Various Forms Of Chromatography Research Articles

Oxytocin Peptide Detection with Carbon Electrodes and Fast Scan Cyclic Voltammetry

Fast scan cyclic voltammetry (FSCV) used in conjunction with carbon fiber-microelectrodes (CFMEs) is an electrochemical technique used to detect neurotransmitters with a sub-section temporal resolution. This allows for a sensitive and selective subsecond analysis of changes in the brain. Typically, this technique is used to detect catecholamines in the brain, but recently, it has branched out into purinergic signaling and peptide neurotransmitters. This technique is not only able to detect neurochemical changes at a quick timescale, but it is able to detect a wide range of molecules with a high spatiotemporal resolution. This results in having chemical selectivity that may not be available in other electrochemical techniques. The nature of CFMEs also allows for the selective targeting of specific brain regions to ensure that precise measurements can be made.The peptide oxytocin is important in understanding stress, depression and anxiety, and it is one of the main components in clarifying signals sent throughout the brain. Oxytocin plays vital roles for women during childbirth as well as for lactation. Oxytocin is typically analyzed by taking a blood sample and running assays that can take a while to obtain the results. Peptides, like oxytocin, can also be detected through methods like mass spectrometry and various forms of chromatography but are sometimes very time consuming and can be destructive. On the other hand, it has been known that there are some amino acids are redox active and can be measured with electrochemical techniques.The detection of tyrosine-containing peptides using electrochemical techniques, such as oxytocin, can be quite difficult. The short amount of time that these molecules are in the extracellular space and its tendency to foul electrodes when oxidized make it hard to collect accurate and consistent data. Also, there is a higher concentration of more electroactive molecules that would make it harder to be specific for these molecules. However, due to the nature of FSCV, it is possible to change the waveform to be able to prevent the fouling of tyrosine on the surface of the electrode and improve the detection of tyrosine-containing peptides in biological samples. Recently, a modified sawhorse waveform (MSW) has been formulated that is able to specifically detect tyrosine in the methionine-enkephalin (M-ENK) neuropeptide using two specific scan rates in the anodic sweep and a short holding period at the switching potential. The MSW waveform was applied at 10 Hz with the holding potential at -0.2 V, then increased to +0.6 V at 100 V/s, and further increased to +1.2 V at 400 V/s. This potential was held in place for 3 ms before sweeping back down to the folding potential of -0.2 V at 100 V/s. This results in high specificity toward tyrosine and eliminates the fouling on the electrode. In this study, we target the tyrosine residue on oxytocin with a MSW waveform and characterize it with carbon fiber microelectrodes and fast scan cyclic voltammetry. We show the first electrochemical detection of oxytocin with FSCV with high sensitivity and spatiotemporal resolution that is distinguished from tyrosine and other amino acids. This work could potentially help further understand oxytocin’s role in vivo and importance in behavioral and disease states.

Flurbiprofen: A Study of the Behavior of the Scalemate by Chromatography, Sublimation, and NMR

2-(2-Fluoro-4-biphenyl) propionic acid (flurbiprofen), from the phenylalkanoic acid family of nonsteroidal anti-inflammatory drugs (NSAID’s), is currently on the pharmaceutical market as a racemate. This racemic compound was tested for its propensity to undergo the self-disproportionation of enantiomers (SDE) phenomenon by various forms of chromatography (SDEvC), such as routine gravity-driven column chromatography, medium-pressure liquid chromatography (MPLC), preparative thin-layer chromatography (PTLC), and size-exclusion chromatography (SEC), as well as by sublimation (SDEvS). Furthermore, examination by nuclear magnetic resonance (NMR) in various solvents found that flurbiprofen exhibited the phenomenon of self-induced diastereomeric anisochronism (SIDA). By measurement of the diffusion coefficient (D), the longitudinal relaxation time (T1), and the transverse relaxation time (T2) using NMR, as well as by electrospray ionization-mass spectrometry (ESI-MS) examinations, the preferred intermolecular association was found to be solvent dependent, e.g., heterochiral association was preferred in toluene, while homochiral association was preferred in more polar solvents. This study also attempted, unsuccessfully, to correlate the NMR measurements of flurbiprofen with chromatographic outcomes for the rationalization and prediction of chromatographic results based on NMR measurements. Because the intermolecular hydrogen bonding of the acid groups in flurbiprofen overwhelmingly predominates over other intermolecular interactions, flurbiprofen seemed to represent a good test case for this idea. The behavior of scalemic samples of flurbiprofen is important, as, although it is currently dispensed as a racemate, clinical applications of the R enantiomer have been investigated. SDEvC and SDEvS both have ramifications for the preparation, handling, and storage of enantioenriched flurbiprofen, and this concern applies to other chiral drugs as well.

A method for screening active components from Chinese herbs by cell membrane chromatography-offline-high performance liquid chromatography/mass spectrometry and an online statistical tool for data processing

Cell membrane chromatography (CMC) has been successfully applied to screen bioactive compounds from Chinese herbs for many years, and some offline and online two-dimensional (2D) CMC-high performance liquid chromatography (HPLC) hyphenated systems have been established to perform screening assays. However, the requirement of sample preparation steps for the second-dimensional analysis in offline systems and the need for an interface device and technical expertise in the online system limit their extensive use. In the present study, an offline 2D CMC-HPLC analysis combined with the XCMS (various forms of chromatography coupled to mass spectrometry) Online statistical tool for data processing was established. First, our previously reported online 2D screening system was used to analyze three Chinese herbs that were reported to have potential anti-inflammatory effects, and two binding components were identified. By contrast, the proposed offline 2D screening method with XCMS Online analysis was applied, and three more ingredients were discovered in addition to the two compounds revealed by the online system. Then, cross-validation of the three compounds was performed, and they were confirmed to be included in the online data as well, but were not identified there because of their low concentrations and lack of credible statistical approaches. Last, pharmacological experiments showed that these five ingredients could inhibit IL-6 release and IL-6 gene expression on LPS-induced RAW cells in a dose-dependent manner. Compared with previous 2D CMC screening systems, this newly developed offline 2D method needs no sample preparation steps for the second-dimensional analysis, and it is sensitive, efficient, and convenient. It will be applicable in identifying active components from Chinese herbs and practical in discovery of lead compounds derived from herbs.

Proceedings of the 2017 Viral Clearance Symposium, Session 2.2: DSP Unit Operations–Purification Unit Operations

The process capability and potential for various forms of chromatography to remove viruses have been discussed extensively in the literature, including the observed variability in performance of some unit operations such as Protein A and cation exchange (CEX). Some unit operations such as anion exchange (AEX) have shown robustness over a wide range of operating conditions. The robustness and effectiveness of the AEX step combined with a detailed understanding of the mechanisms that result in virus and impurity partitioning versus protein partitioning (e.g. conductivity, loading, pH range) support the feasibility of a generic or modular claim for AEX. A more fundamental understanding of the mechanisms for other chromatographic media (Protein A, CEX, and mixed-mode) could lead to more effective and more robust log reduction value (LRV) claims for these steps as well. Specific examples of CEX and mixed-mode chromatography were explored in the session and were also discussed in detail at the 2013 and 2015 Viral Clearance Symposia. Although some gaps remain in the mechanistic understanding of these unit operations, significant progress has been made and was reported at the 2017 Viral Clearance Symposium. It is important to note that recent publications on the mechanisms of viral clearance for mixed-mode chromatography and a framework for measurement of relative hydrophobicity have provided insights and new tools to better define the operating space and critical process parameters. The session also explored the use of next-generation mixed-mode adsorbers and the potential mechanisms contributing to the observed viral clearance. Gaps were also identified (e.g. integrity test when size-based mechanisms are used) and should be addressed to ensure robust viral clearance for these integrated and productive emerging unit operations.LAY ABSTRACT: Preparative chromatography is the most widely used unit operation for purification of therapeutic proteins. This session focused on the potential for various forms of chromatography to remove viruses. To advance to the next level of process, understanding the virus removal mechanism of different types of chromatography was addressed.

Abstract 5544: Attempts to synthesize 5,7-dihydroxy-3H-1,2-dithiolo[4,3-d]- pyrimidine, M-47, a potential new treatment for drug- resistant breast cancer

Abstract M-47, 5,7-Dihydroxy-3H-1,2-dithiolo[4,3-d]pyrimidine, represents a novel class of antineoplastic agents both in structure and apparent mechanism of action. The synthesis of M-47 has been shown to be problematic. Mass spectroscopy and HPLC suggest the presence of certain oxidative byproducts in minor quantities along with the desired analog. Repeated efforts to purify M-47 via crystallization, extraction, chemical modification, and various forms of chromatography have failed. Although enrichment of the sample in excess of 90% is possible, absolute purity has not been achieved. Despite the presence of minor impurities this analog was tested against a variety of cancer cell lines in vitro. Interestingly, M-47 displayed equipotent activity against certain breast cancer lines and their multidrug resistant progeny Furthermore, in vitro testing of various ratios of M-47 with its oxidative byproducts indicated that they possessed similar IC50's suggesting that cellular oxidation of M-47 might be a necessary step in its cytotoxic pathway. The profound activity of M-47, especially against drug-resistant tumor cells, warrants further investigation into the chemistry of these compounds, their biological activity and their possible mechanism of action. Note: This abstract was not presented at the meeting. Citation Format: Canio J. Marasco, Joseph A. Dunn, Paula J. Pera, Alexander E. Macubbin. Attempts to synthesize 5,7-dihydroxy-3H-1,2-dithiolo[4,3-d]- pyrimidine, M-47, a potential new treatment for drug- resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5544. doi:10.1158/1538-7445.AM2015-5544

Dynamics of reactive chromatographic columns of inert core/hollow/film coated spherical packing: An analytical solution and applications

The transient behavior of reactive-diffusive fixed-bed (chromatographic columns or reactors) packed with inert core/shell core/hollow/thin film coated monodisperse spherical rigid particles is investigated. We modeled the reactive sorption system with linear mass exchange law between the flowing and the stationary phases. The coupled governing partial differential equations were integrated analytically using Laplace transformations. The analytical closed form solution involves two infinite integrals. The closed form solution was integrated numerically and the breakthrough curves of the system were studied under the influence of the system parameters. The limiting cases are discussed, a simple asymptotic closed form solution for fixed-bed desorbers with monodisperse spherical particles is obtained, and results are generalized for similar kinetics of reactive chromatographic columns (fixed-beds) for diffusion/absorption into the inert core spherical packing. Further, potential applications of the mathematical model and results in processes such as liquid vapor extraction, various forms of chromatography, reactive sorption, multifunctional reactors, process intensification, and regeneration of ion exchange columns are briefly discussed.

Analysis of Complex Samples by Solvating Gas Chromatography (Supercritical Fluid to Gas Transition)

The various forms of chromatography are primarily determined by differences in the physical state of the mobile phases. The main chromatographic categories include gas chromatography (GC), liquid chromatography, and supercritical fluid chromatography. Adjusting a temperature and pressure will change the mobile phase from liquid to supercritical fluid to gas, with concomitant changes in their physical properties. In this paper, the technique transition-phase chromatography (TPC) is described. In TPC, different mobile phase conditions exist inside the column. This phase transformation within the column results in huge differences in density, solvating power, viscosity, diffusivity, and, as a consequence, in the chromatographic properties of the mobile phase. TPC experiments using capillary columns packed in our laboratory have shown that when the mobile phase is transformed from supercritical fluid to gas, high column efficiencies can be achieved. The transition from supercritical fluid to gas (also called solvating GC), a particular case of the TPC, is evaluated for the separation of complex real samples (environmental, food, and fuels).

Gut sugar analysis in field-caught parasitoids: Adapting methods originally developed for biting flies

The ability to determine the presence and identity of sugars in the guts of adult parasitoids in the field would aid researchers in addressing long-standing problems in parasitoid ecology. Until very recently, however, gut sugar analyses have not been carried out on parasitoids. This is despite the development and use of methodologies for gut sugar analyses in biting flies (mosquitoes, sand-flies, black-flies, horse- and deer-flies, and biting midges) for decades. Methods used have been the cold anthrone test for the detection of gut sugars, and various forms of chromatography for the identification of gut sugars. We review the use of these methods in biting fly research and then describe the nascent field of gut sugar analyses in parasitoids. Both cold anthrone and chromatography tests have begun to be used on field-caught parasitoids, and we describe progress from our own work. We used cold anthrone on the aphid parasitoid Aphelinus albipodus (Hymenoptera: Aphelinidae), and results from one field study show that approximately one-fifth of individuals tested were positive for gut sugars. The characteristics of the field site point to the primary source of these gut sugars as being aphid honeydew. We also analysed the gut contents of Diadegma insulare (Hymenoptera: Ichneumonidae), a parasitoid of diamondback moth. In this case, HPLC analyses showed that over 85% of field-captured individuals had fed upon sugars. These same analyses suggested that honeydew may have been a major source of the gut sugars in this case also, but the sugar profiles suggest some nectar feeding. Understanding the importance of various sugar sources on parasitoid activity and effectiveness will facilitate the incorporation of sugar sources in habitat manipulation programmes as a part of IPM.

Proteolysis and Lipolysis of Goat Milk Cheese

Numerous varieties of goat milk cheeses are produced worldwide. Maturation or ripening of goat and other species milk cheeses is governed by interplay of many different factors. Proteolysis and lipolysis are two major biochemical processes in the multifaceted phenomenon of cheese aging, which involves a variety of chemical, physical, and microbiological changes under controlled environmental conditions. Proteolysis of cheeses in general is influenced by several factors including plasmin, chymosin, protease from starter and nonstarter bacteria, pH and moisture levels of the curds, storage temperature and time, salt content, salt-to-moisture ratio, and humidity. Primary factors affecting lipolysis in cheeses are fatty acid composition, lipolytic enzymes, lipolytic microorganisms, moisture, temperature, storage time, oxygen, and surface area, etc. Several analytical techniques have been used to measure proteolysis of goat and (or) cow milk cheeses during ripening, such as solubility of peptides and amino acids in various solvents or precipitants, liberation of reactive functional groups, various forms of chromatography, and different forms of electrophoresis. Lipolysis of goat milk cheeses has been estimated by acid degree value (ADV), acid value, and free fatty acid concentration, while lipid oxidation of dairy goat products can be determined by peroxide value, thiobarbuturic acid value (TBA). Recent reports have shown that goat cheeses had greater rates of protein degradation than cow counterparts, and that aging time and temperature synergistically elevated most of proteolytic and lipolytic indices in goat cheeses. This paper will further discuss proteolytic and lipolytic characteristics of goat milk cheeses.

Applications of Extraction Chromatography in the Development of Radionuclide Generator Systems for Nuclear Medicine

Numerous methods have been described for the separation and purification of radionuclides for application in diagnostic and therapeutic nuclear medicine, among them ion exchange, solvent extraction, and various forms of chromatography. Although extraction chromatography has previously been shown to provide a means of performing a number of separations of potential use in radionuclide generator systems, the application of the technique to generator development has thus far been limited. Recent work directed at improved methods for the determination of radionuclides in biological and environmental samples has led to the development of a series of novel extraction chromatographic resins exhibiting enhanced metal ion retention from strongly acidic media and excellent selectivity, among them materials suitable for the isolation of 212Bi, 90Y, and 213Bi. These resins, along with extraction chromatographic materials employing functionalized supports to improve their physical stability or metal ion retention prop...

General Equation for Peak Capacity in Column Chromatography

In this study, the peak capacities for packed capillaries used in various forms of chromatography were investigated. Experiments found that the peak width at half-height (w1/2) linearly increased with increasing retention time when operating under isothermal, isobaric, and isocratic conditions in capillary gas chromatography, solvating gas chromatography, supercritical fluid chromatography, and liquid chromatography. From this relationship, a general peak capacity (n) expression was obtained. This expression covers conventional packed, packed capillary, and open tubular column chromatographies. With this general expression, it is possible to directly compare the separating capabilities of the various column types and chromatographic techniques.

Hindered transport of large molecules in liquid‐filled pores

AbstractTransport in liquid‐filled pores of molecular dimensions plays an important role in membrane separations, in various forms of chromatography, and in catalysis, to name a few examples. A frequent observation is that if the pore dimensions are of the same order as those of a solute molecule, the apparent diffusion coefficient of that solute is much lower than in bulk solution. Likewise, rates of convective transport of such solutes are generally lower than the product of bulk concentration and volume flow rate. Thus, solute transport is typically “hindered” or restricted. A key objective of research on hindered transport is to be able to predict the applicable transport coefficients from such fundamental information as the size, shape, and electrical charge of the solutes and pores. The present status of this research is reviewed.

Precipitating carbohydrate antigens of Bacteroides fragilis NCTC 9343.

An antigen fraction isolated by various forms of chromatography from phenol/water extracts of Bacteroides fragilis NCTC 9343 was devoid of phosphorus, fatty acids and amino acids. Rhamnose, fucose, galactose, glucose and a neutral sugar tentatively identified as arabinose were present at molar ratios of 1:1:1.2:9.0.9:0.7. The fraction contained an unidentified reducing and ninhydrin positive substance and trace amounts of glucosamine and galactosamine. The antigen fraction produced three immunoelectrophoretic bands against NCTC 9343 antiserum, and reacted in complement fixation tests.

Potent Immunosuppression by Oxidized Cholesterol

Abstract Sepharose 4B to which ε-dinitrophenyl lysine has been covalently attached behaves as an in vitro T-independent antigen when cultured for 4 days with mouse spleen cells. Almost total inhibition of the plaque-forming cell (PFC) response is obtained when liposomes composed of dimyristoylphosphatidylcholine (DMPC) and 0.05 μmole of a particular sample of recrystallized but aged cholesterol are cultured with 107 viable spleen cells; no inhibition is apparent when liposomes composed only of DMPC are used. After purification on Sephadex LH 20 with methylene dichloride, the cholesterol is no longer inhibitory. Fractionation by various forms of chromatography, together with biologic assays, has led to the conclusion that the major inhibitory component in the impure cholesterol is 25-hydroperoxycholesterol, an oxidation product of cholesterol whose Rf on silica gel, in several solvent systems, is very close to that of cholesterol. The peroxide can be reduced metabolically, or by the action of NaBH4, to give 25-hydroxycholesterol, a compound that is at least as immunosuppressive as its precursor. It is estimated that 25-hydroperoxycholesterol is present in the impure cholesterol sample at a level slightly less than 1%. Liposomes composed of DMPC:purified cholesterol:25-hydroxycholesterol (molor ratio 100:100:1) have been used to study the phenomenon in a better defined system. The biologic activity of such liposomes is essentially identical to that of liposomes containing DMPC:impure cholesterol (molar ratio 1:1). The liposomes are highly inhibitory when added in the first 24 hr of culture, but the system becomes rapidly less susceptible to inhibition during the next 24 hr. By the 3rd day of culture, which is when a rapid onset of measurable PFC becomes apparent, the cells are no longer susceptible to inhibition by 25-hydroxycholesterol. Almost total inhibition of the PFC response is obtained at a concentration of 25-hydroxycholesterol, which would be expressed as 5 × 10-7 M if the compound were in monomolecular dispersion (i.e., 0.5 nanomoles/1.0 ml culture volume). At this concentration there are 3 × 107 molecules of 25-hydroxycholesterol per viable cell originally cultured and the viability of the cells at the time of assay is not significantly different from that of controls lacking liposomes. Suppression of the in vitro primary response to sheep red blood cells (SRBC) by hydrocortisone occurs at about the same minimum concentration of that steroid. It is well known that 25-hydroxycholesterol is a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and hence inhibits cholesterol synthesis from acetate and other precursors of mevalonate in the biosynthetic pathway; however, mevalonate does not abrogate the inhibition of the PFC response by liposomes containing 25-hydroxycholesterol. In the absence of liposomes, the PFC response to dinitrophenylated beads is significantly and reproductibly greater in the presence of mevalonate than it is in its absence. This suggests that the cells are capable of assimilating the exogenous mevalonate. If this is true, a hypothesis that PFC suppression by 25-hydroxycholesterol is mediated by its action on HMG CoA reductase activity per se is not tenable.

Sphingolipid base metabolism sphinganine-1-phosphate lyase: Identification of ethanolamine 1-phosphate as product

Ethanolamine 1-phosphate has been characterized as a product of the action of rat liver microsomal sphinganine 1-phosphate lyase on erythro-sphinganine 1-phosphate. The product was characterized by various forms of chromatography, gas-liquid chromatography-mass spectral analysis of appropriate derivatives, and by conversion to ethanolamine. The results of detailed studies of the mass spectral fragmentation of the tetra-trimethylsilyl derivative of ethanolamine 1-phosphate are also reported.

L-trans-2,3-epoxysuccinate. A new substrate for fumarase

Fumarase (fimLarate hydratase, EC 4.2.1.2) has been found to catalyze the stereospecific hydration of the L-isaner of trans -2,3-epoxysuccinate to yield meso -tartrate. The product was- characterized by various forms of chromatography and by combined gas-liquid chromatography-mass spectrometry. Attempts to dissociate the fiunarate-hydrating activity of the enzyme from the epoxide-hydrating activity were unsuccessful.

A comparative study of the influence of restricted diffusion upon the moving rate of the solute in various forms of chromatography

The conditions under which the moving rate of a solute in chromatography is influenced by restricted diffusion are investigated. It appears that this phenomenon does not occur in practice in gas-liquid and liquid-liquid chromatography. In molecular sieve chromatography, the calculated eluent flow rate for the onset of the phenomenon agrees with the flow rate at which it has been observed.

Chromatography of polyamines

The separation of polyethylene polyamines by various forms of chromatography has been studied. Ethylenediamine is easily separated from the higher polyamines by thin-layer chromatography and by ion-exchange chromatography. Diethylenetriamine, triethylenetetramine and tetraethylenepentamine can be separated by gas-liquid chromatography on Teflon powder coated with 5% SE-30, and on a larger scale by ion-exchange chromatography. The latter technique involves absorption on AG-50X8, development with 2 M hydrochloric acid, and elution with 6% ammonia solution.