Stability studies of stationary phases from poly(methyltetradecylsiloxane) sorbed and immobilized onto metalized and unmodified silicas

Stationary phases for RP-HPLC were prepared from metalized (titanized and zirconized) and unmodified silica particles using sorbed and immobilized poly(methyltetradecylsiloxane) (PMTDS). Different immobilization procedures, such as gamma irradiation and thermal treatments, were used for the preparation of the immobilized PMTDS phases. The stabilities of these stationary phases were evaluated by passing alkaline (pH 10) mobile phase through 60 mm x 3.9 mm columns of the different phases, with periodic tests to evaluate chromatographic performance. The results show that higher stabilities were obtained with stationary phases based on PMTDS immobilized on zirconized silica, these phases being 50% more stable than their titanized silica counterparts and 400% more stable than those based on unmodified silica. These supports provide higher chemical stability to the laboratory-made stationary phases, when compared with chemically bonded silica-based phases.     

Retention of C60 and C70 fullerenes on reversed-phase high-performance liquid chromatographic stationary phases.

The separation of C60 and C70 fullerenes on four different polysiloxane stationary phases was examined. It was determined that polar solvents can be used as mobile phases effectively for the separation of fullerene molecules. Unlike previously published work, a polymeric octadecyl siloxane (ODS) stationary phase provided higher separation factors for C70/C60 than did monomeric ODS stationary phases or phenyl substituted stationary phases. For example, for a methanol-diethyl ether (50:50, v/v) mobile phase and C60, k' approximately 5.0 separation factors, alpha = 3.3, were achieved with polymeric ODS compared to alpha = 2.2, with a monomeric ODS stationary phase. A linear solvation energy relationship (LSER) was used to model the importance of solvent interactions and stationary phase interaction to solute retention.     

Utilization of zirconia stationary phase as a tool in drug control

Zirconia-based stationary phases represent an interesting alternative to silica-based materials. Two zirconia-based stationary phases were studied as an option for use in drug analysis. The different properties of zirconia material, distinct from RP silica-columns, were employed for the development of a novel and rapid stability monitoring HPLC method. This method enables simultaneous control of possible degradation processes of active substance (ibuprofen) as well as antimicrobial excipients (methyl-and propylparaben). The separation of ibuprofen, its two main degradation products 2-(4-isobutyrylphenyl)propionic acid and 4-isobutylacetophenone, parabens, and 4-hydroxybenzoic acid as their degradation product was successfully accomplished on a Zr-CarbonC18 column using a mobile phase consisting of acetonitrile-phosphate buffer (pH 4.8)-propan-2-ol (27:56:17, v/v/v). Detection was performed at 258 nm and the analysis was completed within 17 minutes.     

Linear Solvation Energy Relationships in the Determination of Specificity and Selectivity of Stationary Phases

The retention of fifty structurally different compounds has been studied using linear solvation energy relationships. Investigations were performed with the use of six various stationary phases with two mobile phases (50/50 % v/v methanol/water and 50/50 % v/v acetonitrile/water). Packing materials were home-made and functionalized with octadecyl, alkylamide, cholesterol, alkyl-phosphate and phenyl molecules. This is the first attempt to compare all of these stationary phases synthesized on the same silica gel batch. Therefore, all of them may be compared in more complex and believable way, than it was performed earlier in former investigations. The phase properties (based on Abraham model) were used to the classification of stationary phases according to their interaction properties. The hydrophilic system properties s, a, b indicate stronger interactions between solute and mobile phase for most of the columns. Both e and v cause greater retention as a consequence of preferable interactions with stationary phase by electron pairs and cavity formation as well as hydrophobic bonds. However, alkyl-phosphate phase has different retention properties, as it was expressed by positive sign of s coefficient. It may be concluded that most important parameters influencing the retention of compounds are volume and hydrogen bond acceptor basicity. The LSER coefficients showed also the dependency on the type of organic modifier used as a mobile phase component.

     

Linear Solvation Energy Relationships in the Determination of Specificity and Selectivity of Stationary Phases

The retention of fifty structurally different compounds has been studied using linear solvation energy relationships. Investigations were performed with the use of six various stationary phases with two mobile phases (50/50?% v/v methanol/water and 50/50?% v/v acetonitrile/water). Packing materials were home-made and functionalized with octadecyl, alkylamide, cholesterol, alkyl-phosphate and phenyl molecules. This is the first attempt to compare all of these stationary phases synthesized on the same silica gel batch. Therefore, all of them may be compared in more complex and believable way, than it was performed earlier in former investigations. The phase properties (based on Abraham model) were used to the classification of stationary phases according to their interaction properties. The hydrophilic system properties s, a, b indicate stronger interactions between solute and mobile phase for most of the columns. Both e and v cause greater retention as a consequence of preferable interactions with stationary phase by electron pairs and cavity formation as well as hydrophobic bonds. However, alkyl-phosphate phase has different retention properties, as it was expressed by positive sign of s coefficient. It may be concluded that most important parameters influencing the retention of compounds are volume and hydrogen bond acceptor basicity. The LSER coefficients showed also the dependency on the type of organic modifier used as a mobile phase component.     

Chromatographic evaluation using basic solutes of the silanol activity of stationary phases based on poly(methyloctylsiloxane) immobilized onto silica

The chromatographic behaviors of some basic solutes were evaluated on stationary phases based on poly(methyloctylsiloxane) immobilized onto silica (PMOS-SiO(2)). The test solutes present both hydrophobic and hydrophilic properties. Evaluations of the pH effect used 80:20 v/v methanol/buffered mobile phase over the pH range of 5-11.5 with inorganic buffers such as borate, carbonate and phosphate and with organic buffers such as citrate, tricine and triethylamine. Evaluations in acidic mobile phases used 50:50 v/v and 30:70 v/v methanol/buffer (pH 2.5; 20 mmol/L) mobile phases. The buffer concentration effect used 65:35 v/v methanol/phosphate (pH 7; 20 and 100 mmol/L) mobile phases. The results are compared with those obtained with two chemically bonded stationary phases. The immobilized phases show greater contributions from an ion-exchange mechanism than do the commercial phases. The results indicate that the silanol activity of PMOS-SiO(2) stationary phases can be adequately evaluated by using appropriate basic probes and mobile phases having different pH, using different buffers.     

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography.

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS-methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS-5% methanol (v/v) at 50 degrees C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Comparison of C18 silica bonded phases selectivity in micellar liquid chromatography

The paper describes a new test designed in micellar LC (MLC) to compare the commercial C18 stationary phase properties. This test provides the total hydrophobicity, hydrophilicity, steric selectivity, hydrogen bonding, and ion‐exchange capacity properties calculation of the ODS stationary phases. Both the test compounds and chromatographic separation conditions choice for column characterization in MLC are detailed. The chromatographic performance of several stationary phases that are used in MLC was evaluated with specific chromatographic test comprising nine test compounds, possessing different physico‐chemical properties, which were injected on different supports with two micellar mobile phases: one at pH 7.0 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol), and other at pH 2.7 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol adjusted to pH by TFA). Fundamental column chromatographic properties were obtained under these conditions and were treated by hierarchical cluster analysis. From the results of cluster analysis, two closely related groups of columns are distinguished, and it was shown that the chosen column characteristic parameters allow characterizing both sorbent and micellar chromatographic system properties. Eleven columns were analyzed by this test, which allows a comparison of columns with the aim of the selection of suitable and analogous column for the analysis with MLC.     

Insights into the Retention Mechanism on a Pentafluorophenylpropylsiloxane-Bonded Silica Stationary Phase (Discovery HS F5) in RP-LC

The solvation parameter model is used to elucidate the retention mechanism of neutral compounds on the pentafluorophenylpropylsiloxane-bonded silica stationary phase (Discovery HS F5) with methanol-water and acetonitrile-water mobile phases containing from 10 to 70% (v/v) organic solvent. The dominant factors that increase retention are solute size and electron lone pair interactions while polar interactions reduce retention. A comparison of the retention mechanism with an octadecylsiloxane-bonded silica stationary phase based on the same silica substrate and with a similar bonding density (Discovery HS C18) provides additional insights into selectivity differences for the two types of stationary phase. The methanol-water solvated pentafluorophenylpropylsiloxane-bonded silica stationary phase is more cohesive and/or has weaker dispersion interactions and is more dipolar/polarizable than the octadecylsiloxane-bonded silica stationary phase. Differences in hydrogen-bonding interactions contribute little to relative retention differences. For mobile phases containing more than 30% (v/v) acetonitrile selectivity differences for the pentafluorophenylpropylsiloxane-bonded and octadecylsiloxane-bonded silica stationary phases are no more than modest with differences in hydrogen-bond acidity of greater importance than observed for methanol-water. Below 30% (v/v) acetonitrile selectivity differences are more marked owing to incomplete wetting of the octadecylsiloxane-bonded silica stationary phase at low volume fractions of acetonitrile that are not apparent for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. Steric repulsion affects a wider range of compounds on the octadecylsiloxane-bonded than pentafluorophenylpropylsiloxane-bonded silica stationary phase with methanol mobile phases resulting in additional selectivity differences than predicted by the solvation parameter model. Electrostatic interactions with weak bases were unimportant for methanol-water mobile phase compositions in contrast to acetonitrile-water where ion-exchange behavior is enhanced, especially for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. The above results are compatible with a phenomenological interpretation of stationary phase conformations using the haystack, surface accessibility, and hydro-linked proton conduit models.     

Characterization of stationary phases by a linear solvation energy relationship utilizing supercritical fluid chromatography

Supercritical fluid chromatography was utilized in combination with the Abraham model of linear solvation energy relationship to characterize 11 different HPLC stationary phases. System constants were determined at one supercritical fluid chromatography condition for each stationary phase. The results indicate that several types of silica columns, including type B silica, type C silica, and fused core silica, are very similar in their retention behavior. Several aromatic stationary phases were characterized and it was found that, in contrast to the other phases studied, all of the aromatic stationary phases had positive contributions from the dispersion/cavity (v) term of the linear solvation energy relationship. Several aliphatic phases were characterized and there were several linear solvation energy relationship constants that differentiated the phases from each other, mainly the polar terms (dipolarity and hydrogen bonding). One stationary phase, a fused core pentafluorophenyl (PFP) phase, had very poor regression quality. The column volume of this phase was lower than the others in the study, which may have had some impact on the results of the regression.     

Insights into the retention mechanism of neutral organic compounds on polar chemically bonded stationary phases in reversed-phase liquid chromatography

The solvation parameter model is used to characterize the retention properties of a 3-aminopropylsiloxane-bonded (Alltima amino), three 3-cyanopropylsiloxane-bonded (Ultrasphere CN, Ultremex-CN and Zorbax SB-CN), a spacer bonded propanediol (LiChrospher DIOL) and a multifunctional macrocyclic glycopeptide (Chirobiotic T) silica-based stationary phases with mobile phases containing 10 and 20% (v/v) methanol-water. The low retention on the polar chemically bonded stationary phases compared with alkylsiloxane-bonded silica stationary phases arises from the higher cohesion of the polar chemically bonded phases and an unfavorable phase ratio. The solvated polar chemically bonded stationary phases are considerably more hydrogen-bond acidic and dipolar/polarizable than solvated alkylsiloxane-bonded silica stationary phases. Selectivity differences are not as great among the polar chemically bonded stationary phases as they are between the polar chemically bonded phases and alkylsiloxane-bonded silica stationary phases.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Synthesis and characterization of hypercrosslinked, surface-confined, ultra-stable silica-based stationary phases

The synthesis and chromatographic characterization of a highly crosslinked self-assembled monolayer (SAM) stationary phase whose acid and thermal stability were significantly improved relative to a sterically protected octadecylsilane (ODS) stationary phase were recently described [B.C. Trammell, L. Ma, H. Luo, D. Jin, M.A. Hillmyer, P.W. Carr, Anal. Chem. 74 (2002) 4634]. Unfortunately, this highly crosslinked SAM phase is much more silanophilic than a conventional sterically protected octadecyl silane phase. 29Si CP-MAS NMR analysis shows that the high concentration of silanol groups in the self-assembled monolayer causes the increased retention and poor peak shape of basic solutes. In this work dimethyl-chloromethyl-phenylethylchlorosilane (DM-CMPES), a silane with only a single reactive silyl chloride group was tested as an alternative to chloromethyl-phenyethyltrichlorosilane (CMPES) as the basis for forming the starting phase. Most importantly this "conventional" silanization step (i.e., a non-SAM silanization) was followed by a Friedel-Crafts reaction using aluminum chloride as the catalyst and styrene heptamer as the multi-valent crosslinker to form the surface DM-CMPES groups into a network polymer which is fully confined and attached to the surface. An octyl (C8) derivative of the hypercrosslinked (HC) dimethyl-chloromethyl-phenylethyl (DM-CMPES) surface-confined stationary phase was synthesized to demonstrate the potential of a Friedel-Crafts based approach to making high efficiency, acid and thermally stable polymerized phases on silica with selectivity closer to conventional aliphatic phases. The stability of the retention factors of these phases under very aggressive conditions (5%, (v/v) trifluoroacetic acid and 150 degrees C) are compared to that of a sterically protected octadecylsilane (ODS) phase. The comparisons show that the long term stability of highly crosslinked DM-CMPES phases in acid is superior to the conventional phase. The HC-C8 phase is even more stable in acid than the HC-styrene heptamer DM-CMPES phase on which it is based. Additionally, the efficiency and peak shape of several prototypical bases under acidic (0.1% TFA, pH 2.0) elution conditions are discussed. The column dynamics and thermodynamic characteristics of the HC-C8 phase were investigated to demonstrate the chromatographic utility of this ultra-stable phase. Inverse size exclusion chromatography and flow studies of the HC-C8 and the sterically protected C18 stationary phases indicate the absence of pore plugging and quite good (nearly 100,000 plates/m) chromatographic efficiency. Further chromatographic investigations show that the HC-C8 stationary phase behaves as a typical reversed phase material. The HC-C8 stationary phase offers unique chromatographic selectivity for certain classes of analytes compared to both alkyl and phenyl bonded phases.     

Insights into the Retention Mechanism for Small Neutral Compounds on Silica-Based Phenyl Phases in Reversed-Phase Liquid Chromatography

The system constants of the solvation parameter model are used to prepare system maps for the retention of small neutral compounds on phenylhexylsiloxane- and pentafluorophenylpropylsiloxane-bonded superficially porous silica stationary phases (Kinetex Phenyl-Hexyl and Kinetex F5) for aqueous mobile phases containing 10–70% (v/v) methanol or acetonitrile. Electrostatic interactions (cation exchange) are important for the retention of weak bases for acetonitrile–water mobile phases, but virtually absent for the same compounds for methanol–water mobile phases. The selectivity of the Kinetex Phenyl-Hexyl stationary phase for small neutral compounds is similar to an octadecylsiloxane-bonded silica stationary phase with similar morphology Kinetex C-18 for both methanol–water and acetonitrile–water mobile phase compositions. The Kinetex Phenyl-Hexyl and XBridge Phenyl stationary phases with the same topology but different morphology are selectivity equivalent, confirming that solvation of the interphase region can be effective at dampening selectivity differences for modern stationary phases. Small selectivity differences observed for XTerra Phenyl (different morphology and topology) confirm previous reports that the length and type of space arm for phenylalkylsiloxane-bonded silica stationary phases can result in small changes in selectivity. The pentafluorophenylpropylsiloxane-bonded silica stationary phase (Kinetex F5) has similar separation properties to the phenylhexylsiloxane-bonded silica stationary phases, but is not selectivity equivalent. However, for method development purposes, the scope to vary separations from an octadecylsiloxane-bonded silica stationary phase (Kinetex C-18) to “phenyl phase” of the types studied here is limited for small neutral compounds. In addition, selectivity differences for the above stationary phases are enhanced by methanol–water and largely suppressed by acetonitrile–water mobile phases. For bases, larger selectivity differences are possible for the above stationary phases if electrostatic interactions are exploited, especially for acetonitrile-containing mobile phases.     

Retention properties of acetone-water mobile phases on a biphenylsiloxane-bonded silica stationary phase in reversed-phase liquid chromatography

The solvation parameter model system constants and retention factors were used to interpret retention properties of 39 calibration compounds on a biphenylsiloxane-bonded stationary phase (Kinetex biphenyl) for acetone-water binary mobile phase systems containing 30–70% v/v. Variation in system constants, phase ratios, and retention factors of acetone-water binary mobile phases systems were compared with more commonly used acetonitrile and methanol mobile phase systems. Retention properties of acetone mobile phases on a Kinetex biphenyl column were more similar to that of acetonitrile than methanol mobile phases except with respect to selectivity equivalency. Importantly, selectivity differences arising between acetone and acetonitrile systems (the lower hydrogen-bond basicity of acetone-water mobile phases and differences in hydrogen-bond acidity, cavity formation and dispersion interactions) could be exploited in reversed-phase liquid chromatography method development on a Kinetex biphenyl stationary phase.     

Preparation and enantiomer separation behavior of selectively methylated β-cyclodextrin-bonded stationary phases for high performance chromatography

Native and three selectively methylated β-cyclodextrin (β-CD)-bonded stationary phases without an unreacted spacer arm for liquid chromatography were prepared, where heptakis(2-O-methyl)-β-CD, heptakis(3-O-methyl)-β-CD and heptakis(2,3-di-O-methyl)-β-CD were used as the methylated β-CDs. The enantiomer separation abilities of the resulting β-CD stationary phases for 12 pairs of dansylamino acid enantiomers and six pairs of N-3,5-dinitrobenzoyl amino acid methyl esters as model solutes were investigated. The effects of pH and methanol content of the mobile phase on the retention and resolution were examined to optimize the mobile phase conditions. The optimum resolution for the dansylamino acids was achieved using a mobile phase consisting of 1.0% triethylammonium acetate buffer (pH 5.0)–methanol (v/v 4/6) on the β-CD stationary phase. Heptakis(3-O-methyl)- and heptakis(2,3-di-O-methyl)-β-CD-bonded stationary phases showed little enantiomer separation abilities for the dansylamino acids. The heptakis(2-O-methyl)-β-CD-bonded stationary phase exhibited no enantioselectivities for those solutes.

For the N-3,5-dinitrobenzoyl amino acid methyl esters, the optimum resolution was achieved using a mobile phase consisting of 1.0% triethylammonium acetate buffer (pH 5.0)–methanol (v/v 9/1) on a heptakis(2-O-methyl)-β-CD stationary phase. The heptakis(2,3-di-O-methyl)-β-CD-bonded stationary phases exhibited no enantioselectivities for the N-3,5-dinitrobenzoyl amino acid methyl esters. β-CD and heptakis(3-O-methyl)-β-CD-bonded stationary phases had no enantiomer separation abilities for those solutes except for the N-3,5-dinitrobenzoyl phenylalanine methyl ester.     

Evaluation of the influence of residual silanol groups on the total retention in reversed-phase chromatography

It was demonstrated that the characteristics of stationary phases for reversed-phase chromatography can be compared by analyzing the retention of several compounds belonging to a homologous series during elution with one and the same composition of mobile phase or the retention of a nonpolar compound during elution with several mobile phases of different compositions. The slope of the corresponding linear correlations can be used to characterize the hydrophobicity of the columns (stationary phases) under study. To analyze the polar interactions between the sorbate and the stationary phase it is advantageous to study the retention of a polar substance in several (at least in two) mobile phases of different compositions. The degree of displacement of the linear correlation relative to that for nonpolar substances can be considered a measure of the influence of residual silanol groups on the total retention characteristics.     

漆酚酯键合硅胶液相色谱固定相的制备与应用

以甲基丙烯酸漆酚酯为色谱配体,制备了一种新型色谱固定相。首先以漆酚和甲基丙烯酰氯为原料制备得到甲基丙烯酸漆酚酯,并通过物理吸附涂覆到由3-（甲基丙烯酰氧）丙基三甲氧基硅烷化学修饰的硅胶上,再通过自由基引发与硅烷化硅胶的双键聚合制得漆酚酯键合硅胶固定相（USP）。对固定相进行傅里叶红外光谱（FT-IR）、热重分析（TGA）、扫描电子显微镜（SEM）和元素分析（EA）表征,结果表明通过共聚反应成功地将漆酚酯固定在硅烷化硅胶上,且制备出的固定相具有良好的单分散性。采用匀浆法装柱,以乙腈-0.05%磷酸溶液（3：97,v/v）为流动相,流速为0.4 mL/min,检测波长为220 nm,考察固定相对天麻浸膏的分离性能。以乙腈-水（50：50,v/v）为流动相,流速为0.5 mL/min,检测波长为290 nm,考察固定相对吴茱萸浸膏的分离性能。结果表明该固定相对天麻浸膏和吴茱萸浸膏均具有良好的分离性能,从天麻浸膏中分离出5个色谱峰,从吴茱萸浸膏中分离出2个色谱峰。与商品化C₁₈ 柱相比,USP柱可以从天麻浸膏中分离出更多的有效组分并实现基线分离,分离吴茱萸浸膏的色谱条件更为环保和安全。采用低流速对天麻浸膏和吴茱萸浸膏进行分离,减少了流动相的使用量,分离结果令人满意。以天然产物漆酚制备色谱固定相,既为分离纯化天麻素和吴茱萸碱提供了一种新的方法,又为液相色谱固定相制备提供了新的思路,还拓展了生漆在色谱分离材料方面的应用。     

A simple TLC and HPTLC method for separation of selected steroid drugs

Chromatographic properties of five steroid drugs: cortisone, hydrocortisone, methylprednisolone, prednisolone and norgestrel have been studied by normal-, reversed-phase and hydrophilic neutral cyano-bonded silica stationary phase with five binary mobile phases (acetonitrile-water, acetonitrile-DMSO, acetonitrile-methanol, acetone-petroleum ether, acetone-water) in which the concentration of organic modifier was varied from 0 to 100% (v/v). This study reports the optimization of steroid hormones separation. Chromatographic retention data and possible retention mechanisms are discussed. Separation abilities of mobile and stationary phases were studied using the principal component analysis method. The best separation of methylprednisolone and prednisolone is with a chromatographic system included silica gel as stationary phase and mixture of acetonitrile and DMSO (10:90 v/v). These two anti-inflammatory drugs can be fast separated from norgestrel when CN is used as stationary phase and acetone and water (40:60 v/v) as mobile phase. The highest values of the parameter Δ(ΔG°) and alfa for cortisone and hydrocortisone was observed in case of using CN as stationary phase and water-acetonitryle (40:60 v/v) as mobile phase.