲醇溶液为流动相时,具有游离末端的多肽的1nKw随温度变化关系和Van′t Hoff曲线比末端羧基和氨基分别被酰胺封闭的多肽要复杂,说明具有游离末端的多肽在甲醇溶液中所采取的构象相对较不稳定,易受环境的影响。这些结果进一步证明,113－120序列在Prion蛋白构象变化中可能起着重要作用。     

Comparison of the Separation Characteristics of the Organic–Inorganic Hybrid Octadecyl Stationary Phases XTerra MS C18 and XBridge C18 and Shield RP18 in RPLC

Differences in the system constants of the solvation parameter model, discontinuities in retention factor plots (log k against volume fraction of organic solvent) and retention factor correlation plots are used to study the retention mechanism on XTerra MS C₁₈, XBridge C₁₈ and XBridge Shield RP₁₈ stationary phases with acetonitrile–water and methanol–water mobile phases containing from 10 to 70% (v/v) organic solvent. Wetting of XBridge C₁₈ at 10 and 20% (v/v) acetonitrile is incomplete and is responsible for small changes in the retention mechanism. The intermolecular interactions responsible for retention on XTerra MS C₁₈ and XBridge C₁₈ are similar with minor differences in cavity formation and hydrogen-bonding interactions responsible for small selectivity differences. On the other hand, for bulky solutes there are large changes in retention at low volume fractions of organic solvent (<40% v/v) associated with steric repulsion on the XTerra MS C₁₈ stationary phases that is absent for XBridge C₁₈. Selectivity differences are more apparent for XBridge C₁₈ and XBridge Shield RP₁₈. For acetonitrile-water mobile phases cavity formation in the solvated XBridge Shield RP₁₈ is slightly more difficult and hydrogen-bond acid and base interactions are more important than for XBridge C₁₈. With methanol–water mobile phases it becomes slightly easier to form a cavity in the solvated XBridge RP₁₈ compared with XBridge C₁₈. In addition, the methanol-water solvated XBridge RP₁₈ is a stronger hydrogen-bond base and more dipolar/polarizable than XBridge C₁₈. These variations in selectivity justify the use of XBridge C₁₈ and XBridge Shield RP₁₈ as complementary stationary phases for method development.     

Mesoporous polybutadiene-modified zirconia for high-temperature packed capillary liquid chromatography: column preparation and temperature programming stability

In the present study, three different methods for packing of 3 microm PBD-ZrO2 particles in 0.5 mm i.d. glass-lined stainless steel columns have been examined. The two first methods were based on a traditional downstream high-pressure technique using tetrachloromethane (Method I) or aqueous Triton X-100 (Method II) as slurry solvents, while Method III was an upstream high-pressure flocculating method with stirring, using isopropanol both as the slurry and packing solvent. Method I was found to be superior in terms of efficiency, producing 0.5 mm i.d. x 10 cm columns with almost 90,000 plates m(-1) for toluene (R.S.D. = 8.7%, n = 3), using a slurry concentration of 600 mg ml(-1), ACN-water (50:50 (v/v)) as the packing solvent and a packing pressure of 650 bars. For Method I, the slurry concentration, column i.d., column length and initial packing pressure were found to have a significant effect on column efficiency. Finally, the long-term temperature stability of the prepared columns was investigated. In isothermal mode, using ACN-20 mM phosphate buffer, pH 7 (50:50 (v/v)) as the mobile phase, the columns were found to be stable for at least 3,000 void volumes at 100 degrees C. At this temperature, the solute efficiencies changed about 5-18% and the retention factors changed about 6-8%. In temperature programming mode (not exceeding 100 degrees C), on the other hand, a rapid decrease in both column efficiency and retention factors was observed. However, when the columns were packed as initially described, ramped up and down from 50 to 100 degrees C for 48 h and refilled, fairly stable columns with acceptable efficiencies were obtained. Although not fully regaining their initial efficiency after refilling, the solute efficiencies changed about 19-28% (32-37%) and the retention factors changed about 4-5% (13-17%) after running 3,000 (25,000) void volumes or 500 (3,900) temperature programs.     

Is phospholipid-saturated alkyl column a convenient replacement for immobilized-artificial-membrane?

Three phosphatidylcholine (PC)-saturated C(8)/C(18) stationary phases prepared using biologically representative membrane lipids (purchasable L-alpha-PC and pure 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine) have been developed to compare with IAM (immobilized-artificial-membrane) and C(8)/C(18) columns. These PC-coated stationary phases were found to be stable and reproducible for retention experiments. The retention characteristics of nucleobases on these coated phases deviate significantly from those on the IAM counterpart, but surprisingly similar to those of the underlying C(8)/C(18) columns. An inter-phase model has been proposed and explored for interpretation of the results.