Search

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.

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.

The complex formation between β-cyclodextrin and nitrophenol isomers was studied with the aid of reversed phase liquid chromatography. The effects of temperature, pH, and methanol content on the formation constants of the nitrophenol/β-cyclodextrin complexes were investigated. The complex formation constants were found to decrease with increasing temperatures. This decrease in formation constant was independent of the pH of the mobile phase. From the temperature dependence of the formation constants we were able to extract enthalpies and entropies of the complex formation. The values calculated in this work agree favorably with published values. Three pHs were examined: 3.57, 4.65, and 5.6. The formation constants of the ortho and meta isomers were highest at the lowest pH and leveled off at the two higher pHs. The para isomer showed a minimum in the formation constant at pH 4.65. This behavior was independent of the pH of the mobile phase.

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.

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.

Phase-transfer catalysis is applied for the separation of carboxylate salts in aqueous solutions.

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.

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.

The present work demonstrates a method by which natural, non-UV absorbing penicillins can be derivatized to form phenacyl esters. The reaction is rapid and essentially completed in twenty minutes. A straight line correlation was found between the peak height of a phenacyl ester derivative and the amount of the penicillin, a fact which attests to the applicability of the method for quantisation. The phenacyl esters of the penicillins studied here were separated on a reversed phase column.

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.

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.

A fast and sensitive method for the analysis of tryptophan and some of its metabolites is discussed. A reversed-phase chromatographic system with water mobile phase can separate tryptophan, N-formalkynurenine, kynurenine and 3-hydroxykynurenine in less than 15 min at a flow-rate of 1 ml/min. The application of the method to the analysis of tryptophan and kynurenine in untreated urine of a patient loaded with tryptophan is described. The ease and speed of analysis makes the method very attractive for clinical purposes. Among other things, it was found that tryptophan in untreated urine degrades with time, even if the sample is frozen at −11°.