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In HPLC the mobile phase is pure or mixed solvents as well as solvents with solid modifiers. Chromatographers have a choice among hundreds of solvents for different application of HPLC. A particular selection is usually affected by solvent characteristics such as viscosity, refractive index, noncorrosiveness, toxicity, miscibility, transparency etc...
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Context 1
... seen in expression (1), a number of factors are included, and its use for the calculation of K i is limited. Some values for the elutropic strength ε 0 for solvents on alumina and C18 are given in Table 2. ...Context 2
... instance, acetonitrile is often the solvent of choice for reversed-phase HPLC because it has not only a lower viscosity than methanol or isopropanol but also has good retention characteristics. Viscosity values for several pure solvents are listed in Table 2. ...Context 3
... Refractive Index: When refractive index detector is being used, this is an important parameter for mobile phase selection. RI values of some pure solvents are listed in Table 2. ...Context 4
... all HPLC solvents are miscible. Several problems may result if immiscible solvents are mixed such as unstable baseline, fluctuating pressures, high pressure etc. Miscibility data of some solvents are given in Table 2 [3]. All pairs whose M numbers differ by 15 units or less are miscible in all ratios at 15 0 C. A difference of 17 or more corresponds to immiscibility. ...Citations
... Also, based on the adapted and modified organic solvents from Chen et al. [28], Sanches et al. [29], and Svedström et al. [30], the use of ACN with HOAc as the acid modifier was observed to provide adequate sensitivity and acceptable peak shape of the overall chromatographic results. Acetonitrile is typically selected as the organic component due to its low UV cutoff (190 nm) and lower viscosity which provide better detection performance and narrower chromatographic peaks, respectively [31]. The addition of an acid modifier in the mobile phase is usually done to reduce the degree of adsorption and tailing of basic compounds by keeping the residual silanol in the column packing to be in its undissociated state [32]. ...
Phenolic compounds are natural substances that exhibit different functional bioactivities and provide health-protective actions against chronic illnesses. The vast potential of these compounds in health and other sectors demands the establishment of analytical procedures for their immediate and simultaneous analysis. In this study, a high-performance liquid chromatography with diode-array detection (HPLC-DAD) method was developed and validated for the simultaneous analysis of gallic acid, catechin, epicatechin, rutin hydrate, caffeic acid, syringic acid, ellagic acid, p-coumaric acid, trans-ferulic acid, myricetin, resveratrol, and quercetin. The chromatographic separation of the selected polyphenols was carried out in a reversed-phase Inertsil ODS-3 column (250mm x 4.5mm x 5µm) at a flow rate of 0.8 mL/min, injection volume of 20 µL, and column temperature of 30°C. The detection and quantification of phenolic compounds were done at specific wavelengths (254, 275, 305, and 325 nm) using gradient elution for 40 minutes, with acidified water and acetonitrile solution as mobile phase. Validation of the established analytical procedure showed that the coefficient of determination (R2 > 0.99), limit of detection (0.01 to 0.35 µg/mL), limit of quantitation (0.03 to 1.07 µg/mL), recovery values (98.33 to 101.12%), and repeatability (RSD < 5%) respectively indicated a linear, sensitive, accurate, and precise analytical method for the simultaneous chromatographic analysis of the 12 phenolic compounds. Overall, the developed HPLC-DAD procedure can offer adequate confidence for the identification and quantification of specific polyphenols and can be modified or updated for future analysis of phenolic compounds in different plant extracts.
... There are some notable solvents in the HPLC system including acetone, acetonitrile, chloroform, dimethylformamide, dimethyl sulfoxide, hexane, isopropyl alcohol, methanol, propyl alcohol, tetrahydrofuran, toluene, trifluoroacetic acid and Water (Agrahari et al., 2013). ...
... ➢ Toxicity: Solvent should be free from any toxic elements. If the solvent is associated with any type of chemicals, it should be careful not to arise any toxicity (Agrahari et al., 2013). ...
This review project paper was undertaken to find out the possible solvent system used to analyze metronidazole assay by high-performance liquid chromatography (HPLC) method. This study was carried out with five individual solvent systems by analyzing five review papers. The proposed
solvent systems were ortho phosphoric acid (0.05M): acetonitrile, sodium acetate: acetonitrile:
glacial acetic acid (75:25:1, v/v/v), phosphate buffer and acetonitrile (90:10 % v/v), triethylamine:
acetonitrile: phosphoric acid (0.02:20:80 v/v/v), methanol: acetonitrile (96:4, v/v). The first two
solvent systems listed were used to determine metronidazole tablet as a single, and the remaining
were used to as a combined mixture. In the case of a single assay of metronidazole tablet, the
solvent system of ortho phosphoric acid: acetonitrile with flow rate 1ml/min was effective enough
to establish method validation parameters linearity, accuracy, precision, Limit of Detection (LOD),
Limit of Quantification (LOQ) and robustness. The chromatogram was found to be a well peak
area with a retention time of 5.35 minutes. Excellent linearity was found with an excellent
correlation coefficient of 0.9999. The method was also indicated as high accuracy and precision
was less than 2% within acceptable range followed by ICH guideline. LOD and LOQ values were
0.78 μg mL−1 and 2.37 μg mL−1 respectively. On the other hand, the methanol: acetonitrile
solvent system with a flow rate of 0.6 ml/min was comparatively better for the combination
mixture assay of metronidazole tablets. By using this solvent system, the percentage recovery of
metronidazole tablet was observed as 99.995 % with a retention time of 2.344 minutes. The
correlation coefficient, percent accuracy, and standard deviation of precision were found as 0.999,
100.10% and 0.507. It can be assumed that ortho phosphoric acid: acetonitrile (0.05M) and
methanol: acetonitrile (96:4) were the possible better solvent system to assay metronidazole table
in HPLC analytical technique.
... At higher pH 7.0, the main peak of gliclazide was merging with the peak of impurity F. As pH decreased to 4, there was a marked separation between the main peak of gliclazide and the peak of impurity F. Various pH combinations using potassium dihydrogen phosphate and ammonium acetate with acetonitrile were tried but impurity separation was found maximum at pH 4.0. Finally, the mobile phase selected was 65% ammonium acetate buffer of pH 4.0 + 35% mixture of 10% ammonium acetate buffer of pH 4.0 and 90% acetonitrile [20,21]. ...
Background
For the determination of gliclazide and its three potential impurities quantitatively, the development of a stability-indicating, accurate, simple, and fast, Ultra-Performance Liquid Chromatography (UPLC) method was done.
Results
On Acquity CSH 18 column (50 mm×2.1 mm, 1.7 μ) separation was achieved by the isocratic elution mode using mobile phase (5 mM ammonium acetate buffer of pH 4 and 10% ammonium acetate buffer + 90% acetonitrile, 65/35 v/v). In total, 0.7 mL ⁻¹ was the chosen flow rate and UV detection was carried out at 227 nm.
Conclusion
By analyzing forced degradation products of the sample, the stability-indicating characteristic of the developed method was proved where the separation of the products of degradation from analyte peak was seen along with spectral purity of gliclazide. Validation of the developed UPLC method was done as per the guidelines of the International Conference on Harmonization in terms of system suitability, precision, accuracy, specificity, sensitivity, linearity, and robustness.
An RP-HPLC method with a UV detector was developed for the simultaneous quantification of diclofenac diethylamine, methyl salicylate, and capsaicin in a pharmaceutical formulation and rabbit skin samples. The separation was achieved using a Thermo Scientific ACCLAIMTM 120 C18 column (Waltham, MA, USA, 4.6 mm × 150 mm, 5 µm). The optimized elution phase consisted of deionized water adjusted to pH = 3 using phosphoric acid mixed with acetonitrile in a 35:65% (v/v) ratio with isocratic elution. The flow rate was set at 0.7 mL/min, and the detection was performed at 205 nm and 25 °C. The method exhibits good linearity for capsaicin (0.05–70.0 µg/mL), methyl salicylate (0.05–100.0 µg/mL), and diclofenac diethylamine (0.05–100.0 µg/mL), with low LOD values (0.0249, 0.0271, and 0.0038 for capsaicin, methyl salicylate, and diclofenac diethylamine, respectively). The RSD% values were below 3.0%, indicating good precision. The overall greenness score of the method was 0.61, reflecting its environmentally friendly nature. The developed RP-HPLC method was successfully applied to analyze Omni Hot Gel® pharmaceutical formulation and rabbit skin permeation samples.
High Performance Thin Layer Chromatography (HPTLC) is the most potent and sophisticated type of Thin Layer Chromatography (TLC). It uses chromatographic layers with the highest levels of separation, efficiency and employs high-tech equipment for every step of the process, including accurate sample application, standardised reproducible chromatogram development, and software-controlled evaluation. HPTLC is a concept that incorporates both the use of established methodologies for qualitative and quantitative analysis and a widely standardised methodology founded on scientific facts. The resolution can be increased and more exact quantitative measurements, which satisfies all quality standards for today's analytical needs. Development of an analytical technique based on HPTLC and parameter validation in line with practical assessment. It complies with standards while reducing mistakes and inquiries. Quality Control and Quality Assurance of raw materials of Plant Origin can easily and effectively be done qualitative characterization and quantitative determination of mixtures of substances, Chemical Fingerprinting by High Performance Liquid Chromatography. This review article provides fundamental principles, guidance for proper validation practise, aids in selecting the best mobile phase, and clarifies the processes of the analytical process., protocol, separation, resolution, validation process, current advancements, changes made to TLC that led to HPTLC, optimization, process control, automation, and hyphenation.
Stability indicating a reverse-phase HPLC analytical method for the quantification of tamoxifen citrate (TMX) in the bulk and lipidic nano-vesicles (LNVs) was developed. The optimized method was validated according to the ICH Q2 (R1) guidelines by following a 3-factor interaction Box-Behnken design using Design-Expert® software. The responses measured at 236 nm were retention time (Rt), peak area, tailing factor (TF), and the number of theoretical plates (NTP). TMX was eluted best using the Luna® C18 LC Column along with a mobile phase of methanol (MeOH) and ammonium acetate buffer (AAB pH 4.5) 80:20 v/v mixture at 25±2 °C temperature. The currently developed method was linear in 100-5000ng/mL range with a detection limit of 4.55 ng/mL and a quantification limit of 13.78 ng/mL. The optimized method was utilized to evaluate the stability of TMX in different stress conditions by performing forced degradation studies. The results from the degradation study stipulated that on exposure to various stressors namely acid, alkali, oxidative, thermal, and UV light, the TMX did not show considerable degradation except for UV light exposure. Further, the method was successfully used for the quantification of TMX in lipidic nano-vesicles.
C. albicans causes human diseases, especially in immune-compromised patients. The current study aimed to identify Candida albicans using different techniques. Dimorphism and virulence behaviour were also studied. A Candida albicans strain was firstly identified by biochemical methods using VITEK 2 Compact automated technique and chromogenically using CHROMagar differential media that differentiate between Candida spp. Based on an enzymatic reaction. Molecular identification using ITS primers was also used to confirm Candida albicans identification. Accession number of the identified C. albicans was obtained as OK104215. The enhancement of dimorphism was studied using RPMI 1640 media (Roswell Park Memorial Institute Medium), while monitoring growth at different time intervals under microscope to investigate dimorphic changes. C. albicans showed its optimum dimorphism after 36-66 hours at 37°C. HPLC analysis for the enzyme product S-adenosylmethionine (SAM) was carried out at different time intervals. By increasing time, SAM production increased until optimum production reached after 72h of incubation on RPMI 1640. After that the production of SAM began to decrease.
Background: Chromatography is defined as a set of techniques that are used for the separation of constituents in a mixture. Introduction: High-Pressure Liquid Chromatography or High-Performance Liquid Chromatography (HPLC) is known as a specialized technique in which columns as well as liquid chromatography are used in the separation, characterizationand investigation of the active moieties existing in the mixture. Objective: Current review focuses on the HPLC technique, including its principles, instrumentation, types, applications, advancements, and patents. Result: HPLC technique is important both for quantitative as well as qualitative analysis and is used for the evaluation of biological and
In this study extraction of curcuminoids from turmeric was explored by various methods of extraction. For extraction soxhelet, ultrasonication and distillation methods were employed for separation of curcuminoids from turmeric powder under more or less varying same parameters. The results were analyzed and compared with reference method. The UV-Vis spectroscopy, HPLC and TLC were used for the confirmation and quantification of extract. The TLC analysis showed the three bands denoted the presence of three curcuminoids. The UV-Vis spectral analysis specified the absorption peak at 425nm. The HPLC studies implied the existence of three peaks for sonicated extract. The interpretation of the observation emphasized that the yield of extraction was higher for sonication method than remaining other extraction methods under varying parameters. Hence sonication method was considered as optimized extraction method which was time saving and less energy consuming favorable extraction method. The emphasized characteristics of improved extraction methods including cost-effectiveness (due to much saving in time and energy consumption) and environmentally benign nature make them more favourable extraction methods.
