Characterization of Column Packings in Normal-Phase Liquid Chromatography. II. Retention Behavior of Fat-Soluble Vitamins in Amino- and Cyano-Propyl Bonded Silica Gel and Binary Solvent Systems

Abstract

As continuation of our study on the characterization of column packings in normal-phase HPLC analysis, the retention indices of ten fat-soluble vitamins on aminopropyl and cyanopropyl bonded silica columns were systematically estimated using binary solvents containing ethyl acetate (EtOAc), tetrahydrofuran (THF) or 2-propanol (PrOH) in n-hexane. A linear relationship between the logarithm of the capacity ratio and that of the solvent composition was confirmed. The retentivity and selectivity for these chemically bonded packing columns were determined as follows: the amino-type column has stronger and cyano-type column weaker retentivity than the bare silica column for n-hexane-EtOAc or THF binary systems. Specific adsorption of tocopherol derivatives containing phenolic hydroxyl groups on the amino column was observed. To obtain high efficiency in the separation of fat soluble vitamins, peak sharpness and asymmetry factors were measured using three columns and three binary solvents. The bare silica and PrOH binary solvent generally gave superior peak shape for all vitamin samples.     

In Process Citation

The separation, identification and determination of the fat- and water-soluble vitamins are realized by partition chromatography with a reversed-phase system made by bonding a C(18) group to silica. The water-soluble vitamins are directly separated with the mobile phase 1% acetic acid/acetonitrile (89:11 v/v) and are revealed by an ultraviolet detector, except for pantothenic acid. The separation efficiency and precision of determination of the fat-soluble vitamins depend on the operational conditions. The composition of the excipients and all the constituents of pharmaceuticals (aqueous and oil solutions, injections, dispersions, emulsions) determine the choice of the extraction solvents and the preparation of the solution to be injected; the polarity of the mobile phase (acetonitrile/water 95:5 v/v) can be changed, and the choice depends on the components to be separated. The experimental conditions are specified and some examples are given of application of HPLC to determination of water-soluble vitamins (B1, B2, C, PP, B6) and fat-soluble vitamins (non-saponifiable oils, vitamin A and its esters, cholecalciferol, ergocalciferol, and tocopherol and its acetate) in multivitamin formulations (solutions, suspensions, syrups, fatty excipients etc.).     

Relevance of 1,2,5,6,9,10-hexabromocyclododecane diastereomer structure on partitioning properties,column-retention and clean-up procedures

To optimize clean-up procedures for the analysis of α-, β-, and γ-hexabromocyclododecanes (HBCD) in environmental and biological extracts, their retention behavior on silica gel and florisil was investigated using diverse mobile-phase solvents and accounting for matrix effects. The β-diastereomer, relative to the α- and γ-diastereomers, is substantially retained on both florisil and silica gel regardless of the solvent used. The β-diastereomer is therefore prone to undergo selective loss during clean-up. This sequence is counterintuitive to sequences based on reverse-phase chromatography with a C₁₈-column, in which the α- (and not the β-) isomer is eluted first when using a polar solvent. There has been some discrepancy regarding the structures of these diastereomers in the literature, with structures based on X-ray crystallography only becoming recently available. Based on these X-ray crystal structures, physical–chemical properties (the octanol–water partitioning constant, the Henry's law constant, subcooled liquid vapour pressures and subcooled liquid water solubilities) of the HBCD diastereomers were estimated using the quantum-chemistry based software COSMOtherm, and were found to differ from previously calculated values using different structures (e.g. log K_aw for α-, β-, and γ-HBCD are here estimated to be −8.3, −9.3 and −8.2 respectively). Hypothesis relating differences in structure to physical–chemical properties and retention sequences are presented. The extra retention of the β-diastereomer on silica gel and florisil is likely because it can form both greater specific (i.e. polar) and non-specific (i.e. non-polar) interactions with surfaces than the other diastereomers. Non-specific interactions can also account for the counter-intuitive elution orders with C₁₈-reverse-phase chromatography. These results indicate that care should be taken when isolating HBCDs and other molecular diastereomers from environmental and biological samples, and that reported concentrations of β-HBCD in the literature may be negatively biased.     

Relative Strength of Stronger Solvents for Silica,Amino- and Cyano-alkyl Bonded Silica Columns in Normal-Phase Liquid Chromatography

Abstract

The strength and selectivity of solvents such as ethyl acetate, dioxan and ethyl alcohol in n-hexane binaries were determined using steroids as solutes in normal-phase liquid chromatography of silica gel, amino- and cyano-propyl silica columns. Based on the linear relationship between the logarithm of the capacity ratio and logarithm of solvent composition, the relative strength of solvents was determined from the experimental retention data described in our earlier articles. A micro-computer data base was compiled for filing the retention behavior of the steroids. Using this computer data base, the optimization process of binary solvents for a given sample was improved. An example of the phase system design is described.     

Sorption of catechins under conditions of reverse-phase high-efficiency liquid chromatography

The physico-chemical principles of catechin sorption from various polar solvents onto silica gel modified with octadecyl groups were studied. Thermodynamic characteristics of the sorption were calculated, and the applicability of different models of retention was demonstrated for catechins under the conditions of reverse-phase high-efficiency liquid chromatography.     

Regularities in the retention of nonsubstituted polynuclear aromatic hydrocarbons in liquid chromatography on polar adsorbents

Summary The retention of polynuclear aromatic hydrocarbons (PAH) is studied in liquid chromatography, using columns packed with silica gel with a hydroxylated surface and the same silica gel with surface-bonded amine and nitrile groups. A linear relationship was found between retention and the number of π-electrons in the molecule, the size of the molecule and the different types of carbon atoms in the molecule. The different carbon atoms in a PAH molecule contribute differently to the retention and this contribution depends on the nature of the polar functional groups on the adsorbent's surface and on the nature and concentration of a polar modifier present in a non-polar eluent.     

Determination of vitamins A, D, E, and K in human and bovine serum, and β-carotene and vitamin A palmitate in cosmetic and pharmaceutical products, by isocratic HPLC

Summary An HPLC method has been developed for the determination of fat-soluble vitamins. Ten fat-soluble vitamins were separated simultaneously on a 25 cm×4.6 mm i.d. Hypersil C₁₈ column with acetonitrile-dichloromethane-methanol, 60:20:20 (v/v) as mobile phase at 1.0 mL min⁻¹ with wavelength-programmed ultraviolet-visible-absorbance detection. Total analysis time was 12 min. The limits of detection were 0.03, 0.01, 0.55, 1.84, 0.02, 0.02, 0.01, 0.16, 0.33, 0.01, and 0.01 ng mL⁻¹ for retinol, retinyl acetate, retinyl palmitate, β-carotene, ergocalciferol, cholecalciferol, tocopherol, tocopherol acetate, phylloquinone, menatetrenone, and menadione, respectively. Analysis of human serum 2–7 days after ingestion of oral vitamins and Chinese herbs led to the conclusion that the concentration of vitamins was higher than for control serum.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

Regularities in the retention of isomeric aromatic hydrocarbons in liquid chromatography II. Analysis of polymethyl- and monoalkylbenzenes on silanized silica gel

Summary The regularities of the retention of polymethyl- and monoalkylbenzenes on a nonpolar adsorbent (silanized silica gel) with a polar eluent (23 isopropanol-water) have been studied. In contrast to the retention of the same compounds on a polar adsorbent (silica gel with hydroxylated surface) and with a nonpolar eluent (hexane) polymethylbenzenes are retained on silanized silica gel for a shorter time than the corresponding monoalkylbenzene isomers. The retention order of tert.-, sec.- and n-butylbenzenes is the reverse of that observed on silica gel having a hydroxylated surface. The retention order of xylenes, trimethyl- and tetramethylbenzenes is also the reverse of their retention order on silica gel having a hydroxylated surface. All the mixtures of the corresponding homologues and isomers have been separated.     

Non-aqueous,reversed-phase,high-performance,thin-layer chromatography and column liquid chromatography of metal complexes of porphine

Summary The chromatographic mobility of 21H, 23H-porphine and its Ni(II), Cu(II), Zn(II) and Pd(II) complexes were investigated by high-performance thin-layer chromatography on an octadecyl-bonded, silica gel plate with various polar organic solvents including alcohols, acetonitrile, dimethylsulfoxide and propylenecarbonate. The mobility generally decreases according to the central metal ion of the complex as follows: Zn(II)>(free porphine)>Ni(II)>Pd(II)>Cu(II). Methanol is a good choice of solvent for the separation of these metal porphine complexes. Successful separation of porphine and the four metal complexes is accomplished within 13 min on a LiChrosorb RP-18 column with methanol eluent.     

Underivatized amylose and cellulose as new stationary phases for hydrophilic interaction chromatography

Two polysaccharide stationary phases have been newly suggested for application in hydrophilic interaction chromatography (HILIC). Both columns (amylose‐silica, 250 × 4.6 mm, 5 μm and cellulose‐silica, 250 × 4.6 mm, 5 μm) demonstrated a satisfactory retention of polar compounds. The influence of the mobile‐phase composition (acetonitrile content, pH, salt concentration) on the retention was in agreement with the HILIC concept. The phases showed a very similar behavior, typical efficiency of about 50 000 plates/m, cellulose retained test compounds somewhat more strongly. Under the experimental conditions, electrostatic (non‐HILIC‐type) interactions due to the dissociation of silanol groups on the silica surface did not influence the retention, noticeably. The applicability of polysaccharide stationary phases for the chromatography of polar compounds was proven by the separation of mixtures of sugars (fructose, glucose, saccharose, maltose, trehalose) or vitamins (nicotinamide, pyridoxine, riboflavin, thiamine, nicotinic acid, ascorbic acid).     

Selectivity of liquid-adsorption chromatography on hydroxylated silica gel with nonpolar and polar eluents

Summary The effect of nonpolar (hexane, decane, benzene) and polar (water, isopropanol, water+isopropanol) eluents on separation of aromatic hydrocarbons and their derivatives in liquid-adsorption chromatography on hydroxylated silica gel columns has been studied. The work has revealed that the traditional application areas of hydroxylated silica gel in liquid chromatography may be extended. In particular it has been shown that, first, polar compounds may be separated on hydroxylated silica gel with nonpolar eluents at high temperatures (100–150°C) and that polar and nonpolar compounds may be separated using pure water as eluent. The effect of adsorbed moisture on retention behaviour of silica gel with nonpolar eluents has also been investigated.Presented at the 14th International Symposium on Chromatography London, September, 1982     

High-performance liquid chromatography of fat-soluble vitamins. Simultaneous quantitative analysis of vitamins D2, D3 and E. Study of percentage recoveries of vitamins from cod liver oil

Vitamins D2, D3 and E were resolved and quantified by applying reversed-phase high-performance liquid chromatography to extracts of cod liver oil. The method, using two reversed-phase C18 columns and a ternary mixture of acetonitrile, methanol and water as the eluent resolved all fat-soluble vitamins well, including the pair D2-D3. The extraction procedure was studied; the recoveries, using two different solvents (hexane and diethyl ether) for extractions were 60.6 +/- 1.0 and 77.1 +/- 1.1, 56.9 +/- 1.2 and 74.8 +/- 0.8, and 14.1 +/- 0.7 and 89.8 +/- 1.4% for vitamins D2, D3 and E, respectively.     

Silica gel thin-layer chromatography of acidic phospholipids. II. Chromatographic behaviour of phosphatidylserine and phosphatidic acid applied with different cation composition on adsorbents either free of metal ions or containing a surplus of divalent metal ions.

Different salt forms of phosphatidylserine and phosphatidic acid (two acidic phospholipids) have been subjected to thin-layer chromatography on two commonly used silica adsorbents, one of which (silica gel HR) is practically free of metal ions and the other (silica gel G) contains 13% of calcium sulphate as binder. The chromatographic behaviour was studied in an acidic, a neutral and a basic solvent. Both adsorbents provided usable systems for phosphatidylserine with each of the three solvents, except for silica gel G with the neutral solvent, in which system tailing was prominent. The inclusion of calcium sulphate in the silica gel tended to impair chromatography of phosphatidylserine in acidic and neutral solvents, but improved its chromatography in the basic solvent. In all the systems, the migration was independent of the cation composition of the applied phosphatidylserine samples. For the chromatography of phosphatidic acid, only three of the systems tested were usable, and in those three, the chromatographic behaviour was independent of the cation composition of the samples. The calcium sulphate in an adsorbent increased tailing of phosphatidic acid in acidic and neutral solvents, as it did for phosphatidylserine, whereas with the basic solvent, calcium sulphate in the adsorbent caused phosphatidic acid to remain at the origin. Two one-dimensional thin-layer chromatographic systems previously recommended for the chromatography of acidic phospholipids were unsuitable for the chromatography of phosphatidic acid under the conditions used here. For both phosphatidylserine and phosphatidic acid chromatographed in acidic systems, the solvent must contain water in addition to acetic acid if excessive tailing is to be avoided.     

Solid phase synthesis of the signal sequence of E. coli alkaline phosphatase

A synthetic peptide corresponding to the signal sequence ofE. coli alkaline phosphatase has been synthesized by the solid phase method employing the transesterification method of cleavage from the resin. The protected peptide obtained after cleavage was purified to homogeneity by column chromatography on silica gel, followed by partition chromatography on SephadexLH-20 using organic solvents like chloroform and methanol as eluants.     

Thermal study of chelates of Co(II), Cu(II), Ni(II), Cr(III), Mo(III), and Fe(III) with bis(acetylacetone)ethylenediimine on activated silica gel surface

The divalent copper, nickel, cobalt and trivalent chromium, molybdenium and iron chelate compounds derived from bis(acetylacetone) ethylenediimine were grafted on activated silica gel using a batch process in methanolic solution. The sequence of the maximum retention capacity was Cr(III)>Mo(III)>Fe(III)>Co(II)>Ni(II)>Cu(II). Calorimetric titration was employed to study the interaction of activated silica gel with these series of metal chelate compounds. Exothermic enthalpic results were obtained throughout all interactions process. The spontaneity of these systems was reflected in negative and positive free Gibbs energy from entropic values.     

Characterization and prediction of retention in gradient-elution normal phase HPLC with ternary gradients

Summary Chromatographic behaviour of phenylurea herbicides and phenols on a silica gel column with ternary gradient elution using mobile phases containing dry 2-propanol, n-heptane and dioxane was investigated. With dried solvents and the temperature controlled to ±0.1 °C, the data from repeated experiments measured on the same column after ten months of use were reproducible. Algorithms were suggested for characterization and prediction of retention in ternary gradients at a constant ratio of the two polar solvents (elution strength gradients) and at a constant sum of their concentrations (selectivity gradients). These were based on two- and three-parameter equations describing the dependence of the sample retention factors (isocratic) on the concentration of the polar solvent in binary mobile phases. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Characterization of Amino-, Cyano-alkyl Bonded Silica Columns in Normal-Phase Liquid Chromatography by Using Steroids

Abstract

In order to characterize the chemically bonded phases in HPLC analysis, the retention behavior of fifteen steroids including estrogen, androgen, progestogen and corticoid were systematically examined using dioxan as the stronger component in an n-hexane-binary system. A linear relationship between the logarithm of the capacity ratio and logarithm of the molar concentration of the binary solvent was confirmed for amino- and cyano-type bonded as well as non-bonded silica gel columns. Based on the retention indices of these phases, the retentivity of the packing materials was determined as follows: the amino-type is similar to and the cyano-type is weaker (0.7 times) than bare silica gel when using dioxan as the stronger component. The specific retentivity of an amino column for polar steroids containing phenolic and alcoholic hydroxyl groups suggests a molecular interaction associated with hydrogen bonding between the polar packing surface and solute compounds. The selectivity of amino packing was found to be larger than cyano packing whose retention selectivity is similar to a bare silica gel.