Capillary electrophoresis fingerprints combined with chemometric methods to evaluate the quality consistency and predict the antioxidant activity of Yinqiaojiedu tablet

The quality consistency of Yinqiaojiedu tablets (YQJDTs) was monitored by extracting their electrophoretic fingerprint and marker compound data that were obtained using capillary electrophoresis. To select the suitable background electrolyte, wing‐shape method was proposed. A background electrolyte composed of 103.1 mM boric acid, 51.6 mM sodium borate, 9.8 mM disodium hydrogen phosphate, and 15.6 mM sodium dihydrogen phosphate was used to separate compounds. Under the optimized conditions, the content of three marker compounds was determined in 25 YQJDT samples. The capillary electrophoresis fingerprints were developed simultaneously, then 25 samples from two manufacturers were clearly divided into two clusters based on the principal component analysis. In fingerprint assessments, systematic quantified fingerprint method was adopted for integrative quality discrimination of YQJDTs from both qualitative and quantitative perspectives, by which the qualities of 25 samples were well differentiated. In addition, partial least squares model was established to explore fingerprint–efficacy relationship between the fingerprints and the antioxidant activities in vitro, providing clinically useful information for quality control. The proposed method was reliable and comprehensive, which could be used for a valuable reference to monitor the quality consistency of traditional Chinese medicines.     

Advancement in optimization tactic achieved by newly developed chromatographic response function: application to LC separation of raloxifene and its impurities

In this paper a new chromatographic response function (CRF) is designed and proposed for utilization in the optimization strategies. The function capability to represent the overall quality of a experimentally obtained chromatograms was compared to the other two objective functions and proved to give more accurate and reliable results. The new CRF has improved concept of separation and time term estimation. It reflects all important defects of the chromatogram such as the appearance of asymmetrical or overlapping peaks and prolonged elution time and allows the appropriate weighting of each of them. The LC separation of raloxifene and its four impurities was evaluated through the central composite design experimental plan choosing the new CRF to be the only output of the system. The function demonstrated the ability to judge the impact of the complex interactions of the selected chromatographic parameters (acetonitrile content in the mobile phase, sodium dodecyl sulfate concentration in the water phase, pH of the mobile phase and column temperature) on the mixture behavior and led to the determination of the optimal separation conditions. The newly developed CRF proved to have the advanced performances and it presents the important step forward in the optimization of the chromatographic separation.     

A hevein-like protein and a class I chitinase with antifungal activity from leaves of the paper mulberry

Paper mulberry (Broussonetia papyrifera, syn. Morus papyrifera L.) is a Chinese traditional medicine and its low‐molecular‐weight extracts are reported to have antifungal activity. In this study, two proteins (PMAPI and PMAPII) with activity against Trichoderma viride were obtained from paper mulberry leaves with a fast protein liquid chromatography (FPLC) unit. The purification protocol employed (NH₄)₂SO₄ precipitation, ion‐exchange chromatography and hydrophobic‐interaction chromatography on FPLC. Molecular masses were 18,798 Da for PMAPI, and 31,178 Da for PMAPII determined by Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Peptide mapping fingerprint analysis showed that PMAPI has no peptides similar to PMAPII. N‐terminal amino acid sequencing revealed that PMAPI is a hevein‐like protein, and PMAPII is a class I chitinase. They both had a half‐maximal inhibitory concentration (IC50) of 0.1 µg/µL against T. viride. This is the first report of high‐molecular‐weight extracts with antifungal activity from paper mulberry. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification of volatiles from pineapple (Ananas comosus L.) pulp by comprehensive two-dimensional gas chromatography and gas chromatography/mass spectrometry

Combining qualitative data from the chromatographic structure of 2-D gas chromatography with flame ionization detection (GC×GC-FID) and that from gas chromatography-mass spectrometry (GC/MS) should result in a more accurate assignment of the peak identities than the simple analysis by GC/MS, where coelution of analytes is unavoidable in highly complex samples (rendering spectra unsuitable for qualitative purposes) or for compounds in very low concentrations. Using data from GC×GC-FID combined with GC/MS can reveal coelutions that were not detected by mass spectra deconvolution software. In addition, some compounds can be identified according to the structure of the GC×GC-FID chromatogram. In this article, the volatile fractions of fresh and dehydrated pineapple pulp were evaluated. The extraction of the volatiles was performed by dynamic headspace extraction coupled to solid-phase microextraction (DHS-SPME), a technique appropriate for slurries or solid matrices. Extracted analytes were then analyzed by GC×GC-FID and GC/MS. The results obtained using both techniques were combined to improve compound identifications.     

Effects of Immobilized Metal Ion on Retention Behaviors of Proteins in Metal Chelate Affinity Chromatography

The chromatographic behaviors of proteins on iminodiacetic acid (IDA) column with and without immobilized metal ion were examined in detail. Comparing the effects of pI, solution pH, and salt concentration on retention of proteins in cation‐exchange chromatography (CEC) and metal chelate affinity chromatography (MCAC), the retention mechanism of proteins was investigated in MCAC. By aid of observing the retention characteristics of proteins on naked IDA and metal chelate columns in high concentration salt‐out salt solution, the hydrophobic interaction in MCAC and the influence of metal ion on it were proved. In terms of the comparison of the thermodynamics of proteins in CEC and MCAC, the thermostability, the conformational change entropy Δ(ΔS⁰) and enthalpy Δ(ΔH⁰), compensation temperature β, the driving force and caloritic effect of proteins in MCAC were discussed. The identity of retention mechanism at protein thermal denaturation in CEC and MCAC was demonstrated by using the compensation relationship between ΔH⁰ and ΔS⁰.     

Finding the best separation in situations of extremely low chromatographic resolution

Samples with a large number of compounds or similarities in their structure and polarity may yield insufficient chromatographic resolution. In such cases, however, finding conditions where the largest number of compounds appears sufficiently resolved can be still worthwhile. A strategy is here reported that optimises the resolution level of chromatograms in cases where conventional global criteria, such as the worst resolved peak pair or the product of elementary resolutions, are not able to detect any separation, even when most peaks are baseline resolved. The strategy applies a function based on the number of "well resolved" peaks, which are those that exceed a given threshold of peak purity. It is, therefore, oriented to quantify the success in the separation, and not the failure, as the conventional criteria do. The conditions that resolve the same amount of peaks are discriminated by either quantifying the partial resolution of those peaks that exceed the established threshold, or by improving the separation of peaks below it. The proposed approach is illustrated by the reversed-phase liquid chromatographic separation of a mixture of 30 ionisable and neutral compounds, using the acetonitrile content and pH as factors.     

The statistical overlap theory of chromatography using power law (fractal) statistics

The chromatographic dimensionality was recently proposed as a measure of retention time spacing based on a power law (fractal) distribution. Using this model, a statistical overlap theory (SOT) for chromatographic peaks is developed that estimates the number of peak maxima as a function of the chromatographic dimension, saturation and scale. Power law models exhibit a threshold region whereby below a critical saturation value no loss of peak maxima due to peak fusion occurs as saturation increases. At moderate saturation, behavior is similar to the random (Poisson) peak model. At still higher saturation, the power law model shows loss of peaks nearly independent of the scale and dimension of the model. The physicochemical meaning of the power law scale parameter is discussed and shown to be equal to the Boltzmann-weighted free energy of transfer over the scale limits. The scale is discussed. Small scale range (small β) is shown to generate more uniform chromatograms. Large scale range chromatograms (large β) are shown to give occasional large excursions of retention times; this is a property of power laws where "wild" behavior is noted to occasionally occur. Both cases are shown to be useful depending on the chromatographic saturation. A scale-invariant model of the SOT shows very simple relationships between the fraction of peak maxima and the saturation, peak width and number of theoretical plates. These equations provide much insight into separations which follow power law statistics.     

Quantifying temperature and flow rate effects on the performance of a fixed-bed chromatographic reactor

Chromatographic reactors are based on coupling chemical reactions with chromatographic separation in fixed-beds. Temperature and flow rate are important parameters for the performance of such reactors. Temperature affects mainly adsorption, chemical equilibria, mass transfer and reaction kinetics, whereas flow rate influences residence time and dispersion. In order to evaluate the mentioned effects, the hydrolysis reactions of methyl formate (MF) and methyl acetate (MA) were chosen as case studies. These reactions were performed experimentally in a lab-scale fixed-bed chromatographic reactor packed with a strong acidic ion exchange resin. The chosen reactions can be considered to represent a relative fast (MF) and a relative slow (MA) reaction. The processes which take place inside the reactor were described and simulated using an isothermal equilibrium dispersive model. The essential model parameters were determined experimentally at different temperatures and flow rates. The performance of the chromatographic reactor was evaluated at several discrete constant temperature levels by quantifying product purity, productivity and yield. The work provides insight regarding the influence of temperature and flow rate on values of the model parameters and the performance criteria.