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.; Horner, Anthony R.; Weber, Stephen G.

doi:10.1016/j.chroma.2015.05.071

Cited by 24 publications

(26 citation statements)

References 26 publications

(29 reference statements)

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“…58 Briefly, timed injections were made using an external 6-port Cheminert injection valve (C72x-669D, VICI Valco, Houston, TX) fitted with a 75 µm × 75 cm nanoViper capillary (Thermo) loop. Samples were loaded into the sample loop using the loading pump and autosampler of the NSC-3500 system.…”

Section: Methodsmentioning

confidence: 99%

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Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

2016

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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.

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Section: Methodsmentioning

confidence: 99%

“…57,58 With TASF, the head of the column is cooled to subambient temperatures during injection. This results in focusing of solutes into narrow bands at the head of the column.…”

mentioning

confidence: 99%

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

2016

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“…1. Hardware was similar to that used for single-stage TASF [53, 55]. In this instrument we constructed an array of three independently controlled 1.0 × 1.0 cm, 10.9 W thermoelectric elements (Peltiers, TECs) from Custom Thermoelectric (04801-9330-34RB, Bishopville, MD).…”

Section: Instrumentation and Chromatographic Conditionsmentioning

confidence: 99%

“…Fused silica capillary columns were packed in-house using a previously described procedure [28, 53, 55]. Briefly, Waters Acquity BEH C18, 1.7 µm d p , particles were packed into 150 µm diameter fused silica capillaries from Polymicro Technologies (Phoenix, AZ).…”

Section: Instrumentation and Chromatographic Conditionsmentioning

confidence: 99%

“…Recently, Schoenmakers et al immersed a capillary monolith in ice water to induce an in-column solvent switch [33]. In addition to our work with temperature-assisted on-column solute focusing (TASF) [53–55], Holm et al [56] and Eghbali et al [57] used segmented column cooling to focus solute bands at specific times and locations along the column, the former the column inlet, the latter the outlet. Dynamic changes in column temperature can be used effectively to manipulate solute bands on column and address the volume overload and dispersion problems.…”

Section: Introductionmentioning

confidence: 99%

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Temperature-assisted solute focusing with sequential trap/release zones in isocratic and gradient capillary liquid chromatography: Simulation and experiment

Groskreutz

Weber

2016

Journal of Chromatography A

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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-minute separation peak capacity increased from 75 under isothermal conditions to 146 using single-stage TASF, and 185 for two-stage TASF.

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The characterization of column heating effect in nanoflow liquid chromatography mass spectrometry (nanoLC‐MS)–based proteomics

Guo¹,

Liu

et al. 2019

J Mass Spectrom

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Column heating strategy is often applied in nano-high-performance liquid chromatography-mass spectrometer (nanoHPLC-MS) platform for enhancing the analytical efficiency of peptides or proteins. Nonetheless, the influence effects of column heating in peptides or proteins identification still lack of deep understanding. In this study, a systematic comparison of room temperature (RT) and column heating of nanoHPLC was done. Based on the data, under column heating condition, the backpressure of nanoHPLC can be decreased. Due to the increase of resolution, the peak widths of precursor ion were narrowed. As a result, in MS/MS data acquisition part, more time was spared for MS1 detecting and MS2 fragmenting, which eventually resulted in increased identification of peptides and proteins. Moreover, we also proposed the application scope of column heating by evaluating its influence on sample detection. On one hand, column heating significantly increased the identification of membrane proteins due to more efficient elution of highly hydrophobic peptides compared with RT. On the other hand, heating was not suitable for analyzing short or/and hydrophilic peptides with low retention time, which would be eluted out during sample loading process under high temperature and missed by mass spectrometric detection. In conclusion, our study provides a reference for rational application of column heating in proteomics research.

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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

Cited by 24 publications

References 26 publications

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

Temperature-assisted solute focusing with sequential trap/release zones in isocratic and gradient capillary liquid chromatography: Simulation and experiment

The characterization of column heating effect in nanoflow liquid chromatography mass spectrometry (nanoLC‐MS)–based proteomics

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