Comprehensive Two-dimensional Liquid Chromatography System Research Articles

Enzyme activity- and chemometrics-assisted comprehensive two-dimensional liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry for the analysis of honeysuckle

A unique and effective comprehensive two-dimensional liquid chromatography system was established and applied for the analysis of bioactive components in honeysuckle. Under the optimal conditions, Eclipse Plus C18 (2.1 × 100 mm, 3.5 μm, Agilent) and SB-C18 (4.6 × 50 mm, 1.8 μm, Agilent) columns were chosen for the first dimension (1D) and the second dimension (2D) separation. The optimal flow rates of 1D and 2D were 0.12 mL/min and 2.0 mL/min, respectively. Additionally, the proportion of organic solution was optimized to enhance orthogonality and integrated shift, and full gradient elution mode was adopted to improve chromatographic resolution. Furthermore, a total of 57 compounds were identified by molecular weight, retention time and collision cross-section value obtained from ion mobility mass spectrometry. Based on the data obtained from the principal component analysis, partial least squares discriminant analysis, and hierarchical cluster analysis, the categories of honeysuckle in different regions were significantly different. Moreover, the half maximal inhibitory concentration values of most samples were between 0.37 and 1.55 mg/mL, and most samples were potent α-glucosidase inhibitors, which is better for the evaluation of the quality of drugs from two aspects of substance content and activity.

Determination of the polyphenolic content of berry juices using focusing-modulated comprehensive two-dimensional liquid chromatography coupled to mass spectrometry detection.

In this work, a comprehensive two-dimensional liquid chromatography system, comprised of a ZIC-HILIC and C18 columns in the first and second dimensions, respectively, was tuned and employed for attaining high resolution profiles of the polyphenolic pattern in seven commercial berry juices. The developed HILIC × RP-LC method was validated in terms of linearity range, correlation coefficients, limit of detection, limit of quantification, precision (intra- and inter-day), and recovery. A total of 104 polyphenolic compounds belonging to different chemical classes (hydroxybenzoic and cinnamic acid derivatives, flavone glycosides, flavonols, flavonol glycosides, dihydroflavonols, and anthocyanin glycosides) have been characterized and quantified in the juices investigated. Despite the constituents being similar, a notable quantitative variation among the analyzed berry species was observed. Elderberry contained the highest amount of polyphenols (918 ± 1.10mg 100mL-1), followed by chokeberry (516 ± 0.08mg 100mL-1). On the other hand, raspberry contained the lowest amount (104 ± 1.21mg 100mL-1). Further, total phenolic, flavonoid, and anthocyanin contents were determined spectrophotometrically, yielding consistent results. The free-radical scavenging activity (DPPH test) and reducing power of the juices, expressed as IC50 (μL mL-1) and mg ASE mL-1, varied from 2.79 ± 0.03 (honeyberry) to 31.66 ± 0.02 (blueberry) and from 1.71 ± 0.01 (blueberry) to 8.89 ± 0.12 (chokeberry), respectively. Such a ZIC-HILIC × C18 platform based on focusing modulation, never employed so far for berry juices, showed a remarkable separation capability with high values of corrected peak capacity (up to 1372) and orthogonality (Ao up to 0.80), thus providing a great applicability to be advantageously employed for other complex food samples.

Chiral separation and chemical profile of Dengzhan Shengmai by integrating comprehensive with multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Chemical profile for Chinese medicine formulas composed of several herbs is always a challenge due to a big array of small molecules with high chemical diversity so much as isomers. The present paper develops a feasible strategy to characterize and identify complex chemical constituents of a four-herb traditional Chinese medicine formula, Denzhan Shenmai (DZSM) by integrating comprehensive two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC×LC-qTOF-MS) with multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (MHC-qTOF-MS). DZSM was separated by C8×C18 HPLC column system for comprehensive two-dimensional liquid chromatography system and 283 compounds most of which belonged to phenolic acid, flavonoid, saponin and lignan families were characterized and identified within 75min. Some isomers and compounds at low level were analyzed on C8×Chiral HPLC column system for multiple heart-cutting two-dimensional liquid chromatography system with 1D and 2D optimized gradient elution program. These 1D cutting fractions were successively separated on 2D chiral chromatographic column under extended the 2D gradient elution time from 30s to 5.0min. 12 pairs of isomer compounds were separated with good resolution. The combination of LC×LC and MHC system provides a powerful technique for global chemical profiling of DZSM and provided feasible strategy for other complex systems.

Construction and evaluation of gel filtration/ion exchange comprehensive two-dimensional liquid chromatography system

Construction and evaluation of gel filtration/ion exchange comprehensive two-dimensional liquid chromatography system

Qualitative and quantitative characterization of phenolic and diterpenoid constituents in Danshen (Salvia miltiorrhiza) by comprehensive two-dimensional liquid chromatography coupled with hybrid linear ion trap Orbitrap mass

Danshen is one of the most frequently used traditional Chinese herbs owing to its remarkable and reliable therapeutic effects. Phenolic acids and diterpenoids have proved to be the bioactive substance groups. In order to fully profile its chemical compositions and explore new potential bioactive compounds, a comprehensive two-dimensional liquid chromatography system coupled to DAD detector and hybrid linear ion trap (LTQ) Orbitrap mass spectrometry (LC×LC-DAD–ESI/HRMS/MSn) was set up in this study based on the column combination of Hypersil gold CN (150mm×1mm, 3μm) and Accucore C18 (50mm×4.6mm, 2.6μm). Using the optimal segment gradient program, phenolic acids and diterpenoids were separated into two independent groups and a total of 328 peaks were successfully detected on the contour plot of Danshen. By means of the accurate mass and reliable MSn data, 102 compounds were identified or tentatively identified and 7 of them were discovered from Danshen for the first time. Moreover, the LC×LC-DAD system was validated for the quantitative analysis of 14 bioactive analytes using the contour plot, exhibiting satisfactory linearity (r≥0.9976) and high precision for both peak locating (≤1.07%) and peak volume calculating (0.34%–4.11%). The established method could afford powerful separation capability, reliable identification data and accurate quantitative results, which is very suitable for analysis of complex herbal samples.

Continuous vs. segmented second-dimension system gradients for comprehensive two-dimensional liquid chromatography of sugarcane (Saccharum spp.).

A comprehensive two-dimensional liquid chromatography system in combination with photodiode array and mass spectrometry detection was developed for analysis of polyphenols in sugarcane (Saccharum spp.) leaf extracts. To achieve this, a micro cyano column and a partially porous octodecylsilica column were used in the first and the second dimension, respectively. The choice of the cyano column over other reversed-phase columns tested for the first-dimension separation was due to its lower correlation selectivity with respect to the octodecylsilica column, which was used for the second-dimension separation. Even when reversed-phase mode was used in both dimensions, a satisfactory degree of orthogonality was achieved by use of different gradient elution modes in the second dimension. By means of the setup investigated, 38 polyphenolic compounds were detected, and among them 24 were positively identified by means of complementary data from photodiode array and mass spectrometry detection and an in-house database. This is the first time such a powerful analytical technique has been used for polyphenolic characterization of sugarcane extracts.

Elucidation of steroid glycosides in Anemarrhena asphodeloides extract by means of comprehensive two‐dimensional reversed‐phase/polyamine chromatography with mass spectrometric detection

In the work presented here, a novel approach to comprehensive two-dimensional liquid chromatography is evaluated. Reversed-phase liquid chromatography was employed for the first-dimension separation and polyamine chromatography was chosen for the second-dimension separation mode. The two dimensions are highly orthogonal and the separation efficacy of the developed octadecylsilica × polyamine was tested by separating an extract from Anemarrhena asphodeloides. The steroid glycosides identified by comprehensive two-dimensional liquid chromatography in this experiment were compared to those obtained for monodimensional liquid chromatography. The comprehensive two-dimensional liquid chromatography system, thanks to the complementary separation selectivity and enhanced peak capacity provided by the two columns, allowed to distribute five compounds of low amounts otherwise unachievable by monodimensional liquid chromatography. In addition, four steroid isomers with similar fragmentation characteristics in MS/MS spectra, were newly separated based on their different chemical structures.

Two‐dimensional chromatography of complex polymers, 8. Separation of fatty alcohol ethoxylates simultaneously by end group and chain length

A comprehensive two-dimensional liquid chromatography system was developed to precisely describe the molecular heterogeneity of fatty alcohol ethoxylates. The end-group functionality was analyzed by gradient HPLC while ethylene oxide oligomer distributions were characterized by liquid adsorption chromatography. A baseline separation of all functionality fractions irrespective of the ethylene oxide oligomer chain length was achieved on nonpolar X-Terra C(18) with a methanol-water gradient, whereas an isocratic flow of isopropanol-water on a polar Chromolith Si column gave a separation according to the oligomer chain length without interference of the end-group distribution. The combination of these two methods to conduct online two-dimensional liquid chromatography experiments resulted in a comprehensive two-dimensional picture on the molecular heterogeneity of the sample.

Optimal gradient operation in comprehensive liquid chromatography × liquid chromatography systems with limited orthogonality

A novel strategy is described for designing optimal second dimension ( 2D) gradient conditions for a comprehensive two-dimensional liquid chromatography system where the two dimensions are not fully orthogonal. Using the approach developed here, the initial and final organic modifier content values resulting in the highest coverage of separation space can be derived for each 2D gradient run. Theory indicates that these values can be determined by adapting 2D gradient operation to the degree of orthogonality. The new method is tested on a comprehensive two-dimensional liquid chromatography system that uses reversed phase (RP) columns showing different selectivities in the two dimensions. A comparison between analyses carried out using normal and optimized 2D gradients showed that the latter allow a more efficient use of analysis time. This can result either in an improved peak capacity or in decreasing total analysis time, depending on the final goal of the experiment. In the latter scenario, the number of separated peaks is comparable to that obtained using gradients spanning a wide range of organic modifier but, now, in half the time. As test samples complex mixtures of peptides were analyzed.

One-dimensional and two-dimensional liquid chromatography of sulphonated lignins

One- and two-dimensional separation methods have been developed for the analysis of lignosulphonates and sulphonated kraft lignins. The evaluated sulphonated lignins are all used as dispersants in agrochemical formulations, where some give rise to physical instabilities of formulations. It is of interest to identify the properties of the sulphonated lignins that determine the formulation characteristics. Tetrapentylammonium bromide has been used as an ion-pair reagent in a gradient-elution reversed-phase liquid-chromatographic (IP-RPLC) method, as well as in aqueous size-exclusion chromatography (SEC). Clear differences in the size distribution were observed between different batches of sulphonated lignins. The RPLC and SEC methods were combined in a comprehensive two-dimensional liquid chromatography system. The retention times in the two dimensions were highly correlated. Therefore, the full potential of comprehensive two-dimensional liquid chromatography was not yet realized. However, the results did reveal that retention in IP-RPLC was not determined by the degree of sulphonation of similar-size molecules. Rather, molecules were separated according to size and the degree of sulphonation appears to be approximately constant. The information obtained in this study represents a significant step towards meaningful correlations between the requirements of surfactants within an agrochemical formulation and structural parameters, such as the size and the degree of sulphonation of lignin oligomers.

A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography × liquid chromatography

A novel approach for the selection of the operational parameters (linear velocity, column length) for a comprehensive 2D-LC system is discussed. Starting point for the calculations is a given second dimension ( 2D) separation and a desired peak capacity for the 2D system. Using the theory developed here the optimum settings for the first dimension ( 1D) column can be derived. Theory clearly indicates that the choice of the 1D conditions is basically limited to just one set of column lengths and linear velocities. The new method is tested on a comprehensive two-dimensional liquid chromatography system which uses size-exclusion chromatography (SEC) followed by reversed phase liquid chromatography (RPLC). A novel LC/LC interface, using a six-port valve rather than storage loops, joins the two chromatographic dimensions. From a theoretical comparison of continuous low flow and stop-flow operation the latter method was found to be an attractive mode of interfacing. The common idea that stop-flow operation results in additional band broadening is shown to be incorrect. The new interface design operated in the stop-flow mode permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC. The reversed phase chromatography utilizes a monolithic C-18 modified silica column, which produces fast and efficient analyses. As test samples complex mixtures of peptides were analyzed.

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.

Improved performance of comprehensive two-dimensional HPLC separation of traditional Chinese medicines by using a silica monolithic column and normalization of peak heights

Performance of comprehensive two-dimensional liquid chromatography system is greatly improved than we reported previously by using a silica monolithic column as for the second dimensional separation. Due to the increase of the elution speed on the second dimensional monolithic column, the first dimensional column efficiency and analysis rate can be greatly improved as comparing with conventionally second dimensional column. The developed system was applied to analysis of methanol extraction of two umbelliferae herbs Ligusticum chuanxiong Hort. and Angelica sinensis (Oliv.) Diels by using CN column as for the first dimensional separation and a silica monolithic ODS column for the second dimensional separation, and the obtained three-dimensional chromatograms were treated by normalization of peak heights with the value of the highest peak or setting a certain value using a software written in-house. It was observed that much more peaks for low-abundant components in TCM extract can clearly be detected here than we reported before, due to the large difference for the amount of components in TCMs’ extract. With the above improvements in separation performance and data treatment, totally about 120 components in methanol extraction of Rhizoma chuanxiong and 100 in A. sinensis were separated with UV detection within 130 min. This result meant that both the number of peaks detected increase twice but the analysis time decease twice if comparing with the previously reported result.