Evaluation of RPLC stationary phases for tocopherol and tocotrienol positional isomer separation: Method development and profiling.

Reversed-phase separation of tocopherols (Ts) and tocotrienols (Ts) using C18 stationary phases results in the coelution of ß and γ positional isomers, leading to identification errors. This study investigates the potential of alternative stationary phase chemistries to effectively resolve tocochromanols, specifically focusing on the critical pair of ß and γ positional isomers. Initial screening of seven different stationary phases (C18, C18-PFP, C30, PFP, 5PYE, πNAP, and RP-Amide) was conducted. Linear solvent strength (LSS) studies were performed to assess the impact of the organic modifier (methanol) and temperature on the chromatographic performance parameters. Five columns were found to be suitable for the tocochromanol separation and two different chromatographical conditions per column were proposed. Elution order of tocochromanols was unique for 5PYE, πNAP and C30 columns in comparison to RP-Amide and PFP. Method development for the quantitative analysis of four tocopherol and four tocotrienol homologues was performed. The optimised method employed the RP-Amide (150 × 4.6 mm, 2.6 µm dp) superficially porous particle column, mobile phase of methanol:water of 92:8, v/v, with a flow rate of 1.0 mL/min, column oven temperature of 40 °C and fluorescence detection (λex 295 nm, λem 330 nm). The analysis run time was 10.5 min with 13.6 MPa back pressure. The method was validated and the obtained LOQs were found to be 1.30-3.13 µg/mL. The method developed was successfully applied for the determination of tocochromanols in twenty samples with unique tocochromanol profiles. Principal component analysis illustrated three distinct groups based on the tocochromanol profile.

Using HPLC with In-Column Derivatization to Authenticate Coffee Samples.

Coffee is one of the world's most popular beverages, with the global coffee capsule market worth over USD 4 billion and growing. The incidence of coffee fraud is estimated to be up to one in five coffees being contaminated with cheaper blends of coffee. Given the worsening extent of climate change, coffee crop yields are harder to maintain, while demand is increasing. The 2021 Brazil frost delaying or destroying many coffee crops is an example. Hence, the incidence of coffee fraud is expected to increase, and as the market becomes more complex, there needs to be faster, easier, and more robust means of real-time coffee authentication. In this study, we propose the use of novel approaches to postcolumn derivatization (termed herein as in-column derivatization) to visualize the antioxidant profiles of coffee samples, to be later used as indicators for authentication purposes. We propose three simple mathematical similarity metrics for the real-time identification of unknown coffee samples from a sample library. Using the CUPRAC assay, and these metrics, we demonstrate the capabilities of the technique to identify unknown coffee samples from within our library of thirty.

A simplified guide for charged aerosol detection of non-chromophoric compounds-Analytical method development and validation for the HPLC assay of aerosol particle size distribution for amikacin.

Amikacin, an aminoglycoside antibiotic lacking a UV chromophore, was developed into a drug product for delivery by inhalation. A robust method for amikacin assay analysis and aerosol particle size distribution (aPSD) determination, with comparable performance to the conventional UV detector was developed using a charged aerosol detector (CAD). The CAD approach involved more parameters for optimization than UV detection due to its sensitivity to trace impurities, non-linear response and narrow dynamic range of signal versus concentration. Through careful selection of the power transformation function value and evaporation temperature, a wider linear dynamic range, improved signal-to-noise ratio and high repeatability were obtained. The influences of mobile phase grade and glassware binding of amikacin during sample preparation were addressed. A weighed (1/X2) least square regression was used for the calibration curve. The limit of quantitation (LOQ) and limit of detection (LOD) for this method were determined to be 5µg/mL and 2µg/mL, respectively. The method was validated over a concentration range of 0.05-2mg/mL. The correlation coefficient for the peak area versus concentration was 1.00 and the y-intercept was 0.2%. The recovery accuracies of triplicate preparations at 0.05, 1.0, and 2.0mg/mL were in the range of 100-101%. The relative standard deviation (Srel) of six replicates at 1.0mg/mL was 1%, and Srel of five injections at the limit of quantitation was 4%. A robust HPLC-CAD method was developed and validated for the determination of the aPSD for amikacin. The CAD method development produced a simplified procedure with minimal variability in results during: routine operation, transfer from one instrument to another, and between different analysts.

Sterols and squalene in apricot (Prunus armeniaca L.) kernel oils: the variety as a key factor.

The profile of sterols and squalene content in oils recovered from the kernels of 15 apricot (Prunus armeniaca L.) varieties were investigated. Nine sterols (campesterol, ß-sitosterol, Δ5-avenasterol, 24-methylene-cycloartanol, cholesterol, gramisterol, Δ7-stigmasterol, Δ7-avenasterol and citrostadienol) were identified in apricot kernel oils. The ß-sitosterol was the predominant sterol in each cultivar and consisted of 76-86% of the total detected sterols. The content of total sterols and squalene were significantly affected by the variety and ranged between 215.7-973.6 and 12.6-43.9 mg/100 g of oil, respectively.

Impact of Species and Variety on Concentrations of Minor Lipophilic Bioactive Compounds in Oils Recovered from Plum Kernels.

The profile of bioactive compounds (carotenoids, tocopherols, tocotrienols, phytosterols, and squalene) in oils recovered from the kernels of 28 plum varieties of hexaploid species Prunus domestica L. and diploid plums Prunus cerasifera Ehrh. and their crossbreeds were studied. Oil yields in plum kernels of both P. cerasifera and P. domestica was in wide ranges of 22.6-53.1 and 24.2-46.9% (w/w) dw, respectively. The contents of total tocochromanols, carotenoids, phytosterols, and squalene was significantly affected by the variety and ranged between 70.7 and 208.7 mg/100 g of oil, between 0.41 and 3.07 mg/100 g of oil, between 297.2 and 1569.6 mg/100 g of oil, and between 25.7 and 80.4 mg/100 g of oil, respectively. Regardless of the cultivar, ß-sitosterol and γ-tocopherol were the main minor lipophilic compounds in plum kernel oils and constituted between 208.5 and 1258.7 mg/100 g of oil and between 60.5 and 182.0 mg/100 g of oil, respectively. Between the studied plum species, significant differences were recorded for δ-tocopherol (p = 0.007), 24-methylenecycloartanol (p = 0.038), and citrostadienol (p = 0.003), but they were insufficient for discrimination by PCA.

Lipophilic bioactive compounds in the oils recovered from cereal by-products.

BACKGROUND: The by-products of seven different cereal grains were investigated as a source of extractable oil, rich in lipophilic bioactive compounds. RESULTS: Oil yields (g kg(-1) DW) recovered from cereal by-products were as follows: 189 (rice bran) > 112 (wheat germ) > 74 (corn bran) > 58 (oat bran) > 41 (buckwheat bran) > 39 (spelt bran) > 33 (wheat bran) > 27 (rye bran). The main fatty acids identified in the studied oil samples were palmitic acid (11.39-17.23%), oleic acid (11.76-42.73%), linoleic acid (35.54-62.65%) and α-linolenic acid (1.05-9.46%). The range of total tocochromanols and phytosterols in the obtained oils was 0.369-3.763 and 1.19-35.24 g kg(-1) of oil, respectively. The oils recovered from buckwheat and corn bran, and wheat germ were dominated by tocopherols (99.9, 84.2 and 96.5%, respectively), whereas the oat, rice, rye, spelt, wheat bran oils were rich in tocotrienols (73.9, 79.6, 78.1, 90.6 and 73.8%, respectively). The campesterol and ß-sitosterol constituted 10.1-32.5 and 30.4-63.7%, respectively, of total phytosterols contents identified in all of the studied samples. CONCLUSION: The present study demonstrated that oils recovered from the cereal by-products are richer sources of bioactive compounds, compared with traditional oils. © 2015 Society of Chemical Industry.

Comparison of core-shell particles and sub-2µm fully porous particles for use as ultrafast second dimension columns in two-dimensional liquid chtomatography.

The peak capacity of small columns packed with 2.7µm core-shell particles and 1.8µm fully porous particles were compared at high temperatures using very steep (fast) gradient conditions and quite high linear velocities using the same instrument configuration as used to transfer first dimension effluent to the second dimension column as done in on-line comprehensive two-dimensional liquid chromatography. The experimental peak capacities of small columns (2.1mm×30mm) packed with both types of particles were measured with fast gradients (9s to 2min) at high temperature (95°C) using both the same flow rate (1.75mL/min) and then at different flow rates at the same pressure (400bar). Equal or slightly better peak capacities were achieved with the core-shell particle columns as compared to the fully porous particle columns at the same backpressure or the same flow rate. However, core-shell particles offer a real advantage over the smaller, fully porous particles because they can be operated at higher flow rates thus gradient mixer flush out and column reequilibration can be done in less time thereby allowing a greater fraction of the second dimension cycle time to be dedicated to the gradient time.

Tocochromanols composition in kernels recovered from different apricot varieties: RP-HPLC/FLD and RP-UPLC-ESI/MS(n) study.

Composition of tocochromanols in kernels recovered from 16 different apricot varieties (Prunus armeniaca L.) was studied. Three tocopherol (T) homologues, namely α, γ and δ, were quantified in all tested samples by an RP-HPLC/FLD method. The γ-T was the main tocopherol homologue identified in apricot kernels and constituted approximately 93% of total detected tocopherols. The RP-UPLC-ESI/MS(n) method detected trace amounts of two tocotrienol homologues α and γ in the apricot kernels. The concentration of individual tocopherol homologues in kernels of different apricots varieties, expressed in mg/100 g dwb, was in the following range: 1.38-4.41 (α-T), 42.48-73.27 (γ-T) and 0.77-2.09 (δ-T). Moreover, the ratio between individual tocopherol homologues α:γ:δ was nearly constant in all varieties and amounted to approximately 2:39:1.

Assessing the performance of curtain flow first generation silica monoliths.

Analytical scale active flow technology first generation silica monolithic columns kitted out in curtain flow mode of operation were studied for the first time. A series of tests were undertaken assessing the column efficiency, peak asymmetry and detection sensitivity. Two curtain flow columns were tested, one with a fixed outlet ratio of 10% through the central exit port, the other with 30%. Tests were carried out using a wide range in inlet flow segmentation ratios. The performance of the curtain flow columns were compared to a conventional monolithic column. The gain in theoretical plates achieved in the curtain flow mode of operation was as much as 130%, with almost Gaussian bands being obtained. Detection sensitivity increased by as much as 250% under optimal detection conditions. The permeability advantage of the monolithic structure together with the active flow technology makes it a priceless tool for high throughput, sensitive, low detection volume analyses.

Investigating retention characteristics of a mixed-mode stationary phase and the enhancement of monolith selectivity for high-performance liquid chromatography.

The synthesis and chromatographic behavior of an analytical size mixed-mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3-cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance π-type bonding within a single separation environment. Selectivity studies were undertaken using n-alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the π-type selectivities afforded by the two different ligands on the mixed-mode column.

Enhancing the separation performance of the first-generation silica monolith using active flow technology: parallel segmented flow mode of operation.

Active flow technology (AFT) columns are designed to minimise inefficient flow processes associated with the column wall and radial heterogeneity of the stationary phase bed. This study is the first to investigate AFT on an analytical scale 4.6mm internal diameter first-generation silica monolith. The performance was compared to a conventional first-generation silica monolith and it was observed that the AFT monolith had an increase in efficiency values that ranged from 15 to 111%; the trend demonstrating efficiency gains increasing as the volumetric flow to the detector was decreased, but with no loss in sensitivity.

Effect of parallel segmented flow chromatography on the height equivalent to a theoretical plate III--influence of the column length, particle diameter, and the molecular weight of the analyte on the efficiency gain.

The effects of column length on performance in segmented flow chromatography were tested. Column efficiencies were measured for 4.6mm I.D. 3, 5, 7.5 and 10 cm long columns packed with 3.0 µm Hypurity-C18 fully porous particles and of 4.6mm I.D. 5, 10, 15 and 25 cm long columns packed with 5 µm Hypersil GOLD C18 particles. For each column length and particle type, two different configurations were tested: (1) both the inlet and outlet column endfittings were standard and (2) the inlet endfitting was standard but the outlet endfitting allowed parallel segmentation of the exiting flow into a central and a peripheral coaxial region. The segmentation flow ratio was set at 45% (for 3 µm) and at 43% or 21% (for 5 µm). Four samples were used, naphthalene, toluene, butylbenzene, and insulin, which has a ten times smaller diffusion coefficient than the small molecules. The column performance for the low molecular weight compound is significantly improved at velocities above the optimum value when the outlet flow rate is segmented because longitudinal diffusion and mass transfer resistance of this compound in the stationary phase are negligible sources of band broadening at reduced linear velocities between 5 and 25. At high flow rate (4 mL/min), the long-range eddy dispersion terms are about 3.9, 3.2, 2.6, and 1.8h unit lower for the 3, 5, 7.5 and 10 cm long columns, respectively. The longer the column, the lower the efficiency improvement because the border effects are smaller. This result was not systematically observed for the columns packed with 5 µm particles because the transverse dispersion is larger. In contrast, the gain in column efficiency is marginal for insulin because the mass transfer mechanism of this compound is mostly controlled by the slow diffusivity of insulin across Hypurity-C18 particles.

Optimization of gradient reversed phase chromatographic peak capacity for low molecular weight solutes.

A general protocol for optimizing peak capacity for the separation of low molecular weight molecules under gradient elution conditions has not yet been developed. By studying the effects of gradient time, flow rate, temperature, final eluent composition, and column length on peak capacity, a protocol has been developed for the optimization of a separation of small molecules such as those seen in metabolomic studies. The strategy developed employs the Linear-Solvent-Strength Theory (LSS Theory) to predict retention, building on an approach for the optimization of the peak capacity of large molecules (peptides) in fixed column format separations.

Cyano bonded silica monolith--development of an in situ modification method for analytical scale columns.

This study investigates the synthesis and chromatographic behaviour of an analytical size cyanopropyl "cyano" bonded silica monolith. Surface modification was undertaken by treating a neat silica monolith with chloro(3-cyanopropyl)dimethyl silane in dry heptane over a two day period. The resulting monolith showed stability over the duration of the testing program that involved flushing the column with more than 2000 column volumes of mobile phase. Efficiency measurements before and after sylation verified that the integrity of the silica monolith itself was not affected by the modification process, the highest number of theoretical plates (N/m) using anisole was 81,650. A brief selectivity test was then undertaken to assess methylene selectivity and phenyl selectivity. Elemental analysis was used to determine the homogeneity of the carbon load throughout the monolithic bed, and was compared to two commercial C18 and one 'self' modified C18 silica monoliths. The development of the in situ modification is also discussed.

Pi-selective stationary phases: (I) Influence of the spacer chain length of phenyl type phases on the aromatic and methylene selectivity of aromatic compounds in reversed phase high performance liquid chromatography.

Phenyl type stationary phases of increasing spacer chain length (phenyl, methyl phenyl, ethyl phenyl, propyl phenyl and butyl phenyl, with 0-4 carbon atoms in the spacer chain, respectively) were synthesised and packed in house to determine the impact that the spacer chain length has on the retention process. Two trends in the aromatic selectivity, q(aromatic), were observed, depending on whether the number of carbon atoms in the spacer chain is even or odd. Linear log k' vs phi plots were obtained for each stationary phase and the S coefficient was determined from the gradient of these plots. For the phenyl type phases, the S vs n(c) plots of the retention factors of linear polycyclic aromatic hydrocarbons vs the number of rings exhibit a distinct discontinuity that between 3 and 4 rings, which increases with increasing spacer chain length for even phases but decreases for odd phases. Accordingly, we suggest that the retention factors depend differently on the number of carbon atoms in the spacer chain depending on whether this number is even or odd and that this effect is caused by different orientations of the aromatic ring relative to the silica surface.

Pi-selective stationary phases: (III) Influence of the propyl phenyl ligand density on the aromatic and methylene selectivity of aromatic compounds in reversed phase liquid chromatography.

The retention characteristics of phenyl type stationary phases for reversed phase high performance liquid chromatography are still largely unknown. This paper explores the retention process of these types of stationary phases by examining the retention behaviour of linear PAHs and n-alkylbenzenes on a series of propyl phenyl stationary phases that have changes in their ligand density (1.23, 1.31, 1.97, 2.50 micromol m(-2)). The aromatic and methylene selectivities increased with increasing ligand density until a point where a plateau was observed, overall the propyl phenyl phases had a higher degree of aromatic selectivity than methylene selectivity indicating that these columns are suitable for separations involving aromatic compounds. Also, retention characteristics relating to the size of the solute molecule were observed to be influenced by the ligand density. It is likely that the changing retention characteristics are caused by the different topologies of the stationary phases at different ligand densities. At high ligand densities, the partition coefficient became constant.

Phenyl-type and C1 stationary phases for environmentally friendlier chromatography.

C1 and phenyl-type stationary phases were assessed in terms of their environmental impact on separations using as test solutes polycyclic aromatic hydrocarbons. Methanol (MeOH) and acetonitrile (ACN) mobile-phase gradients were employed. These stationary phases were examined to determine if different physical and chemical properties possessed by these surfaces decreased the organic solvent consumption, and yet maintained peak capacity. The cumulative energy demand (CED) was used to gauge the environmental impact of the separations. The separation of the polycyclic aromatic hydrocarbon test mixture using current methodologies (i.e. a C18/ACN combination) had a CED of 1.13 MJ-eq, and a peak capacity of 27 peaks (resolving 7 of 12 peak pairs with R(s)>1). In comparison, a butyl phenyl stationary phase with a methanol mobile phase had a peak capacity of 26, but with a CED of 0.670 MJ-eq. Monolithic columns containing C18 and C1 phases were also tested. A monolithic C18 column with MeOH had the lowest CED at 0.675 MJ-eq, a peak capacity of 28 peaks and good resolving power (resolving ten peak pairs with R(s)>1), suggesting that this is a viable option with respect to reducing environmental impact for these types of analyses.