High performance liquid chromatography of chlortetracycline and related substances on poly(styrenedivinylbenzene)copolymer

Summary An isocratic high performance liquid chromatographic method has been developed for assay and purity control of chlortetracycline, which is separated from all related compounds. The stationary phase is poly(styrene-divinylbenzene)copolymer (PSDVB), which is heated at 60°C. The mobile phase is 2-methyl-2-propanol-1.0 M perchloric acid (5.0 ml)-water (up to 100 ml). The amount of alcohol modifier varies between 2.5 and 6.5% m/v depending upon the brand and porosity of PSDVB used. The flow rate is 1.0 ml/min and UV detection is at 254 nm. The total analysis time does not exceed 40 min. Although the method has been shown to be applicable with several brands of 100 Å PSDVB e.g. PLRP-S, PRP-1, RoGeL and TSK-Gel, the best separations were obtained on the wide pore material PLRP-S 100 Å.     

Modification of porous poly(styrene-divinylbenzene) beads by friedel-crafts reaction

Summary The influence of Friedel-Crafts reaction on the properties of poly(styrene-divinylbenzene) porous copolymers was studied. Two porous copolymers containing 0.7 and 0.9 mole fractions of divinylbenzene were used for modification. The aim of the process was to increase the copolymer crosslinking. As a crosslinker, tetrachloromethane in the presence of aluminium chloride was used. Reactions were monitored by FTIR and XRF analyses. The main result of the modification process is the change of the copolymer porous structure. Modification also has an influence on the chromatographic properties of the copolymers, especially selectivities.     

Isocratic separation of erythromycin, related substances and degradation products by liquid chromatography on XTerra RP18

Summary A simple, sensitive, selective and robust isocratic LC method is described for the analysis of erythromycin on XTerra RP₁₈. The main component, erythromycin A, is separated from all known related substances and degradation products. Several unknown impurities are also separated. Acetonitrile-0.2 MK₂HPO₄pH7.0-water, (35∶5∶60, v/v) was used as a mobile phase at 1.0 mL min⁻¹. UV detection was at 215 nm. The robustness of the method was evaluated by a full-factorial experimental design.     

Reversed-phase and cation-exchange chromatography on a new poly(styrene-divinylbenzene) high capacity,weak cation-exchanger

Summary A weak cation-exchanger for high-performance liquid chromatography is obtained by oxidation of either poly(methylstyrene-divinylbenzene) or of poly(chloromethylstyrene-divinylbenzene). Reaction conditions were optimised to yield an exchange capacity of about 4meqg^–1 dry resin. The material was evaluated chromatographically as a function of pH, organic modifier, temperature and flow rate. A combination of ionexchange and hydrophobic interaction between the solutes and the packing material was observed. This could be used to provide more options for realising chromatographic separations. Some chromatograms of heterocyclic bases, nucleosides, nucleotides and amino acids are shown.     

Separation of erythromycin and related substances by high-performance liquid chromatography on poly(styrene-divinylbenzene) packing materials

A comparative evaluation of three brands of poly(styrene-divinylbenzene) copolymers, Hamilton PRP-1 (10 micron), Rogel (8 micron) and TSK-Gel (10 micron), as column packing materials for high-performance liquid chromatographic separation of erythromycins is presented. Erythromycins A, B and C, anhydroerythromycin A, erythromycin A enol ether, N-demethylerythromycin A, anhydro N-demethylerythromycin A and N-demethylerythromycin A enol ether were chromatographed. The effects of column temperature, concentration of organic modifier in the mobile phase, concentration of phosphate buffer, the addition of quaternary ammonium salts and pH are described. The best separations were obtained on TSK-Gel with the mobile phase acetonitrile-methanol-0.2 M tetramethylammonium hydroxide pH 8.0-0.2 M phosphate buffer pH 8.0-water (30:15:25:5:25). PRP-1 and Rogel gave equally good separations but with higher retention volumes.     

Characterization of some commercial poly (styrene-divinylbenzene) copolymers for reversed-phase HPLC1

Summary The properties of the poly(styrene-divinylbenzene) copolymers PRP-1 and PLRP-S have been studied by infrared spectroscopy and chromatographic techniques. The following results were obtained: PRP-1 and PLRP-S are spectroscopically very similar. Their surfaces are chemically neither homogeneous nor stable during use. Retained nonpolar solutes should be eluted with a mobile phase containing tetrahydrofuran. Uncharged acids and anions can be chromatographed without difficulty. Cations of amines should be chromatographed only with a mobile phase of low pH. Uncharged amino-alcohols cannot be chromatographed without the addition of a competitor. Presented at the 16th International Symposium on Chromatography, Paris, France, September 1986     

Liquid chromatography of josamycin on poly(styrene-divinylbenzene)

An isocratic liquid chromatographic method for the assay and purity control of josamycin using wide-pore poly(styrene-divinylbenzene) as the stationary phase is presented. Josamycin has been well separated from all the related leucomycins, present as impurities in commercial samples. Wide-pore PLRP-S 8 m 1000 Å has shown the best selectivity. The separation has been carried out at 65°C with a mobile phase of 2-methyl-2-propanol/0.2 mol/l potassium phosphate buffer pH 10.5/water (24.5: 5: 70.5, V/V) and a flow rate of 1.0 ml/min. UV detection has been performed at 232 nm. The total analysis time has been about 40 min. The method has been used to analyse commercial bulk samples and tablets containing josamycin base.Dedicated to Professor Dr. Dr. h.c. mult. J.F.K. Huber on the occasion of his 70th birthday     

Liquid chromatographic determination of erythromycins in fermentation broth

Summary A simple and sensitive isocratic LC method is described for the determination of erythromycins in fermentation broths. A simple technique utilizing acetone-methyl ethyl ketone, 1∶1, as extraction solvent was coupled with suitable chromatographic conditions—compounds were separated on a 250 mm×4.6 mm i.d., 5 μm, reversed-phase column at 65°C with acetonitrile-0.2m K₂HPO₄ pH7.0-water, 35:5:60 (v/v), as mobile phase at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 215 nm. Separation of erythromycin F from polar components of the fermentation liquid was sufficient. Erythromycins A, B, C, D, and E, andN-desmethylerythromycin A were also separated, as were known decomposition products of erythromycin A and several unknown components. The method is suitable for monitoring the progress of erythromycin fermentation.     

Determination of Iodide and Thiocyanate in Seawater by Liquid Chromatography with Poly(ethylene glycol) Stationary Phase

Novel, simple, rapid, highly sensitive, and direct determination of iodide and thiocyanate ions in seawater has been performed by liquid chromatography (LC) with UV detection at 220 nm. The separation was achieved on a C₃₀ column of conventional size (150 mm × 4.6 mm i.d.) modified with poly(ethylene glycol); an aqueous solution of 300 mM sodium sulfate and 50 mM sodium chloride was used as mobile phase. Detection limits (S/N=3) obtained by injecting a 20-L sample were 0.5 and 6 ng mL^–1 for iodide and thiocyanate, respectively. The method was successfully used for rapid and direct determination of iodide and thiocyanate in seawater samples, collected from the coasts of Japan, without any extra pretreatment.Dedicated to Professor K. Jinno on the occasion of his 60^th birthday     

Chromatography of inorganic anions on poly(vinyl alcohol) gel columns with acidic eluents

Summary Poly(vinyl alcohol) (PVA) gel shows ionic retention properties for common inorganic anions when an acidic eluent is used. The ionic property of the PVA gel is due to the proton-acceptable nitrogen atoms of the cross-linking agent and the carboxylic residues being comprised in the gel matrix. The extent of the net charge on the gel surface depends on the pH of the eluent. At a pH ranging from 2.3 to 5.3, the PVA gel behaves as a weak anion exchanger with very low ion-exchange capacity. At these conditions four UV-absorbing inorganic anions (bromate, bromide, nitrate, and nitrite) are separated by eluting with aqueous sulfuric acid. Alkyl groups introduced on the gel surface hinder the ionized solute molecules from accessing to the positively charged functional groups on the gel surface. A neutral solute (HNO₂) is retained with non-ionic interactions.     

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.     

Preparation of magnetite/poly(styrene-divinylbenzene) nanoparticles for selective enrichment-determination of fenitrothion in environmental and biological samples

In the present study, a cross-linked nano-sized spherical magnetic poly(styrene-divinylbenzene) is synthesized and used as an adsorbent for enrichment-determination of fenitrothion. A miniemulsion polymerization procedure was used to prepare the adsorbent. The magnetic adsorbent was characterized by FT-IR, SEM and TEM. The prepared magnetic adsorbent nanoparticles were mixed with magnetite nanoparticles for faster and more efficient magnetic precipitation. The reduced fenitrothion was coupled with 3-methyl-2-benzothiazolinone hydrazone and then the blue colored complex was extracted. The blue derivative of fenitrothion was eluted by a 1 mL aliquot of 1-propanol prior to spectrophotometry at 571 nm. Beer's law was obeyed in the range of 2–230 ng mL⁻¹ of fenitrothion with relative standard deviation and recovery in the ranges of 0.9–5.1% and 97.2–100.0%, respectively. Selectivity of the method was evaluated, and the method was successfully applied to the determination of fenitrothion in various water, soil, urine and human plasma samples.     

Determination of metacycline and related substances by column liquid chromatography on poly(styrene-divinylbenzene).

Isocratic column liquid chromatography on poly(styrene-divinylbenzene) copolymer allowed complete separation of metacycline, 4-epimetacycline, oxytetracycline, doxycycline and 6-epidoxycycline. 2-Acetyl-2-decarboxamidometacycline was eluted on the tail of metacycline. The mobile phase was 2-methyl-2-propanol-0.2 M phosphate buffer (pH 9.0)-0.01 M sodium ethylenediaminetetraacetate (pH 9.0)-water (2.5:10:10:77.5, m/v/v/v). The flow-rate was 1.0 ml/min and detection was performed at 254 nm. Official standards were compared and a number of commercial bulk samples and specialties were analysed. 2-Acetyl-2 decarboxamidometacycline, 6-epidoxycycline and doxycycline were the main impurities, while 4-epimetacycline and oxytetracycline were minor impurities.     

Poly(methylglutamate)-coated surfaces in HPLC and CE

Summary Thermal polymerization of the N-carboxyanhydride of glutamic acid-5-methylester was used to coat microparticulate silica and the surface of capillaries with poly(methylglutamate) (PMG). By increasing the thickness of the PMG layer the hydrophobicity of the stationary phases could be increased. However, total shielding of surface silanols was not achieved. The PMG-coated silicas for HPLC showed unexpected and strange behavior in the chromatography of proteins. Nevertheless, capillaries coated with PMG proved to be excellent for highly efficient CE separations of proteins at medium pH values.     

Liquid chromatography of gramicidin

Summary A simple, selective method is described for separation of more than 10 gramicidin components on Spherisorb ODS B, 5 μm,250×4.6 mm I.D. column, maintained at 50°C. The mobile phase comprised methanol-water (71:29) at a flow rate of 1.0 mL min⁻¹. Detection was by UV at 282 nm. Valine gramicidins A, B and C were very well separated from the isoleucine gramicidins A, B and C. Four new gramicidin components were also resolved and their structures determined by liquid chromatography/ mass spectrometry. The names 10-methionine valine gramicidin C, 4-methionine valine gramicidin A, valine gramicidin A hydroxypropyl and isoleucine gramicidin A hydroxypropyl were proposed. Robustness of the liquid chromatography method was evaluated by performing a full factorial design experiment. The method also showed good repeatability, linearity and sensitivity.     

反相高效液相色谱法同时测定甘草酸单铵盐原料药主成分及有关物质含量

建立了同时测定甘草酸单铵盐原料药中主成分18α-甘草酸、18β-甘草酸及其有关物质A、有关物质B含量的高效液相色谱法,并用于其质量标准的建立。采用Durashell C₁₈色谱柱(250 mm×4.6 mm, 5 μm),以10 mmol/L高氯酸铵(氨水调节pH 8.20)-甲醇(48:52, v/v)为流动相,流速0.80 mL/min,检测波长254 nm,柱温50 ℃,进样量10 μL。在优化的色谱条件下,18α-甘草酸、18β-甘草酸、有关物质A、有关物质B在0.50~100 mg/L范围内线性关系良好(r>0.9999),检出限分别为0.15、0.10、0.10、0.15 mg/L,平均回收率在97.32%~99.33%之间(n=3),相对标准偏差(RSD)在0.05%~1.06%之间。本方法检测灵敏、重现性好,结果准确可靠,可用于甘草酸单铵盐原料药主成分及有关物质的检测分析,有利于其原料药的质量控制。     

Liquid chromatography of carbon clusters

Summary A comparison of three binary mobile phases in LC separation of C₆₀ and C₇₀ fullerences on chemically bonded 2,4-dinitroanilinopropyl (DNAP) stationary phase was carried out, n-Hexane-benzene has been found to be the best mobile phase for efficient separation of the all-carbon molecules permitting high loads in preparative LC.     

离子色谱法测定三磷酸腺苷二钠制剂中主成分及有关物质的含量

建立了用于三磷酸腺苷二钠制剂中主成分及有关物质含量测定的离子色谱方法。采用IonPac AS11-HC色谱柱,以KOH溶液为淋洗液,梯度洗脱,流速为1.0 mL/min,进样10 μL,以Dionex AERS 500 4-mm抑制器的电导检测器检测,三磷酸腺苷二钠(ATP-Na₂)的含量按峰面积以外标法计算,二磷酸腺苷二钠(ADP-Na₂)及单磷酸腺苷二钠(AMP-Na₂)按加校正因子的主成分自身对照法计算,未知杂质按主成分自身对照法计算。ATP-Na₂、ADP-Na₂及AMP-Na₂的线性范围分别为0.000146~1.83 g/L、0.000484~1.51 g/L及0.000426~0.804 g/L,相关系数分别为0.9997、0.9996及0.9999;对照品溶液在24 h内的稳定性良好(峰面积RSD分别为1.3%、1.4%、2.5%);ATP-Na₂、ADP-Na₂、AMP-Na₂的方法定量限(S/N=10)分别为1.5 ng、4.8 ng、4.3 ng,检出限(S/N=3)分别为0.58 ng、1.21 ng、1.28 ng;ATP-Na₂在3个水平的加样回收率分别为96.50%、96.57%和96.77%。本方法适用于三磷酸腺苷二钠制剂的质量控制。     

High-performance liquid chromatography of sugars on copper (II) modified silica gel

Summary A sensitive method of sugar analysis by HPLC is described in which a copper (II) modified silica gel as stationary phase is used. Detection is based on UV absorption of a complex formed between the sugar, copper, and ammonia.