Time optimization for routine separations, using high-speed microbore HPLC

Dezaro, Derek A.; Dvorn, D.; Horn, Carl K.; Hartwick, Richard A.

doi:10.1007/bf02280603

Cited by 7 publications

(6 citation statements)

References 24 publications

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“…The determination of capacity factors by WCD measurement of linear velocity is more precise, and potentially more accurate, than the conventional method of retention time measurements k'= (t, -t0)/ t0 (3) Since linear velocity is measured as Ad/At between each pair of adjacent diodes, it is not subject to errors resulting from a delay between injection of the sample and recorder start time. The precision of the k' measurement is improved by the use of multiple detection cells along the column, in this case 14. In order to achieve the same precision using conventional methods, several chromatographic runs would be required.…”

Section: Rate Of Resolutionmentioning

confidence: 99%

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Whole column detection: application to high-performance liquid chromatography

Rowlen¹,

Duell²,

Avery³

et al. 1989

Anal. Chem.

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Section: Rate Of Resolutionmentioning

confidence: 99%

“…Optimization continues to be of major concern to chromatographers. One may find a large variety of strategies for optimization in the literature (12)(13)(14)(15)(16)(17)(18)(19). There are graphical approaches, such as the window-diagram method (13) and overlapping resolution mapping (18), as well as mathematical approaches (15).…”

mentioning

confidence: 99%

Whole column detection: application to high-performance liquid chromatography

Rowlen¹,

Duell²,

Avery³

et al. 1989

Anal. Chem.

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“…However, it has been shown that accurately calculating the variance of these extra-column peaks can sometimes prove difficult due to detection limitations [28], challenges selecting proper integration limits for peak moment analysis [31][32][33][34], and discrepancies in the pressure when either the column or ZDV fitting is being measured [35]. An alternative to this "subtraction" method to determine extra-column effects is the use of linear extrapolation [18,[36][37][38][39][40][41], where the variance of several analyte peaks with different retention factors are extrapolated back to an intercept that describes the extra-column variance. Others have reported the calculation of extra-column band broadening through graphical analysis or deconvolution of the peak shapes eluted from the column [42,43].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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Abstract:As column volumes continue to decrease, extra-column band broadening has become an increasingly important consideration when determining column performance. Combined contributions due to the injector and connecting tubing in a capillary LC system were measured and found to be larger than expected by Taylor-Aris theory. Variance from sigma-type and tau-type broadening was isolated from eluted peaks using the Foley-Dorsey Exponentially Modified Gaussian peak fitting model and confirmed with computational fluid dynamics. It was found that the tau-type contributions were the main cause for the excessive broadening because of poorly-swept volumes at the connection between the injector and tubing. To reduce tau-type contributions (and peak tailing), a timed pinch mode could be used for analyte injection.

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“…Jung, et al 9, using a 100 μm inside diameter (ID) column, performed dopamine (DA) determinations in microdialysate with 0.5 μL samples. The use of capillary columns with much smaller peak volumes than standard or microbore (1 mm diameter) HPLC columns minimizes the dilution of small volume samples10. However, neither of the DA separations mentioned above approached the one-minute timeframe.…”

mentioning

confidence: 99%

Capillary Ultrahigh Performance Liquid Chromatography with Elevated Temperature for Sub-One Minute Separations of Basal Serotonin in Submicroliter Brain Microdialysate Samples

Liu

Zhang

et al. 2010

Anal. Chem.

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Improving the time resolution in microdialysis coupled to high performance liquid chromatography (HPLC) requires that the volume of the separation system be decreased. A lowvolume separation permits smaller microdialysate volumes to be injected without suffering a sensitivity loss from dilution. Thus, improved time resolution can be achieved with offline analysis simply by decreasing the separations system volume. For online (near real-time) analysis, there is a further requirement. The separation speed must be at least as fast as the sampling time. Here, the combined use of high column pressures and temperatures, sub-2-μm stationary phase particles, capillary columns, and sensitive, low dead-volume detection resulted in a retention time for the neurotransmitter serotonin of less than one min in a 500 nL dialysate sample volume. Two sensitive detectors, photoluminescence following electron transfer (PFET) and electrochemical, were used for the detection of sub-nanomolar concentrations of serotonin in brain microdialysate samples. The general principles developed are applicable to a wide range of separations with the additional advantages of increases in sample throughput and decreases in mobile phase usage.Monitoring extracellular levels of serotonin (5-hydroxytryptamine; 5-HT) in the central nervous system with higher temporal resolution than typically practiced (~ 10-30 min) will be a key to understanding how the dynamics of serotonergic signaling modulate normal and disease-associated brain function 1 . The most commonly used method for in vivo measurement of extracellular serotonin concentration is microdialysis, a sampling method, coupled to high performance liquid chromatography (HPLC) 2 . Newton and Justice reported that one-minute time resolution was possible for microdialysis sampling of dopamine (DA) 3 . Indeed, recently, Wang et al. demonstrated 2-s microdialysis time resolution 4 . Thus, microdialysis is not currently the limiting factor in defining the time resolution of microdialysis/HPLC. High speed has recently been attained by HPLC. Approaches to improving speed include the use of high pressure to increase the linear velocity of the mobile phase, elevating column temperatures, and using shorter columns packed with smaller particles 5 . However, these applications relied on typical injection volumes of several μL. In microdialysis, the sample volume is defined by the product of dialysate flow rate and the sampling time. Because typical microdialysis flow rates are in the low-or sub-μL/min range, a sampling time (and thus time resolution) of 10-30 min is generally required to Jung, et al. 9 , using a 100 μm inside diameter (ID) column, performed dopamine (DA) determinations in microdialysate with 0.5 μL samples. The use of capillary columns with much smaller peak volumes than standard or microbore (1 mm diameter) HPLC columns minimizes the dilution of small volume samples 10 . However, neither of the DA separations mentioned above approached the oneminute timeframe. The shortest reported...

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Time optimization for routine separations, using high-speed microbore HPLC

Cited by 7 publications

References 24 publications

Whole column detection: application to high-performance liquid chromatography

Whole column detection: application to high-performance liquid chromatography

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

Capillary Ultrahigh Performance Liquid Chromatography with Elevated Temperature for Sub-One Minute Separations of Basal Serotonin in Submicroliter Brain Microdialysate Samples

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