Trifluoroacetic Acid Concentration Research Articles

Optimum concentration of trifluoroacetic acid for reversed-phase liquid chromatography of peptides revisited

Trifluoroacetic acid (TFA) remains the dominant mobile phase additive for reversed-phase high-performance liquid chromatography (RP-HPLC) of peptides after more than two decades since its introduction to this field. Generally, TFA has been employed in a concentration range of 0.05–0.1% (6.5–13 mM) for the majority of peptide separations. In order to revisit the question as to whether such a concentration range is optimum for separations of peptide mixtures containing peptides of varying net positive charge, the present study examined the effect of varying TFA concentration on RP-HPLC at 25 and 70 °C of three groups of synthetic 10-residue synthetic peptides containing either one (+1) or multiple (+3, +5) positively charged groups. The results show that the traditional range of TFA concentrations employed for peptide studies is not optimum for many, perhaps the majority, of peptide applications. For efficient resolution of peptide mixtures, particularly those containing peptides with multiple positive charges, our results show that 0.2–0.25% TFA in the mobile phase will achieve optimum resolution. In addition, the use of high temperature as a complement to such TFA concentration levels is also effective in maximizing peptide resolution.

Selectivity differences in the separation of amphipathic $alpha;-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0Effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of α-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac–EAEKAAKEXEKAAKEAEK–amide (with position X in the centre of the hydrophobic face of the α-helix), where position X is substituted by l- or d-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.

Haloacetic acids in the aquatic environment. Part II: ecological risk assessment

Haloacetic acids (HAAs) are environmental contaminants found in aquatic ecosystems throughout the world as a result of both anthropogenic and natural production. The ecological risk posed by these compounds to organisms in freshwater environments, with a specific focus on aquatic macrophytes, was characterized. The plants evaluated were Lemna gibba, Myriophyllum spicatum and M. sibiricum and the HAAs screened were monochloroacetic acid (MCA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), trifluoroacetic acid (TFA) and chlorodifluoroacetic acid (CDFA). Laboratory toxicity data formed the basis of the risk assessment, but field studies were also utilized. The estimated risk was calculated using hazard quotients (HQ), as well as effect measure distributions (EMD) in a modified probabilistic ecological risk assessment. EMDs were used to estimate HAA thresholds of toxicity for use in HQ assessments. This threshold was found to be a more sensitive measure of low toxicity than the no observed effect concentrations (NOEC) or the effective concentration (EC10). Using both deterministic and probabilistic methods, it was found that HAAs do not pose a significant risk to freshwater macrophytes at current environmental concentrations in Canada, Europe or Africa for both single compound and mixture exposures. Still, HAAs are generally found as mixtures and their potential interactions are not fully understood, rendering this phase of the assessment uncertain and justifying further effects characterization. TCA in some environments poses a slight risk to phytoplankton and future concentrations of TFA and CDFA are likely to increase due to their recalcitrant nature, warranting continued environmental surveillance of HAAs.

Evaluation of an embedded polar C 4 phase for hydrophobic protein analysis by reversed-phase liquid chromatography

A C 4 column (InertsilWP300 C 4) was developed for a protein analysis in reversed-phase chromatography. Polar groups were embedded on this phase. And this column was evaluated with low concentration of trifluoroacetic acid (TFA) and formic acid. Test C 4 phases that controlled carbon loading and commercially available columns were compared with this embedded C 4 column. The test column with higher carbon loading showed good separations of proteins at low concentrations of TFA, although hydrophobic proteins were broadened. On the other hand, embedded polar C 4 packings showed ideal retention behavior for proteins, which is independent of ion-pairing effects of additives in mobile phase. The reason was concluded to be the shielding effects of polar groups on the sorbents, which reduce silanophilic interactions with polar groups of proteins.

Data preprocessing by wavelets and genetic algorithms for enhanced multivariate analysis of LC peptide mapping

Peptide mapping by means of liquid chromatography is a powerful technique used for the characterisation and analysis of the primary structure of proteins. Subtle changes in the covalent structure of the protein can be detected by means of the chromatographic profile (fingerprint). Chromatographic methods, however, display variations in the chromatographic profile even at identical instrumental settings and sample conditions. These variations may be due to changes of the chromatographic conditions, e.g. slight shifts in column temperature, and degradation or alterations of the stationary phase or small changes in the trifluoroacetic acid (TFA) concentration. Such variations may result in varying retention times and peak shapes of the analytes and differences in the chromatographic baseline, thereby having a detrimental impact on the results obtained on multivariate analysis of peptide maps. In order to reduce the non-sample-related variations and to be able to more fully extract the information in peptide mapping, approaches for achieving this objective are outlined in the present study. These methods are denoising and data compression of the chromatograms by wavelets, baseline corrections by linear interpolation, and peak shift alignments towards a target chromatogram by means of a genetic algorithm. Visual inspections of preprocessed chromatograms and principal component analysis (PCA) score plots demonstrate the efficiency of the methodology used. Furthermore, deliberately added changes, e.g. insertions of small Gaussian peaks (outliers), are more easily detected by the proposed methods than from the original chromatograms by multivariate analysis.

Airborne haloacetic acids.

Haloacetic acid (HAA) concentrations were measured in air samples from a semi-rural and a highly urbanized site in southern Ontario throughout 2000 to investigate their sources and gas-particle partitioning behavior. Denuders were efficient for collection of gaseous HAAs, and the particle phase was collected on a downstream quartz filter with negligible breakthrough. Total HAA concentrations (i.e., gas + particles) ranged between <0.025 and 19 ng m(-3) for individual HAAs at both sites. The dominant airborne HAA was monochloroacetic acid (MCA), followed in decreasing order by dichloroacetic acid (DCA), trifluoroacetic acid (TFA), and trichloroacetic acid (TCA). Difluoroacetic acid (DFA), monofluoroacetic acid (MFA), and chlorodifluoroacetic acid (CDFA) were also frequently detected at lower concentrations. Between sites, TFA, DFA, MFA, and TCA concentrations were significantly higher in Toronto, while CDFA concentrations were higher in Guelph. HAAs were primarily in the gas phase all year; however, during colder months, particle-phase HAA concentrations increased relative to the gas phase. Trichloroacetic acid had the highest particle fraction (phi) for all detected HAAs, with a mean phi of 0.51 and 0.56 for Guelph and Toronto, respectively, and both vapor pressure and acid strength appeared to influence gas-particle partitioning. Temporal trends at both sites were partially explained by temperature, short-wave radiation, and particle mass (PM10), leading to indications of the respective sources. A simple deposition model indicated that dry deposition of TFA and TCA should not be neglected in temperate mid-latitude environments and that precipitation concentrations can be successfully predicted by the Henry's law constant.

Changes in tropospheric composition and air quality due to stratospheric ozone depletion.

Increased UV-B through stratospheric ozone depletion leads to an increased chemical activity in the lower atmosphere (the troposphere). The effect of stratospheric ozone depletion on tropospheric ozone is small (though significant) compared to the ozone generated anthropogenically in areas already experiencing air pollution. Modeling and experimental studies suggest that the impacts of stratospheric ozone depletion on tropospheric ozone are different at different altitudes and for different chemical regimes. As a result the increase in ozone due to stratospheric ozone depletion may be greater in polluted regions. Attributable effects on concentrations are expected only in regions where local emissions make minor contributions. The vertical distribution of NOx (NO + NO2), the emission of volatile organic compounds and the abundance of water vapor, are important influencing factors. The long-term nature of stratospheric ozone depletion means that even a small increase in tropospheric ozone concentration can have a significant impact on human health and the environment. Trifluoroacetic acid (TFA) and chlorodifluoroacetic acid (CDFA) are produced by the atmospheric degradation of hydrochlorofluorocarbons (HCFCs) and hydrofluorocarbons (HFCs). TFA has been measured in rain, rivers, lakes, and oceans, the ultimate sink for these and related compounds. Significant anthropogenic sources of TFA other than degradation HCFCs and HFCs have been identified. Toxicity tests under field conditions indicate that the concentrations of TFA and CDFA currently produced by the atmospheric degradation of HFCs and HCFCs do not present a risk to human health and the environment. The impact of the interaction between ozone depletion and future climate change is complex and a significant area of current research. For air quality and tropospheric composition, a range of physical parameters such as temperature, cloudiness and atmospheric transport will modify the impact of UV-B. Changes in the chemical composition of the atmosphere including aerosols will also have an impact. For example, tropospheric OH is the 'cleaning' agent of the troposphere. While increased UV-B increases the OH concentration, increases in concentration of gases like methane, carbon monoxide and volatile organic compounds will act as sinks for OH in troposphere and hence change air quality and chemical composition in the troposphere. Also, changes in the aerosol content of the atmosphere resulting from global climate change may affect ozone photolysis rate coefficients and hence reduce or increase tropospheric ozone concentrations.

Electrochemical Polymerization of Thianaphthene in Mixed Electrolytes of Boron Trifluoride Diethyl Etherate and Trifluoroacetic Acid

AbstractSubstrate‐supported polythianaphthene (PTN) films have been electrochemically synthesized by direct oxidation of thianaphthene on platinum or stainless steel electrode in mixed electrolytes of boron trifluoride diethyl etherate (BFEE) and trifluoroacetic acid (TFA). The oxidation potentials of the monomer in these media are considerably lower than those in neutral solvents. Increasing the content of TFA in the mixed electrolytes can effectively decrease the oxidation potential and increase the coupling rate of the monomer. On the other hand, a high TFA concentration can result in a negative effect on the properties of the as‐formed polymer. The mixed electrolyte of BFEE + 16.7% TFA was found to be the most suitable medium for the electrosyntheses of PTN. PTN in dedoped state is soluble in usual strong polar organic solvent such as dimethyl sulfoxide (DMSO). Fluorescent spectral studies indicate that the polymer is a blue light emitter. magnified image

The effect of triethanolamine on the formation of sol–gel derived fluoroapatite/hydroxyapatite solid solution

Fluoroapatite/hydroxyapatite (FA/HA) solid solution shows good bioactivity and stability, and is one of promising biomaterials. In this work, 1.67Ca(NO 3) 2–PO(OEt) x (OH) 3− x ethanol solutions with different concentrations of trifluoroacetic acid and triethanolamine were mixed. The mixed solutions were transformed into the as-prepared powder on a hot plate. After the as-prepared powder was calcined at 500 or 900 °C, a pure FA/HA phase could be obtained. The fluorine contents in the FA/HA strongly depended on the ratios of triethanolamine/trifluoroacetic acid. The presence of triethanolamine in the mixed solutions enhances the dissociation of trifluoroacetic acid and accelerates the formation of unevaporable Ca(OOCF 3C) x (NO 3) 2− x complex species, as a result, more fluorine could be incorporated into the apatite to form the solid solution.

Distribution of haloacetic acids in the water columns of the Laurentian Great Lakes and Lake Malawi.

Haloacetic acids (HAAs) are persistent and mildly phytotoxic compounds that have been detected in many aquatic environments, including the waters of the Great Lakes. Sources of HAAs, especially of trifluoroacetic acid (TFA), are not well understood. In this study we assessed the influence of urbanization on the concentrations and profiles of HAAs in the Laurentian Great Lakes and in Lake Malawi, an African Great Lake. Vertical depth profiles for these compounds were taken for each of the Great Lakes with additional profiles taken 2 years later for Lakes Erie and Ontario. The results showed that while TFA was relatively constant throughout the water column, the chloroacetic acids (CAAs) varied with depth. There was a trend of increasing TFA proceeding from Lake Superior to Lake Ontario (18-150 ng/L). Total CAA concentrations were relatively constant throughout the lakes (approximately 500 ng/L) with dichloroacetic acid being the most abundant. No bromoacetic acids were detected. In the Detroit River, a connecting channel between Lakes Huron and Erie, the TFA values were similar to those in Lake Huron, but the CAAs levels were higher than in the upstream lakes and dependent on location, indicating inputs from urban areas along the river. These results were compared to those from Lake Malawi, which has a high population density within the watershed but no heavy industry. CAAs were nondetectable, and TFA concentrations were just at the detection limit (1 ng/L). Total HAA in the water column of Lakes Superior and Huron was compared to annual precipitation inputs at a site situated near both lakes. For Lake Huron, precipitation was a minor contributor to the total HAA inventory of the lake, but for Lake Superior precipitation could be the major contributor to the mass of HAA in this lake. Generally, high HAA levels paralleled the degree of industrial activity in the adjacent waters.

EFFECTS OF HCFC-123 EXPOSURE TO MATERNAL AND INFANT RHESUS MONKEYS ON HEPATIC BIOCHEMISTRY, LACTATIONAL PARAMETERS AND POSTNATAL GROWTH

ABSTRACTPeroxisome proliferators are a class of nongenotoxic rodent hepatocarcinogens that cause peroxisome proliferation and liver tumors when administered to rats and mice; but other species, including guinea pigs, dogs, and primates are less sensitive or refractory to the induction of peroxisome proliferation. Therefore, rodent peroxisome proliferators are not believed to pose a hepatocarcinogenic hazard to humans. Some peroxisome proliferators produce developmental toxicity in rats that is expressed as suppressed postnatal growth. To evaluate the relevance of the rat developmental effect to primates, groups of 4 lactating female Rhesus monkeys and their infants were exposed for 6 h/day, 7 days/week for 3 weeks to air or 1000 ppm HCFC-123. Animals were evaluated for clinical signs, body weights, clinical pathology parameters, and biochemical and pathological evaluations of liver biopsy samples. The effect of HCFC-123 exposure on milk quality (protein and fat concentration) was evaluated. The concentrations of HCFC-123 and the major metabolite, trifluoroacetic acid (TFA), were measured in the blood of the mothers and infants and in the milk. Exposure of monkeys to 1000 ppm HCFC-123 did not result in exposure-related clinical observations, or changes in body weight, appetence and behavior. There were no exposure-related effects on serum triglycerides, cholesterol, or glucose levels. HCFC-123 and TFA were present in milk, although maternal HCFC-123 exposure did not affect milk protein and fat content. In general, HCFC-123 was not detected in maternal or infant blood. TFA was detected in the majority of the mothers and TFA levels in infants ranged from 2 to 6 times higher than levels in the corresponding maternal blood. A pharmacokinetic analysis in a maternal animal indicated a peak concentration of TFA at approximately 1 h post-exposure, with a half-life of approximately 20 h. Liver microsomal P450 and peroxisome oxidase activities showed exposure-related decreases in CYP4A1 and CYP2E1 and acyl-CoA oxidase for animals exposed to HCFC-123. Microscopic evaluation of maternal liver from HCFC-123 exposed animals revealed mild to moderate centrilobular hepatocyte vacuolation, trace to mild centrilobular necrosis, and trace to mild subacute inflammation. The histopathological damage and altered hepatic biochemical activities produced by HCFC-123 in monkeys are not consistent with the HCFC-123 peroxisome proliferation response observed in rat livers. These findings demonstrate that HCFC-123 is not a peroxisome proliferator in adult Rhesus monkeys and postnatal exposure to HCFC-123 does not affect body weight of nursing infant monkeys.

Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography.

This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E. coli. protein (ECP) impurities. The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem. Several operational parameters were investigated to study their effects on the yield of the displacement process. The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification. Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step. The process was successfully scaled-up with respect to particle and column diameter. The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.

Solvent Dependence of Electronic Relaxation in All-trans Retinal Studied by One- and Two-Photon Induced Transient Absorption

One- and two-photon excited ultrafast transient absorption measurements are reported for all-trans retinal in polar, nonpolar, and hydrogen-bonding solvents. A comparison of the response following one- and two-photon excitation allows assignment of the observed decays to the 1Bu+-like and 1Ag--like excited states. In the polar, hydrogen-bonding solvent ethanol, the transient absorption changes following one-photon excitation are dominated by a 2-ps response observed both in the decay of transient absorption and in the decay of stimulated emission observed in the region from 600 to 750 nm. Because of the strong stimulated emission gain, this response can be assigned to decay of the strongly optically allowed “1Bu+” state. With two-photon excitation of all-trans retinal in ethanol, a 200−300 fs decay component appears as well and is assigned to decay of the “1Ag-” state, which has a large two-photon cross section. In contrast, for all-trans retinal in hexane, both 200−300 fs and 2-ps decay components are observed following one-photon excitation, and no stimulated emission is detected. A comparison of one- and two-photon induced transient absorption leads to an assignment of the decay events. The ultrafast responses of all-trans retinal in the polar, aprotic solvents acetonitrile and propionitrile are found to be similar to the response in ethanol, indicating that protic solvents act through solvent polarity rather than a specific effect of hydrogen bonding. The effect of hydrogen bonding was studied further for all-trans retinal in mixed solvents with varying concentrations of trifluoroacetic acid, a strong hydrogen-bond donor, in hexane. As the ratio of trifluoroacetic acid to retinal increased from 1:5 to 10:1, the ultrafast response changed smoothly from “hexane-like” to “ethanol-like”, with an increasingly strong stimulated emission component having a 2-ps decay, in agreement with the increased fluorescence quantum yield observed in polar and hydrogen-bonding solvents. A model is proposed incorporating two populations of retinal having different electronic relaxation pathways to explain the solvent-dependent photophysics of all-trans retinal.

Trifluoroacetic acid in ancient freshwater

Substantial concentrations (typically 10–100 ng/l) of trifluoroacetic acid (TFA) have been reported in contemporary saltwater and freshwater. It is well established that TFA is produced as the result of industrial activities. It is unclear whether there are any significant natural sources of this compound. To provide insight into the likely magnitude of possible natural sources of TFA we collected five samples of pre-industrial (>2000 year old) freshwater from Greenland and Denmark; there was no detectable TFA (<2 ng/l) in any of these samples. The available data suggest that TFA is not a naturally occurring trace component of the freshwater environment.

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

Trichloroacetic acid and trifluoroacetic acid-induced unfolding of cytochrome c: stabilization of a native-like folded intermediate

A systematic investigation of trichloroacetic acid (TCA) and trifluoroacetic acid (TFA)-induced equilibrium unfolding of native horse cytochrome c has been carried out using a combination of optical spectroscopy and electrospray ionization mass spectroscopy (ESI MS). In the presence of an increasing concentration of TCA the native cytochrome c does not undergo significant unfolding but stabilization of a partially folded intermediate is observed. This TCA-induced partially folding intermediate of cytochrome c had an enhanced secondary structure and slightly disrupted tertiary structure compared to native protein and undergoes extensive unfolding in the presence of TFA. However, in the presence of an increasing concentration of TFA, cytochrome c was found to undergo extensive unfolding characterized by a significant breakdown of the secondary and tertiary structure of protein. The TFA-unfolded cytochrome c was found to undergo folding in the presence of TCA and low guanidine hydrochloride (GdmCl) resulting in the stabilization of the partially folded intermediate. The effectiveness of TCA as compared to TFA in the stabilization of intermediates was further supported by the observation that low concentrations of TCA were found to induce refolding of HCl-denatured cytochrome c whereas, under similar concentrations of acid, no significant effect on the unfolded structure of protein was observed in the presence of TFA. ESI MS studies indicated that the trichloroacetate anion has a greater affinity for cytochrome c compared to trifluoroacetate anion, which might be the reason for the stabilization of the native-like folded intermediate during TCA-induced denaturation of cytochrome c as compared to extensive unfolding observed in the presence of TFA.

N- and C-Attacks on aromatics of phenylnitrenium ion generated from N-phenylhydroxylamine in the presence of trifluoroacetic acid containing polyphosphoric acid or trifluoroacetic anhydride

A phenylnitrenium ion formed from N-phenylhydroxylamine in the presence of trifluoroacetic acid (TFA) containing polyphosphoric acid (PPA) interacts with the counterion −O2CCF3 and the unshared electron-pair of H2O, showing Hammett’s ρ values −5.2 and −4.0 for N- and C-attacks on aromatics PhX (X = H, Me, Et, Ph and Cl), respectively. The ratio N-/C-attack for this nitrenium ion is lower than for the nitrenium ion interacting with only the counterion; the latter nitrenium ion is generated by the use of trifluoroacetic anhydride (TFAA) instead of PPA or by reaction of phenyl azide with aromatics in TFA. The ratio for the former nitrenium ion is affected by the aromatic substituent X: X = Me > X = Et > X = Ph > X = H > X = OMe at 20 and 50 °C. The order for X = Cl is between those of X = H and X = Ph at 20 °C, but highest at 50 °C. The ratio is increased by higher reaction temperature and by decreased concentration of TFA. The results are demonstrated from the mechanistic viewpoint for the nitrenium ions.

Simplified Method for Trace Analysis of Trifluoroacetic Acid in Plant, Soil, and Water Samples Using Headspace Gas Chromatography

A simple and sensitive analytical procedure was developed to determine the concentration of trifluoroacetic acid (TFA) in plant, soil, and water samples. The analysis involves extraction of TFA by sulfuric acid and methanol followed by derivatization to the methyl ester of TFA (MTFA). This is accomplished within a single vial without complex extraction procedures. The highly volatile MTFA is then analyzed using headspace gas chromatography. The spike recovery trials from all media ranged from a low of 86.7% to a high of 121.4%. The relative standard deviations (RSDs) were typically below 10%, although some tests had higher RSDs due to low concentrations near the detectable limit. The minimum detectable limit (MDL) for the method was 34 ng/g for dry plant material, 0.20 ng/g for soil, and 6.5 ng/L for water. This is the first practical method for low-level analysis of TFA in plants and soil, and it is a significant improvement over previous methods for TFA in water.

Determination of selenoamino acids by high-performance liquid chromatography-hydraulic high pressure nebulization-atomic fluorescence spectrometry

For the analysis of the most important selenoamino acids [selenomethionine (SeMet), selenoethionine (SeEt) and selenocystine (SeCys)] a HPLC-hydraulic high pressure nebulization (HHPN)-AFS system was assembled. The separation was accomplished on a C 18 column with the help of trifluoroacetic acid (TFA) and methanol in water as eluent. The effects of methanol and TFA concentrations on the retention time were investigated. The argon and hydrogen gas flow rate on the AFS signal intensity was optimized. The detection limit and the retention time for SeCys, SeMet and SeEt were 50, 42 and 71 ng ml –1 and 2.1, 3.9 and 6.9 min, respectively.

Isolation of Colistin A and B Using High-Speed Countercurrent Chromatography

High-speed countercurrent chromatography was successfully applied to the isolation of colistin-A and colistin-B from a commercial colistin preparation. As the first step. TLC and HPLC analysis conditions for the colistin components were established. Using these techniques, a two-phase solvent system composed of n-butanol-0.04M aqueous trifluoroacetic acid (TFA) (1:1) was selected for high-speed CCC, where the concentration of TFA was the major factor in controlling the partition coefficients of the colistin components. Yields of colistin-A and colistin-B were 9 mg each from 20 mg of the commercial sample, and the purity of each component was over 90%. Fast atom bombardment (FAB) mass spectrometry was utilized to confirm the nature of the isolated components.