High-performance Liquid Chromatographic Behaviour Research Articles

Reversed-phase high-performance liquid chromatographic behaviour of some metal 1-(2-pyridylazo)-2-naphthol chelates

The HPLC behaviour of Cu(II), Co(III), Ni(II) and FE(II) chelates with 1-(2-pyridylazo)-2-naphthol (PAN) on alkyl-bonded silica gel stationary phase was studied in detail. Peaks of [Ni II(PAN) 2] and [Fe II(PAN) 2] were observed with the mobile phase acetonitrile-water (70:30, v/v). The cationic chelates, [Cu II(PAN)] + and [CoIII(PAN) 2] +, were found to be adsorbed on the stationary phase as a result of interaction with the residual silanol groups. A sharp peak of [Co III(PAN) 2] + chelate was obtained by adding one of several organic or inorganic salts to the mobile phase, but a [Cu II(PAN)] + chelate peak could only be obtained on addition of ammonium thiocyanate. The retention of the CO(III) chelate depended to a large extent on the alkyl chain length of the stationary phase and the species of an organic modifier in the mobile phase.

Dipicolinic acid as eluent for the high-performance liquid chromatographic determination of transition metals using iminodiacetic acid-bonded silica

The high-performance liquid chromatographic behaviour of Co2+, Zn2+, Cu2+, Cd2+ and Pb2+ on a column packed with iminodiacetic acid-bonded silica (250 × 3 mm i.d.) was studied using solutions of dipicolinic acid as eluent. Optimum separation was achieved when the concentration of dipicolinic acid in the eluent was 1 × 10–3 mol l–1 and the eluent pH was 3.0. The possibility of the sensitive indirect spectrophotometric detection of transition metals at 295 nm is demonstrated. The detection limits for the investigated metals are in the range 0.1–1 µg ml–1. The determination of zinc and lead in industrial waste waters was performed.

High Performance Liquid Chromatographic Separation of Sensitive Fluorescent Derivatives of Bile Acids with Cyclodextrin-Containing Mobile Phase

Abstract The high-performance liquid chromatographic behavior of new bile acid fluorescent derivatives, the 7-methoxy-1,4-benzoxazin-2-one-3-methy 1 esters, with a cyclodextrin-containing mobile phase is compared to those of the bile acid-pyrenacyl and -anthroyl esters. Compared with conventional methods, inclusion chromatography gives a much more satisfactory separation of the bile acid fluorescent derivatives in a short time, but the chromatographic behavior of these derivatives has not been much influenced by the fluorophore used. The detection limits of the new derivatives are in the range of 10–20 fmol at a signal to noise ratio of 5. The application of this method for the separation of g1ycine-conjugated bile acids in human bile is also described.

High-performance liquid chromatography of amino acids, peptides and proteins CXXVIII. Effect of d-amino acid substitutions on the reversed-phase high-performance liquid chromatography retention behaviour of neuropeptide Y[18–36] analogues

The reversed-phase high-performance liquid chromatographic (RP-HPLC) gradient elution behaviour of a series of peptides related to Neuropeptide Y (NPY) has been investigated. The peptides studied included NPY, NPY[13–36], NPY[18–36] and a series of 16 analogues of NPY [18–36], each with a single d-amino acid substitution. Chromatographic parameters which relate to the interactive contact area and the binding affinity have been evaluated with two different stationary phase ligands and two organic modifiers. The results demonstrate that d-amino acid substitutions in the sequence region encompassing amino acid residues NPY [27–31] of these NPY [18–36] peptides significantly influence the interactive behaviour of these peptides relative to the unsubstituted NPY [18–36] molecule, while substitutions in the N- and C-terminal regions had little effect. Further, these results indicate that, in the hydrophobic environments, NPY [18–36] adopts a significant degree of secondary structure which is severely disrupted by the presence of the d-amino acids in the central portion of the molecule.

Reversed-phase high-performance liquid chromatography of phospholipids with fluorescence detection

Reversed-phase high-performance liquid chromatographic (HPLC) behavior of fluorescent labeled phospholipids (PLs) was studied. Molecular species of phosphatidylethanolamine (PE) derivatized with fluorescein-, thiocarbamoyl-, pyrenesulfonyl-, and dimethylaminonaphthalenesulfonyl (dansyl)-fluorophores were separated on octadecylsilica with a mobile phase of acetonitrilemethanol- water in the presence of tetraalkylammonium phosphates (TAAPs). Under similar HPLC conditions, dansylated phosphatidylserines and PE plasmalogens (ether-linked PLs) were also resolved. Incorporation of the fluorescein moiety to the parent PE appeared to facilitate further resolution of its subcomponents. Subcomponents of dansylated PE derived from egg phosphatidylcholines were quantifiable. Effects of the type and concentration of TAAP on capacity factors of PL solutes were indicative of an ion-pair separation processes. HPLC with high-molecular-mass TAAPs favored the separation of the components that remained unresolved with mobile phases containing low-molecular- mass TAAPS. The HPLC-fluorescence detection method provided a useful approach to quantitative analyses of various PL structures. Compositions of PL subcomponents were determined directly from peak areas.

High-performance liquid chromatographic retention behaviour of ring-substituted aniline derivatives on a porous graphitized carbon column

The retentions of sixteen ring-substituted aniline derivatives were determined on a graphitized carbon column using unbuffered acetonitrile-water and methanol-water eluent mixtures at various organic phase concentrations. Principal component analysis calculated from both the correlation and covariance matrices was used to detect the similarities and dissimilarities between the retention behaviours of the aniline derivatives. Each aniline derivative showed narrow and symmetrical peaks in each eluent. The high percentage of variance explained in the first principal component suggests that the eluents have common elution characteristics, but according to the second principal component they showed slightly different selectivities. The retention of aniline derivatives is not governed by their lipopholicity and bulkiness. The data indicate that a mixed type of retention mechanism accounts for the retention.

High-Performance Liquid Chromatography of Iproplatin, an Antitumor Quadrivalent Platinum Complex

Iproplatin [cis-dichloro-trans-dihydroxo-bis(isopropylamine)platinum(IV), CHIP] is a potent antitumor platinum complex with axial hydroxy ligands. The high-performance liquid chromatographic behavior of CHIP was investigated on a poly(styrene-divinylbenzene)-based cation exchange column with a dipotasshim hydrogen phosphate eluent and an octadecylsilica column with a phosphate buffer/methanol eluent containing a sodium alkylsulfonate. On both cation exchange and octadecylsilica columns, the capacity factor of CHIP increased, the lower was the eluent pH. However, this was not found to be the case in the absence of an alkylsulfonate. Using the cation exchange column, the CHIP capacity factors at various pH values were plotted (on log-log graph paper) as a function of the eluent potassium concentration. Straight lines were obtained with slopes of —0.65 at pH 4.0 and —0.81 at pH 2.5. The observed charges on CHIP evaluated from these slopes were 0.65 at pH 4.0 and 0.81 at pH 2.5, indicating that a large portion of CHIP was present as a monovalent cation in both cases. This study shows that CHIP is protonated and, consequently, can be chromatographed on cation exchange and octadecylsilica columns as a cationic compound, under acidic conditions.

Phosphinobenzyl- and aryl-silanes and their triosmium cluster carbonyl derivatives

The Grignard reagents MgR(Br)(R = C6H4CH2SiMe2H-o or C6H4CH2SiMeH2-o) have been found to undergo unusual reactions with Ph2PCl to afford o-Ph2PC6H4CH2SiMe2H I and o-HMe2SiC6H4CH2PPh2II and o-Ph2PC6H4CH2SiMeH2III and o-H2MeSiC6H4PPh2IV respectively. Reactions of the respective bidentate ligands I, II, III and IV with [Os3(CO)10(MeCN)2] gave the novel triosmium cluster [Os3(µ-H)(CO)10(L–L)]1, 2, 3 and 4 involving cleavage of one Si–H bond. Similar reactions of ligands II and IV with [Os3(µ-H)2(CO)10] afforded two new clusters [Os3(µ-H)3(CO)8(L–L)]5 and 6 respectively. The structures of 1, 2, 4 and 5 have been determined by single-crystal X-ray diffraction techniques. In each case, the ligand assumes a chelating mode across the long edge of the osmium triangle which is also bridged by a hydride. The high-performance liquid chromatographic behaviour of these clusters has also been determined and correlated with the size and nature of the ligands.

Mass spectrometric and gas and high-performance liquid chromatographic behaviour of an impurity in 2,5-hexanedione

The analysis of 2,5-hexanedione, a metabolic compound of several industrial solvents, is normally carried out using gas chromatographic (GC) or GC-mass spectrometric (MS) techniques. This work, with the aim of verifying the possibility of determining the diketone by means of a high-performance liquid chromatographic (HPLC) method with UV detection, illustrates the importance of the choice of a 2,5-hexanedione standard for the determination of the diketone response factor. In some commercial diketone samples the presence of an impurity, which may interfere with the analysis of the target analyte, was ascertained. This impurity showed GC and HPLC behaviour similar to that of 2,5-hexanedione, but gave a very different UV response. This impurity was identified as 3-methylcyclopent-2-enone by means of MS, GC-MS, HPLC-photodiode-array detection, IR and UV spectrometry. The structure was confirmed by comparing the chromatographic, mass and ultraviolet data of the unknown compound with those of a synthesized reference sample. The well known difficulty in determining 2,5-hexanedione by HPLC with UV detection was reconfirmed owing to its low molar absorptivity.

Applicability of high-performance liquid chromatography-continuous-flow fast atom bombardment mass spectrometry for simultaneous quantitation of multiple neurochemicals

A method is described for the analysis of multiple neurochemically important compounds. The technique involves high-performance liquid chromatographic separation in combination with ultraviolet and continuous-flow fast atom bombardment mass spectrometric detection. High-performance liquid chromatographic elution behaviour and relative detection responsiveness as correlated with chemical structure are also presented.

High-Performance Liquid Chromatographic Behavior of 2-,4- and 6-Hydroxylated Bile Acid Stereoisomers

Abstract High-performance liquid chromatographic behavior has been investigated for 40 stereoisomeric 2-, 4- and 6-hydroxylated bile acids, most of which possess a vicinal glycol structure, as their 4-nitrophthalimidomethyl (NPM) ester derivatives on a reversed-phase column using methanol-water systems as a mobile phase. The bile acid NPM esters were further derivatized to the so-called “mixed” acetonide-NPM esters, whereby the stereochemical relationships (α, α-and β,β-cis, diaxial trans or diequatorial trans) of 2,3-, 3,4-and 6,7-diol groups in the molecules were reflected to changes in the capacity factors of bile acids.

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata : I. Effect of the mobile phase

The high-performance liquid chromatographic (HPLC) behaviour of two different styrene-divinyl-benzene-based reversed-phase (RP) columns was evaluated using crude acetic acid extracts from normal and diabetic human pancreata as samples. Acetic acid gradients in water and acetonitrile gradients in triethylammonium phosphate (TEAP) and trifluoroacetic acid (TFA) were used as mobile phases, and comparisons were made with a silica-based C 4 column. When two different polymeric RP columns were eluted with acetic acid gradients in water, surprisingly similar HPLC profiles of the pancreatic extracts were obtained. Elution of the polymer-based columns with acetonitrile gradients in TFA or TEAP resulted in changes in the polypeptide selectivity of these columns, in parallel with that of a silica-based C 4 column eluted under similar conditions, indicating the general usability of polymeric columns for RP-HPLC of peptides and proteins. The pronounced difference in composition between normal and diabetic samples, which also was demonstrated after size-exclusion chromatography (SEC) on a silica-based and an agarosebased high-performance SEC column, was found to be related to the different ischaemia times for the two types of pancreata.

Relationships between nucleotide incorporation rates and molecular parameters obtained by molecular modelling and chromatography

Deoxyuridine derivatives play an important role in pharmaceutical chemistry as they are potential antiviral and antitumour agents. Their pharmacological activity depends on their ability to incorporate into DNA in their triphosphate forms. High-performance liquid chromatographic (HPLC) retention behaviour of a series of 5-alkyl, alkenyl and alkynyl substituted deoxyuridine derivatives were investigated on reversed-phase stationary phase using various mixtures of methanol and water as mobile phases. The slope and the intercept values of the linear relationships between the logarithmic capacity ratio (log k') values and methanol concentration have been calculated. Non-polar, non-polar unsaturated and polar surface areas, surface energies, dipole moments, van der Waals radii of the derivatives have been calculated on the bases of molecular mechanics by PC Model approach. The correlation study of the above-mentioned parameters revealed that hydrophobic and hydrophilic surface areas and the electronic effects of the substituents determine not only the retention behaviour of the derivatives but also their incorporation rate into DNA in their triphosphate forms.

Factors influencing the retention of rare earth—tetraphenylporphine complexes in reversed-phase high-performance liquid chromatography

The high-performance liquid chromatographic (HPLC) retention behaviour of the complexes of tetraphenylporphine (TPP) with twelve rare earths (REs), viz., Y(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(III) and Lu(III), on an octadecyl-bonded silica gel column is described. All these metal complexes can be chromatographed without undesirable demetallation in their migration process along the column with a methanol-water mixture containing a small amount of acetyl-acetone and an amine. The elution sequence for the RE complexes depends on the amine added to the mobile phase. With trialkylamines and dialkylamines possessing branched alkyl structures, the capacity factors of the RE-TPP complexes increase monotonously in the order of increasing atomic number of the REs. With di- n-alkylamines, the capacity factor tends to decrease in the order of increasing atomic number of the REs for light to moderate lanthanides, whereas the reverse tendency occurs for the complexes of heavier lanthanides. In all instances, the capacity factor of the Y(III) complex lies between those of the Dy(III) and Ho(III) complexes. The effects of the amine are discussed in terms of the adduct complex formation of RE-TPP with the amine. Successful separation of the Nd, Gd, Tb, Dy, Ho, Er and Lu complexes in 15 min using triethylamine is demonstrated.

High Performance Liquid Chromatographic Behavior of Difluoroborane Derivatives of β-Diketones

β-Diketones react with boron trifluoride (BF3) under mild reaction conditions to give their difluoroborane (BF2) chelates quantitatively. The complexes gave sharp peaks on both normal- and reversed-phase high performance liquid chromatography (HPLC), while the unreacted β-diketones gave broad peak and poor reproducibility probably due to keto-enol interconversion during the separation. Fe(III) chelate of acetylacetone (Haa) was also found to react with BF3 to give the BF2 chelates quantitatively.

Reversed-phase high performance liquid chromatographic behaviour of homologous series

The retention model for reversed-phase high-performance liquid chromatography (RP-HPLC) based on interaction indices was applied to separation of the homologous series members. The validity of the equations describing relationship between retention (log k) and the number of methylene groups in alkyl chain (Cn) and composition of mobile phase (φ) by using statistical methods was verified. The regression coefficients of a quadratic term for relationship log k vs Cn and log k vs φ can be neglected. The regression coefficient of interaction term Cnφ is statistically significant.

Modification of the high-performance liquid chromatographic retention behaviour of β-diketones by the inclusion of metal ions in the mobile phase

The effect of transition metals as components of the mobile phase in high-performance liquid chromatography on the retention of analytes containing a β-diketone chelating group has been examined. A model for the changes has been developed which takes into account the secondary chemical equilibria in solution, including the formation constants of the complexes, the pH of the model phase and the concentration of the metal ions.

Prediction of the high-performance liquid chromatographic retention behaviour of some benzodiazepine derivatives by thin-layer chromatography

The reversed-phase retention behaviour of eighteen benzodiazepine derivatives was studied using high-performance liquid chromarography (HPLC) and thin-layer chromatography (TLC). In each instance the retention decreased with increasing concentration of the organic mobile phase in the eluent, that is, no anomalous effect was observed. In reversed-phase TLC the R M value of benzodiazepine derivatives depended linearly on the organic phase concentration and logarithmically on the buffer concentration in the eluent. A highly significant correlation was found between the HPLC log k 0 value and the reversed-phase TLC parameters, suggesting that TLC can be used for predicting the HPLC retention behaviour of benzodiazepine derivatives. As the TLC parameters explained about 75% of the total variance, the predictive power of TLC is limited.

Expert system for the selection of initial high-performance liquid chromatographic conditions for the analysis of pharmaceuticals

The high-performance liquid chromatographic (HPLC) behavior of basic pharmaceutical substances is strongly influenced by the type of column packing, the pH of the mobile phase and the concentration and type of buffer ions. This results in many choices to be made by the chromatographer. In order to assist the chromatographer, an expert system has been developed for the selection of initial HPLC conditions. For this purpose, HPLC data for about 600 basic compounds were used. These compounds belong to the class of CNS-active or cardiovascular drugs. On the basis of this knowledge, which was completed with literature data, rules were defined and a knowledge base was built. The knowledge was implemented in KES (Knowledge Engineering System), a mid-sized expert system shell which runs on an IBM-PC. The system asks for information with respect to the substance(s) to be analysed. This information is given in the form of a table of structural elements. The output of the system specifies the chromatographic characteristics which should be used, i.e., type of stationary phase, mobile phase composition, buffer pH, flow-rate and method of detection.

Comparison of reversed-phase thin-layer and high-performance liquid chromatography of flavonoid compounds

The reversed-phase high-performance liquid (HPLC) and thin-layer chromatographic (TLC) behaviour of flavonoids with methanol, tetrahydrofuran and acetonitrile as organic modifiers was compared. Twenty-six different correlation cases, together with their statistical parameters, are presented and discussed. Both TLC and HPTLC plates were considered. The dependence of TLC incremental R m , values from different group substitutions on solvent type, solvent composition and type of plate is considered. A method for establishing HPLC gradient elution conditions by using TLC data is discussed.