Optimization of gradient elution conditions in multicomponent preparative liquid chromatography

Gradient elution is widely applied in analytical chromatography to reduce the separation time and/or to improve the selectivity. Increasingly the potential of modulating the solvent strength during gradient operation is exploited in preparative liquid chromatography. The purpose of this paper is to investigate theoretically the effect of optimizing free parameters available in gradient chromatography (extents and shapes of gradients) on the productivity of isolating a target component in a multicomponent mixture. An equilibrium stage model was used to quantify and compare different modes of operation (isocratic and various variants of gradient elution). By combining experimental design and artificial neural network concepts, optimal conditions were identified for the production of the second eluting component in a ternary mixture. The strong impact of the shape of gradients on process performance is elucidated.     

Continuous stationary phase gradients for planar chromatographic media

A simple, elegant method for the formation of a continuous stationary phase gradient for use in chromatographic separations is described. Its applicability to separation science is demonstrated using thin-layer chromatography as a test case. Gradient stationary phases were formed on activated High Performance Thin-Layer Chromatography (HP-TLC) plates using a newly developed methodology termed "controlled rate infusion". Specifically, the SiOH groups on the activated HP-TLC plates were reacted with 3-aminopropyltriethoxysilane (APTEOS) in a time dependent fashion by using a programmable syringe pump to control the rate of APTEOS infusion into the deposition reservoir. The shape (profile) of the gradient was controlled by the rate of infusion and imaged by taking advantage of the concentration-dependent color formation reaction between amine groups and ninhydrin. The advantages of such gradients in optimizing the retention and separation of various components in different mixtures were illustrated using mixtures of (1) four weak acids and bases and (2) three widely used over-the-counter drugs. The separation of the individual components on the gradient stationary phase was clearly improved relative to those on either traditional normal-phase TLC plates or uniformly amine-modified TLC plates. Precise control over component retention and separation was also demonstrated by strategically modifying the steepness of the gradient.     

Retention prediction of a set of amino acids under gradient elution conditions in hydrophilic interaction liquid chromatography

The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed‐phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous‐organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.     

Improvement of an overloaded, multi-component, solvent gradient bioseparation through multiobjective optimization

Solvent gradient chromatography is quite often used in analytical studies for decreasing the analysis time of samples having components with widely different retention behaviour. Several studies, both theoretical and experimental, have been reported on the optimization of gradient profiles in improving analytical separation performance, suggesting various linear and non-linear gradients. In preparative chromatography, on the other hand, though solvent gradient is being increasingly used (especially in bioseparation) to improve the product yield and productivity, there is a dearth of literature and clearer understanding of the effect(s) of modifier gradients on the separation performance. For this, the gradients used in applications are of relatively simple profiles like step or linear gradients, obtained through hand optimization based on experience and intuition. Significant improvements, however, can be expected using the state-of-the art modelling of chromatographic processes and optimization routines running on widely available hi-speed desktop computers. In this work we are reporting such an optimization procedure to improve the purification of an industrial multi-component mixture, containing 65.8% of Calcitonin as the main product, in an overloaded reversed-phase column. The work comprises both theoretical simulations and their experimental validation using multilinear gradients as optimization variable. The study produced interesting insights for modifier gradient design, like using peak deformation of the target peptide to increase yield and productivity, and improved our understanding of the effect of modifier gradients in non-linear separations.     

The study of properties of HPLC determination of polycyclic aromatic nitrogen heterocycles

The study of the separation of polycyclic aromatic nitrogen heterocycles (PANHs) by reverse-phase liquid chromatography with an octadecyl stationary phase is presented. The retention behaviour of a mixture of PANHs was studied under different chromatographic conditions. A mixture of phosphate buffer/acetonitrile was used as mobile phase in isocratic and gradient modes. The effect of different pH mobile phase in the range from 2.5 to 6.5 has been investigated to describe retention changes of PANHs as a function of their acid/base properties. Different concentrations of phosphate buffer as a component of the mobile phase were used to study the effect of ionic strength. Very good RP-HPLC separation of 24 PANHs and 16 EPA polycyclic aromatic hydrocarbons (PAHs) was obtained without a pre-separation step in a test mixture and the extract of a real soil sample. Limits of detection of PANHs obtained by two detection techniques, ultraviolet-diode array detection (UV-DAD) and fluorescence detector (FD), are compared. The proposed method is tested with a real soil sample.     

Protein separation and enrichment by counter-current chromatography using reverse micelle solvent systems

A protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by high-speed counter-current chromatography using a two-phase aqueous/reverse micelle-containing organic solvent system. About 50% stationary phase retention ratio was obtained in most chromatographic experiments. Separations were manipulated mainly by pH gradients that controlled the electrostatic interactions between the protein molecules and reverse micelles. Separations were further improved by incorporating an ionic strength gradient along with the pH gradient. Control of ionic strength in the aqueous solution helped fine-tune protein partitioning between the stationary and mobile phases. Although non-specific protein interactions affected baseline resolution, recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions.     

Nylon-6 capillary-channeled polymer (C-CP) fibers as a hydrophobic interaction chromatography stationary phase for the separation of proteins

Nylon-6 capillary-channeled polymer (C-CP) fibers are used as the stationary phase for the hydrophobic interaction chromatography (HIC) separation of a synthetic protein mixture composed of ribonuclease A, lysozyme, and holotransferrin. Nylon is a useful polymer phase for HIC as it has an alkyl backbone, while the amide functionality is hydrophilic (in fact ionic) in nature. The combination of a nonporous polymer surface of the fiber phases and high column permeability yields very efficient mass transfer characteristics, as exhibited by narrowing of peak widths with increases in mobile phase linear velocity. Retention factors and resolution were evaluated at flow rates ranging from 0.5 to 9 mL/min (linear velocities of ca. 2 to 15 mm/s) and at gradient slopes between 3.3 and 20 %B/min. Optimum resolution was achieved by employing fast flow rates (9 mL/min) and slow gradients (3 %B/min), also resulting in the highest peak capacities.     

Reversed-phase chromatography of proteins with diphenyl-silica column and hydro-organic eluents containing two organic solvents

High-performance liquid chromatography of proteins with silica bonded diphenyl stationary phase and hydro-organic mobile phases with linear gradient elution has been carried out with binary organic modifiers. The use of a mixture of 2-propanol and 1-butanol facilitated elution at total organic modifier concentrations significantly lower than with the use of 2-propanol alone. Furthermore higher protein recovery and retention of biological activity was obtained with the binary organic modifier because the increase in eluent strength with binary organic modifier was greater than that of the denaturing strength of the eluent. The use of a short, 3 cm long, column, relatively high flow-rates and steep gradients was also advantageous in attenuating protein denaturation. The results were interpreted within the framework of a theoretical treatment of the combined effect of the retention process and denaturation reaction simultaneously occurring in the column.     

Separation of different oligomers by supercritical fluid chromatography using gradient elution

Summary In supercritical fluid chromatography (SFC), the solubilizing nature of the mobile phase enables the use of composition gradients (gradient elution) like in liquid chromatography. With this gradient technique, oligomer separations have been achieved over wide molecular weight ranges. For separating oligomers which absorb only at low wavelengths, a carbon dioxide/acetonitrile mobile phase was used which allows UV detection down to 200 nm. As an ideally suited detection assembly for the chromatography of oligomers, a combination of mass-sensitive and evaporative light-scattering detection is suggested.

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Trennung von verschiedenen Oligomeren durch Chromatographie mit überkritischen fluiden Phasen unter Verwendung der Gradientenelution

     

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.     

Fast, generic gradient high performance liquid chromatography coupled to fourier transform ion cyclotron resonance mass spectrometry for the accurate mass analysis of mixtures

Fast gradient high performance liquid chromatography (HPLC) has been combined with a commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer for the routine and high performance analysis of mixtures. With this combination we were able to separate and detect, under high mass accuracy conditions, a six-component drug mixture in less than 5 minutes. The fast gradients described are now possible due to the development of mechanically robust, ultra pure silica packing materials, which allow relatively high flow rates (ca. 1 mL/min for a 2 mm diameter column). For the six compounds present in the model mixture, relative mass errors of less than 1 ppm were obtained (based on an external calibration) providing sufficient mass accuracy to make unequivocal assignments of empirical formulae. Preliminary results of fast gradient HPLC/FTICR-MS/MS are also shown for the same six-component mixture. Copyright 2000 John Wiley & Sons, Ltd.     

Separation of non-steroidal anti-inflammatory agents using supercritical fluid chromatography.

Supercritical fluid chromatography (SFC) was investigated for the separation of non-steroidal anti-inflammatory agents (NSAIs). Three different stationary phases (SB-methyl-100, SB-biphenyl-30, and SB-cyanopropyl-50) were compared for the separation of the compounds. Baseline separation of a flufenamic acid, mefenamic acid, fenbufen and indomethacin mixture was achieved on the SB-biphenyl-30 column using a pressure gradient. A mixture containing flufenamic acid, mefenamic acid, acetylsalicylic acid, ketoprofen and fenbufen and another mixture containing ibuprofen, fenoprofen, naproxen, ketoprofen and tolmetin were well separated on the SB-cyanopropyl-50 column using pressure gradients. Typical analysis time for a mixture of NSAIs on the biphenyl or cyanopropyl column was approximately 20-25 min. Application of the method using the biphenyl column to the determination of NSAIs present in selected commercial dosage forms was demonstrated.     

Reversed-phase high-performance liquid chromatography fingerprint profiles of thirty-nine mosses with chemometric

Thirty-nine land and aquatic mosses were extracted by double maceration and ultrasonic extraction techniques using the mixture of 80% ethanol and water. Obtained extracts were analyzed using the reversed-phase-high-performance liquid chromatography–diode array detector with the Kinetex C18 chromatographic column and mobile phase consisting of acetonitrile–water–0.1% formic acid mixture (gradient 5–100%, 60 min) with detection wavelength of 280 nm. Next, obtained chromatograms were preliminary processed with the smoothing, noise reduction, background subtraction, and alignment using the SpecAlign program (version 2.4.1). The chemometric analysis was performed to obtain the fingerprint chromatograms of selected mosses. The principal component analysis and the cluster analysis (with paired group algorithm and correlation coefficient—r, as similarity measure) confirm the chemical similarity or differences between studied Bryophyta sp.     

Numerical modeling of elution peak profiles in supercritical fluid chromatography. Part I—Elution of an unretained tracer

When chromatography is carried out with high-density carbon dioxide as the main component of the mobile phase (a method generally known as “supercritical fluid chromatography” or SFC), the required pressure gradient along the column is moderate. However, this mobile phase is highly compressible and, under certain experimental conditions, its density may decrease significantly along the column. Such an expansion absorbs heat, cooling the column, which absorbs heat from the outside. The resulting heat transfer causes the formation of axial and radial gradients of temperature that may become large under certain conditions. Due to these gradients, the mobile phase velocity and most physico-chemical parameters of the system (viscosity, diffusion coefficients, etc.) are no longer constant throughout the column, resulting in a loss of column efficiency, even at low flow rates. At high flow rates and in serious cases, systematic variations of the retention factors and the separation factors with increasing flow rates and important deformations of the elution profiles of all sample components may occur. The model previously used to account satisfactorily for the effects of the viscous friction heating of the mobile phase in HPLC is adapted here to account for the expansion cooling of the mobile phase in SFC and is applied to the modeling of the elution peak profiles of an unretained compound in SFC. The numerical solution of the combined heat and mass balance equations provides temperature and pressure profiles inside the column, and values of the retention time and efficiency for elution of this unretained compound that are in excellent agreement with independent experimental data.     

A Computer Program for the Selection of Gradient Elution in High Performance Liquid Chromatography

Abstract

A computer program is presented for the selection of a gradient mobile phase that will give the same resolution for all the component pairs in the mixture. Each pair is treated as a separate experiment. The computer is used to compile these experiments and to recommend an optimum gradient.     

Comparison of the sequential simplex method and linear solvent strength theory in HPLC gradient elution optimization of multicomponent flavonoid mixtures

Summary Using a camomile flavonoid extract as the sample and four different reversed-phase partition systems, the ability of the Simplex procedure to produce optimum gradient separation of unknown multicomponent mixtures was checked against the linear solvent strength (LSS) gradient elution theory. On the same partition systems the mean solvent strenghts were measured by experimentally determined logk vs. mobile phase composition plots. These mean solvent strengths are compared to those inferred from the optimum gradients and the usefulness of LSS theory in multicomponent mixture gradient elution optimization is discussed.Dadicated to Prof. Dr. A Liberti on the occasion of his 70th birthday.     

Optimal gradient operation in comprehensive liquid chromatography × liquid chromatography systems with limited orthogonality

A novel strategy is described for designing optimal second dimension (²D) gradient conditions for a comprehensive two-dimensional liquid chromatography system where the two dimensions are not fully orthogonal. Using the approach developed here, the initial and final organic modifier content values resulting in the highest coverage of separation space can be derived for each ²D gradient run. Theory indicates that these values can be determined by adapting ²D gradient operation to the degree of orthogonality. The new method is tested on a comprehensive two-dimensional liquid chromatography system that uses reversed phase (RP) columns showing different selectivities in the two dimensions. A comparison between analyses carried out using normal and optimized ²D gradients showed that the latter allow a more efficient use of analysis time. This can result either in an improved peak capacity or in decreasing total analysis time, depending on the final goal of the experiment. In the latter scenario, the number of separated peaks is comparable to that obtained using gradients spanning a wide range of organic modifier but, now, in half the time. As test samples complex mixtures of peptides were analyzed.     

Analysis of mixtures containing free fatty acids and mono-, di- and triglycerides by high-performance liquid chromatography coupled with evaporative light-scattering detection

Commercial monostearates of glycerol, generally used as antistatic agents for thermoplastic polymers, consist of a complex mixture of mono-, di- and trisubstituted glycerides and the corresponding fatty acids. Thus far, the glycerides and the fatty acids have been analyzed separately by high-performance liquid chromatography (HPLC). In fact, the simultaneous analysis of glycerides and fatty acids requires esterification of the acids in order to avoid overlapping of the chromatographic peaks. This paper describes an HPLC method which allows the direct separation and identification, simultaneously and in a single run, of the variously substituted glycerides, and also the corresponding saturated fatty acids that are found as by-products in commercial glycerol monostearates. The procedure is based on the use of a ternary gradient HPLC instrument equipped with an evaporative light-scattering detector; the stationary phase was a reversed-phase RP-8 end-capped (Merck) column; the mobile phase was two consecutive binary gradients consisting of acetonitile-water plus acetic acid (0.1%, v/v) and acetonitrile-methylene chloride. The method is fast and shows high sensitivity and selectivity, being able to separate also the positional isomers of mono- and digycerides in addition to the mixed di- and triglycerides.     

Theoretical opportunities and actual limitations of pH gradient HPLC

In a series of reports published recently by our laboratory comprehensive theory and experimental conditions were established for reversed-phase high-performance liquid chromatography (RP HPLC) employing the programmed pH gradient of mobile phase. A procedure was developed providing, rapidly and conveniently, the acidity (pK_a) of weak acids and bases and their lipophilicity (hydrophobicity) log k_w. The basis of the double-gradient RP HPLC, employing simultaneous gradients of organic modifier content and mobile phase pH, was also elaborated. The fundamentals of the approach are presented briefly and systematically and its advantages and limitations are discussed. It is demonstrated that the newly introduced pH gradient method increases the analytical versatility of RP HPLC and our understanding of its physicochemical basis.