Demonstration of the Feasibility of Radially Compressed Microbore HPLC Columns

Abstract

This work involved the development of radially compressed, microbore high performance liquid chromatography (HPLC) columns. The design of the overall system and the column are described, and the problems associated with the design features are reported. Variables examined during the course of this work included the column material, column length, packing method, flow rate, radial compression pressure, and internal column pressure. Efficiencies (expressed as plates/meter) are shown for various combinations of those variables and are compared to those obtained using a commercial, steel microbore column.     

Semimicro Size Exclusion Chromatography For Oligomers. I. Column Packing Procedure

Abstract

Polystyrene gels of a particle diameter 10 ± 2 μm for the use in oligomer separation were packed into 1.5 mm i.d. × 25 cm length columns by the balanced density slurry-packing technique under a constant flow rate of 500 μL/min. The slurry solvent was a mixture of toluene and chloroform (50.5/49.5, v/v). The example of the number of theoretical plates (N) of these columns was 8600 plates/25 cm (HETP = 29.1 μm) at flow rate of 40 μL/min by injecting 1 μL of 0.5% benzene solution. Sixteen columns were connected and the overall value of N was 103000 plates/4 m. A typical example of oligomer separation was demonstrated. A constant-flow technique is preferable to a constant-pressure technique. When two or three column blanks were packed together, the columns located at the outlet of the packer-column assembly had higher values of N. Optimum flow rate of the slurry solvent when three column blanks were packed together lay between 400 and 500 μL/min. The packing efficiency, that is, the probability of getting valid columns was about 60%. Viscous slurry solvents were not effective to get efficient columns. To pack gels in the less swollen state gave sometimes efficient columns. Pressure monitoring in progress of packing was very effective to foresee the column efficiency.     

A Portable Slurry Packing Apparatus for High Performance Liquid Chromatography

Abstract

A portable apparatus for slurry packing high performance microparticulate liquid chromatographic columns is described. The packer offers lab to lab portability and ease of operations. Columns up to 50 cm in length can be accommodated with the described configuration. Material costs per column are $40 – $70, offering a substantial saving over commercially prepared columns, in situations where labor costs are minimal. Efficiencies of columns packed with this apparatus have been equivalent to or better than those obtained with commercially available columns. Columns are packed using a staged pressurization/depressurization method. This method also leads to longer column life. Evaluation of relative column efficiency is also discussed.     

Spherical Versus Irregular-Shaped Silica Gel Particles in HPLC

Abstract

With optimised packing procedures, spherical shaped silica gel particles produce 1.5 to 2 times more plates in HPLC than irregular shaped silica gel particles. The lowest reduced plate height obtained by us so far is for 5 μm ROSiL-C₁₈-HL-D and is h: 1.62 for k': 4.5. It is suggested to transform h into 100/h% and to name this the “Chromatographic efficiency”, or a % of the ideal 100% limit. This limit would be an h value equal to the mean particle diameter. Spherical and irregular silica gel particles of 5 and 10 μm particle diameter and with similar physical characteristics have the same permeability in HPLC columns.

Whether a correct column packing procedure is used can be shown by the constancy of plate number and column permeability in function of different packing pressures.     

Mixed Packings in High Performance Liquid Chromatography. II. Mixed Packings vs. Mixed Ligands

Abstract

The use of mixed packing of different selectivities for the separation of antidepressants and anticonvulsants was studied. The results show that columns packed with mixed ligand supports (C₈ and cation exchange), gave the better resolution and peak shapes than the physically mixed C,₈/cation exchange and serially connected columns. Also, the retention times obtained on the mixed ligands column, the physically mixed supports column and the two columns in series were different in each case.     

High Performance Liquid Chromato-Graphy Separations of Nitrosamines. II. Acyclic Nitrosamines

Abstract

The separation of acyclic nitrosamines by HPLC using a β-cyclodextrin bonded silien gel column and a C₁₈ reversed phase column was evaluated. Four groups of nitrosamines were used to evaluate the utility of both columns: (1.) methylmethyl-, methylethyl-, methylpropyl and methylbutyl-; (2.) ethylmethyl-, ethylethyl-, and ethylpropyl-; (3.) propylmethyl-, propylpropyl-, and propylbutyl-; and (4.) butylmethyl-, butylethyl-, butylpropyl-, and butylbutyl nitrosamines.

The results show that both columns performed well in separating the nitrosamines in each group, with the C₁₈ column requiring a higher percentage of organic modifier (methanol) than the β-cyclodextrin column. The β-cyclodextrin column was superior in separating all the E to Z isomers of short chain alkyl nitrosamines, while the C₁₈ column was superior in separating long alkyl chain nitrosamines and their E and Z isomers.

It, was also found that, the longer the alkyl chain the longer the retention time, while nitrosamines having the same number of carbons, for example ethylbutyl-and propylpropyleluted at virtually the same time, using both columns.     

High Pressure Liquid Chromatographic Determination of Parabens in Pharmaceutical Preparations Containing Hydroxyquinolines

Abstract

A high pressure liquid chromatographic (HPLC) procedure for the analysis of methyl paraben (MP) and propyl paraben (PP) in pharmaceutical preparations containing a halogenated hydroxyquinoline (HHQ) is described. The method involves a separation of the phenolic constituents, MP, PP and HHQ with a Bio-Rad AG 1–X8 anion exchange resin, elution of the phenols with methanol after acidification and a reverse phase HPLC separation of the parabens using methanol-pH 6.5 buffer (60/40) mobile phase, a 30 cm × 3.9 mm (i.d.) column packed with Waters μBondapak C₁₈ packing and a guard column packed with Waters Bondapak C₁₈/Corasil packing. Recovery, precision, specificity and interference data along with the application of the proposed method for some commercial formulations both with and without a hydroxyquinoline are described.     

Separtion of Menthol Isomers by Normal Phase High Performance Liquid Chromatography (1)

Abstract

An HPLC method has been developed for the separtion of the four isomers of methol using isocaratic and normal phase ethyl acetate/isooctane systems. This method has used to detect and measure these isomers in peppermint techniques. It is more rapid than GC which in addition requires unstable columns for similar analysis. Beacause solvent and column are normal phase and isocratic, the method and sample preparation are very simple.     

Separation of Xanthine Derivates by High Pressure Liquid Chromatography and Application to Plasma Analysis

Abstract

High pressure liquid chromatography (HPLC) methods were developed for separation and plasma analysis of ten xanthine derivates. Separation was evaluated on silica column and on three different reverse phase columns, with optimum conditions obtained on C₆ spherisorb column using isocratic elution with phosphate buffer 10^?2 M, pH 2.7 - acetonitrile mixture (80/20 V/V). Determination of these xanthine derivates in plasma for therapeutic control was studied.     

Effect of Alkyl Chain Length of Bonded Silica Phases on Separation,Resolution and Efficiency in High Performance Liquid Chromatography

Abstract

A comparative study of alkyl bonded phases was carried out under optimum solvent conditions for each phase. Three columns, RP-2, RP-8 and RP-18, were tested for their efficiency and resolving power using three groups of compounds in three binary organic-water mobile phases. The organic solvents were acetonitrile, methanol and tetrahydrofuran which are widely used as solvent modifiers.

The results indicate that each of the three factors, i.e. solvent, solute and bonded alkyl chain length, play an important role, with the solvent being the most significant. When tetrahydrofuran-water was used as the mobile phase, the ratio of THF/H₂O did not vary by much when an RP-2, RP-8 or RP-18 column was used to separate naphthalene from biphenyl, dimethylphthalate from diethylphthalate or anthraquinone from methyl, anthraquinone and ethyl anthraquinone. When acetonitrile-water and methanol-water were used the ratio of organic modefier to water changed so as to accomodate the hydrophobic properties of the columns. The efficiency of the columns, expressed as theoretical plates per meter (TPM) was highest when acetonitrile-water was used as the mobile phase. Although there were variations in TPM and resolution from column to column, the three columns gave good separation of the components of the three groups of compounds.     

High Performance Size Exclusion Chromatography Using Porous Glass Packing Materials

Abstract

Porous glass packing materials of average particle diameter 5 μm have been packed into a 7.2 mm i.d. x 25 cm column by viscousslurry packing parocedure. Average pore diameters of porous glasses were 170 Â, 500 Â, 1000 A, and 2000 A. The numbers of theoretical plates were between 7000 and 8000 per a column for porous glasses of pore diameters of 170, 500, and 1000 A, and 5000 for that of 2000 A. The retention volumes of narrow molecular weight-distribution polystyrene standards have been determined using tetrahydrofuran as mobile phase for the construction of calibration curves. Separations of polystyrene over molecular weight ranges of 1000 and 4,000,000 have been obtained by combining all four porous glass columns in series. Molecular weight averages of NBS 706 polystyrene have been measured and compared with the values determined with polystyrene gel columns. Both results were equivalent to the manufacturer's data. Porous glasses thus appear to be a useful packing materials for HPSEC.     

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.     

High Pressure Liquid Chromatographic Determination of Isoniazid and Acetylisoniazid in Human Plasma

Abstract

A quantitative high pressure liquid chromatographic (HPLC) assay has been developed for the determination of isoniazid (INH) and acetylisoniazid (ACINH) in human plasma. Plasma samples were taken from a patient after oral administration of INH (with proven tuberculosis infection). A C₁₈ reversed phase radial compression column was used to separate INH and ACINH from other plasma components. The analysis takes 10 minutes per sample and the lower limit of detection for each compound is 0.10 ug/ml plasma.     

Fast separation of light hydrocarbons by gas chromatography on monolithic capillary columns based on silica gel

Monolithic capillary columns based on silica gel were tested in the course of high-speed gas-chromatographic separations of a five-component mixture of C₁–C₄ hydrocarbons. It was found that short-length monolithic columns could be used because of their high specific efficiency; this allowed us to shorten the column dead time and the duration of analysis. The column performance of about 1000 theoretical plates per second was reached. The test sorbate mixture was completely separated on a 58.5-cm column with an efficiency of about 18 700 theoretical plates in a time shorter than 17 s. It was noted that CO₂ and N₂O should be predominantly used as carrier gases.     

Serum Injection of the HPLC Column for Carbamazepine Assay

Abstract

Serum was injected directly on an HPLC column packed with C₁, 6.5 μm particle size, 300 Å pore-packing material for carbamazepine determination. The use of a wide-pore column with low hydrophobicity eliminated excessive pressure buildup in the column from protein precipitation which is usually caused by the high concentration of organic solvents in the mobile phase. This simple approach can be utilized for the determination of other drugs and endogenous compounds in serum.     

The Purification of Xanthene Dyes by Reverse Phase High Performance Liquid Chromatography

Abstract

A reliable method for the separation of fluorescein dyes from their impurities was developed using high performance liquid chromatography and involved a μBondapak C₁₈ reverse phase column and mixtures of methanol and ammonium acetate buffer. This technique was used to verify the purity of commercial products as well as to aid in the development of an empirical theory related to retention of halogenated fluorescein dyes by reverse phase columns.     

High Performance Liquid Cbromatographic Separation of Globin Chains on a Large-Pore C4 Column

Abstract

Excellent resolution of human and baboon globin chains may be obtained by HPLC on a Vydac large-pore C₄ column. The procedure is rapid and uses a gradient between aqueous trifluoroacetic acid and trifluoroacetic acid in acetonitrile. The common human γ chains are easily separable from each other as are some α- and β-chain variants from the normal chains and from each other.     

Modification of Selectivity in Reversed-Phase Liquid Chromatography of Polycyclic Aromatic Hydrocarbons Using Mixed Stationary Phases

Abstract

Monomeric and polymeric C₁₈ materials provide significantly different selectivities for polycyclic aromatic hydrocarbons (PAH) in reversed-phase liquid chromatography. Selectivity factors vary in a regular manner with respect to surface concentration of C₁₈ groups on different C₁₈ columns. In this study, we investigated the feasibility of “customizing” a C₁₈ column to provide an intermediate selectivity by mixing 5-μm polymeric C₁₈ material from two different lots with high and low C₁₈ surface concentrations. Polymeric C₁₈ materials from different production lots were mixed in ratios of 30/70, 50/50, and 70/30 (w/w). Selectivity factors for these columns were found to be similar to those predicted by the linear addition of the selectivities of the two individual phases. The PAH selectivities on these mixed columns were also found to be comparable to data obtained from coupled short columns of appropriate lengths each containing one of these different C₁₈ materials. These studies indicate that columns of specific selectivity can be prepared by either mixing two different C₁₈ materials or by coupling columns containing each of these different phases. The use of mixed phase columns is illustrated for the analysis of a fraction containing five condensed ring PAH isomers (molecular weight 278) isolated from an air particulate sample.     

Preparation and evaluation of tandem stationary phases for μ-HPLC and capillary electrochromatography

Tandem separation beddings were prepared within the capillary tube with a photopolymerized monolith initially formed by sol-gel technology in combination with microspheric octadecylsilane material by slurry packing. The chromatographic performance of the tandem stationary phases was evaluated in detail by varying the flow rate and the composition of the mobile phase using a self-installed capillary HPLC system. For the tandem stationary phases with a forepart monolithic length of 11 cm and 1 cm the lowest theoretical plate height for the retained component was 26 µm and 34 µm, respectively. After evaluation by capillary electrochromatography, enhanced chromatographic performance was obtained using a column with 1 cm monolithic inlet frit with a theoretical plate height up to 7.20 µm. A scanning electron micrograph with different cross-sections of the column showed that a porous network formed in the center of the capillary and a homogenous slurry packing of C₁₈ was obtained at the back part.     

Column Switching Technique for Group-Type Separation of Different Pah Classes by use of C18-Modified Silica and Polystyrene Packings

Abstract

A column switching technique was developed to realize a group-type separation of PAHs and nitrogen containing PAHs (N-PAHs) applying a C₁₈-immobilized polystyrene packing as well as a C₁₈-modified silica stationary phase. On the first column the group-type separation and also the separation of the N-PAH fraction in single compounds was performed. After backflush and transfer to a second column, the separation of the PAH fraction could be achieved.