Peak Asymmetry Factor Research Articles

A new standard reference material for column evaluation in reversed‐phase liquid chromatography

AbstractA new Standard Reference Material (SRM) for use in characterizing general aspects of liquid chromatographic column performance is described. SRM 870 Column Performance Test Mixture for Liquid Chromatography contains five components: uracil, toluene, ethylbenzene, quinizarin, and amitriptyline, and is intended to provide indications of column void volume, hydrophobicity, methylene selectivity, activity toward organic bases, and activity toward metal chelators. The test is performed under neutral, buffered conditions. The measurement of retention and peak asymmetry factors for the components facilitates column classification. It is anticipated that this material will be useful (1) in column selection during method development, (2) as a control material to monitor column performance over time, and (3) in quality control in column manufacturing.

Methacrylate monolithic columns of 320 μm I.D. for capillary liquid chromatography

Monolithic capillary columns (320 μm I.D.) were prepared for capillary liquid chromatography (CLC) by radical polymerization of butylmethacrylate (BMA) and ethylenedimethacrylate (EDMA) in the presence of a porogen solvent containing propan-1-ol, butane-1,4-diol and water. The influence of the contents of the porogen solvent and EDMA in the polymerization mixture on the monolith porosity and column efficiency was investigated. The composition of the polymerization mixture was optimized to attain a minimum HETP of the order of tens of μm for test compounds with various polarities. The separation performance and selectivity of the most efficient monolithic column prepared was characterized by van Deemter curves, peak asymmetry factors and Walters hydrophobicity and silanol indices. It was demonstrated that the 320-μm I.D. monolithic column exhibited CLC separation performance similar to that observed for 100- and 150-μm I.D. monolithic columns reported in the literature; moreover, the 320-μm I.D. column was easier to operate in CLC and exhibited a higher sample loadability.

Affinity screening by packed capillary high performance liquid chromatography using molecular imprinted sorbents: II. Covalent imprinted polymers

This study concentrates on the production of covalent molecular imprint polymers (MIPs) as highly selective sorbents for nortriptyline (NOR), a representative tricyclic antidepressant (TCA). The functionalized template contains a polymerizable 4-vinylphenyl carbamate moiety used to bind the template molecule to the polymer matrix. Polymerization with a cross-linker followed by hydrolytic cleavage of the labile carbamate functionality leaves an MIP with selective binding sites capable of binding template through hydrogen bonding interactions. Demonstrated chromatographically through a “selection index”, these MIPs showed high selectivity for the template molecule (NOR) among a library of structurally similar compounds. The recognition was found to correlate with structural similarity to the template compound. A direct comparison between covalent and non-covalent molecular imprinting strategies reveals a great deal of improvement in the peak shape of the retained compound resulting from covalent imprinting (evidenced by peak asymmetry factors A s).

Use of principal component analysis for investigation of factors affecting retention behaviour of ceramides on porous graphitized carbon column

The potentiality of a porous graphitized carbon stationary phase was assessed for ceramide analysis in nonaqueous reversed phase chromatography with an evaporative light scattering detection. The influence of the nature of the mobile phase was first performed under isoeluotropic isocratic conditions. Eight mobile phase compositions in the presence or absence of triethylamine/formic acid were tested with a set of structurally relevant ceramides. Values obtained under these conditions for the peak heights, retention factors, plate numbers, asymmetry factors, relative retentions and Time-Corrected Calibrated Normalised Resolution Product were analysed by Principal Component Analysis. Principal Component Analysis was effective in attributing relationships between the chromatographic behaviour and analytical conditions. The relationship between the increase of the detector response and the presence of triethylamine/formic acid has been confirmed, and the main change of selectivity has been attributed to interactions provided by the weak solvent of the mobile phase. These conclusions have allowed the development of gradient conditions for the analysis of complex ceramide mixtures on porous graphitized carbon column.

Characterization of a Range of Alkyl-Bonded Silica HPLC Stationary Phases: Correlation of Quantitative Surface Analysis Data with the Retention Behavior of Neutral, Acidic, and Basic Solutes

An investigation was made of the correlation between quantitative surface analytical data obtained by XPS and static SIMS and the chromatographic performance of a range of n-alkyl-bonded silica (C1-C18) packing materials. A series of acidic, basic, and neutral solutes was used to study the retention behavior. For comparison, analysis of bulk total percentage carbon (%C) and alkyl surface density of the bonded silica particulates were also included. Significant correlations were observed, in the majority of cases, between the retention factor (k) and the XPS C:Si atomic ratio, which was similar to that obtained between k and the bulk %C or k and the bonded alkyl chain length. Similar significant correlations were also obtained between k and the static SIMS alkyl:Si ion peak area ratios. XPS alkyl:Si atomic ratios were calculated as an estimate of alkyl surface coverage of the silica support, and these correlated well with the surface density calculated from the bulk %C and the surface area of the packing material. The XPS alkyl:Si ratio also demonstrated a significant correlation with the peak asymmetry factor derived for basic solutes. These studies confirm that both XPS and static SIMS can generate surface chemical data from chromatography particulates, which has direct relevance to the prediction of chromatographic behavior. We believe that these techniques will prove to be effective tools to assist in the characterization of chromatographic supports and stationary phases.

Influence of analyte stereochemistry and basicity on peak shape of basic compounds in high-performance liquid chromatography with reversed-phase columns, using pyridine and alkyl-substituted derivatives as probe compounds

Column efficiency, peak asymmetry and retention factor of a series of sixteen compounds comprising pyridine and some alkyl-substituted derivatives, with p K a values ranging from 5.17 to 6.74 is reported on a RP-HPLC column previously shown to be especially suitable for the analysis of basic compounds. Compound stereochemistry in close vicinity of the basic group is found to influence peak shape strongly: however, stereochemistry at more remote positions is also of some significance. The effect of p K a on peak shape is discussed in the light of measurements of the degree of protonation of the compounds in the methanolic mobile phase as well as water.

Retention of basic drugs on porous polymers in high-performance liquid chromatography

The behaviour of basic drugs on two polymeric high-performance liquid chromatographic columns in the presence of alkaline mobile phases was studied. The packings were PRP-1 and di(methacryloyloxymethyl)naphthalene-divinylbenzene copolymer. Using the alkyl aryl ketone scale, the retention indices for ephedrine, clonidine, lidocaine, benzocaine, procaine, propranolol, diazepam, strychnine, cyproheptadine, amitriptyline and promethazine were calculated. To determine the influence of the pH of the mobile phase on the retention of these compounds, peak asymmetry factors and reduced plate heights were measured.

Prediction of initial high-performance liquid chromatographic conditions for selectivity optimization in pharmaceutical analysis by an expert system approach

Initial mobile phase compositions can be selected from any physicochemical properties of solutes which can be correlated with their high-performance liquid chromatography (HPLC) retention. In this study, octanol—water partition coefficients (log P values) calculated from molecular increment are converted to HPLC retention data to achieve capacity factors ( k′) within a range 0.5–5. The eluent compositions are calculated to produce k′ values of 0.5 ( OP min) and 5 ( OP max) for all solutes of interest. From the various eluent compositions obtained as OP min values, the highest value (the strongest eluent composition, OP min,ST) and as OP max values, the lowest value (the weakest eluent composition, OP max,WE), are chosen to calculate the optimum composition for the initial experiments ( OP IN). Using the suggested mobile phase composition, OP IN, the k′ values of each component are predicted and checked. The first guess experiment can be started with the suggested eluent composition. Pass—fail criteria have been established to evaluate the experimental data; the capacity factors of all components should be within the range 0.5–10, and the peak asymmetry factors should be between 0.8 and 1.8. For the direction of the second and third guess experiments, special rules followed by different actions are formulated. The applicability of the developed expert system is demonstrated through the separation of a model mixture containing solutes with widely different chemical structures.

General expression for the peak asymmetry factor in amplitude spectra

General expression for the peak asymmetry factor in amplitude spectra

Characterization of some styrene-divinylbenzene sorbents for reversed-phase chromatography

The preparation and characterization of laboratory made polymeric columns of Chromosorb 101 and Porapak Q are described, with reference to a PRP-1 column packed under the same conditions. Column parameters such as permeability, efficiency and peak asymmetry factor were calculated. The swelling propensity of the materials was measured and the dependence of peak shape on the type of organic solvent used was investigated. The efficiencies of Chromosorb 101 and Porapak Q columns were satisfactory but the spherical material PRP-1 gave better results. Porapak Q exhibited very good physical properties such as high rigidity and permeability. The swelding of polymers in acetonitrile is higher than in methanol, so a better column performance is obtained when acetonitrile is employed as the eluent.

Gas Chromatographic Relative Retention Data for Pesticides on Nine Packed Columns: II. Organophosphorus and Organochlorine Pesticides, Using Electron-Capture Detection

The retention time relative to parathion, absolute retention time, concentration range, peak asymmetry factor, and peak shape class are given for each of 42 organophosphorus pesticides and 28 organochlorine pesticides analyzed by gas chromatography (GC) on 9 different packed columns. The packing materials used were 3% SP-2100, 1% Dexsil-300, 3% OV-17, 1.5% OV-17 + 1.95% QF-1, 4% SE-30 + 6% QF-1, 3% OV-17 + 3% OV-210, 5% DC-200 + 7.5% QF-1,3% Carbowax-20M, and 4% Reoplex-400. Retention data were determined at 200 degrees C with a carrier gas flow at uopt, using a 63Ni electron-capture detector. Results should be useful for preliminary identification of environmental samples and also for single or multiple pesticide residue analysis.

Chromosorb 101 as a packing material for reversed-phase chromatography

A procedure for preparing a Chromosorb 101 liquid chromatographic column is described. Some column parameters such as the permeability, efficiency and peak asymmetry factor were calculated. The elution of some benzene and phenol derivatives from the polymeric column with different mobile phases was performed. The results showed that some gas chromatographic adsorbents such as Chromosorb 101 can be used as packings for high-performance liquid chromatographic columns. The behaviour of these styrene copolymers resembles that of a C 18 silica bonded phase.

Gas Chromatographic Relative Retention Data for Pesticides on Nine Packed Columns: I. Organophosphorus Pesticides, Using Flame Photometric Detection

Abstract The retention time relative to parathion, absolute retention time, concentration range, peak asymmetry factor, and peak shape class are given for each of 50 organophosphorus pesticides analyzed by gas chromatography (GC) on 9 different packed columns. The packing materials used were 3% SP-2100, 1% Dexsil-300, 3% OV-17, 1.5% OV-17 + 1.95% QF-1, 4% SE-30 + 6% QF-1,3% OV-17 + 3% OV- 210, 5% DC-200 + 7.5% QF-1,3% Carbowax 20M, and 4% Reoplex- 400. Retention data were determined at 200°C with a carrier gas flow at uopt, using a flame photometric detector. The results should be useful for choosing the most suitable column for single or multiple pesticide residue analysis.

Characterization of OV‐1/FFAP‐mixture coated glass capillary columns used for the separation of free fatty acids

AbstractAs an alternative to acid pretreated UCON and FFAP capillaries for the analysis of wide‐boiling free fatty acid mixtures, OV‐1 : FFAP phase mixtures were used on high‐temperature silylated inert glass capillary columns. The HETP‐carrier gas velocity curves, peak asymmetry factors, coating efficiency, gas phase and stationary phase contributions to the mass transfer resistance were determined for various OV‐1 : FFAP ratios. Mixed‐phase capillaries showed optimum performance at a 2 : 1 OV‐1 : FFAP ratio. The thermal and long‐term stability of OV‐1‐stabilized FFAP columns surpassed those of the UCON and pure FFAP reference column used.

Separation of proteins by high-performance anion-exchange chromatography

The chromatographic separation of four proteins, cytochrome c, α 1-acid glycoprotein, ovalbumin, and β-lactoglobulin, was achieved on a 4.6 × 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-μm particles, 330-Å pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 m potassium phosphate, pH 6.80, to 0.50 m potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 m potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 × 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.

Solvent extraction of tricyclic amines from blood plas- ma and liquid chromatographic determination

The extraction of seven tricyclic antidepressant amines from human plasma at different pH values was investigated for dichloromethane, diethyl ether and hexane—1-pentanol (95:5). The amines were extracted as bases and back-extracted to sulphuric acid, 0.10 mol/l, prior to the separation by bonded-phase liquid chromatography. Ether and hexane— 1-pentanol (95:5) were most suitable, tertiary amines being best extracted at pH 8, and secondary amines at pH 10. Using ether, both tertiary and secondary amines required 30 min extraction time for a quantitative yield while 15 rain was sufficient for hexane— -1-pentanol (95:5). UV detection allowed concentrations down to 10 ng in 1 ml of plasma to be determined. Three ammonium ions—octylammonium, dimethylammonium, and trimethylammonium - were added as modifiers to the mobile phase containing acetonitrile in phosphoric acid, 0.10 mol/l. In the concentration interval 0.010–0.030 mol/l all of the amine modifiers gave on Polygosil C 8 peak asymmetry factors of sufficiently low magnitude, while on Li- Chrosorb RP-18 this was so only for di- and trimethylammonium in a concentration of 0.030 mol/l.

Evaluation of glass capillary columns with different pre-coating layers

An evaluation is made of glass capillary columns pre-coated with dispersions of various materials (Silanox, Carbopack, Chromosorb, perlite and Porapak) and then coated with polyethylene glycol (PEG 20M) at various concentrations as stationary phase by analysing alkanes and alcohols in terms of the peak-asymmetry factor, separation factor and kinetic characteristics. Pre-coating with Silanox, Carbopack and Chromosorb yielded similar results, but with different amounts of stationary phase. Use of Carbopack for pre-coating offers the advantage that, according to the concentration of stationary phase, separation can be achieved by gas-liquid or gas-liquid-solid chromatography.