Separation of Natural Food Pigments in Saponified and Un-Saponified Paprika Oleoresin by Ultra High Performance Supercritical Fluid Chromatography (UHPSFC)

The natural pigments in paprika were rapidly and efficiently separated by ultra high performance supercritical fluid chromatography. The separation of both un-saponified and saponified mixtures of paprika oleoresin were optimized, with run times of 10.6 min. Three different C18 columns, a cyano, silica and diol column, all 3 × 100 mm, with 1.8 μm particles were compared. The best separation for the un-saponified sample was found with an SB-C18 column, while the saponified samples were best separated on a bare silica, RX-Sil column. A SB-CN column allowed near optimum separation of both the unsaponified, and saponified samples, with similar run times. The best mobile phase was carbon dioxide (CO₂) modified with isopropyl alcohol (IPA), with a composition gradient. Fingerprints of several commercial pepper products indicated that one appeared to be colored with artificial dyes, while the color of a chili powder may have been enhanced with a paprika extract. Spectra, using CO₂ with IPA as modifier, produced a single maximum at 453 nm, which appears to represent up to a 30 nm solvatochromic shift from the maxima in most organic solvents. Acetonitrile (ACN) as modifier produced spectra with two maxima and a similar solvatochromic shift. These results appear to be the first on saponified paprika oleoresin samples using SFC. It is also the first detailed report on the separation of un-saponified samples. The results are up to six times faster than comparable results by HPLC. It appears that SFC is a viable, superior alternative to HPLC for the analysis of this important commercial product, without using ACN, or chlorinated solvents.     

Separation of enantiomers of ibuprofen on chiral stationary phases by packed column supercritical fluid chromatography.

A packed column supercritical fluid chromatography (SFC) method for the separation of ibuprofen enantiomers on a chiral stationary phase and CO2 with modifier as mobile phase has been developed at an analytical scale. Among 11 different stationary phases the Kromasil CHI-TBB phase showed by far the best separation properties. The influence of different modifiers, injection solvents, temperature, and pressure, and density of the fluid, respectively, on the separation behavior has been studied. It was found that the separation behavior strongly depends on the type of modifier and the modifier content. Temperature and pressure are of less influence.     

Pharmaceutical analysis by supercritical fluid chromatography: Optimization of the mobile phase composition on a 2-ethylpyridine column

The separation of neutral, acidic, and basic pharmaceuticals with diverse physicochemical properties by packed column supercritical fluid chromatography (pSFC) on a 2-ethylpyridine column (25 cmx4.6 mm id, 3 mum particles) is presented. The optimization strategy involved separations at 100% methanol (MeOH) and at 50% MeOH/50% ACN while keeping the peak symmetry additives formic acid (FA) and isopropylamine (IPA) at constant levels of 0.25% v/v. By plotting the adjusted retention times as a function of the MeOH/ACN ratio, an optimal modifier ratio composition of 65% MeOH/35% ACN was found. The total set of 26 neutral, acidic, and basic pharmaceuticals was analyzed and the optimal composition experimentally verified. This mobile phase composition is currently used in pharmaceutical method development and open-access generic screening environments.     

Rapid, Direct Quantitation of the Preservatives Benzoic and Sorbic Acid (and Salts) Plus Caffeine in Foods and Aqueous Beverages Using Supercritical Fluid Chromatography

The preservatives benzoate and sorbate, plus caffeine were rapidly separated and quantified, in just over 2 min, in a wide range of beverages and foods, using supercritical fluid chromatography (SFC). Fifteen beverages and 10 semi-liquid foods were evaluated. The benzoate and sorbate were originally present in the samples as the acid, or the sodium, or potassium salt. The aqueous samples were diluted 3:1 with acidified methanol, to insure the acids were protonated, then directly injected. The solutes were isocratically eluted from a 4 × 250 mm, 5 μm Diol column with 3.5 mL min^?1 of 8.5 % methanol containing 0.3 % acetic acid at 50 °C and a column outlet pressure of 150 bar. The real samples exhibited remarkably little interference. All the beverages were accurately labeled. However, many of the foods, such as salad dressings, mustard, etc., were mislabeled. The method was linear over a wide range with correlation coefficients for all three solutes >0.999. RSD’s were generally less than 1 %. The results agreed with the caffeine content on the labels within a few percent. Surprisingly, this appears to be the first published separation of benzoic and sorbic acid preservatives in food, and beverages using SFC, and one of a very few SFC applications where aqueous samples were simply diluted and injected. Compared to published references, the SFC method was found to be up to 7 times faster than HPLC, and eliminated the use of acetonitrile.     

Novel Data on the Effect of Tetrahydrofuran as an Organic Co-Modifier in RP-HPLC

The aim of the study was to characterise the unique chromatographic properties of tetrahydrofuran (THF) based on hydrogen-bonding interactions with weakly acidic compounds (pK _a = 7.4–12.48) including steroids with phenolic hydroxyl groups, their substituted derivatives and heterocyclic amides having different polar functional groups (log P = 1.15–4.78). The results suggested that the organic modifier does not simply affect retention by changing the hydrophobicity of eluent, but rather specifically modifies the nature of the analyte–stationary phase interaction. In the water/isopropanol (IPA)/THF eluent mixture THF forms a hydrogen-bonded complex with the phenolic steroid compounds. The apparent formation of the THF–analyte complex depended on the proportion of components in the ternary mobile phase employed (from 70:30:0 to 70:0:30 v/v/v). The weakly acidic model compounds showed an increasing retention time with increasing THF concentration. This effect of THF was found to be a solvent-specific interaction, which was only observed in the presence of IPA. The systematic modification of the phase ratio of organic modifiers exerts a great influence on retention time and changes the separation processes over a wide range. In the case of other protic solvents (methanol, ethanol) we could not observe this selective chromatographic behaviour. From the point of view of chromatographic practice, the use of THF–IPA co-modifiers may increase the selectivity and provide excellent possibilities for separation of weakly acidic compounds including the large family of phenolic compounds.     

A systematic investigation of the effect of sample diluent on peak shape in hydrophilic interaction liquid chromatography

The aim of this study was to evaluate the importance of sample diluents to improve peak shapes in hydrophilic interaction liquid chromatography (HILIC), using low molecular weight (<1000 Da) analytes as well as peptides (with MW ranging between 1000 and 6000 Da) as model compounds. Various solvents were tested including water, acetonitrile, methanol, ethanol, propan-2-ol, dimethyl sulfoxide, and a number of combinations of them. For the analysis of small MW compounds, best peak shapes were obtained with sample dissolved in pure ACN but, IPA or a mixture of ACN/IPA (50:50, v/v) could represent a viable alternative in the case of solubility issues with pure ACN. For drug discovery applications, DMSO can be employed but in combination with at least 80% of ACN. For peptides analysis, acetonitrile, EtOH and IPA as sample diluents, provided similar chromatographic profiles, but pure EtOH or IPA were recommended to limit denaturation and samples solubility issues. Finally, whatever the nature of the compounds, it is recommended to add the lowest amount of water to the sample diluent, to maintain suitable peak shapes.     

High-resolution analysis of polyprenols by supercritical fluid chromatography

A high-resolution analysis of polyprenol mixtures was achieved by supercritical fluid chromatography (SFC). The separation of polyprenols was examined on an octadecylsilane-packed column with liquid carbon dioxide as the mobile phase and ethanol as modifier. Using this chromatography system, the resolution of separation (Rs) between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) was two times higher than that using conventional reversed-phase high-performance liquid chromatography. Our SFC technique allows the advantage of baseline separation of polyprenol samples containing hydrophobic components such as terpenes or fatty acids that are unfavorable for good separation. This method is very useful for the analysis of structurally close polyprenol analogues of rubber plant metabolites.     

Chromatographic resolution of closely related species: Drug metabolites and analogs

In this study, we investigate the separation of a variety of mixtures of drugs, metabolites, and related analogs including representatives of the carbamazepine, methylated xanthine, steroid hormone, nicotine, and morphine families using several automated chromatographic method development screening systems including ultra high performance liquid chromatography, core–shell HPLC, achiral supercritical fluid chromatography (SFC), and chiral SFC. Of the 138 column and mobile phase combinations examined for each mixture, a few chromatographic conditions afford the best overall performance, with a single achiral SFC method (4.6 × 250 mm, 3.0 μm GreenSep Ethyl Pyridine, 25 mM isobutylamine in methanol/CO₂) affording good separation for all samples. Four of these mixtures were also resolved by achiral SFC on the Luna HILIC and chiral SFC Chiralpak IB columns using methanol or ethanol with 25 mM isobutylamine as polar modifiers. Modifications of standard chromatography screening conditions afforded fast separation methods (from 1 to 5 min) for baseline resolution of all components of each of these challenging sets of closely related compounds.     

Separation of 9 Sulfonamide Drugs in ≈4 Min by Ultra-High Performance Supercritical Fluid Chromatography (UHPSFC): with a Feasibility Study for Detection in Milk

Nine widely used veterinary sulfonamide drugs were baseline separated (R _s ≥1.5) in just over 4 min using a 3 × 100 mm, 1.8 μm RX-Sil column, with 9.2 % methanol in carbon dioxide, at 110 bar and 30 °C, with direct UV detection at 260 nm using a 3 mm, 2 μL tapered flow cell. Pressure drop was only 172 bar. Optimization was difficult due to the similarity in structures. Small changes in modifier concentration, temperature and pressure, each tended to improve the resolution of some peak pairs but degraded the resolution of others. There were four critical pairs, each responding differently to changes in conditions. Optimization was performed by plotting resolution between pairs as a function of modifier concentration first, temperature second, and outlet pressure third. Retention time was then minimized by changing flow rate. The estimated limit of quantitation (LOQ, S/N >10), for direct injections, was ≈200–400 ng/g of each, inadequate for regulatory requirements. Solid phase extraction (SPE) attempted to pre-concentrate samples spiked with sulfamethazine by ≈20:1. From water, the limit of detection (LOD) was ≈2.7 ng/mL with LOQ ≈9 ng/mL using UV at 260 nm. The LOD for milk was 6.2 ng/mL, and LOQ was 20.1 ng/mL. A better pre-concentration step or a more sensitive detector such as MS–MS is required. Even with these inadequacies, SFC was shown to be a feasible, faster, “greener” alternative to HPLC for the separation of these drugs.     

Separation of 9 Sulfonamide Drugs in ≈4 Min by Ultra-High Performance Supercritical Fluid Chromatography (UHPSFC): with a Feasibility Study for Detection in Milk

Nine widely used veterinary sulfonamide drugs were baseline separated (R _s ≥1.5) in just over 4 min using a 3 × 100 mm, 1.8 μm RX-Sil column, with 9.2 % methanol in carbon dioxide, at 110 bar and 30 °C, with direct UV detection at 260 nm using a 3 mm, 2 μL tapered flow cell. Pressure drop was only 172 bar. Optimization was difficult due to the similarity in structures. Small changes in modifier concentration, temperature and pressure, each tended to improve the resolution of some peak pairs but degraded the resolution of others. There were four critical pairs, each responding differently to changes in conditions. Optimization was performed by plotting resolution between pairs as a function of modifier concentration first, temperature second, and outlet pressure third. Retention time was then minimized by changing flow rate. The estimated limit of quantitation (LOQ, S/N >10), for direct injections, was ≈200–400 ng/g of each, inadequate for regulatory requirements. Solid phase extraction (SPE) attempted to pre-concentrate samples spiked with sulfamethazine by ≈20:1. From water, the limit of detection (LOD) was ≈2.7 ng/mL with LOQ ≈9 ng/mL using UV at 260 nm. The LOD for milk was 6.2 ng/mL, and LOQ was 20.1 ng/mL. A better pre-concentration step or a more sensitive detector such as MS–MS is required. Even with these inadequacies, SFC was shown to be a feasible, faster, “greener” alternative to HPLC for the separation of these drugs.

     

Two Minute Separation of the cis- and trans-Isomers of Vitamin K1 without Heptane, Chlorinated Solvents, or Acetonitrile

Resolution of the cis-, trans-isomers of vitamin K₁ (phylloquinone) is important in nutritional analysis since only the trans-isomer is biologically active, while artificial vitamin K₁ may contain significant amounts of the inactive cis-isomer. The cis-, trans-isomers of vitamin K₁ were separated, with a resolution of 3.4, using pure carbon dioxide (CO₂) as the mobile phase, with a run time ≈6 min, on a 4.6 × 150 mm, 3.5 μm RX-Sil bare silica column, using supercritical fluid chromatography (SFC). With 2.5 % ethyl acetate in CO₂, R_s = 2.77, with a run time <4 min. On a 3 × 100 mm, 1.8 μm RX-Sil column R_s remained at 2.69, with a run time just over 2 min. This is the first report on the apparent separation of these isomers by SFC, and is up to 3–5 times faster than separations using high performance liquid chromatography (HPLC) in the literature. Unlike most HPLC separations, no dichloromethane, acetonitrile, or hexane/heptane was used. The CO₂ was beverage grade, at 1/20th to 1/50th the cost of the replaced organics, is recycled, and is considered “green”.     

Supercritical fluid chromatographic solvatochromic modifier polarity studies

To understand the chromatographic process as a whole, whether it be for gas chromatography (GC), liquid chromatography (LC), or supercritical fluid chromatography (SFC), one needs to know the chemical and physical nature of the mobile and stationary phases and also the interactions that take place between analytes (solutes) and the two phases. An approach towards Investigating the ways that stationary and mobile phases contribute to chromatographic retention Involves exploring the effects of solvent polarity on the strength of the mobile phase. In SFC this could involve determining the polarity of several different modifier/carbon dioxide mobile phases. In this paper, the use of a solvatochromic indicator to learn more about the effects of SFC modifier/mobile phase polarity will be investigated and discussed using several different modifiers and a diolmodified silica column.     

Separation of Chlorophylls and Their Degradation Products using Packed Column Supercritical Fluid Chromatography (SFC)

Complex mixtures of chlorophyll degradation products may arise during processing and storage of vegetable oil and green plant materials like broccoli and spinach. Determination of these compounds is important in the area of food chemistry. Therefore a method using packed column supercritical fluid chromatography (SFC) has been developed. The method comprises chromatography using a simple gradient of methanol in carbon dioxide at constant column back pressure of 30 MPa and a column temperature of 40°C. Effects of pressure and mobile phase composition showed the importance of applying a modifier gradient for optimal separation of the chlorophyll products. The method permits separation of 15 chlorophyll derivatives including chlorophyll a and b, pheophytins, and pyropheophytins on a C₁₈ column in about 20 minutes. Identifications of the individual peaks were based on reference compounds, the retention order of the compounds, and their absorption spectra.     

Separation and purification of six components from the roots of Rheum officinale Baill. by supercritical fluid chromatography

The dried roots of Rheum officinale Baill., named Dahuang in Chinese, has many pharmacological effects and has been widely used for the treatment of many diseases. To develop a harmless and eco-friendly method for the separation of components in Dahuang is of great interest for quality control and pharmacological study of Dahuang. A method for separation and purification of components in Dahuang using supercritical fluid chromatography (SFC) is established in this work. Samples were prepared by extraction of Dahuang powders with 20% H₂SO₄ and benzene under reflux. The extracts were obtained by evaporating benzene extracts and separating by SFC on YMC-Diol column using supercritical CO₂ with CH₃OH 4% (v/v) as the modifier. The flow rate of the mobile phase was 15?mL/min, the column pressure was 13?MPa, and the column temperature was 318?K. Cinnamic acid and five kinds of hydroxyanthraquinones including rhein, emodin, aloe emodin, chrysophanol, and physcion were obtained by SFC. The purities of the obtained compounds were all above 97% as determined by high performance liquid chromatography and their chemical structures were identified by nuclear magnetic resonance and mass spectrum. The thermodynamics of chromatographic process was also studied and it revealed that the SFC separation process of these compounds on YMC-Diol column was controlled by enthalpy.     

超临界流体色谱对吴茱萸中吲哚类生物碱的快速分析

建立了超临界流体色谱快速分析吴茱萸中吲哚类生物碱的方法。以标准品混合物和复杂样品为对象比较4种色谱柱的分离效果,进行色谱柱的筛选;考察了进样体积、改性剂、添加剂、温度和背压对保留行为的影响。结果表明,进样体积对峰形影响显著;添加剂对保留时间和色谱峰形影响有限;改变改性剂能使保留时间显著改变;降低温度,升高背压,保留时间减小。经过优化,确定采用Waters ACQUITY UPC² BEH色谱柱,以甲醇为改性剂,在35 ℃柱温和2.07×10⁷ Pa背压条件下,15 min内完成复杂样品的分析。同时采用超高效液相色谱完成复杂样品的快速分析。结果表明,超临界流体色谱可用于天然产物的高效快速分析,同时该方法与超高效液相色谱在分离选择上的差异有助于天然产物分析方法的拓展。     

Validated LC Method for Determination of Enantiomeric Purity of Apremilast Using Polysaccharide-Type Stationary Phases in Polar Organic Mode

Enantioseparation of an anti-psoriatic agent, apremilast (APR), was performed by HPLC using polysaccharide-type chiral stationary phases in polar organic mode for the first time. The separation capability of six different chiral columns (Chiralpak AD, Chiralpak IA, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) was investigated using neat MeOH and ACN. During the preliminary experiments the best results were obtained on Chiralpak IA column with ACN (R_s?=?5.4). The effects of binary mobile phases on the resolutions and retention factors were also investigated containing different percentages of MeOH:ACN. U-shaped retention pattern was obtained when plotting the retention factors of the APR enantiomers versus the MeOH content of the binary mobile phases on Chiralpak IA column. For further method optimizations an L25 orthogonal array table was employed altering the concentration of MeOH in ACN, column temperature, and flow rate. The best result was achieved on Chiralpak IA column with 80/20 (v/v%) MeOH/ACN with 0.7 mL min^?1 flow rate at 25 °C (R_s?=?5.4, t₂?=?7.45 min). Thermodynamic analysis revealed an enthalpy-driven enantioseparation. The developed HPLC method was validated according to the ICH guideline Q2(R1) and proved to be reliable, linear, precise and accurate for the determination of 0.1% R-enantiomer as chiral impurity in S-APR as well as quantification of the S-enantiomer.

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Direct determination of tocopherols in paprika and paprika oleoresin by liquid chromatography

A method for the determination of tocopherols and tocopherol esters, quantified as tocopherol acetate, in paprika and paprika oleoresin is reported. Paprika samples were extracted with ethyl acetate and aliquots of the extracts were directly injected into a liquid chromatograph. Reverse phase high-performance liquid chromatography with spectrophotometric detection at 280 nm was used. Gradient elution was applied, allowing the determination of tocopherols and tocopherol esters in the presence of carotenoids. The method does not need previous separation steps, so is useful for the routine determination of vitamin E in commercial samples.     

Quantitative LC Analysis of Cyclosporine A in Ocular Samples

An isocratic reversed-phase HPLC method with ultraviolet detection at 205 nm has been developed for analysis of cyclosporine A (CyA) in rabbit ocular samples. Neither internal standard nor extraction was needed for sample preparation. Acetonitrile (ACN; 1 mL) was added to 250 μL aqueous and vitreous samples to precipitate proteins. The supernatant was dried and the residue was reconstituted in mobile phase and injected for HPLC analysis. Chromatography was performed on an octadecyl silane-A (ODSA) C₁₈ (4.6 × 250 mm, 5 μm) column. The column temperature was fixed at 70 °C and the mobile phase was ACN 65%, methanol 20% and water 15% at a flow rate of 1.5 mL min^?1. The calibration curve for CyA in rabbit ocular samples was linear over the concentration range 0.2 and 10 μg mL^?1 with a correlation coefficient of 0.9992. Intra-day and inter-day precision were 4.61–7.83% and 5.27–10.70%, respectively. Intra-day and inter-day accuracy were 89.2–108% and 83.4–111%, respectively. The limits of detection (LOD) and quantification (LOQ) were 5.7 and 38 ng mL^?1, respectively. The method was successfully used for analysis of CyA in real aqueous and vitreous humor samples from New Zealand albino rabbits. The method is therefore suitable for analysis of CyA in ocular samples.     

Fast cleanup method for the analysis of Sudan I-IV and para red in various foods and paprika color (oleoresin) by high-performance liquid chromatography/diode array detection: focus on removal of fat and oil as fatty acid methyl esters prepared by transesterification of acylglycerols

A fast and effective cleanup method was developed for the analysis of Sudan I, II, III, IV, and Para Red (Sudan dyes) in various foods and paprika color (oleoresin) by high-performance liquid chromatography (LC) with a diode array detector (DAD). Removal of fat or oil in fatty sample was a critical point for reducing the volume of the final sample solution in order to obtain a sufficient level of the analytes. Separation of fat or oil from the dyes with a silica gel solid-phase extraction (SPE) column seemed unfeasible, because elution profiles of oil, fat, and the dyes were similar. Finally, fat and oil were separated from the dyes by elution from the SPE column with n-hexane, not as intact compounds but as fatty acid methyl esters prepared by direct transesterification of acylglycerols in fat and oil, leaving the dyes on the column. The dyes were eluted with n-hexane-diethyl ether (9 + 1). Gradient elution with water and tetrahydrofuran was used for separation on a C18 column by LC. Measurement of spectral of 0.5 microg/g of Sudan dyes in foods and 1 microg/g in paprika color (oleoresin) with the DAD was achieved.     

反相液相制备色谱法结合超临界流体制备色谱法分离纯化海风藤中的化合物

建立了基于反相液相制备色谱和超临界流体制备色谱的组合方法,用于分离纯化醇提水沉后石油醚层中的海风藤。首先以甲醇作为改性剂,采用醇提水沉法去除海风藤甲醇提取物中的叶绿素,加入硅藻土后用石油醚回流富集目标成分。选用反相C18制备色谱柱将其分为18个组分,然后将组分在SFC模式下进行制备。选用酰胺色谱柱,以甲醇为改性剂,在柱温30℃、背压15.0 MPa的条件下进行分离。基于反相色谱和超临界流体色谱不同的分离选择性,最后分离得到6个高纯度化合物。该法展示了反相制备色谱和超临界流体制备色谱在海风藤分离纯化方面的优势,特别是超临界流体色谱在天然产物的分析和制备方面的巨大潜力。