High-performance Liquid Chromatography Retention Research Articles

Rapid HPLC Method Development of Polynuclear Aromatic Hydrocarbons Separation Based on Quantitative Structure Retention Relationships

Previous studies demonstrated that quantitative structure retention relationships (QSRR) combined with the linear solvent strength (LSS) model allow for prediction of gradient reversed phase high performance liquid chromatography (HPLC) retention time for any analyte of a known molecular structure under defined HPLC conditions. The QSRR model derived at the selected gradient time was tested at the same gradient time. Presently, in the first step, experimental retention data for model sets of just 5 analytes were used to derive appropriate QSRR models at two gradient times. Additionally, a new molecular modeling approach based on a more accurate ab initio method was here proposed. Those QSRR models were used to further predict gradient retention times for sets of 16 test analytes belonging to polynuclear aromatic hydrocarbons (PAHs), at two selected gradient times. Then, applying linear solvent strength (LSS) theory, only predicted retention times for PAHs were used to find the optimal gradient conditions to separate them. Satisfactory predictions of gradient retention times for PAHs were obtained. Contrary to the previous achievements, the proposed QSRR provides the chance to predict retention of PAHs with the appropriate selectivity achieving the same sequence of analytes eluted in the experiment and during the simulation performed on the computer screen.

The influence of the structure of some aromatic heterocyclic derivatives on their retention in reversed-phase high-performance liquid chromatography

The chromatographic behavior of aromatic heterocyclic derivatives in reversed-phase high-performance liquid chromatography was investigated. The retention characteristics of the substances under the conditions of chromatography with water-acetonitrile mobile phases (retention factors, relative retentions, distribution coefficients, Henry adsorption constants, differences in the differential molar energy of sorption, and Gibbs energies of sorption) were determined. It was shown that the chromatographic retention of the sorbates depended on their molecular structure. The influence of the nature of heteroatoms and their number on the sorption of heterocyclic compounds was discussed.

Identification and Quantitation of theN-Acetyl-L-cysteineS-Conjugates of Bendamustine and Its Sulfoxides in Human Bile after Administration of Bendamustine Hydrochloride

We recently reported the detection of mercapturic acid pathway metabolites of bendamustine, namely, cysteine S-conjugates in human bile, which are supposed to subsequently undergo further metabolism. In this study, we describe the identification and quantitation of consecutive bendamustine metabolites occurring in human bile using authentic reference standards and the synthesis and structural confirmation of these compounds. Mass spectrometry data along with high-performance liquid chromatography retention data (fluorescence detection) of the synthetic reference standards were consistent with those of the metabolites found in human bile after administration of bendamustine hydrochloride to cancer patients. Analysis of the purified synthetic reference compounds showed a purity of at least 95%. Structural confirmation was achieved by one- and two-dimensional proton as well as carbon-13 NMR spectroscopy and mass spectrometry. A total of 16 bendamustine-related compounds were detected in the bile of patients, 11 of them were recovered as conjugates. Eight conjugates have been structurally confirmed as novel mercapturic acids and sulfoxides. Biliary excretion of the sulfoxides was twice that of the mercapturate precursors. Glutathione S-conjugates of bendamustine have not been detected in bile samples, indicating rapid enzymatic cleavage in humans. Both the lack of glutathione (GSH) conjugates and occurrence of diastereomeric sulfoxides emphasize species-related differences in the GSH conjugation of bendamustine between humans and rats. The total amount recovered in the bile as the sum of all conjugates over the period of 24 h after dosing averaged 5.2% of the administered dose. The question of whether the novel metabolites contribute to urinary excretion should be a target of future investigations.

Application of LC–MS to the analysis of dyes in objects of historical interest

High-performance liquid chromatography (HPLC) with photodiode array and mass spectrometric detection permits dyes extracted from objects of historical interest or from natural plant or animal dyestuffs to be characterized on the basis of three orthogonal properties: HPLC retention time, UV–visible spectrum and molecular mass. In the present study, we have focused primarily on yellow dyes, the bulk of which are flavonoid glycosides that would be almost impossible to characterize without mass spectrometric detection. Also critical for this analysis is a method for mild extraction of the dyes from objects (e.g., textiles) without hydrolyzing the glycosidic linkages. This was accomplished using 5% formic acid in methanol, rather than the more traditional 6 M HCl. Mass spectroscopy, besides providing the molecular mass of the dye molecule, sometimes yields additional structural data based on fragmentation patterns. In addition, coeluting compounds can often be detected using extracted ion chromatography. The utility of mass spectrometry is illustrated by the analysis of historical specimens of silk that had been dyed yellow with flavonoid glycosides from Sophora japonica (pagoda tree) and curcumins from Curcuma longa (turmeric). In addition, we have used these techniques to identify the dye type, and sometimes the specific dyestuff, in a variety of objects, including a yellow varnish from a 19th century Tibetan altar and a 3000-year-old wool mortuary textiles, from Xinjiang, China. We are using HPLC with diode array and mass spectrometric detection to create a library of analyzed dyestuffs (>200 so far; mostly plants) to serve as references for identification of dyes in objects of historical interest.

Identification and Characterization of Anthocyanins by High-performance Liquid Chromatography–Electrospray Ionization–Mass Spectrometry in Herbaceous Peony Species

Petal anthocyanins were systematically identified and characterized by high-performance liquid chromatography (HPLC)–electrospray ionization–mass spectrometry (MS) coupled with diode array detection among nine wild herbaceous peony (Paeonia L.) species (15 accessions). Individual anthocyanins were identified according to the HPLC retention time, elution order, MS fragmentation patterns, and by comparison with authentic standards and published data. Six main anthocyanins, including peonidin-3,5-di-O-glucoside, peonidin-3-O-glucoside-5-O-arabinoside (Pn3G5Ara), peonidin-3-O-glucoside, pelargonidin-3,5-di-O-glucoside, cyanidin-3,5-di-O-glucoside, and cyanidin-3-O-glucoside (Cy3G), were detected. In addition to the well-known major anthocyanins, some minor anthocyanins were identified in herbaceous peony species for the first time. Detection of the unique anthocyanins cyanidin-3-O-glucoside-5-O-galactoside and pelargonidin-3-O-glucoside-5-O-galactoside in both Paeonia anomala L. and P. anomala ssp. veitchii (Lynch) D.Y. Hong & K.Y. Pan indicated these two species should belong to the same taxon. Pn3G5Ara was found only in European wild species and subspecies suggesting different metabolic pathways between European and Chinese accessions. Anthocyanins conjugated with galactose and arabinose were observed in the genus Paeonia for the first time. The North American species, Paeonia tenuifolia L., had high Cy3G content in flower petals. This anthocyanin composition is distinct from the anthocyanin composition in Asian and European species and possibly is responsible for the vivid red coloration in flowers.

Biodegradable fumarate‐based drug‐delivery systems for ophthalmic applications

The function of a photocrosslinked poly(propylene fumarate) (PPF)/poly(N-vinyl pyrrolidone) (PVP) matrix for the sustained release of three ophthalmic model drugs, acetazolamide (AZ), dichlorphenamide (DP), and timolol maleate (TM), was investigated. The drugs differ in molecular weight and degree of dissociation in aqueous environments; both are parameters that significantly influence drug diffusivity. AZ, DP, and TM-loaded cylindrical rods (10 mm length, 0.6 mm diameter) were fabricated by photoinduced cross-copolymerization of PPF and N-vinyl pyrrolidone (NVP) in molds. The released amounts of AZ, DP, TM, and NVP were determined by high-performance liquid chromatography (HPLC). The effects of drug properties and loading on the release kinetics were investigated. The in vitro release of AZ, DP, and TM was well sustained from the polymer matrices over a period of approximately 210, 270, and 250 days, respectively. The release kinetics correlated with the HPLC retention profiles of the different drugs. Following a small initial burst release (<10%), a dual modality release controlled by diffusion and bulk erosion was found for all drugs. Drug release rates of up to 4 microg/day were reached. Matrix drug loading generally affected the extent of the burst release, release kinetics, as well as the matrix water content and matrix degradation that were determined gravimetrically. Microcomputed tomography was used to image structural and dimensional changes of the devices. A preliminary rabbit implantation study revealed promising ocular biocompatibility of drug-free PPF/PVP matrices. All results indicate the potential of photocrosslinked PPF-based matrices as polymeric carriers for long-term ophthalmic drug delivery.

Alkyl sulfur chlorophyll derivatives: Preparation and liquid chromatography-multistage tandem mass spectrometric characterisation of analogues of naturally occurring sedimentary species

A series of synthetic sulfur-containing chlorophyll derivatives with alkyl sulfur groups attached to the C-3 1 position, the same as in structures proposed for natural alkyl sulfur chlorophyll derivatives, has been prepared from the chlorophyll a derivative, methyl pyrophaeophorbide a. The synthetic compounds exhibit shifts in high performance liquid chromatography (HPLC) retention times and online UV/vis absorption maxima relative to methyl pyrophaeophorbide a, consistent with those observed between the natural alkyl sulfur chlorophyll derivatives and their non-sulfurised counterparts. The 1H nuclear magnetic resonance and multistage tandem mass spectra of the synthetic and natural alkyl sulfur chlorophyll derivatives show close agreement. The prominent loss of the C-3 1 alkylsulfur group as a radical during multistage tandem MS has been confirmed as a diagnostic feature of the compounds.

Temperature-Modulated Interaction Changes with Adenosine Nucleotides on Intelligent Cationic, Thermoresponsive Surfaces1

Thin layer poly(N-isopropylacrylamide-co-n-butyl-methacrylate-co-N,N-dimethylaminopropylacrylamide) (IBD) copolymer gels are covalently introduced to initiator immobilized silica bead surfaces to create thermally sensitive intelligent cationic surfaces. The surface shows thermoresponsive changes in charge density as well as hydrophilic/hydrophobic character. The polymer chains dehydrate and inter-/intra-molecular aggregation occurs due to weakly deprotonated cationic amino groups in the hydrophobized circumstances, resulting in the surface charge density decrease. This was corroborated by the thermoresponsive pKa shift of dimethylamino side groups in the copolymers as well as the surface potential changes at elevated temperature. The unique characteristics of the IBD copolymer-immobilized surfaces were applied to regulate adenosine nucleotides retention in high-performance liquid chromatography using aqueous mobile phase by thermal stimulus. At lower temperature, adenosine nucleotides showed higher retention which was primarily driven by ionic interaction with positively charged surfaces. With increasing temperatures, their retention was shortened and a drastic change was observed above the polymer transition temperatures. This is strong evidence that the solute interaction is being regulated by the thermoresponsive surface charge density changes and hydrophobic alterations. Furthermore, we confirmed the modulation of nucleotide retention by step-temperature gradient without changing mobile phase composition. These findings should be beneficial in utilizing this stimuli responsive surface for the separation of bioactive compounds in aqueous system and environmental impact.

Predictions of Peptide Retention in HPLC with the use of Amino Acid Retention Data Obtained in a TLC System

A thin‐layer chromatography (TLC) experiment was used to find a new structural descriptor based on empirical data, which can be useful for prediction of high performance liquid chromatography (HPLC) retention of peptides. The optimization of TLC separation of a series of twenty naturally occurring amino acids was performed and, finally, the mobile phase comprising water and ethanol 95° (20:80 v/v) appeared to be an optimal one. The designed TLC experiment enabled obtaining different values of retardation factor, R f , and to further calculate R M values for individual amino acids. The sum of calculated R M values, corresponding to the individual amino acids in the appropriate peptide, and four other descriptors calculated from the peptides' structural formulas using molecular modeling methods, were used in quantitative structure retention relationships (QSRR) analysis to predict retention times of a series of structurally diversified peptides chromatographed in a reversed phase HPLC system.

Characterization and identification of isomeric flavonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-flight mass spectrometry

Flavonoid O-diglycosides are important bioactive compounds from genus Citrus. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior of six flavonoid O-diglycosides from genus Citrus was investigated using ion trap mass spectrometry in negative electrospray ionization (ESI) with loop injection. For the flavonoid O-rutinosides, [M−H−308]− ion was typically observed in the MS2 spectrum, suggesting the loss of a rutinose. The fragmentation patterns of flavonoid O-neohesperidosides were more complicated in comparison with their rutinoside analogues. A major difference was found in the [M−H−120]− ion in the MS2 spectrum, which was a common feature of all the flavonoid O-neohesperidosides. The previous literature for naringin located the loss of 120Da to the glycan part, whereas the present study for naringin had shown that the [M−H−120]− ion was produced by a retro-Diels-Alder reaction in ring C, and this fragmentation pattern was confirmed by the accurate mass measurement using an orthogonal time-of-flight mass spectrometer. Combined with high performance liquid chromatography (HPLC) and diode array detection (DAD), the established approach to the structural identification of flavonoid O-diglycosides by ion trap mass spectrometry was applied to the analysis of extracts of two Chinese medicines derived from genus Citrus, namely Fructus aurantii and F. aurantii immaturus. According to the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by multistage mass spectrometry (MSn) spectra, 13 flavonoid O-glycosides in F. aurantii and 12 flavonoid O-glycosides in F. a. immaturus were identified rapidly.

A new method for assessing the effect of residual silanol groups on total retention in reversed-phase HPLC

A new approach is proposed to assess the effect of residual silanol groups (hydrophilicity) on the retention of polar substances in reversed-phase high-performance liquid chromatography (HPLC). It is shown that the numerical characteristic of the relative position of trend lines in the coordinates logk(B) vs. logk(A) of the relative retention parameters (at different mobile phase compositions) for any stationary phase relative to the stationary phase selected as a standard is preferable to the separation selectivity of the same pair of substances. Full information can be obtained by determining the retention of corresponding adsorbates with two different mobile phase compositions for each column under study. It was shown that the pair of adsorbates p-toluidine-p-cresol exhibits higher sensitivity to a change in the hydrophilicity of stationary phases as compared to the pair aniline-phenol. However, the use of a nonpolar substance as a reference compound is more informative.

Identification of Urinary Metabolites of Orally Administered N,N-Dimethyl-p-Toluidine in Male F344 Rats*

The metabolism of orally administered N,N-dimethyl-p-toluidine (DMPT) in male F344 rats was investigated. The rat urinary metabolite profile was determined by analytical reverse-phase high performance liquid chromatography (HPLC). Four radiolabeled peaks were observed, isolated, and purified by solid-phase extraction (SPE) and preparative HPLC methods. The 4 peaks were identified as p-(N-acetylhydroxyamino)hippuric acid (M1), DMPT N-oxide (M2), N-methyl-p-toluidine (M3), and parent DMPT. Metabolites M1 and M2 were identified by spectrometric and spectroscopic methods, including mass fragmentation pattern identification from both liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry, and from chemical analysis of nuclear magnetic resonance spectra. Structural confirmation of metabolite M2 was accomplished by comparison with a synthetic standard. Peaks M3 and the peak suspected to be DMPT were identified by comparison of their HPLC retention times and mass fragmentation patterns with authentic standards of N-methyl-p-toluidine and DMPT, respectively. DMPT metabolism is similar to that reported for N,N-dimethylaniline.

Identification and Quantitation of Asparagine and Citrulline Using High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (μMol m−1/μMol ml−1)], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [ Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides.

Proteomic analysis of the venom from the scorpion Tityus stigmurus: Biochemical and physiological comparison with other Tityus species

The venom from the Brazilian scorpion Tityus stigmurus was fractionated by high performance liquid chromatography (HPLC) and the corresponding components were used for molecular mass determination using electrospray ion trap mass spectrometry. One hundred distinct components were clearly assigned showing molecular masses from 216.5 to 44,800.0 Da. Fifteen new components were isolated and sequenced, four of them to completion: Tst-3 (similar to Na+ channel specific scorpion toxins), Tst-17 (a K+ channel blocking peptide similar to Tc1), TstβKTx (a peptide with identical sequence as that of TsTX-Kβ toxin earlier described to exist in T. serrulatus venom) and finally a novel proline-rich peptide of unknown function. Among the eleven components partially sequenced were two enzymes: hyaluronidase and lysozyme. The first enzyme has a molecular mass of 44,800.0 Da. This enzyme showed high activity against the substrate hyaluronan in vitro. Amino acid sequence of the second enzyme showed that it is similar to other known lysozymes, with similar molecular mass and sequence to that of bona fide lysozymes reported in public protein data banks. Finally, this communication reports a correlation among HPLC retention times and molecular masses of folded scorpion toxins as well as a comparative structural and physiological analysis of components from the venom of several species of the genus Tityus.

Identificação de antocianidinas em acerolas do Banco Ativo de Germoplasma da Universidade Federal Rural de Pernambuco

The red color of mature acerola is due to the presence of anthocyanins. These pigments were extracted and purified from 12 genotypes cultivated at the Active Germoplasm Bank of the Rural Federal University of Pernambuco in order to determine their composition. The aglycon forms of the anthocyanins were revealed by acid hydrolysis. Anthocyanins and anthocyanidins were separated by using high-performance liquid chromatography (HPLC) using a C18 reversed phase analytical column. Anthocyanidins were identified by comparing HPLC elution order and retention times relative to authentic standards and to anthocyanidins obtained from acid hydrolysis of Isabel, Patricia and Red Globe grapes, plum, red onion, strawberry and Tommy Atkins mango skin. The chromatograms showed that the acerola genotype anthocyanins profile is simple, showing three to five peaks, similar between them. After the acid hydrolysis, the chromatograms revealed the presence of two aglycons which were identified as cyanidin and pelargonidin. By comparing chromatograms of anthocyanins and anthocyanidins and analyzing the retention times, the presence of anthocyanins with different glycosylation patterns and the absence of acylation in their molecules could be clearly seen. The aglycons identified (cyanidin and pelargonidin) were found in different proportions in these acerola genotypes.

154 POSTER Novel produrugs of SN38 generated by Multi-Arm Poly(ethylene glycol)

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k′) and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.

Optimization of Analysis Method of Kudzu Root Crude Extract by High Performance Liquid Chromatography

Kudzu root is an important traditional Chinese herb. Its crude extract has been used in the treatment of hypertension and angina pectoris in China. Several parameters for high performance liquid chromatography (HPLC) analytical method for kudzu root extract, including HPLC model, mobile phase, additive and gradient conditions have been optimized. The HPLC retention parameters a and c, and the peak shape parameters sigma and tau of 25 chromatographic peaks were obtained accurately and rapidly using five linear gradients and were calculated using CSASS software. The CSASS software was then used to simulate the gradient conditions in the experiments, and the optimized condition was obtained. The comparison of the simulated and real chromatogram profiles showed that the potential of simulation using the CSSASS software as compared to that of the real experimental conditions was precise. Reversed-phase HPLC model and the mobile phase of (A) acetonitrile containing 0.1% (v/v) acetic acid and (B) water containing 0.1% (v/v) acetic acid were used. The optimized gradient profile showed a linear increase as follows: from 5% A to 35% A at 0 - 33 min; from 35% A to 100% A at 33 - 50 min, and then the HPLC system was held for 5 min. The reproducibility and precision of the method were investigated. The relative standard deviations (RSDs) of retention time, peak height, and peak area were less than 0.25%, 11%, and 4.9%, respectively, which showed that the method was stable, reliable, and reproducible.

High-performance liquid chromatography retention mechanisms and their mathematical descriptions

Different mechanisms of analyte behavior in HPLC columns are considered from the point of view of analyte distribution between the adsorbent and the bulk liquid phase. Expressions describing the analyte retention volume are derived for each selected retention mechanism. Methodology of the incorporation of multi-equilibrium processes in the analyte retention expression is suggested for ideal linear chromatography conditions. The description of eluent components’ adsorption is discussed on the basis of the excess adsorption process. The applicability of the excess adsorption description without introduction of the adsorbed layer model or any other convention to the chromatographic process is shown.

Synthesis of Ermanin, 5,7-Dihydroxy-3,4'-dimethoxyflavone from Kaempferol, 3,5,7,4'-Tetrahydroxyflavone with Two O-Methyltransferases Expressed in E. coli

Enzymatic modifications of natural compounds have been drawn attention because they provide regioselectivity and chiral selectivity, which are hardly achievable with chemical synthesis. Using a mobilized enzyme or a whole cell containing a particular gene delivers useful tools for modification of natural compounds. In addition, using a whole cell, known as biotransformation, has advantage of saving expensive cofactors. Flavonoids and alkaloids are secondary metabolites produced mainly in plants. Their biological activities have an impact on human health so that they serve as target molecules to develop new drugs. One of common modification reactions found in these two groups of compounds is O-methylation due to free hydroxyl groups. Various Omethylation reactions have been reported and a number of genes (O-methyltransferases: OMTs) that medicated Omethylation have been cloned and characterized. Some of OMTs that use flavonoids as substrates are highly specific and thus they could be used for the regioselective modification of flavonoids. O-methylation of flavonoids resulted in reduction of chemical reactivity and increase of antimicrobial activity. So far, O-methylated flavonoids have been mainly isolated from plants, which could be a limitation for biological assays. Kaempferol (3,5,7,4'-tetrahydroxyflavone) is one of commonly found flavonoids in nature. Even though several biological activities of kaempferol have been established, its dimethoxy form, ermanin (5,7-dihydroxy-3,4'dimethoxyflavone) was known to have several activities including antiviral, anti-inflammatory, cytotoxic, and antibacterial activity. Lists of its biological activity might be extended if large amount of ermanin is supplied. Previously, we cloned and characterized two OMT genes, SOMT-2(soybean O-methyltransferase-2) and ROMT-9 (rice O-methyltrasnferase-9). The transgenic E. coli expressing SOMT-2 transferred a methyl group to 4'-hydroxyl group of flavonoids. ROMT-9 expressed in E. coli showed different regioselectivity depending on the availability of 3'hydroxyl groups. It transferred a methyl group to 3'hydroxyl group if flavonoids have 3'-hydroxyl group. But, when 3'-hydroxyl group is not present, it methylated a 3hydroxyl group. Here we report the biological synthesis of ermanin from kaempferol with two OMTs expressed in E. coli. ROMT-9 and SOMT-2 were cloned in one expression vector and both proteins were induced from E. coli containing both ROMT-9 and SOMT-2 by adding IPTG at 100 μM. In addition, ROMT-9 and SOMT-2 were induced separately from E. coli containing either ROMT-9 or SOMT2. After 4 hr induction, the cells from each culture were harvested and resuspended in LB containing ampicillin (50 μg/mL). 100 μM of kaempferol (3,5,7,4'-tetrahydroxyflavone) was added. The mixture was further incubated for 15 hrs at 28 C. Analysis of culture filtrates from three reactions using high performance liquid chromatography (HPLC) revealed that kaempferol was converted into a new product which had different HPLC retention time from kaempferol itself. ROMT-9 produced a new peak at 15.2 min (Fig. 1B) and SOMT-2 produced a new peak at 19.9 min

Prediction of Chromatographic Retention Times for Aromatic Hydrocarbons

A quantitative model for predicting the retention times of various single- and multi-ring aromatic hydrocarbons (AH) and heteroatom-containing molecules in high-performance liquid chromatography (HPLC) is presented. The retention time behavior of 44 aromatic molecules containing one-, two-, and three-ring structures on a [3-(2,4-dinitroanilino)]propyl-silica column was investigated under subambient separation conditions. Substituent effects (their number, type, and position on the ring) on the retention behavior of these aromatic ring structures were also investigated. A quantitative structure−property relationship (QSPR) model elucidating the retention behavior of AH molecules was developed based on the obtained experimental data. It was found that the electronic properties of the molecule and its molecular geometry play a pivotal role in determining its retention in the HPLC column. The important electronic and geometric descriptors were identified using partial least squares analysis. The electronic properties of the molecule were quantified by its ionization potential and electron affinity computed from the optimized solute geometry using the PM3 energy function. The molecular geometry was characterized by the number of rings, molecular weight, and the valence connectivity indices. The mathematical form of the QSPR model was derived using genetic algorithms, and a log-linear form of the model was found to best correlate the HPLC retention time data with the solute molecular descriptors. The QSPR model was also used to predict the retention behavior of many other AH molecules that are commonly present in most heavy petroleum streams. The results obtained in this study enhance our understanding of the AH retention behavior and are of significance in predicting the identity of chromatographic peaks and quantify the extent of overlap based solely on parameters derived from solute structure.