Copper fractionation by SEC-HPLC and ETAAS: study of breast milk and infant formulae whey used in lactation of full-term newborn infants

This method will allow the determination of bound copper to low relative molecular mass compounds in milk. The milk whey obtained by ultracentrifugation was submitted to fractionation by size exclusion chromatography (SEC) on a TSK-Gel2000 (Toso Haas) column with a mobile phase of 0.2 M NH4NO3 + NH3, pH 6.7. Fractions of effluent corresponding to the protein peaks were collected and the copper content was determined by ETAAS. The method was sensitive (LOD 0.4 microgram l-1 and LOQ 1.5 micrograms l-1 in the fraction; LOQ 7.5-22.5 micrograms l-1 referred to the milk sample and depended on fraction volume) and precise (RSD +/- 10%). Media sample recoveries from the column were 101.2%. Cu was predominantly present in fractions corresponding to relative molecular mass 76 and 15 kDa of breast milk while copper was mostly found in fractions corresponding to 14 and 38 kDa of cow's milk-based infant formulae; moreover, copper was eluted in the relative molecular mass region < 6 kDa.     

Synthesis and rapid enantiomeric separation of the chiral mixed ligand [5-(4-hydroxybutyl)-5′-methyl-2,2′-bipyridine]-bis(1,10-phenanthroline)-ruthenium(II) complex by electrokinetic chromatography

The chiral complex [5-(4-hydroxybutyl)-5′-methyl-2,2′-bipyridine]-bis(1,10-phenanthroline)ruthenium(II)-bis(hexafluoroantimonate) was successfully synthesized and fully characterized by two-dimensional ¹H and ¹³C{¹H} NMR techniques (COSY and HMQC) as well as EA- and FAB-MS. A very fast separation of the Δ and Λ enantiomers with excellent efficiency and resolution was achieved by electrokinetic chromatography using anionic carboxymethyl-β-cyclodextrin as a chiral mobile phase additive. The optimum separation conditions were obtained with 50 mM borate buffer at pH 9 and 10 mg/ml of the chiral selector at 20°C. Attempts to separate the well known unmodified tris(2,2′-bipyridine)ruthenium(II) [Ru(bpy)₃] complex into its enantiomers under the same conditions were unsuccessful.     

Enhanced sensitivity and selectivity in the detection of polycyclic aromatic hydrocarbons using sequential simplex optimization, the addition of an organic modifier and wavelength programming

Optimized separation of a mixture of 16 priority pollutant polycyclic aromatic hydrocarbons (PAHs) by high performance liquid chromatography (HPLC) using the sequential simplex optimization method was accomplished by varying the starting and ending compositions of acetonitrile and water, linear gradient time, mobile phase flow rate, column temperature and holding time of the final mobile phase composition. Focusing on the two sets of difficult-to-separate pairs (acenaphthene–fluorene and benzo[g,h,i]perylene–indeno[1,2,3-c,d]pyrene), analysis time was reduced by about ten percent through the use of an organic modifier (isopropanol or methanol), under both optimum and near-optimum conditions, while maintaining good separation of the remaining PAHs. High sensitivity for all of the 16 PAHs was achieved by wavelength programming during elution using five wavelengths (224, 235, 254 270 and 296 nm), depending upon the molar absorptivities of the individual compounds. Detection limits (DLs) ranging from 0.002 (benzo[a]pyrene) to 0.140 μg ml⁻¹ (acenaphthene) were achieved for this set of 16 standard compounds.     

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-μm diol-silica particles. The CSP mixed with 5-μm amino silica particles (3:1) was packed into 75-μm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1–3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor R_s=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)–water–acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)–water–methanol–acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12–20 min).     

On-line flow injection solvent extraction for electrothermal atomic absorption spectrometry: Determination of nickel in biological samples

A flow injection (FI) on-line solvent extraction system for electrothermal atomic absorption spectrometry (ETAAS) was developed with nickel as a model trace element. The nickel pyrrolidine-dithiocarbamate chelate was extracted on line into isobutyl methyl ketone, which was delivered into the FI system by a peristaltic pump equipped with poly(tetrafluoroethylene) tubing. The organic phase was separated from the aqueous phase by a novel gravity phase separator with a small conical cavity, and stored in a collector tube, from which 50 μl organic concentrate was introduced into the graphite tube by an air flow. ETAAS determination of the analyte was performed in parallel with the extraction process. An enrichment factor of 25 was obtained in comparison with 50 μl direct introduction while achieving a detection limit of 4 ng l⁻¹ (3σ), and a precision of 1.5% relative standard deviation for 1.0 μg l⁻¹ nickel (n = 11). The proposed method was successfully applied to the determination of nickel in body fluids and other biological samples.     

Selective flow injection analysis of ultra-trace amounts of Cr(VI), preconcentration of it by solvent extraction, and determination by electrothermal atomic absorption spectrometry (ETAAS)

A rapid, robust, sensitive and selective time-based flow injection (FI) on-line solvent extraction system interfaced with electrothermal atomic absorption spectrometry (ETAAS) is described for analyzing ultra-trace amounts of Cr(VI). The sample is initially mixed on-line with isobutyl methyl ketone (IBMK). The Cr(VI) is complexed by reaction with ammonium pyrrolidine dithiocarbamate (APDC), and the non-charged Cr(VI)–PDC chelate formed is extracted into IBMK in a knotted reactor made from PTFE tubing. The organic extractant is separated from the aqueous phase by a gravity phase separator with a small conical cavity and delivered into a collector tube, from which 55 μl organic concentrate is subsequently introduced via an air flow into the graphite tube of the ETAAS instrument. The operations of the FI-system and the ETAAS detector are synchronously coupled. A significant advantage of the approach is that matrix constituents, such as high salt contents, effectively are eliminated. The extraction procedure was optimized by a simplex approach. A central composite design was subsequently employed to verify the estimated operational optimum. An 18-fold enhancement in sensitivity of Cr(VI) was achieved after preconcentration for 99 s at a sample flow rate of 5.5 ml min⁻¹, as compared to direct introduction of 55 μl of sample, yielding a detection limit (3σ) of 3.3 ng l⁻¹. The sampling frequency was 24.2 samples h⁻¹. The proposed method was successfully evaluated by analyzing a NIST Cr(VI)-reference material, synthetic seawater and waste waters, and waste water samples from an incineration plant and a desulphurization plant, respectively.     

Cloud point extraction for the determination of As(III) in water samples by electrothermal atomic absorption spectrometry

Cloud point extraction was applied as a preconcentration step for electrothermal atomic absorption spectrometry (ETAAS) determination of As(III) in aqueous solutions. After complexation with ammonium pyrrolidinedithiocarbamate, the analyte was quantitatively extracted to the surfactant-rich phase in the non-ionic surfactant octylphenoxypolyethoxyethanol (Triton X-114) after centrifugation. 0.1 mol L⁻¹ HNO₃ in methanol was added to the surfactant-rich phase before ETAAS determination. The precision (R.S.D.) for 11 replicate determinations of 5.0 μg L⁻¹ of As(III) was 3.0%. The concentration factor, which is defined as the concentration ratio of the analyte in the final diluted surfactant-rich extract ready for ETAAS determination and in the initial solution, was 36 for As(III). The linear concentration range was from 0.1 to 20 μg L⁻¹. The developed method was applied to the determination of As(III) in lake water and river water.     

Emulsification using micro porous glass (MPG): surface behaviour of milk proteins

To investigate the emulsifying properties and adsorption behaviour of high molecular amphiphilic substances such as proteins, it is important to maintain the native status of the used samples. The new method of micro porous glass (MPG) emulsification could offer an opportunity to do this because of the low shear forces. The oil-in-water emulsions were produced by dispersing the hydrophobic phase (liquid butter fat or sunflower oil) through the MPG of different average pore diameters (d_p=0.2 or 0.5 μm) into the flowing continuous phase containing the milk proteins (from reconstituted skim milk and buttermilk). The emulsions were characterised by particle size distribution, creaming behaviour and protein adsorption at the hydrophobic phase. The particle size distribution of protein-stabilised MPG emulsions is determined by the pore size of MPG, the velocity of continuous phase (or wall shear stress σ_w) and the transmembrane pressure. A high velocity of =2 m s⁻¹ (σ_w=13.4 Pa) and low pressure (pressure of disperse phase slightly exceeded the critical pressure Δp_TM=4.5 bar of 0.2 μm-MPG) led to the smallest droplet diameter. As a consequence of average droplet diameters of d₄₃>3.5 μm creaming was observed without centrifugation in all MPG emulsions after 24 h, but no coalescence of the oil droplets occurred. The study of protein adsorption showed that the MPG emulsification at low shear forces resulted in lower protein load values (2.5±0.5 mg m⁻²) than pressure emulsification (11.5±1.0 mg m⁻²). In addition, the various emulsification conditions (MPG or pressure homogenization) led to differences in the relative proportions of casein fractions, whey proteins and milk fat globule membranes (MFGM) at the fat globule surfaces.     

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R_F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection (λ_ex=330 nm; λ_em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.     

Selective complex formation of saccharides with europium(III) and iron(III) ions at alkaline pH studied by ligand-exchange chromatography

At high pH, saccharides become negatively charged by deprotonation of one or several hydroxylic groups and they are highly and selectively retained by ligand-exchange chromatography. The systems consist of a sulphonated polystyrene strong cation exchanger in europium(III) or iron(III) form and sodium hydroxide as mobile phase. The degree of complex formation is dependent on solute character and concentration, metal ion and pH, the reaction being of second order as confirmed by breakthrough studies. Rapid desorption of the solutes is performed by the introduction of an acidic mobile phase. Monosaccharides, and especially sugar alcohols, are selectively retained by a column in Fe(III) form whereas all saccharides are strongly retained as Eu(III) complexes, e.g., the capacity factor for the breakthrough of 10 μM glucose, in 0.1 M NaOH as mobile phase, was ca. 3500 The systems are proposed to be highly selective for the analysis of sugars.     

A liquid chromatographic method, with fluorometric detection, for the determination of enrofloxacin and ciprofloxacin in plasma and endometrial tissue of mares

The aim of this work was to develop and validate a simple and sensitive analytical method for determining enrofloxacin (EFX) and ciprofloxacin (CFX) in equine plasma and endometrial tissue samples, as a precursor to conducting pharmacokinetic/pharmacodynamic studies on equine endometritis This was achieved in the form of a liquid chromatographic procedure, with fluorometric detection, which also gave good separation of other fluoroquinolones including marbofloxacin (MFX), danofloxacin (DFX) and ofloxacin (OFX). Analytes were separated on a C₁₈ reversed phase column using an acidified mobile phase. The exact composition of the mobile phase differed for plasma (16% acetonitrile:methanol [13:1,v/v] 84% water containing 0.4% triethylamine and 0.4% phosphoric acid [35%]) and endometrial tissue (14% acetonitrile, 86% water, without methanol) samples. EFX and CFX were both detected at excitation and emission wavelengths of 294 and 500 nm, respectively. Prior to chromatography, EFX and CFX were purified by solid phase extraction from plasma, and a combination of solvent/solid phase extraction from endometrial tissue.

Mean absolute recoveries for EFX and CFX from plasma were 94.1 and 78.0%, respectively, and from endometrial tissue, 78.0 and 57.8%, respectively, with a percentage residual standard deviation (%R.S.D.) <10% in each case. Mean relative recoveries for EFX and CFX from plasma were 91.3 and 119.4%, respectively, and from endometrial tissue, 80.2 and 108.0%, respectively, with a %R.S.D. <20% in each case.

Standard curves constructed using blank plasma and endometrial tissue samples, spiked with authentic EFX and CFX in the ranges 0.005–10.0 μg mL⁻¹ and 0.05–10.0 μg g⁻¹, respectively, all showed acceptable linearity with correlation coefficients, r² ≥ 0.977. Mean intra- and inter-day precision (expressed as %R.S.D.) was <6 and <13%, respectively, with an associated accuracy (expressed as percentage relative error, %R.E.) of <20% for both analytes in both matrices. Acceptable precision and accuracy was also demonstrated at the pre-assigned LOQs of 0.005 μg mL⁻¹ for both EFX and CFX in plasma, and 0.05 μg g⁻¹ for both drugs in endometrial tissue. EFX and CFX were stable in both plasma and endometrial tissue for at least 60 days at −20 °C.     

Separation of ternary systems of hydrophilic ionic liquid with miscible organic compounds by RPLC with refractive index detection

A rapid and simple analytical method was developed for the simultaneous and quantitative determination and separation of hydrophilic imidazolium ionic liquids (ILs) (1-butyl-3-methylimidazolium chloride, [C(4)mim]Cl; 1-hexyl-3-methylimidazolium chloride, [C(6)mim]Cl; 1-octyl-3-methylimidazolium chloride, [C(8)mim]Cl; 1-allyl-3-methylimidazolium chloride, [Amim]Cl; or 1-allyl-3-methylimidazolium bromide, [Amim]Br) with miscible ethyl acetate and EtOH and their mixtures using reverse phase liquid chromatography coupled with refractive index detection (RPLC-RI). The influence of 60 to 100% (volume percentage) methanol in the mobile phase on the IL systems ([C(4)mim]Cl, [C(6)mim]Cl, [C(8)mim]Cl, [Amim]Br, or [Amim]Cl)-ethyl acetate-EtOH was investigated. The optimum mobile phase for the system [C(8)mim]Cl-ethyl acetate-EtOH, [C(4)mim]Cl-ethyl acetate-EtOH, [Amim]Br-ethyl acetate-EtOH and [Amim]Cl-ethyl acetate-EtOH was methanol/water (60:40, v/v), and methanol/water (70:30, v/v) for [C(6)mim]Cl-ethyl acetate-EtOH. Under optimum mobile phase conditions for each system, the RSD of the retention time ranged from 0.02 to 0.04%, and the RSDs of the peak area percent ranged from 0.23 to 1.85%, which showed good reproducibility of the RPLC-RI method. The RPLC-RI method can determine IL, ethyl acetate, and EtOH simultaneously in 5 min, and the analytes, especially IL, can be eluted completely. The results show that the RPLC-RI method can be used to separate and determine ILs in mixtures with organic compounds simultaneously and quantitatively.     

A dynamic continuous-flow dialysis system with on-line electrothermal atomic-absorption spectrometric and pH measurements for in-vitro determination of iron bioavailability by simulated gastrointestinal digestion

A dynamic continuous-flow dialysis (CFD) method with on-line electrothermal atomic absorption spectrometric (ETAAS) and pH measurements for study of simulated gastrointestinal digestion has been developed for prediction of iron bioavailability. The method used to estimate mineral bioavailability was based on gastric digestion in a batch system then dynamic continuous-flow intestinal digestion. The intestinal digestion was performed in a dialysis bag placed inside a chamber containing a flowing stream of dialyzing solution. Mineral concentration and dialysate pH were monitored by ETAAS and use of a pH meter, respectively. The amount of dialyzed minerals in the intestinal digestion stage was used to evaluate the dialyzability. The dialysis profile and pH change can be used to understand or examine differences between the dialyzability of different food samples. To test the proposed system it was used to estimate the iron dialyzability of different kinds of milk. Iron dialyzability for powdered cow milk, cereal milk, and two brands of soymilk was found to be 1.7, 20.4, 24.9, and 37.7%, respectively. The developed CFD–ETAAS–pH system is a simple, rapid, and inexpensive tool for bioavailability studies, especially for minerals at ultratrace levels.     

Off-line coupling of pressurized liquid extraction and LC/ED for the determination of retinyl acetate and tocopherols in infant formulas

An analytical methodology including pressurized liquid extraction (PLE) as sample treatment to isolate retinyl acetate and tocopherols from infant formulas has been developed. The milk extracts were kept at −18 °C for 30 min and after filtration could be injected directly into the chromatographic system. Thus, a rapid and simple routine control method of these products is possible.

The parameters affecting both the extraction process and the liquid chromatography (LC) system were optimized. PLE was performed using one cycle of extraction during a static time of 5 min. Methanol was chosen as the extraction solvent for a temperature of 50 °C. Chromatographic separation was accomplished using a RP-18 column; the mobile phase used was methanol–water (94:6, v/v) containing 2.5 mM acetic acid/sodium acetate buffer. Electrochemical detection in amperometric mode with a glassy carbon electrode at +1100 mV was applied. The proposed methodology was successfully used for the determination of retinyl acetate, δ-tocopherol, (β + γ)-tocopherol and -tocopherol in different infant formulas. The analytes were evaluated in the same chemical form present in the samples. Recoveries were between 92 and 106%. A certified reference material of milk powder was also analyzed.     

Simultaneous determination of sulfaquinoxaline, sulfamethazine and pyrimethamine by liquid chromatography

A liquid chromatography method is described to determine sulfaquinoxaline (SQX), sulfamethazine (SMT), and pyrimethamine (PMT), by using a Kromasil C₁₈ column and a 40 mM NaH₂PO₄ buffer solution, containing 10 mM NaClO₄ (pH 3.0)–acetonitrile (65:35) as mobile phase. The mobile phase flow-rate and sample volume injected were 1.5 ml/min and 20 μl, respectively and the samples were dissolved in the mobile phase. The limits of quantification were found to be about 180 μg/l (3.6 ng) for each compound. The method was applied in veterinary commercial formulations. Analyses were made by means of the standard addition method, whose results were compared with those obtained by preparing “tests” (from the stock solutions) and with those obtained by a capillary electrophoresis method. Both methods showed similar results, and then it was proved that some commercial claimed levels were not in agreement with the obtained results by using our analytical method, as they were in other cases.     

Separation of tanshinones from Salvia miltiorrhiza bunge by multidimensional counter-current chromatography

Non-equilibrium chromatography (NEC) is a chromatographic mode for the rapid separation of polymers. The retention behavior of various proteins (human, chicken, bovine serum albumin) and supercoiled circular double-stranded DNA (plasmids) was investigated using a phosphate buffer as a mobile phase at different velocities and column temperatures with a C₁ column with very low-packing particle diameter as a stationary phase. It was shown that the two factors (temperature and velocity) constituted important parameters in the retention mechanism of plasmids and proteins in NEC. The protein was retained more than the plasmid. At all the temperatures (5, 10, 15, 20, 25 °C) the plasmid retention increased over the entire flow-rate range (0.02–1.8 ml/min). For the protein, the retention curve presented a decrease in the relative retention time until a critical value of the mobile phase flow-rate, followed by an increase. The transition between the two well known NEC methods, slalom chromatography and hydrodynamic chromatography was clearly visualized for proteins at the lowest temperature, but did not appear for plasmids due to their strong compact structure.     

亲水作用毛细管整体柱的制备及其用于奶制品中三聚氰胺的加压毛细管电色谱分析

以甲基丙烯酸丁酯(BMA)和3-［N,N-二甲基-［2-(2-甲基丙-2-烯酰氧基)乙基］铵］丙烷-1-磺酸内盐(SPE)为单体,制备了新型的亲水作用毛细管整体柱,并通过三聚氰胺在此柱上的保留行为证明其具有亲水性。以加压毛细管电色谱(pCEC)技术为平台,优化了整体柱基于亲水作用分离分析奶制品中三聚氰胺的色谱条件。当流动相中乙腈与10 mmol/L磷酸盐缓冲液的体积比为80:20, pH为3.0,电压为3 kV,检测波长为215 nm时,三聚氰胺能获得很好的分离。方法学考察结果表明,合成的亲水整体柱具有良好的重现性和渗透性,建立的pCEC分析方法的检出限为0.05 mg/L。该方法简单方便,回收率较高,而且流动相中无需添加离子对试剂,适合于奶制品中三聚氰胺的定量测定。     

Preparation and characterization ofp-tert-butyl-calix[6]arene-bonded silica gel stationary phase for high-performance liquid chromatography

Summary For the first time calix[6]arene has been chemically combined with silica gel via a longer spacer to prepare calix[6]arene-bonded silica gel stationary phase for high-performance liquid chromatography (HPLC). The separation of positional isomers and polycyclic aromatic hydrocarbons on the calix[6]arene-bonded phase was achieved with methanol-water as mobile phase. Some nucleosides were also separated on the bonded phase. The reversed-phase chromatographic performance of the bonded phase was studied. The results showed that the calix[6]arene-bonded phase was highly hydrophobic. A possible separation mechanism has been proposed.     

Phase behaviour of oppositely charged protein and surfactant mixtures: DOTAC-BLG-Water

The interactions between the negatively charged protein, β-lactoglobulin (BLG) and the cationic surfactant dodecyltrimethylammonium chloride (DOTAC) in water have been investigated by determining the phase equilibria of the ternary system within the concentration range of 20 wt.% of both protein and surfactant. Three main regions are formed—an isotropic solution phase, a white precipitation region and a blueish, isotropic, highly viscous gel phase. The protein solution can solubilize 1 mole surfactant, [DOTAC] per mole protein, [BLG] prior to precipitation. The protein-surfactant precipitate complex is neutral and consists of 8 [DOTAC]/[BLG]. The net charge of the protein in water at aqueous pH is −7 and this agrees with the determined composition. The pH is, however, decreased on addition of DOTAC, but this does not seem to affect the composition of the precipitate significantly. The amount of precipitate reaches a maximum at about 8 [DOTAC]/[BLG] and thereafter a plateau region occurs where no more precipitate seems to be formed. On further increasing the surfactant concentration the precipitate redissolves either into a solution phase directly or into a solution phase via a gel phase depending on the protein concentration. On decreasing salt concentration the ternary system shows similar phase behaviour, but the stability of the regions are different. It is also observed that oppositely charged protein-surfactant systems show similar phase behaviour irrespective of nature of the net charge on the protein.     

1,3-Alternate 25,27-dibenzoiloxy-26,28-bis-[3-propyloxy]-calix[4]arene-bonded silica gel as a new type of HPLC stationary phase

A new 1,3-alternate 25,27-dibenzoiloxy-26,28-bis-[3-propyloxy]-calix[4]arene-bonded silica gel stationary phase (1,3-Alt CalixBn) has been prepared and used for the separation of aromatic positional isomers by high-performance liquid chromatography (HPLC). The effect of organic modifier content and pH of the mobile phase on retention and selectivity of these compounds were studied. Application examples were provided for separation of purine and pyrimidine bases and non-steroidal anti-inflammatory drugs.