Separation and purification of isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze by counter-current chromatography comparing two kinds of solvent systems

The first preparative separation of a flavonoid sulphate isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze by counter-current chromatography (CCC) was presented. Two kinds of solvent systems were used. A conventional organic/aqueous solvent system n-butanol-ethyl acetate-water (4:1:5, v/v) was used, yielding isorhamnetin 3-sulphate 2.0 mg with a purity of 93.4% from 83 mg of pre-enriched crude extract obtained from 553 mg ethanol extract by macroporous resin. A one-component organic/salt-containing system composed of n-butanol-0.25% sodium chloride aqueous solution (1:1, v/v) was also used, and the LC column packed with macroporous resin has been employed for desalination of the target compound purified from CCC. As a result, 2.1 mg of isorhamnetin 3-sulphate with a purity of over 97% has been isolated from 402 mg of crude extract without pre-enrichment. Compared with the conventional organic/aqueous system, the one-component organic/salt-containing aqueous system was more suitable for the separation of isorhamnetin 3-sulphate, and purer target compound was obtained from the crude extract without pre-enrichment using the new solvent system. The chemical structure was confirmed by ESI-MS and (1)H, (13)C NMR. In summary, our results indicated that CCC using one-component organic/salt-containing aqueous solution is very promising and powerful for high-throughput purification of isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze.     

Determination of substituted benzenes in water samples by fiber-in-tube liquid phase microextraction coupled with gas chromatography

A method for determination of toluene, ethylbenzene, p-xylene, o-xylene, 1,3,5-trimethylbenzene and 1,2,4-trimethylbenzene in water samples was developed by a fiber-in-tube liquid phase microextraction technique (fiber-in-tube LPME) coupled with GC-flame ionization detector (FID). The method used a tube packed with polytetrafluoroethylene (PTFE) fibers as an extraction medium, improving the stableness of the solvent and the performance of extraction. Certain amounts of curled PTFE fibers were packed into a section of PTFE tube. Because the fibers were curled, they formed network structure in the tube. The fiber packed tube was firstly immersed into organic solvent to be filled with organic solvent and then was exposing to an aqueous solution to extract the target compounds. The extract was then retracted by a conventional GC microsyringe and analyzed by GC-FID. Extraction of the analytes in 8 ml aqueous solution for 15 min yielded enrichment factors of 224-361. The precision (R.S.D., n = 5) was 3.6-8.1% for peak area. The limit of detection (LOD, S/N = 3) for the six substituted benzenes were in the range of 0.3-5.0 μg l⁻¹.     

Preparative isolation of novel antioxidant flavonoids of alfalfa by stop-and-go counter-current chromatography and following on-line liquid chromatography desalination

In this work, we have established a new stop-and-go two-dimensional chromatography coupling of counter-current chromatography and liquid chromatography (2D CCC × LC) for the preparative separation of two novel antioxidant flavonoids from the extract of alfalfa (Medicago sativa L.). The CCC column has been used as the first dimension to purify the target flavonoids using a solvent system of isopropanol and 20% sodium chloride aqueous solution (1:1, v/v) with the stop-and-go flow technique, and the LC column packed with macroporous resin has been employed as the second dimension for on-line absorption, desalination and desorption of the targeting effluents purified from the first CCC dimension. As a result, two novel flavonoids, 6,8-dihydroxy-flavone-7-O-β-D-glucuronide (15.3 mg) and 6-methoxy-8-hydroxy-flavone-7-O-β-D-glucuronide (13.7 mg), have been isolated from 126.8 mg of crude sample pre-enriched by macroporous resin column. Their structures have been identified by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) and one- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR). Further antioxidant assays showed that the first component possess a strong antioxidant activity. All the results demonstrated that the stop-and-go 2D CCC × LC method is very efficient for the separation of flavonoids of alfalfa and it can also be applied to isolate other comprehensive multi-component natural products.     

Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography

Polar betacyanin pigments together with betaxanthins from ripe cactus fruits of Hylocereus polyrhizus (Cactaceae) were fractionated by means of preparative ion-pair high-speed countercurrent chromatography (IP-HSCCC) also using the elution–extrusion (EE) approach for a complete pigment recovery. HSCCC separations were operated in the classical ‘head-to-tail’ mode with an aqueous mobile phase. Different CCC solvent systems were evaluated in respect of influence and effectiveness of fractionation capabilities to separate the occurring pigment profile of H. polyrhizus. For that reason, the additions of two different volatile ion-pair forming perfluorinated carboxylic acids (PFCA) were investigated. For a direct comparison, five samples of Hylocereus pigment extract were run on preparative scale (900 mg) in 1-butanol–acetonitrile–aqueous TFA 0.7% (5:1:6, v/v/v) and the modified systems tert.-butyl methyl ether–1-butanol–acetonitrile–aqueous PFCA (2:2:1:5, v/v/v/v) using 0.7% and 1.0% trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) in the aqueous phase, respectively. The chemical affinity to the organic stationary CCC solvent phases and in consequence the retention of these highly polar betalain pigments was significantly increased by the use of the more lipophilic fluorinated ion-pair reagent HFBA instead of TFA. The HFBA additions separated more effectively the typical cacti pigments phyllocactin and hylocerenin from betanin as well as their iso-forms. Unfortunately, similar K_D ratios and selectivity factors α around 1.0–1.1 in all tested solvent systems proved that the corresponding diastereomers, 15S-type pigments cannot be resolved from the 15R-epimers (iso-forms). Surprisingly, additions of the stronger ion-pair reagent (HFBA) resulted in a partial separation of hylocerenin from phyllocactin which were not resolved in the other solvent systems. The pigments were detected by means of HPLC-DAD and HPLC-electrospray ionization–MS using also authentic reference materials.     

Advantages of a small-volume counter-current chromatography column

Counter-current chromatography (CCC) works with a support-free liquid stationary phase. This allows for preparative separations and purifications. However, there are serious technical constraints because of the need to keep a liquid stationary phase in a column. Centrifugal fields are used. A new commercial hydrodynamic 18 mL column made with a narrow-bore 0.8 mm Teflon tubing was evaluated by comparing it with older hydrodynamic CCC columns and a similar 19 mL column but made with 1.6 mm Teflon tubing. A small-volume CCC column allows for reliable and fast solute partition coefficient determination. When resolution is required, both high efficiency and liquid stationary phase retention are needed. Unfortunately, these two requirements bear technical contradictions. A column coiled with a narrow tubing bore will provide a high chromatographic efficiency while a column containing wider tubing bore will achieve higher stationary phase retention. In all cases, increasing the magnitude of the centrifugal field also increases the stationary phase retention. The solution is to build centrifuges able to produce high fields that will provide acceptable liquid phase retention with narrow-bore tubes. The new 18 mL 0.8 mm tubing bore column is able to rotate as fast as 2100 rpm generating a 240 × g field. The two older CCC columns cannot compete with the new one. However, the small 19 mL column with 1.6 mm bore tubing can be useful when fast results are desired without top resolution.     

Rapid and preparative separation of traditional Chinese medicine Evodia rutaecarpa employing elution-extrusion and back-extrusion counter-current chromatography: Comparative study

Traditional Chinese medicines (TCMs) have attracted much attention in recent years. Elution-extrusion and/or back-extrusion counter-current chromatography (EECCC/BECCC) both take full advantage of the liquid nature of the stationary phase. They effectively extend the solute hydrophobicity window that can be studied and rendered the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, a popular traditional Chinese medicine, Evodia rutaecarpa, was used as the target complex mixture for extrusion CCC separations. With a carefully selected biphasic liquid system (n-hexane/ethyl acetate/methanol/water, 3/2/3/2, v/v) and optimized conditions (V_CM = V_C, mobile phase flow rate: 3 mL/min in descending mode, sample loading: 100 mg), five fractions could be obtained in only 100 min on a 140-mL capacity CCC instrument using both elution- and back-extrusion methods. Each fraction was analyzed and identified compared with the data of major standards using LC/MS. Moreover, the performance of both extrusion protocols was systematically compared and summarized. EECCC could be operated continuously and was found extremely suitable for high-throughput separation; however, post-column addition of a clarifying reagent is recommended to smooth the UV-signal during the extrusion process. Considering BECCC, the practical operation is very simple by just switching a 4-port valve to change the flow direction. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode so that solutes with small and medium distribution constants have been eluted. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column, mix together and produce a broad peak showing in the mobile phase eluting after the stationary phase extrusion. Compared with classical CCC or other preparative separation tools, extrusion CCC approaches exhibit distinguished superiority in the modernization process of traditional Chinese medicines.     

Two-dimensional counter-current chromatography: 1st traditional counter-current chromatography, 2nd acid-base elution counter-current chromatography

An on-line column-switching counter-current chromatography (CCC) with solid-phase trapping interphase is presented in this paper. The large volume injection is avoided using solid-phase trapping interphase. Thereby, totally different chemical composition biphasic solvent system can be utilized to enhance system orthogonality. In the present work, a 140 mL-capacity CCC instrument was used in the first dimension, and a parallel three-coil CCC centrifuge (40 mL each coil) was used in the second dimensional separation allowing three injections at the same time. With biphasic solvent system composed of n-hexane: ethyl acetate: methanol: water (1:1:1:1, v/v), five well-separated fractions were obtained in the first dimension. Two fractions were online concentrated and further separated in the second dimension with solvent system composed of methyl tert-butyl ether: acetonitrile: water (2:2:3, v/v), where trifluoroacetic acid (10 mM) was added to the upper organic phase as a retainer and triethylamine (10mM) to the aqueous mobile phase as an eluter. Four hydroxyanthraquinones were successfully separated and purified from Chinese medicinal plant Rheum officinale in only one step.     

Protein separation using toroidal columns by type-J synchronous counter-current chromatography towards preparative separation

Separation of large bioactive molecules such as proteins, DNAs and RNAs using aqueous two-phase systems (ATPSs) and liquid–liquid partition-based counter-current chromatography (CCC) can avoid risks of sample loss and denaturation, and greatly reduce processing time. We have constructed toroidal columns (length 26–140 m, column volume 51–280 ml, bore size 1.6 mm) suitable for mounting onto a commercially available preparative CCC apparatus. With the use of an ATPS containing 12.5% (w/w) PEG1000 and 12.5% (w/w) K₂HPO₄ and at a rotational speed of 800 rpm for the rotor of the CCC device, the lower phase (i.e. the phosphate-enriched phase) has been used as the mobile phase and a pair of proteins, myoglobin and lysozyme, as model proteins for demonstrating the separation capability of the CCC system. For a toroidal column with a length of 53.5m and a column volume of 107.5 ml, and operated for the Coriolis force parallel flow mode at 0.62 ml/min, protein sample loading (containing 2.2 mg/ml myoglobin and lysozyme, respectively) at 1.7% and 7.4% to the column volume led to peak resolution (with theoretical plate number TP and stationary phase retention S_f shown in the parenthesis) of R_s = 1.5 (N = 211 and N = 113 TP for myoglobin and lysozyme, respectively, and S_f = 45.0%), and R_s = 1.4 (218 and 152 TP, and S_f = 34.0%). However, further increase of the loading to 13% failed to separate the two proteins. Although proteins eluted at positions predictable from the distribution coefficients, they showed broader peaks when compared with small dipeptides under identical CCC operating conditions. This confirms that the molecular weight of the partitioned species is an important factor causing peak broadening on CCC chromatograms. These results paved the way for further scaling-up toroidal CCC columns for processing larger quantities of samples containing large biomolecules.     

Two-dimensional preparative liquid chromatography system for preparative separation of minor amount components from complicated natural products

An on-line comprehensive two-dimensional preparative liquid chromatography system was developed for preparative separation of minor amount components from complicated natural products. Medium-pressure liquid chromatograph (MPLC) was applied as the first dimension and preparative HPLC as the second one, in conjunction with trapping column and makeup pump. The performance of the trapping column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure from the view of medium pressure liquid chromatograph. Satisfactory trapping efficiency can be achieved using a commercially available 15 mm × 30 mm i.d. ODS pre-column. The instrument operation and the performance of this MPLC × preparative HPLC system were illustrated by gram-scale isolation of crude macro-porous resin enriched water extract of Rheum hotaoense. Automated multi-step preparative separation of 25 compounds, whose structures were identified by MS, ¹H NMR and even by less-sensitive ¹³C NMR, could be achieved in a short period of time using this system, exhibiting great advantages in analytical efficiency and sample treatment capacity compared with conventional methods.     

Separation of epigallocatechin and flavonoids from Hypericum perforatum L. by high-speed counter-current chromatography and preparative high-performance liquid chromatography

High-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were successively used for the separation of epigallocatechin and flavonoids from Hypericum perforatum L. The two-phase solvent system composed of ethyl acetate–methanol–water (10:1:10, v/v) was used for HSCCC. About 900 mg of the crude extract was separated by HSCCC, yielding 7.8 mg of quercitrin at a purity of over 97%, 12.6 mg of quercetin at a purity of over 93%, and 38.9 mg of a mixture of hyperoside, isoquercitrin and miquelianin constituting over 97% of the fraction. A mixture of epigallocatechin and avicularin pooled from three HSCCC runs, a total amount of 54.3 mg, was further separated by prep-HPLC yielding 23.4 mg of epigallocatechin and 15.3 mg of avicularin each at a purity of over 97%.     

A new high-speed hollow fiber based liquid phase microextraction method using volatile organic solvent for determination of aromatic amines in environmental water samples prior to high-performance liquid chromatography

A new and fast hollow fiber based liquid phase microextraction (HF-LPME) method using volatile organic solvents coupled with high-performance liquid chromatography (HPLC) was developed for determination of aromatic amines in the environmental water samples. Analytes including 3-nitroaniline, 3-chloroaniline and 4-bromoaniline were extracted from 6 mL basic aqueous sample solution (donor phase, NaOH 1 mol L⁻¹) into the thin film of organic solvent that surrounded and impregnated the pores of the polypropylene hollow fiber wall (toluene, 20 μL), then back-extracted into the 6 μL acidified aqueous solution (acceptor phase, HCl 0.5 mol L⁻¹) in the lumen of the two-end sealed hollow fiber. After the extraction, 5 μL of the acceptor phase was withdrawn into the syringe and injected directly into the HPLC system for the analysis. The parameters influencing the extraction efficiency including the kind of organic solvent and its volume, composition of donor and acceptor phases and the volume ratio between them, extraction time, stirring rate, salt addition and the effect of the analyte complexation with 18-crown-6 ether were investigated and optimized. Under the optimal conditions (donor phase: 6 mL of 1 mol L⁻¹ NaOH with 10% NaCl; organic phase: 20 μL of toluene; acceptor phase: 6 μL of 0.5 mol L⁻¹ HCl and 600 m mol L⁻¹ 18-crown-6 ether; pre-extraction and back-extraction times: 75 s and 10 min, respectively; stirring rate: 800 rpm), the obtained EFs were between 259 and 674, dynamic linear ranges were 0.1-1000 μg L⁻¹ (R > 0.9991), and also the limits of detection were in the range of 0.01-0.1 μg L⁻¹. The proposed procedure worked very well for real environmental water samples with microgram per liter level of the analytes, and good relative recoveries (91-102%) were obtained for the spiked sample solutions.     

Absorption and emission behaviour of trans-p-coumaric acid in aqueous solutions and some organic solvents

The absorption and fluorescence behaviour of trans-p-coumaric acid (trans-4-hydroxycinnamic acid) is investigated in buffered aqueous solution over a wide range from pH 1 to pH 12, in un-buffered water, and in some organic solvents. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and degrees of fluorescence polarisation are measured. p-Coumaric acid exists in different ionic forms in aqueous solution depending on the pH. There is an equilibrium between the neutral form (p-CAH₂) and the single anionic form (p-CAH⁻) at low pH (pK_na ≈ 4.9), and between the single anionic and the double anionic form (p-CA²⁻) at high pH (pK_aa ≈ 9.35). In the organic solvents studied trans-p-coumaric acid is dissolved in its neutral form. The fluorescence quantum yield of trans-p-coumaric acid in aqueous solution is ?_F ≈ 1.4 × 10⁻⁴ for the neutral and the single anionic form, while it is ?_F ≈ 1.3 × 10⁻³ for the double anionic form. For trans-p-coumaric acid in organic solvents fluorescence quantum yields in the range from 4.8 × 10⁻⁵ (acetonitrile) to 1.5 × 10⁻⁴ (glycerol) were measured. The fluorescence spectra are 7700–10,000 cm⁻¹ Stokes shifted in aqueous solution, and 5400–8200 cm⁻¹ Stokes shifted in the studied organic solvents. Decay paths responsible for the low fluorescence quantum yields are discussed (photo-isomerisation and internal conversion for p-CA²⁻, solvent-assisted intra-molecular charge-transfer or ππ^∗ to nπ^∗ transfer and internal conversion for p-CAH₂ and p-CAH⁻). The solvent dependence of the first ππ^∗ electronic transition frequency and of the fluorescence Stokes shift of p-CAH₂ is discussed in terms of polar solute–solvent interaction effects. Thereby the ground-state and excite-state molecular dipole moments are extracted.     

Separation of proanthocyanidins isolated from tea leaves using high-speed counter-current chromatography

The proanthocyanidin extract from tea (Camellia sinensis) leaves was purified for the further study of the biological role of proanthocyanidins in blister blight leaf disease of tea, which is caused by the fungus Exobasidium vexans. An aqueous acetone extract of proanthocyanidins prepared from healthy tea leaves was partially purified using Sephadex LH-20 chromatography. The crude proanthocyanidin extract obtained was fractionated with high-speed counter-current chromatography (HSCCC) using the solvent system n-hexane–EtOAc–MeOH–water (1:5:1:5). The purity of the each isolated fraction after a single HSCCC run was evaluated by high-performance liquid chromatography (HPLC). Seven fractions of high purity were isolated. The identity of the compound present in each fraction isolated was established using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. Five proanthocyanidins and two flavanol digallates, (−)-epigallocatechin digallate (EGCDG) and (−)-epicatechin digallate (ECDG) were isolated. Comparison of spectral data of the proanthocyanidins isolated with those previously reported indicated that all five were known B-type proanthocyanidins with 2,3-cis stereochemistry in both the upper (u-unit) and the terminal (t-unit) units, and 4R configuration of the C-ring in the u-unit. The proanthocyanidins were established to be dimers composed of (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG) and (−)-epiafzelechin gallate (EAG) units with the following structures: EGCG-(4β → 6)-EGCG, ECG-(4β → 6)-EGCG, EGCG-(4β → 6)-ECG, EAG-(4β → 6)-EGCG, ECG-(4β → 6)-ECG by analysis of spectral data. Therefore HSCCC offers a powerful method for the separation of a group of closely related naturally occurring compounds.     

Simultaneous measurement of trace monoadenosine and diadenosine monophosphate in biomimicking prebiotic synthesis using high-performance liquid chromatography with ultraviolet detection and electrospray ionization mass spectrometry characterization

The method for simultaneous separation and determination of trace monoadenosine and diadenosine monophosphate (i.e. 2′-AMP, 3′-AMP, 5′-AMP and 3′-5′ ApA) in biomimicking prebiotic synthesis was developed using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) identification. The separation was performed on a Supelco C₁₈ column with a gradient elution (solvent A: 10 mM NH₄Ac aqueous solution; solvent B: MeOH). The flow rate was set at 1.0 ml/min. The quantitative determination was achieved by HPLC with UV detection at 260 nm. The linearity ranged from 0.5 to 100 μg/ml for each nucleotide. The limits of detection (LODs) for the four nucleotides were less than 0.30 μg/ml. The recovery ranged from 95.2 to 100.7%. The intra-day relative standard deviations (RSDs) of the retention times were between 0.7 and 1.1%. Both full-scan ESI-MS and -MS² for the four nucleotides under both positive and negative polarity were carried out and the possible cleavage pathways of them were depicted. The specific ions, [AMP + H]⁺ at m/z 348 and [ApA + H]⁺ at m/z 597, were chosen to characterize the four nucleotides in biomimicking prebiotic synthesis between N-(O,O-diisopropyl) phosphoryl amino acid (Dipp-aa) and adenosine. Using the proposed HPLC/UV/ESI-MS method, the concentration of 2′-AMP, 3′-AMP, 5′-AMP and 3′-5′ ApA in the biomimicking prebiotic synthesis samples were determined.     

Rapid and high-throughput purification of salvianolic acid B from Salvia miltiorrhiza Bunge by high-performance counter-current chromatography

A large-scale purification of salvianolic acid B from Salvia miltiorrhiza Bunge is presented. The method development began with selection of the solvent system, then optimization of the operating parameters and ended up with linear scale-up from an analytical to a preparative instrument. Three factors were used for method optimization and scale-up estimation: purity, process throughput and process efficiency. Preparation was achieved using a two-phase solvent system comprising hexane–ethyl acetate–methanol–acetic acid–water (1:5:1.5:0.00596:5, v/v). This preparation yielded 475 mg of salvianolic acid B with a purity of 96.1% from 1.5 g of crude extract. The process throughput of crude was 2.23 g/h while process efficiency per gram of target compound was 0.769 g/h. Two factors—process environmental risk factor and process evaluation factor were used for evaluation of the separation process.     

Extraction and determination of organophosphorus pesticides in water samples by a new liquid phase microextraction-gas chromatography-flame photometric detection

A rapid, sensitive and efficient liquid phase microextraction (LPME) method was developed to determine trace concentrations of some organophosphorus pesticides in water samples. This method combines liquid phase microextraction with gas chromatographic (GC) analysis in a simple and inexpensive apparatus involving very little organic solvent consumption. It involves exposing a floated drop of an organic solvent on the surface of aqueous solution in a sealed vial. Experimental parameters which control the performance of LPME such as type of organic solvent, organic solvent and sample volumes, sample stirring rate, sample solution temperature, salt addition and exposure time were investigated and optimized. Finally, the enrichment factor, dynamic linear range (DLR), limit of detection (LOD) and precision of the method were evaluated by the water samples spiked with organophosphorus pesticides. Using optimum extraction conditions, very low detection limits (0.01-0.04 μg L⁻¹) and good linearities (0.9983 < r² < 0.9999) were achieved. The LPME was performed for determination of organophosphorus pesticides in different types of natural water samples and acceptable recoveries (96-104%) and precisions (3.5 < R.S.D.% < 8.9) were obtained. The results suggested that the newly proposed LPME method is a rapid, accurate and effective sample preparation method and could be successfully applied for extraction and determination of organophosphorus pesticides in water samples.     

Determination of methylamines and trimethylamine-N-oxide in particulate matter by non-suppressed ion chromatography

An ion chromatography method with non-suppressed conductivity detection was developed for the simultaneous determination of methylamines (methylamine, dimethylamine, trimethylamine) and trimethylamine-N-oxide in particulate matter air samples. The analytes were well separated by means of cation-exchange chromatography using a 3 mM nitric acid/3.5% acetonitrile (v/v) eluent solution and a Metrosep C 2 250 (250 mm × 4 mm i.d.) separation column. The effects of the different chromatographic parameters on the separation were also investigated. Detection limits of methylamine, dimethylamine, trimethylamine, and trimethylamine-N-oxide were 43, 46, 76 and 72 μg/L, respectively. The relative standard deviations of the retention times were between 0.42% and 1.14% while the recoveries were between 78.8% and 88.3%. The method is suitable for determining if methylamines and trimethylamine-N-oxide are a significant component of organic nitrogen aerosol in areas with high concentration of these species.     

Simple hollow fiber renewal liquid membrane extraction method for pre-concentration of Cd(II) in environmental samples and detection by Flame Atomic Absorption Spectrometry

A hollow fiber renewal liquid membrane (HFRLM) extraction method to determine cadmium (II) in water samples using Flame Atomic Absorption Spectrometry (FAAS) was developed. Ammonium O,O-diethyl dithiophosphate (DDTP) was used to complex cadmium (II) in an acid medium to obtain a neutral hydrophobic complex (ML₂). The organic solvent introduced to the sample extracts this complex from the aqueous solution and carries it over the poly(dimethylsiloxane) (PDMS) membrane, that had their walls previously filled with the same organic solvent. The organic solvent is solubilized inside the PDMS membrane, leading to a homogeneous phase. The complex strips the lumen of the membrane where, at higher pH, the complex Cd-DDTP is broken down and cadmium (II) is released into the stripping phase. EDTA was used to complex the cadmium (II), helping to trap the analyte in the stripping phase. A multivariate procedure was used to optimize the studied variables. The optimized variables were: sample (donor phase) pH 3.25, DDTP concentration 0.05% (m/v), stripping (acceptor phase) pH 8.75, EDTA concentration 1.5 × 10⁻² mol L⁻¹, extraction temperature 40 °C, extraction time 40 min, a solvent mixture N-butyl acetate and hexane (60/40%, v/v) with a volume of 100 μL, and addition of ammonium sulfate to saturate the sample. The sample volume used was 20 mL and the stripping volume was 165 μL. The analyte enrichment factor was 120, limit of detection (LOD) 1.3 μg L⁻¹, relative standard deviation (RSD) 5.5% and the working linear range 2-30 μg L⁻¹.     

Determination of 3-nitroaniline in water samples by directly suspended droplet three-phase liquid-phase microextraction using 18-crown-6 ether and high-performance liquid chromatography

Liquid–liquid–liquid microextraction (LLLME) with directly suspended droplet in high-performance liquid chromatography (HPLC) has been applied as a new, rapid and easy method for the determination of 3-nitroaniline in environmental water samples. The target compound was extracted from the aqueous sample solution (donor phase, pH 13) into an organic phase and then was back-extracted into a directly suspended droplet of an acidic aqueous solution (acceptor phase, pH 0.3). In this method, without using a microsyringe as supporting device, an aqueous large droplet is freely suspended at the top-center position of an immiscible organic solvent, which is laid over the aqueous sample solution while being agitated. Then, the droplet was withdrawn into the microsyringe and directly was injected into the HPLC system with UV detection at 227 nm. Up to 148-fold enrichment of the analyte could be obtained under the optimal conditions [i.e. donor phase: 0.1 M sodium hydroxide solution (4.5 mL); organic phase: o-xylene/1-octanol (90:10, v/v; 250 μL); acceptor phase: 0.5 M hydrochloric acid and 500 mM 18-crown-6 ether (6 μL); extraction time: 60 s; back-extraction time: 6 min and stirring rate: 600 rpm]. The limit of detection was 1 μg/L (n = 7) and the relative standard deviation (RSD, n = 5) was 4.9 at S/N = 3. The calibration graph was linear in the range of 5–1500 μg/L with r = 0.9983. All experiments were carried out at room temperature (22 ± 0.5 °C).     

Isolation of the retinal isomers from the isomerization of all-trans-retinal by flash countercurrent chromatography

The isolation of the retinal isomers from all-trans-retinal was performed by flash countercurrent chromatography. In each separation, isomerization reaction solution of 200 mg all-trans-retinal could be loaded on a 1200 mL of high-speed countercurrent chromatographic column with 5 mm bore, eluted by a mobile phase flow rate of 25 mL/min, resulting in 63 mg of 11-cis-retinal, 24 mg of 13-cis-retinal and 26 mg of 9-cis-retinal with purities more than 95%. n-Hexane–acetonitrile (3:1) was used as the solvent system which possesses the advantages of simplicity, re-use of the solvent and multiple injections. This method could be used to prepare 13-cis-retinal, 11-cis-retinal and 9-cis-retinal for the photoisomerization investigation, such as the effect of 11-cis-retinal in the visual system.