Mobile Phase Selection Research Articles

Protocol for Oligonucleotides Characterization Using Hydrophilic Interaction Chromatography.

Oligonucleotides (ONs) are an increasingly popular category of molecules in the pharmaceutical landscape, particularly attractive for the treatment of genetic and rare diseases. However, analyzing these molecules presents significant challenges, due to their highly hydrophilic nature, multiple negative charges, and the presence of closely related impurities resulting from the complex solid-phase synthesis process. Ion pairing reverse-phase liquid chromatography (IP-RPLC) is the preferred technique for ONs analysis but is not ideal for mass spectrometry (MS) coupling. Consequently, there is a growing interest in exploring alternative strategies with hydrophilic interaction chromatography (HILIC) emerging as one of the most promising options. As HILIC is not yet fully established for the analysis of ONs, we have prepared this protocol paper to facilitate entry into this field. It not only provides best practices, opportunities, and potential advantages but also caveats and other important considerations for using HILIC to characterize ONs. The paper addresses the selection of stationary and mobile phases, optimization of gradient conditions, MS coupling, and key aspects to consider when manipulating ON samples. We hope this protocol will help establish HILIC as a more universal solution for ONs analysis.

Separation of weak acids, neutral compounds, and permanent anions using sequential elution liquid chromatography with tandem columns

This paper discusses the development of an analytical method by an alternative separation approach, sequential elution liquid chromatography (SE-LC), to separate permanently charged ions (anions), weak acids, and neutral compounds using anion exchange and reversed-phase columns in tandem. SE-LC separates classes of compounds by group by employing two or more elution modes. Advantages to using SE-LC over conventional HPLC are a greater peak capacity and a reduced separation disorder. Importantly, the same HPLC as used for a conventional HPLC separation may be used to afford a successful SE-LC separation. Mobile phase selection and gradient optimization are integral for a successful SE-LC class separation of permanent anions, weak acids, and neutral compounds and will be discussed in detail in this paper. The most successful (best resolution and repeatability) SE-LC separation was achieved by applying isocratic elution at low pH to elute the weak acids, followed by an acetonitrile gradient to elute the neutral compounds, and last a sodium methanesulfonate gradient to elute the anionic compounds using a superficially porous C18 column coupled with a strong anion exchange (SAX) column. Repeatability (RSD) in the retention times and peak areas of the analytes was less than 0.25 % and 1.5 %, respectively.

Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility-aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N-piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho-, meta-, and para-methyl fentanyl), increased significantly for others (e.g., cis-isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho- and para-methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues.

Quality by Design Approach for the Stability Indicating Method Development and Validation of Selpercatinib Drug Formulation by using RP-HPLC

In method development quality design approach is useful for method goal identification evaluation, method selection and risk assessment. Selpercatinib is a drug used in the treatment of certain types of cancer. The method development includes selecting appropriate chromatographic conditions such as the mobile phase composition, column, and selection wave length. The aim is to achieve optimum separation and quantification of selpercatinib in the dosage form. Optimizing chromatographic conditions by using design expert software. The mobile phase methanol and 0.1% Acetic acid are used. The mobile phase range is 80:20 v/v. The range of this mobile phase is 80:20 .0.7 mL/min found flow rate. The retention time of selpercatinib is 3.20 minutes. The assay was found to be 99.01% for selpercatinib. Optimization was performed. Factorial design performed, i.e., flow rate and percentages of methanol. Analysis of variance confirmed that method parameters. UV detection at 220 nm. The International Council of Harmonization (ICH) guidelines ensure the accurate, precise and reliable results to validate the parameters is an acceptable range. Stress studies reveal that drugs were degraded acidic conditions, alkaline conditions, neutral conditions, oxidative conditions, and photolytic conditions. Hence the proposed method was stability, indicating using quality by design (QbD) approach all the method parameters were better understood. This systematic approach ensures a thorough understanding of the assay, facilitates process optimization, and enables effective control strategies to reduce variability and enhance product quality.

Simultaneous Analysis of Anti-Hypertensive Drugs by RP-HPLC: An Overview

High performance liquid chromatography (HPLC) is an important qualitative and quantitative technique, generally used for the estimation of pharmaceutical and biological samples. In now a day pharmaceutical market, many newer antihypertensive combination drugs are available to control hypertension as well as to keep society healthy and stress free. All presently available old drugs have frequent dosing produces various side effects. So, there is need to analyse such antihypertensive drugs. The aim of this review is to analysed such commonly used antihypertensive combination drugs by using reverse phase high performance liquid chromatography (RP-HPLC). Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase. The drugs like Amlodipine besylate and Valsartan, Irbesartan and Hydrochlorothiazide, Atenolol and Chlorthalidone, Hydrochlorothiazide and Candesartan, Ramipril and Amlodipine, Hydrochlorothiazide and Enalapril, Quinapril and Hydrochlorothiazide etc. About fifteen combination drugs are analysed by using RP-HPLC. This review assists in appropriate selection of column, mobile phase, pH, flow rate, detector and form of such combination drugs.

Application of molecular simulation in mobile phase development of HPLC

The selection of mobile phase is significant for the separation result of HPLC, for it may have influence on the quantitative analysis. During HPLC analysis, engineers usually use (water-acetonitrile) mobile phase system to separate aldehydes and ketones, however, the existence of acetone will affect the separation effect of other substances. The paper researches one new (water-acetonitrile-tetrahydrofuran) mobile phase system to separate aldehydes and ketones. By analyzing the standard curves and test results of unknown samples, the new mobile phase can meet the analyzing needs, also it can eliminate the influence of acetone in the analysis process. What’s more, the paper uses molecular simulation[1–3] (MS) to calculate the interactions between the mobile phase and the aldehydes and ketones, mean square displacement value (MSD) and radial distribution function value (RDF), and the separation phenomenon can be well explained. In this paper, MS technology is first proposed for the development of new mobile phase, which can predict new unknown mobile phase, improve the efficiency of method development, also it can benefit the accurate qualitative analysis and prediction of peak out behavior of unknown substances.

HSPiP, Computational Modeling, and QbD-Assisted Optimized Method Validation of 5-Fluorouracil for Transdermal Products.

This study addressed the simplest and most efficient HPLC (high-performance liquid chromatography) method for the estimation of 5-fluorouracil (5-FU) from rat blood plasma by implementing the Hansen solubility parameters (HSP), computation prediction program, and QbD (quality by design) tool. The mobile phase selection was based on the HSP predictions and experimental data. The Taguchi model identified seven variables (preoptimization) to screen two factors (mobile phase ratio as A and column temperature as B) at three levels as input parameters in "CCD (central composite design)" optimization (retention time as Y1 and peak area as Y2). The stability study (freeze-thaw cycle and short- and long-term stability) was conducted in the rat plasma. Results showed that HSPiP-based HSP values and computational model-based predictions were well simulated with the experimental solubility data. Acetonitrile (ACN) was relatively suitable over methanol as evidenced by the experimental solubility value, HSP predicted parameters (δh of 5-FU - δh of ACN = 8.3-8.3 = 0 as high interactive solvent whereas δh of 5-FU - δh of methanol = 8.3-21.7 = -13.4), and instrumental conditions. CCD-based dependent variables (Y1 and Y2) exhibited the best fit of the model as evidenced by a high value of combined desirability (0.978). The most robust method was adopted at A = 96:4 and B = 40 °C to get earlier Y1 and high Y2 as evidenced by high desirability (D) = 0.978 (quadratic model with p < 0.0023). The estimated values of LLOD and LLOQ were found to be 0.11 and 0.36 μg/mL, respectively with an accuracy range of 94.4-98.7%. Thus, the adopted method was the most robust, reliable, and reproducible methodology for pharmacokinetic parameters after the transdermal application of formulations in the rat.

Hplc Method Development and Validation for the Simultaneous Estimation of Pitavastatin and Telmisartan

Method validation of both the compounds was done on the basis of ICH guidelines. Validation by HPLC method the wavelength was selected at the isobestic point at which the two drugs can be detected using UV detectors. The selected wavelength was 250nm. Optimized liquid chromatographic condition was obtained by performing many trials for the selection of mobile phase and column, for the separation of Pitavastatin &amp; Telmisartan. The method development was conducted with C18, 250 × 4.6 mm, 5µm particle size, Phenomenex with the flow rate of 1.0mL/min. the optimized mobile phase conditions were Acetonitrile and 10mM Ammonium acetate buffer containing 0.1% formic acid in the ratio of 65:35 v/v. Data of Simultaneous shows retention time for Pitavastatin 5.408 &amp; Telmisartan was 7.183. The method found to be linear, accurate, rugged and robust for validated parameters. The linearity range was determined by external standard calibration method in the concentration range of 10μg/ml to 60μg/ml. The amount of recovery was calculated as 98% – 101% and it was observed that all the values are within the limits. Further the precision of the method was confirmed by the repeatable analysis of sample. The results were found to be precise due to low values of the %RSD. It indicated that the method has good precision. Limit of quantification for Pitavastatin &amp; for Telmisartan 1.554μg/ml and 4.709μg/ml respectively. Similarly limit of detection for Pitavastatin &amp; for Telmisartan 0.647μg/ml and 1.959μg/ml respectively. In the robustness study %RSD obtained for change of flow rate and wavelength and ruggedness for change of analyst was found to be below 2, which was within the acceptance criteria. So, simple, sensitive, accurate, precise RP- HPLC methods were developed and validated for the simultaneous estimation of Pitavastatin &amp; Telmisartan.

A Review on Method Development of High Performance Liquid Chromatography (HPLC) and its Applications

The most common method for identifying, separating, and measuring the medication is HPLC.To improve the approach, a variety of chromatographic factors including sample pretreatment, mobile phase selection, column selection, and detector selection were examined. Reviewing the technique creation, optimization, and validation is the article's main goal. The chemistry of the molecules, the synthetic pathway, solubility, polarity, pH and pKa values, and the activity of the functional groups are all factors that affect the creation of an HPLC technique. Information on several stages and properties including accuracy, specificity, linearity, limit of detection, and limit of quantification are provided through the validation of HPLC methods in accordance with ICH Guidelines1.

Mobile phase and column chemistry selection for high sensitivity non-targeted LC/ESI/HRMS screening of water

Systematic selection of mobile phase and column chemistry type can be critical for achieving optimal chromatographic separation, high sensitivity, and low detection limits in liquid chromatography electrospray high resolution mass spectrometry (LC/MS). However, the selection process is challenging for non-targeted screening where the compounds of interest are not preselected nor available for method optimization. To provide general guidance, twenty different mobile phase compositions and four columns were compared for the analysis of 78 compounds with a wide range of physicochemical properties (logP range from −1.46 to 5.48), and analyte sensitivity was compared between methods. The pH, additive type, column, and organic modifier had significant effects on the analyte response factors, and acidic mobile phases (e.g. 0.1% formic acid) yielded highest sensitivity. In some cases, the effect was attributable to the difference in organic modifier content at the time of elution, depending on the mobile phase and column chemistry. Based on these findings, 0.1% formic acid, 0.1% ammonia and 5.0 mM ammonium fluoride were further evaluated for their performance in non-targeted LC/ESI/HRMS analysis of wastewater treatment plan influent and effluent, using a data dependent MS2 acquisition and two different data processing workflows (MS-DIAL, patRoon 2.1) to compare number of detected features and sensitivity. Both data-processing workflows indicated that 0.1% formic acid yielded the highest number of features in full scan spectrum (MS1), as well as the highest number of features that triggered fragmentation spectra (MS2) when dynamic exclusion was used.

Understanding mobile phase buffer composition and chemical structure effects on electrospray ionization mass spectrometry response

Mobile phase selection is of critical importance in liquid chromatography – mass spectrometry (LC-MS) based studies, since it affects retention, chromatographic selectivity, ionization, limits of detection and quantification, and linear dynamic range. Generalized LC-MS mobile phase selection criteria, suitable for a broad class of chemical compounds, do not exist thus far. Here we have performed a large-scale qualitative assessment of the effect of solvent composition used for reversed-phase LC separations on electrospray ionization (ESI) response for 240 small molecular weight drugs, representing various chemical compound classes. Of these 240 analytes 224 were detectable using ESI. The main chemical structural features affecting ESI response were found to all be surface area or surface charge-related. Mobile phase composition was found to be less differentiating, although for some compounds a pH effect was noted. Unsurprisingly, chemical structure was found to be the dominant factor for ESI response for the majority of the investigated analytes, representing about 85% of the replicating detectable complement of the sample data set. A weak correlation between ESI response and structure complexity was observed. Solvents based on isopropanol, and those containing phosphoric or di- and trifluoracetic acids, performed relatively poorly in terms of chromatographic or ESI response, whilst the best performing ‘generic’ LC solvents were based on methanol, acetonitrile using formic acid and ammonium acetate as buffer components, consistent with current practice in many laboratories.

A strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part I: Selection of columns and mobile phases

The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW <10 kDa.Retention data for 29 diverse peptides (1 < MW < 14 kDa, 3.7 < pI < 12.5) measured in this study using a variety of column and mobile phase conditions (i.e., 30 in total) are consistent with the classification of these various chromatographic conditions using the previously reported Peptide RPC Column Characterisation Protocol. For the investigated peptides trifluoroacetic acid was found to reduce selectivity differences between columns of diverse properties, probably due to its potential to form ion-pairs with peptides. Trifluoroacetic acid often improves peak shape for very large peptides (i.e. MW > 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape.The reported work here constitutes part one of a series of two papers. The second paper focuses on the use of retention modelling for rapid and accurate selection of the shallow gradients (i.e., << 1% ACN/min) required to obtain sufficient peptide isomer retention and separation in the second dimension. The overall results presented in this series of papers provides the guidance needed to develop a 2D-LC-MS method from start to finish for the analysis of main peak purity of therapeutic peptides.

Two-dimensional reversed phase-normal phase liquid chromatography for simultaneous achiral-chiral analysis to support high-throughput experimentation

Typical chromatographic analysis of chiral compounds requires the use of achiral methods to evaluate impurities or related substances along with separate methods to evaluate chiral purity. The use of two-dimensional liquid chromatography (2D-LC) to support simultaneous achiral-chiral analysis has become increasingly advantageous in the field of high-throughput experimentation where low reaction yields or side reactions can lead to challenging direct chiral analysis. Advancements in multi-dimensional chromatography have led to the development of robust 2D-LC instrumentation with reversed phase solvent systems (RPLC-RPLC) enabling this simultaneous analysis, eliminating the need to purify crude reaction mixtures to determine stereoselectivity. However, when chiral RPLC cannot separate a chiral impurity from the desired product, there are few viable commercial options. The coupling of NPLC to RPLC (RPLC-NPLC) continues to remain elusive due to solvent immiscibility between the two solvent systems. This solvent incompatibility leads to lack of retention, band broadening, poor resolution, poor peak shapes, and baseline issues in the second dimension. A study was conducted to understand the effect of various water-containing injections on NPLC and applied to the development of robust RPLC-NPLC methods. Following thoughtful consideration and modifications to the design of a 2D-LC system in regards to mobile phase selection, sample loop sizing, targeted mixing, and solvent compatibility, proof of concept has been demonstrated with the development of reproducible RPLC-NPLC 2D-LC methods to perform simultaneous achiral-chiral analysis. Second dimension NPLC method performance proved comparable to corresponding 1D-NPLC methods with excellent percent difference in enantiomeric excess results ≤ 1.09% and adequate limits of quantitation down to 0.0025 mg/mL for injection volumes of 2 µL, or 5 ng on-column.

Understanding the mechanism of CO2-Assisted electrospray ionization for parameter optimization in supercritical fluid chromatography mass spectrometry

Supercritical fluid chromatography (SFC) is often coupled with electrospray ionization mass spectrometry (ESI-MS) for analyte detection because of its detection capability to a wide range of chemical properties. However, MS sensitivity is highly dependent on the chromatographic conditions, so that it is important to understand the ionization mechanism to determine the optimal chromatographic conditions. The ionization mechanism in SFC/ESI-MS is different to that of liquid chromatography because of the use of CO2 as a mobile phase. Some studies have suggested that alkoxycarbonic acids are formed in the mixture of CO2 and the alcohol modifier, and these species contribute to ionization in CO2-assisted SFC/ESI-MS. Therefore, in this study, we investigated CO2-assisted ESI to test this hypothesis, and we confirmed that methoxylcarbonic acid is generated in CO2/methanol mixtures and contributed to ion generation and detection because it acts as a proton donor in positive-ion mode. However, methoxylcarbonic acid interfered with ionization in negative-ion mode. Addition of ammonium acetate, which is often added to the modifier for negative ion detection in SFC/MS analysis, did not contribute to the recovery of MS sensitivity, although it tended to suppress the formation of metoxylcarbonic acid. This is likely due to ion suppression and neutralization of the negative sites of the analytes by anions or cations derived from ammonium acetate in the negative ion mode. Thus, additive-free methanol/CO2 was the most suitable mobile phase for obtaining high sensitivity in SFC/MS. To demonstrate the practicality of these findings, we tested our optimal mobile phase selection for pesticide analysis. In addition, we tested the addition of 0, 1, and 5 mM ammonium formate to the modifier and make-up solvent, and found that the addition of 1 mM ammonium formate gave the best results in pesticides analysis. In SFC/MS, salt is often added to improve separation or prevent desorption, but our findings suggest that the concentration of salt must be kept as low as possible to achieve highly sensitive MS detection. The results of this study reveal the best selection of the optimal conditions for the modifier and make-up solvent for CO2-assisted SFC/MS analysis and will be useful for the method development in SFC/MS.

Single-run UHPLC-MS/MS method for simultaneous quantification of endogenous steroids and their phase II metabolites in serum for anti-doping purposes

Anti-doping rule violations related to the abuse of endogenous anabolic androgenic steroids can be currently discovered by the urinary steroidal module of Athlete Biological Passport. Since this powerful tool is still subjected to some limitations due to various confounding factors altering the steroid profile, alternative strategies have been constantly proposed. Among these, the measurement of blood concentrations of endogenous steroid hormones by LC-MS is currently of increasing interest in anti-doping, bringing significant advantages for the detection of testosterone abuse in females and in individuals with deletion of UGT2B17 enzyme. Although various research groups have made significant efforts in method development, there is currently no accepted or harmonized anti-doping method for quantitative analysis of the various testosterone doping markers in blood. In this study we present a UHPLC-MS/MS method for the quantification of major circulating steroid hormones together with an extended panel of glucuro- and sulpho-conjugated phase II metabolites of androgens. Chromatographic setup was optimized by comparing the performance of three different C18 stationary phases and by the careful selection of mobile phases with the aim of separating all the target steroids, including numerous isomeric/isobaric compounds. MS parameters were fine-tuned to obtain the sensitivity needed for measuring the target analytes, that show specific serum concentrations ranging from low pg/mL for less abundant compounds to μg/mL for sulpho-conjugated steroids. Finally, sample preparation protocol was developed for the extraction of steroid hormones from 200 μL of serum and the performance was evaluated in terms of extraction recovery and matrix effect. The final method was then applied to authentic serum samples collected from healthy volunteers (40 males and 40 females) at the Blood Bank of the City of Health and Science University Hospital of Turin. The analysis of these samples allowed to obtain results on serum concentrations of the targeted steroids, with particular emphasis on previously undiscovered phase II metabolites, such as the isomers of 5-androstane-3,17-diol glucuronide. This preliminary application also enabled measuring dihydrotestosterone sulphate in male samples, efficiently separating this analyte from its isomer, epiandrosterone sulphate, which circulates in blood at high concentrations.The promising results of this study are encouraging for the measurement of blood steroid profile markers in serum and plasma samples for Athlete Biological Passport purposes.

Analytical Methods of Antidiabetic Drugs – Sitagliptin, Saxagliptin, Linagliptin, Alogliptin, Gemifibrozil, Troglitazone, Pioglitazone and Rosiglitazone: A Review

Analytical techniques used to determine chemical or physical property of analyte, chemical substance, chemical element or mixture. There are different types of analytical techniques used in pharmaceutical field for qualitative and quantitative estimation of drugs in biological fluids such as human plasma, human serum, human urine etc. and its formulations such as tablet, capsule, bulk drugs etc. This review work aims to provide most of existing analytical methods for analysis of Dipeptidyl peptidase-4 (DPP4) inhibitor and Peroxisome proliferator activated (PPAR) agonist. Estimation of Sitagliptin, Saxagliptin, Alogliptin, Gemifibrozil, Troglitazone, Pioglitazone, Linagliptin, and Rosiglitazone in active pharmaceutical ingredients, biological fluids and in various formulations which is reported in various research articles are reviewed. The analytical techniques which are utilised for the estimation of these drugs involves RP-HPLC, HPTLC, UPLC-MS/MS, LC-MS/MS, MALDI-TOF mass spectrometry, Raman spectroscopy, Liquid chromatography and Tandem mass spectrometry, TLC-densitometry with fluorescent detector, LC-ESI-MS-MS etc. are involved. The review assist in appropriate selection of analytical technique, mobile phase, column and detector based on available analytical instruments and chemicals by referring tabulated extensive condition. DPP4 inhibitor used in treatment of type 2 diabetes, hypoglycemia, weight loss etc. PPAR agonist also used in treatement of type 2 diabetes, in metabolic syndrome mainly for lowering triglycerides and blood sugar etc.

Selection of Stationary Phase and Mobile Phase in High Performance Liquid Chromatography

Selection of ideal mobile phase and stationary phase is very important to get accurate separation of mixtures or impurities as a whole. There indeed are parameters which are explained in this article, important to be kept in mind while a method development of HPLC method. But as such there is no list of such parameters and their accurate limits can be applied while method development. In this article, there have been mention of certain parameters which are generally looked for, and not exact, but optimal application of such has been discussed. When we talk about stationary phase, parameters which we tend to optimize very often include mostly commonly the pH of the analyte as well the mobile phase used and stability of the column packing material over a range of temperature. Choosing of stationary phase greatly depends on the nature of analyte. Stationary phase will depend and vary simultaneously if analyte is lipophilic or an ionic compound. In order to increase separation and increase mass transfer there have been new addition to column packing material apart from the widely used silica. New advances result into greater column loading, better flow, reduced plate height and reduced back pressure. Leaving alone stationary phase, mobile phase selection is also a task of its own. Very commonly used mobile phase solvents include acetonitrile, methanol, THF, water and buffer of salts like acetate, phosphate, etc. Apart from these traditional choices, there have been use of pressurized hot water and ionic liquids as mobile phases which are termed as “Environmental-friendly” because these solvents are devoid of any organic counterpart or organic modifier in them, which produces harmful fumes on getting heated. Apart from the environmentally friendly mobile phase options, we have some specific chemicals to be used when chemical property of the analyte is specific.

A Review : Method of Development of HPTLC and Application in Pharma Industry

This review provides knowledge on the development and validation parameters of HPTLC-based analytical methods in line with actual evaluation. Meet the criteria and minimize errors and investigations. This review assists in the selection of mobile thermal phases and provides guidelines for understanding the steps of good validation practices and analytical procedures

Advanced Development of Supercritical Fluid Chromatography in Herbal Medicine Analysis.

The greatest challenge in the analysis of herbal components lies in their variety and complexity. Therefore, efficient analytical tools for the separation and qualitative and quantitative analysis of multi-components are essential. In recent years, various emerging analytical techniques have offered significant support for complicated component analysis, with breakthroughs in selectivity, sensitivity, and rapid analysis. Among these techniques, supercritical fluid chromatography (SFC) has attracted much attention because of its high column efficiency and environmental protection. SFC can be used to analyze a wide range of compounds, including non-polar and polar compounds, making it a prominent analytical platform. The applicability of SFC for the separation and determination of natural products in herbal medicines is overviewed in this article. The range of applications was expanded through the selection and optimization of stationary phases and mobile phases. We also focus on the two-dimensional SFC analysis. This paper provides new insight into SFC method development for herbal medicine analysis.

Two-dimensional high-performance thin-layer chromatography for the characterization of milk peptide properties and a prediction of the retention behavior – a proof-of-principle study

High-performance thin-layer chromatography (HPTLC) is a suitable method for the analysis of peptides and proteins due to a wide selection of stationary and mobile phases and various detection options. Especially, two-dimensional HPTLC (2D-HPTLC) enables a higher resolution compared to one-dimensional HPTLC in the separation of complex peptide mixtures. Similar to 2D electrophoresis, characteristic peptide patterns can be obtained, allowing a differentiation of ingredients based on varying protein origins.The aim of this study was to evaluate 2D-HPTLC with regard to its suitability for the characterization of proteins/peptides and to verify whether it is possible to predict the retention behavior of peptides based on their properties. As models, the five most abundant milk proteins α-lactalbumin, β-lactoglobulin, α-, β-, and κ-Casein were used. In order to determine the repeatability of the peptide separation by 2D-HPTLC, each tryptic protein hydrolyzate was separated eight times. The standard deviations of the retardation factors for the separated peptides varied between 1.0 and 11.1 mm for the x-coordinate and 0.5–7.3 mm for the y-coordinate. It was also shown that after the chromatographic separation, peptides of the individual protein hydrolyzates were located in specific areas on the HPTLC plate, so that a clustering could be obtained for the whey proteins‘ as well as the caseins‘ hydrolyzates. For establishing correlations between the properties of the peptides and their retardation factors, 51 of 85 selected peptides were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). On this basis, statistically significant correlations (α = 0.05) between the retardation factors of the peptides and their isoelectric points, as well as the percentage of anionic and non-polar amino acids in the peptides were established. Finally, it was investigated, whether the retardation factors for peptides can be predicted on the basis of a linear regression of the percentage of non-polar amino acids in a peptide. For this purpose, a mixture of artifical (synthetic) peptides (n = 14) was separated by 2D-HPTLC and the measured retardation factors were compared with the corresponding retardation factors calculated. Absolute deviations of 0.3–17.9 mm were obtained. In addition, the universal applicability of the method to other protein sources other than milk proteins (animal protein) was tested using a mixture of pea peptides (plant protein, n = 3) resulting in absolute deviations of 0.7–8.6 mm.