A vacuum assisted dynamic evaporation interface for two-dimensional normal phase/reverse phase liquid chromatography

A vacuum assisted dynamic solvent evaporation interface for coupling of two-dimensional normal phase/reverse phase liquid chromatography was developed and evaluated. A normal-phase liquid chromatographic (NPLC) column of a 250 mm × 4.6 mm I.D. 5 μm CN phase was used as the first dimension, and a reversed-phase liquid chromatographic (RPLC) column of 250 mm × 4.6 mm I.D. 5 μm C₁₈ phase was used as the second dimension. The eluent from the first dimension flowed into a fraction loop, and the solvent in the eluent was dynamically evaporated and removed by vacuum as it was entering the fraction loop of the interface. The non-evaporable analytes was retained and enriched in about 5–25 μL solution within the loop. Up to 1 mL/min of mobile phase from the first dimension can be evaporated and removed dynamically by the interface. The mobile phase from the second dimension then entered the loop, and dissolved the concentrated analytes retained inside the loop, and carried them onto the second dimension column for further separation. The operation conditions of the two dimensions were independent from each other, and both dimensions were operated at their optimal chromatographic conditions. We evaluated the interface by controlling the loop temperature in a water bath at normal temperature, and investigated the sample losses by using standard samples with different boiling points. It was found that the sample loss due to evaporation in the interface was negligible for non-volatile samples or for components with boiling point above 340 °C. The interface realizes fast solvent removal of mL volume of fraction and concentration of the fraction into tenth of μL volume, and injection of the concentrated fraction on the secondary column. The chromatographic performance of the two-dimensional LC system was enhanced without compromise of separation efficiency and selectivity on each dimension.     

Two‐dimensional liquid chromatography with active solvent modulation for studying monomer incorporation in copolymer dispersants

A pseudo‐comprehensive two‐dimensional liquid chromatography approach with size exclusion chromatography in the first dimension and gradient reversed‐phase liquid chromatography in the second dimension was successfully developed for the characterization of vinyl acetate/acrylic acid copolymers and vinyl acetate/itaconic acid/acrylic acid terpolymers. Active solvent modulation was exploited to prevent the polymer breakthrough in the second dimension separation caused by the strong solvent used in the first dimension. The conditions of the active solvent modulation valve were optimized to achieve sufficient on‐line dilution and to completely prevent polymer breakthrough without adding excessive time to the modulation cycle. Using this approach, copolymers made with different monomer ratios and processes were studied. Heterogeneous composition distribution due to insufficient monomer incorporation was detected in some of the copolymer samples. We demonstrated that with active solvent modulation, the two‐dimensional liquid chromatography approach is no longer limited to water‐soluble polymers and can be used for a broader range of polymers and copolymers.     

Two-dimensional liquid chromatography analysis of synthetic polymers using fast size exclusion chromatography at high column temperature

In recent years, two-dimensional liquid chromatography (2D-LC) has been used increasingly for the analysis of synthetic polymers. A 2D-LC analysis provides richer information than a single chromatography analysis at the cost of longer analysis time. The time required for a comprehensive 2D-LC analysis is essentially proportional to the analysis time of the second dimension separation. Many of 2D-LC analyses of synthetic polymers have employed size exclusion chromatography (SEC) for the second-dimension analysis due to the relatively short analysis time in addition to the wide use in the polymer analysis. Nonetheless, short SEC columns are often used for 2D-LC analyses to reduce the separation time, which inevitably deteriorates the resolution. In this study, we demonstrated that high temperature SEC can be employed as an efficient second-LC in the 2D-LC separation of synthetic polymers. By virtue of high temperature operation (low solvent viscosity and high diffusivity of the polymer molecules), a normal length SEC column can be used at high flow rate with little loss in resolution.     

Influence of temperature on peak shape and solvent compatibility: implications for two-dimensional liquid chromatography

Solvent compatibility is a limiting factor for the success of two-dimensional liquid chromatography (2-D LC). In the second dimension, solvent effects can result in overpressures as well as in peak broadening or even distortion. A peak shape study was performed on a one-dimensional high-performance liquid chromatography (HPLC) system to simulate the impact of peak distorting solvent effects on a reversed-phase second dimension separation operated at high temperatures. This study includes changes in injection volume, solute concentration, column inner diameter, eluent composition and oven temperature. Special attention was given to the influence of high temperatures on the solvent effects. High-temperature HPLC (HT-HPLC) is known to enhance second dimension separations in terms of speed, selectivity and solvent compatibility. The ability to minimise the viscosity contrast between the mobile phases of both dimensions makes HT-HPLC a promising tool to avoid viscosity mismatch effects like (pre-)viscous fingering. In case of our study, viscosity mismatch effects could not be observed. However, our results clearly show that the enhancement in solvent compatibility provided by the application of high temperatures does not include the elimination of solvent strength effects. The additional peak broadening and distortion caused by this effect is a potential error source for data processing in 2-D LC.     

Quantitative aspects of gradient HPLC of copolymers from styrene and ethyl methacrylate

Summary Prerequisite of quantitative evaluation in chromatography is equivalence of sample composition and detector signal. This includes complete retention and proper elution of all sample constituents. In polymer HPLC, complete retention requires a poor starting eluent, a sufficiently active column, and a low ratio of injection volume to column volume. On small pore columns, insufficient retention caused the polymer to elute either in the interstitial volume (sample exclusion), together with the sample solvent, or immediately after the solvent plug.Stat-copoly(styrene/ethyl methacrylate) samples are more difficultly retained thanstat-copoly(styrene/acrylonitrile) specimes. With the former copolymer it could be shown that incomplete retention did not cause sample demixing. In order to gain complete retention, non-exclusion HPLC of polymers should be performed with columns whose solvent volume is at least 50 times as large as the injection volume. This consequence is of practical importance in chromatographic cross-fractionation where rather large volumes of SEC eluate are injected into the apparatus for gradient HPLC.     

Reduced sample recovery in liquid chromatography at critical adsorption point of high molar mass polystyrene

Low sample recovery may represent an important drawback in liquid chromatography at the critical adsorption point (LC-CAP) if the critical eluent is not carefully fitted to the system. So far, this problem was often overlooked and only few experimental examples can be found in literature. We showed that in the case of polystyrene (PS) in a tetrahydrofuran (THF)/n-hexane critical mixed eluent, PS with molar masses higher than 100 kg mol⁻¹ were not eluted from a tandem of two columns packed by bare silica gels with 30 nm and 100 nm pore size, respectively. The polymer trapped within the columns was well recovered after injection of a small volume of pure THF as demonstrated using 2D chromatography. We studied PS conformations by means of small angle neutron scattering and found that the THF/n-hexane critical eluent is in fact a theta solvent for PS. By replacing it by a CH₂Cl₂/n-hexane critical mixture, which is a good solvent for PS, the limits of reduced sample recovery was displaced towards far higher molar masses. Thus, thermodynamic quality of eluent - theta or good solvent - plays an important role on the phenomenon of sample recovery.     

A trapping column for the coupling of reversed-phase liquid chromatography and capillary gas chromatography

A new technique for coupling reversed-phase liquid chromatography (RPLC) with gas chromatography is described. A fraction eluting from an RPLC column is trapped on a short column packed with polymeric adsorbent. After the mobile phase has been displaced with water, the analytes are desorbed with ethyl acetate. Following a delay time to enable the water to be flushed to waste, the ethyl acetate containing the analytes is introduced into the gas chromatograph under conditions suitable for partially concurrent solvent evaporation, i.e. below the solvent boiling point and at a rate just exceeding the evaporation rate. Post-column addition of water to the RPLC eluent helps to prevent breakthrough of compounds which are only modestly retained on the trapping column. The relationship between the capacity factors of the analytes on the trapping column and the required dilution factor is discussed. Polycyclic aromatic hydrocarbons are used as test compounds to study the system.     

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.     

Column selection for comprehensive multidimensional ion chromatography

This paper discusses the selection of ion chromatography (IC) columns for use in comprehensive multidimensional ion chromatography (IC x IC). First, a single number was determined for a wide range of anions (one number for each anion) using the linear solvent strength model. These numbers were then used to compare the column selectivity characteristics for five different columns. Principal component analysis was used to illustrate selectivity differences between columns. Dionex AS16 and AS20 columns were selected for use in the development of an IC x IC method for the separation of ten anions. To achieve the required speed of analysis in both the first and second separation dimensions, custom column lengths were packed in-house. The use of an eluent suppressor between the first and second columns permits a relatively low flow ratio regime of only <1:20 in the first and second dimensions, respectively, which reduces dilution effects common in comprehensive multidimensional LC. Selection of the second dimension eluent conditions was aided by the development of a spreadsheet based on the linear solvent strength model.     

HPLC of synthetic polymers characterization of polystyrenes by high performance precipitation liquid chromatography (HPPLC)

Summary The advantages and disadvantages of high performance precipitation liquid chromatography have been demonstrated for polystyrene homopolymers. Depending on the mobile phase composition at the dissolution point of the polymeric sample and surface properties of the stationary phase, elution is governed either by a solution process or by adsorption. A contribution by adsorption was noticed on silica as well as on reversed phases based on silica with a normal phase gradient of increasing polarity (heptane to dichloromethane). Elution was solely governed by solubility of the polymers on both types of stationary phase for polystyrenes with a molecular weight above 35 000 and reversed phase gradient of decreasing polarity (methanol to dichloromethane). Under these conditions an identical dependence of elution solvent composition on sample size was found as for turbidity titrations. Due to differences in the velocity of the eluent front and the polymeric sample with porous stationary phases the polymers can be eluted as colloidal solutions Non-porous stationary phases are superior in this respect because the velocities of eluent and solutes are identical.     

A comprehensive two-dimensional normal-phase × reversed-phase liquid chromatography based on the modification of mobile phases

A comprehensive orthogonal two-dimensional liquid chromatography (2D-LC) based on the modification of mobile phases was developed with a sample loop–valve interface. To improve the compatibility of mobile phases and analysis speed, some special solvents were chosen as the mobile phases, and the column temperature was elevated to decrease the viscosity of mobile phases of reversed-phase liquid chromatography (RPLC). Based on this principle, the first dimension was normal-phase liquid chromatography (NPLC) with a SiO₂ column, and the second dimension was reversed-phase liquid chromatography containing two tandem C18 columns. 1,4-Dioxane was used in the NPLC mobile phase, and isopropyl alcohol was employed in the RPLC mobile phase. Moreover, the elevated column temperature enabled the reduction of the backpressure and using tandem C18 columns to improve the resolving power in RPLC. The new comprehensive 2D-LC system and applied strategy offered a novel idea for construction of 2D-LC system. A traditional Chinese medicine, Zhengtian pill, was used as the test sample to evaluate the constructed 2D-LC system. 876 peaks were detected, and the peak capacity reached 1740.     

On-line coupling of counter-current chromatography and macroporous resin chromatography for continuous isolation of arctiin from the fruit of Arctium lappa L.

In this work, we have developed a novel hybrid two-dimensional counter-current chromatography and liquid chromatography (2D CCC × LC) system for the continuous purification of arctiin from crude extract of Arctium lappa. The first dimensional CCC column has been designed to fractionalize crude complex extract into pure arctiin effluent using a one-component organic/salt-containing system, and the second dimensional LC column has been packed with macroporous resin for on-line adsorption, desalination and desorption of arctiin which was effluent purified from the first CCC dimension. Thus, the crude arctiin mixture has been purified efficiently and conveniently by on-line CCC × LC in spite of the use of a salt-containing solvent system in CCC separation. As a result, high purity (more than 97%) of arctiin has been isolated by repeated injections both using the ethyl acetate–8% sodium chloride aqueous solution and butanol–1% sodium chloride aqueous solution. By contrast with the traditional CCC processes using multi-component organic/aqueous solvent systems, the present on-line CCC × LC process only used a one-component organic solvent and thus the solvent is easier to recover and regenerate. All of used solvents such as ethyl acetate, n-butanol and NaCl aqueous solution are low toxicity and environment-friendly. Moreover, the lower phase of salt-containing aqueous solution used as mobile phase, only contained minor organic solvent, which will save much organic solvent in continuous separation. In summary, our results indicated that the on-line hybrid 2D CCC × LC system using one-component organic/salt-containing aqueous solution is very promising and powerful tool for high-throughput purification of arctiin from fruits of A. lappa.     

Molecularly imprinted polymers for on-line preconcentration by solid phase extraction of pirimicarb in water samples

The synthesis and performance of a molecularly imprinted polymers (MIPs) as a selective solid phase extraction sorbent for the preconcentration of the carbamate pirimicarb from water samples is described. The MIP was prepared using pirimicarb as the template, methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross-linking monomer, and using chloroform as the solvent. The detection of pirimicarb was carried out by differential pulse voltammetry (DPV) at a hanging mercury drop electrode (HMDE) in 0.1 mol l⁻¹ HCl. Solvents of different polarities were checked for the polymer synthesis, and different experimental variables (sample pH, selection of the eluent used, eluent volume, analyte and eluent flow rates and sample volume) associated with the rebinding/extraction process were optimised. For a 25 ml sample, the process took about 13 min and resulted in a nominal enrichment factor of 50 (eluent MeOH:H₂O:HAc, 7:2:1; 0.5 ml) for pirimicarb. A limit of detection of 4.1 μg l⁻¹ was obtained, and a good reproducibility of the measurements using different MIP microcolumns was found. Furthermore, the MIP selectivity was evaluated by checking several substances with similar and different molecular structures to that of pirimicarb. As an application, pirimicarb was determined in water samples of diverse origin which were spiked at a concentration level of 71.5 μg l⁻¹.     

Characterization of complex copolymers by two-dimensional Liquid Chromatography

Complex polymers were characterized by combinations of different chromatographic separation mechanisms: liquid adsorption chromatography (LAC), liquid chromatography under critical conditions (LCCC), and liquid exclusion-adsorption chromatography (LEAC). These techniques were combined off-line and on-line in two-dimensional separations. Fatty acid ethoxylates, fatty esters of polyethylene glycol (PEG) and polysorbates were analyzed by two-dimensional liquid chromatography with normal phase LAC as the first and liquid chromatography at critical conditions (LCCC) or liquid exclusion adsorption chromatography (LEAC) as the second dimension. A full separation of all oligomers to the baseline could be achieved in both dimensions. In two-dimensional separations, the offline approach is compared to comprehensive chromatography, and the scope and limitations of both techniques are discussed.     

Separation of branched polystyrene by comprehensive two-dimensional liquid chromatography

Branched polystyrenes (PS) featuring a bivariate distribution in the molecular weight and in the number of branches were characterized by comprehensive two-dimensional liquid chromatography (2D-LC). The branched PS were prepared by anionic polymerization using n-butyl Li as an initiator and a subsequent linking reaction with p-(chlorodimethylsilyl)styrene (CDMSS). The n-butyl Li initiator yields polystyryl anions with broad molecular weight distribution (MWD) and the linking reaction with CDMSS yields branched PS with different number of branches. For the first dimension (1st-D) separation, reversed-phase temperature gradient interaction chromatography (RP-TGIC) was employed to separate the branched polymer according to mainly the molecular weight. In the second dimension (2nd-D) separation, the effluents from the RP-TGIC separation are subjected to liquid chromatography at chromatographic critical conditions (LCCC), in which the separation was carried out at the critical condition of linear homo-PS to separate the branched PS in terms of the number of branches. The 2D-LC resolution of RP-TGICxLCCC combination worked better than the common LCCCxsize-exclusion chromatography (SEC) configuration due to the higher resolution of RP-TGIC in molecular weight than SEC. Furthermore, by virtue of using the same eluent in RP-TGIC and LCCC (only the column temperature is different), RP-TGICxLCCC separation is free from possible 'break through' and large system peak problems. This type of 2D-LC separation could be utilized efficiently for the analysis of branched polymers with branching units distinguishable by LC separation.     

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.     

High-pressure liquid chromatography of drugs: II. An evaluation of a microparticulate cation-exchange column

A microparticulate cation-exchange column has been evaluated for the chromatography of thirty compounds selected as representative of a wide variety of drug substances. Although the column exhibited a strong partition effect besides the expected ion-exchange mechanism, the retention of drugs could be predictably influenced by variation of the eluent ionic strength and organic solvent content.

For acidic drugs the column showed little selectivity (although a salting-out effect increased retention at high eluent ionic strengths), but for basic substances Partisil SCX may afford a useful separative medium offering reasonable chromatographic efficiency (HETP ≈ 0.1 mm). The column longevity, however, is at present questionable.     

Aqueous chromatography system using temperature-responsive polymer-modified stationary phases

Extensive research has been carried out on functional polymers which are currently playing important roles in various fields such as medicine and engineering. Such functional polymers which respond to various kinds of stimuli are termed 'intelligent materials'. Poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer, was utilized as a chromatography column matrix modifier for a novel chromatographic approach in which only aqueous media are used as a mobile phase. The ability of the developed temperature-responsive chromatography system to separate solutes without using an organic solvent is advantageous from the point of view of maintaining the structure and activity of bioactive compounds. Recently, we designed and synthesized a new pH- and temperature-responsive copolymer as a representative of such environment-responsive polymers and grafted it onto aminopropyl silica beads. The products were evaluated as HPLC packing materials for separation systems based on a new concept, according to which the properties of the stationary phase surface are altered by external stimuli such as pH and temperature. This chromatography system utilizing the PNIPAAm copolymer is very useful for the separation of bioactive substances, such as proteins and peptides, because separation in the aqueous mobile phase is controlled solely by changing the temperature. This analytical system reduces organic waste because no organic solvent is used to separate the solutes and can therefore be classified as environmentally friendly. Future medical and pharmaceutical applications are expected.     

Liquid exclusion-adsorption chromatography: a new technique for isocratic separation of non-ionic surfactants. V. Two-dimensional separation of fatty acid polyglycol ethers

Fatty acid polyglycol esters can be fully characterized using two-dimensional liquid chromatography with liquid chromatography under critical conditions (LCCC) as the first and liquid exclusion-adsorption chromatography (LEAC) as the second dimension. LEAC is run under isocratic conditions, which allows the use of the refractive index detector, and thus accurate quantitation. Fractions from LCCC are transferred to LEAC using the full adsorption-desorption technique, by which they are focussed and reconcentrated before injection into the second dimension. This is achieved by increasing the water content of the mobile phase behind the LCCC column. Monoester oligomers of up to 20 oxyethylene units can be resolved to the baseline. Diester oligomers are partially separated in the first dimension (LCCC).     

A new method for the determination of mobile phase volume in normal and reversed-phase liquid chromatography

Summary A new method for the determination of the mobile phase volume (V _m) in liquid chromatography is presented based on the model regarding the retention of ionic solutes in the presence of eluent electrolytes. TheV _m value can be determined by measuring the retention volumes of two ions that have the same charge in two eluent electrolyte systems. Compared with the methods using isotopically labelled eluent components or inorganic salts asV _m markers, the method presented is proved to give more reasonableV _m values for both normal and reversed-phase liquid chromatography. As well as in binary mixed solvent systems, theV _m values in single solvent systems can be determined by this method.