Eluotropic Strength Research Articles

Formation of Micelles by Nonionic Detergent Molecules Leads to the Breakthrough Peak in Reversed-Phase Ultraperformance Liquid Chromatography (UPLC).

A peculiar phenomenon known as "breakthrough" occurs under reversed-phase ultraperformance liquid chromatography (UPLC) conditions and has been under scrutiny for decades. This effect takes place when a large volume of analyte solution, prepared in a solvent with an eluotropic strength significantly higher than that of the initial mobile phase solvent, is injected. According to the literature, under specific experimental conditions, a substantial portion of solutes is carried by the mobile phase and detected near the dead time of the chromatographic system. This phenomenon is typically observed when the injected volume of a particular analyte is sufficiently large. However, the underlying physicochemical principles governing this phenomenon have remained elusive. We present evidence demonstrating that breakthroughs can occur even when injecting a sample of a neat solvent devoid of any solute. By mass spectrometric analysis, we identified the breakthrough peak to represent the nonionic detergent Triton. When columns are equilibrated with water, Triton molecules, present as impurities in filtered water, accumulate on the nonpolar stationary phase. Upon the introduction of a solvent with a stronger elution strength, Triton molecules retained on the stationary phase are removed. As detergents, these Triton molecules aggregate into micelles featuring a hydrophobic inner core and a hydrophilic outer shell. These hydrophilic micelles are carried by the polar mobile phase and detected as the breakthrough peak at the dead time of the chromatographic system. When analytes are present, a portion of the injected solutes is captured by the micelles and transported with the breakthrough plug. This assertion was verified and confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis of a methanolic solution of perfluorooctanoic acid (PFOA). The mass spectra corresponding to the breakthrough plug featured a peak for the PFOA anion (m/z 413) in addition to those for Triton.

Possibilities and Limitations of Aqueous Temperature Responsive Liquid Chromatography in Comprehensive 2D–LC

In theory, comprehensive two-dimensional liquid chromatography (2D-LC) allows for significant enhanced peak capacity compared to one-dimensional high performance LC (1D-HPLC). However, reaching such a separation performance while also obtaining robust, easily implementable, and sensitive methods proves challenging. Because it can hinder the broader use of 2D-LC, there is a need for developing easier, more trouble-free approaches that feature the benefits of LC×LC while not compromising with what can be done with 1D-HPLC. Commercial 2D-LC interfaces are based on two-position multiport valves composed of two loops, which are alternatingly used either to collect the effluent from the first dimension (1D), or to inject their content to the second dimension (2D). This design implies that if large sampling volumes transferred to the second column need to be avoided, a comparatively (much) higher flow rate and broader column i.d. is required for the second column. However, doing so can lead to a loss in sensitivity because of dilution and impractical analytical chromatography as a result of the high flow rates involved. In most LC×LC column combinations, this problem is exacerbated thanks to the high eluotropic strength of transferred loop volumes. However, when the elution strength of the transferred solvent is very small, refocusing the analytes can be obtained, which allows the user to overcome such issues. For example, this is the case when a purely aqueous separation mode is combined with reversed-phase LC (RPLC) in the 1D and 2D, respectively. Temperature responsive LC (TRLC), which is an emerging LC mode requiring only water as the mobile phase and whereby retention is controlled via temperature only, is promising in this context. In this second installment about TRLC, we illustrate the unique benefits of the combination of this separation mode with RPLC in comprehensive 2D-LC. The potential of the approach is shown through the analysis of representative standard mixtures, active pharmaceutical ingredients, synthetic impurities and phenolics in natural products.

Enhanced Sensitivity in Comprehensive Liquid Chromatography: Overcoming the Dilution Problem in LC × LC via Temperature-Responsive Liquid Chromatography.

In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension (1D), which lead to peak broadening in the second dimension (2D). In the majority of the current LC × LC column combinations, analytes are less than optimally refocused upon transfer to the second column, which negatively affects sensitivity. Furthermore, the typical combination of 1 or 2.1 mm columns in the 1D paired with a 3 mm (or broader) column in the 2D leads to at least a 9- or 4-fold dilution and a corresponding loss of sensitivity when using concentration-sensitive detectors. This occurs due to the enhanced radial dilution of the analytes in a broader column, while the sensitivity problem is further exacerbated in LC × LC due to the high flow operated 2D. In this paper, we introduce a solution to neutralize and inverse this dilution problem through a reconcentrating solution using temperature-responsive liquid chromatography (TRLC) in the 1D, which is a purely aqueous separation mode. Full solute refocusing at the 2D column head is thereby obtained when TRLC is combined with reversed-phase liquid chromatography (RPLC). This is shown for the combination of a 2.1 mm I.D. TRLC column with decreasing RPLC column diameters (3-2.1-1 mm) operated at the same linear velocities, hence a resulting decrease in dilution, respectively. Ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection were used to determine the experimental detection limits. Sensitivity improvements with UV detection were somewhat lower than expected, but represent ∼1.5- and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the 2D, respectively. This is attributed to extra-column dispersion and the poorer performance of 1 mm I.D. columns. A major benefit of the use of 1 mm I.D. columns in the 2D is that it allows split-free coupling of 2D effluent with ESI-MS (at 450 μL/min), making the coupling robust and simple. When using ESI-MS even better, albeit more variable, sensitivity enhancements were obtained on the narrower columns. The benefits of the methodology are demonstrated for paraben test solutes and for phenolic compounds in a blueberry extract by TRLC × RPLC-UV-ESI-TOF-MS.

Speeding up temperature-responsive × reversed-phase comprehensive liquid chromatography through the combined exploitation of temperature and flow rate gradients

Comprehensive two-dimensional liquid chromatography (LC×LC) can provide enhanced resolving power and higher peak capacities for the separation of complex samples. The transfer of fractions of too high eluotropic strength from the first dimension, however, can lead to peak broadening. This process is related to the column dimensions, the flow rates, mobile phase compositions, and stationary phase compatibility. Temperature-responsive LC (TRLC) uses a smart polymer coupled to silica (poly(N-isopropylacrylamide), pNIPAAm), that exhibits a change in polarity upon modest variations in column temperature. Retention is thus modulated by temperature and not by organic solvents, allowing for the use of purely aqueous mobile phases. As these aqueous mobile phases depict a very low eluotropic strength on a reversed-phase column, it facilitates band refocusing at the second-dimension column head in TRLC×RPLC. One of the remaining obstacles of TRLC is the long analysis time. In this research, the potential of this column combination in terms of analyte refocusing will be exploited. First, it is shown that upwards flow gradients can be implemented in the first dimension of TRLC×RPLC. As the flow in the second dimension is maintained at a constant level, a first-dimension flow gradient does not lead to impaired sensitivity and has no negative effects on the resulting peak size. Then, the novel combination of a downwards temperature gradient with an upwards flow gradient will be introduced to speed up the analyses further. Analysis time was, depending on the method used, reduced by 36–54%, as demonstrated by the analysis of mixtures of food additives, phenolic compounds, and small molecule pharmaceuticals mimicking impurity analysis at a 0.05% level.

Comprehensive two-dimensional temperature-responsive × reversed phase liquid chromatography for the analysis of wine phenolics

Phenolic compounds are an interesting class of natural products because of their proposed contribution to health benefits of foods and beverages and as a bio-source of organic (aromatic) building blocks. Phenolic extracts from natural products are often highly complex and contain compounds covering a broad range in molecular properties. While many 1D-LC and mass spectrometric approaches have been proposed for the analysis of phenolics, this complexity inevitably leads to challenging identification and purification. New insights into the composition of phenolic extracts can be obtained through online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to photodiode array and mass spectrometric detection. However, several practical hurdles must be overcome to achieve high peak capacities and to obtain robust methods with this technique. In many LC × LC configurations, refocusing of analytes at the head of the 2D column is hindered by the high eluotropic strength of the solvent transferred from the 1D to the 2D, leading to peak breakthrough or broadening. LC × LC combinations whereby a purely aqueous mobile phase is used in the 1D and RPLC is used in the 2D are unaffected by these phenomena, leading to more robust methods. In this contribution, the combination of temperature-responsive liquid chromatography (TRLC) with RPLC is used for the first time for the analysis of phenolic extracts of natural origin to illustrate the potential of this alternative combination for natural product analyses. The possibilities of the combination are investigated through analysis of wine extracts by TRLC × RPLC-DAD and TRLC × RPLC-ESI-MS.

Novel Strategy for Mining and Identification of Acylcarnitines Using Data-Independent-Acquisition-Based Retention Time Prediction Modeling and Pseudo-Characteristic Fragmentation Ion Matching.

It is a challenging work to screen, identify, and quantify acylcarnitines in complex biological samples. A method, based on the retention time (RT) prediction and data-independent acquisition strategies, was proposed for the large-scale identification of acylcarnitines using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Relative cumulative eluotropic strength was introduced as a novel descriptor in building a linear prediction model, which not only solves the problem that acylcarnitines with long carbon chains cannot be well predicted in traditional models but also proves its robustness and transferability across instruments in two data sets that were acquired in distinct chromatography conditions. The accessibility of both predictive RT and MS2 spectra of suspect features effectively reduced about 30% false-positive results, and consequently, 150 and 186 acylcarnitines were identified in the rat liver and human plasma (NIST SRM 1950), respectively. This method provides a new approach in large-scale analysis of acylcarnitine in lipidomic studies and can also be extended to the analysis of other lipids.

A new strategy for development of eco-friendly RP-HPLC method using Corona Charged Aerosol Detector and its application for simultaneous analysis of risperidone and its related impurities

Green analytical chemistry is primarily directed towards minimization of the amount of waste associated with either the sample preparation or analysis. Among different chromatographic methods, liquid chromatography is considered the least green, allowing for various possibilities for greening. Using green solvents such as ethanol or acetone in RP-HPLC, as an alternative to acetonitrile, is recently attracting an attention. Both ethanol and acetone are characterized with low toxicity, with certain drawbacks limiting their regular use in RP-HPLC. Ethanol has low eluotropic strength and causes high backpressures, while acetone shows high UV cut-off, making it unsuitable for UV/Vis detection. To overcome the existing problems, Corona Charged Aerosol Detector was employed for development of RP-HPLC methods for separation of risperidone and its structurally related impurities with either ethanol or acetone as organic modifier. The methods were optimized by experimental design methodology, while optimal conditions for separation were determined using Derringer's desirability function. Detailed assessment of 3D surface plots of Derringer's desirability function enabled selection of 0.6 mL min−1 flow rate and 20% (v/v) organic modifier content as optimal when using ethanol, while in case of acetone mobile phase flow rate was 0.8 mL min−1 and organic modifier content 17% (v/v). Methods were validated and their eco-friendly character was confirmed through Green Analytical Procedure Index (GAPI). Although both methods are ecologically acceptable, the main drawback is reflected in the fact that no recycling or another waste treatment method exist. In the end, acetone was prioritized over ethanol, due to lower health hazard and decreased amount of generated waste.

Experimental evaluation of solvent elution strength in reversed-phase TLC on medium polarity phases

Seven chromatographic grade solvents miscible with water (acetone, acetonitrile, dioxane, ethanol, isopropanol, methanol, tetrahydrofuran), as well as water itself were investigated in the context of their eluotropic strength in reversed-phase chromatography on four medium-polar adsorbents (cellulose, CN, NH2 and DIOL). The test set for this research consisted of 35 model compounds. A modern chemometric mixture design concept allowed to estimate each solvent effect separately with a relatively small error (about 0.05 of RM value for all adsorbents). The solvent effects were analyzed chemometrically in multivariate manner and a final model was fitted to achieve the solvent strength. The estimation error was computed both from regression and bootstrap approach.

A twin‐column recycling chromatography with solvent gradient for reinforcing the isolation of minor impurities

A twin‐column recycling chromatography with solvent gradient was developed to isolate the minor impurities in the drugs. Two columns were connected through two 6‐port valves, and the solute band was diverted from one column into another by switching the valves. During the circulation of the solute band, a modifier was constantly added to the middle between the two columns so that the eluotropic strength of the downstream liquid was reduced. Thereby, the front of the band moved slower than the tail, bringing about a band compression effect to offset the band broadening so as to improve the separation and enhance the concentration of fraction without reducing the feeding amount. Such modified scheme was successfully used to purify a proprietary intermediate, displaying great advantages over the conventional scheme without solvent gradient in terms of recovery and fraction concentration. © 2018 American Institute of Chemical Engineers AIChE J, 65: 702–711, 2019

Acetonitrile as tops solvent for liquid chromatography and extraction

This article deals with acetonitrile physico-chemical properties and its mixtures with water. It covers the information about isotherms of such parameters of the acetonitrile-water binary system as density, viscosity, permeation coefficient, refraction index, optic density, a boiling point isobar. Authors suggest a generalized criterion of acetonitrile polarity. The article also discusses means of polarity assessment and eluotropic strength of mobile phases on the basis of acetonitrile mixtures with water with application of generalized criteria. Special attention is paid to the options of acetonitrile application in various extraction methods of chromatographic sample preparation. The article also reveals advantages and problems of acetonitrile application as a mobile phase modifying agent in the HPLC reversed-phase and as a hydrophilic extra-agent in liquid-liquid extraction.

Experimental evaluation of solvent elution strength in reversed-phase thin-layer chromatography on RP2 and RP8 plates

The eluotropic strength on RP2 and RP8 plates was investigated for 7 chromatographic grade solvents miscible with water as a diluent (acetone, acetonitrile, dioxane, ethanol, isopropanol, methanol, and tetrahydrofuran) as well as for water itself. Thirty-five model compounds were used as a test set in the investigation. The use of a modern chemometric mixture design concept allowed separate estimation of each solvent effect from the other results with a relatively small error (about 0.05 of RM value for both adsorbents). Principal component analysis was used as an additional tool and revealed that only one parameter (mean eluotropic strength) is satisfactory to explain the overall variability in the retention datasets. The bootstrapping approach was also used to check the distribution and uncertainty of the obtained values. It can be concluded that all solvents have quite similar elution strength for both adsorbents. The obtained values can be used as a reference during the optimization of thin-layer chro...

Fast and accurate separation of the paclitaxel from yew extracum by a pseudo simulated moving bed with solvent gradient

A pseudo simulated moving bed (SMB) with solvent gradient was used to trap and separate paclitaxel from yew extracum. This SMB process consisted of three steps: feeding, purification and recovery. In comparison with methanol/water as an eluent, acetonitrile/water could give a better separation but had a poor dissolubility of the yew extracum, and therefore methanol/water was used in the feeding followed by acetonitrile/water in the purification. In the first two steps, water was deliberately added into zone III to modulate the eluotropic strength of the liquid entering zone III, so as to make paclitaxel separation from impurities be more efficient. Once most of impurities discarded, the columns were in turn eluted to recover the trapped paclitaxel of 98% yield with a purity of 78% from the yew extracum containing 1.5% paclitaxel. Afterward, an additional operation of crystallization improved the purity further to 97.8% with the yield of 95%.

Natural deep eutectic solvents as the major mobile phase components in high-performance liquid chromatography-searching for alternatives to organic solvents.

Over the past six decades, acetonitrile (ACN) has been the most employed organic modifier in reversed-phase high-performance liquid chromatography (RP-HPLC), followed by methanol (MeOH). However, from the growing environmental awareness that leads to the emergence of "green analytical chemistry," new research has emerged that includes finding replacements to problematic ACN because of its low sustainability. Deep eutectic solvents (DES) can be produced from an almost infinite possible combinations of compounds, while being a "greener" alternative to organic solvents in HPLC, especially those prepared from natural compounds called natural DES (NADES). In this work, the use of three NADES as the main organic component in RP-HPLC, rather than simply an additive, was explored and compared to the common organic solvents ACN and MeOH but additionally to the greener ethanol for separating two different mixtures of compounds, one demonstrating the elution of compounds with increasing hydrophobicity and the other comparing molecules of different functionality and molar mass. To utilize NADES as an organic modifier and overcome their high viscosity monolithic columns, temperatures at 50°C and 5% ethanol in the mobile phase were used. NADES are shown to give chromatographic performances in between those observed for ACN and MeOH when eluotropic strength, resolution, and peak capacity were taken into consideration, while being less environmentally impactful as shown by the HPLC-Environmental Assessment Tool (HPLC-EAT) metric. With the development of proper technologies, DES could open a new class of mobile phases increasing the possibilities of new separation selectivities while reducing the environmental impact of HPLC analyses. Graphical abstract Natural deep eutectic solvents versus traditional solvents in HPLC.

Increasing Flexibility in Two-Dimensional Liquid Chromatography by Pulsed Elution of the First Dimension: A Proof of Concept.

This work demonstrates the development of an online two-dimensional liquid chromatography (2D-LC) method where the first dimension column is eluted by a sequence of pulses of increasing eluotropic strength generated by the LC pumps (pulsed-elution 2D-LC). Between the pulses, the first dimension is kept in a no-elution state using low eluent strength. The eluate from the first dimension is actively modulated using trap columns and subsequently analyzed in the second dimension. We demonstrate that by tuning the length and eluotropic strength of the pulses, peaks with retention factors in water, kw, above 150 can be manipulated to elute in 3-4 pulses. The no-elution state can be kept for 1-10 min with only minor changes as to which and how many pulses the peaks elute in. Pulsed-elution 2D-LC combined with active modulation tackles three of the main challenges encountered in 2D-LC and specifically online comprehensive 2D-LC: undersampling, difficulties in refocusing, and lack of flexibility in the selection of column dimensions and flow rates because the two dimensions constrain each other. The pulsed-elution 2D-LC was applied for the analysis of a basic fraction of vacuum gas oil. Peak capacity was 4018 for a 540 min analysis and 4610 for a 1040 min analysis.

The experimental design approach to eluotropic strength of 20 solvents in thin-layer chromatography on silica gel

The eluotropic strength on thin-layer silica plates was investigated for 20 chromatographic grade solvents available in current market. 35 model compounds were used as test subjects in the investigation. The use of modern mixture screening design allowed to estimate each solvent as a separate elution coefficient with an acceptable error of estimation (0.0913 of RM value). Additional bootstrapping technique was used to check the distribution and uncertainty of eluotropic estimates, proving very similar confidence intervals to linear regression. Principal component analysis proved that the only one parameter (mean eluotropic strength) is satisfactory to describe the solvent property, as it explains almost 90% of variance of retention. The obtained eluotropic data can be good appendix to earlier published results and their values can be interpreted in context of RM differences.

Investigation of the effect of mobile phase composition on selectivity using a solvent-triangle based approach in achiral SFC

Defining a method development methodology for achiral drug impurity profiling in SFC requires a number of steps. Initially, diverse stationary phases are characterized and a small number of orthogonal or dissimilar phases are selected for further method development. In this paper, we focus on a next step which is the investigation of the modifier composition on chromatographic selectivity. A solvent-triangle based approach is used in which blends of organic solvents, mainly ethanol (EtOH), propanol (PrOH), acetonitrile (ACN) and tetrahydrofuran (THF) mixed with methanol (MeOH) are tested as modifiers on six dissimilar stationary phases. The tested modifier blends were composed to have equal eluotropic strengths as calculated on bare silica. The modifier leads to minor changes in terms of elution order, retention and mixture resolution. However, varying only the modifier composition on a given stationary phase does not lead to the creation of dissimilar systems. Therefore the modifier composition is an optimization parameter, with the stationary phase being the factor determining most the selectivity of a given mixture in achiral SFC.

Rapid sequential separation of sedimentary lipid biomarkers via selective accelerated solvent extraction

Accelerated solvent extraction (ASE) can be adapted for selective and sequential separation of saturated hydrocarbon, unsaturated/aromatic hydrocarbon and polar lipid fractions from complex organic extracts in environmental matrices. ASE extraction cells were packed with a layer of Ag+ impregnated and activated silica gel, followed by an aliquot of the total lipid extract, and topped with another layer of activated silica gel. A fraction containing saturated hydrocarbons (FSAT) was eluted with hexane. Then, cells were inverted for reversed solvent flow and compounds of increasing polarity were eluted with solvent of increasing eluotropic strength to yield an unsaturated/aromatic hydrocarbon fraction (FARO) separated from a polar lipid fraction (FPOL). Gas chromatography–mass spectrometry analysis demonstrated high recovery of standards in FSAT (90±3%), FARO (82±4%) and FPOL (87±3%). Average compound recovery and efficiency of separation between lipid fractions were significantly improved (p<0.05) relative to separation with gravity column chromatography using similar stationary phases. Overall, this selective extraction method affords reliable, semi-automated separation of compound classes in total lipid extracts according to saturation and polarity and is well suited for molecular characterization and compound-specific isotopic analysis.

Development of HPLC analysis methods for therapeutic monoclonal antibodies

Therapeutic monoclonal antibody (mAb) preparations are produced from cultured cells; therefore, detailed and multidimensional analyses of their heterogeneities are required. We analyzed five commercially available mAb preparations by high-temperature reversed-phase LC using a wide-pore core-shell column for pluralistic quality assessment. At a highly elevated column temperature, isopropanol with high eluotropic strength coefficients and a wide-pore core-shell type octyl column showed good peak resolution of the investigated mAbs and their related constituents. We used this method to estimate the residual rate of intact mAbs after a heat aggregation treatment and conducted fragmentation analysis by analyzing their pepsin digests. Each peak component was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. All results were compared with those of reversed-phase and size exclusion analyses.

Analysis of fatty acid samples by hydrophilic interaction liquid chromatography and charged aerosol detector

HILIC/CAD techniques were used in analysis of samples containing fatty acids. Amine base column appeared to be the more retentive stationary phase compared to zwitterionic and BEH silica. The retention decreased with pH mobile phases changing from 3 to 5. Acetonitrile and acetone organic modifier were compared. Acetone gave higher eluotropic strength and better peak symmetry whereas acetonitrile led to higher efficiency. The retention decreased when ammonium acetate concentration increased from 5 to 20mM. The use of sub-2μm column did not show flat Van Deemter curves at high flow rates.A rapid separation of PGI2 and its main degradation product, 6-keto prostaglandin F1α was obtained in 1.6min with a Hypersil GOLD, 50mm×2.1mm, 1.9μm with; acetonitrile/acetate ammonium pH 5 at 20mM (85/15; v/v at 0.7ml/min).

Analysis of non-phthalates plasticizers on porous graphitic carbon by supercritical fluid chromatography using evaporative light scattering detection

The analysis of several plasticizers, widely used in the production of medical devices, was investigated on porous graphitic carbon (PGC) stationary phase in supercritical fluid chromatography (SFC) with an evaporative light scattering detector (ELSD). Due to strong interaction of compounds with the PGC support, solvents of strong eluotropic strength were added to the CO2 supercritical fluid. The effect of alkyl chain (pentane, hexane, heptane) and chlorinated (CH2Cl2, CHCl3, CCl4) solvents was studied on the retention and on the ELSD detection of plasticizers. A co-solvent mixture composed of CHCl3/heptane, eluted under gradient mode, allowed a significant improvement of the ELSD response compared to the use of each solvent individually. Then, a central composite design (CCD) was implemented to optimize both the separation and the detection of plasticizers. The parameters involved were the outlet pressure, the gradient slope, the co-solvent composition and the drift tube temperature of the ELSD. After optimization, baseline separation of plasticizers was achieved in 7min and best signal-to-noise ratios were obtained with outlet pressure and drift tube temperature of ELSD set at 200bar and 31°C, respectively. The co-solvent mixture was also composed of CHCl3/heptane (35/65 v/v) and a gradient from 15 to 60% of co-solvent in 2.2min was employed. The results demonstrated that CCD is a powerful tool for the optimization of SFC/ELSD method and the response surface model analysis can provide statistical understandings of the significant factors required to achieve optimal separation and ELSD sensitivity.