Refractive index detection in microbore column liquid chromatography

Summary An existing commercial refractive index detector was modified for use with microbore column LC systems. The detector utilizes the Fresnel method. The effect of band dispersion and dilution at the detector side is of extreme importance in connection with the miniaturized LC system. In the modified model the original heat-exchanger tube was removed and a stainless steel capillary was used for heat-exchanging. Gaskets having different cell volumes were also examined with respect to band broadening and sensitivity. The detection limit was 10ng for di-n-pentyl phthalate. The examples include the detection of phthalates, alcohols, n-paraffins, and kerosine.     

Relationship between column efficiency and column length in chromatography: Efficiency of small-bore HPLC columns of different length

Summary The relationship between column length and efficiency is theoretically derived. Based on experimental data, the validity of the relationships between peak width and retention time, and between plate height, column length and the capacity ratio is shown.     

Electrodialytic sample treatment coupled on-line with high-performance liquid chromatography

Summary An electrodialytic sample treatment method coupled on-line with high-performance liquid chromatography (EDIST-HPLC) is discussed in this paper. The performance of EDIST as a function of the donor-phase (sample solution) flow rate, the voltage applied over the electrodialysis block, and the time of dialysis has been studied using the basic drug ephedrine as a model compound. Enrichment of the analyte by a factor of 10–20 was possible. The determination of human plasma spiked with ephedrine is briefly discussed.     

Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Summary Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 m particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (7525, v:v) and switched to a CN column (10 cm long, 5 m particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 m particle diameter) with acetonitrile-0.088 M acetate buffer (7525, v:v) as mobile phase.Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (_ex= 225 nm; _em > 320 nm).Presented at the 14th International Symposium on Chromatography London, September, 1982     

Influence of the eluent composition on column efficiency in reversed-phase high-performance liquid chromatography

Summary The dependence of column efficiency on the eluent (MeOH/H₂O) composition in a reversed-phase liquid chromatography system within a wide concentration range has been systematically examined. It is shown that when the intracolumn effect of mass transfer and diffusion is the main factor controlling band broadening the column efficiency decreases with the increase of the viscosity of the MeOH/H₂O mixture; on the other hand, when the extra-column effect is the main factor an increase in the viscosity of the eluents will help in improving column efficiency. The column efficiency is also related to the properties of the sample.     

Fluorometric detection with a packed flow cell in micro high-performance liquid chromatography

Summary A packed flow cell was used for fluorometric detection in micro high-performance liquid chromatography. The flow cell consisted of fused-silica tubing packed with the same material as the separation column. A focusing effect of the stationary phase on the signal intensity was observed, leading to an improvement of the mass detection limit, as achieved by on-column detection.     

An examination of simple injection valve coupling techniques in high-performance liquid chromatography

Summary The performance of injection valves with different column couplings and linking tubes has been evaluated using reverse-phase octadecylsilane columns and a standard test mixture. The results using three different injection valves are compared. A configuration and couplings which give optimum efficiency and ease of use are recommended.     

Determination of ephedra alkaloids by high-performance liquid chromatography

Summary Two simple methods were developed for the simultaneous determination of six alkaloids (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine and methyl-pseudoephedrine) inEphedrae Herba by high-performance liquid chromatography. The first method was carried out by using a Cosmosil 5C₁₈-MS column with a gradient solvent system consisting of a phosphate buffer and acetonitrile, and detection at 210 nm. The contents of alkaloids in non-pretreated ephedra herb extracts could be determined easily in 50 min. Alternatively, the alkaloids could be determined within 35 minutes by using a Cosmosil 5C₁₈-MS column with an isocratic solvent system of a sodium dodecyl sulfate-acetonitrile solution. The two methods are compared and discussed.     

Comparison of provitamin A determination by normal-phase gravity-flow column chromatography and reversed-phase high performance liquid chromatography

Summary The provitamin A content of some food samples was determined by methods involving MgO: Hyflosupercel gravityflow column chromatography (GFCC) and reversed phase high performance liquid chromatography (HPLC), the quantitation being done by external standardization (HPLC-ES) or internal standardization (HPLC-IS) with Sudan. The results obtained with - and -carotene in carrots, -carotene and -cryptoxanthin in papaya and -carotene in tomato and kale agreed well, showing that any of the these techniques can be used, provided the analysis is done under optimum conditions. Good separation of the different provitamins using GFCC depends on the analyst's skill and visual acuity. HPLC-ES required a constant supply of provitamin standards, thus the varying purity of commercially available standards and the high instability of these compounds could pose grave problems. Due to the stability of Sudan, HPLC-IS appeared to be the method of choice although passage of the extract through a MgO: Hyflosupercel minicolumn was required prior to injection to separate chlorophylls, dihydroxy- and polyoxycarotenoids which would otherwise elute with Sudan. Nonconformity of the Sudan structure to those of the provitamins did not effect the quantitative results. The chromatographic separation, identity and quantification of the provitamins could be more easily established by using HPLC-IS, complemented with GFCC.     

A comparative study of large volume injection techniques for microbore columns in HPLC

Summary This paper focuses attention on the potentially larger signal-to-noise ratios produced by microbore columns in comparison with conventional columns. The increased chromatographic signals by the application of microbore columns are due to the lower chromatographic dilution of elution profiles which are proportional to the square of the column inner radius. Generally less than 1μl sample should be injected into microbore systems to obtain the full benefit of the column performance. However, since more sample can be loaded on conventional columns compared to microbore columns the advantage of improved signal-to-noise ratio can only be realised in situations where very little sample is available. To inject more than 1μl sample, at the same time avoiding extra band-broadening effects, suitable injection techniques must be available. In this study three injection methods for microbore systems that meet this condition, are studied and compared.     

Retention behavior of dipeptide isomers in reversed-phase high-performance liquid chromatography

Summary The effects of eluent pH and organic modifier concentration on the capacity factor (k) and selectivity of dipeptide isomers were investigated. It has been observed that the variation in the logarithm of the capacity factor of the dipeptide isomers is linearly dependent on the organic modifier concentration (C_b), however, the selectivity is almost independent of it. Both capacity factor and selectivity were seriously affected by the pH of the eluent. Both the capacity factor and the intercept of the ln k vs. C_b plot increased with increasing van der Waals volume of the non-polar amino acid subunit of the dipeptides.     

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

Summary An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C₁₈ column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min⁻¹. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL⁻¹). The linearity of response was good (r ²≥0.999) over the concentration range 1–250 ng mL⁻¹.     

Process scale high-performance liquid chromatography Part I: An optimisation procedure to maximise column efficiency

Summary Scale up of analytical High-Performance Liquid Chromatography (HPLC) to the process scale offers promise as a preferred separation method for many high value fine chemicals, both for development purposes and continuous production. It's early adoption is curtailed by the widely held perception that preparative HPLC is a low efficiency high cost technique of limited application. This view has largely beeen generated by the generalised use of large particle size, low quality angular silica supports, and difficulties with packing methodologies. A systematic study of a new silica, in new axially compressed equipment using Plackett-Burman statistics, has demonstrated that very high efficiences, leading to high capacities, can be attained. This work was conducted in the Chemical Technology Unit, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester, M60 1QD, UK.     

Non-aqueous,reversed-phase,high-performance,thin-layer chromatography and column liquid chromatography of metal complexes of porphine

Summary The chromatographic mobility of 21H, 23H-porphine and its Ni(II), Cu(II), Zn(II) and Pd(II) complexes were investigated by high-performance thin-layer chromatography on an octadecyl-bonded, silica gel plate with various polar organic solvents including alcohols, acetonitrile, dimethylsulfoxide and propylenecarbonate. The mobility generally decreases according to the central metal ion of the complex as follows: Zn(II)>(free porphine)>Ni(II)>Pd(II)>Cu(II). Methanol is a good choice of solvent for the separation of these metal porphine complexes. Successful separation of porphine and the four metal complexes is accomplished within 13 min on a LiChrosorb RP-18 column with methanol eluent.     

Determination of myristicin in rat serum samples using microbore high performance liquid chromatography with column-switching

Summary A microbore high-performance liquid chromatographic method with column-switching was developed for the analysis of myristicin from rat serum without prepurification. Deproteinization, fractionation, concentration and separation of analyte were carried out by appropriate switching of columns and using solvent mixtures. The method showed excellent precision, accuracy and speed with a detection limit of 10 ng mL⁻¹ from 25 μL of serum. The total analysis time per sample was 25 min and the coefficients of variation for intra- and inter-assay were less than 1.8%.     

Analysis of aldehydes by micro high-performance liquid chromatography with post-column derivatization on enzyme-immobilized glass beads

Summary Aldehydes in liquors were analyzed by micro high-performance liquid chromatography with post-column derivatization on the enzyme-immobilized glass beads. β-nicotinamide adenine dinucleotide (NAD) was converted to its reduced form (NADH) on the enzyme-immobilized column in the presence of an aldehyde and a thiol, followed by fluorometric detection. A detection limit of 2.5 ppm (50 pg) was achieved for acetaldehyde.     

Column selectivity in reversed-phase liquid chromatography. VII. Cyanopropyl columns

Eleven cyanopropyl ("cyano") columns were characterized by means of a relationship developed originally for alkyl-silica columns. Compared to type-B alkyl-silica columns (i.e., made from pure silica), cyano columns are much less hydrophobic (smaller H), less sterically restricted (smaller S*), and have lower hydrogen-bond acidity (smaller A). Because sample retention is generally much weaker on cyano versus other columns (e.g., C8, C18), a change to a cyano column usually requires a significantly weaker mobile phase in order to maintain comparable values of k for both columns. For this reason, practical comparisons of selectivity between cyano and other columns (i.e., involving different mobile phases for each column) must take into account possible changes in separation due to the change in mobile phase, as well as change in the column.     

Multidentate phenyl-bonded phases in high-performance liquid chromatography

Summary New multidentate phenyl-bonded phases (MPBPs) were synthesized and evaluated the chromatographic retention behaviour with polycyclic aromatic hydrocarbons as sample probes in high-performance liquid chromatography. The new MPBPs show different retention characteristics from the previously synthesized MPBP, designated TP. Nevertheless, the results indicate that the retention mechanism is still the same as the TP phase: the size and shape of the solute molecule can be recognized by a cavity-like space formed by the methyl groups and phenyl rings of MPBPs.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Analysis of ofloxacin in plasma samples by high-performance liquid chromatography

Summary A sensitive HPLC method has been developed for determination of ofloxacin (OFL) in biological fluids. Sample preparation was performed by adding phosphate buffer (pH 7.4, 0.1m) then extraction with trichloromethane. OFL and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column with aqueous phosphate solution-acetonitrile, 80∶20, as mobile phase. The fluorescence of the column effluent was monitored at λ_ex 338 and λ_em 425 nm. The retention times were 2.66 and 4.24 min for OFL and SAR, respectively, and the detection and quantitation limits were 8 and 15 ng mL⁻¹, respectively. Plots of response against ofloxacin concentration were linear in the range 8 to 2000 ng mL⁻¹. Recovery was 92.9% for OFL.     

Fast determination of tetracycline antibiotics in different media by high-performance liquid chromatography

Summary The use of high-performance liquid chromatography (HPLC) for the identification and determination of tetracycline antibiotics is reviewed. HPLC chromatograms provide fast identification by retention time, t_R, and precise quantitation by measurement of peak height or peak area. For separation of tetracycline compounds, most HPLC methods use reversed-phase C₁₈ or C₈ columns and UV detection. The HPLC solvent system should have a pH of about 6 to prevent steric changes in the tetracycline molecule. For accurate quantitation it is necessary to avoid tailing and this is accomplished by adding a zwitter ion to the solvent system. Methanol and acetonitrile are frequently used as organic modifiers in these solvent systems. In a single analysis, HPLC methods can be used to separate as many as nine or ten commercially used tetracycline compounds and to determine four to five tetracyclines in commercial tetracycline preparations or in biological fluids.