Stationary Phase Gradients Research Articles

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.

Separation of positional and geometrical fatty acid isomers as 2-nitrophenylhydrazide derivatives by high-performance liquid chromatography

Previously, following derivatization to their 2-nitrophenylhydrazide (2-NPH) derivatives, fatty acid (FA) abundances have been evaluated using high-performance liquid chromatography (HPLC). Although the method was sensitive, resolution was insufficient for many of the biologically important FAs. We have developed an enhanced separation of 24 FAs by use of different column temperature, stationary phase, and mobile phase gradient conditions. We applied this method to analysis of mouse skeletal muscle phospholipid and triglyceride. This further development of the chromatographic separation of 2-NPH FAs may lead to greater utility of this HPLC approach.

TLC with an adsorbent gradient for the analysis of taxol inTaxus baccataL.

TLC with a stationary-phase gradient has been used for purification and quantitative analysis of taxol in the needles and stems of female and male specimens of Taxus baccata L. harvested during the vegetative period of the plant. Parts of different TLC plates were connected by use of a Migas device. Separated taxol-containing zones were moved 1 cm, with methanol, on to another plate. The lipophilic substance zone was cut out after separation of the sample on a TLC Si60 F 254s plate with n -heptane-ethyl acetate 5:5 ( v/v ) as mobile phase. Further separation of taxol from accompanying hydrophilic substances was carried out on RP18W F 254s HPTLC plates with methanol-water 8:2 ( v/v ) as mobile phase. The taxol-containing fraction was finally separated on Si60 F 254s TLC plates with chloroform-acetone 15:5 ( v/v ) as mobile phase. The optimized method for taxol separation was used for quantitative densitometric analysis. The results revealed differences in the amounts of taxol in the needles and stems of Ta...

Fabrication and characterisation of capillary polymeric monoliths incorporating continuous stationary phase gradients

Polymeric monoliths in capillary formats have been fabricated incorporating a gradient of charged functional groups along their length. Scanning capacitively coupled contactless conductivity detection (sC(4)D) was then used to measure the conductive response of the stationary phase and characterise the relative axial distribution of functional groups along the column length. Gradients of 2-acrylamido-2-methyl-1-propanesulphonic acid were prepared using either photografting methods or by filling a capillary column with segmented plugs of monomer mixtures each containing incrementally higher concentrations of the functional monomer. The utility of sC(4)D as a rapid and non-invasive tool for assessing the slope of a variety of gradient configurations is demonstrated. Repeatability of the sC(4)D measurements was <1.7% RSD. Columns with a gradient of covalently bonded iminodiacetic acid were also produced. Changes in the gradient slope were observed after chelation of copper on the stationary phase via a reduction of the conductive response. The effect upon the observed gradient profile of changing the co-monomer composition during column fabrication was studied.

Perturbation analysis of the Kuramoto phase-diffusion equation subject to quenched frequency disorder

The Kuramoto phase-diffusion equation is a nonlinear partial differential equation which describes the spatiotemporal evolution of a phase variable in an oscillatory reaction-diffusion system. Synchronization manifests itself in a stationary phase gradient where all phases throughout a system evolve with the same velocity, the synchronization frequency. The formation of concentric waves can be explained by local impurities of higher frequency which can entrain their surroundings. Concentric waves in synchronization also occur in heterogeneous systems, where the local frequencies are distributed randomly. We present a perturbation analysis of the synchronization frequency where the perturbation is given by the heterogeneity of natural frequencies in the system. The nonlinearity in the form of dispersion leads to an overall acceleration of the oscillation for which the expected value can be calculated from the second-order perturbation terms. We apply the theory to simple topologies, like a line or sphere, and deduce the dependence of the synchronization frequency on the size and the dimension of the oscillatory medium. We show that our theory can be extended to include rotating waves in a medium with periodic boundary conditions. By changing a system parameter, the synchronized state may become quasidegenerate. We demonstrate how perturbation theory fails at such a critical point.

Preparative separation of multi-component mixtures using stationary phase gradients

Solvent gradients have been widely applied in analytical and preparative chromatography to enhance selectivity and improve productivity. Recently, an alternative concept using solid phase gradients was introduced in analytical chromatography [Nyiredy, Szucs, Szepesy, Chromatographia 63 (2006) 3; Nyiredy, Szucs, Szepesy, J. Chromatogr. A 1157 (2007) 122]. This concept is based on connecting columns filled with different stationary phases in series, applying a constant mobile phase composition. With the availability of column connecting devices with low dead volumes [K. Bischoff, S. Nyiredy, Z. Szücs, Elements for separating substances by distributing between a stationary and a mobile phase, and method for the production of a separating device. WO/2006/125564; PCT/EP2006/004744, 2006.], the concept can be easily implemented. The application of sequentially connected columns with different properties introduces new degrees of freedom like the type, number, relative lengths and positions of individual segments. In this study, an attempt is made to analyze if such stationary phase gradients could be exploited favorably in preparative chromatography for separating multi-component mixtures. A theoretical study is carried out for a connection of two different types of column segments based on numerical solutions of the equilibrium dispersive model assuming specific adsorption isotherm parameters for each segment. The influence of relative segment lengths and the order of the arrangement are evaluated with respect to the performance of separating intermediately eluting components from multi-component mixtures.

Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography

There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.

A zinc(II) complex‐conjugated polymer for selective recognition and separation of phosphates

AbstractA new polymer, Zn2+–cyclen complex‐conjugated polymer 4 (cyclen = 1,4,7,10‐tetraazacyclododecane), was synthesized by copolymerization of a Zn2+–(4‐vinyl)benzylcyclen complex (3, ZnL2) with ethylene glycol dimethacrylate (EGDMA). The contents of the zinc(II) ions in Zn2+–cyclen polymer 4 were determined as 36–42 μmol g−1, utilizing a Zn2+‐selective fluorophore, dansylamidoethylcyclen 10 (L3). It was found that 31% of deoxyadenosine 5′‐monophosphate (5′‐dAMP) (50 μM) is adsorbed on 4 (corresponding to 50 μM) in 20 mM HEPES (pH 7.0) with I = 0.1 (NaNO3) at 25°C. The apparent complexation constants for the 1:1 complex of ZnL2 on 4 with 5′‐dAMP and 4‐NPP, logKapp(ZnL–S2−) (S2− denotes a phosphomonoester dianion), at pH 7.0 and 25°C were determined as 4.1±0.1, which was larger than that (3.5) for the 1:1 complex of 3 (ZnL2) with 5′‐dAMP in homogeneous aqueous solution at pH 7.0 and 25°C. Deoxyadenosine (dA) and adenosine 3′,5′‐cyclic‐monophosphate (3′,5′‐cAMP), a cyclic phosphodiester monoanion, were negligibly adsorbed on Zn2+–cyclen polymer 4, implying that 4 interacts selectively with phosphomonoester dianions. For reference, Zn2+‐free polymer 8 (prepared from 4) and control polymer 9 (prepared by copolymerization of styrene with EGDMA) hardly adsorbed 5′‐dAMP, dA and 3′,5′‐cAMP. High‐performance liquid chromatography (HPLC) of mononucleotides such as 5′‐dAMP, cytidine 5′‐monophosphate (5′‐CMP), guanosine 5′‐monophosphate (5′‐GMP), and thymidine 5′‐monophosphate (5′‐dTMP) was achieved using 4 as a stationary phase and continuous gradient elution with 10:90 MeOH–1 mM HEPES (pH 7.0) and 10:90 MeOH–1 mM HEPES+10 mM (NH4)2HPO4 (pH 7.0). As expected, the retention times of mononucleotides were larger than those of the corresponding nucleosides, owing to the Zn2+‐phosphate interactions. These results indicated that Zn2+–cyclen‐conjugated polymer 4 affords a strong methodology to detect and separate biologically important phosphates. Copyright © 2004 John Wiley &amp; Sons, Ltd.

Convenient purification of tritylated and detritylated oligonucleotides up to 100-mer

Oligomers from crude phosphoramidite synthesis mixtures have been purified by reversed-phased high-performance liquid chromatography by exploiting the chromatographic variables of stationary phase pore size, chain length, and gradient shape. Chromatography was performed on oligomers up to 100-mer with mobile phases containing triethylammonium acetate/acetonitrile mixtures. Convenient guidelines are offered to enrich or purify synthetic oligomers. Tritylated oligomers up to 25 bases in length are best purified on C 18 or C 18, 80 å columns with moderate strength mobile phases using a combination of isocratic delays and shallow gradients. For oligomers longer than 25-mer, C 3, 300 å columns provide adequate fast purification in as little as 5 min, while 300 å, C 8 columns with long, slow gradients gave substantially increased purity. Chromatography of detritylated oligomers requires a modified approach. Up to 25-mer they are best purified on 80 å, C 18 columns with much lower organic concentrations and shallower gradients than those used for tritylated oligomers. Detrytilated oligomers greater than 25-mer can be enriched on both C 3 and C 8, 300 å columns using the same conditions described for shorter detrytilated oligomers.

Mechanistic Considerations on the Reversed Phase Liquid Chromato-Graphic Separation of Proteins

Abstract The effect of column length, stationary phase chemistry, flow rate and gradient variation and direction on the separation of seven proteins was evaluated. In general, resolution increased with shorter columns and higher flow rates. Nontraditional reverse gradients (from acetonitrile to buffer) sometimes produced better protein separations than traditional reversed phase gradients. Bonded phase chemistry has little effect on gradient protein separations, but silica gel properties (e.g., surface area, pore size, etc.) did. Both retention and resolution varied considerably with the nature of the gradient used.