High Performance Liquid Chromatography Separations Using Columns Packed with Spherical ODS Particles. III. Effect of Column Dimensions on the Resolution of a Complex Mixture

Abstract

Separations on short columns, (3 and 5 cm, packed with 3 μ ODS spherical materials) and somewhat larger ones (10 cm and 20 cm columns having 2.1 mm and 4.6 mm diameters packed with 5 μ ODS spherical materials) were compared using Aroclor 1254. With simple mixtures, the results showed that short columns can give separations comparable with those on longer columns when the percentage of the organic modifier in the mobile phase is adjusted. This was not so with more complex mixture. The results also showed that columns which have a comparable volume do not produce comparable separation. The longer column, 200 mm × 2.1 mm gave better resolution than the shorter 50 mm × 4 mm column. Also a shorter column, (100 mm × 4.6 mm), which had double the volume of a longer column. (200 mm × 2.1 mm), gave better resolution of the Aroclor 1254 test solution.     

High Performance Liquid Chromatography Separations Using Short Columns Packed with Spherical and Irregular Shaped ODS Particles

Abstract

In a comparative study of the separation of a mixture of polycyclic aromatic hydrocarbrons on 5 cm and 10 cm columns packed with 3μ and 5μ ODS spherical particles, the separations achieved on the 10 cm column were not significantly better than those on the 5 cm column. Although, columns packed with 3μ ODS spherical particles gave slightly better resolution than those packed with 5μ ODS spherical particles having the same physical properties, differences were observed when columns packed with ODS spherical particles were compared with columns packed with ODS irregular shaped particles of the same size.

The results show that separations on a 5μ or 3μ packed columns takes place at the first few centimeters.     

Effect of Alkyl Chain Length of Bonded Silica Phases on Separation,Resolution and Efficiency in High Performance Liquid Chromatography

Abstract

A comparative study of alkyl bonded phases was carried out under optimum solvent conditions for each phase. Three columns, RP-2, RP-8 and RP-18, were tested for their efficiency and resolving power using three groups of compounds in three binary organic-water mobile phases. The organic solvents were acetonitrile, methanol and tetrahydrofuran which are widely used as solvent modifiers.

The results indicate that each of the three factors, i.e. solvent, solute and bonded alkyl chain length, play an important role, with the solvent being the most significant. When tetrahydrofuran-water was used as the mobile phase, the ratio of THF/H₂O did not vary by much when an RP-2, RP-8 or RP-18 column was used to separate naphthalene from biphenyl, dimethylphthalate from diethylphthalate or anthraquinone from methyl, anthraquinone and ethyl anthraquinone. When acetonitrile-water and methanol-water were used the ratio of organic modefier to water changed so as to accomodate the hydrophobic properties of the columns. The efficiency of the columns, expressed as theoretical plates per meter (TPM) was highest when acetonitrile-water was used as the mobile phase. Although there were variations in TPM and resolution from column to column, the three columns gave good separation of the components of the three groups of compounds.     

Large-Scale Preparative Counter-Current Chromatography with a Coil Planet Centrifuge

Abstract

Development of the large-scale preparative countercurrent chromatographic schemes has been continued by increasing the diameter of the separation column. A 0.55 cm i.d. FEP tube was coaxially coiled around the holder (7.5 cm, 10 cm or 15 cm in diameter) of a horizontal flow-through coil planet centrifuge (15 cm revolutional radius). Performance of each column was evaluated on the separation of dinitrophenyl amino acid samples with a two-phase solvent system composed of chloroform, acetic acid, and 0.1N hydrochloric acid (2:2:1) by using both aqueous and nonaqueous phases as the mobile phase. Experiments with the short preliminary columns (114 ml capacity) revealed that the hydrodynamic distribution of the two solvent phases was sensitively affected by the helical diameter of the column. However, by choosing the proper elution mode of the mobile phase, satisfactory results were obtained with the helical diameters of 7.5 cm and 15 cm at a high flow rate of 500 ml/h under a moderate revolutional speed of 300 rpm. With the long coiled columns (750 ml capacity), the preparative capability of the present scheme was successfully demonstrated on separations of the 1g-quantity sample mixture under optimized operational conditions. Overall results indicated that the sample-loading capacity of the present scheme can be further increased by the use of longer and/or larger-diameter columns.     

Nonaqueous electrochromatography on continuous bed columns of sol–gel bonded large-pore C18 material: separation of retinyl esters

A nonaqueous electrochromatographic reversed-phase separation method for retinyl esters using continuous bed columns has been developed. The packing material 7 μm Nucleosil 4000 Å C₁₈ was sol–gel bonded in 180 μm I.D. capillaries. The mobile phase used was 2.5 mM lithium acetate in N,N-dimethylformamide–acetonitrile–methanol (2+7+1, v/v). At 350 V/cm and 30°C, this mobile phase composition gave rise to an electroosmotic flow of 1 mm/s. No Joule heating nor bubble formation were observed even at 625 V/cm (17 μA). With a 36 cm L_eff column complete separation of the commercially available and synthesized standards (all-trans-retinyl acetate, palmitate, heptadecanoate, stearate, oleoate, and linoleoate) was obtained within 10 min. The within-day and between-day variations of retention times of all-trans-retinyl palmitate were <0.3% relative standard deviation (RSD) (n=3) and <2% RSD (n=6), respectively. The within-day and between-day variations of peak areas were both <2% (both n=3). The columns were used for more than 1 month without degradation. Liver extracts from arctic seal were analyzed.     

The High Pressure Liquid Chromatogrpaphy of Metal Chelates of Diethyldithiocarbmic Acid and Dithizone

Abstract

The HPLC behavior of Ni(II), Co(III), Cu(II), Zn(II), Cd(II), Hg(II) and Pb(II) chelates of diethyldithiocarbamic acid, H(DDTC), and dithizone, H(D_z), was studied on silica gel columns with toluene as the mobile phase. Calibration plots gave linear results for plots of peak area vs. amount of metal injected over the low nanogram to milligramrange. Calibration plots for the above cations were determined on the basis of 3 to 5 replicate determinations for 8 to 10 different concentration levels over the above range with a relative standard deviation less than 5.0% for all metals studied. A variable wavelength photometric detector was used and the detection limits at 270 nm were in the range of 5 to 200 ng depending on the metal species. The best separations were obtained on a1/4″ × 35 cm stainless-steel packed with 10 micron μ-Porasil.     

Comparison of Various Columns for the High-Speed HPLC Analysis of Drugs of Forensic Interest

Abstract

A comparison of the use of various commercially available columns for the high-speed reverse-phase ion-pair high performance liquid chromatographic separation of drugs of forensic interest is discussed. The columns include a Partisil 5 ODS-3 RAC, a Partisil 5 C8 RAC, a Radial Pak microBondapak C18 cartridge, a Perkin-Elmer HS/5 C18 and a Perkin-Elmer HS/3 C18. The mobile phases employed contain water, acetonitrile, phosphoric acid, and sodium hydroxide, with or without hexylamine. When a mobile phase without an amine modifier is employed, retention times were at least halved, except with a HS/3 C18 column, over those obtained with conventional columns. Basic drugs did not elute when the above mobile phase is used with a HS/3 C18 column. In addition, the selectivities of the other high speed columns were similar. Further reductions in retention times and different selectivities were obtained when an amine modifier is utilized. Column performance parameters such as n, V and v are presented for the colupns examined. A new column performance parameter S which is (n/V)^1/2 is introduced and discussed.     

Retention Behavior of Benzodiazepines in Normal-Phase HPLC. Silica,Cyano, and Amino Phases Comparison

Abstract

The retention data for 16 benzodiazepines was evaluated on different normal-phase HPLC columns using the Soczewinski-Snyder model. The slopes and intercepts of the linear relationships between log k' and the mobile phase composition have been discussed in relation to solute and stationary phase characteristics. The good correlation between substituent chromatographic contribution and substituent electronic constant showed that the benzodiazepine molecule acts as a proton donor towards the adsorbent sites of the stationary phases investigated.     

Liquid Chromatographic Analysis of Antiepileptic Drugs: An Overview

Abstract

Extraction of antiepileptic drugs (AEDs) can be carried out by different procedures which include: (i) protein precipitation, (ii) liquid-liquid and (iii) liquid-solid extraction. The latter yields a cleaner sample and shows efficiency comparable to the other procedures; recovery ranges from 90 to 105% for most AEDs.

Types of column include: (i) conventional, (ii) microbore and (iii) high-speed. Compared with conventional and high-speed columns, microbore columns, which have a diameter of 1–2 mm, allow to achieve the highest sensibility (up to double values) and to reduce solvent consume (up to 70 %). High-speed columns, characterized by a length of 3–10 cm and by a reduced packing particle size (? 3μm), make the analysis time more rapid by at least 50%.

Reversed phase chromatography is the most versatile technique as compared to the normal phase and to the precision and accuracy. These parameters, in fact, resulted close to those of the fluorescence polarization immunoassay technique (FPIA), which was the most precise and accurate among the methods compared (79).     

Effect of Column Dimensions on HPLC Separations Using Constant Volume Columns

Abstract

The effect of column dimension on resolution, sample capacity, retention time, efficiency and mobile phase composition were studied, using both constant flow rate and constant linear velocity. The four columns selected (A = 238 × 3.2 mm, B = 153 × 4.0 mm, C = 116 × 4.6 mm and D = 50 × 7 mm) had the same volume. K¹ values were found to be constant, within experimental error, for all columns. At constant linear velocity, the retention time was found to be a linear function of column length, while at constant flow rate retention time was constant for all columns. The longest column (A) generated the largest N values while columns 3 and C gave the lowest H values, for dilute solutions, while they decreased with decreasing column length. On the other hand, it was observed that as the sample size increased, N generated by column A decreased more rapidly and eventually fell below the values generated by columns B and C. These two columns (B & C) can tolerate a larger sample size with less reduction in N value than the longest column. It is important to note that although there were minor differences in performance between columns B and C, there were significant differences between them (B and C) and the other two columns (A and D). Column A offered the highest sensitivity (narrower peaks) for dilute solutions, while columns B and C offered higher loadability. The volume of organic modifier in the mobile phase affected the retention equally in the four columns. It was also found that equal separation (a) was obtained for each column at constant flow rate and constant linear velocity, except with the latter the retention times were longer.     

Semimicro Size Exclusion Chromatography For Oligomers. I. Column Packing Procedure

Abstract

Polystyrene gels of a particle diameter 10 ± 2 μm for the use in oligomer separation were packed into 1.5 mm i.d. × 25 cm length columns by the balanced density slurry-packing technique under a constant flow rate of 500 μL/min. The slurry solvent was a mixture of toluene and chloroform (50.5/49.5, v/v). The example of the number of theoretical plates (N) of these columns was 8600 plates/25 cm (HETP = 29.1 μm) at flow rate of 40 μL/min by injecting 1 μL of 0.5% benzene solution. Sixteen columns were connected and the overall value of N was 103000 plates/4 m. A typical example of oligomer separation was demonstrated. A constant-flow technique is preferable to a constant-pressure technique. When two or three column blanks were packed together, the columns located at the outlet of the packer-column assembly had higher values of N. Optimum flow rate of the slurry solvent when three column blanks were packed together lay between 400 and 500 μL/min. The packing efficiency, that is, the probability of getting valid columns was about 60%. Viscous slurry solvents were not effective to get efficient columns. To pack gels in the less swollen state gave sometimes efficient columns. Pressure monitoring in progress of packing was very effective to foresee the column efficiency.     

Factors Affecting the Interpretation of Selectivity on Synergi Reversed-Phase Columns

The solvation parameter model is used to establish the contribution of cohesion, dipole-type, and hydrogen-bonding interactions to the retention mechanism on Synergi Hydro-RP, Fusion-RP, and Polar-RP reversed-phase columns with methanol–water mobile phases containing from 10–70% (v/v) methanol. Large changes in relative retention on the compared columns can result from steric resistance, differences in the phase ratios, and from dewetting at low methanol compositions while changes in intermolecular interactions are responsible for smaller changes at a fixed mobile phase composition. For Synergi Hydro-RP and Polar-RP changing methanol for acetonirile is more powerful for affecting changes in retention order than changing the stationary phase. The three Synergi columns show useful selectivity differences for method development when compared with 13 other modern reversed-phase columns representing a selection of different stationary phase chemistries. The results from this study indicate the limitations of classifying reversed-phase columns by the retention of prototypical compounds to define specific retention mechanisms.     

A Comparison of Radially-Compressed and Stainless Steel Columns for the Reversed-Phase Ion-Pair Separation of Phencyclidine Synthetic Mixtures

Abstract

The reversed-phase ion-pair HPLC separation of phencyclidine synthetic mixtures was optimized utilizing Radial-Pak radially compressed columns. Variables examined in the optimization included column type (C-18, C-8, or CN), pairing ion (methane-, pentane-, hexane-, or octane sulfonates) and mobile phase composition (varying concentrations of methanol or acetonitrile in water). The chromatographic behavior of the phencyclidine mixtures in the various systems utilizing radially compressed columns is compared and contrasted to a similar previous study which examined similar variables on stainless steel columns. The optimum system for radially compressed columns was found to consist of a Radial-Pak C-18 column and a mobile phase of 85:15 MeOH:H₂O, 2.5% acetic acid, 1% triethylamine and 5mM sodium hexane sulfonate.     

Nonaqueous electrochromatography on continuous bed columns of sol-gel bonded large-pore C18 material: separation of retinyl esters

A nonaqueous electrochromatographic reversed-phase separation method for retinyl esters using continuous bed columns has been developed. The packing material 7 μm Nucleosil 4000 Å C₁₈ was sol–gel bonded in 180 μm I.D. capillaries. The mobile phase used was 2.5 mM lithium acetate in N,N-dimethylformamide–acetonitrile–methanol (2+7+1, v/v). At 350 V/cm and 30°C, this mobile phase composition gave rise to an electroosmotic flow of 1 mm/s. No Joule heating nor bubble formation were observed even at 625 V/cm (17 μA). With a 36 cm L_eff column complete separation of the commercially available and synthesized standards (all-trans-retinyl acetate, palmitate, heptadecanoate, stearate, oleoate, and linoleoate) was obtained within 10 min. The within-day and between-day variations of retention times of all-trans-retinyl palmitate were <0.3% relative standard deviation (RSD) (n=3) and <2% RSD (n=6), respectively. The within-day and between-day variations of peak areas were both <2% (both n=3). The columns were used for more than 1 month without degradation. Liver extracts from arctic seal were analyzed.     

Determination of Organic Acids in Apple and Cider by Liquid Chromatography with ordinary and narrow-bore columns. A Comparative Study

Abstract

Two narrow-bore columns packed with octadecylsilane of various particle sizes were used to compare their efficiency for the separation of organic acids in apple and cider with that of ordinary columns. The best simultaneous resolution of quinic, malic, shikimic, lactic, acetic, citric and succinic acid was accomplished by using a 100 × 2.1 mm ID, 3-μm Spherisorb ODS-2 column and a phosphate buffer as the mobile phase. This chromatographic system provided a separation efficiency comparable to that afforded by an ordinary 250 × 4.6 mm ID, 5-μm Spherisorb ODS-2 column, plus greater rapidity (30%) and economy, all of which allowed the accurate, precise determination (CV=3%) of the above-mentioned compounds. Finally, the performance of an ordinary UV detector and that of a rapid spectral detector in this type of determination were critically compared.     

Comparison of Column Packings for Isocratic High-Performance Liquid Chromatographic Analysis of Carotenoids Using Non-Aqueous Reversed-Phase

Abstract

Ten different columns are compared for the isocratic non-aqueous reversed-phase separation of carotenoids, using solvent mixtures of ethyl acetate-acetonitrile both with and without 0.1% n-decanol as modifier. Conditions were established for separation of a mixture of alfalfa carotenoids containing mainly neoxanthin, violaxanthin, lutein and β-carotene. The best material for use for rapid isocratic separation of all major components was a high carbon loading non-endcapped material with ODS functionality, although one endcapped C8 material gave similar results. The use of n-decanol as mobile phase modifier is imperative to rapidly condition new columns to give optimum peak shape and definition and system linearity.     

Use of Ethoxy-Homologs as Internal Standards for Determination of Urinary Vanillylmandelic Acid and Normetanephrine in Man by High Performance Liquid Chromatography

Abstract

Synthesis of the ethoxy-homologs of vanillylmandelic acid (3-ethoxy-4-hydroxymandelic acid, EMA) and normetanephrine (3-ethoxy-4-hydroxyphenylethanolamine, EHPEA) and their use in minor modifications of existing assays on human urine are described. For analysis of vanillylmandelic acid, the homologous internal standard is added to an aliquot of urine, organic acids are then extracted into ethyl acetate and back extracted into 10% K₂CO₃, and finally extracted a second time into ethyl acetate. After evaporation of the ethyl acetate, the residue is redissolved in the mobile phase to be used in the chromatography, and injected onto a reverse phase column. Comparison of results with a gas chromatography-mass spectrometry (GCMS) assay gave almost identical results (96% ± 2%, mean ± SE, n=10). Analysis of normetanephrine is performed by addition of internal standard to an aliquot of urine before heat hydrolysis of amine conjugates. The amines are adsorbed onto columns of Bio Rex 70, and then eluted with a solution of 1 M NH₄OH, which is then evaporated to dryness. After residue is redissolved in borate buffer, the amines are extracted into toluene:isoamyl alcohol and back extracted into 0.1 M acetic acid before injection onto a reverse phase column. Correlation with a GCMS assay gave similar results (102% ± 4%, mean ± SE, n=9).     

Determination of Steroids in Human Urine by Micellar Liquid Chromatography

Abstract

A simple and sensitive method for the determination of steroids using micellar liquid chromatography is described. The steroids, including hydroxycorticosterone. corticosterone, northisterone, testosterone, mexdroprogesterone acetate and progesterone, were separated by reversed-phase using a micelles mobile phase following UV detection at 245 nm. The parameters affecting retention of the test solutes such as the concentration of sodium dodecyl sulfate (SDS) and n-butanol-1 in the mobile phase were investigated. It was found that the retention of the solutes was dependent on the composition of mobile phase. The linear calibration plots range from 0.1 to 10 μg ml^?1 in mobile phase containing 5.0 × 10^?2 mol l^?1 SDS/9 % n-butanol-1 at pH 6.0, and the detection limit in order of 0.1 μg ml^?1 was obtained. The proposed method was used for the determination of steroids in urine using direct injection of samples without previous treatment.     

A New Technique for Simultaneous Assay of Biogenic Amines and Their Metabolites in Unpurified Mouse Brain

Abstract

We developed a chromatographic system which separates cate-cholamines and indolamines as well as a large number of their metabolites (acids, aminoacids and glycols). Waters Radial Pak columns compressed radially to avoid formation of cavities in the packing, were used. These columns must be washed with methanol in order to obtain reproducible results when an ion-pairing mobile phase is employed. The conditioning of bhe columns was not affected by washing with triethylamine or variations in radial pressure. Good resolution of 13 samples was obtained in 28 minutes with a mobile phase composed of 0.1 M KH²PO⁴, 0.1 mM EDTA and 5 mM heptane sulfonic acid in water and Me OH (85:15), pH = 3.6. Products were identified with the use of a Metrohm 64l electrochemical detector.

The technique was used for rapid, simultaneous determination of NA, DA, DOPAC, HVA, 5HT and 5HIAA in deproteinated mouse brain samples.     

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography has been investigated in the present study. Columns with inner diameters of 0.32, 0.53, 3.2 and 4.6 mm were compared with respect to retention and efficiency characteristics using temperature gradients from 30 to 90 degrees C with temperature ramps of 1, 5, 10 and 20 degrees C min(-1). The columns were all of 15 cm length and were packed with 3 microm Hypersil ODS particles. Alkylbenzenes served as model compounds, and the mobile phase consisted of acetonitrile-water (50:50, v/v). The study revealed that the column ID is not a critical limiting factor when performing temperature programming in LC, at least for columns narrower than 4.6 mm inner diameter in the temperature interval 30-90 degrees C. The retention times for all components on all columns were highly comparable, with similar peak profiles without any signs of peak splitting. The use of mobile phase pre-heating when using the larger bore columns was avoided by starting the temperature gradients close to ambient. However, the relative apparent efficiency was inversely proportional to column inner diameter, making the capillary columns generally more functional towards temperature gradients than the larger bore columns with respect to chromatographic efficiency. In addition, the capillary columns possessed higher robustness towards temperature programming than the conventional columns.