Highly fluorinated polymers with sulfonate,sulfamide and N,N‐diethylamino groups for the capillary electromigration separation of proteins and steroid hormones

Highly fluorinated polymers are promising materials in separation methods due to their combination of high chemical and thermally stability, hydro‐ and oleophobicity, and weak intermolecular forces. However, application of these polymers in chromatography is limited because of their low solubility in aqueous‐organic solvents. In our research, the highly fluorinated water soluble polymers with –SO₃⁻N(Et)₄⁺, –SO₂NH₂, and –N(Et)₂ terminal groups were synthesized and applied as additives to the background electrolyte for the separation of steroid hormones and proteins by capillary electromigration methods. It is shown that highly fluorinated polymers can be used both as pseudo‐stationary phases in electrokinetic chromatography for high separation efficiency (N  ∼ 200 × 10³) and selectivity (α  ∼  1.1) of uncharged analytes (e.g., steroid hormones), and as dynamic modifiers of fused silica capillary walls. The highest separation efficiency (N  ∼ 1 × 10⁶) and selectivity (α  ∼ 1.3) of steroid hormones was achieved by combination of sodium dodecyl sulfate and fluoropolymer with sulfonate groups in background electrolyte with pH 2. Dynamic wall coatings based on fluoropolymer with –SO₂NH₂ (which are easier and faster to create and wash off) exhibit significantly higher separation efficiency and selectivity compared to capillary electrochromatography on capillary columns based on polymethacrylate polymers.     

Facile extraction of berberine from different plants,separation, and identification by thin-layer chromatography,high-performance liquid chromatography,and biological evaluation against Leishmaniosis

The extraction of berberine was carried out from Berberis vulgaris, Berberis aquifolium, and Hydrastis canadensis plants using ethanol and water (70:30, v/v). The extracted berberine was characterized by ultraviolet-visible and Fourier-transform infrared spectroscopy. The purity of berberine was ascertained by thin-layer chromatography using n-propanol-formic acid-water (95:1:4) and (90:1:9) solvents. hR_f values were in the range of 44–49 with compact spots (diameter 0.2–0.4 cm). HPLC was carried out using ammonium acetate buffer and acetonitrile in gradient mode with Zodiac (4.6 × 150 mm, 3 μm) column. The flow rate was 1.0 mL/min and detection was at 220 nm. The values of separation and resolution factors of the standard and the extracted berberine were in the range of 1.13–1.16 and 1.40–1.71, respectively. A comparison has shown that both thin-layer chromatography and high-performance liquid chromatography (HPLC) methods found applications in different situations and requirements. The extracted berberine samples were used to treat Leishmaniosis and the results showed better activity of berberine in comparison to the standard drug Amphotericin B. Briefly, the reported research is a novel and may be used to extract berberine from plants, separation and identification of berberine by thin layer chromatography and HPLC and to treat Leishmaniosis.     

Direct determination of tri-<Emphasis Type="Italic">n</Emphasis>-butyl phosphate by HPLC and GC methods

The high performance liquid chromatography and gas chromatography methods were investigated for their applicability in determining micro-level concentrations of tri-n-butyl phosphate (TBP). A high performance liquid chromatograph (HPLC) equipped with refractive index detector was used in determining TBP up to 2 ppm concentration level in the aqueous nitric acid solutions. The gas chromatography incorporated with Thermionic Detector (NPD) and Flame Photometric Detector (FPD) were examined for their potential in analyzing TBP in organic phase up to sub-ppm level. The results indicated that HPLC-RI technique is well suited for direct analysis of aqueous phase. For organic phase analysis, gas chromatographic methods with the TID and FPD were suitable but performance of detectors deteriorated often due to fouling.     

Comparison of microemulsion electrokinetic chromatography with high-performance liquid chromatography for fingerprint analysis of resina draconis

Microemulsion electrokinetic chromatography (MEEKC) has been developed for fingerprint analysis of resina draconis, a substitute for sanguis draconis in the Chinese market. The microemulsion as the running buffer was made up of 3.3% (w/v) sodium dodecyl sulfate (SDS), 6.6% (w/v) n-butanol, 0.8% (w/v) n-octane, and 10 mmol/L sodium tetraborate buffer (pH 9.2), which was also used as the solvent for ultrasonic extraction of both water- and fat-soluble compounds in the traditional Chinese medicine samples. Four batches of resina draconis obtained from different pharmaceutical factories located in different geographic regions were used to establish the electrophoretic fingerprint. MEEKC was performed using a Beckman PACE/MDQ system equipped with a diode-array detector and with monitoring at 280 nm. The fingerprint of resina draconis comprised 27 common peaks within 100 min. The relative standard deviations of the relative migration time of these common peaks were less than 2.1%. Through repetitive injection of the sample solution six times in 24 h, all relative standard deviations of the migration time and peak area of loureirin A and loureirin B were less than 2.5 and 3.8%, which demonstrated that the method had good stability and reproducibility. The relative peak areas of these common peaks in the electropherograms of four batches of resina draconis were processed with two mathematical methods, the correlation coefficient and the interangle cosine, to valuate the similarity. The values of the similarity degree of all samples were more than 0.91, which showed resina draconis samples from different origins were consistent. On the other hand, high-performance liquid chromatography (HPLC) coupled with photodiode-array detection was also applied to establish the fingerprint of resina draconis. The samples were separated with a LiChrospher C₁₈ column using acetonitrile (solvent A) and water containing 0.1% H₃PO₄ (solvent B) as the mobile phase in linear gradient elution mode at a flow rate of 0.6 mL/min and detection was at 280 nm. There were only 20 common peaks in the HPLC fingerprint, and the values of the similarity degree of all samples were also more than 0.91. Though the similarity results of fingerprint analysis seemed to be the same, MEEKC resulted in more common peaks and higher separation efficiency for a variety of polarities of the components than HPLC. So, MEEKC was more suitable for development of the fingerprint of resina draconis.     

Generic approach to the method development of intact protein separations using hydrophobic interaction chromatography

We describe a liquid chromatography method development approach for the separation of intact proteins using hydrophobic interaction chromatography. First, protein retention was determined as function of the salt concentration by isocratic measurements and modeled using linear regression. The error between measured and predicted retention factors was studied while varying gradient time (between 15 and 120 min) and gradient starting conditions, and ranged between 2 and 15%. To reduce the time needed to develop optimized gradient methods for hydrophobic interaction chromatography separations, retention‐time estimations were also assessed based on two gradient scouting runs, resulting in significantly improved retention‐time predictions (average error < 2.5%) when varying gradient time. When starting the scouting gradient at lower salt concentrations (stronger eluent), retention time prediction became inaccurate in contrast to predictions based on isocratic runs. Application of three scouting runs and a nonlinear model, incorporating the effects of gradient duration and mobile‐phase composition at the start of the gradient, provides accurate results (improved fitting compared to the linear solvent‐strength model) with an average error of 1.0% and maximum deviation of –8.3%. Finally, gradient scouting runs and retention‐time modeling have been applied for the optimization of a critical‐pair protein isoform separation encountered in a biotechnological sample.     

Application of probe sonication extraction for the determination of linear alkylbenzene sulfonates from sewage sludge. Comparison with other extraction methods

A new method based on probe sonication extraction (USP) prior to high performance liquid chromatography (HPLC) has been developed for the determination of linear alkylbenzene sulfonates (LAS) from sewage sludge. The optimized method was designed to be cost effective compared to existing extraction methods (ultrasonic assisted extraction, Soxhlet or pressurized liquid extraction) which may require large quantities of organic solvents, or costly instrumentation or equipment.The main factors affecting the extraction efficiency (extractant volume, ultrasounds power and extraction time) were optimized using compost sludge. The detection limit of total LAS in the sludge was 10 mg kg^− 1. The extraction of C₁₀-C₁₃ homologues is carried out using an extraction time of 7 min with 10 mL of methanol. Liquid chromatography with fluorescence (FL) detector is used for determination of LAS homologues. A mobile phase acetonitrile-water containing 0.1 M NaClO₄ (65:35) and isocratic elution was used. Compounds were eluted over 6 min at a flow rate of 1 mL/min. Polar interferences are eluted between 0 and 2 min and no purification of the samples is required prior to the final determination by high performance liquid chromatography (HPLC). The recoveries of LAS in spiked sewage sludge were between 84.0% and 97.0%, which reflect the efficiency of the method for extraction of these analytes from sewage sludge. Concentration levels found were between 11,858 mg kg^− 1 for digested sludge and 2379 mg kg^− 1 for compost sludge.     

A fast method for screening and/or quantitation of tetrahydrocannabinol and metabolites in urine by automated SPE/LC/MS/MS

Marijuana is one of the most commonly used illicit substances. The high usage of this substance results in it being commonly encountered in clinical samples throughout the USA and Europe. Due to its wide availability and use, marijuana is also commonly encountered in forensic toxicology laboratories. The proposed method utilized an automated solid phase extraction (SPE) coupled to liquid chromatography/mass spectrometry (LC/MS). The automated SPE procedure was developed using Hysphere C8-EC sorbent, and the high performance liquid chromatography (HPLC) separation was performed using an Xterra MS C₁₈ column with a total runtime of 10 min. The standard curves linearity generally fell between 6 and 500 ng/mL. The limits of detection ranged from 2 to 4 ng/mL, and the limits of quantitation ranged from 8 to 12 ng/mL. The bias and imprecision were determined using a simple analysis of variance (single factor). The results demonstrate bias as <11% and percent imprecision as <12% for all components at four quality control levels. This method has been in use for over 2 years and has been applied to numerous forensic samples. When compared to other published methods, it exceeds others in its simplicity and speed of analysis. This method takes advantage of robotics and automation for a total analysis time of 10 min, including sample preparation, separation, and detection.     

Miniaturized capillary ion chromatograph with UV light‐emitting diode based indirect absorbance detection for anion analysis in potable and environmental waters

A miniaturized, flexible, and low‐cost capillary ion chromatography system has been developed for anion analysis in water. The ion chromatography has an open platform, modular design, and allows for ease of modification. The assembled platform weighs ca. 0.6 kg and is 25 × 25 cm in size. Isocratic separation of common anions (F^–, Cl^–, NO₂^–, Br^–, and NO₃^–) could be achieved in under 15 min using sodium benzoate eluent at a flow rate of 3 μL/min, a packed capillary column (0.150 × 150 mm) containing Waters IC‐Pak 10 μm anion exchange resin, and light‐emitting diode based indirect UV detection. Several low UV light‐emitting diodes were assessed in terms of sensitivity, including a new 235 nm light‐emitting diode, however, the highest sensitivity was demonstrated using a 255 nm light‐emitting diode. Linear calibration ranges applicable to typical natural water analysis were obtained. For retention time and peak area repeatability, relative standard deviation values ranged from 0.60–0.95 and 1.95–3.53%, respectively. Several water samples were analysed and accuracy (recovery) was demonstrated through analysis of a prepared mixed anion standard. Relative errors of –0.36, –1.25, –0.80, and –0.76% were obtained for fluoride, chloride, nitrite, and nitrate, respectively.     

Post column derivatisation analyses review. Is post-column derivatisation incompatible with modern HPLC columns?

Post Column derivatisation (PCD) coupled with high performance liquid chromatography or ultra-high performance liquid chromatography is a powerful tool in the modern analytical laboratory, or at least it should be. One drawback with PCD techniques is the extra post-column dead volume due to reaction coils used to enable adequate reaction time and the mixing of reagents which causes peak broadening, hence a loss of separation power. This loss of efficiency is counter-productive to modern HPLC technologies, -such as UHPLC. We reviewed 87 PCD methods published from 2009 to 2014. We restricted our review to methods published between 2009 and 2014, because we were interested in the uptake of PCD methods in UHPLC environments. Our review focused on a range of system parameters including: column dimensions, stationary phase and particle size, as well as the geometry of the reaction loop. The most commonly used column in the methods investigated was not in fact a modern UHPLC version with sub-2-micron, (or even sub-3-micron) particles, but rather, work-house columns, such as, 250 × 4.6 mm i.d. columns packed with 5 μm C18 particles. Reaction loops were varied, even within the same type of analysis, but the majority of methods employed loop systems with volumes greater than 500 μL.     

Standardization and validation of phytometabolites by UHPLC and high-performance thin layer chromatography for rapid quality assessment of ancient ayurvedic medicine,Mahayograj Guggul

Mahayograj Guggul is an ancient ayurvedic medicine, prescribed for various joint disorders like arthritis, gout, and rheumatism. The present research was envisaged to develop a simple, sensitive, and comprehensive analytical method for standardization of Mahayograj Guggul. The analysis was conducted for gallic acid, protocatechuic acid, vanillic acid, cinnamic acid, piperine, guggulsterone-E, and guggulsterone-Z by high-performance thin-layer chromatography, and additionally ferulic acid, ellagic acid, and picroside I by ultra high-performance liquid chromatography. These developed methods were validated as per international guidelines, and were found linear (r² > 0.99), sensitive, precise (relative standard deviation < 5%), and accurate with recovery values (85–105%). The limit of detection and quantification were in the range of 0.11–23.6 and 0.33–71.51 μg/g. Gas chromatography tandem mass spectrometry was used to develop Mahayograj Guggul fingerprinting profile and to identify mid-polar or nonpolar compounds. Proximate analysis was used to ascertain the functional groups present in Mahayograj Guggul. Ultra high-performance liquid chromatography and gas chromatography tandem mass spectrometry were further employed to authenticate quality reproducibility in the active ingredients of Mahayograj Guggul in six commercial batches. Taken together, these analytical methods provide a scientific basis and reference for quality control evaluation of Mahayograj Guggul and similar traditional broad-spectrum formulations.     

Separation of diastereomers of phosphinic pseudopeptides by capillary zone electrophoresis and reverse phase high‐performance liquid chromatography

Capillary zone electrophoresis (CZE) and reverse phase high‐performance liquid chromatography (RP‐HPLC) were used for separation of diastereomers of phosphinic pseudopeptides in achiral separation media. A set of phosphinic pseudopeptides, i. e. peptides with one peptide bond substituted by phosphinic acid moiety ‐PO₂^–‐CH₂‐ derived from the structure N‐Ac‐Val‐AlaB(‐CH₂)Leu‐His‐NH₂ synthesized as a mixture of four diastereomers was used. Separations of diastereomers by CZE were carried out in Tris‐phosphate background electrolytes in the pH range 1.1–3.2 and at least partial separation of the four diastereomers of each pseudopeptide was achieved. A routinely used RP‐HPLC method (C₁₈‐silica column and water/acetonitrile/trifluoroacetic acid mobile phase) was also capable of resolving the diastereomers. In addition, since individual diastereomers of majority of the pseudopeptides were isolated by RP‐HPLC it was possible to check the purity of these RP‐HPLC separated diastereomers and to compare the migration order of the diastereomers in CZE with their elution order in RP‐HPLC. The results obtained by CZE and RP‐HPLC demonstrate a complementarity of both methods in analysis and separation of phosphinic pseudopeptides including their diastereomers.     

Determination of Total Radiochemical Purity of [18F]FDG and [18F]FET by High-Performance Liquid Chromatography Avoiding TLC Method

The goal of this work was to present two high-performance liquid chromatography (HPLC) method that could be applied for the determination of the total radioactive purity of 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) and O-(2-[¹⁸F]fluoroethyl)-L-tyrosine ([¹⁸F]FET). The separation of [¹⁸F]fluoride ions, [¹⁸F]FET and [¹⁸F]FET intermediate was accomplished on LiChrosper RP-18, 250?×?4 mm, 5 µm (Merck) analytical column. For mobile phase 10 mM potassium dihydrogen phosphate buffer at pH7 (A) and acetonitrile (B) was used: 0–2 min: 15% B; 2–12 min: 85% B; 12–15 min: 15% B, respectively. Analysis of [¹⁸F]FDG was performed using LiChrosper 100 NH₂, 250?×?4.5 mm, 5 µm (Merck) analytical column. The initial mobile phase composition was 10 mM KH₂PO₄ buffer (pH7) and acetonitrile (15:85, v/v) and the acetonitrile ratio was decreased to 15% at 2 min after the sample injection and held for 5 min. Complete elution of [¹⁸F]fluoride ions from stationary phases could be achieved by adding 10 mg/mL K[¹⁹F]F to radioactive samples in a ratio 1:1 during the sample preparation. Recovery of [¹⁸F]fluoride ions ranged from 99.5 to 100.6%. The validation of the developed methods showed good results for linearity (r²?=?0.9981–0.9996), specificity (R_S?=?3.7–10.2), repeatability (%Area RSD_%?=?1.2–4.3%) and limit of quantitation (LOQ?=?1.6–4.5 kBq). During the cross-validation similar radiochemical purity values were obtained by the novel HPLC methods and thin layer chromatography performed according to the recommendations of the Ph. Eur. monographs.

     

Simultaneous detection and quantification of parecoxib and valdecoxib in canine plasma by HPLC with spectrofluorimetric detection: development and validation of a new methodology

Parecoxib is the injectable prodrug of valdecoxib, a cicloxygenase-2 selective drug, currently used in human medicine. Recent studies have suggested both its excellent clinical effectiveness and wide safety profile. The aim of the present study was to develop and validate a new high-performance liquid chromatography (HPLC) with spectrofluorimetric detection method to quantify parecoxib and valdecoxib in canine plasma. Several parameters both in the extraction and the detection method were evaluated. The applicability of the method was determined by administering parecoxib to one dog: the protocol provided the expected pharmacokinetic results. The final mobile phase was acetonitrile: AcONH₄ (10 mM; pH 5.0) 55:45, v/v, with a flow rate of 0.4 mL min⁻¹, and excitation and emission wavelengths of 265 and 375 nm, respectively. The analytical column was a reverse-phase C18 ODS2 3-μm particle size. Protein precipitation in acidic medium followed by two successive liquid–liquid steps was carried out. The best extraction solvent was cyclohexane:Et₂O (3:2, v/v) that gave recoveries ranging from 81.1% to 89.1% and from 94.8% to 103.6% for parecoxib and valdecoxib, respectively. The limits of quantification were 25 and 10 ng mL⁻¹ for parecoxib and valdecoxib, respectively. The chromatographic runs were specific with no interfering peaks at the retention times of the analytes, as confirmed by HPLC–mass spectrometry experiments. The other validation parameters were in agreement with the European Medicines Evaluation Agency and International Conference on Harmonisation guidelines. In conclusion, this method (extraction, separation and applied techniques) is simple and effective. This is the first time that use of a HPLC with spectrofluorimetric detection technique to simultaneously detect parecoxib and valdecoxib in plasma has been reported. This technique may have applications for pharmacokinetic studies.     

Comparison of two different isolation methods of benzimidazoles and their metabolites in the bovine liver by solid-phase extraction and liquid chromatography–diode array detection

A new analytical method that uses high performance liquid chromatography–diode array detector (HPLC–DAD) was developed for the analysis of 14 benzimidazoles residues, including metabolites, in bovine liver. Samples were extracted using two different extraction procedures: with phosphate buffer after enzymatic hydrolysis (method A) or using organic solvent, i.e. acetonitrile (method B). Then, samples were purified on a strong cation exchange (SCX) cartridge and analyzed in HPLC/DAD. The recovery percentages, obtained spiking the matrix (liver) at concentrations of 500 and 100 μg kg^?1 with a standard mixture of benzimidazoles, were in the range 6–101% and 80–102% for methods A and B, respectively. The repeatability of the methods was assessed in all cases by the % of correlation value (CV) that was lower than 19%. The limits of quantification (LOQs) in the matrix for methods A and B were in the range 40–60 and 20–50 μg kg^?1, respectively. The best of the two methods, method B, was used for the analysis of 10 bovine liver samples.     

HPLC and GC Methods for Determination of Lubricants and Their Evaluation in Analysis of Real Samples of Polyethylene

High performance liquid chromatography (HPLC) and gas chromatography (GC) are introduced for analysis of polymer lubricants (stearamide, oleamide and erucamide). In the HPLC method, a reverse phase octadecylsilane (ODS) column along with acetonitrile/methanol (60:40) as a mobile phase were used. Detection of analytes was performed by a UV detector at 202 nm. The analysis time was less than 8 min. In the GC method, polar capillary column and flame ionization detector (FID) were used for separations and detection, respectively. The analysis time by GC was longer than HPLC and was about 30 min. Limits of detection, linear range and repeatability of both methods are similar, but determination of oleamide in real samples by HPLC method is difficult due to complexity of the initial part of HPLC chromatogram in polyethylene samples. That problem is not observed in the GC method. Detection limits in both methods for all analytes are lower than 0.003% which are much lower than the amount of lubricants in commercial polymers (0.05–0.2%).     

Development of a micellar electrokinetic capillary chromatography method for the determination of four naphthalenediols in cosmetics and a comparison with a HPLC method

A micellar electrokinetic capillary chromatography (MEKC) method with ultraviolet visible (UV) detection was used for the determination of 1,7-naphthalenediol, 2,3-naphthalenediol, 1,5-naphthalenediol, and 2,7-naphthalenediol in cosmetics. The current method for their determination in various cosmetics is high-performance liquid chromatography (HPLC). Separation conditions affecting the MEKC method were optimized as 20 mM Na₂B₄O₇–50mM SDS, pH 9.8, with 22 kV applied voltage and UV detection at 230 nm. Under optimal conditions, electrophoretic analysis was completed in less than 6 min, with limit of detection (LOD) of 0.070–0.19 μg/mL and limit of quantitation (LOQ) of 0.23–0.63 μg/mL. A good linear relationship (r² > 0.99) was obtained at the range of 0.75–20 μg/mL. Recoveries for the four naphthalenediols in lotion, loose powder, and sun cream are between 91.2–107.2% with relative standard deviation (RSD) less than 4.04%. The method has been successfully applied to the determination of the four naphthalenediols in different kinds of cosmetics. A comparison with HPLC-UV method was also carried out according to the National Standards of the People's Republic of China. The results obtained by MEKC and HPLC methods are comparable, but the proposed MEKC method can help us obtain a much shorter detection time and low cost.     

Determination of theophylline in serum by high performance liquid chromatography/mass spectrometry with atmospheric pressure chemical-ionization (APCI HPLC/MS)

 A method for the determination of theophylline (TH), without derivatization, in serum by isotope dilution mass spectrometry using labelled [1, 3-¹⁵N₂-2-¹³C]theophylline (LTH) as internal standard is described. After deproteinization, the analyte is directly injected into a high performance liquid chromatography – mass spectrometer operating with atmospheric-pressure chemical-ionization (APCI HPLC/MS). The concentrations of TH in sera measured by APCI HPLC/MS are compared with results from gas chromatography – isotope dilution mass spectrometry (GC-ID/MS), high performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the APCI HPLC/MS and GC-ID/MS methods are discussed. The coefficient of variation (CV) determined from duplicate samples was less than 2%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1. Received: 17 January 1996/Revised: 26 March 1996/Accepted: 5 April 1996     

2H/1H Ratio Analysis of Flavor Compounds by On‐Line Gas Chromatography Pyrolysis Isotope Ratio Mass Spectrometry (HRGC‐P‐IRMS): Benzaldehyde

Isotope ratio monitoring gas chromatography‐mass spectrometry of the ²H/¹H ratio by pyrolysis isotope ratio mass spectrometry (P‐IRMS) was used to analyze benzaldehyde originating from various sources. Based on the δ²H_SMOW value of an authentic reference sample determined with an elemental analyzer (EA), the range of reproducibility and linearity was checked. Correct (EA related) and reproducible data were obtained for sample amounts >0.6 μg benzaldehyde (on column). In another series of experiments, the influence of sample preparation, i. e. simultaneous distillation‐extraction (SDE) was found to be negligible. The following ranges of δ²H_SMOW values were determined for benzaldehyde using five types of samples, i. e. (i) synthetic (δ²H_SMOW –78 to –85‰, ex benzal chloride; +420 to +668‰, ex toluene) and ‘natural’ (including ‘ex‐cassia’) references (δ²H_SMOW –83 to –144‰); (ii) bitter almond oils (δ²H_SMOW –113 to –148‰); (iii) fruits (δ²H_SMOW –111 to –146‰); (iv) kernels (δ²H_SMOW –115 to –188‰); and (v) leaves (δ²H_SMOW –165 to –189‰).     

Separation of Anthraquinones by Capillary Electrophoresis and High‐Performance Liquid Chromatography

The separation and determination of twelve anthraquinones, viz. anthraquinone 1, chrysphanol 2, aloe‐emodin 3, alizarin 4, anthraquinone‐2‐carboxylic acid 5, purpurin 6, sennoside B 7, sennoside A 8, emodin 9, quinalizarin 10, rhein 11, and anthraflavic acid 12, were achieved by capillary electrophoresis (CE) and high‐performance liquid chromatography (HPLC). Detection at 260 nm with a buffer solution containing 30 mM sodium borate (adjusted to pH = 10.56 with 0.05N NaOH) and acetonitrile (9 : 1) in CE or with a linear gradient elution containing 20 mM KH₂PO₄ with 0.05% phosphoric acid (pH = 2.91) and methanol in HPLC was found to be the most suitable approach for this separation. Contents of six components (2, 3, 7, 8, 9, 11) in crude Rhei Rhizoma extract could easily be determined within 39 min by CE or 63 min by HPLC. The effects of buffers on this separation and the validation of the two methods were studied.     

Nuclear magnetic resonance-based solvent system selection for counter-current chromatography separation of compounds present in the same high-performance liquid chromatography peak: Flavonoids in barley seedlings as an example

Partition coefficient is a key parameter for counter-current chromatography separation. High-performance liquid chromatography (HPLC) is the most commonly used tool for the screening of partition coefficients. However, HPLC technology is not applicable to the compounds present in the same chromatographic peak. Nuclear magnetic resonance (NMR) technology could easily distinguish compounds according to their characteristic absorption even if they exist in the same HPLC peak. In this study, two flavonoids present in the same HPLC peak were successfully purified by counter-current chromatography with a solvent system screened by NMR to show the great potential of NMR technology in the screening of the partition coefficient of co-efflux compounds. Through NMR screening, an optimized ethyl acetate/n-buthanol/water (7:3:10, v/v/v) system was applied in this study. As a result, two flavonoids, including 4.8 mg of 3′-methoxyl-6′’’-O-feruloylsaponarin and 9.8 mg of 6′’’-O-feruloylsaponarin were separated from 15 mg of the mixture. There is only one methoxy group difference between the two flavonoids. This study provides a new strategy for the screening of counter-current chromatography solvent systems and broadens the application scope of counter-current chromatography.