Gradient high performance liquid chromatography of block copolymers of styrene andt-butyl methacrylate II. Association phenomena in mixtures of block copolymer with polystyrene

Summary Block copolymers of styrene andt-butyl methacrylate can be analysed by methanol/tetrahydrofuran gradients on C18 or phenyl bonded phase columns. On both of these columns, retention increases with styrene content of the samples. At 50°C, the retention of PS or a block copolymer containing 45% styrene was longer on the phenyl than the C18 columns. This indicates the contribution of adsorption to retention on phenyl bonded phase columns. Lowering the temperature from 50 to 30°C caused earlier elution of part of the sample from the phenyl phase. On a C18 phase the same drop in temperature improved the shape of the peak, which also started later than at 50°C. This effect of temperature is generally observed in polymer retention due to an adsorption mechanism, whereas increasing retention with decrease in temperature is characteristic of a precipitation mechanism.The block copolymer investigated contained 15% free polystyrene precursor which could not be separated from the block copolymer under the conditions employed. The addition of 20% PS homopolymer with a molecular weight similar to that of the styrene block in the copolymer showed that the polystyrene eluted together with the block copolymer, whereas the addition of PS homopolymer with a much higher molecular weight caused an extra peak at the expected elution time.Part I see Ref. [1].Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Separation of copoly(styrene/acrylonitrile) samples according to composition under reversed phase conditions

Summary Copoly(styrene/acrylonitrile) samples (S/AN) have been repeatedly separated according to composition by gradient HPLC with alkane hydrocarbons as a starting eluent A and dichloromethane (DCM) or tetrahydrofuran (THF) as a solvent B. In these systems, retention increased with AN content of the copolymers. The chemical nature of the column packings used had almost no influence on the retention of S/AN samples. The present paper shows thatn-pentane andn-heptane, when used in a given volumetric gradient with DCM+20% methanol as a solvent B, lead to identical solution characteristics of S/AN on silica columns. A similar result was obtained on C18 columns withn-heptane or cyclohexane, whereas gradient elution with toluene as a starting eluent caused insufficient resolution. Reversed phase separation of S/AN copolymers could be achieved on polystyrene gel columns through gradients with methanol as a starting eluent and DCM or THF as a solvent B. In both systems, retention decreased with increasing AN content of the copolymers. The elution characteristics were almost linear in the range 0–20 wt% AN. This behaviour can be understood in the context of polymer solubility: in both systems, the solubility borderline of S/AN has a distinct maximum at about 25 wt% AN. Reversed phase separation was achieved at the lefthand slope of these curves where the dissolution of a sample with a higher AN content requires less DCM or THF solvent than the dissolution of copolymers which are poorer in AN. This idea predicts that samples with more than 25 wt% AN should elute later than S/AN whose composition is near to the solubility maximum. This indeed was found with a copolymer containing 36.2 wt% AN.     

Unusual signal enhancement in reversed-phase chromatography of polystyrene monitored by UV detector

Summary Gradient elution of polystyrene standards on reversed phase C18 columns by methanol/tetrahydrofuran or methanol/dichloromethane mixtures yielded a strange effect of the molecular weight of the sample on the specific peak are (mAUs per g sample injected). The effect did not occur when pure dichloromethane was used as an eluent. Further to this, dependence of specific peak area on flow rate was observed in gradient elution with methanol/tetrahydrofuran mixtures. It was found that these effects were due to polymer elution at the verge of precipitation. Depending on the dwell time of the sample in the column, opalescence occurred that added to the UV signal used for monitoring the elution.     

Synthesis and chromatographic evaluation of phenyl/tetrazole bonded stationary phase based on thiol‐epoxy ring opening reaction

A silica‐based reversed‐phase stationary phase bonding with phenyl and tetrazole groups was synthesized by thiol‐epoxy ring opening reaction. The bonded groups could not only provide hydrophobic interaction, but also π–π, hydrogen bonding, electrostatic interactions, and so on. The results of characterization with elemental analysis and solid‐state ¹³C cross‐polarization magic‐angle‐spinning NMR spectroscopy indicated the successful preparation of phenyl/tetrazole sulfoether bonded stationary phase. Chromatographic evaluation revealed that phenyl/tetrazole sulfoether bonded stationary phase behaved well under the reversed‐phase mode. The column parameters (H, S*, A, B, and C) showed different selectivity compared with some typical commercial columns, and it was validated by the separation of estrogen, ginsenoside, alkaloid samples. Based on the different selectivity between phenyl/tetrazole sulfoether bonded stationary phase and C18 columns, phenyl/tetrazole sulfoether bonded stationary phase also showed potential to construct a 2D reversed‐phase liquid chromatography system with C18. And it was verified by the separation of corydalis tuber and curcuma zedoary extracts.     

Liquid chromatography at the critical condition: Thermodynamic significance and influence of pore size

Liquid chromatography at the critical condition (LCCC) is a high performance liquid chromatography (HPLC) technique that lies between size exclusion chromatography and adsorption-based interaction chromatography, where the elution of polymers becomes independent of polymer molecular weight. At LCCC, the balance between the entropic exclusion and the enthalpic adsorption interactions between polymers and stationary phases results in the simultaneous HPLC elution of polymers regardless of molecular weight. Using C18-bonded silica chromatographic columns with 5 μm particle size and different average pore size (diameter = 300 Å, 120 Å, 100 Å, and 50 Å), we report (1) the thermodynamic significance of LCCC conditions and (2) the influence of column pore size on the determination of critical conditions for linear polymer chains. Specifically, we used mixtures of monodisperse polystyrene samples ranging in molecular weight from 162 to 371,100 g/mol and controlled the temperature of the HPLC columns at a fixed composition of a mobile phase consisting of 57(v/v)% methylene chloride and 43(v/v)% acetonitrile. It was found that, at the fixed mobile phase composition, the temperature of LCCC (T_LCCC) is higher for C18-bonded chromatographic columns with larger average pore size. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 2533–2540, 2009     

Control of adsorption and solubility in gradient high performance liquid chromatography 1. Principles of sudden transition gradients and elution characteristics of copolymers from styrene and methacrylates

Summary Proper retention of polymers in high performance liquid chromatography often requires injection into a starting eluent which is not a solvent for the sample under investigation. In this case, the polymer is precipitated at the top of the column. Subsequent gradient elution has to be performed by addition of an eluent with sufficient chromatographic strength and solvent power. In normal phase chromatography, it must be a solvent of high polarity. With the gradient elutions reported so far, polarity and dissolution power were simultaneously increased.The present paper reports the separate control of solvent strength and chromatographic power by applying gradient programs which include sudden addition of a moderately polar solvent. The amount of the latter does not suffice for elution, which is performed by subsequent, controlled addition of a highly polar nonsolvent. Sudden transition gradients of this kind work with, e.g.,iso-octane as a nonpolar starting eluent, tetrahydrofuran as a solvent of intermediate polarity, and methanol as a strongly polar nonsolvent. They have been applied to copolymers from styrene and ethyl methacrylate, methyl methacrylate, or methoxyethyl methacrylate.     

Rapid determination of molecular parameters of synthetic polymers by precipitation/redissolution high‐performance liquid chromatography using “molded” monolithic column

Rapid high‐performance liquid chromatography (HPLC) of polystyrenes, poly(methyl methacrylates), poly(vinyl acetates), and polybutadienes using a monolithic 50 × 4.6 mm i.d. poly(styrene‐co‐divinylbenzene) column have been carried out. The separation process involves precipitation of the macromolecules on the macroporous monolithic column followed by progressive elution utilizing a gradient of the mobile phase. Depending on the character of the separated polymer, solvent gradients were composed of a poor solvent such as water, methanol, or hexane and increasing amounts of a good solvent such as THF or dichloromethane. Monolithic columns are ideally suited for this technique because convection through the large pores of the monolith enhances the mass transport of large polymer molecules and accelerates the separation process. Separation conditions including the selection of a specific pair of solvent and precipitant, flow rate, and gradient steepness were optimized for the rapid HPLC separations of various polymers that differed broadly in their molecular weights. Excellent separations were obtained demonstrating that the precipitation‐redissolution technique is a suitable alternative to size‐exclusion chromatography (SEC). The molecular weight parameters calculated from the HPLC data match well those obtained by SEC. However, compared to SEC, the determination of molecular parameters using gradient elution could be achieved at comparable flow rates in a much shorter period of time, typically in about 1 min. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2767–2778, 2000     

Problems in the size exclusion chromatography of poly(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide) on styragel columns

The molecular weights of poly(N-isopropylacrylamide) (PNIPA), calculated according to polystyrene calibration standards upon the elution of THF on styragel columns, appear to be much lower than their actual values determined using independent approaches. This is likely due to interactions between the nitrogen-containing units of PNIPA polymer chains and the sorbent, so the polymer is eluted in the mode intermediate between exclusion and critical. An effective exclusion mode during the elution of PNIPA on a styragel column can be achieved by using an eluent more polar than tetrahydrofuran (particularly, 1-methylpyrrolidone).     

Molecular weight characterization of soluble high performance polyimides. 1. Polymer-solvent-stationary phase interactions in size exclusion chromatography

Soluble, fully cyclized m-amino phenyl acetylene terminated polyimides based on several anhydride/diamine monomers were prepared in N-methylpyrrolidine (NMP) and cyclized by solution imidization to controlled molecular weight. The polyimides and a polyamic acid precursor were successfully analyzed by size exclusion chromatography (SEC) utilizing online parallel coupled refractive index and differential viscometer detectors. The calculated M _nvalues were varied from 3,000 to 20,000 daltons. N-methylpyrrolidone (NMP), tetrahydrofuran (THF), and chloroform served as mobile phases for the cross-linked polystyrene gel packings. Normal retention behavior of the polyimides was observed in chloroform, THF, and NMP containing LiBr, or in NMP stirred over P₂O₅ before use. Values of Mark-Houwink-Sakurada exponents for narrow distribution linear polystyrene indicate that pure NMP and NMP with 0.06 M LiBr are good solvents for polystyrene standards at 60°C. In contrast, SEC behavior of polyimides in pure NMP leads to splitting of the peaks with the major portion observed to pass through the columns at the exclusion limit. In contrast to strong polymeric chain expansion of the polyamic acid in dilute solution, presumably due to a polyelectrolyte effect, no increase of intrinsic viscosity of polyimide samples in pure NMP was observed. This exclusion effect of polyimides analyzed in NMP is discussed in terms of possible ion-exclusion from pores of the stationary phase. Differences in polystyrene calibration in NMP with or without additives and the temperature dependence of calibration curves in these mobile phases is discussed as well. ©1995 John Wiley & Sons, Inc.     

Study of molecular motion of block copolymers in solution by high-resolution proton magnetic resonance

Molecular motions of hydrophobic–hydrophilic water-soluble block copolymers in solution were investigated by high-resolution proton magnetic resonance (NMR). Samples studied include block copolymers of polystyrene–poly(ethylene oxide), polybutadiene–poly(ethylene oxide), and poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide). NMR measurements were carried out varying molecular weight, temperature, and solvent composition. For AB copolymers of polystyrene and poly(ethylene oxide), two peaks caused by the phenyl protons of low-molecular-weight (M?_n = 3,300) copolymer were clearly resolved in D₂O at 100°C, but the phenyl proton peaks of high-molecular-weight (M?_n = 13,500 and 36,000) copolymers were too broad to observe in the same solvent, even at 100°C. It is concluded that polystyrene blocks are more mobile in low-molecular-weight copolymer in water than in high-molecular-weight copolymer in the same solvent because the molecular weight of the polystyrene block of the low-molecular-weight copolymer is itself small. In the mixed solvent D₂O and deuterated tetrahydrofuran (THF-d₈), two peaks caused by the phenyl protons of the high-molecular-weight (M?_n = 36,000) copolymer were clearly resolved at 67°C. It is thought that the molecular motions of the polystyrene blocks are activated by the interaction between these blocks and THF in the mixed solvent.     

Spectrometry: Speciation of Parts PER Billion of Metal Ions Using Silica and C18-Bonded Silica Columns and Graphite Furnace Atomic Absorption Spectrometry

Abstract

It is demonstrated that silica gel columns will quantitatively adsorb free Cu²⁺ and Pb²⁺ ions at pH > 8. These are eluted with 0.1 M HNO₃ but not with methanol. Negatively charged EDTA chelates are not adsorbed. Neutral APDC chelates are partially adsorbed on silica columns, but are quantitatively adsorbed on C18-bonded columns, and are eluted with methanol. The metal ions are partially adsorbed on C18-bonded columns, due to residual silanol groups. A microcolumn (1 mm i.d., 5 cm length) manifold system is described for automatic delivery of eluant (0.12 ml) to a heated atomic absorption graphite atomizer, using either methanol or 0.1 M HNO₃ in methanol eluant, allowing speciation and measurement of parts per billion of metals. These studies demonstrate that by using a mixed column or sequential columns of silica gel and C18-bonded silica, cationic and neutral metal species could be adsorbed, followed by sequential elution and measurement using methanol and then 0.1 M HNO. Negatively charged species could be measured directly in the sample eluant or obtained by difference from a total metal measurement.     

Effect of Alkyl Chain Length of Bonded Silica Phases on Separation,Resolution and Efficiency in High Performance Liquid Chromatography

Abstract

A comparative study of alkyl bonded phases was carried out under optimum solvent conditions for each phase. Three columns, RP-2, RP-8 and RP-18, were tested for their efficiency and resolving power using three groups of compounds in three binary organic-water mobile phases. The organic solvents were acetonitrile, methanol and tetrahydrofuran which are widely used as solvent modifiers.

The results indicate that each of the three factors, i.e. solvent, solute and bonded alkyl chain length, play an important role, with the solvent being the most significant. When tetrahydrofuran-water was used as the mobile phase, the ratio of THF/H₂O did not vary by much when an RP-2, RP-8 or RP-18 column was used to separate naphthalene from biphenyl, dimethylphthalate from diethylphthalate or anthraquinone from methyl, anthraquinone and ethyl anthraquinone. When acetonitrile-water and methanol-water were used the ratio of organic modefier to water changed so as to accomodate the hydrophobic properties of the columns. The efficiency of the columns, expressed as theoretical plates per meter (TPM) was highest when acetonitrile-water was used as the mobile phase. Although there were variations in TPM and resolution from column to column, the three columns gave good separation of the components of the three groups of compounds.     

Enantioseparation of planar chiral ferrocenes on cellulose-based chiral stationary phases: Benzoate versus carbamate pendant groups

In this study, the enantioseparation of 14 planar chiral ferrocenes containing halogen atoms, and methyl, iodoethynyl, phenyl, and 2-naphthyl groups, as substituents, was explored with a cellulose tris(4-methylbenzoate) (CMB)-based chiral column under multimodal elution conditions. n-Hexane/2-propanol (2-PrOH) 95:5 v/v, pure methanol (MeOH), and MeOH/water 90:10 v/v were used as mobile phases (MPs). With CMB, baseline enantioseparations were achieved for nine analytes with separation factors (α) ranging from 1.24 to 1.77, whereas only three analytes could be enantioseparated with 1.14 ≤ α ≤ 1.51 on a cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC)-based column, used as a reference for comparison, under the same elution conditions. Pendant group–dependent reversal of the enantiomer elution order was observed in several cases by changing CMB to CDMPC. The impact of analyte and chiral stationary phase (CSP) structure, and MP polarity on the enantioseparation, was evaluated. The two cellulose-based CSPs featured by different pendant groups were also compared in terms of thermodynamics. For this purpose, enthalpy (ΔΔH°), entropy (ΔΔS°) and free energy (ΔΔG°) differences, isoenantioselective temperatures (T_iso), and enthalpy/entropy ratios (Q), associated with the enantioseparations, were derived from van ’t Hoff plots by using n-hexane/2-PrOH 95:5 v/v and methanol/water 90:10 v/v as MPs. With the aim to disclose the functions of the different substituents in mechanisms and noncovalent interactions underlying analyte–selector complex formation at molecular level, electrostatic potential (V) analysis and molecular dynamics simulations were used as computational techniques. On this basis, enantioseparations and related mechanisms were investigated by integrating theoretical and experimental data.     

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18相似文献   

填充柱超临界流体色谱系统中的溶剂效应

 考察了填充柱超临界流体色谱法 (SFC)中的样品溶剂及连续进样等因素对化合物保留行为变化的影响规律。以超临界 CO2 或含低体积分数甲醇的 CO2 为流动相时 ,氨基柱上组分的保留时间随着样品溶剂的极性增大而增大 ,而溶剂对 C1 8柱上组分的保留时间影响不大 ;在 C1 8柱上 ,溶剂对连续进样的后续效应不强 ;而在氨基柱上 ,甲醇溶液的后续效应比丙酮、氯仿溶液的后续效应强。当甲醇的体积分数大于 1 .0 %时 ,溶剂的效应明显减弱。这种变化规律对填充柱 SFC的合理进样并获得重现性良好的色谱数据具有实际意义。     

Performance comparison between packed column supercritical fluid chromatography (SFC) and HPLC using cyclandelate as the model analyte

A reproducible and fast method has been developed for the assay of cyclandelate in bulk and drug forms using packed column supercritical fluid chromatography using dicyclohexyl phthalate (DCHP) as internal standard. The drug and the internal standard were resolved by elution with supercritical fluid carbon dioxide doped with 14.29% (v/v) methanol on an RP-C₁₈ column and detected spectrophotometrically at 228 nm. Chromatographic figures of merit using C₈, C₁₈, cyano and phenyl columns have been assessed. Parallel experiments have been performed by HPLC and the data have been compared. Supercritical fluid extraction using CO₂ modified with a small amount of methanol was found to give quantitative analytical recoveries of cyclandelate from a dosage form. SFC has been shown to be a viable, faster alternative technique to HPLC generating less disposable waste.     

Investigating retention characteristics of a mixed‐mode stationary phase and the enhancement of monolith selectivity for high‐performance liquid chromatography

The synthesis and chromatographic behavior of an analytical size mixed‐mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3‐cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance π‐type bonding within a single separation environment. Selectivity studies were undertaken using n‐alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the π‐type selectivities afforded by the two different ligands on the mixed‐mode column.     

Performance comparison between packed column supercritical fluid chromatography (SFC) and HPLC using cyclandelate as the model analyte

A reproducible and fast method has been developed for the assay of cyclandelate in bulk and drug forms using packed column supercritical fluid chromatography using dicyclohexyl phthalate (DCHP) as internal standard. The drug and the internal standard were resolved by elution with supercritical fluid carbon dioxide doped with 14.29% (v/v) methanol on an RP-C₁₈ column and detected spectrophotometrically at 228 nm. Chromatographic figures of merit using C₈, C₁₈, cyano and phenyl columns have been assessed. Parallel experiments have been performed by HPLC and the data have been compared. Supercritical fluid extraction using CO₂ modified with a small amount of methanol was found to give quantitative analytical recoveries of cyclandelate from a dosage form. SFC has been shown to be a viable, faster alternative technique to HPLC generating less disposable waste. Received: 20 June 1997 / Revised: 20 October 1997 / Accepted: 26 October 1997     

Comparison of a C30 Bonded Silica Column and Columns with Shorter Bonded Ligands in Reversed-Phase LC

The Carotenoid S is a new C₃₀ bonded silica stationary phase, intended for reversed-phase chromatographic applications, which is more hydrophobic and consequently shows stronger retention in comparison to conventionally used C₁₈ stationary phases. We compared the non-polar selectivities of the columns for homologous alkylbenzenes in acetonitrile—water and methanol–water mobile phases and polar reversed-phase selectivities employing the interaction indices and the Linear Free Energy Relationship models. Further, we investigated possibilities of separations of structurally closely related compounds in the groups of phenolic acids, flavones, phthalic acids and related compounds and of acylglycerols on the new C₃₀ column and with different types of columns for reversed-phase chromatography, including shorter alkyl C₄, C₈, C₁₈ and phenyl bonded stationary phases. The C₃₀ column has in some aspects properties similar to the non-endcapped Nova-Pak column for separation of some acylglycerols with equal equivalent carbon numbers, but enables separations of longer chain triacylglycerols in a single gradient run.

     

Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques

The overall peak capacity in comprehensive two-dimensional liquid chromatographic (LC x LC) separation can be considerably increased using efficient columns and carefully optimized mobile phases providing large differences in the retention mechanisms and separation selectivity between the first and the second dimension. Gradient-elution operation and fraction-transfer modulation by matching the retention and the elution strength of the mobile phases in the two dimensions are useful means to suppress the band broadening in the second dimension and to increase the number of sample compounds separated in LC x LC. Matching parallel gradients in the first and second dimension eliminate the necessity of second-dimension column re-equilibration after the independent gradient runs for each fraction, increase the use of the available second-dimension separation time and can significantly improve the regularity of the coverage of the available retention space in LC x LC separations, especially with the first- and second-dimension systems showing partial selectivity correlations. Systematic development of an LC x LC method with parallel two-dimensional gradients was applied for separation of phenolic acids and flavone compounds. Several types of bonded C18, amide, phenyl, pentafluorophenyl and poly(ethylene glycol) columns were compared using the linear free energy relationship method to find suitable column combination with low correlation of retention of representative standards. The phase systems were optimized step-by-step to find the mobile phases and gradients providing best separation selectivity for phenolic compounds. The optimization of simultaneous parallel gradients in the first and second dimension resulted in significant improvement in the utilization of the available two-dimensional retention space.