Dependence of the retention volumes of additional streaming current responses and of the column void volume on mobile phase composition

Summary The influence of the alcohol content of the mobile phase and water, acetic acid and aniline as mobile phase additives on the generation and shape of two additional changes of the streaming current, generated inside the liquid chromatography column by injection of any sample and recorded before the responses of retained solutes, was studied in a normal-phase system using silica gel as the stationary phase. The mobile phases were based on a n-heptane-1-propanol mixtures. Under the same conditions the relationships between the column interparticle volume, the column void volume and the total liquid volume in the column and the retention volumes of these two streaming current responses, having the form of chromatographic peaks, were studied. The column void volume was identified with the retention volume of n-octane. The total liquid volume in the column (column hold-up) was calculated from the weight loss of the column wetted with water at first and then dried in nitrogen stream. The retention volume of the first streaming current response equals the column interparticle volume disregarding the mobile phase composition. If the 95∶5 n-heptane-1-propanol mobile phase contains water up to 80% of its saturated concentration (up to 0.114% by vol.), the retention volume of the second response agrees with the total volume of the liquid in the silica gel column, with a precision better than 2%. At a higher relative water saturation the retention volume of the second response increases, while the column void volume decreases. Both changes are explained by the spontaneous formation of a highly polar stagnant liquid in the pores of the silica gel.     

Statistical evaluation of the validity of a method for characterizing stationary phases for reversed-phase liquid chromatography based on retention of homologous series

Summary The validity of a method for characterizing stationary phases for reversed-phase, liquid chromatography, based on the use of homologous series, has been evaluated. The method is based on a retention model which describes the dependence of the logarithm of the capacity factor on mobile phase composition and the carbon number of specific homologous series. A first-order as well as a second-order version of this model was investigated. The second-order model proved to be a significant improvement on the first-order model, even for smaller mobile-phase ranges. Nevertheless both models showed a significant lack of fit, reflecting the incompleteness of these models. Therefore, it is very questionable whether this method is suitable to describe HPLC-column characteristics like hydrophobicity and hydrophylicity.     

The calculation of retention and selectivity in reversed-phase liquid chromatography II. Methanol-water eluents

Summary To calculate retention in reversed-phase, high-performance liquid chromatography a method based on the molecular structure of the analyte and the characteristics of sorbents and mobile phases has been employed. Characteristics of different ODS-columns in water-methanol eluents have been determined.     

Increased sample throughput in HPLC using sample switching

Summary Column switching methods are described which demonstrate an alternative approach to the reduction of analytical time in HPLC. Although most methods use a combination of techniques, it has been possible to group them into major categories involving column cleanup, sample cleanup, multicolumn methods and boxcar chromatography. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Peak parking technique for the simultaneous determination of anions and cations

An ion chromatography method is described for the simultaneous determination of anions (Cl^–, NO₃^–, and SO₄^2–) and cations (Na⁺, NH₄⁺, K⁺, Mg²⁺, and Ca²⁺) using a single pump, a single eluent and a single detector. An anion-exchange column modified with chondroitin sulfate C facilitated the elution of the above three anions using 5 mM tartaric acid as the eluent in isocratic mode, whereas the same eluent facilitated the separation of the above five cations on a commercially-available cation-exchange column. The separation columns were connected in series via two six-port switching valves, so the required cation-exchange or anion-exchange separation could be carried out by selecting the appropriate positions for the switching valves. The separations were completed in 30 min.     

Determination of void/dead volume of liquid chromatographic columns containing β-cyclodextrin as the stationary phase. Part I. Existing methods for the determination of LC column dead volume—A survey of literature

Summary A brief critique on the existing methods for the determination of LC column dead volume is presented. The dispute and controversy among scientists regarding the definition of column void volume, as well as the methods for its determination are pointed out. Difficulties arising in connection with the determination of the dead volume of cyclodextrin columns are discussed; it seems that the applicability of conventional methods is questionable. The necessity for further research in this field is stressed.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

新型微填充柱-毛细管柱二维色谱系统的理论研究(Ⅱ)──双柱条件对柱效的影响

从色谱动力学角度探讨了改变微填充柱-毛细管柱二维色谱系统中双柱条件对柱效的影响规律,进一步证实了短填充柱接长毛细管柱及高预柱柱温、快预柱载气线速有利于提高双柱系统柱效的结论;同时,通过对主柱柱温、载气线速及柱间分流比的讨论,从理论和实验上也证实了相对较低的主柱柱温、较慢主柱线速以及稍大的分流比有益于柱效利用率的提高。     

Determination of void/dead volume of liquid chromatographic columns containing β-cyclodextrin as the stationary phase. Part II. A new graphical method

Summary A new graphical method is proposed for the determination of the dead/void volume of liquid chromatographic columns with -cyclodextrin stationary phase. Two different approaches are presented which lead to very similar dead volume values for the cyclodextrin columns. The validity of the proposed method is discussed on the basis of column porosity values, as well as the resulting linear relationship between the logarithm of the capacity factor and the number of carbon atoms in the n-alkanol homologs. The method was applied to study the influence of various experimental parameters on the dead volume of cyclodextrin columns.     

Catecholamine: „On-Line” probenaufarbeitung und nachsäulenderivatisierung

Summary We developed an automated, two-column HPLC-method that can be used routinely to quantify the catecholamines norepinephrine and epinephrine in body fluids. The method is based on a new, laboratory-prepared SEC-HPAC column material, on the application of a microprocessor-controlled column-switching technique, and on the use of an integrated reaction system for postcolumn derivatization and trihydroxyindol-fluorescence monitoring. It allows the direct injection (upto 500 μl) of an appropriate biological fluid and distinguishes on its integrated sample-processing mode, its practicability, its chemoselectivity and-specivity, its detection limit (2 pg), its within- and between-run precision and its speed of analysis.

     

Selective and sensitive analysis of 1-nitropyrene in diesel exhaust particulate extract by multidimensional HPLC

Summary A multicolumn (MC) HPLC method for the determination of 1-nitropyrene (1-NP) at trace-levels via on-line reduction to 1-aminopyrene and fluorescence detection is presented. On the first column, packed with a pyrenebutyric acid amide stationary phase, the nitro-derivatives of PAHs are strongly retained and separated from other matrix components. The nitro-PAHs-containing fractions are transferred onto a RP₁₈-column via stepwise gradient elution and finally separated according to their various lipophilicities and sizes. To increase the overall selectivity and sensitivity of the multidimensional method (MD-HPLC system) post-column, on-line reduction of the nitro-PAHs to the respective amino-PAHs via a short catalysis column is performed thus enhancing the sensitivity significantly (to low pg levels). The applicability of this method for the determination of trace amounts of 1-NP in real samples (diesel particulate extracts) is demonstrated.     

Direct determination of Benzo[a]pyrene in oil distillates by on-line two-dimensional HPLC with column switching

Summary A selective and sensitive HPLC method for the determination of Benzo[a]pyrene (B[a]p) in oil fractions by means of column switching is described. The diluted oil samples were injected directly onto a silica column with isooctane as eluent. After fast elution of the main part of the sample matrix, the B[a]p containing fraction was transferred on-line to a dinitro-aryl-modified silica column for final separation with isooctane/tetrahydrofuran. A detection limit of 50 ppt B[a]p was found when using fluorescence detection.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

A column switching technique for the determination of lidocaine and its metabolites in horse urine

Summary A rapid method is described for the determination of lidocaine and its metabolites in horse urine using a column switching technique and HPLC analysis. This procedure offers a sensitive assay without the need for time consuming extractions.     

Syntheses of spherical silica and titania from alkoxides on a laboratory scale

Summary A process to synthesize silica and titania as spherical packing materials has been investigated on the laboratory scale by the sol-gel method. The silica and titania obtained were tested under normal-phase separation conditions for comparison of their retention characteristics silica witha a commercial. The silica was found to be similar in its retention behaviour to the commercial silica. The titania showed basic properties and strongly retained acidic compounds.     

Coupled column liquid chromatography for the rapid determination of N-methylcarbamates and some of their main metabolites in water

Summary A sensitive and selective coupled-column liquid chromatography (LC-LC) method was developed for the trace level determination of some N-methylcarbamates and some of their main metabolites as aldicarb, aldicarb-sulphoxide, aldicarb-sulphone, carbofuran and 3-hydroxicarbofuran in drinking and ground waters. The limit of determination can be reduced to 0.1 μg.L⁻¹ by solid phase extraction with a subsequent evaporation step. Environmental samples spiked at 0.1 μg.L⁻¹ were preconcentrated off-line with graphite carbon and then analyzed by LC-LC with UV detection yielding average recoveries between 81–109% (n=5) with RSD between 5–9%. The overall procedure allowed a sample throughput of up to 30 samples per day.     

Theoretical study of the effect of frit quality on chromatography using computational fluid dynamics

Summary Frits at both ends of a chromatographic column, especially for a preparative column, have significant influence on the flow distribution within the column and thus the column efficiency. However, frits have received little attention from chromatographers in the past. Here a theoretical study was conducted with the aid of CFD software FLUENT to investigate the effect of frits on the performance of homogeneous and heterogeneous chromatographic columns. A dimensionless number,FQ, was applied to characterize frit quality. This study visualized how frit quality affects the flow distribution and the concentration band, the shape of eluted pulse at the colum exit and column efficiency. Simulation results show that the development length of the flow distribution is related toFQ but has nothing to do with the packing heterogeneity. The curvature of the concentration band in a column depends onFQ and packing quality. This study shows column efficiency can be improved significantly by increasingFQ and/or frit permeability.     

An advanced solventless column test for capillary GC columns

Manufacturing skills for capillary GC columns have improved to a point where the commonly used tests no longer distinguish between "adequate" and "excellent" columns. A more stringent test mixture, coupled with a more exacting procedure, was proposed for testing capillary columns in 2004. The solutes were less sterically hindered and less retained, permitting the test to be run isothermally at lower temperatures where sorptive forces are stronger. To avoid masking active sites by solvent flooding, the test used a higher boiling solvent that eluted last. This test mixture, used under the prescribed conditions, differentiated adequate from excellent columns, but removal of the late-eluting solvent prolonged run times to as long as 1 h. The new test uses the same probes proposed in 2004, but entirely eliminates the solvent. Injections utilize a plunger-in-needle microvolume syringe, and the "gas saver" feature of a contemporary gas chromatograph. The latter serves as a dynamic diluter to deliver nanogram quantities of undiluted solutes to the column. The test can be conducted isothermally at a lower temperature in less than 15 min for most of the columns. This paper summarizes the analytical approach used, and presents method performance data and test results obtained on contemporary capillary columns from leading manufacturers.     

Enantioselective analysis of (<Emphasis Type="Italic">R</Emphasis>)- and (<Emphasis Type="Italic">S</Emphasis>)-carvedilol in human plasma by high-performance liquid chromatography

Summary An HPLC column-switching method has been developed and validated for the enantioselective determination of (R)- and (S)-carvedilol in human plasma. Sample preparation was performed either off-line, by extraction with trichloromethane and back-extraction into 0.01m aqueous citric acid which was injected on to a LiChrosorb RP 8 column, or on-line, by injecting diluted (0.1m formic acid) plasma on to a LiChrosorb ADS column. In both instances separation was performed by gradient elution and on-line transfer of the fraction containing, the carvedilol on to an enantioselective Teicoplanin column. The enantiomers of carvedilol were separated isocratically by use of methanol-acetonitrile-triethylammonium acetate, 70:30:0.05 (v/v/w), as mobile phase. With fluorescence detection the limits of quantitation were 0.30 ng mL⁻¹ for (R)-carvedilol and 0.26 ng mL⁻¹ for (S)-carvedilol; these were sufficient to enable investigation of the effect of exercise on plasma concentrations of (R)- and (S)-carvedilol after oral administration of either the racemate or the pure enantiomers. Although the operating conditions were optimized for sample preparation by on-line deproteination on a LiChrospher RP 18 ADS column, the complete method was insufficiently rugged for analysis of large numbers of plasma samples—the enantioselectivity of the Teicoplanin column deteriorated too rapidly because of the transfer of enantioselectivity-poisoning interferences which could not be suppressed sufficiently. In contrast the liquid-liquid sample-extraction procedure combined with column switching resulted in a analytical method with long-term stability. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Effect of solute association on the apparent adsorption isotherm. A model of the separation of non-racemic mixtures of enantiomers in achiral chromatographic systems

Summary A computer spreadsheet application has been developed for the optimization of step-gradient elution conditions as applied in coupled-column RPLC for online clean-up and separation in the analysis of pesticide residues. The procedure is based on the experimentally determined retention behaviour of the analytes as a function of mobile phase composition. Retention and peak volumes of the analytes eluting under isocratic and one- and/or two-step gradient conditions can be calculated. For a given set of analytes, the spreadsheet systematically calculates the resolution of the least efficiently separated pair of peaks in the space defined by the eluotropic strengths of the mobile phases and the time at which the step-gradient takes place.The performance of the proposed procedure was tested and evaluated for the analysis of nine polar pesticides for which in a previous study adequate conditions had been found by computer-aided chromatogram simulation. the optimum conditions predicted by the procedure were in good agreement with those established experimentally and by simulation. The simultaneous production of data on retention, peak volume and resolution for a group of analytes under selected one- and/or two step-gradient elution conditions makes the calculation procedure highly productive and flexible in searching for and/or adjusting appropriate separation conditions.