Total Peak Capacity Research Articles

Comprehensive Study on the Optimization of Online Two-Dimensional Liquid Chromatographic Systems Considering Losses in Theoretical Peak Capacity in First- and Second-Dimensions: A Pareto-Optimality Approach

A method to optimize different objectives (total analysis time, total peak capacity, and total dilution) has been applied to comprehensive two-dimensional liquid chromatography. The approach is based on Pareto-optimality, and it yields optimal parameters (column particle sizes, column diameters, and modulation times). Losses in the peak capacities in the first dimension (due to undersampling) and in the second dimension (due to high injection volumes) have been taken into account. The first effect (detection band broadening) reduces the original peak capacity by about a half, the second effect can reduce the total peak capacity by an additional half. Thus, the total loss in peak capacity may be 75% of its theoretical value. Analytical expressions to calculate these effects under gradient and isocratic conditions are derived. Of particular interest is the study of the optimal modulation times, which corresponded to between 2 and 3 two-dimensional runs per one-dimensional peak. The effects of using gradient or isocratic elution, conventional (40 MPa) or "ultra-high" (100 MPa) pressures, and focusing between the first and second dimensions were also studied. A trade-off between total peak capacity, total analysis time, and total dilution can be established.

On-line separation of native proteins by two-dimensional liquid chromatography using a single column

This paper reports the on-line separation of native (N) proteins by two-dimensional liquid chromatography (2D-LC) using a single column with one phase (called 2D column). The 2D column exhibits excellent resolution, selectivity, and retention of proteins in the N state and functions in two retention modes—hydrophobic interaction chromatography (HIC) and weak-cation exchange chromatography (WCX). We describe a new approach to on-line buffer exchange and collection of fractions from the first retention mode and their quantitative re-injection into the same column, followed by re-separation in the second retention mode. Thus, liquid chromatography in a closed system and in an on-line manner could be successfully carried out. This method was termed on-line protein separation by 2D-LC using only a single column (on-line 2D-LC-1C). The applicability of this method was experimentally demonstrated using standard proteins and a human serum sample. The total hypothetical maximum possible peak capacity n c,total and total sample peak capacity n c , total * of the 2D column were 329 and 199, respectively. By comparison against several popular commercially available columns, it was found that the 2D column had not only comparable resolution and better selectivity but also some unique characteristics. This 2D-LC-1C method could be applied to the fast purification of intact proteins in the N state, such protein drugs from natural products, and recombinant proteins and also for the fast pre-fractionation of intact proteins in the “top-down” MS strategy in proteomics.

Dependence of Effective Peak Capacity in Comprehensive Two-Dimensional Separations on the Distribution of Peak Capacity between the Two Dimensions

One of the basic tenets of comprehensive two-dimensional chromatography is that the total peak capacity is simply the product of the first- and second-dimension peak capacities. As formulated, the total peak capacity does not depend on the relative values of the individual dimensions but only on the product of the two. This concept is tested here for the experimentally realistic situation wherein the first-dimension separation is undersampled. We first propose that a relationship exists between the number of observed peaks in a two-dimensional separation and the effective peak capacity. We then show here for a range of reasonable total peak capacities (500-4000) and various contributions of peak capacity in each dimension (10-150) that the number of observed peaks is only slightly dependent on the relative contributions over a reasonable and realistic range in sampling times (equal to the first-dimension peak standard deviation, multiplied by 0.2-16). Most of this work was carried out under the assumption of totally uncorrelated retention times. For uncorrelated separations, the small deviations from the product rule are due to the "edge effect" of statistical overlap theory and a recently introduced factor that corrects for the broadening of first-dimension peaks by undersampling them. They predict that relatively more peaks will be observed when the ratio of the first- to the second-dimension peak capacity is much less than unity. Additional complications are observed when first- and second-dimension retention times show some correlation, but again the effects are small. In both cases, deviations from the product rule are measured by the relative standard deviations of the number of observed peaks, which are typically 10 or less. Thus, although the basic tenet of two-dimensional chromatography is not exact when the first dimension is undersampled, the deviations from the product rule are sufficiently small as to be unimportant in practical work. Our results show that practitioners have a high degree of flexibility in designing and optimizing experimental comprehensive two-dimensional separations.

Calculating Optimal Modulation Periods to Maximize the Peak Capacity in Two-Dimensional HPLC

Theoretical calculations are presented to optimize modulation period for maximum total peak capacity in comprehensive two-dimensional HPLC (2D-HPLC) taking into account the effect of modulation on the apparent peak capacity of the first-dimension (1D) separation. Results indicate that modulation periods are most favorable when they are adjusted to approximately 2.2-4 times the standard deviation of a 1D peak in order to avoid excessively short run times at the second dimension (2D). Data are presented that effective peak capacities of several thousand in 60 min can be expected for practical 2D-HPLC conditions, utilizing 1D gradient elution followed by 2D isocratic elution, that remain at approximately 50-70% of the theoretical maximum peak capacity. This work suggests that lower modulation frequencies and longer 2D separation times than previously proposed are favorable under realistic chromatographic conditions, alleviating some practical problems associated with 2D-HPLC.

On-line parallel reversed phase two-dimensional liquid chromatography for high-throughput analysis of complex proteomic samples

A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is constructed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by reversed phase chromatography (Hypersil BDS C18, 15 mm×4.6 mm I.D.) to separate the complex peptides from globin peptic hydrolysate. After the sample was loaded on the SCX column, the phosphate buffer (pH 4.0) was used to elute the peptides. Then, elutes flowed through the interface and the peptides focused on the head of the trapping columns (Hypersil BDS C18, 15 mm×4.6 mm I.D.) but salt passed into the waste. After the valve was switched, the samples were flushed with a backward flow into the RP analytical column. The peptides on the SCX were eluted with 12 discontinuous steps linearly increasing salt concentrations. The peptides enriched on the trapping column were desalted and separated by the RP columns. The resolution and the resolved peaks of the 2D SCX/RP system were greatly increased and the total peak capacity reached as high as 2280.

Off-line comprehensive two-dimensional high-performance liquid chromatography system with size exclusion column and reverse phase column for separation of complex traditional Chinese medicine Qingkailing injection

A comprehensive two-dimensional liquid chromatography system was constructed on the combination of size exclusion chromatography (SEC) and reverse phase liquid chromatography (RPLC). The first dimension used a SEC column with 300 mm × 8 mm i.d., packed with Toyopearl HW-40S. The column was eluted with 0.05 mol/l Tris–HCl (pH 6.9) at a flow rate of 0.4 ml/min. The second dimension used a RPLC column with 100 mm × 4.6 mm i.d., which was operated in gradient form at a flow rate of 1.0 ml/min. An automatic switching valve was used to collect the effluent of the SEC column every 3 min, and the effluent was automatically injected into the RPLC column. Mass spectrometer was used for peak identification. This system was used to separate Qingkailing injection, a traditional Chinese medicine (TCM). The result showed that the total peak capacity of this system could reach 1134 and the qualitative analysis of seven chemical components of the Qingkailing injection was accomplished by this system. The results show that comprehensive two-dimensional liquid chromatography system is of great importance and high value in the separation of complex TCM.

Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LC × LC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the “analytic dynamic range” problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.

Two-dimensional reversed-phase liquid chromatography using two monolithic silica C18 columns and different mobile phase modifiers in the two dimensions

Comprehensive two-dimensional (2D) HPLC in the reversed-phase liquid chromatography (RPLC) mode using C18 silica monolith columns at first dimension (1st-D) (10 cm × 4.6 mm I.D.) and second dimension (2nd-D) (5 cm × 4.6 mm I.D.) was carried out successfully. A mixture of water and tetrahydrofuran (THF) was used as a mobile phase in the 1st-D separation, and a mixture of water and methanol (CH 3OH) in the 2nd-D separation. Sample fractions from 1st-D column were directly loaded into an injection loop of the 2nd-D HPLC equipped with two injector valves for one column. The fractionation time at the 1st-D that was equal to the separation time at the 2nd-D was 45 or 60 s. Total peak capacity up to 900 was obtained in about 60 min for the isocratic mode separation of aromatic compounds in this system. Gradient elution mode applied to both 1st-D and 2nd-D separations resulted in shorter separation time and better separation efficiencies than the isocratic mode. It was demonstrated that 2D-HPLC systems employing popular C18 stationary phases with different organic modifiers in mobile phases for each dimension could produce large peak capacity. The different selectivities were provided by the difference in polar interactions between a solute and the organic modifier existing in the stationary phase.

Rapid sequential heart-cut multidimensional gas chromatographic analysis

A method is described which allows the sequential fast analysis of heart-cuts taken during the first column elution of an essential oil sample in a multidimensional gas chromatography experiment. In this investigation, a relatively short, narrow bore capillary column is used in the second dimension, with fast cryogenic modulation permitting rapid delivery of cryofocussed heart-cuts into the second column of the multidimensional arrangement. In this implementation, the total analysis time on the second column is of the order of 30 s, which is less than the duration of the sampled heart-cuts (1 min), with peak theoretical efficiencies of about 7000 m −1. Thus, the method should allow on-line heart-cutting of as many discrete heart-cuts as one may require from the first dimension chromatogram, and so two-dimensional separation can be achieved for almost the whole sample in one single analysis without the need for repeat injections or cycling of the oven temperature. The method is demonstrated by the transfer and rapid re-injection of 1 min heart-cuts taken from an initial separation stage of peppermint essential oil. The cryofocussing and fast analysis step is shown to increase signal response by up to 40-fold. Total peak capacity in the described system is measured to be of the order of 1800, with capacities of about 20–30 for each 30 s second dimension analysis. By increasing the frequency of sampling to remove excessive (unused) time to develop the 2 D separation, it should be possible to achieve peak capacities of 5000 or more baseline resolved peaks.

Packed Capillary Reversed-Phase Liquid Chromatography with High-Performance Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Proteomics

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.

Peak capacity, peak-capacity production rate, and boiling point resolution for temperature-programmed GC with very high programming rates

Recent advances in column heating technology have made possible very fast linear temperature programming for high-speed gas chromatography. A fused-silica capillary column is contained in a tubular metal jacket, which is resistively heated by a precision power supply. With very rapid column heating, the rate of peak-capacity production is significantly enhanced, but the total peak capacity and the boiling-point resolution (minimum boiling-point difference required for the separation of two nonpolar compounds on a nonpolar column) are reduced relative to more conventional heating rates used with convection-oven instruments. As temperature-programming rates increase, elution temperatures also increase with the result that retention may become insignificant prior to elution. This results in inefficient utilization of the down-stream end of the column and causes a loss in the rate of peak-capacity production. The rate of peak-capacity production is increased by the use of shorter columns and higher carrier gas velocities. With high programming rates (100-600 degrees C/min), column lengths of 6-12 m and average linear carrier gas velocities in the 100-150 cm/s range are satisfactory. In this study, the rate of peak-capacity production, the total peak capacity, and the boiling point resolution are determined for C10-C28 n-alkanes using 6-18 m long columns, 50-200 cm/s average carrier gas velocities, and 60-600 degrees C/min programming rates. It was found that with a 6-meter-long, 0.25-mm i.d. column programmed at a rate of 600 degrees C/min, a maximum peak-capacity production rate of 6.1 peaks/s was obtained. A total peak capacity of about 75 peaks was produced in a 37-s long separation spanning a boiling-point range from n-C10 (174 degrees C) to n-C28 (432 degrees C).

Temperature Programming for High-Speed GC

Fast temperature programming (20-50 °C/min) is used with relatively short separation columns to achieve high-speed separations of mixtures covering a wide boiling point range. A cryofocusing inlet is used to obtain narrow injection plugs. High-speed temperature-programmed chromatograms are evaluated by considering local peak capacity as a function of carbon number and boiling point for the normal alkanes in the range C(8)-C(19). The peak capacity generation rate (peaks per second) as a function of carbon number and the total cumulative peak capacity as a function of time are also considered for various column lengths and carrier gas flow rates. Column lengths in the range 3.6-25.4 m and average carrier gas velocity values in the range 50-200 cm/s are considered. For a 6.8-m-long, 0.25-mm-i.d. column operated at an average carrier gas velocity of about 100 cm/s and using a nominal programming rate of 50 °C/min, C(19) elutes in 178 s with a total peak capacity of 168 peaks. If the programming rate is reduced to 20 °C/min, the C(19) elution time more than doubles but the total peak capacity increases by only 20%. For a 25.4-m-long column using a nominal 50 °C/min programming rate, the C(19) retention time is 262 s with a peak capacity of 279 peaks. The use of average carrier gas flow rates greater than about 100 cm/s, which is common in isothermal high-speed GC, results in a considerable loss in total peak capacity with remarkably little reduction in analysis time.