Temperature effects in Microcolumn Size Exclusion Chromatography

Abstract

Effects of column temperature on the column efficiency, retention time and stability of analytes were studied in microcolumn size-exclusion chromatography. Larger theoretical plates were achieved at column temperatures betwen 70 and 100 °C. In the constant-flow mode the retention time of analytes decreased with increasing column temperature, which was due mainly to thermal expansion of the mobile phase. When the column temperature was around or higher than the critical temperature of the mobile phase, the retention times of the analytes observed under supercritical pressure conditions still indicated dominancy of the size-exclusion mechanism, while another retention mechanism was involved under subcritical pressure conditions. In the case of the analysis of saccharides, the column temperature should be lower than 100 °C because oligosaccharides were decomposed at higher temperatures.     

Anion Exchange-Adsorption on Low Capacity Anion Exchangers: Separation of Organic Acids,Amino Acids,Small Chain Peptides

Abstract

The variables that influence the retention of organic analyte anions on a macroporous, high surface area polystyrenedivinyl-benzene copolymer that is chemically modified by attaching tetraalkylammonium groups to the copolymer surface are identified and studied as a function of anion exchange capacity. A combined adsorption-anion exchange retention of the organic analyte anion is possible providing the analyte has both a hydrophophic center and an anionic charge site. As the column anion exchange capacity (0 to 173 μeq of anion exchange sites/column was studied) increases, analyte retention due to adsorption decreases and retention due to anion exchange increases. The factors influencing organic analyte anion retention by adsorption are low anion exchange capacity and mobile phase solvent composition, type of organic modifier, and pH for analytes that are weak organic acids. For anion exchange the major factors are a high anion exchange     

Peak Reversals of Organics on Bentone-34 Modified Gas Chromatographic Columns

Abstract

Enthalpies of solution are reported for 22 different organics chromatographed on a 5% SP 200, 1.75% Bentone 34 gas chromatographic column. Reversal of retention order was observed for butanol and 2,2,5 trimethylhexane as the column temperature was increased. This was probably due to their similar boiling points with different enthalpy of solutions.     

Separation of Sulphonium Salts by Ion Chromatography: Five- and Six-Membered Cyclic Sulphonium Salts

Abstract

Several cyclic 5-and 6-membered sulphonium compounds were separated by ion pair chromatography on a opti-dbb ion pair column. The salts show specific retention behavior when using different mixtures of acetonitrile and aqueous sodium perchlorate solutions as eluents. The increase of retention times with extending chain length of the alkyl group R and with ring size is due to an increase of the solute molecular surface areas which were also calculated. The obtained retention parameters give additional information for structural determination.     

α-Amylase Purification and Separation from Gluco-Amylase by Affinity Chromatography on Cross-Linked Amylose(CL-Amylose)

Abstract

Using a CL-amylose column it is possible to purify α-amylase from other protein contaminants (e.g. albumin, haemoglobin, etc.) and from interfering exoamylases, by its specific retention on CL-amylose. α-Amylase is eluted off the column in the second step by a mixture containing 0.1 M NaCl in acetic acid 0.01 M and CaCl₂ 0.015 M. The recoveries of glucoamylase and α-amylase were 74% and 65% respectively. The retention capacity of the stationary phase is 0.5 mg of α-amylase/ml bed volume.     

Theory of Open Capillary Chrcmatography Using Electrical Diffuse Layer as the Virtual Stationary Phase for Charged Macro Mxecular Separations

Abstract

Electrical diffuse layers established by arrangement of ions along a metal capillary column can be used as the virtual “stationary phase” for charged macromolecular separations and determinations. Preliminary studies have indicated that resolution depends on solvent flow rate, pH, column diameter, etc., as well as the applied potential. In this communication we report a theory which underlies the mechanism of retention of charged macro-molecules, e.g., proteins, on the column. The utility and difficulties of this novel fractionation technique will also be discussed.     

The Separation of Organic Analyte Cations on a Low-Capacity Cation Exchange Column Using Indirect UV Detection

Abstract

The retention of organic analyte cations on a low-capacity cation exchange column using indirect UV detection was studied. It was found that a combination of cation exchange/reversed-phase interactions affected the retention of organic analyte cations provided the analytes have both a cationic charge site and a hydrophobic center. The factors that influenced the organic analyte cation retention were: concentration of organic modifier, concentration of UV absorbing analyte, pH, and mobile phase ionic strength. Elution orders for several of the organic analytes studied on the low-capacity cation exchange column were different than those observed on silica-based strong cation exchange columns.     

A Method of Correcting for Flow-Rate Fluctuations in Size Exclusion Chromatography Calculations: Applications to Methylene Chloride/Hexafluoroisopropanol Solvent System

Abstract

The mixture of methylene chloride/hexafluoroisopropanol (70/30, v/v) is an excellent polyester solvent, but its low boiling point causes unstable flow when it is used for size exclusion chromatography (SEC). In high-performance SEC experiments, retention time is normally used to measure elution volume; however, unstable flow makes it difficult to calibrate an SEC column set or calculate molecular weight parameters from a chromatogram. We have devised a simple and inexpensive method to compensate for the effect of unstable flow in SEC calculations. A calibration marker injected along with each sample is used to indicate flow-rate variations. The ratio of the sample retention time to the marker retention time is invariant to flow-rate changes and is used in place of retention time as a measure of elution volume in the universal calibration technique. Calibrating a column set and analyzing chromatograms by this method result in a large improvement in the accuracy and precision of calculated molecular weight parameters.     

Chromatographic Properties of Metallothioneins on a Gel Permeation Column: Changes Induced by Replacement of Cadmium with Cuprous and Cupric Ions

Abstract

In vitro experiments were conducted to find whether or not a similar elution profile to rat kidney metallothionein with high copper content was obtained on a gel permeation column by replacement of cadmium and/or zinc in rat liver metallothionein with copper. Stepwise replacement of cadmium in rat liver metallothionein with cuprous ion did not cause any shifts of retention times from those of the original proteins on a gel permeation column (SW 3000 column). In contrast to cuprous ion, stepwise replacement of cadmium with cupric ion induced shifts of retention times to larger values than the original ones for the two isometallothioneins on a SW column. Replacement of zinc in zinc-thionein with cupric ion but not with cuprous ion caused a retardation of elution volume on a Sephadex G-75 column. The decreases of cadmium peaks were accompanied by the increases of copper peaks in the case of replacement of cadmium in metallothionein with cuprous ion. Although stepwise decreases of cadmium peaks were observed by the replacement of cadmium in metallothionein with cupric ion, concomitant increases of copper peaks were not observed. Although the relative peak heights of isometallothionein peaks were different from those of rat kidney metallothionein, the third peak with the same retention time as that of rat kidney metallothionein was observed for the replacement of cadmium with cupric but not with cuprous ion.     

Determination of Tryptophan-Human Serum Albumin Binding from Retention data and Separation of Tryptophan Enantiomer by High Performance Liquid Chromatography

Abstract

The retention volume of a ligand, injected onto a size exclusion chromatographic column and eluted by a macromolecular complexing solution, is theoretically analysed for evaluating the binding between the solute and the macromolecule. The binding of D- and L- tryptophan to human serum albumin is given as an example, and the separation of these enantiomers is then achieved     

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.     

Effect of Column Dimensions on HPLC Separations Using Constant Volume Columns

Abstract

The effect of column dimension on resolution, sample capacity, retention time, efficiency and mobile phase composition were studied, using both constant flow rate and constant linear velocity. The four columns selected (A = 238 × 3.2 mm, B = 153 × 4.0 mm, C = 116 × 4.6 mm and D = 50 × 7 mm) had the same volume. K¹ values were found to be constant, within experimental error, for all columns. At constant linear velocity, the retention time was found to be a linear function of column length, while at constant flow rate retention time was constant for all columns. The longest column (A) generated the largest N values while columns 3 and C gave the lowest H values, for dilute solutions, while they decreased with decreasing column length. On the other hand, it was observed that as the sample size increased, N generated by column A decreased more rapidly and eventually fell below the values generated by columns B and C. These two columns (B & C) can tolerate a larger sample size with less reduction in N value than the longest column. It is important to note that although there were minor differences in performance between columns B and C, there were significant differences between them (B and C) and the other two columns (A and D). Column A offered the highest sensitivity (narrower peaks) for dilute solutions, while columns B and C offered higher loadability. The volume of organic modifier in the mobile phase affected the retention equally in the four columns. It was also found that equal separation (a) was obtained for each column at constant flow rate and constant linear velocity, except with the latter the retention times were longer.     

High Performance Liquid Chromatography Separations of Nitrosamines. I. Cyclic Nitrosamines

Abstract

The separation of cyclic nitros amines by HPLC was achieved using a cyclobond I column and a mobile phase of methanol/water. The effect of in-the-ring and on-the-ring substitutions on retention times were studied. The results show that the longer the chain of the substituent, the longer the retention time, which is consistent with the inclusion mechanism of separation. Also, the larger the ring, the longer the retention time. The results also show that the in-the-ring substitution has a dramatic effect on retention.     

Cellulose Derivatives Used as Chiral Stationary Phases in Capillary Gas Chromatography

Abstract

In this work, we present the first separation of enantiomers in gas chromatography (GC) using a fused‐silica capillary column containing cellulose triacetate, cellulose triphenylcarbamate, or cellulose tris(3,5‐dimethylphenylcarbamate) as the new chiral stationary phase. The separated solutes included alcohols, amine, ketone, ether, ester, and amino acid. Their column efficiency, polarity, and chiral selectivity were studied. The retention mechanism was discussed. The results showed that those derivatives had relatively high chiral recognition abilities and can be used as the chiral stationary phases in GC.     

Enantiomer Separations by Normal HPLC Systems with Permethylated β-Cyclodextrin Dynamically Coated on Silica Solid Supports

Abstract

The paper describes the technique of solvent-generated liquid-solid chromatography with a chiral stationary phase consisting of permethylated β-cyclodextrin coated on a bare silica surface. The chiral adsorption layer was generated dynamically by pumping diluted solutions of permethylated β-cyclodextrin in binary (hexane/different alcohols) eluents through a column packed with microparticulate silica. This technique leads to column with excellent time stability and good reproducibility of the enantioselectivity. It was found that retention characteristics e. g. enantioselectivity and capacity depend very strongly on the type and concentration of the alcohol used as mobile phase component.     

Reversed Phase High Performance Liquid Chromatography Using Carboxylic Acids in the Mobile Phase

Abstract

This report describes the use of different carboxylic acids as mobile phase modifiers. The effect on retention of acid chain length, pH, and eluent composition for a series of phenylalkanols, phenol, and the amines aniline, N-methylaniline, and benzylamine is discussed. The retention of both neutral and positively charged compounds is influenced by the dissociation equilibrium of the carboxylic acid in the mobile phase. By using l-pentanol to coat excess exposed silanol groups on the reversed phase column used, the inflection in the retention of both neutral and charged solutes as pH is changed occurs at the pK_a of the acid in the mobile phase. In addition, by using an acid and amine with the same or similar pK_a values, selective ion-pairing of this pair over others with dissimilar pK_a values can be promoted. Application of this technique to the selective retention of amino acids and peptides was unsuccessful.     

High-Performance Liquid Chromatography of T-RNA

Abstract

Fractionation of amino acid specific tRNAs by descending salt gradient on a reversed-phase high-performance liquid chromatography column is demonstrated. Stationary phase variables such as n-alkyl chain length and silica type are discussed in terms of stability and selectivity. Mobile phase parameters including salt concentration, organic modifier, pH, and temperature are investigated. The concentration of organic modifier to adjust elution profiles can be critical in terms of tRNA retention.     

Optimization Based on Retention Prediction and Information Theory for Liquid-Chromatographic Analysis of Alkylbenzenes

Abstract

The mobile phase composition and column length are optimized for analyses of six alkylbenzenes in reversed-phase liquid chromatography with the aid of retention prediction and information theory. Optimal conditions selected according to the resolution Rs and information theory are evaluated from the viewpoint of the precision and analytical efficiency (rapidity) of chromatography. The combination of the information-theoretical optimization with the retention prediction will accelerate the development in the automation of liquid-chromatographic analysis.     

HPLC of Metal Chelates: Separation of NiII and FeII as 2,2′-Bipyridine Complexes with Ion-Pair Chromatography

Abstract

The separation of 2,2′-bipyridine and its trischelate complexes of Ni^II and Fe^II was achieved on a μ-Bondapak-CN column using NCS as counter ion in the mobile phase (methanol-water mixtures). The retention volumes for the Ni chelate were found to vary exponentially with the concentration of KNCS in the mobile phase. The concentration of the metals in the effluents was checked by means of atomic absorption spectroscopy.     

High-Performance Liquid Chromatographic Determination of Atenolol in Tablets

Abstract

A reversed phase high-performance liquid chromatographic method was developed for the determination of atenolol in four oral 100 mg atenolol preparations.

An aliquot of the sample is dissolved in a mobile phase consisting of 0.0612 M potassium hydrogen phosphate - isopropanol-tetrahydrofuran (84:10:6) v/v). The pH was adjusted to 6.7 with phosphate buffer. Nicotinamide was used as internal standard and chromatographed on a Pinkerton column ISRP (GFF-S5–80) 5 μm, 150 × 4.6 mm i.d. The applied column is convenient for the assay at least 90 samples of atenolol without degrading column performance. The detection was performed at 272 nm. The retention time for atenolol was 5.07 min.

The proposed HPLC method was found to be suitable for the rapid and precise routine analysis of atenolol in tablets.