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A new chromatographic stationary phase obtained by coating a reversed phase amide column with phosphatidylcholine based liposomes solution to yield a phospholipid modified column (PLM). The modification is achieved by the dynamic coating method which recycles the coating solution through the column in a closed loop for a period of 24 h. The chromatographic properties of the new column have changed significantly as compared to the original amide column due to the phospholipid coating. A good correlation was observed between n-octanol/water log P values and the logarithm of the retention factor obtained on the PLM column for a large number of solutes. In addition the PLM column was characterized using the linear solvation energy relationship (LSER). The values of the LSER system constants for the PLM column were calculated and were found to be very close to those of the n-octanol/water extraction system thus suggesting that the PLM column can be used for the estimation of n-octanol/water partition coefficient and serve as a possible alternative to the shake-flask method for lipophilicity determination. In addition, the results suggest that the PLM column can provide an alternative to other phospholipid-based column such as the IAM and the DPC columns.

In this study, we show that the addition of n-octanol to the mobile phase improves the chromatographic determination of lipophilicity parameters of xenobiotics (neutral solutes, acidic, neutral and basic drugs) on a Phenomenex Gemini C18 column. The Gemini C18 column is a new generation hybrid silica-based column with an extended pH range capability. The wide pH range (2–12) afforded the examination of basic drugs and acidic drugs in their neutral form. Extrapolated retention factor values, log k ′ w ( o ) , obtained on the above column with the n-octanol-modified mobile phase were very well correlated (1:1 correlation) with literature values of log P (logarithm of the partition coefficient in n-octanol/water) of neutral compounds and neutral drugs (69). In addition, we found good linear correlations between measured log k ′ w ( o ) values and calculated values of the logarithm of the distribution coefficient at pH 7.0 (log D7.0) for ionized acidic and basic drugs (r2 = 0.95). The Gemini C18 phase was characterized using the linear solvation energy relationship (LSER) model of Abraham. The LSER system constants for the column were compared to the LSER constants of n-octanol/water extraction system using the Tanaka radar plots. The comparison shows that the two methods are nearly equivalent.

While studying the properties of some potential, UV-detection compatible, mobile phase modifiers for reversed-phase liquid chromatography (RPLC) we encountered serious detector baseline problems. The most severe problem was a periodic saw-tooth-shaped drift. This drift was most noticeable when using water-THF and water-dioxolane mobile phases. We demonstrate that the baseline problems are related to temperature fluctuation in a water bath used to maintain a constant column temperature. The temperature fluctuations change the refractive index (RI) of the mobile phase, which in turn affects the amount of light reaching the detector. Our results show that the magnitude of the baseline drift is related to the RI value of the mobile phases. The baseline problems can be alleviated using a different temperature controller whose feedback mechanism assures smoother operation. Since there is no single and complete source of information in the literature concerning the RI values of typical mobile phases in RPLC, we decided to measure them as a function of the % modifier (between 0 and 100%) and the temperature (between 25 degrees C and 50 degrees C). Knowing the RI values allows us to calculate the excess volume of a binary liquid system.

A study was made to establish whether the size of system peaks, which occur in chromatography with multi-component mobile phases, is a linear function of the concentration of the components. In the two chromatographic systems studied, it was found that the size of the system peaks is proportional to concentration. Using the fact that the injection of concentrations lower than that in the mobile phase results in negative system peaks whereas the injection of higher concentrations yields positive peaks, a simple method to determine the concentration of the components in the mobile phase is described. The approach seems to be robust although the retention times of some mobile phase components might be affected by the concentration of other components. The results presented indicate that system peaks can be used to monitor continuously various streams such as rivers, waste waters and physiological fluids.

The results of the present work show that the physical shape of the injection end of the capillary in CE has a major effect on the observed efficiencies. Straight-edge capillaries yield much more efficient electropherograms than do slanted edge capillaries. Also, injections into straight-edge capillaries give more symmetrical peaks. Indications are that the effect of the inlet shape is independent of molecular size. Moments analysis shows that the increase in size of the injected plug. due to the slanted shape of the capillary inlet, is not sufficient to explain the large decrease in efficiency.

Electroosmotic flow in capillary zone electrophoresis must be controlled for precise and reproducible performance. We found that the addition of any one of several classes of compounds such as amines, amino acids or organic acids to the running buffer stabilizes electroosmotic flow. With the additives in the buffer, the precision of migration times improved to better than 1%. The reproducibilities improved to better than 5%. The system is very stable at various conditions for small molecules and for proteins at very acidic or very basic conditions.

An expression is derived for the plate height in capillary electrophoresis for cases where hydrostatic flow (HF) is present. HF will occur whenever the buffer levels at both ends of the capillary are not at the same height. Whereas the plate height equation for an HF-free system has only a molecular diffusion term, the plate equation in the presence of HF has an additional term which is a resistance to mass transfer term. The second term is a function of capillary radius, the hydrostatic velocity, the solute diffusion coefficient and the electrophoretic velocity. Unlike in chromatography, the mass transfer term usually does not increase with increasing solute velocity. Nonetheless, the contribution of this additional term to the total plate height can be substantial for wide capillaries and large solute molecules. We calculated maximum allowable buffer height differences, Δ h max , for a given loss in plate height. It was found that for large molecules and wide capillaries, Δ h max , can be less than 1 mm, making severe demands on the instrumental design. With small solutes and narrow capillaries, the requirement for extract buffer levelling at both ends of the capillary is less acute.

Selectivity and retention surfaces were developed for the rapid high-performance liquid chromatographic separation of dideoxyribonucleosides which are promising antiretroviral agents. In this study, the dideoxyribonucleosides 2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine, 2′,3′-dideoxycytidine and 3′-deoxythymidine were used as model compounds and 2′-azido-3′-deoxythymidine (AZT) and AZT-5′-phosphate (AZT-5′-P) as experimental text compounds. The interdependence of the experimental variables, column temperature, mobile phase pH and percentage of methanol, and the effects of these variables on the reversed-phase liquid chromatographic (RPLC) behavior of the dideoxy compounds were investigated. The condition for optimal resolution of these compounds, as determined from the surfaces, were mobile phases of pH 5.0 and either 14% methanol at 40°C or 15% methanol at 30°C. These surfaces served as guidelines in determining conditions for preparative and analytical scale RPLC separations of AZT from AZT-5′-P.

The analysis of inorganic anions found in residues of low explosives present a difficult forensic problem. Current methods are neither quantitative nor sensitive. While the liquid chromatographic separation of these anions is relatively easy, their detection is difficult since they cannot be detected by conventional high-performance liquid chromatography detectors. We report a method that a can detect inorganic anions, which are commonly found in combustion residues of low explosives, by reversed-phase ion-pair liquid chromatography, using indirect UV detection. The ion-pair reagent is benzyltributylammonium chloride. In addition, the phosphate buffer mobile phase contains hexane sulfonate. There is little sample preparation since no interferences were encountered while testing real samples from explosive residues. Detection limits are low and selectivity is high compared with existing techniques. Analysis time is short and the separation can be repeated immediately.

We have modified a reversed-phase (RP8) column by passing through it an aqueous solution of phosphatidylcholine-based liposomes. The phospholipids from the liposomes adsorb onto the octyl chain of the stationary phase, thus altering the nature of the stationary phase and of the chromatographic interactions. The properties of the phospholipid-modified column were investigated using solutes belonging to several chemical classes. We found that the retention factors of negatively and positively charged solutes decreased as the amount of phospholipid in the column was increased. For the solutes studied here the extent of the decrease was smaller for the positive solutes. With neutral solutes, the retention factors of some (benzenediols) increased markedly while with others (ketones) the retention factors decreased. The selectivities between the various solutes on the phospholipid-modified column were different than on the original reversed-phase column. The retention behavior of the solutes can be explained in terms of (1) effective shielding of the hydrophobic part of the stationary phase by the polar head groups of the phospholipids and (2) hydrogen bond formation between the solutes and the carbonyl oxygens as well as the non-ester phosphate oxygens in the polar head groups of the phospholipids.

Summary In order to assess the viability of the photodiode array detector, its noise characteristics and linear range have been studied. It was found that at worst the peak noise is ±0.001 a.u. but more typical values are ±6·10 −4 a.u. With increased signal averaging, the noise values can be decreased to ±2·10 −4 a.u. The detector was found to have a linear range between 2·10 3 and 6.5·10 3 , depending on the wavelength. The versatility of the diode array detector was demonstrated by deconvoluting a multisolute chromatographic peak and determining the amounts of each solute in the mixture. The agreement between the amounts injected and those calculated was within several percent.

A new bonded phase, containing a Co(en)33+ moiety, was prepared in order to study the chromatographic behavior of solutes capable of forming outersphere complexes with cobalt compounds. The test solutes were nucleotides and nucleosides. It was found that solute—statoinary phase interactions could be quite strong resulting in long retention times. This was particularly so with triphosphate nucleotide. To overcome this difficulty Mg(II) was added to the mobile phase. Under these conditions selectivities, efficiencies and analysis times were greatly improved. The behavior of the Co(en)33+ phase was compared to that of a C18 column and a diamine system. The importance of adding Mg(II) to the mobile phase is demonstrated on all systems.

The theory of resolution optimization by isochronal analysis is further extended to the cases involving simultaneous changes in mobile phase velocity and column temperature, and in mobile phase composition and temperature. In each case, the resolution equation, when written in terms of isochronal conditions, shows that improvements can be obtained without sacrificing analysis time. Several subcases, which relate to the dependence of the selectivity factors on the changed parameters, are described. For each system, the contributions of the various parameters to the resolution are examined. The results of these discussions are plotted as resolution surfaces, on which lines of constant analysis time are indicated. The experimental routes that chromatographers must take in order to apply isochronal analysis are described.

A new diode array detector for liquid chromatography is described. The low-volume flow cell of the detector and the real time signal averaging of the spectra allow operation at 0.005 a.u.f.s. with noise levels of 0.0005 a.u. peak to peak. Since the detection is done over a large range of wavelengths, the detector allows identification of various solutes and peak deconvolution. The paper demonstrates how first derivative spectra can be gainfully utilized in a unique way to maximize the information obtained from the detector.

By plotting capacity factors and selectivity factors as functions of temperature and pH or methanol concentration of the mobile phase, retention and selectivity surfaces of four deoxyribonucleosides were generated. Although changes in the methanol content had little effect on selectivity, changes in pH and temperature could be used to improve selectivity. However, changes in pH or temperature can be coupled with changes in methanol concentration to optimize the analysis time. In addition, by using lines of constant analysis time (isochrons), it was found that several sets of conditions will result in nearly identical retention times. These isochrons can be used to optimize the resolution of the solutes.

The reversed-phase chromatographic behaviour of a homologous series has been investigated as a function of the mobile phase composition and temperature. In general, it was found that the methylene selectivity decreased with increasing temperature and decreasing amount of water in the mobile phase. The dependence of the calculated data on the correct determination of the hold-up time is discussed. The methylene group contribution to ΔH of transfer was calculated using two different methods and the agreement was excellent. In addition, the agreement with one other reported value was excellent. Using the homologous series data, the solvent strength of the water-methanol mobile phases was calculated at different temperatures. The results were compared with the data obtained on pyrocarbon supports and the agreement was very good. Linearization methods for calculating to are discussed and the difficulties in using such procedures are described.

The chromatographic separation of N-methylamino acids can be accomplished using a reversed-phase column with an aqueous mobile phase containing copper ions. The N-methylamino acid—Cu complex absorbs UV radiation with λ max around 235 nm. Thus copperions ifluence the retention times and allows the detection of the above solutes. Unsubstituted amino acids also absorb radiation at 235 nm. The molar absorptivities of the two types of solutes seem to be of the same order of magnitude and the detection limits are therefore also close. The method can be utilized in the simultaneous analysis of amino acids and their N-methyl derivatives. Although the method cannot compete with the sensitivity of amino acid analysers, its simplicity and its detection limits make it useful in such research fields as peptide synthesis.

Amino acids can be separated with a simple isocratic liquid chromatographic system in which no pre- or post-column derivatization of the amino acids is needed. Copper ions and alkylsulphonate additives are used to effect both the selectivity and the retention of the solutes. The effect of several operating conditions on the performance of the system was examined, especially the nature and concentration of the alkylsulphonate, the concentration of copper ions, the ionic strength of the buffer and temperature. Guidelines are given for optimization of the separation. the deleterious effects of system peaks and means of overcoming them are discussed.

Solute identification by retention data alone does not yield a positive identification for two or more solutes with approximately the same retention times. In such cases, two columns with two different stationary phases are needed for solute verification. For this reason, the concentration profiles of gas chromatographic peaks have been studied using the empirically derived eight-parameter model of Chesler and Cram. A non-linear least squares fit of the data to this model has made it possible to derive parameters which allow homologous class recognition and specific solute identification. Two methods for determining the initial estimates are presented. In one method the initial estimates were calculated from the data in a purely empirical manner, while the other is based mathematically on the model. The fitted values of the parameters from the two methods are compared for the purpose of illustrating the dependence of the fit on the choice of initial estimates. Some of the parameters are believed to have physical significance. For example, an almost linear relationship between one of the fitted parameters, C 2 , and the diffusion coefficient, D AB , of the solute in the carrier gas was found. Similar results were found for C 7 versus D AB , the capacity ratio k ′ and C 2 , and k ′/(1 + k ′) 2 and C 2 .

We report the separation of amino acids on reversed-phase columns using aqueous mobile phases containing copper ions. Cu(II) forms complexes with the amino acids and therefore influences their retention times. More importantly the Cu—amino acid complex absorbs UV radiation with λ max around 230 nm. Thus the solutes can be detected at relatively long wavelengths. The linearity of the detector signal and the detection limits were studied. Ssamples as small as 10 ng per 10 μI can be detected and the useful range of detection is oer four orders of magnitude. The effects of the pH of the mobile phase were also studied: retention increases with pH, at least over the range investigated.

A dithiocarbamate column is used to resolve diphosphate and triphosphate nucleotides with Mg(II) in the mobile phase. A retention mechanism is proposed to account for the retention behavior in the present system. Using this retention model, a linear correlation is found between the concentration of Mg(II) and the K ′ values of nucleotides. Complex formation constants can also be obtained by this method showing good agreement with those reported in the literature.

Bonded optically active tripeptides have been applied as stationary phases for liquid chromatography. Significant retention variations are shown for some UV absorbing amino acids when compared to a silica gel column using the same mobile phase. The separation of certain isomeric dipeptides has been accomplished using the bonded optically active tripeptide stationary phases. It is shown that these separations are superior to those possible on silica gel, although the efficiencies of the column are low. Phenylthiohydantoin (PTH)-amino acids have also been analyzed using the bonded tripeptide L -Val- L -Ala- L -Ser on silica gel CT and 1% citric acid-water as the mobile phase. No two PTH-amino acids showed the same capacity ratio out of 25 tested and the separation of 15 in one isocratic run is presented. The implications and future for the use of bonded optically active peptides as stationary phases for liquid chromatography are discussed.

In order to decrease the analysis time of a given chromatographic system, many parameters may need to be changed. These changes can adversely affect the chromatographic performance: in particular, the peak capacity may decrease. The peak capacity is a measure of the number of components that can be resolved in a given time. Using this definition of the peak capacity, various options to obtain shorter analysis times were investigated. It was found that such parameters as particle size, column diameter, mobile phase velocity and column length can be changed so that the analysis time decreases without loss of peak capacity. Optimum procedures for that purpose are given. In the course of the analysis, a new efficiency criterion—the rate of peak capacity production—is defined and examined.

The calculation of the higher central moments of chromatographic peaks has been a long-time burden of the chromatographer. The calculation of these quantities experimentally is not often accomplished since the integration process is extremely sensitive to noise and the limits of integration, causing the results to vary over a large range. Owing to these discrepancies, the purpose of this work was to derive the moments of the predetermined eight-parameter peak shape model of Chesler and Cram after fitting the equation to experimental data, and to offer a comparison of the results to the direct integration of the profile. In some cases, the experimental moments are in close agreement with those predicted by the model, whereas in others the difference is quite substantial.

The ternary complex between a nucleotide, a metal cation and an amino acid can be viewed as a simple model of the active centre of certain enzymatic sy

The present paper investigates the behavior of p-(p-ethoxyphenylazo)phenyl undecylenate (EPAPU) liquid crystal as a stationary phase coated on glass beads in gas chromatography under a variety of conditions. The columns studied were 0.69% and 0.10% (w/w) mesophase coated on neutral glass beads and 0.11% (w/w) on acid-treated beads. Unlike previous studies no hysteresis was found in the capacity ratio, k′, and the plate height, H, as a function of the temperature. However, around the chromatographically observed melting point, k′ and H varied with time at a given temperature. The same behavior was found around the solidification point. Surface orientation effects and the “swarm” model were used in trying to explain the above phenomena. When comparing EPARU coated on acid-treated glass beads and on neutral beads different kinetics behavior (in terms of H) around the melting point was observed. The diffusion coefficients of p-xylene in EPARU and activation energies in the nematic range were obtained. The lightly loaded columns gave very similar results which differed from the heavily loaded columns.

An expression for the radial temperature profile in capillary zone electrophoresis was derived, taking into account the temperature dependence of the buffer electrical conductivity and the polyimide coating of the quartz capillary. Calculations show that in typical capillary zone electrophoresis experiments; i.e. , capillaries with 50–100 μm I.D., 375 μm O.D., and up to 5 W power input, the temperature profile rigorously derived is nearly identical to a parabolic profile. At high input powers, the parabolic approximation underestimates the temperature at the capillary center.

Phenacyl and benzyl derivatives of some biologically significant dicarboxylic acids have been prepared in a quantitative manner using a crown ether catalyst. They were separated using reversed-phase chromatography. The column used was packed with Corasil II to which a C9 phase (nonyl) was bonded. Water-methanol (68:32) was used as the mobile phase. Detection limits in the range of 5--15 ng have been obtained for a non-optimized system. The ease of preparation of these derivatives, coupled with their excellent chromatographic properties, makes this a very attractive procedure for the investigation of mixtures containing biologically significant acids. The implications and future of this technique are discussed.

A new source of zone broadening in capillary zone electrophoresis (CZE) associated with electrokinetic and hydrostatic injection techniques has been identified. The actual insertion of the capillary into or withdrawal of the capillary from the sample solution results in the extraneous injection of sample into the capillary. Examination of the sample plug resulting from this extraneous injection indicates that its length can exceed the maximum value permitted to realize the high separation efficiency of CZE. Experiments show that this mode of injection is present even under conditions where hydrostatic flow should occur from the capillary back into the sample vial.

The effect of the carrier velocity on the optimum length in time normalization chromatography (or analysis at constant retention time) was investigated. It was found both theoretically and experimentally that at any given carrier velocity (or analysis time) the resolution can be optimized. The length at which the optimization occurs is a function of the plate height dependence on the temperature. In general, an absolute maximum resolution occurs when operating at carrier velocities around the Van Deemter plot minimum. It appears that a prediction of the column length can still be made using teh approach suggested by us previously. Theoretically, in the case of a capillary column, one can operate at twice the minimum velocity without a large loss in the resolution but a saving in the analysis time.

Nucleotides, nucleosides and their bases are known to form complexes with metal cations, some of which are essential for the proper functioning of thse compounds. The cations have several possible roles, all of which cause some changes in the parent molecule's spacial arrangement and/or charge. Hence chromatographic retention, being sensitive to these two parameters, should differ for complexed and uncomplexed nucleotides. The retentions were obtained using mobile phases containing various concentrations of one of four metal cations, Mg(II), Ni(II), Nz(II) and Cu(II). These were then compared with values obtained without the cations. As expected, the nature and concentration of the metal ion in the mobile phase affected the chromatographic behaviour. In all instances Mg(II) caused a decrease in the retention times, as measured from the capacity ratios (k'. The presence of Ni(II) or Zn(II) in the mobile phase caused an initial decrease and then, as the concentration of these metal ions increased further, k' increased. The nucleotide retentions increased when Cu(II) was added to the mobile phase. The possible formation of an intramolecular macrochelate can be related to the pattern of the retention times. In addition, it is shown that the capacity ratios can be used to predict properties such as base acidity and dephosphorylation.

The resolution of amino acid enantiomers in liquid chromatography using chiral eluents is examined in light of the three “points” association model. The resolution is found to be a function of the alkyl chain length of the resolving agent, which is an alkyl amide derivative of aspartic acid complexed with copper(II). For example, mobile phases with l -aspartylethylamide copper(II) chiral reagent gave lower resolution than with l -aspartyl-n-hexylamide copper(II). The pH also influences the resolution. In general, k′ values and selectivity factors increase with increasing pH. A mobile phase containing a derivative of glutamic acid could not resolve enantiomers. Analysis of the results shows that the three “points” contact is between the α-carboxy and α-amino groups of the amino acid and the copper(II) and the side-chain of the amino acid and the alkylamide moiety of the aspartyl derivative.

A new method is described for the analysis of organic additives in copper plating baths. Liquid chromatography, utilizing an ion-pair reversed-phase technique, was found to be most suited for this purpose. Ion pairing neutralizes the brightener components, thereby enabling the use of moderate conditions during analysis, I.e. , a pH of 4–5 and isocratic elution. Preceding the chromatography, a solid-phase extraction process was used to decrease the copper ion concentration and the acidity of the bath. Active copper plating baths were investigated and the results are given and explained.

The work described in this paper tries to demonstrate that the formation of diastereomers, needed for the resolution of enantiomers of amino acids, takes place in the stationary phase, when aspartylalkylamides are used as chiral reagents. The chromatographic behavior of some amino acid enantiomers are studied under various chromatographic conditions. Resolution of the isomers occurs even under extreme conditions, such as low pH or very low resolving agent concentration. The same elution order was observed whether the chiral reagent was in the mobile phase or only in the stationary phase. The temperature dependence of the resolution was studied. it was found that quite frequently the entropy of the D -diastereomer is larger than that of the L -isomer. The behavior of the capacity ratios as a function of the chiral reagent concentration allowed the calculation of the diastereomer formation constant.

On fait deplacer la colonne de separation dans une colonne concentrique contenant la phase mobile (comme une seringue). La pression obtenue evite d'utiliser une pompe

A method for the fluorescence labeling of fatty acids with 4-bromomethyl-7-methoxycoumarin using 18-crown-6 as a catalyst is described. The procedure is rapid, simple and requires little or no experience in derivatization techniques. The amounts of the derivatives are linearly related to the amount of the starting fatty acids and the procedure can therefore be used in the quantitative analysis of these solutes. Using reversed-phase chromatography, detection levels as low as 9 pmole could be realized. The separations of several derivatives using a reversed-phase system are discussed.

The theoretical aspects of isochronal optimization (time normalization) are verified experimentally in the present communication. The variant examined here utilizes simultaneous changes in mobile phase composition and velocity. Two empirical relationships for the capacity factors were examined. From these relationships, predicted resolution surfaces were generated and these were compared with the experimental ones. It was found that quadratic relationships between the natural logarithm of k ′ and the mobile phase composition yielded surfaces that resembled the experimental ones most closely. Moreover, it was found that only three experiments, with three different mobile phases, were sufficient to calculate the resolution surface with adequate accuracy. Isochronal lines (resolution lines with constant analysis times) were generated from the predicted data and compared with experimental isochrons. The agreement between the two sets of lines was very good. Based on three experiments it is possible to predict experimental conditions that will yield better resolution without increasing the analysis time.

The chromatographic behavior of liquid crystal stationary phases has almost never been studied. In the present papaer, retention data, efficiency and mass ransfer data were investigated usingp-(p-ethoxyphenylazo)phenyl undecylenate (EPAPU) coated on glass beads. Glass beads were chosen as the support for several reasons: their geometry is known, they tend to orient liquid crystals, and diffusion coefficients can be studied when the liquid crystal is coated on them. The preliminary rresults obtained have shown the following. The resistance to mass transfer in EPAPU is rahter large due to the viscosity of the liquid crystal. In general, as the column temperature increases the minimum velocity (in the Van Deemter sense) increases. HETPversus temperture results seem to indicate that the resistance to mass transfer of a solute in the nematic EPAPU depends on whether the column is being heated or cooled. At the same time, the capacity ratio is independent of temperature in the nematic range. Also, it was found that EPAPU can be supercooled when coated on glass beads.