The Clever Use of Pressure in Column Liquid Chromatography

The pressure limits of a liquid chromatographic instrument can be maxed out by running the separation at the highest possible flow rate. This approach reduces analysis time but it does not save solvent and the separation will be poorer due to the properties of the van Deemter curve. Thus, it is better to use a shorter column with smaller particle size because both analysis time and solvent consumption will decrease while the resolution will remain constant. This paper shows how to utilize the pressure which is offered by a certain LC instrument in a clever way. It explains the algebraic background and illustrates the validity of the approach with two analytical problems, namely the separation of seven doping agents and of six drugs.     

Use of Alkaline Degradation for the Analysis of Dihydrotriazines via High Pressure Liquid Chromatography

Abstract

An HPLC method is described for the determination of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3′,4′-dichlorobenzyloxy)-1,3,5-triazine hydrochloride (WR 38839). The procedure required the isomerization of the drug sample by alkaline treatment with sodium hydroxide, as the parent compound was retained by the column. The reaction product of the drug was analyzed by HPLC using a strong cation exchange resin as the stationary phase and glycine buffer, pH 10.4 as the mobile phase. The product was isolated and identified by TLC, UV, IR, mass spectroscopy and elemental analysis. The postulated mechanism indicates that this would be a general analytical method for dihydrotriazine compounds. This technique, developed for the assay of the dihydrotriazine in an aqueous system, was successfully applied to rat urine samples spiked with the drug.     

The Identification of Drugs of Abuse in Urine Using Reverse Phase High Pressure Liquid Chromatography

Abstract

A rapid, isocratic HPLC procedure for the identification of drugs of abuse in urine is described. The procedure utilizes a reverse phase μC₁₈ column, a methanol/water mobile phase, buffered to pH 7.5, and a UV detector operating at 254 nm. Using indole as an external standard, 13 common drugs of abuse can be differentiated in less than 15 minutes. Nine different drugs of abuse were identified in actual drug screen urine samples, confirming TLC results. Reproducibility and quantitative capabilities of this method were also demonstrated. Due to apparent interferences from drug metabolites this method must at present be used in conjunction with another established method such as TLC or GC for positive drug identification.     

Preparation of High Efficiency Columns for High Performance Liquid Chromatography

Abstract

This method employs a specially constructed slurry reservoir, which features an internal taper and a quick-disconnect pump fitting. A major innovation is the use of a three-solvent system to maintain a slurry of packing material as a ‘slug’ within the reservoir. A measured amount of carbon tetrachloride is poured into the empty reservoir-column assembly. The column packing material is introduced as an isopropanol slurry and the reservoir is filled with isooctane. The assembly is then connected to a Haskel DSTV 122C pump, and the column is packed with isooctane at 10,000 psi pressure.

This procedure provides HPLC columns with efficiencies of 40,000 plates per meter for 10-micron silica, 30,000 plates per meter for 10-micron C₁₈, and 70,000 plates per meter for 5-micron C₁₈ packing materials. We have used this method for preparation of over 35 HPLC columns during the past two years.     

高效液相色谱法同时测定食品中7种非食用色素

建立了高效液相色谱法同时测定食品中7种非食用色素(碱性嫩黄O、碱性橙、酸性橙Ⅱ、酸性金黄、玫瑰红B、对位红、苏丹红Ⅰ)的方法。依次采用乙腈、甲醇和碱性甲醇提取豆制品和肉制品;采用乙腈和70%乙腈依次提取调味品。采用SunFireTMC18色谱柱(250 mm×4.6 mm,5μm),以甲醇-50 mmol/L乙酸铵水溶液(H3PO4调至pH 4.5)为流动相,梯度洗脱,流速1 mL/min,测定波长为450和520 nm。7种非食用色素的在各自相应浓度范围内线性相关系数均大于0.998;检出限(LOD)在0.01～0.1 mg/L之间;定量限(LOQ)在0.18～1.2 mg/kg之间。平均回收率均大于80%;相对标准偏差(RSD,n=3)在2.0%～5.7%之间。     

On Line Detection of Plasma Borne Vasoconstrictor by the Use of High Pressure Liquid Chromatography

Abstract

An on line detection system for a plasma borne vasoconstrictor was developed using a rat heart bioassay and a reversed-phase high pressure liquid chromatograph (HPLC). Partially purified plasma borne vasoconstrictor, which is yet to be characterized, was fractionated by HPLC, and the output from the unit was introduced into the rat heart bioassay system directly. The on line HPLC-rat heart bioassay system detected the active fraction from approximately 20 substances in the partially purified plasma. This system enabled rapid and reproducible identification of the active vasoconstrictor in plasma.     

High Pressure Liquid Chromatography of Selected Sulfur Compounds

Abstract

High pressure liquid chromatography was used to separate and determine quantitatively the following groups of sulfur compounds: thiols, sulfides, disulfides, sulfones, isothiocyanates, thioamides, and thioureas. Amperometric and UV detectors were compared; for thiols, thioureas, isothiocyanates, and thioamides, the former was generally more sensitive. With the exception of alkyl and cycloalkyl sulfides, the liquid chromatographic method can be used for the analysis of the investigated sulfur compounds below the ppm range. The method developed was compared to gas chromatography with flame photometric detection. The latter was found to be superior for the analysis of alkyl and cycloalkyl thiols, sulfides and disulfides of molecular weight below 200, whereas the former was more suitable for the analysis of aromatic thiols, sulfides and disulfides, as well as thioamides, isothiocyanates, and thioureas. Both methods were equivalent for the analysis of aromatic sulfones.     

High Pressure Liquid Chromatography of Thyromimetic Iodoamino Acids

Abstract

The chromatographic properties of 16 thyromimetic iodoamino acids and related compounds on microparticulate non-polar stationary phases have been examined and conditions determined which allow optimised resolution with analysis time ca. 60 minutes. These compounds elute in order of increasing hydrophobicity which correlates with the progressive increase in the number of iodogroups present in the tyrosine or thyronine aromatic nucleus. The reverse isomers, e.g. rT3, have consistently greater k' values than their corresponding analogues, e.g. T3. Conditions for the direct application of the rapid HPLC analyses of the iodoamino acids in biological or pharmaceutical samples have been examined.     

An Organic Nitrogen Specific Detector for High Pressure Liquid Chromatography

Abstract

The Thermal Nitrogen Analyzer (TNA), a new highly sensitive and nitrogen-selective detector for high pressure liquid chromatography, is described.     

The Determination of Metribuzin and Its Metabolites by High Pressure Liquid Chromatography

Abstract

A method for the determination of metribuzin and its metabolites in plant tissues has been developed using High Pressure Liquid Chromatography (HPLC). The system used involves reversed-phase chromatography on a C-18 HPLC column and a 62:38 methanol/0.05 M acetic acid mobile phase. Under these conditions, metribuzin and the three known metabolites [deaminated metribuzin (DA), deaminated diketometribuzin (DADK) and diketometribuzin (DK)] are completely resolved from each other. Detection is by UV absorbance at 254 nm.     

Use of High Pressure Liquid Chromatography and Thin Layer Chromatography for the Separation and Detection of Testosterone and its Metabolites from in vitro Incubation Mixtures

Abstract

Testosterone and its 6β-, 7α-, and 16α-hydroxylated metabolites were resolved by high pressure liquid chromatography and thin layer chromatography. Separation by HPLC was achieved in less than 45 min on a microparticulate silica gel column using isocratic elution with isopro-panol:tetrahydrofuran:hexane (5:15:80) as the mobile phase. TLC systems utilizing silica gel on glass and plastic plates, and polysilicic acid on glass fiber sheets are presented. The monohydroxylated metabolites of testosterone formed during incubation of (¹⁴C)-testosterone with liver postmitochondrial preparations from adult male rats pre-treated with phenobarbital or Aroclor 1254 were separated and quantitated by both HPLC and TLC. The results using both techniques are compared with those obtained by paper chromatography.     

新型离子交换硅胶键合相的制备及评价

二甲基氯硅烷与硅胶表面反应,形成牢固的ＳｉＨ键之后,连接上活泼的中间体——烯丙基缩甘油醚作为柔软的分子臂,最后接上二乙基氨基,由此制得了新型的离子交换硅胶键合相。经漫反射红外光谱、元素分析和高效液相色谱法对键合相进行了鉴定和评价。结果表明：键合反应按预定路线进行,键合相具有较好的色谱性能。此种方法可有效地运用于无孔硅胶填料的制备。     

球形氧化锆液相色谱柱填料的制备和评价

采用改进油乳液-溶胶-凝胶法制备了不同粒径的色谱用氧化锆微球。用X射线衍射、扫描电镜、比表面测定仪进行了结构、比表面积、孔径、孔体积和形貌表征。讨论了影响颗粒大小和颗粒分布均匀的制备因素。实验证明:有机相性质、有机相与水相的体积比、乳化剂的性质和浓度、助乳化剂的存在、搅拌速度是决定最终制备氧化锆颗粒大小和均匀的主要因素。     

Synergistic Use of Countercurrent Chromatography and High Performance Liquid Chromatography for the Purification of Synthetic Peptides

Abstract

Examples are given demonstrating how countercurrent chromatography (CCC) and high performance liquid chromatography (HPLC) can be used together to purify synthetic peptides. In one example, CCC provided a preliminary purification of Met-Arg-Asp-Val-Val-Leu-Phe-Glu-Lys by enabling separation of ultraviolet absorbing, ninhydrin-negative material from the desired peptide. Final purification was achieved with HPLC without risk of harming the HPLC column. In a second example Tyr-Ala-Ala-Nle-Ala-Ala was completely purified by CCC with the CCC separation rapidly and conveniently monitored by HPLC. CCC appears to be a very useful technique for the peptide chemist.     

痕量神经肽的高效液相色谱

本文选用芴甲氧羰基氯(FMOC-CL)作为肽的荧光衍生试剂,建立了两段淋洗分离测定神经肽RP-HPLC的方法。衍生反应简便、快速、衍生物稳定。FMOC-脑啡肽、FMOC-P物质在18min内全部被洗脱。本方法在1～100pmol具有良好的定量线性关系。当信噪比为2.5:1时,检测极限:FMOC-甲硫脑啡肽为405fmol,FMOC-亮脑啡肽337fmol,FMOC-P物质为500fmol。并且能够容易地脱去衍生基团得到反应前的神经肽。     

Separation of Methylated Purines by High Pressure Liquid Chromatography

Abstract

This paper reports a method for the separation and measurement of methylated purines of interest to carcinogenesis studies by high-pressure liquid chromatography (HPLC) following their column chromatographic isolation from collected urine samples. HPLC was evaluated on three different cation-exchange columns, with optimum conditions obtained on a Partisil 10-SCX column employing isocratic elution with 0.25M (NH₄)H₂PO₄ at pH 4.0. This column was also found to be useful for the separation of mono-methylguanine isomers. Application is shown to the analysis of rat urine following animal treatment with methyl methanesulfonate.     

Analysis of Hydroxybenzoic Acids by High Pressure Liquid Chromatography

Abstract

Good separation of a range of hydroxybenzoic acids was achieved by HPLC on a μBondapak phenyl column using 5% v/v acetic acid in water as the eluting solvent.     

Determination of Amikacin Isomers by High Pressure Liquid Chromatography

Abstract

A high-pressure liquid chromatographic (HPLC) method for quantitative monitoring of amikacin isomers is described. Four isomers, BB-K8, BB-K29, BB-K6 and BB-K11 were applied to a silica gel column. While adsorbed, the isomers were derivatized with o-phthalaldehyde and the derivatized products eluted with ethanol. A decrease in the fluorescence of the derivatized products with time was observed. Heating at 50°C for 5 min produced products with stable fluorescence for at least three hours. Using the fluorescent properties of the amikacin derivative for detection, the four isomers of amikacin were separated by reverse phase (HPLC). A linear relationship from 1 to 10 μg/mL was obtained for all four isomers.     

Separation of Biological Pyridines by High Pressure Liquid Chromatography

Abstract

The separation of the six pyridine compounds which comprise the pyridine nucleotide cycle, nicotinamide adenine dinucleotide phosphate and para-aminobenzoic acid, a compound biologically related to these pyridines, can be achieved rapidly utilizing high pressure liquid chromatography. Optimum separation is accomplished using ion-ion pairing in reverse phase chromatography with a C₁₈ stationary phase and an aqueous mobile phase of 5mM pentanesulfonic acid and 25 mM KH₂PO₄. The effect of temperature on the separation is minimal. As little as 10 ng of these compounds is detected via absorption of ultraviolet light at a wavelength of 254 nm.     

Determination of Pentachlorophenol Laurate by High Pressure Liquid Chromatography

Abstract

Pentachlorophenol Laurate (PCPL) in canvas is determined by extraction and chromotography on silica gel. Conditions are chosen to eliminate peak splitting due to the presence of different “laurate” fatty acids. The determination is faster and more specific than previously reported methods.