Measurement and Modeling of Extra-Column Effects Due to Injection and Connections in Capillary Liquid Chromatography

Grinias, James P.; Bunner, Bernard; Gilár, Martin; Jorgenson, James W.

doi:10.3390/chromatography2040669

Cited by 36 publications

(21 citation statements)

References 101 publications

(149 reference statements)

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“…It allows for the precise injection of volumes between 1 and 100 µL, and conventional (lidless) 1.5 mL reaction tubes (such as from Eppendorf, Germany) can be used (with an approximate dead volume of 5 µL). Care was taken to select connecting tubes leading to the SAXS capillary of the smallest feasible diameter, to avoid band broadening and a subsequent loss of resolution [23].…”

Section: Chromatography Systemmentioning

confidence: 99%

Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

et al. 2020

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We describe the updated size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) set-up used at the P12 bioSAXS beam line of the European Molecular Biology Laboratory (EMBL) at the PETRAIII synchrotron, DESY Hamburg (Germany). The addition of size exclusion chromatography (SEC) directly on-line to the SAXS capillary has become a well-established approach to reduce the effects of the sample heterogeneity on the SAXS measurements. The additional use of multi-angle laser light scattering (MALLS), UV absorption spectroscopy, refractive index (RI), and quasi-elastic light scattering (QELS) in parallel to the SAXS measurements enables independent molecular weight validation and hydrodynamic radius estimates. This allows one to address sample monodispersity as well as conformational heterogeneity. The benefits of the current SEC-SAXS set-up are demonstrated on a set of selected standard proteins. The processed SEC-SAXS data and models are provided in the Small Angle Scattering Biological Data Bank (SASBDB) and are labeled as “bench-marked” datasets that include the unsubtracted data frames spanning the respective SEC elution profiles and corresponding MALLS-UV-RI-QELS data. These entries provide method developers with datasets suitable for testing purposes, in addition to an educational resource for SAS data analysis and modeling.

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Section: Chromatography Systemmentioning

confidence: 99%

Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

et al. 2020

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“…Most studies only focus on the combined contribution of the different parts of the chromatographic system (injector, connection tubing, preheaters, valves, detector), because they only need the total extra-column dispersion to correct the measured total dispersion to determine the "column-only" band broadening. Some studies went a step further and attempted to separate the effect of pre-and post-column contributions, or the effects of individual aspects, such as injection volume [1,3,4,12,23,24]. Understanding extra-column band broadening and, more specifically, the variance contribution of injector valves and sample loops is also critical for the design of improved multi-dimensional LC systems [25,26].…”

Section: Introductionmentioning

confidence: 99%

“…The dispersion volumetric contribution (V 2 ,inj.vol) is generally related to the square of the injection volume via a dummy factor 1/inj [1,3,7,9,12,16,17,23,29,[32][33][34] (also denoted as 1/D², 1/K², 1/k²).…”

Section: Introductionmentioning

confidence: 99%

On-tubing fluorescence measurements of the band broadening of contemporary injectors in ultra-high performance liquid chromatography

Broeckhoven

Vanderlinden

Guillarme

et al. 2018

Journal of Chromatography A

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We report on a detailed study of the injection contribution to band broadening in contemporary UHPLC-instruments, using either flow-through needle or fixed loop injection (full loop). Using on-tubing fluorescence measurements at the outlet of the injector valve, very localized and undisturbed measurements were obtained. Varying both the flow rate and the injected volume allowed to split the injection variance (σ) in a volumetric component (related to the amount injected) and a hydrodynamic component (related to the flow rate). For the flow-through needle injector and for the small injection volumes (<2 μL) typically used in UHPLC, it was found that the volumetric contribution (i.e. the part of σ, that increases with increasing injection volume) is given by a value of σ = 0.8 to 1·V rather than by the value of 0.125 to 0.2·V that is normally assumed in literature. For the hydrodynamic contribution to σ (i..e, the part which remains present even for very small injection volumes), a clear increase in dispersion with flow rate is found, reaching a plateau around 0.8ml/min of 0.6 μL² or 1.2 μL² for the 75 μm and 120 μm needle seat capillaries respectively. The difference between both shows the clear advantage of using a low dispersion 75 μm injection needle seat capillary. For a loop-type injector operated in a full-loop mode, the increase in peak variance with the injection volume is much less pronounced, leading to a total injector variance given by σ = 0.34 μL² + 0.12·V over the entire range of investigated injection volumes of 1.1 μL up to 4.5 μL when using 120 μm or narrower ID loops. This expression was nearly completely independent of the flow rate. For larger ID sample loops, a clear increase of peak variance with flow rate at fixed injection volume was observed (σ increases with 20% for a 170 μm ID loop and with 70% for a 220 μm ID loop from 0.3 to 1 ml/min).

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“…where t capillary is the residence time of the analyte in the capillaries, d the diameter of the capillaries and D m the molecular diffusion coefficient [12][13][14]. On the Shimadzu equipment, the inner diameter of the capillaries is…”

Section: Taylor-aris Disperson Of Macromoleculesmentioning

confidence: 99%

Flow-Reversal Experiments with Macromolecules to Measure Column End Efficiency and Bed Heterogeneity

Zelenyánszki

Felinger

2019

Chromatographia

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A flow-reversal method combined with peak parking has been introduced recently to determine the band broadening occurring at the two respective column ends and in the bed of the packing material. Flow-reversal has a peak compression effect, therefore, the peaks observed are always narrower and more symmetrical than the peaks obtained without reversing the flow. This phenomenon can originate from the compensation of the multipath dispersion effects. In the present study, peak parking and flow-reversal measurements were extended to macromolecules and carried out with human insulin. We observed that the peaks of insulin are always narrower with reversed flow than without reversing the flow, and the compression effect can be significantly larger than it is for small molecules. The contributions of the column inlet and outlet to the total band variance have been characterized.

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Measurement and Modeling of Extra-Column Effects Due to Injection and Connections in Capillary Liquid Chromatography

Cited by 36 publications

References 101 publications

Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

On-tubing fluorescence measurements of the band broadening of contemporary injectors in ultra-high performance liquid chromatography

Flow-Reversal Experiments with Macromolecules to Measure Column End Efficiency and Bed Heterogeneity

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