Total Peak Capacity Research Articles

Comprehensive two-dimensional countercurrent chromatography × gas chromatography characterization of Artemisia argyi essential oil

A comprehensive two-dimensional (2D) countercurrent chromatography (CCC) × gas chromatography (GC) was investigated for characterization of chemical constituents of Artemisia argyi essential oil, and orthogonality for the 2D chromatographic system was evaluated. A solvent system composed of n-hexane/acetonitrile/methanol (2:2:1, v/v/v) was selected for first dimensional separation of Artemisia argyi essential oil. Then all CCC fractions were analyzed by GC, which provided a wealth of information regarding the composition of the essential oil. Visualization of chemical compositions obtained from the comprehensive 2D CCC × GC separation was achieved by creation of a 2D contour plot map. Total peak capacity was evaluated and approximately 1392 peaks were obtained through a comprehensive 2D CCC × GC separation. A high spatial coverage and a low linear correlation coefficient were achieved. Meanwhile, all compounds were identified by GC-MS. The obtained 2D contour plot could be divided into six zones to show the characteristic chemical compositions. Six zones could be divided into different component groups, including monoterpenes, sesquiterpenes, monoterpene alcohols, phenols, aldehydes, ketones and esters, which could be used to identify compounds that have not been reported, and to predict the structure of unknown compounds in Artemisia argyi essential oil and comprehensively characterize fingerprint peak.

Off-line comprehensive two-dimensional reversed-phase countercurrent chromatography with high-performance liquid chromatography: Orthogonality in separation of Polygonum cuspidatum Sieb. et Zucc.

Off-line comprehensive two-dimensional reversed-phase countercurrent chromatography with high-performance liquid chromatography was investigated in separation of crude ethanol extract from traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. Two-dimensional contour plots for countercurrent chromatography with high-performance liquid chromatography was obtained after comprehensive separation was completed. Total peak capacity was evaluated and approximately 810 peaks were obtained through a comprehensive two-dimensional separation. A highly orthogonality of 52.23% and a large separation space occupancy of 88.86% were achieved. Meanwhile, it was found that several components could be well separated by countercurrent chromatography while they could not be separated by high-performance liquid chromatography, and vice versa, which further indicated the orthogonality of the two separation methods. The off-line comprehensive two-dimensional countercurrent chromatography with high-performance liquid chromatography provided a promising and powerful method for separation of complex natural products.

Column selection approach to achieve a high peak capacity in comprehensive three-dimensional gas chromatography

The separation power of comprehensive three-dimensional gas chromatography (GC3) is substantially enhanced through proper selection of the phase volume ratio, β, of each column relative to each other on successive dimensions. Consideration and application of the ratio of phase volume ratios, βr, or β ratio, between successive dimensions has been a relatively un-studied approach to maximize separating power in comprehensive multidimensional GC instrument design. Herein, proper selection of βr in multidimensional GC is shown to control the elution temperature, Te, of analytes throughout a 40 min primary (1D) column separation, and thus better control width-at-base, W, on all three dimensions. Specifically, between the 1D and secondary (2D) columns, a βr of 0.45 was applied, and between the 2D and tertiary (3D) columns a βr of 1.0 was applied. A total ideal peak capacity of 30,600, or a peak capacity production of ~770 peaks/min, was accomplished with the GC3 instrument with the reconfigured parameters. Additionally, due to the complex nature of this three-dimensional data, a novel approach to “slicing” the chromatographic run into user-defined time intervals is shown. This novel way to view the data still elicits a traditional GC×GC chromatograms, but with the focus on 2D × 3D separations. Moreover, due to proper βr selection, every 2 s window (i.e. every 1D modulation period) is shown to have a peak capacity of ~50–100 for each 2D × 3D separation. This high overall peak capacity (30,600) and peak capacity per 1D modulation (~50–100), courtesy of proper column selection, is demonstrated to hold great promise to physically separate truly complex mixtures.

Implications of phase ratio for maximizing peak capacity in comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

The relationship between the phase ratio, β, of the primary (1D) and secondary (2D) separation dimensions of comprehensive two-dimensional (2D) gas chromatography (GC×GC) separations, and the implications of β on realization of maximal 2D peak capacity, nc,2D, are examined. A GC×GC chromatographic system with time-of-flight mass spectrometry, TOFMS, was otherwise held constant for the separation of a multi-component test mixture spanning a range of chemical functionalities, while only the β of the two analytical columns were changed, 1β for 1D and 2β for 2D. Six column sets were studied using common, commercially available β values. The β ratio, βR=1β/2β, is defined as a quantitative metric to facilitate this study. It is demonstrated that βR plays a key role in maximizing nc,2D. Overall, βR substantially affected nc,2D by influencing retention factors on the 2D column, 2k, and thereby changing the modulation period, PM, necessary for proper 2D column separations. The necessary changes to PM modify the modulation ratio, MR, which affects the 1D column peak widths and 1nc due to the impact of undersampling. Through changes to 1β, the range of 2k can be controlled, with subsequent effects to both 2nc and 1nc. These effects were opposite in direction, such that improvements to 2nc may result in declines in 1nc. It is observed that due to the pseudo-isothermal nature of the 2D separation, there are diminishing returns to extending the 2nc at the cost of 1nc. In this particular study, column set 3 (1D: 20m length, 250μm i.d., 0.25μm film; 2D: 2m, 180μm i.d., 0.2μm film; βR=1.11) with a PM of 3s provided the highest theoretical nc,2D of ∼8200, though this was at a relatively low MR of ∼1.8. Column set 2 (1D: 20m length, 250μm i.d., 0.5μm film; 2D: 2m, 180μm i.d., 0.2μm film; βR=0.56) with a PM of 1.5s provided a high theoretical nc,2D of ∼5800, at a much higher MR of ∼3.7. Though column set 2 had a lesser total peak capacity than column set 3, its higher MR suggests that by improving the 1D column efficiency (i.e., narrowing the 1D column peak widths) to improve 1nc, can result in an increased theoretical nc,2D.

Comprehensive Three-Dimensional Gas Chromatography with Time-of-Flight Mass Spectrometry.

Development of comprehensive, three-dimensional (3D) gas chromatography with time-of-flight mass spectrometric detection (GC3/TOFMS) is described. This instrument provides four dimensions (4D) of chemical selectivity and includes significant improvements to total selectivity (mass spectrometric and chromatographic), peak identification, and operational temperature range relative to previous models of the GC3 reported. The new instrumental design and data output are evaluated and illustrated via two samples, a 115-component test mixture and a diesel fuel spiked with several compounds, for the purpose of illustrating the chemical selectivity benefits of this instrumental platform. Useful approaches to visualize the 4D data are presented. The GC3/TOFMS instrument experimentally achieved total peak capacity, nc,3D, ranging from 5000 to 9600 (x̅ = 7000, s = 1700) for 10 representative analytes for 50 min separations with component dimensional peak capacities averaging 406, 3.6, and 4.9 for 1D, 2D, and 3D, respectively. Particularly, GC3/TOFMS achieved a combined 2D × 3D peak capacity ranging from 10 to 26 (x̅ = 17.6, s = 5.0), which is similar to what is achieved by 2D alone in a GC × GC operating at equivalent modulation period conditions. The analytical benefits of employing three varied chemical selectivities in the 3D separation coupled with TOFMS are illustrated through the separation and detection of 1,6-dichlorohexane and cyclohexyl isothiocyanate as part of the diesel fuel analysis.

Longitudinal On-Column Thermal Modulation for Comprehensive Two-Dimensional Liquid Chromatography.

Longitudinal on-column thermal modulation for comprehensive two-dimensional liquid chromatography is introduced. Modulation optimization involved a systematic investigation of heat transfer, analyte retention, and migration velocity at a range of temperatures. Longitudinal on-column thermal modulation was realized using a set of alkylphenones and compared to a conventional valve-modulator employing sample loops. The thermal modulator showed a reduced modulation-induced pressure impact than valve modulation, resulting in reduced baseline perturbation by a factor of 6; yielding a 6-14-fold improvement in signal-to-noise. A red wine sample was analyzed to demonstrate the potential of the longitudinal on-column thermal modulator for separation of a complex sample. Discrete peaks in the second dimension using the thermal modulator were 30-55% narrower than with the valve modulator. The results shown herein demonstrate the benefits of an active focusing modulator, such as reduced detection limits and increased total peak capacity.

A novel four-dimensional analytical approach for analysis of complex samples.

A two-dimensional LC (2D-LC) method, based on the work of Erni and Frei in 1978, was developed and coupled to an ion mobility-high-resolution mass spectrometer (IM-MS), which enabled the separation of complex samples in four dimensions (2D-LC, ion mobility spectrometry (IMS), and mass spectrometry (MS)). This approach works as a continuous multiheart-cutting LC system, using a long modulation time of 4min, which allows the complete transfer of most of the first - dimension peaks to the second - dimension column without fractionation, in comparison to comprehensive two-dimensional liquid chromatography. Hence, each compound delivers only one peak in the second dimension, which simplifies the data handling even when ion mobility spectrometry as a third and mass spectrometry as a fourth dimension are introduced. The analysis of a plant extract from Ginkgo biloba shows the separation power of this four-dimensional separation method with a calculated total peak capacity of more than 8700. Furthermore, the advantage of ion mobility for characterizing unknown compounds by their collision cross section (CCS) and accurate mass in a non-target approach is shown for different matrices like plant extracts and coffee. Graphical abstract Principle of the four-dimensional separation.

Analyses of Total Alkaloid Extract of Corydalis yanhusuo by Comprehensive RP × RP Liquid Chromatography with pH Difference.

A comprehensive two-dimensional (2D) reverse phase (RP) liquid chromatography (LC) method is developed for alkaloid analysis. This offline comprehensive 2D method is developed using different pH values. With a pH value of 10.5, most alkaloids appear in the form of neutral molecules possessing high retention factors based on their polarity, while the alkaloid polarity order is changed when the pH value decreased to 3.0. The performance of pH modulated 2D LC is demonstrated with 8 alkaloid standards which resulted in orthogonal separation. The developed method is then applied to total alkaloid separation in Corydalis yanhusuo. The first-dimension separation is carried out using methanol and water containing 1.0% ammonium hydroxide and a strong base-resistant RP column, which afforded a peak capacity of 94. The second-dimension analysis is carried out with a surface positive charge column providing a peak capacity of 205 using a mobile phase consisting of acetonitrile and water with 0.15% formic acid. 2D analyses of total alkaloid extract from C. yanhusuo afford a total peak capacity of 9090. Sixteen compounds were tentatively identified based on their ultraviolet spectrum and MS/MS analyses. The proposed method provides an alternative approach to achieve high peak capacity for analysis of alkaloid extract.

Multi-Dimensional Liquid Chromatography and Metabolomics, Are Two Dimensions Better Than One?

While data processing methods in metabolomic studies often work with 'n' number of dimensions, analytical techniques, with the notable exception of NMR, have mostly stuck only to one. Peak overlap continues to be a problem and there is an ever-present demand to maximize the number of metabolites that can be separated and identified in a single run. One method that might help to overcome these issues is multidimensional liquid chromatography, which uses two columns of different phases. A sequential collection of aliquots is made from the first column and reinjected onto a second, and the resulting data are then plotted in 2D or 3D space. The total peak capacity of such a system is the combined peak capacities of each column. The 'offline' version of this technique, using a fraction collector, was introduced over 30 years ago but with recent advances in instrumentation and software, particularly the 'online' approach using automated switching valves, has led to increasing interest in the technique. Both offline and online methods can be carried out as a comprehensive procedure, or via 'heart-cutting', in which only specific peaks are analysed in the second dimension. Past applications include proteomics, natural product chemistry, forensic science and pharmaceutical analysis. These successes are likely to be built on in the future as new column chemistries and bio-informatic approaches are developed. In this review an overview of the theory of twodimensional liquid chromatography is presented, its potential in the field of metabolomics is assessed and predictions for future research directions are made.

Multidimensional capillary electrophoresis.

Multidimensional separation where two or more orthogonal displacement mechanisms are combined is a promising approach to increase peak capacity in CE. The combinations allow dramatic improvement of analytical performance since the total peak capacity is given by a product of the peak capacities of all methods. The initial reports were concentrated on the construction of effective connections between capillaries for 2D analysis. Today, 2D and 3D CE systems are now able to separate real complex biological or environmental mixtures with good repeatability, improved resolution with minimal loss of sample. This review will present the developments in the field of multidimensional CE during the last 15 years. The endeavors in this specific field were on the development of interfaces, interface-free techniques including integrated separations, microdevices, and on-line sample concentration techniques to improve detection sensitivity.

Development of online conventional array-based two-dimensional liquid chromatographic system for proteins separation in human plasma

Human plasma is one of the proteins-containing samples most difficult to characterize on account of the wide dynamic concentration range of its intact proteins. Herein, we developed a high-throughput conventional array-based two-dimensional liquid chromatographic system for proteins separation in human plasma in online mode. In the system, a conventional strong-anion exchange chromatographic column was used as the first separation dimension and eight parallel conventional reversed-phase liquid chromatographic columns were integrated as the second separation dimension. The fractions from the first dimension were sequentially transferred into the corresponding reversed-phase liquid chromatographic precolumns for retention and enrichment using a 10-port electrically actuated multi-position valve. The second dimensional solvent flow was directly and identically split into 8 channels. The fractions were concurrently back-flushed from the precolumns into the 8 conventional RP columns and were separated simultaneously. An 8-channel fraction collector was refitted to collect the reversed-phase liquid chromatographic fractions for further investigation. Bicinchoninic acid (BCA) dyein solution was conveniently used for high-abundance protein location. Two separation dimensions were relatively independent parts, as well as each channel of the second dimensional array separation. Therefore, the new system could improve the separation throughput and total peak capacity. The system was successfully applied for the separation of human plasma intact proteins. The results indicated the established system is an effective method for removing high abundance proteins in plasma and in-depth research in plasma proteomics.

Study on the performance of different types of three-dimensional chromatographic systems

The maximum achievable performance of possible types of three-dimensional chromatographic systems (LC×LC×LC) has been investigated. The Pareto-optimization approach was applied to establish a trade-off between three main objectives (total peak capacity, analysis time and dilution of the sample) and Pareto-front values were obtained. The performances of xLC×xLC×xLC (three-dimensional separation in space), tLC×tLC×tLC (three-dimensional separation in time) and the hybrid xLC×xLC×tLC system were compared mutually and with two-dimensional chromatographic systems. It was found that xLC×xLC×xLC performs best in terms of maximum achievable peak capacity in shortest analysis time. Based on current thin-layer-chromatography performance it should be possible to obtain a peak capacity of 50,000 within 20min. If contemporary column-packing standards can be upheld the achievable limit is approximately 50% higher. However, in an xLC×xLC×xLC chromatographic system analytes remain in the separation domain after the analysis, which complicates the detection. Use of an xLC×xLC×tLC system with elution in the last dimension alleviates the detection problem. The maximum achievable peak capacity in the same analysis time is lower for xLC×xLC×tLC than for xLC×xLC×xLC. Using the same (reasonable) length of the separation domain (e.g. a cube 200×200×200mm) for both systems, it is possible to achieve peak capacities of 78,000 for xLC×xLC×tLC operated in the gradient mode, which is twice higher than for an xLC×xLC×xLC system. A three-dimensional (three-column) time-based tLC×tLC×tLC system does not greatly improve the performance of tLC×tLC in terms of (maximum) peak capacity and (minimum) analysis time. Dilution factors in tLC×tLC×tLC are very high. Decreasing the dilution has a detrimental influence on the peak capacity. The trade-off between these objectives is of crucial importance. The influence of several parameters (length of the separation domain, particle size, etc.) on the performance of chromatographic systems was investigated, optimal ranges were found.

High throughput analysis of atmospheric volatile organic compounds by thermal injection – isothermal gas chromatography – time-of-flight mass spectrometry

Sixty one volatile organic compounds (VOCs) from a standard gas mixture were separated via isothermal gas chromatography coupled with time-of-flight mass spectrometry (GC–TOFMS) in a ∼35s separation time window (∼45s separation). The VOCs in the standard gas mixture were selected based on the EPA TO-15 methodology. The high throughput separation was achieved with a relatively high total peak capacity (nc∼114), by simultaneously minimizing both on-column and off-column peak width broadening. The on-column contributions to peak width broadening were minimized by taking into account and applying GC separation theory for the selection of column dimensions and carrier gas flow rate conditions. Both fast cryogenic focusing and re-injection of compounds (implemented via a commercially available thermal modulator and referred to herein as thermal injection (TI) and fast TOFMS detection (100scans/s)) were applied to reduce off-column sources of peak width band broadening (sometimes referred to as off-column band broadening). Cryogenic focusing during TI and minimal band broadening-based dilution during separation resulted in preconcentration factors for the detected peaks ranging from 78 (1,4-dichlorobenzene) to 420 (propylene). Since the injected volume for preconcentration was 500μl, and based on the detected noise levels at selected m/z for each analyte compound, the concentration limit of detection (LOD) ranged from 67ppbv (parts per billion by volume) for propylene, to 4ppbv for freon-12. While application of standard VOC analysis conditions leads to separation times typically ranging from ∼30 to 50 min, the isothermal GC–TOFMS method reported herein represents a 40-fold improvement in analysis time while maintaining peak capacity and detection sensitivity that is comparable to traditional GC–MS VOC analysis.

Ion mobility separation of variant histone tails extending to the "middle-down" range.

Differential ion mobility spectrometry (FAIMS) can baseline-resolve multiple variants of post-translationally modified peptides extending to the 3-4 kDa range, which differ in the localization of a PTM as small as acetylation. Essentially orthogonal separations for different charge states expand the total peak capacity with the number of observed states that increases for longer polypeptides. This potentially enables resolving localization variants for yet larger peptides and even intact proteins.

Metrics of separation performance in chromatography: Part 2. Separation performance of a heating ramp in temperature-programmed gas chromatography

Metrics of separation performance described in Part 1 of this series were applied here to theoretical evaluation of performance of a heating ramp in temperature-programmed GC. As in Part 1, the dependence of the separation (Δs) of two arbitrarily spaced peaks (the number of σ-slots between them) on operational parameters of GC analysis was treated here as the main building block for construction of other performance metrics. Simple expressions describing Δs of two peaks eluting during isobaric linear heating ramp and dependence of Δs on the heating rate, on column efficiency and on the peak spacing were derived. The use of dimensionless heating rate and other dimensionless operational parameters made these expressions universally suitable for different column dimensions, heating rates, carrier gas types, flow rates, etc. The use of dimensionless parameters also made it possible to express Δs as a simple function of the earlier introduced chromatographically meaningful characteristic parameters of thermodynamics of solute–column interactions. The expressions for Δs developed here, together with the ones described in Part 1 were used for theoretical prediction of total separation capacity (sc), total peak capacity (nc), and total number of resolved peaks in temperature-programmed analysis controlled by a balanced temperature program consisting of a linear heating ramp preceded by specially designed balanced temperature plateau. A numerical example of these parameters for a particular column, heating rate, carrier gas, and its flow rate is also provided. It has been shown that nc obtainable in temperature-programmed analysis is 2–3 times larger than that obtainable in equally long isothermal analysis using the same column and gas. Comparison of this improvement with potentially more than an order of magnitude larger peak capacity of GC×GC has been discussed. Also described are the speed of analysis (the number of σ-slots per unit of time), and other characteristics of the separation performance of a heating ramp. In many respects, metric Δs is similar to the widely known metric of resolution (Rs). Advantages of Δs over Rs as a metric of the separation performance, as well as the advantages of other metrics utilized here over their widely known counterparts are discussed.

A simple way to configure on-line two-dimensional liquid chromatography for complex sample analysis: Acquisition of four-dimensional data

An on-line comprehensive two-dimensional liquid chromatography (HPLC×UPLC-TOF MS) was set up just using the injection valve of the ultra performance liquid chromatography (UPLC) as the interface through which the effluent of high performance liquid chromatography (HPLC) was injected automatically to UPLC coupled to time-of-flight mass spectrometry (TOF MS). As a demonstrative application, a complex sample of Traditional Chinese Medicine, Qingkailing was analyzed. As a result, a four-dimensional (4D) data containing 2D retention times, peak intensity and m/z ratios was plotted, where 398peaks were counted and low concentration components were distinguished from the high concentration ones with a total peak capacity of 1090. Comparing with traditional 3D data acquired by HPLC×HPLC, the 4D data generated by HPLC×UPLC-TOF MS can increase the number of recognized components by three times, reduce the analysis time by 75%. Such a configuration of HPLC×UPLC-TOF MS can realize easily upon commercial chromatographs while exhibited enhanced separation efficiency, high sensitivity, huge peak capacity and great potential in complex sample analysis.

High-speed cryo-focusing injection for gas chromatography: Reduction of injection band broadening with concentration enrichment

In order to maximize peak capacity and detection sensitivity of fast gas chromatography (GC) separations, it is necessary to minimize band broadening, and in particular due to injection since this is often a major contributor. A high-speed cryo-focusing injection (HSCFI) system was constructed to first cryogenically focus analyte compounds in a 6cm long section of metal MXT column, and second, reinject the focused analytes by rapidly resistively heating the metal column via an in-house built electronic circuit. Since the cryogenically cooled section of column is small (∼750nl) and the direct resistive heating is fast (∼6000°C/s), HSCFI is demonstrated to produce an analyte peak with a 6.3ms width at half height, w1/2. This was achieved using a 1m long column with a 180μm inner diameter (i.d.) operated at an absolute head pressure of 55psi and an oven temperature of 60°C, with a 10V pulse applied to the metal column for 50ms. HSCFI was also used to demonstrate the head space sampling and fast GC analysis of an aqueous solution containing six test analytes (acetone, methanol, ethanol, toluene, chlorobenzene, pentanol). Using Henry’s law constants for each of the analytes, injected mass limits of detection (LODs) were typically in the low pg levels (e.g., 1.2pg for acetone) for the high speed separation. Finally, to demonstrate the use of HSCFI with a complex sample, a gasoline was separated using a 20m×100μm i.d. column and the stock GC oven for temperature programming, which provided a separation time of 200s and an average peak width at the base of 440ms resulting in a total peak capacity of 460 peaks (at unit resolution).

Fast, High Peak Capacity Separations in Gas Chromatography–Time-of-Flight Mass Spectrometry

Peak capacity production (i.e., peak capacity per separation run time) is substantially improved for gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and applied to the fast separation of complex samples. The increase in peak capacity production is achieved by selecting appropriate experimental conditions based on theoretical modeling of on-column band broadening, and by reducing the injection pulse width. Modeling to estimate the on-column band broadening from experimental parameters provided insight for the potential of achieving GC separations in the absence of off-column band broadening, i.e., the additional band broadening not due to the on-column separation process. To optimize GC-TOFMS separations collected with a commercial instrumental platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a commercially available thermal modulator is adapted and applied (referred to herein as thermal injection) to provide a narrow injection pulse, while the TOFMS provided a data collection rate of 500 Hz, initially averaged to 100 Hz for data storage. The use of long, relatively narrow open tubular capillary columns and a 30 °C/min programming rate were explored for GC-TOFMS, specifically a 20 m, 100 μm inner diameter (i.d.) capillary column with a 0.4 μm film thickness to benefit column capacity, operated slightly below the optimal average linear gas velocity (at ~2 mL/min, due to the flow rate constraint of the TOFMS). Standard autoinjection with a 1:100 split resulted in an average peak width of ~1.2 s, hence a peak capacity production of 50 peaks/min. Metabolites in the headspace of urine were sampled by solid-phase microextraction (SPME), followed by thermal injection and a ~7 min GC separation (with a ~6 min separation time window), producing ~660 ms peak widths on average, resulting in a total peak capacity of ~550 peaks (at unit resolution) and a peak capacity production of ~90 peaks/min (~2-fold improvement relative to standard autoinjection with the 1:100 split). This total peak capacity production achieved is equivalent to, or greater than, that currently utilized in metabolomics studies using GC/MS, but with much slower separations, on the order of 40 to 60 min, corresponding to a 5-fold or greater GC/MS analysis throughput rate.

Pareto-optimality study into the comparison of the separation potential of comprehensive two-dimensional liquid chromatography in the column and spatial modes

The expected performance of spatial (“flat-bed”) two-dimensional liquid chromatography (xLC×xLC) has been calculated using the Pareto-optimality strategy. This approach allowed different objectives (total peak capacity, total analysis time, and total dilution) to be considered simultaneously and to establish optimal parameters (pressure drop, particle size, bed length, and initial spot size). The performance of spatial two-dimensional chromatographic systems was compared with that of conventional on-line, real-time two-dimensional column-liquid-chromatography systems (tLC×tLC). The potential gain in peak capacity and/or analysis time of the spatial configuration was confirmed. By restricting the spatial parameters to realistic chromatographic conditions (limiting the stress, as counterbalance for the pressure drop through the sorbent bed, to 2500kg) it was found that xLC×xLC is attractive for very fast analysis of complex samples, rather than for extremely efficient separations. For example, a peak capacity of 780 may be achieved in only 2.7min using a 100×100mm sorbent bed of a quality currently encountered thin-layer chromatography. Furthermore, if beds can be packed as efficiently as contemporary columns, the predicted peak capacity increases to around 1000, corresponding to a peak-production rate of about 6.3peaks/s. Possibilities to boost the performance of xLC×xLC further are briefly discussed. Unless we can overcome the severe stress requirements of high-performance xLC×xLC, conventional tLC×tLC may be more amenable to very complex separations, thanks to the very high peak capacities. However, tLC×tLC separations will require long analysis times (e.g. 10,000 peaks in 37h, corresponding to 0.075peaks/s at a pressure drop of 40MPa). The best trade-off between total peak capacity, total analysis time, and total dilution under restricted (realistic) conditions was obtained using high pressures, small chromatographic beds, small particles, and relatively large sample spots.

Estimation and optimization of the peak capacity of one-dimensional gradient high performance liquid chromatography using a long monolithic silica capillary column

An estimation of the performance and optimization of gradient HPLC conditions using various columns for maximum total peak capacity was studied for “one-shot proteomics”[1] which involves one-dimensional gradient HPLC, connected to an electrospray ionization (ESI)-mass spectrometry (MS), using a monolithic silica-C18 capillary column (350cm long and 100μm internal diameter) and an over 40h shallow gradient elution with one injection. Optimization of such special one-dimensional HPLC has been a tedious task if carried out with a trial-and-error approach due to the extremely long analysis time for each run. Here, the optimized separation conditions including the column type, either particle-packed or monolithic, and the column length with a fixed gradient time are proposed by calculating the peak capacity obtainable using a long column and a long gradient time that may promote the “one-shot proteomics” approach. For instance, conventional conditions at less than 20MPa can be adapted for a 40h gradient elution for the proteomics experiment, and a ca. 3m long monolithic silica-C18 capillary column was identified as the optimized medium indicated by our model with peak capacity theory. To verify this model experimentally, the numbers of identified peptides and proteins were investigated with a nano LC/MS/MS system coupled with a 3m monolithic silica-C18 capillary column by using various elution times. The experimental results showed that the numbers of identified peptides and proteins were maximized and reached a plateau with a gradient time of several tens of hours, which indicated that our model to optimize one dimensional HPLC conditions with a long column could be verified and useful.