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Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing.

ABSTRACT: On-column focusing is essential for satisfactory performance using capillary scale columns. On-column focusing results from generating transient conditions at the head of the column that lead to high solute retention. Solvent-based on-column focusing is a well-known approach to achieve this. Temperature-assisted on-column focusing (TASF) can also be effective. TASF improves focusing by cooling a short segment of the column inlet to a temperature that is lower than the column temperature during the injection and then rapidly heating the focusing segment to the match the column temperature. A troublesome feature of an earlier implementation of TASF was the need to leave the capillary column unpacked in that portion of the column inside the fitting connecting it to the injection valve. We have overcome that problem in this work by packing the head of the column with solid silica spheres. In addition, technical improvements to the TASF instrumentation include: selection of a more powerful thermo-electric cooler to create faster temperature changes and electronic control for easy incorporation into conventional capillary instruments. Used in conjunction with solvent-based focusing and with isocratic elution, volumes of paraben samples (esters of p-hydroxybenzoic acid) up to 4.5-times the column liquid volume can be injected without significant bandspreading due to volume overload. Interestingly, the shapes of the peaks from the lowest volume injections that we can make, 30nL, are improved when using TASF. TASF is very effective at reducing the detrimental effects of pre-column dispersion using isocratic elution. Finally, we show that TASF can be used to focus the neuropeptide galanin in a sample solvent with elution strength stronger than the mobile phase. Here, the stronger solvent is necessitated by the need to prevent peptide adsorption prior to and during analysis.

SUBMITTER: Groskreutz SR

PROVIDER: S-EPMC4488902 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing.

Groskreutz Stephen R SR Horner Anthony R AR Weber Stephen G SG

Journal of chromatography. A 20150608

On-column focusing is essential for satisfactory performance using capillary scale columns. On-column focusing results from generating transient conditions at the head of the column that lead to high solute retention. Solvent-based on-column focusing is a well-known approach to achieve this. Temperature-assisted on-column focusing (TASF) can also be effective. TASF improves focusing by cooling a short segment of the column inlet to a temperature that is lower than the column temperature during t ...[more]

PMID: 26091787

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Project description:Capillary HPLC (cLC) with gradient elution is the separation method of choice for the fields of proteomics and metabolomics. This is due to the complementary nature of cLC flow rates and electrospray or nanospray ionization mass spectrometry (ESI-MS). The small column diameters result in good mass sensitivity. Good concentration sensitivity is also possible by injection of relatively large volumes of solution and relying on solvent-based solute focusing. However, if the injection volume is too large or solutes are poorly retained during injection, volume overload occurs which leads to altered peak shapes, decreased sensitivity, and lower peak capacity. Solutes that elute early even with the use of a solvent gradient are especially vulnerable to this problem. In this paper, we describe a simple, automated instrumental method, temperature-assisted on-column solute focusing (TASF), that is capable of focusing large volume injections of small molecules and peptides under gradient conditions. By injecting a large sample volume while cooling a short segment of the column inlet at subambient temperatures, solutes are concentrated into narrow bands at the head of the column. Rapidly raising the temperature of this segment of the column leads to separations with less peak broadening in comparison to solvent focusing alone. For large volume injections of both mixtures of small molecules and a bovine serum albumin tryptic digest, TASF improved the peak shape and resolution in chromatograms. TASF showed the most dramatic improvements with shallow gradients, which is particularly useful for biological applications. Results demonstrate the ability of TASF with gradient elution to improve the sensitivity, resolution, and peak capacity of volume overloaded samples beyond gradient compression alone. Additionally, we have developed and validated a double extrapolation method for predicting retention factors at extremes of temperature and mobile phase composition. Using this method, the effects of TASF can be predicted, allowing determination of the usefulness of this technique for a particular application.

Project description:In this work we characterize the development of a method to enhance temperature-assisted on-column solute focusing (TASF) called two-stage TASF. A new instrument was built to implement two-stage TASF consisting of a linear array of three independent, electronically controlled Peltier devices (thermoelectric coolers, TECs). Samples are loaded onto the chromatographic column with the first two TECs, TEC A and TEC B, cold. In the two-stage TASF approach TECs A and B are cooled during injection. TEC A is heated following sample loading. At some time following TEC A's temperature rise, TEC B's temperature is increased from the focusing temperature to a temperature matching that of TEC A. Injection bands are focused twice on-column, first on the initial TEC, e.g. single-stage TASF, then refocused on the second, cold TEC. Our goal is to understand the two-stage TASF approach in detail. We have developed a simple yet powerful digital simulation procedure to model the effect of changing temperature in the two focusing zones on retention, band shape and band spreading. The simulation can predict experimental chromatograms resulting from spatial and temporal temperature programs in combination with isocratic and solvent gradient elution. To assess the two-stage TASF method and the accuracy of the simulation well characterized solutes are needed. Thus, retention factors were measured at six temperatures (25-75°C) at each of twelve mobile phases compositions (0.05-0.60 acetonitrile/water) for homologs of n-alkyl hydroxylbenzoate esters and n-alkyl p-hydroxyphenones. Simulations accurately reflect experimental results in showing that the two-stage approach improves separation quality. For example, two-stage TASF increased sensitivity for a low retention solute by a factor of 2.2 relative to single-stage TASF and 8.8 relative to isothermal conditions using isocratic elution. Gradient elution results for two-stage TASF were more encouraging. Application of two-stage TASF increased peak height for the least retained solute in the test mixture by a factor of 3.2 relative to single-stage TASF and 22.3 compared to isothermal conditions for an injection four-times the column volume. TASF improved resolution and increased peak capacity; for a 12-min separation peak capacity increased from 75 under isothermal conditions to 146 using single-stage TASF, and 185 for two-stage TASF.

Project description:Recent years have seen the initial success of a variational implicit-solvent model (VISM), implemented with a robust level-set method, in capturing efficiently different hydration states and providing quantitatively good estimation of solvation free energies of biomolecules. The level-set minimization of the VISM solvation free-energy functional of all possible solute-solvent interfaces or dielectric boundaries predicts an equilibrium biomolecular conformation that is often close to an initial guess. In this work, we develop a theory in the form of Langevin geometrical flow to incorporate solute-solvent interfacial fluctuations into the VISM. Such fluctuations are crucial to biomolecular conformational changes and binding process. We also develop a stochastic level-set method to numerically implement such a theory. We describe the interfacial fluctuation through the "normal velocity" that is the solute-solvent interfacial force, derive the corresponding stochastic level-set equation in the sense of Stratonovich so that the surface representation is independent of the choice of implicit function, and develop numerical techniques for solving such an equation and processing the numerical data. We apply our computational method to study the dewetting transition in the system of two hydrophobic plates and a hydrophobic cavity of a synthetic host molecule cucurbit[7]uril. Numerical simulations demonstrate that our approach can describe an underlying system jumping out of a local minimum of the free-energy functional and can capture dewetting transitions of hydrophobic systems. In the case of two hydrophobic plates, we find that the wavelength of interfacial fluctuations has a strong influence to the dewetting transition. In addition, we find that the estimated energy barrier of the dewetting transition scales quadratically with the inter-plate distance, agreeing well with existing studies of molecular dynamics simulations. Our work is a first step toward the inclusion of fluctuations into the VISM and understanding the impact of interfacial fluctuations on biomolecular solvation with an implicit-solvent approach.

Dataset Information

Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing.

Publications

Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing.

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