Reversed-phase HPLC Separation Research Articles

Inducing α-Helicity in Peptides by Silver Coordination to Cysteine.

Short peptide sequences consisting of two cysteine residues separated by three other amino acids display complete change from random coil to α-helical secondary structure in response to addition of Ag+ ions. The folded CXXXC/Ag+ complex involves formation of multinuclear Ag+ species and is stable in a wide pH range from below 3 to above 8. The complex is stable through reversed-phase HPLC separation as well as towards a physiological level of chloride ions, based on far-UV circular dichroism spectroscopy. In electrospray MS under acidic conditions a peptide dimer with four Ag+ ions bound was observed, and modelling based on potentiometric experiments supported this to be the dominating complex at neutral pH together with a peptide dimer with 3 Ag+ and one proton at lower pH. The complex was demonstrated to work as a N-terminal nucleation site for inducing α-helicity into longer peptides. This type of silver-mediated peptide assembly and folding may be of more general use for stabilizing not only peptide folding but also for controlling oligomerization even under acidic conditions.

Hydrophobicity and molecular mass-based separation method for autoantibody discovery from mammalian total cellular proteins.

Serum autoantibody profiles are unique to individuals and reflect the level and history of autoimmunity and tumor immunity. The identification of autoantibody biomarkers is critical for the development of immune monitoring systems for immune-related disorders. Here, we present a practical method for large-scale autoantibody discovery using total cellular proteins from cultured mammalian cells. We found that nucleic acid-free and fully denatured water-soluble total cellular proteins from mammalian cells were superior, allowing precise separation by reversed-phase HPLC after preparing a large set of homogeneous total cellular proteins. After separating the proteins based on hydrophobicity, the fractionated samples were subjected to molecular mass analysis using conventional SDS-PAGE. The resulting two-dimensional gel electrophoresis was successfully employed for immune blotting and LC-MS/MS analysis. All procedures, including TRIzol-based total cellular protein extraction, solubilization of denatured proteins, reversed-phase HPLC separation, and SDS-PAGE, were highly reproducible and easily scalable. We propose this novel two-dimensional gel electrophoresis system as an alternative proteomics-based methodology suitable for large-scale autoantibody discovery.

Tailoring two white chromatographic platforms for simultaneous estimation of ritonavir-boosted nirmatrelvir in their novel pills: degradation, validation, and environmental impact studies.

As the COVID-19 pandemic is not yet over, Pfizer has launched the novel pill Paxlovid® (Nirmatrelvir (NMV) co-packaged with ritonavir (RIT)) as an effective medication for hospitalized and non-hospitalized patients. Making pharmaceutical analysis greener and more sustainable has lately become the main direction of the research community. In this context, two fast, green, and stability-indicating chromatographic methods were designed for the neat quantitative determination of NMV and RIT in their bulk and dosage forms. Method I is deemed the first electro-driven attempt for the assay of Paxlovid®. Herein, the optimized conditions of the Micellar Electrokinetic Chromatographic (MEKC) method were 50 mM borate buffer at pH 9.2 with 25 mM sodium lauryl sulfate (SDS) being used as the background electrolyte (BGE) on a deactivated fused silica capillary (50 cm effective length × 50 μm id). Method II was an isocratic reversed-phase HPLC separation method using Zorbax-Eclipse C18 (4.6 × 250 mm, 5 μm particle size) column and 50 mM ammonium acetate buffer at pH 5 and acetonitrile as mobile phase constituents at a flow rate of 1 mL min-1. For the sake of simplicity and increasing sensitivity, a single wavelength of 210 nm was used for the two methods to assay both drugs. Linear correlations between peak areas and concentration were observed in the ranges of 10-200 μg mL-1 for NMV and 5-100 μg mL-1 RIT in both methods. The impact of versatile stress conditions such as hydrolysis, oxidation, and photolysis on the stability of NMV and RIT was studied. Fortunately, both methodologies were able to separate both drugs from their degradants. Thus, the stability indicating power of the methods was proved. The derived methods were statistically validated in agreement with the ICH guidelines. Furthermore, the environmental friendliness and sustainability of these methods were investigated and compared with the cited methods using the holistic multicriteria evaluation tools namely Hexagon, AGREE, and RGB12 metrics. Conclusively, the proposed methods offered reliable, feasible, economic, white, and stability-indicating alternatives to the cited chromatographic methods.

Supramolecular solvents based on quaternary ammonium salts and perfluorinated compounds for dispersive liquid-liquid microextraction of phthalates

In this study, eight hydrophobic supramolecular (SUPRA) solvents composed of tetrabutylammonium chloride and tetradecyltrimethylammonium bromide as amphiphiles and four perfluorinated compounds (hexafluoroisopropanol, trifluoroacetic acid, pentafluoropropionic acid and heptafluorobutyric acid) as coacervation-inducing agents were developed. The SUPRA phase volumes were determined as a function of individual concentrations of the system components. The extraction properties of the SUPRA solvents for the extraction of phthalates from the model aqueous solutions were investigated and their applicability as sample diluents for the reversed-phase HPLC separations of phthalates was tested. Finally, the solvent consisting of tetradecyltrimethylammonium bromide and hexafluoroisopropanol was applied for dispersive liquid-liquid microextraction of phthalates from wastewater samples prior to HPLC analysis. Under optimised extraction conditions, the enrichment factors were around 75. The calibration curves were linear (R2 ≥ 0.9965) for the concentration level between 1.00 and 50.0 μg/L and the detection limits were in the range 0.3–0.6 μg/L. The recoveries of the phthalates of the developed method for the spiked wastewater samples were 93.1–104.4%, with the relative standard deviation values less than 7.4%.

Characterising diflunisal as a transthyretin kinetic stabilizer at relevant concentrations in human plasma using subunit exchange

Transthyretin (TTR) dissociation is the rate limiting step for both aggregation and subunit exchange. Kinetic stabilisers, small molecules that bind to the native tetrameric structure of TTR, slow TTR dissociation and inhibit aggregation. One such stabiliser is the non-steroidal anti-inflammatory drug (NSAID), diflunisal, which has been repurposed to treat TTR polyneuropathy. Previously, we compared the efficacy of diflunisal, tafamidis, tolcapone, and AG10 as kinetic stabilisers for transthyretin. However, we could not meaningfully compare diflunisal because we were unsure of its plasma concentration after long-term oral dosing. Herein, we report the diflunisal plasma concentrations measured by extraction, reversed phase HPLC separation, and fluorescence detection after long-term 250 mg BID oral dosing in two groups: a placebo-controlled diflunisal clinical trial group and an open-label Japanese polyneuropathy treatment cohort. The measured mean diflunisal plasma concentration from both groups was 282.2 M 143.7 M (mean standard deviation). Thus, quantification of TTR kinetic stabilisation using subunit exchange was carried out at 100, 200, 300, and 400 concentrations, all observed in patients after 250 mg BID oral dosing. A 250 M diflunisal plasma concentration reduced the wild-type TTR dissociation rate in plasma by 95%, which is sufficient to stop transthyretin aggregation, consistent with the clinical efficacy of diflunisal for ameliorating transthyretin polyneuropathy.

Chromatographic behaviour of peptides modified with amine-reacting tags for relative protein quantitation in proteomic applications

Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts ΔHI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs.

Three-dimensional liquid chromatography with parallel second dimensions and quadruple parallel mass spectrometry for adult/infant formula analysis

Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, “constructive wraparound”) elution, which employed flow rate programming. In the 1D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.

Development and Validation of a Novel LC-MS/MS Method for the Quantitation of 19 Fingerprint Phytochemicals in Salvia Species: A Chemometric Approach.

Being traditionally utilized mainly as appetizers and herbal teas as well as used to ease abdominal pains, colds and gastrointestinal issues, the genus Salvia L. has gained significant consideration owing to its remarkable phytochemicals and industrial importance. The present study aimed to develop and validate an LC-MS/MS method for the qualitative and quantitative investigation of 19 fingerprint phytochemicals in six endemic Salvia species. The validation parameters of the developed LC-MS/MS method were repeatability (intermediate precision), recovery (accuracy), limits of detection and quantification, linearity and uncertainty (U% at 95% confidence level (k = 2)). Reversed-phase HPLC separation and mass spectrometry parameters were optimized for each analyte. Ethanol extracts of the studied Salvia species collected in three consecutive years were screened for their fingerprint phytochemicals by using the developed and validated LC-MS/MS method. Moreover, studied Salvia species were subjected to multivariate analysis such as principal component analysis techniques to demonstrate the variabilities in phytochemical contents by years and parts of the samples. Roots, flowers, leaves, branches and whole plant of the Salvia species collected in 2015, 2016 and 2017 were used for the analyses. It was observed that the roots and branches of Salvia species were similar in terms of their salvianolic acid A, caffeic acid, and 6,7-dehydroroyleanone components. Accordingly, apigenin, rosmarinic acid, luteolin 7-O-glucoside, caffeic acid, salvianolic acid B, and 6,7-dehydroroyleanone were notable phytochemicals that were present in the studied Salvia species.

Identification, Characterization and Antihypertensive Effect In Vivo of a Novel ACE-Inhibitory Heptapeptide from Defatted Areca Nut Kernel Globulin Hydrolysates.

The areca (Areca catechu L.) nut kernel (ANK) is a good potential protein source for its high protein content of 9.89–14.62 g/100 g and a high yield of around 300,000 tons per year in China. However, utilization of the areca nut kernel is limited. To expand the usage of ANK in pharmaceutical or foods industries, areca nut kernel globulin was extracted and angiotensin-I converting enzyme (ACE) inhibition peptides were prepared and identified using gel chromatography, reversed phase HPLC separation, UPLC-ESI-MS/MS analysis and in silico screening. Finally, a novel ACE-inhibitory heptapeptide (Ala–Pro–Lys–Ile–Glu–Glu–Val) was identified and chemically synthesized. The combination pattern between APKIEEV and ACE, and the inhibition kinetics, antihypertensive effect and endothlein-1 inhibition activity of APKIEEV were studied. The results of the molecular docking demonstrated that APKIEEV could bind to four active sites (not the key active sites) of ACE via short hydrogen bonds and demonstrated high ACE-inhibitory activity (IC50: 550.41 μmol/L). Moreover, APKIEEV exhibited a significantly lowering effect on both the systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rats, and had considerable suppression ability on intracellular endothelin-1. These results highlight the potential usage of APKIEEV as ingredients of antihypertensive drugs or functional foods.

Functional Characterization of Mung Bean Meal Protein-Derived Antioxidant Peptides.

The aim of this work was to characterize the antioxidant properties of some of the peptides present in bromelain mung bean meal protein hydrolysate (MMPH). The MMPH was subjected to two rounds of bioassay-guided reversed-phase HPLC separation followed by peptide identification in the most potent fractions using tandem mass spectrometry. Twelve antioxidant peptides, namely, HC, CGN, LAN, CTN, LAF, CSGD, MMGW, QFAAD, ERF, EYW, FLQL, and QFAW were identified and assayed for antioxidant properties. CTN, HC, CGN, and CSGD were the most potent (p < 0.05) DPPH radical scavengers with EC50 values of 0.30, 0.29, 0.28, and 0.30 mg/mL, respectively, which are lower than the 0.03 mg/mL obtained for reduced glutathione (GSH). CTN, HC, CGN, and CSGD exhibited the most potent (p < 0.05) scavenging activities against hydroxyl and superoxide radicals with EC50 values that are similar to those of GSH. The cysteine-containing peptides also had stronger ferric reducing antioxidant power and metal chelation activity than peptides devoid of cysteine. In contrast, MMGW, ERF, and EYW had poor radical scavenging and metal chelation activities. We conclude that the availability of the sulfhydryl group may have enhanced antioxidant potency while the presence of bulky groups such phenylalanine and tryptophan had an opposite effect.

Unequivocal Identification of Aspartic Acid and isoAspartic Acid by MALDI-TOF/TOF: From Peptide Standards to a Therapeutic Antibody.

Aspartic acid (Asp) to isoaspartic acid (isoAsp) isomerization in therapeutic monoclonal antibodies (mAbs) and other biotherapeutics is a critical quality attribute (CQA) that requires careful control and monitoring during the drug discovery and production processes. The unwanted formation of isoAsp within biotherapeutics and resultant structural changes in the peptide backbone may negatively impact the efficacy, potency, and safety of the molecule or become immunogenic, especially if the isomerization occurs within the mAb complementarity determining region (CDR). Herein we describe a MALDI-TOF/TOF mass spectrometry method that affords unequivocal identification of the presence and the exact position of the isoAsp residue(s) in peptide standards ranging in size from a tripeptide to a docosapeptide (22 residues). In general, the peptide bond immediately N-terminal to the isoAsp residue is more susceptible to MALDI-TOF/TOF fragmentation than its unmodified counterpart. In some of the peptides evaluated in this study, fragmentation of the peptide bond C-terminal to the isoAsp residue (the aspartate effect) is also enhanced when compared to the control. Relative quantification by MALDI-TOF/TOF of this chemical modification is dependent upon a successful reversed-phase HPLC (rpHPLC) separation of the control and modified peptides. This method has also been validated on a therapeutic mAb that contains a well-documented isoAsp residue in the heavy chain CDR3 after forced degradation. Moreover, we also demonstrate that higher energy C-trap dissociation of only the singly charged species, and not the multiply charged form, of the isoAsp containing peptide, separated by rpHPLC, results in LC-MS/MS fragmentation that is highly consistent to that of MALDI-TOF/TOF.

Using a 3-hydroxyflavone derivative as a fluorescent probe for the indirect determination of aminothiols separated by ion-pair HPLC.

Homocysteine, cysteine, cysteinyl-glycine, and glutathione are significant biological aminothiols (ATs) that are marker-molecules in Down syndrome, Alzheimer's disease, or have been implicated as risk factors in atherosclerosis and other vascular diseases, and therefore rapid determination of these molecules is desirable. After reduction of the disulfides, a widely used method utilizes derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) as a fluorogenic probe prior to reversed-phase HPLC separation followed by fluorescence detection. The traditional HPLC determination of ATs is time consuming and economically expensive. We have developed an ion-pair HPLC method coupled with indirect fluorescence detection after post-column reaction with a 2,4-dinitrobenzenesulfonate derivative of a 3-hydroxyflavone. The accuracy, precision, post-column temperature and residence time, and limit-of-detection were evaluated. Sample throughput and reduced sample preparation time of over an hour for the existing methods to less than 20 minutes for the new method is also demonstrated. No statistical differences in HCy, Cys, or Cys-Gly determinations in plasma samples were observed between our method and the traditional HPLC method.

Bioavailability, tissue distribution and excretion studies of a potential anti-osteoporotic agent, medicarpin, in female rats using validated LC–MS/MS method

Medicarpin, one of the active constituents isolated from the extract of Butea monosperma, has been shown to have various pharmacological activities including potent anti-osteoporotic properties. The aim of this study was to investigate the oral pharmacokinetics, tissue distribution and excretion of medicarpin following single oral dose administration in female rats. Oral pharmacokinetics was explored at 5 and 20 mg/kg while tissue distribution, urinary and fecal excretion were studied following 20 mg/kg oral dose. Medicarpin was quantified in rat plasma, urine, feces and tissue samples using a validated LC–MS/MS method following reverse-phase HPLC separation on RP18 column (4.6 mm × 50 mm, 5.0 μm) using methanol and 10 mM ammonium acetate (pH 4.0) as mobile phase in the ratio of 80:20 (v/v) at a flow rate of 0.8 mL/min. The oral bioavailability of medicarpin was found to be low with low systemic levels. The concentration in tissues was significantly higher than plasma. Highest tissue concentrations were found in the liver followed by bone marrow. Urinary and fecal excretion of medicarpin was < 1 %. In conclusion, medicarpin was found to be highly distributed in body tissues and minimally excreted via urine or feces.

Kinetics of acetylcholinesterase inhibition by hemp seed protein-derived peptides.

The aim of this work was to enhance the acetylcholinesterase (AChE)-inhibitory activity of a pepsin-produced hemp seed protein hydrolysates (HPH) through reverse-phase HPLC separation followed by identification of peptide sequences present in the most active fraction. The HPH was separated into eight fractions (F1-F8) with F7 exhibiting significantly (p<0.05) the strongest (97.5%) in vitro inhibition of electric eel AChE (eeAChE) activity in comparison to 53.8% for HPH. The HPH consisted mostly of low molecular weight peptides of<11 amino acid residues and most contained at least one hydrophobic amino acid. Kinetics of enzyme inhibition revealed a mixed-type inhibition of eeAChE activity by HPH whereas F7 acted through an uncompetitive mode; in contrast inhibition of human AChE by HPH and F7 was uncompetitive. The stronger inhibitory potency of the F7 peptides fraction against both enzymes was confirmed through reduced maximal velocity, catalytic efficiency, and inhibition constant values when compared to the HPH. PRACTICAL APPLICATIONS: The use of natural products for the prevention or treatment of human diseases continues to be an area of intense research activities. Food protein-derived peptides obtained through enzymatic hydrolysis of hemp seed proteins were shown in vitro to be strong inhibitors of activities of both the eel and human forms of acetylcholinesterase (AChE). AChE is an important therapeutic target because excessive activity of this enzyme is a causative factor of neurodegenerative diseases such as dementia and Alzheimer's. This work showed that peptides in the most active fraction are small in sizes, which may favor their absorption into blood circulation and possible permeation of the blood-brain barrier. Therefore, the hemp seed peptides are potential agents that can be used to formulate functional foods and nutraceuticals against neurodegenerative diseases.

Olive mill wastewaters: quantitation of the phenolic content and profiling of elenolic acid derivatives using HPLC-DAD and HPLC/MS2 with an embedded polar group stationary phase

Olive mill wastewaters (OMWs) are important by-products of olive oil production. The wide availability and the forbidding economic costs of OMWs disposal have generated interest in their possible exploitation as a sustainable source of polyphenols. The development and optimisation of improved analytical methods for the detailed characterisation of polyphenol molecular profiles after production and during storage conditions are thus required. A new analytical method based on reverse-phase HPLC separation of polyphenols in OMWs on an embedded-polar group (EPG) stationary phase is proposed as an alternative to conventional C18 columns. The procedure was used for the quantitation of 11 polyphenols, using diode-array detection (DAD), and achieving quantitation limits equal to or lower than 0.1 µg g−1. The same chromatographic set-up, coupled with high resolution tandem mass spectrometry (HPLC/ESI-Q-ToF), allowed us to identify hydroxytyrosyl esters of elenolic acid, whose relative abundance is proposed for monitoring the ageing of OMWs during storage.

Application of Central Composite Design for the Optimization of Reverse-Phase HPLC/DAD Separation of the cis- and trans- Isomers of Long-Chain Unsaturated Fatty Acids

The study deals with the optimization of reverse-phase HPLC separation of cis-/trans- isomers of long-chain unsaturated fatty acids by the assessment of the central composite design (CCD) of response surface methodology (RSM). The optimized conditions were also applied for the analysis of fatty acids in functional cold-pressed oils. The data obtained from experimental applications of CCD was used to model the parameters that significantly affect separations. The independent variables chosen were flow rate (X 1 ), column temperature (X 2 ), and acetonitrile content in mobile phase (X 3 ). A second-order polynomial model was used to estimate the impact of variables on separation efficiencies of the C30 and C18 stationary phases. The proposed CCD models were also validated with the ANOVA. The predicted values were in good agreement with experimental data, advising expert application of CCD as an option to obtain maximum information for the modeling of reverse-phase HPLC separation with little number of experiments. The optimal values of method parameters for the efficient C18 and C30 column separations as part of the k′ response value were found to be flow rate of 1.10 and 0.42 mL min−1, temperature of 3.6 and 9.4 °C, and acetonitrile content in mobile phase of 100 and 77.4%, respectively. ANOVA test results also illustrate that the CCD models can be successfully used to predict the optimum method parameters. To maximize both sensitivity and precision of the methods, the validation procedure was also performed and the higher correlation coefficients (r = 0.9149–0.9993) were determined for all fatty acid methyl esters. Thus, the proposed experimental designs were shown to offer considerable advantages over traditional method optimization approaches.

HPLC Method for Measurement of Human Salivary \u03b1-Amylase Inhibition by Aqueous Plant Extracts

Control of hyperglycemia is an important treatment in metabolic disorders such as type II diabetes and obesity. α-Amylase, as the first enzyme of glucose release from dietary polysaccharides, is a potential target to identify new sources of novel anti-obesity and anti-diabetic drugs. In this work, different herbal extracts as α-amylase inhibitors were studied by measuring the rate of the cleavage of a maltooligomer substrate 2-chloro-4-nitrophenyl-β-D-maltoheptoside. Measurement of chromophore containing products after reversed phase HPLC separation was used for α-amylase activity measurement. Rates of hydrolysis catalysed by human salivary α-amylase were determined in the presence and absence of lyophilised water extracts of eleven herbs. Remarkable bioactivities were found for extracts of Cinnamomum zeylanicum Blume (bark), Camellia sinensis L. (leaf), Ribes nigrum L. (leaf), Laurus nobilis L. (leaf), Vaccinium macrocarpon Aiton (fruit) and Syzygium aromaticum L. (bud). Determined IC50 values were in 0.017–41 μg/ml range for these six selected plant extracts. Our results confirm the applicability of this HPLC-based method for the quick and reliable comparison of plants as α-amylase inhibitors.

In-Depth, Reproducible Analysis of Human Plasma Using IgY 14 and SuperMix Immunodepletion.

Identification of cancer and other disease biomarkers in human plasma has been exceptionally challenging due to the complex nature of plasma and the presence of a moderate number of high- and medium-abundance proteins which mask low-abundance proteins of interest. As a result, immunoaffinity depletion formats combining multiple antibodies to target the most abundant plasma proteins have become the first stage in most plasma proteome discovery schemes. This protocol describes the use of tandem IgY 14 and SuperMix immunoaffinity depletion to reproducibly remove >99% of total plasma protein. This greatly increases the depth of analysis of human plasma proteomes. Depleted plasma samples can then be analyzed in a single high-resolution LC-MS/MS run on a Q Exactive Plus mass spectrometer, followed by label-free quantitation. If greater depth of analysis is desired, the depleted plasma can be further fractionated by separating the sample for a short distance on a 1D SDS gel and cutting the gel into uniform slices prior to trypsin digestion. Alternatively, the depleted plasma can be reduced, alkylated, and digested with trypsin followed by high-pH reversed-phase HPLC separation.

Integrating Elemental Analysis and Chromatography Techniques by Analyzing Metal Oxide and Organic UV Absorbers in Commercial Sunscreens

A series of undergraduate laboratory experiments that utilize reversed-phase HPLC separation, inductively coupled plasma spectroscopy (ICP), and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) are described for the analysis of commercial sunscreens. The active ingredients of many sunscreen brands include zinc or titanium oxide in addition to organic acids. Students determine the zinc content using ICP, and the chemical composition as well as particle sizes using SEM-EDS. The organic UV absorbers octocrylene and oxybenzone are quantified using HPLC. With the incorporation of these interesting characterization techniques in second or fourth-year chemistry courses, and by having students analyze sunscreen samples that are medically relevant in terms of health effects, students engage in timely research and at the same time gain exposure to a variety of instruments in the analysis of a familiar household product.

Computer simulation and optimization for reversed-phase HPLC separation: A novel algorithm simulating and optimizing the non-linear and non-ideal separation process in analytical chromatography

A novel algorithm simulating and optimizing separation process in analytical chromatography is developed. The non-linearity of analytical chromatography was based on Langmuir isotherm equation. Meanwhile, non-ideal chromatography shows non-equilibrium distribution of the solutes between the two phases. Then, a new concept named “phase transfer probability factor” was defined to describe the non-equilibrium distribution extent. Subsequently, the algorithm can be used to fit the reversed-phase HPLC (RP-HPLC) experimental and simulated chromatograms associated with the aid of genetic algorithm (GA). Moreover, based on the method, the RP-HPLC separation process of the mixture samples including phenol, hydroquinone, resorcinol, and 4-nitrophenol was simulated and optimized with the aid of multiple layer perceptron artificial neural networks (MLP-ANNs) and GA. The proposed method can easily be generalized to other kinds of analytical chromatographic techniques.