Search

A green approach of using supercritical CO 2 in a silica-packed column via adsorption and desorption steps has been developed to enrich β-carotene in the 10% β-carotene concentrate made from crude palm oil. In the adsorption stage, the yield of the β-carotene poor fraction with the content of 3% was 82.2% when the pressure, temperature, CO 2 flow rate and the time were 25 MPa, 60 ℃, 3 kg/h, and 180 min, respectively. The rich β-carotene fraction adsorbed in the column was thoroughly extracted out by supercritical CO 2 when the pressure, temperature, flow rate and time were 35 MPa, 70 ℃, 3 kg/h, and 120 min, respectively, in which the β-carotene content reached 27% and the yield reached 22.3%. This two-step process is an efficient way to prepare natural β-carotene with high content and has potential applications in the concentration of value-added components. [ABSTRACT FROM AUTHOR]

It is very common in India that with low priced cow milk adulterated high priced buffalo milk. Various techniques are available in market to identify the origin of milk but all these techniques have their own limitations. Thus, DNA based technologies are preferred now a days to identify the origin of food products from animal sources due to high sensitivity and specificity. Currently a PCR based method was developed to identify cow milk in raw buffalo milk. DNA was isolated from milk by DNeasy Mericon food kit (Quiagen,USA) which resulted in DNA of requisite quality for downstream applications. A bovine specific primer targeting D-loop (displacement) of mt- DNA (mitochondrial) was selected and standardized to amplify cow DNA. Specificity of primer was tested across the species in the genomic DNA isolated from both milk and blood. The protocol can be sensitive to detect upto 5% level of cow milk in the buffalo milk. Designed protocol was efficient, robust and sensitive and could be used as a platform test in routine quality analysis laboratory. Hence, it could be a great alternative for other protein based methods to identify cow milk in buffalo milk. [ABSTRACT FROM AUTHOR]

Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds. The method was successfully used to extract high quality RNA from citrus ( Citrus aurantifolia ), grapevine ( Vitis vinifera ), peach ( Prunus persica ), pear ( Pyrus spp. ), taro ( Colocosia esculenta ) and tobacco ( Nicotiana benthamiana ) samples. The method was comparable to conventional CTAB method in RNA isolation efficiency, but it was more sample-adaptable and cost-effective than commercial kits. High quality RNA isolated using silica spin column-based method was successfully used for the RT-PCR and/or multiplex RT-PCR amplification of woody fruit tree viruses and a viroid. The study provided a useful tool for the detection and characterization of plant viruses. [ABSTRACT FROM AUTHOR]

A simple, precise and fast Gas Chromatographic method has been developed first time for the simultaneous determination of Methyl Salicylate, Menthol, Thymol and Camphor in an ointment formulation. Chromatographic separations of the four drugs were performed on fused silica column of length 30 meters, diameter 0.53mm with ethylene glycol film thickness 1μm as stationary phase. Nitrogen was flown through the column as carrier gas at a flow rate of 5.0mL/min. The initial oven temperature was 120°C (5 min) and raised at the rate of 15°C per minute to final temperature 200°C(3min.). Detector and injection temperatures were fixed at 270°C and 230°C respectively. The injection volume was 1 μl with run time 20 minutes. The linearity of Methyl Salicylate, Menthol, Thymol and Camphor were in the range of 0.1-0.15 mg/ml, 0.1-0.15 mg/ml, 0.02-0.03 mg/ml and 0.2-0.3 mg/ml respectively. The recovery was calculated by standard addition method and the average recovery was found to be 100.629%, 100.417%, 100.086% and 100.045% for Methyl Salicylate, Menthol, Thymol and Camphor respectively. The proposed method is simple, accurate, precise and rapid for the simultaneous estimation of these four actives. [ABSTRACT FROM AUTHOR]

(-)-Epigallocatechin gallate (EGCg)-rich extract (EGCg >700 mg g-1) was prepared from green tea leaves through a three-stage process consisting of liquid-liquid extraction and silica column purification. Crude tea extract was dissolved in ethyl acetate. After filtration, the solution was extracted by 10 gL-1 citric acid solution twice, and then passed through silica column. The catechins compounds in the ethyl acetate eluate were back extracted to the aqueous phase, then extracted with a mixed solution of n-hexane/ethyl acetate (2/5, v/v) 3 times, concentrated, and freeze dried. 12.8 g EGCg-rich extract containing 709 mg g-1 EGCg and 965 mg g-1 total catechins was obtained from 300 g green tea leaves, with an EGCg recovery of 26.1% and a yield of 4.3%. This method was suitable for bulk preparation of EGCg-rich catechins from green tea leaves. [ABSTRACT FROM AUTHOR]

In the present study, a simple, sensitive, and rapid hydrophilic interaction chromatographic (HILIC) method with ultraviolet (UV) detection for the analysis of oxytetracycline (OTC) was developed and applied to the determination of the antibiotic in environmental waters. OTC was extracted and purified from water samples by solid-phase extraction (SPE) employing Oasis HLB cartridge and analyzed on a Kromasil silica column with the mobile phase of acetonitrile-sodium oxalate (10 mM, pH 2.5) (90:10, v/v). The analyte was detected at 365 nm, and the total analysis time was ∼7 min. Method validation was accomplished by examination of the linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). More importantly, in a linear range of 0.15-10 mg L-1, OTC had high repeatability with good peak shape and no irreversible adsorption was found. The results of this study demonstrated that bare silica column could be used in the determination of tetracycline antibiotics in HILIC mode with no peak tailing, providing a different understanding from that previously found in literature. [ABSTRACT FROM AUTHOR]

Abstract: Different RP-HPLC columns (phenyl, conventional ODS, cross-linked C18 and special end-capped C8 and C18 phases) were used to investigate the separation of four basic ionizable isomers. Using ACN/20mM NH4OAc aq., a separation was observed exclusively on RP columns with higher silanol activity at unusual high ACN concentration, indicating cation-exchange as main retention mechanism. Using MeOH/20mM NH4OAc aq., another separation at low MeOH concentrations was observed on both, RP columns with higher as well as RP columns with lower silanol activity, which is mainly based on hydrophobic interactions. The isomers were also separated on a bare silica column at higher MeOH content using NH4OAc. Since cation-exchange governs this retention, the elution order was different compared to the RP phases. A strong retention on the silica column was observed in ACN, which could be attributed to partition processes as additional retention mechanism. [ABSTRACT FROM AUTHOR]

Five column experiments have been carried out to investigate the effect of flow rate on the breakthrough curves (BTCs) of phosphate, fulvic acid, and uranium(VI) onto a silica column. Both BTCs of phosphate and fulvic acid, and three BTCs of uranium(VI) in the presence and absence of phosphate or fulvic acid at high flow rate published in the previous paper [] were compared with corresponding initial parts of BTCs at low flow rate in this paper. Each BTC in this paper was expressed as both C/Co–t and C/Co–V/Vo plots, where C and Co are the concentrations in the influent and the effluent respectively, t and V are the time and the effluent volume from the start of injection of pulse solution respectively, Vo is the pore volume of the SiO2 column. Based on the experimental results and the relationship among V, t, and flow rate F, it was found that there are advantages to using C/Co–V/Vo plot as BTC to study the effect of flow rate. Based on these comparisons of C/Co–V/Vo plots at different flow rates and the theoretical analysis from the Bohart–Adams sorption model, it was found that the right shift (increase in V/Vo of breakthrough), the left shift (decrease in V/Vo of breakthrough), and the non-shift (non-change in V/Vo of breakthrough) of initial parts of BTCs with increasing flow rate are certain to occur instead of only left shift and that three different trends of shifts can be mainly attributed to different rate-controlling mechanisms of sorption process. [ABSTRACT FROM AUTHOR]

Abstract: An LC–MS/MS method for the determination of isoniazid in human plasma was developed and validated. Human plasma aliquots of 100μL were used for analysis. The assay used nialamide as the internal standard. The calibration curve concentration range was 50–10,000ng/mL. Sample preparation utilized protein precipitation, and the supernatant was directly injected onto silica column without reconstitution. The recovery was over 90% and matrix effect was negligible. The method is simple and fast, which is advantageous in respect to instability of isoniazid in human plasma and loss on reconstitution due to its low molecular weight. [Copyright &y& Elsevier]

Abstract: The possible presence of a mobile phase layer rich in water on the surface of silica columns used under conditions typical in hydrophilic interaction chromatography was investigated by the injection of a small hydrophobic solute (benzene) using acetonitrile–water mobile phases of high organic content. Benzene does not partition into this layer and is thus partially excluded from the pores of the phase up to a water content of about 30%, after which hydrophobic retention of the solute on siloxane bonds is observed. In 100% acetonitrile, the retention volume of benzene was smaller than that estimated either by pycnometry or by calculation from the basic physical parameters of the column. This result might be attributable to the larger size of the benzene molecule: the elution volume of a molecule is the pore volume minus a surface layer half the diameter of the analyte molecule. However, some influence of strongly adsorbed water that remains on the surface of the phase even after extensive purging with dry acetonitrile cannot be entirely discounted. The results suggest that about 4–13% of the pore volume of a silica phase is occupied by a water-rich layer when using acetonitrile–water containing 95–70% (v/v) acetonitrile. [Copyright &y& Elsevier]

The retention behaviors and efficiencies of the chromatographic separation of dextromethorphan (DM) and its metabolites (dextrorphan (DX), 3-methoxymorphinan (MM), and 3-hydroxymorphinan (HM)), on three different silica columns (Inertsil, μ-Porasil, and Lichrospher), were compared using a simple aqueous mobile phase consisting of an organic solvent (methanol or acetonitrile) and water at different volume ratios (1:9 to 9:1) containing triethylamine (TEA) and acetic acid (ACH). Results demonstrated that the retention capacities for basic compounds with the same ionization conditions of the silanol group were the largest for the Lichrospher column, followed by the μ-Porasil one, with the Inertsil column exhibiting the lowest level. Based on their physical characteristics, the larger retention capacities for DM and its three metabolites in the LiChrospher column compared to those of the Inertsil and μ-Porasil columns may have been because the LiChrospher column has the largest surface area for interaction. However, since the surface areas of the Inertsil and μ-Porasil columns are similar, the greater retention capacities for DM and its three metabolites in the μ-Porasil column can probably be attributed to the existence of a greater number of silanol groups than in the Inersil column. This also demonstrates that the interaction of tertiary amines with ionized silanol groups is greater than that of secondary amines resulting in the elution order being MM>DM and HM>DX. However, O-demethylation to expose metabolites with a phenolic hydroxy group seemed to decrease the retention capacity, thus yielding the elution order of DX>DM (tertiary amine) and HM>MM (secondary amine). This might be attributable to the negative charge repulsion between phenoxyl groups (PhO-) and ionized silanol groups (SiO-) decreasing the affinity of basic compounds. [ABSTRACT FROM AUTHOR]

Abstract: The separation of acidic, neutral and particularly basic solutes was investigated using a bare silica column, mostly under hydrophilic interaction chromatography (HILIC) conditions with water concentrations >2.5% and with >70% acetonitrile (ACN). Profound changes in selectivity could be obtained by judicious selection of the buffer and its pH. Acidic solutes had low retention or showed exclusion in ammonium formate buffers, but were strongly retained when using trifluoroacetic acid (TFA) buffers, possibly due to suppression of repulsion of the solute anions from ionised silanol groups at the low of TFA solutions of aqueous ACN. At high buffer pH, the ionisation of weak bases was suppressed, reducing ionic (and possibly hydrophilic retention) leading to further opportunities for manipulation of selectivity. Peak shapes of basic solutes were excellent in ammonium formate buffers, and overloading effects, which are a major problem for charged bases in RPLC, were relatively insignificant in analytical separations using this buffer. HILIC separations were ideal for fast analysis of ionised bases, due to the low viscosity of mobile phases with high ACN content, and the favourable Van Deemter curves which resulted from higher solute diffusivities. [Copyright &y& Elsevier]

Abstract: Maillard reaction of glucose with amino acids and peptides has become a very important experimental model in the food flavor and pharmaceutical industries for better understanding the mechanism of food flavor generation and drug stability. Because of the amino acid and sugar functional groups present in their structures, most of the reaction components formed during the initial stages of Maillard reaction as well as the substrates are relatively polar. These compounds are poorly retained on a conventional reversed phase column. While polar stationary phases like HILIC column do provide better retention for these polar components, method selectivity could still be a challenge due to the structural similarity between these analytes. In this report, parameters such as pH, mobile phase composition and temperature were investigated using different brands of bare silica columns in order to separate glycine (G), diglycine (DG), triglycine (TG), and the corresponding Amadori compounds of glucose–glycine (GG), glucose–diglycine (GDG) and glucose–triglycine (GTG). An excellent separation for glycine, glycine peptides and their Amadori compounds was obtained on a bare silica column at an elevated temperature. [Copyright &y& Elsevier]

Abstract: Trifluoroacetic acid (TFA) is a commonly used additive in HPLC and LC–MS analysis of basic compounds. It is also routinely added to aqueous–organic mobile phases utilized in the hydrophilic interaction chromatography–electrospray tandem mass spectrometry (HILIC–ESI/MS/MS) technique used in our laboratories for bioanalysis. However, TFA is known to suppress the ESI signals of analytes due to its ability to form gas-phase ion pairs with positively-charged analyte ions. The most common method to overcome this problem involves the post-column addition of a mixture of propionic acid and isopropanol. However the post-column addition setup requires additional pumps and is not desirable for continuous analysis of large amounts of samples. In this paper we present a simple yet very effective means of minimizing the negative effect of TFA in bioanalysis by direct addition of 0.5% acetic acid or 1% propionic acid to mobile phases containing either 0.025 or 0.05% TFA. A factor of two- to five-fold signal enhancement was achieved for eight basic compounds studied. Furthermore, chromatography integrity was maintained even with the addition of acetic acid and propionic acid to existing TFA mobile phases. This method has been successfully applied to the HILIC–ESI/MS/MS high-throughput analysis of extracted biological samples to support pre-clinical and clinical studies. [Copyright &y& Elsevier]

Abstract: The influence of the average column pressure (ACP) on the elution volume of thiourea was measured on two RPLC columns, packed with Resolve-C18 (surface coverage 2.45  mol/m2) and Symmetry-C18 (surface coverage 3.18  mol/m2), and it was compared to that measured under the same conditions on an underivatized silica (Resolve). Five different methanol–water mixtures (20, 40, 60, 80 and 100% methanol, v/v) were used. Once corrected for the compressibility of the mobile phase, the data show that the elution volume of thiourea increases between 3 and 7% on the C18-bonded columns when the ACP increases from 50 to 350 bar, depending on the methanol content of the eluent. No such increase is observed on the underivatized Resolve silica column. This increase is too large to be ascribed to the compressibility of the stationary phase (silica + C18 bonded chains) which accounts for less than 5% of the variation of the retention factor. It is shown that the reason for this effect is of thermodynamic origin, the difference between the partial molar volume of the solute in the stationary and the mobile phase, , controlling the retention volume of thiourea. While is nearly constant for all mobile phase compositions on Resolve silica (with  mL/mol), on RPLC phases, it significantly increases with increasing methanol content, particularly above 60% methanol. It varies between 5 mL/mol and 17 mL/mol on Resolve-C18 and between 9 mL/mol and 25 mL/mol on Symmetry-C18. The difference in surface coverage between these two RP-HPLC stationary phases increases the values of by about 5 mL/mol. [Copyright &y& Elsevier]

Abstract: In this work a monolithic silica column operated in the hydrophilic interaction chromatography (HILIC) mode in conjunction with an evaporative light scattering detector (ELSD) was investigated. Lithium, sodium and potassium were used as the test counter-ions for this evaluation. Chromatographic properties of this column operated in the HILIC mode were determined by varying key mobile phase parameters, such as pH, flow rate, buffer strength, acid and organic modifier. As organic content was increased from 60 to 90% acetonitrile, retention time increased on average by a factor of seven for the test cations listed above. Buffer concentration and pH were also observed to have an effect, although not as significant as the HILIC effect that was observed by changing organic content. Flow rates up to 5mL/min were utilized to perform counter-ion separations in less than 3min. After examining the changes in retention, resolution, and peak shape an optimized method was established and then further evaluated for linearity, reproducibility, and limit of detection (LOD) for sodium. Linearity was acceptable with an R 2 value of 0.999 across the working-standard range and a LOD of 0.1μg/mL was calculated. The reproducibility on the counter-ion determination from pharmaceutical sodium salts was 1.6% R.S.D. on average, and the accuracy of the counter-ion prediction was approximately 3% from theory when salt content was corrected for potency. [Copyright &y& Elsevier]

Abstract: The compressibilities of aqueous solutions of methanol or acetonitrile containing 0, 20, 40, 60, 80 and 100% (v/v) organic solvent were measured with a dynamic chromatographic method. The elution volumes of thiourea samples (2  L) in these solutions were measured at different average column pressures, adjusted by placing suitable capillary restrictors on-line, after the detector. The reproducibility of the measurements was better than 0.2%. In the range of average pressures studied (10–350 bar), the maximum change in elution volume of thiourea is 1.3% (in pure water) and 4.0% (in pure methanol). This difference is due to the different compressibilities of these pure solvents. For mixtures, the plots of the elution volume of thiourea versus the pressure are convex downward, which is inconsistent with the opposite curvature predicted by the classical Tait model of liquid compressibility. This difference is explained by the variation of the amount of thiourea adsorbed with the pressure. The deconvolution of the two effects, adsorption of thiourea and solvent compressibility, allows a fair and consistent determination of the compressibilities of the methanol–water mixtures. A column packed with non-porous silica particles was also used to determine the compressibility of methanol–water and acetonitrile–water mixtures. A negative deviation by respect to ideal behavior was observed. [Copyright &y& Elsevier]

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) was developed and validated for the simultaneous analysis of antihistamine drug loratadine (LOR) and its active metabolite descarboethoxy-loratadine (DCL) in human plasma. Deuterated analytes, i.e. LOR-d3 and DCL-d3 were used as the internal standards (I.S.). Analytes were extracted from alkalized human plasma by liquid/liquid extraction using hexane. The extract was evaporated to dryness under nitrogen, reconstituted with 0.1% (v/v) of trifluoroacetic acid (TFA) in acetonitrile, and injected onto a 50×3.0 mm I.D. 5 μm, silica column with an aqueous–organic mobile phase consisted of acetonitrile, water, and TFA (90:10:0.1, v/v/v). The chromatographic run time was 3.0 min per injection and flow rate was 0.5 ml/min. The retention time was 1.2 and 2.0 min for LOR and DCL, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor→product ion transitions: 383→337 (m/z) for LOR, 311→259 (m/z) for DCL, 388→342 (m/z) for LOR-d3, and 316→262 (m/z) for DCL-d3. The low limit of quantitation (LLOQ) was 10 pg/ml for LOR and 25 pg/ml for DCL. The inter-day precision of the quality control (QC) samples was 3.5–9.4% relative standard deviation (R.S.D.). The inter-day accuracy of the QC samples was 99.0–107.9% of the nominal values. [Copyright &y& Elsevier]