Evaluation of liquid chromatography column retentivity using macromolecular probes. III. Partition properties of C18 phases traced by polymers

Application of polymeric probes was proposed for evaluation of partition properties of the high performance liquid chromatographic stationary phases. The approach was tested with selected silica gel C-18 column packings. Polystyrene (PS) and poly(n-butyl methacrylate) (PnBMA) narrow molar mass standards of low polarity were applied to avoid adsorption of macromolecules on silanols and other polar groups present within column packings. Polar eluent components further reduced contingency of silanophilic interactions. The major eluent component was dimethylformamide (DMF), a thermodynamically poor solvent for polymer probes, which strongly promoted enthalpic partition of macromolecules in favor of the C18 bonded phase. Methyl ethyl ketone (MEK) and diethyl malonate (DEM) were also tested as the partition promoting eluent components. With polystyrenes, MEK was rather inefficient as a partition promoter while DEM was similarly active as DMF. A thermodynamically good solvent for polymer probes, viz. tetrahydrofuran (THF) was added to eluent to reduce and control the extent of partition. The differences in elution behavior of column tested indicate their unlike partition properties.     

Studies on high-performance size-exclusion chromatography of synthetic polymers. I. Volume of silica gel column packing pores reduced by retained macromolecules

Macromolecules, which stay adsorbed within the active size-exclusion chromatography (SEC) column packings may strongly reduce effective volume of the separation pores. This brings about a decrease of retention volumes of the non-retained polymer samples and results in the increased apparent molar mass values. The phenomenon has been demonstrated with a series of poly(methyl methacrylate)s (PMMA) and a polyethylenoxide (PEO) fully retained by adsorption within macroporous silica gel SEC column from toluene or tetrahydrofuran, respectively. The non-retained probes were polystyrenes (PS) in toluene and both PS and PMMA in THF eluents. The errors in the peak molar mass values determined for the non-retained polymer species using a column saturated with adsorbed macromolecules and considering calibration curves monitored for the original "bare" column packing assumed up to several hundreds of percent. Errors may appear also in the weight and number averages of molar masses calculated from calibration dependences obtained with columns saturated with adsorbed macromolecules. Moreover, the SEC peaks of species eluted from the polymer saturated columns were broadened and in some cases even split. These results demonstrate a necessity not only to periodically re-calibrate the SEC columns but also to remove macromolecules adsorbed within packing in the course of analyses.     

Evaluation of high-performance liquid chromatography column retentivity using macromolecular probes I

The application of macromolecular probes is proposed for evaluation of HPLC column retentivity. The idea is tested with a set of different commercial silica C₁₈ reversed-phases. For comparison, porous glass C₁₈ and polystyrene/divinylbenzene column packings are also included. Polar, mainly silanophilic interactions are evaluated. The retention volumes of a series of narrow molar mass distribution polystyrenes (PS) and poly(methyl methacrylate)s (PMMA) in toluene eluent are compared. Toluene is a weak mobile phase concerning silica gel surface and it promotes adsorption of PMMA on silanols, while PS is not adsorbed from toluene. Simultaneously, toluene is a thermodynamically good solvent for both polymers so that extensive partition in favour of stationary phase is not probable. Differences in retention behaviour of PS and PMMA indicate presence of abundant free silanols on the surface of some reversed-phases. These silanols are accessible even for large macromolecules of PMMA. Pore diameter and pore volume of the column packing can be semiquantitatively evaluated from the elution data of PS in toluene in the course of retentivity tests.     

Enthalpy assisted size exclusion chromatography. Part 2. Adsorption retention mechanism

A novel high performance liquid chromatographic method for separation of synthetic polymers has been tested. It involves combination of the enthalpic and entropic retention mechanisms, resulting in increased selectivity of separation within a specific molar mass range. In this present case, the enthalpic retention mechanism is adsorption of macromolecules on a bare silica gel column packing. Under critical conditions of enthalpic interactions, homopolymers are known to elute irrespective of their molar mass. However, in the vicinity of critical conditions, a situation can be identified when retention volumes (V(R)) rapidly decrease with increasing molar mass. Typically, this happens for polymer species close to or above their exclusion limit observed with the same column in the absence of enthalpic interactions between macromolecules and packing, that is near "ideal SEC" conditions. The dependence of polymer retention volume on molar mass closely resembles size exclusion conditions. However, the witnessed rate of change in V(R )with polymer molar mass is more pronounced, thus indicating increased selectivity of separation. This situation not only offers the benefit of more selective separation according to molar mass but efficient discrimination of macromolecules possessing different nature and interactivity with the column packing can be accomplished as well.     

Liquid chromatography of macromolecules at the critical adsorption point. II. Role of column packing: Bare silica gel

Liquid chromatography of macromolecules at the critical adsorption point (LC CAP) presents a potentially very powerful method for molecular characterization of complex polymers. However, LC CAP applicability is limited due to various experimental problems. The pore sizes and surface chemistry of the column packings belong to the most important weak points of the method. The LC CAP behavior of poly(methyl methacrylate)s was investigated using bare silica gels of 6, 12, and 100 nm pore sizes and with various amounts of surface silanols. Tetrahydrofuran as the adsorption suppressing liquid and toluene as the adsorption promoting liquid were mixed to form the “nearly critical” eluents. Both pore size and surface chemistry of silica were found to strongly influence the retentive characteristics of the system in the critical adsorption area. Macromolecules that were large enough to be excluded from the packing pores hardly followed the LC CAP rules: their retention volumes changed irregularly with the polymer molar mass and their recovery dropped sharply. The narrow pore silica gel-packed column governed the elution patterns of the whole column set composed of silica gels with different pore sizes. This makes the conventional LC CAP characterization of common polymers with broader molar mass distribution impractical and even not feasible. A hybrid column system was proposed containing narrow pore nonadsorptive column added in series to the meso- and macroporous LC CAP silica gels. This narrow pore column would allow separation of gas, impurities, and system peaks from the polymer peaks. The possible successive changes of the surface of silica gel, e.g., due to formation of silanols by hydrolysis or due to irreversible adsorption of some admixtures from the sample or eluent may make the LC CAP irrepeatable. Pronounced peak broadening was observed in the critical adsorption area and this effect increased strongly with the polymer molar mass. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 1363–1371, 1998     

Liquid chromatography of synthetic polymers under critical conditions of enthalpic interactions 4: Sample recovery

Reduced sample recovery is a frequent feature of LC of macromolecules under critical conditions of enthalpic interactions (LC CC). Several methods of assessment of LC CC sample recovery are compared. A novel approach is based on an online combination of the. The LC CC column with a noninteractive SEC column. It provides not only the amount but also the molar mass of the eluted/withheld polymer. The procedure was tested with poly(methyl methacrylate), bare silica gel column packings, and “critical eluent” tetrahydrofuran/toluene. It was shown that macromolecules with higher molar masses were preferentially trapped within the LC CC column packing so that the eluted part of the sample was no longer representative. The incomplete polymer elution can make the LC CC polymer analyses susceptible to significant experimental errors.     

Role of adsorption in liquid chromatography of macromolecules and potential of liquid chromatography in assessing adsorption of macromolecules onto solid surfaces

Many liquid chromatographic (LC) separations of macromolecules are influenced or directly based on adsorption of solutes on column packing. In the case of well known size exclusion chromatography (SEC), adsorption effects are usually unwanted and therefore suppressed. Still they appear in many SEC systems and may badly affect precision of results obtained. In other LC methods applicable to high polymers, adsorption is deliberately combined with exclusion. The aim is to discriminate complex polymer systems which exhibit more than one single distribution of their molecular characteristics. The main goals of such combinations include either a controlled increase or a full suppression of separation selectivity according to one molecular characteristics. Most important so far known exclusion-adsorption compensation methods allowing to suppress dependence of LC retention volumes on polymer molar mass are reviewed. The discussion is accomplished with a presentation of newly developed full adsorption - desorption (FAD) method which can be combined with various LC procedures. A very useful combination represents the on-line FAD/SEC procedure which enables also to study adsorption and desorption phenomena in the systems solid surface - solvent - macromolecules.     

Liquid chromatography of polymers under limiting conditions of desorption II. Tandem injection and quantitative molar mass determination

Liquid chromatography under limiting conditions of desorption (LC LCD) is a method which allows molar mass independent elution of various synthetic polymers. A narrow, slowly moving zone of small molecules, which promotes full adsorption of one kind of polymer species within column (an adsorli) acts as an impermeable barrier for the fast moving macromolecules. The latter accumulate on the barrier edge and elute nearly in total volume of liquid within column. At the same time, transport of less adsorptive macromolecules is not hampered so that these are eluted in the size exclusion (SEC) mode. As result, polymers differing in their polarity and adsorptivity can be easily separated without molar mass interference. Three methods of barrier creation are discussed and compared. It is shown that a fraction of sample may elute unretained if the adsorli sample solvent is used as a barrier in connection with a narrow-pore column packing. One part of excluded macromolecules likely breaks-out from the adsorli zone and this results in partial loss of sample and distortion of the LC LCD peaks. This problem can be avoided if the adsorli zone is injected immediately before sample solution. Applicability of the LC LCD method for polymer separation has been demonstrated with a model mixture of poly(methyl methacrylate) (adsorbing polymer) and polystyrene (non adsorbing polymer) using bare silica gel as a column packing with a combination of tetrahydrofuran (a desorption promoting liquid -a desorli) and toluene (adsorli). It has been shown that the LC LCD procedure with tandem injection allows simple and fast discrimination of polymer blend components with good repeatability and high sample recovery. For quantitative determination of molar masses of both LC LCD and SEC eluted polymers, an additional size exclusion chromatographic column can be applied either in a conventional way or in combination with a multi-angle light scattering detector. A single eluent is used in the latter column, which separates the mixed mobile phase, system peaks and the desorli zone from the polymer peaks so that measurements are free from disturbances caused by the changing eluent composition. The resulting LC LCD x SEC procedure has been successfully applied to poly(methyl methacrylate) samples.     

Adsorption and desorption of macromolecules on solid surfaces studied by on-line size exclusion chromatography 2. Preferential adsorption and exchange processes

Preferential and exchange adsorption of polymers differing in molar mass and/or chemical nature under dynamic conditions were investigated using on-line size-exclusion chromatography (SEC). The sample investigated dissolved in an appropriate solvent was injected into a small adsorption–desorption column packed with nonporous silica. A nonadsorbed or desorbed fraction of the polymer was directed into an SEC column for determination of both the amount and the molecular characteristics. This approach is in many aspects superior to other techniques for studies of polymer adsorption onto solid surfaces due to its low sample and time consumption. At a low degree of surface coverage, adsorption and desorption of macromolecules were rapid and were affected by the rate of supply of macromolecules to the adsorbent surface. The exchange between macromolecules at the stage of surface saturation was found to depend on the mean molar masses of preadsorbed and displacing polymer species and possibly also on the chain flexibility of the macromolecules. It was shown that the preferential adsorption driven by the chain-length difference upon saturation of the adsorbent surface was more noticeable if the preadsorbed macromolecules were smaller. Received: 7 April 1999 Accepted in revised form: 21 July 1999     

Liquid chromatography of polymer mixtures applying a combination of exclusion and full adsorption mechanisms. 5. Six-component blends of chemically similar polymers

The separation of six-component blends of chemically similar homopolymers utilising the full adsorption-desorption (FAD) process is presented. The main advantage of the FAD approach over other methods represents the successive and independent size- exclusion chromatography (SEC) characterisation of all blend components. The method is based on the full adsorption and retention of all n or n−1 components of the polymer blend from an adsorption promoting liquid (ADSORLI) in a small FAD column. Nonadsorbed macromolecules are forwarded directly into SEC for molecular characterisation. Next, appropriate displacers are successively applied to the FAD column to selectively release preadsorbed blend constituents into the on-line SEC column. Dynamic integral desorption isotherms for single constituents, as well as for polymer blends to be analysed, allow identification of optimal displacer compositions to release just one kind of macromolecule. Model polymer blends containing polystyrene (PS), poly(lauryl methacrylate), poly(butyl methacrylate), poly(ethyl methacrylate), poly(methyl methacrylate) and poly(ethylene oxide) (PEO) or, alternatively, PS, poly(2-ethylhexyl acrylate), poly(butyl acrylate), poly(ethyl acrylate), poly(methyl acrylate) and PEO of similar molar masses can be separated and characterised in one multistep run using nonporous silica FAD packing, toluene as an ADSORLI and its mixtures with a desorption promoting liquid such as ethyl acetate, tetrahydrofuran or dimetylformamide to form displacers with appropriate desorption strength. Received: 9 September 1998 Accepted in revised form: 16 November 1998     

Liquid chromatography of polymers under limiting conditions of adsorption. IV. Sample recovery

The high performance liquid chromatography of polymers under limiting conditions of adsorption (LC LCA) separates macromolecules, either according to their chemical structure or physical architecture, while molar mass effect is suppressed. A polymer sample is injected into an adsorption-active column flushed with an adsorption promoting eluent. The sample solvent is a strong solvent which prevents sample adsorption. As a result, macromolecules of sample elute within the zone of their original solvent to be discriminated from other, non-adsorbing polymer species, which elute in the exclusion mode. LC LCA sample recovery has been studied in detail for poly (methyl methacrylate)s using a bare silica gel column and an eluent comprised toluene (adsorli) and tetrahydrofuran (desorli). Sample solvent was tetrahydrofuran. It was found that a large part of injected sample may be fully retained within the LC LCA columns. The amount of retained polymer increases with decreasing packing pore size and with higher sample molar masses and, likely, also with the column diameter. The extent of full retention of sample does not depend of sample volume. An additional portion of the injected desorli sample solvent (a tandem injection) does not fully eliminate full retention of the sample fraction and the reduced recovery associated with it. The injected sample is retained along the entire LC LCA column. The reduced sample recovery restricts applicability of many LC LCA systems to oligomers and to discrimination of the non-adsorbing minor macromolecular components of complex polymer mixtures from the adsorbing major component(s). The full retention of sample molecules within columns may also complicate the application of other liquid chromatographic methods, which combine entropic and enthalpic retention mechanisms for separation of macromolecules.     

含酯基包覆聚合物液相色谱柱填料

以丙烯酸甲酯或辛酯和二乙烯苯为原料，在溶液中用游离基聚合法制备了一系列含酯基包覆聚合物反相液相色谱柱填料．用傅立叶红外光谱、电子显微镜和元素分析鉴定了聚合物层，并评价了诸如硅羟基、柱压降、柱效和峰对称性等特性．该类填料适合于含氨基和羟基化合物的分离，作为应用实例，对洛伐他汀（Lovastatin）的分析展示其优良的色谱性能。     

Enthalpic Partition-Assisted Size Exclusion Chromatography: 1. Principle of Method

A novel high performance liquid chromatographic (HPLC) method viz. “enthalpic partition assisted size exclusion chromatography” deliberately combines entropic and enthalpic partition mechanisms. It enables separation of homopolymers according to their molar mass with increased selectivity, as well as discrimination of polymer species differing in their nature/composition. Enthalpic partition of macromolecules takes place between the mobile phase and the stationary “liquid” of a different chemical nature, which is immobilized within pores of an appropriate carrier (a bonded phase). The extent of enthalpic partition depends on the accessibility of bonded phase for macromolecules and on the difference of polymer solubility in the mobile phase and in the solvated bonded phase. The enthalpic partition in favor of column packing arises from better solubility of polymer solutes in the solvated stationary phase compared to the mobile phase. Macromolecules are “pushed” into the solvated stationary phase and their retention volumes (V_R) increase. In the area of high molar masses, the extent of enthalpic partition as rule raises with the increasing size of macromolecules. However, under properly chosen experimental conditions the enthalpic partition may rapidly diminish with the sample molar mass (M), likely due to the solubility changes and/or due to partial exclusion of macromolecules from the pores. As result, the corresponding retention volumes sharply drop within a narrow range of M with the increasing size of macromolecules. This results in the log M vs. V_R dependences, which resemble in their form that for size exclusion chromatography but are much more flat indicating highly selective separations of homopolymers according to their molar masses. In this way, enthalpic partition “assists” entropic partition (size exclusion). Polymer species, which do not undergo enthalpic partition, elute from the HPLC column in the conventional size exclusion mode and can be discriminated from the partitioning species. Enthalpic partition assisted size exclusion chromatography can be utilized in separation and characterization of various homopolymers, and polymer blends.     

高分子覆盖型硅胶填料的制备与色谱保留行为

 采用甲基丙烯酸甲酯 (MMA)或MMA 亚乙基二甲基丙烯酸酯 (EDMA)在硅胶表面聚合的方法 ,制备生成了新型高分子覆盖型硅胶填料C或D。借助红外光谱、元素分析、尺寸排阻色谱和反相液相色谱分析对反应过程、覆盖程度、交联剂的影响和填料的色谱保留行为进行了评价和讨论。结果显示 ,在合成时可以通过控制聚合单体的量控制生成高分子层的厚度 ,而合成中加入交联剂可以改变填料表面的微孔构造。通过考察这种色谱填料的疏水性和对芳香族化合物的分离性能 ,认为其柱效和分离效果接近C18填料的性能。     

Packed column supercritical fluid chromatography with light-scattering detection. II. Retention behaviour of squalane and glucose with mixed mobile phases

Summary Evaporative light scattering detectors can be used to detect organic substances without chromophoric groups in packed column supercritical fluid chromatography (SFC). A detector of this type has been used to detect squalane and glucose after SFC with various packed columns and binary mobile phases. In this study, the amount of organic modifier in carbon dioxide/modifier mixtures was varied. The results give further insight into the mechanisms that influence retention behaviour in packed column separations with super- and subcritical mobile phases. Squalane is an ideal non-polar test solute which shows long retention times on non-polar columns while its elution can be accelerated by non-polar modifiers in carbon dioxide. Glucose is an extremely polar solute containing hydroxyl groups. Elution of this sugar can be improved with polar modifiers. Column packings with polar end groups lead to high capacity ratios and long retention times for glucose. Most columns used in this study contained silica-based packing materials. For purposes of comparison, a polymeric packing (HEMA RP-18) was also employed.     

Reversed Phase High-Performance Liquid Chromatography of Basic Drugs on a Silanol Deactivated Support

Abstract

When polar and non-polar basic drugs were separated by reversed phase HPLC, a short-chain silanol deactivated (SCD-100) reversed phase column gave superior results over a standard C18 reversed phase column. The nature of silane, the type of silica and the chemistry of endcapping influenced chromatographic behavior. The peaks in the chromatogram obtained from this column had excellent peak shapes and eluted at predictable retention times, indicating that no silanols remained on the surface. The mobile phase was composed of phosphate buffer at pH 3.5, with variable amounts of methanol. All applications were isocratic without the addition of silanol suppressing reagents.     

Retention and selectivity effects caused by bonding of a polar urea-type ligand to silica: a study on mixed-mode retention mechanisms and the pivotal role of solute-silanol interactions in the hydrophilic interaction chromatography elution mode

The separation properties of five silica packings bonded with 1-[3-(trimethoxysilyl)propyl]urea in the range of 0–3.67 μmol m⁻² were investigated in the hydrophilic interaction chromatography (HILIC) elution mode. An increase of the ligand surface density promoted retention of non-charged polar compounds and even more so for acids. An opposite trend was observed for bases, while the amphoteric compound tyrosine exhibited a U-shaped response profile. An overall partitioning retention mechanism was incompatible with these observations; rather, the substantial involvement of adsorptive interactions was implicated. Support for the latter was provided by column-specific changes in analyte retention and concomitant selectivity effects due to variations of salt concentration, type of salt, pH value, organic modifier content, and column temperature. Silica was more selective for separating compounds differing in charge state (e.g. tyramine vs. 4-hydroxybenzoic acid), while in cases where structural differences of solutes resided in non-charged polar groups (e.g. tyramine vs. 5-hydroxydopamine, nucleoside vs. nucleobase) more selective separations were obtained on bonded phases. Hierarchical cluster analysis of the home-made urea-type and three commercial amide-type bonded packings evinced considerable differences in separation properties. The present data emphasise that the role of the packing material under HILIC elution conditions is hardly just the polar support for a dynamic coating with a water-enriched layer. Three major retention mechanisms are claimed to be relevant on bare silica and the urea-type bonded packings: (i) HILIC-type partitioning, (ii) HILIC-type weak adsorption such as hydrogen bonding between solutes and ligands or solutes and silanols (potentially influenced by individual degrees of solvation, salt bridging, etc.), (iii) strong electrostatic (ionic) solute–silanol interactions (attractive/repulsive). Even when non-charged polar bonded phases are used, solute–silanol interactions should not be discounted, which makes them a prime parameter to be characterised by HILIC column tests. Multi/mixed-mode type separations seem to be common under HILIC elution conditions, associated with a great deal of selectivity increments. They are accessible and controllable by a careful choice of the type of packing, the mobile phase composition, and the temperature.     

Separation of Proteins on Polar Bonded Phases by Hydrophobic Interaction Chromatography

Abstract

Hydrophobic interaction chromatography (HIC) with a polar bonded phase (“Acetamide”) developped for size exclusion chromatography (SEC) is described. Retention of proteins depends on the surface area of the stationary phase, the pH and ionic strength of the eluent. For efficient separation the pore diameter should be 25 nm or more. The surface area should be large to achieve retention even at low ionic strength. Separation is only possible with a gradient from high to low ionic strength. Gradient volumes of 10 empty column volumes with column lengths above 15 cm are recommended. Selectivity can be optimized via pH adjustment. The advantage of this column packing is its applicability for two different separation modes: SEC and HIC.     

HPLC of nucleotides. II. General methods and their development for analysis and preparative separation. An approach to selectivity control

In order to develop efficient separation methods for nucleotides according to their size and heterocyclic composition, the application of ion-exchange, reverse-phase, and normal-phase adsorption HPLC has been studied. The comparative investigation of retention power and selectivity of various packings (non-polar bonded-phase and amino silicas) in relation to nucleotide length and composition yields data which enable suitable packings to be selected and a method of preparing the new packing for a particular separation problem to be formulated. Thus a new anion exchanger with high selectivity and dynamic mass transfer has been prepared for fractionation of large oligonucleotides. The effect of the eluent pH and composition (organic modifier, salt) on retention, selectivity, and resolution in ion-exchange and reverse-phase HPLC has been studied. The optimum separation conditions comprise elution with oppositely directed gradients of the salt and the modifier, use of a precolumn packing that provides the best protection for the main column without loss of its efficiency, and the optimum gradient program for the desired retention of the component of interest. The relation between loading and sample concentration has been studied and the system for gradient elution improved. Our work shows that two-dimensional separation is the most reliable and informative method for preparation of homogeneous oligonucleotides. The hydrophobic-pair ion-exchange mechanism is proposed for ion-pair chromatography. Protected and partially deblocked oligonucleotides, chemically synthesized for genetic engineering studies, have been separated with high selectivity by adsorption (normal-phase) HPLC which is efficient for gradient elution with isohydric eluents. The analysis of a monomeric composition of nano-(pico-) molar amounts of oligonucleotides has been developed; the procedure involves microcolumn digestion of the oligonucleotides with immobilized enzymes followed by microcolumn separation of the nucleoside-mononucleotide mixture. Also, a new slurry method for packing stable HPLC columns with a tightly consolidated, nonshrinkable bed of particles has been developed.