Statistical mixture design optimization of extraction media and mobile phase compositions for the characterization of green tea

The influence of different solvents on the extraction medium and the RP-HPLC mobile phase composition were investigated by statistical mixture designs to optimize solvent proportions to prepare the fingerprint of a medicinal herbal extract. For modeling, the number of peaks was used as a measure of fingerprint information. The optimum compositions of solvent to extract chemical substances from green tea and for mobile phase chromatographic analysis were ethyl acetate/ ethanol/dichloromethane (20:5:75 v/v/v) and MeOH/ACN/water (7.5:57.5:35 v/v/v), respectively. This system results in 26 peaks in the chromatographic fingerprint. These results show that an incorrect choice of modifiers for mobile phase composition and solvent extraction hampers the detection of a maximum number of peaks and produces a poor chromatographic fingerprint.     

Mixture-mixture design for the fingerprint optimization of chromatographic mobile phases and extraction solutions for Camellia sinensis

A composite simplex centroid-simplex centroid mixture design is proposed for simultaneously optimizing two mixture systems. The complementary model is formed by multiplying special cubic models for the two systems. The design was applied to the simultaneous optimization of both mobile phase chromatographic mixtures and extraction mixtures for the Camellia sinensis Chinese tea plant. The extraction mixtures investigated contained varying proportions of ethyl acetate, ethanol and dichloromethane while the mobile phase was made up of varying proportions of methanol, acetonitrile and a methanol-acetonitrile-water (MAW) 15%:15%:70% mixture. The experiments were block randomized corresponding to a split-plot error structure to minimize laboratory work and reduce environmental impact. Coefficients of an initial saturated model were obtained using Scheffe-type equations. A cumulative probability graph was used to determine an approximate reduced model. The split-plot error structure was then introduced into the reduced model by applying generalized least square equations with variance components calculated using the restricted maximum likelihood approach. A model was developed to calculate the number of peaks observed with the chromatographic detector at 210 nm. A 20-term model contained essentially all the statistical information of the initial model and had a root mean square calibration error of 1.38. The model was used to predict the number of peaks eluted in chromatograms obtained from extraction solutions that correspond to axial points of the simplex centroid design. The significant model coefficients are interpreted in terms of interacting linear, quadratic and cubic effects of the mobile phase and extraction solution components.     

Computer-assisted optimization for the selection of mobile phase in semi-preparative HPLC

A computer-assisted method is presented for optimization for the selection of mobile phasecomposition in semi-preparative HPLC.The optimization for the expected separation is based ona polynomial estimation from five preliminary runs.Statistical scanning technique was used foroptimization.Double criteria simulation system (DCSS) is established for chromatographic perfor-mance measurement in this method.The validity of the optimization strategy is confirmed by applyingit to a technical Cypermethrin separation.Excellent agreement is obtained between the predictedand experimental results.     

Experimental design for optimization of microwave-assisted extraction of benzodiazepines in human plasma

A simple and fast microwave-assisted-extraction (MAE) method has been evaluated as an alternative to solid-phase extraction (SPE) for the determination of six benzodiazepines widely prescribed in European countries (alprazolam, bromazepam, diazepam, lorazepam, lormetazepam and tetrazepam) in human plasma. For MAE optimization a Doehlert experimental design was used with extraction time, temperature and solvent volume as influential parameters. A desirability function was employed in addition to the simultaneous optimization of the MAE conditions. The analysis of variance showed that the solvent volume had a positive influence on the extraction of all the analytes tested, achieving a statistically significant effect. Also, the extraction time had a statistically significant effect on the extraction of four benzodiazepines. The selected MAE conditions—89 °C, 13 min and 8 mL of chloroform/2-propanol (4:1, v/v)—led to recoveries between 89.8 ± 0.3 and 102.1 ± 5.2% for benzodiazepines using a high performance liquid chromatography method coupled with diode-array detection. The comparison of MAE and SPE shows better results for MAE, with a lower number of steps in handling the sample and greater efficiency. The applicability of MAE was successfully tested in 27 plasma samples from benzodiazepine users.     

Statistical experimental design for bioprocess modeling and optimization analysis

The statistical design of experiments (DOE) is a collection of predetermined settings of the process variables of interest, which provides an efficient procedure for planning experiments. Experiments on biological processes typically produce long sequences of successive observations on each experimental unit (plant, animal, bioreactor, fermenter, or flask) in response to several treatments (combination of factors). Cell culture and other biotech-related experiments used to be performed by repeated-measures method of experimental design coupled with different levels of several process factors to investigate dynamic biological process. Data collected from this design can be analyzed by several kinds of general linear model (GLM) statistical methods such as multivariate analysis of variance (MANOVA), univariate ANOVA (timesplit-plot analysis with randomization restriction), and analysis of orthogonal polynomial contrasts of repeated factor (linear coefficient analysis). Last, regression model was introduced to describe responses over time to the different treatments along with model residual analysis. Statistical analysis of biprocess with repeated measurements can help investigate environmental factors and effects affecting physiological and bioprocesses in analyzing and optimizing biotechnology production.     

Solid phase extraction and analysis of horse urine for aminocaproic acid

Summary A rapid and efficient procedure is described for extraction and determination of aminocaproic acid in horse urine. Urine was extracted by passing through a bonded silica column (Bond-Elut). The adsorbed drug was washed free of endogenous materials before being eluted. The extract was then examined by thin-layer chromatography and HPLC. The purity of the extract was determined by gas chromatography-mass spectrometry.     

A simple procedure for the rapid optimization of reversed-phase separations with ternary mobile phase mixtures

Summary A simple rapid procedure is described for estimating optimum compositions of ternary mobile phase mixtures for the separation of samples by reversed-phase liquid chromatography (RPLC). Retention data in two iso-eluotropic binary mobile phase mixtures (mixtures with equal retention times) are required to initiate the procedure. The logarithm of the capacity factor is assumed to vary linearly with the composition of isoeluotropic ternary mixtures formed by mixing the two limiting binaries. Using the product of resolution factors of adjacent peaks as the criterion, an optimum ternary composition is then calculated. After a chromatogram has been obtained with the predicted optimum ternary mobile phase, the procedure is repeated until no further improvement can be achieved. Examples of the application of the present procedure are described to illustrate its effectiveness.     

Bonded stationary phases for reversed phase liquid chromatography with a water mobile phase: application to subcritical water extraction

Reversed phase high-performance liquid chromatography (RP-HPLC) is demonstrated for hydrophobic analytes such as aromatic hydrocarbons on a chemically bonded stationary phase and a mobile phase consisting of only water. Reversed phase liquid chromatography separations using a water-only mobile phase has been termed WRP-LC for water-only reversed phase LC. Reasonable capacity factors are achieved through the use of a non-porous silica substrate resulting in a chromatographic phase volume ratio much lower than usually found in RP-HPLC. Two types of bonded WRP-LC columns have been developed and applied. A brush phase was synthesized from an organochlorosilane. The other phase, synthesized from an organodichlorosilane, is termed a branch phase and results in a polymeric structure of greater thickness than the brush phase. A baseline separation of a mixture containing benzaldehyde, benzene, toluene, and ethyl benzene in less than 5 min is demonstrated using a water mobile phase with 12 000 plates generated for the unretained benzaldehyde peak. The theoretically predicted minimum reduced plate height is also shown to be approached for the unretained analyte using the brush phase. As an application, subcritical water extraction (SWE) at 200°C is combined with WRP-LC. This combination allows for the extraction of organic compounds from solid matrices immediately followed by liquid chromatographic separation of those extracted compounds all using a solvent of 100% water. We demonstrate SWE/WRP-LC by spiking benzene, ethyl benzene, and naphthalene onto sand then extracting the analytes with SWE followed by chromatographic separation on a WRP column. A sand sample contaminated with gasoline was also analyzed using SWE/WRP-LC. This extraction process also provides kinetic information about the rate of analyte extraction from the sand matrix. Under the conditions employed, analytes were extracted at different rates, providing additional selectivity in addition to the WRP-LC separation.     

Orthogonal array design for the optimization of supercritical fluid extraction of tanshinones from Danshen

Different from the west medicine, the therapeutic effect of the traditional Chinese medicine is usually based on multifarious essential components or the combination of them instead of only one component. In this paper, a novel supercritical fluid extraction (SFE) method has been developed for extracting tanshinones (dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone II(A)) from Danshen, the dried root of Salvia miltiorrhiza. Various experimental conditions were investigated to optimize the SFE. Under the appropriate conditions, extracting at 40 MPa, and 50 degrees C and with CO(2) flow rate of 25 L/h for 1 h, SFE can achieve a better yield as well as the recoveries of the tanshinones than the conventional extraction using methanol. Moreover, the target compounds were analyzed by HPLC with a C(18) RP column by gradient elution using ACN and water as mobile phase at a flow rate of 1.0 mL/min and with UV detection at 270 nm. Four calibration equations were then established and good linear relationships were shown (r(2) >0.999) in the investigated concentration range. The recoveries, measured at three concentration levels, varied from 97.2 to 103.8%. The method provided in this article could be applied as an improved quality control method for Danshen products.     

An improved optimization procedure for the selection of mixed mobile phases in reversed phase liquid chromatography

Summary The stepwise procedure described previously [1] for the selection of optimal mobile phase compositions in reversed phase liquid chromatography (RPLC) is improved in two ways. The optimization criterion is modified to account for variations in analysis time. The iterative approach to the optimal solvent composition makes a more efficient use of previous data. An example illustrates the resulting gain in speed and accuracy.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Effect of the lectin of Bauhinia variegata and its recombinant isoform on surgically induced skin wounds in a murine model

Lectins are a structurally heterogeneous group of highly specific carbohydrate-binding proteins. Due to their great biotechnological potential, lectins are widely used in biomedical research. The purpose of the present study was to evaluate the healing potential of the lectin of Bauhinia variegata (nBVL) and its recombinant isoform (rBVL-1). Following surgical creation of dorsal skin wounds, seven groups of mice were submitted to topical treatment for 12 days with lectin, D-galactose, BSA and saline. The animals were anesthetized and euthanized on POD 2, 7 and 12 in order to evaluate the healing potential of each treatment. The parameters considered included wound size, contraction rate, epithelialization rate and histopathological findings. Wound closure was fastest in animals treated with rBVL-1 (POD 7). nBVL was more effective than the controls. All skin layers were reconstructed and keratin deposition increased. Our findings indicate that the lectin of Bauhinia variegata possesses pro-healing properties and may be employed in the treatment of acute skin wounds.     

Partial least squares model and design of experiments toward the analysis of the metabolome of Jatropha gossypifolia leaves: Extraction and chromatographic fingerprint optimization

A major challenge in metabolomic studies is how to extract and analyze an entire metabolome. So far, no single method was able to clearly complete this task in an efficient and reproducible way. In this work we proposed a sequential strategy for the extraction and chromatographic separation of metabolites from leaves Jatropha gossypifolia using a design of experiments and partial least square model. The effect of 14 different solvents on extraction process was evaluated and an optimized separation condition on liquid chromatography was estimated considering mobile phase composition and analysis time. The initial conditions of extraction using methanol and separation in 30 min between 5 and 100% water/methanol (1:1 v/v) with 0.1% of acetic acid, 20 μL sample volume, 3.0 mL min^?1 flow rate and 25°C column temperature led to 107 chromatographic peaks. After the optimization strategy using i‐propanol/chloroform (1:1 v/v) for extraction, linear gradient elution of 60 min between 5 and 100% water/(acetonitrile/methanol 68:32 v/v with 0.1% of acetic acid), 30 μL sample volume, 2.0 mL min^?1 flow rate, and 30°C column temperature, we detected 140 chromatographic peaks, 30.84% more peaks compared to initial method. This is a reliable strategy using a limited number of experiments for metabolomics protocols.     

Voltammetry and electrochemical stripping analysis for antimony in aqueous and nonaqueous media after extraction

Cyclic voltammetry of antimony was studied in aqueous media (HCl-LiCl) and in nonaqueous media after extraction with 20% tri-n-butylphosphate in toluene, with a rotating glassy carbon disc electrode. Reduction of antimony to the element in aqueous media is nearly reversible, but irreversible in nonaqueous media. Anodic stripping voltammetric and chronopotentiometric determinations were also studied in nonaqueous media; methanol and LiCI, NH₄SCN or NH₄NO₃, were used as base electrolytes. In nonaqueous media, antimony can be determined down to concentrations of 1O⁻⁸ M by stripping voltammetry, and lO⁻⁷ M by stripping chronopotentiometry. Electrochemical stripping determinations of 10⁻⁶ M antimony(III) were not affected by Co²⁺, Ni²⁺, Cd²⁺, Zn²⁺ or As³⁺ (5 · 10⁻³ M), ag⁺ (4 · 10⁺⁴ M in stripping voltammetry or 10⁻³ M in stripping chronopotentiometry), Hg²⁺ (5 · 10⁻⁴M), Pb²⁺ (3 · 10⁻⁴ M), Cu²⁺ (1.5 · 10⁻⁴ M)Sn²⁺ and Sn⁴⁺ (7 · 10⁻⁴ M), Fe³⁺ (4 · 10⁻⁴ M), Au³⁺ (5 · 10⁻⁵ M) and Bi³⁺ (1.5 · 10⁻⁵ M). Thestripping chronopotentiometric determination showed better selectivity.     

Increasing the sensitivity of an LC-MS method for screening material extracts for organic extractables via mobile phase optimization

Organic extractables (substances extracted from materials used in pharmaceutical packaging) are discovered, identified, and quantified via screening of extracts with analytical methods including liquid chromatography with mass spectrometric detection (LC-MS). Because extractables include a large number of diverse compounds that are typically present in plastic extracts at low levels, the LC-MS methods must be broad scope and sensitive. To accomplish these objectives, screening studies typically couple gradient reversed-phase separations with electrospray MS detection (both positive and negative ion modes). While such methods are generally applicable for a number of extractables, they are not optimal for some commonly encountered extractables due to either poor chromatographic performance (e.g., peak tailing) or poor MS response. Modifications to mobile phase composition (e.g., pH adjustment) were examined to improve the performance of an LC-MS screening method. The use of 0.1% acetic acid with 1 mM ammonium acetate (pH 3.6) as the aqueous portion of the mobile phase provided favorable sensitivities for a number of extractables both in positive and negative ion modes. In positive ion mode, the acidic mobile phase improved responses for moderately weak basic compounds by increasing their degree of protonation. For very weak basic compounds such as amides, ammonium ions in the mobile phase promoted proton adduct responses. In negative ion mode, an acidic mobile phase containing acetate anion improved ESI responses for acidic compounds, primarily due to gas phase effects.     

Optimization by isochronal analysis. II. Changes in mobile phase velocity and temperature, and in mobile phase composition and temperature

The theory of resolution optimization by isochronal analysis is further extended to the cases involving simultaneous changes in mobile phase velocity and column temperature, and in mobile phase composition and temperature. In each case, the resolution equation, when written in terms of isochronal conditions, shows that improvements can be obtained without sacrificing analysis time. Several subcases, which relate to the dependence of the selectivity factors on the changed parameters, are described. For each system, the contributions of the various parameters to the resolution are examined. The results of these discussions are plotted as resolution surfaces, on which lines of constant analysis time are indicated. The experimental routes that chromatographers must take in order to apply isochronal analysis are described.     

The principle of optimization of binary mobile phase composition of multi-step linear gradient elution

The basic principle of optimal method called “moving overlapping resolution mapping method” to select the optimal binary mobile phase composition of multi-step linear gradient liquid chromatography is discussed with simultaneously considering effects of position of solute inside the column and mobile phase composition on peak resolution and retention value, then a BASIC program based on this principle is developed in IBM-PC computer. The validities of both principle of optimization and BASIC program are confirmed by separation of samples containing bile acids and PAHs in RP-HPLC.