Separtation of metal tetrakis(p-tolyl)porphine complexes by reversed-phase high-performance liquid chromatography

Summary The feasibility of reversed-phase high-performance liquid chromatography for the separation of several metal complexes ofmeso-tetrakis(p-tolyl)porphine (TTP) is described. A combination of an octadecyl-bonded stationary phase with a non-aqueous polar mobile phase, such as an acetone-acetonitrile mixture, has proved effective for the separation. Thus, the TTP complexes of Mg, VO, Ni, Cu, Zn, and Pd and also TTP free acid were successfully separated in about 10min on a Li-Chrosorb RP-18 column (7m, 250×4mm i.d.) with a 7030 (vol/vol) mixture of acetone and acetonitrile at a flow-rate of 1 mlmin^–1.     

Monitoring of the synthesis ofcis andtrans ruthenium macrocyclic complexes by reversed-phase high-performance liquid chromatography

Summary The synthesis of the complex [Ru(cyclam)Cl₂]Cl (cyclam=1,4,8,11-tetraazacyclotetradecane) has been monitored by reversed-phase high-performance liquid chromatography. The analytical results obtained during the reaction have shown that it is feasible to identify and isolate the two isomerscis- andtrans- [Ru(cyclam)Cl₂]Cl. The use of an octadecylsiloxy preparative column enabled the separation and purification of these two isomers and the compounds have been obtained in high purity. The use of reversed-phase high-performance liquid chromatography has afforded complete analytical control of the syntheses of saturated nitrogendonor macrocyclic complexes of ruthenium, enabling identification of thecis andtrans isomers of the complex [Ru(cyclam)Cl₂]Cl.     

Monitoring of the synthesis ofcis andtrans ruthenium macrocyclic complexes by reversed-phase high-performance liquid chromatography

Summary The synthesis of the complex [Ru(cyclam)Cl₂]Cl (cyclam=1,4,8,11-tetraazacyclotetradecane) has been monitored by reversed-phase high-performance liquid chromatography. The analytical results obtained during the reaction have shown that it is feasible to identify and isolate the two isomerscis- andtrans- [Ru(cyclam)-Cl₂]Cl. The use of an octadecylsiloxy preparative column enabled the separation and purification of these two isomers and the compounds have been obtained in high purity. The use of reversed-phase high-performance liquid chromatography has afforded complete analytical control of the syntheses of saturated nitrogendonor macrocyclic complexes of ruthenium, enabling identification of thecis andtrans isomers of the complex [Ru(cyclam)Cl₂]Cl.     

Determination of cortisol in guinea pig urine by high performance thin-layer chromatography, high performance liquid chromatography, and thin-layer chromatography/radioimmunoassay

Summary In order to collect urinary samples from unrestrained guinea pigs, animals were kept in their familiar home cages with wood shavings for bedding. Cortisol was removed from shavings by a simple washing step, and an attempt was made to measure its concentrations by high performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC), or thin layer chromatography/radioimmunoassay (TLC-RIA). After intramuscular administration of 25 mg cortisol, cortisol excretion increased from about 20–30 μg/day to 400–500 μg/day (HPTLC: 531 μg/day, HPLC: 493 μg/day; TLC-RIA: 394 μg/day). Similarly, the treatment of the animals with 20 IU ACTH resulted in an augmented cortisol excretion, with mean values of 294 μg/day (HPTLC), 256 μg/day (HPLC) and 143 μg/day (TLC-RIA), respectively. The present study shows, for the first time, that cortisol excretion in unrestrained laboratory animals can be determined. Whilst the cortisol values measured by HPTLC and HPLC agree, the amounts measured by TLC-RIA were significantly lower. These differences are probably due to the presence of substances in urine or shavings which interfere with the radioimmunological determination. Hence, cortisol should be determined either by HPTLC or HPLC. Beside having a desirable specificity, these methods are more suited than TLC/RIA for steroid analysis since they confer the possibility of measuring additional steroids (e.g. precursors and/or metabolites of cortisol) in a single urine extract. This is especially the case for the HPTLC method since substances can be transformed into fluorescent derivatives.     

Retention behaviour of metal complexes with 5-sulphoquinoline-8-ol in reversed-phase ion-pair liquid chromatography

Summary The reversed-phase ion-pair liquid chromatographic behaviour of several metal complexes with 5-sulphoquinoline-8-ol (HQS) on an octadecyl-modified silica column was investigated by using a fluorescence detector. The aluminum complex gave two peaks on the chromatograms when eluents containing phosphate buffer were used. The effects of the pH of the eluent, HQS concentration, the nature of anions in the eluent, and the column temperature on the retention behaviour of the aluminum complex are discussed on the basis of the equilibria and the kinetics of the complexation of the aluminum ion with HQS and anions in the eluent. The equilibrium constant for the interconversion reaction between the two Al(III)-HQS species which correspond to the two peaks observed and the second stepwise formation constant for the Zn(II)-HQS complex were evaluated from the HPLC data obtained.     

Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

On-column endcapping and derivatization in reversed-phase high-performance liquid chromatography

Summary On-column endcapping and derivatization or regeneration of C₈ and C₁₈ reversed-phase HPLC columns with newly introduced reagents were studied. These treatments can increase column life expectancy by restoring retention times and original chromatographic characteristics of the columns. This is illustrated by examples. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Separation of polypeptide antibiotics by reversed-phase high-performance liquid chromatography

Summary The influence of different reversed-phase packings and the addition of acidic modifiers to the mobile phase was observed on the separation of basic and neutral polypeptide antibiotics by gradient elution. A dependence of pore size, coverage, reaction type and endcapping of the packings was not observed. Nevertheless, not all reversed-phase packings were suitable for the separation of polypeptides, especially of basic molecules. The addition of phosphoric or perchloric acid to the mobile phase prevented adsorption of the basic polypeptide antibiotics on the stationary phase.     

Monitoring the preparation of NAD analogs by reversed-phase high-performance liquid chromatography

Summary Pig brain NAD glycohydrolase immobilized on Affi-Gel 10 or nylon 6 was used for the conversion of NAD into 3-acetylpyridine adenine dinucleotide (APAD) or 3-aminopyridine adenine dinucleotide (AAD). A reversed-phase chromatographic system consisting of a C₁₈ Resolve column and phosphate buffer (pH 6.2)-methanol as the mobile phase was used to monitor the production of APAD and AAD.     

Comparison of provitamin A determination by normal-phase gravity-flow column chromatography and reversed-phase high performance liquid chromatography

Summary The provitamin A content of some food samples was determined by methods involving MgO: Hyflosupercel gravityflow column chromatography (GFCC) and reversed phase high performance liquid chromatography (HPLC), the quantitation being done by external standardization (HPLC-ES) or internal standardization (HPLC-IS) with Sudan. The results obtained with - and -carotene in carrots, -carotene and -cryptoxanthin in papaya and -carotene in tomato and kale agreed well, showing that any of the these techniques can be used, provided the analysis is done under optimum conditions. Good separation of the different provitamins using GFCC depends on the analyst's skill and visual acuity. HPLC-ES required a constant supply of provitamin standards, thus the varying purity of commercially available standards and the high instability of these compounds could pose grave problems. Due to the stability of Sudan, HPLC-IS appeared to be the method of choice although passage of the extract through a MgO: Hyflosupercel minicolumn was required prior to injection to separate chlorophylls, dihydroxy- and polyoxycarotenoids which would otherwise elute with Sudan. Nonconformity of the Sudan structure to those of the provitamins did not effect the quantitative results. The chromatographic separation, identity and quantification of the provitamins could be more easily established by using HPLC-IS, complemented with GFCC.     

Computer-assisted transposition of conditions in high-performance thin-layer chromatography to high-performance liquid chromatography for the separation of pesticides

Summary A computer-assisted method is presented for mobile phase selection for the optimal separation of pesticides by HPTLC and HPLC. The system is based on a plot of solute retention value and separation criterion vs. binary mobile phase composition for graphic optimization. The result of HPTLC can be transposed to HPLC for optimal separation. The transposition equation is given.     

Reversed-phase,high-performance,thin-layer chromatography of metal tetrakis (p-tolyl) porphine complexes on octadecylbonded stationary phase

Summary The metal complexes ofmeso-tetrakis (p-tolyl) porphine (TTP) are chromatographed on octadecyl-bonded silica gel thin-layer with various organic solvents of relatively high polarity used as the eluent. The mobility of the metal-TTP complex depends on its centralmetal, increasing in the order of Cu(II)<Pd(II)NI(II)<TTP free acid<V(IV) as vanadyl<Zn(II)<Mg(II), although some irregularities are found with the different developing solvents used. The concept of Hildebrand's solubility parameter is applied to the discussion of the mobility of each compound, on the solvents used as the developer. Successful separation of the TTP compounds is performed by using a mixture of acetone and acetonitrile as the developer, although the Ni- and Pd-complexes are not completely resolved.     

Retention behavior of dipeptide isomers in reversed-phase high-performance liquid chromatography

Summary The effects of eluent pH and organic modifier concentration on the capacity factor (k) and selectivity of dipeptide isomers were investigated. It has been observed that the variation in the logarithm of the capacity factor of the dipeptide isomers is linearly dependent on the organic modifier concentration (C_b), however, the selectivity is almost independent of it. Both capacity factor and selectivity were seriously affected by the pH of the eluent. Both the capacity factor and the intercept of the ln k vs. C_b plot increased with increasing van der Waals volume of the non-polar amino acid subunit of the dipeptides.     

Analysis of fatty acids in mouse cells using reversed-phase high-performance liquid chromatography

Summary A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to analyze various fatty acids in recombinant mouse L cells. These fatty acids were the metabolites of oleic acid. A process was developed to extract fatty acids from the cell samples before RP-HPLC analysis. The samples were first saponified with 0.5 M NaOH in 96% ethanol then extracted with acidified ethyl acetate. After extraction, the sample was dried and dissolved in HPLC-grade methanol. After centrifugation to remove insoluble impurities, the sample was applied to a C₁₈RP-HPLC column using a gradient of acetonitrile (ACN)-H₂O. The eluted fatty acids were monitored by ultraviolet (UV) absorption at 195 nm and identified by retention time and adsorption spectrum comparison. This method successfully resolved various fatty acids and provided a tool for the elucidation of the fatty acid metabolic pathway in the cells.     

Influence of injection parameters on column performance in nanoscale high-performance liquid chromatography

Summary The influence of the injection volume and the sample solvent on column efficiency has been evaluated in packed nano liquid chromatography using columns 150μ i.d. Evaluation of column performance was by means of reduced plate height (h) versus reduced velocity (v) for four polyaromatic hydrocarbon test compounds (PAHs). When compounds are dissolved in a weak solvent (such as MeCN: H₂O, 30∶70), and whatever the injection volume −60 or 200 nL-a gain in efficiency can be observed due to the well-known on-column focusing phenomenon, but keeping constant solute retention factors. Under optimized conditions (flow rate: 150 nLmin⁻¹, solvent sample MeCN: H₂O, 30∶70, injection volume 200 nL), a reduced plate height of 1.83 has been obtained for a 15 μm C₁₈ packing corresponding to 36000 plates m⁻¹, which illustrates the absence of any extracolumn band broadening under nano LC conditions.     

Determination of thiopental in human serum and plasma by reversed-phase high-performance liquid chromatography

Summary A high-performance liquid chromatographic method for the analysis of thiopental in 100l of human serum or plasma is described. The procedure involves protein precipitation with acetonitrile. The supernatant is directly injected into a chromatograph containing a reversed-phase CLC-ODS (Shimadzu) column. A 5050 (v/v) mixture of water-acetonitrile, at a flow-rate of 1.0ml/min is used as the mobile phase. Detection is carried out ata wavelength of 280nm. Total analysis time per sample is 10min. The assay was found to be linear in the range of 0.1 to 120g/ml. Reproducibility was good, with intra-assay coefficients of variation from 1.780 to 3.208% and inter-assay coefficients of variation from 3.241 to 4.860%. The absolute recoveries were 97.4 to 101,4%. Other drugs were tested for potential interference with the assay, but none was found.     

Porous graphitic carbon (PGC) high-performance liquid chromatography of diphosphine-bridged complexes with heteronuclear Au-M (M=Mn, Re) bonds

Summary The isocratic high-performance liquid chromatography of 1,1′-bis(diphenylphosphino)ferrocene (dppf), 1,1′-bis(diphenylphosphino)ruthenocene (dppr), bis(diphenylphosphino)methane (dppm) and triphenylphosphine (PPh₃)-substituted heterometallic Au-Mn or Au-Re carbonyl complexes is reported. A column packed with PGC (porous graphitic carbon) was used after preliminary experiments had shown that silica- and bonded-phase (silica-based) stationary phases were unsatisfactory for separation. The PGC column exhibited unique selectivity for the complexes studied. The mobile phases used were water-acetonitrile, dichloromethanehexane and tetrahydrofuran-hexane. The retention behaviour of the compounds was governed by the polar character and size of molecules, and influenced by metal-metal bond polarity. Separation of isomorphous structures with different metallocenyl moieties was achieved.     

Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Summary Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 m particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (7525, v:v) and switched to a CN column (10 cm long, 5 m particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 m particle diameter) with acetonitrile-0.088 M acetate buffer (7525, v:v) as mobile phase.Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (_ex= 225 nm; _em > 320 nm).Presented at the 14th International Symposium on Chromatography London, September, 1982     

Synthesis and characterization of new organometallic complexes bonded stationary phases for high-performance liquid chromatography

Summary The preparation, characterization and potential liquid chromatographic applications of various organometallic iron complexes silica stationary phases are presented. These new supports are synthesized by covalently linking ferrocene, as well as some of its cationic derivatives, to appropriately derivatized silica support matrices. These columns exhibit moderate to high selectivity towards the separation of polycyclic aromatic hydrocarbons (PAHs). A charge transfer retention mechanism has been proposed. A comparison with a reference stationary phase, 3,5-dinitrobenzamide (DNB), to quantify the acceptor power of the new stationary bonded phases, is also reported. Finally, the effect of varying the derivatives of the bonded metallocene on PAHs retention is discussed.     

On the chromatography mechanism of reversed-phase liquid chromatography

Summary An equation is derived which can describe how the retention of solutes is influenced by the composition of the mobile phase in reversed-phase liquid chromatography, the retention of solutes in alkyl bonded stationary phase regarded as the complexation between solute molecule and the active sites on the surface of the stationary phase. When the stationary phase is not fully saturated by the organic modifier, the activity of the active sites, the activity coefficient of the adsorbed solute as well as the activity coefficient of the solute in the mobile phase depend on the composition of the mobile phase. However, when the stationary phase is fully saturated, the composition of the mobile phase mainly influences the activity coefficient of the solute in the mobile phase. In addition, the selectivity of retention is discussed in terms of the derived equation.