Packed capillary HPLC: An attractive separation technique for small organic molecules

The feasibility of using packed capillary HPLC for the analysis of small organic molecules has been demonstrated by three examples: the separation of glucose and sucrose telomers, the separation of the different components of a solvent extraction reagent, and the separation of mono-, di-, and trioleins, all of which have been achieved with packed capillary columns of 0.32 mm inner diameter and eluent flow rates of 3 microliters per minute. Application to the analysis of the solvent extraction reagents has shown that this technique can be used as a quantitative tool in just the same way as any conventional HPLC method. The feasibility of a direct packed capillary HPLC – mass spectrometry interface has been demonstrated for the glucose telomer separation; direct coupling is only possible because of the microliter flow rates employed by the technique.     

Identification and Determination of Nicorandil and its Degradation Products by HPLC and GC/MS

Nicorandil [N-(2-hydroxyethyl)-nicotinamide nitrate NIC] is a novel kind of compound in the treatment of angina pectoris. NIC can be degraded easily in storage. The degradation products include N-(2-hydroxyethy) nicotinamide (HN), nitrate ion (NI), and ni…     

Resolution of complex wax ester mixtures by glass capillary GC

A glass capillary GC method providing high resolution analysis of complex wax ester mixtures is described. The two key parameters needed for evaluating a wax ester mixture for industrial utility – the total carbon chain length and the number of carbon-carbon double bonds present in each component – are simply and rapidly obtained by this method.     

Studying the size/shape separation and optical properties of silver nanoparticles by capillary electrophoresis

This paper describes the feasibility of employing capillary electrophoresis (CE) to separate silver particles in nanometer regimes. We have found that the addition of an anionic surfactant, sodium dodecyl sulphate (SDS), to the running electrolyte prevents coalescence of the silver particles during the process, which improves the separation performance; the concentration of SDS required for optimal silver nanoparticle separation is ca. 20 mM. By monitoring the electropherograms using a diode-array detection (DAD) system, we have also investigated the separation of suspended silver nanorods with respect to their shapes. Our results demonstrate that the combination of CE and DAD is a powerful one for the separation and characterization of various silver nanoparticles.     

Analysis of free and esterified sterols in vegetable oil methyl esters by capillary GC

The introduction of quality standards for vegetable oil methyl esters is gaining in importance due to their increased use as diesel fuel substitutes and as technical products. Free and esterified sterols, the main constituents of the unsaponifiable matter in vegetable oils, are recovered in vegetable oil methyl esters and may influence the technical properties of vegetable oil methyl ester products. A rapid gas chromatographic method for the qualitative and quantitative determination of free and esterified sterols in vegetable oil methyl esters has therefore been developed. The concentration of the free sterols as well as their qualitative and quantitative composition and the concentration of the sterol esters have been determined in rape seed oil methyl ester samples by GC–FID. Prior to analysis, the free sterols were silylated with N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane; betulinol was used as an internal standard. Calibration was performed by analysis of standard solutions containing β-sitosterol, cholesteryl stearate, and betulinol. The reproducibility of the quantitative results has been evaluated by repeated injections of the same test solution and by repeated complete analysis of the same sample.     

Advances in the preparation and evaluation of GC capillary columns

The preparation of (glass) capillary columns of high quality is still not without difficulties. Six problems in this context are highlighted. Column material choice(l), glass wall modification(2) and coating(3) are considered to have reached a satisfactory level. Deactivation(4) is still in a state of flux. Quantitation(5) is the least satisfactory aspect of capillary GC. Scope expansion(6) will be helped by the introduction of new phases of high thermal stability and by the adoption of solutions exclusively applicable to capillary GC. Evaluation of (glass) capillary GC columns is a rapidly developing field. Hopefully the number and complexity of test or polarity mixtures will not expand much further and it will be possible to make a clear and generally acceptable choice. The preparation and evaluation of ever-improving capillary columns is important to an ever-widening group of chromatographers. Our opinion on selected topics in this already vast field is summarized in the following overview.     

A new type of two-dimensional packed + capillary column GC system. II. System and applications

Summary A packed column containing immobilized SE-54 liquid phase on pellicular silica beads ZIPAX and having a high efficiency and high mass transfer rate has been successfully used in a two-dimensional packed+capillary column system without a cold trap. The application of this system is demonstrated by the analysis of C₆-C₈ aromatic hydrocarbons and lowboiling hydrocarbons present in natural oils, and of highboiling components present in low concentrations in a low-boiling solvent.     

Determination of azelaic and sorbic acid in pharmaceduticals by capillary electrophoresis

Summary Capillary electrophoresis with indirect UV detection at 254 nm was applied to simultaneous determination of ∼20% of azelaic acid and ∼0.1% of sorbic acid in AKNOREN cream. The acids were separated in fused silica capillary (45 cm × 50 μm) at 30 kV. Optimised back-ground electrolyte was 30 mM benzoate buffer (pH∼6, adjusted with TRIS) containing 7 mM β-cyclodextrin and 5% of methanol; internal standard was 2-hydroxysobutyric acid (HIBA). Rectilinear calibration ranges were 0.4–4 mg mL⁻¹ for azelaic acid and 2–20 μg mL⁻¹ for sorbic acid and the recoveries were 97.2–100.5%. A single analysis took <15 min.     

Identification of dimethyl- and ethylphenanthrenes in HPLC-fractions of rock and coal extracts by capillary gas chromatography and high resolution spectrofluorometry at 15K

Summary Total-aromatics fractions from an oil shale and a coal extract were fractionated in a three-step HPLC procedure. C₂-phenanthrenes in the triaromatic subfraction from the first step were tentatively identified on the basis of retention indices and GC-MS data. Successive fractionation of triaromatics on reverse-phase columns yielded 20 subfractions that were amenable to a positive identification of individual C₂-phenanthrene isomers by using high resolution spectrofluorometry. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Gas-and liquid chromatographic separation of naphthalenophanes of the [2.2] and [3.3] type

Summary Separations of various isomeric naphthalenophanes have been performed in different LC-systems, including those containing Ag⁺ in the mobile phase, and also by high temperature capillary GC. Ag⁺-complexation in the mobile phase of LC-systems can be observed with several of the strained aromatic systems, but most of the separations of practical interest can also be achieved without Ag⁺-complexation. For solubility reasons purification and isolation of certain isomeric species have been performed in normal phase LC-systems only.Dedicated to Prof. Dr. I. Halász to his 60th birthday.     

HPLC of nucleotides. II. General methods and their development for analysis and preparative separation. An approach to selectivity control

In order to develop efficient separation methods for nucleotides according to their size and heterocyclic composition, the application of ion-exchange, reverse-phase, and normal-phase adsorption HPLC has been studied. The comparative investigation of retention power and selectivity of various packings (non-polar bonded-phase and amino silicas) in relation to nucleotide length and composition yields data which enable suitable packings to be selected and a method of preparing the new packing for a particular separation problem to be formulated. Thus a new anion exchanger with high selectivity and dynamic mass transfer has been prepared for fractionation of large oligonucleotides. The effect of the eluent pH and composition (organic modifier, salt) on retention, selectivity, and resolution in ion-exchange and reverse-phase HPLC has been studied. The optimum separation conditions comprise elution with oppositely directed gradients of the salt and the modifier, use of a precolumn packing that provides the best protection for the main column without loss of its efficiency, and the optimum gradient program for the desired retention of the component of interest. The relation between loading and sample concentration has been studied and the system for gradient elution improved. Our work shows that two-dimensional separation is the most reliable and informative method for preparation of homogeneous oligonucleotides. The hydrophobic-pair ion-exchange mechanism is proposed for ion-pair chromatography. Protected and partially deblocked oligonucleotides, chemically synthesized for genetic engineering studies, have been separated with high selectivity by adsorption (normal-phase) HPLC which is efficient for gradient elution with isohydric eluents. The analysis of a monomeric composition of nano-(pico-) molar amounts of oligonucleotides has been developed; the procedure involves microcolumn digestion of the oligonucleotides with immobilized enzymes followed by microcolumn separation of the nucleoside-mononucleotide mixture. Also, a new slurry method for packing stable HPLC columns with a tightly consolidated, nonshrinkable bed of particles has been developed.     

Separation of metal cations in acidic solution by capillary electrophoresis with direct and indirect UV detection

Methods for the detectionn of metal cations under acidic conditions, near PH 2, in capillary electrophoresis (CE) were investigatged. Conditions for direct UV detection of UV absorbing metal cations such as Cr³⁺, Cu²⁺, Fe³⁺, UO₂²⁺, VO²⁺, and VO²⁺ were established With aqueous HCl or HClO₄ as the electroyte carrier. The speciation of vanadium(IV) and vanadium(V) at PH 2.3 by CE was achieved with direct detection at 185 nm. With the strong absorbance at 185 nm, no complexation was needed to detect the metal cations. An indirect UV detrection scheme for acidic conditions was also investigated. Several background carrier electolytes (BCES) were studied including 4-methylbenzylamince, nicontinamide, pyridazine, guanidine, 3-picoline, and chromium (III) to determine their effectivencess under very acidic conditions. The efect of ioni c surfactants and the nonionic surfactant, Trition X-100, on the peak heights and N Values was also studied.     

Group separation of non-sulphated and 3-sulphated bile acids by disposable anion-exchange cartridges

Summary A rapid and simple method has been developed for the group fractionation of the major unsulphated and mono-sulphated bile acids in human body fluids. After extraction with Bond Elut C₁₈ cartridges, the bile acids are separated into the unconjugated, glycine-, taurine- and sulphate-conjugated forms on pre-packed Bond Elut SAX columns by increasing the ionic strength of the methanol-acetate buffer eluent. The procedure was found to be accurate and reproducible and to give complete resolution between the different groups. The levels of 3-sulphate bile acids in human serum and urine from patients with liver disease were determined by high-performance liquid chromatography, after group separation and solvolysis of the sulphate fraction.     

Quantitative yields of underivatized peptides by HPLC

New methods for peptide separation are being developed. Resolution of peptide mixtures on HPLC with good reproducibility indicates the progress that has already been made in development of supports with useful separation capabilities. At present adsorptive characteristics of the stationary phases may place limitations on the applicability of the technique to underivatized peptides. Hopefully, the application of ionpairing techniques and the development of new support materials will expand the range of application for protein and peptide purification.