Reversed-phase Liquid Chromatography Column Research Articles

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatographic columns: II. Results obtained with Symmetry C 18 columns

The long-term repeatability, the column-to-column and the lot-to-lot reproducibilities of the retention and profile of the peaks obtained with 15 different Symmetry C 18 columns were investigated. This packing material is an octadecyl bonded silica for RP-LC. Data characterizing the retention, the steric selectivity, the hydrogen bonding capacity, the hydrophobic interaction selectivity, the column efficiency, and the peak asymmetry are reported for a group of 30 neutral, acidic and basic compounds selected as probes. These data were acquired under five different sets of chromatographic conditions, using an HP 1100 liquid chromatograph and following an experimental protocol previously described. Statistical comparison of five different columns of one batch and of columns representative of 10 different batches are based on the high precision data obtained. For example, for 28 of the 30 components, the R.S.D.s of the retention factors measured on 10 columns from as many different batches were better than 2%. The R.S.D.s of the separation factors for 27 of these 30 components were below 1.4%. Such a level of column reproducibility is most impressive.

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns: I. Experimental protocol

A procedure is described for the determination of the short-term and long-term repeatabilities and of the column-to-column and lot-to-lot reproducibilities of the retention and profile characteristics of the peaks obtained with a number of different reversed-phase liquid chromatography (RPLC) C 18-bonded silica columns of several commercial brands. Data characterizing the retention, the steric selectivity, the hydrogen bonding capacity, the hydrophobic interaction selectivity, the column efficiency and the peak asymmetry will be acquired for all the probe compounds. These include 30 neutral, acidic and basic compounds distributed into five groups to be eluted under as many different sets of chromatographic conditions. The data will be obtained using an HP 1100 liquid chromatograph. The compounds and experimental conditions selected were similar to those previously used by different authors for comparison purposes. Careful attention will be paid to minimization of external error contributions by adhering to a strict operational procedure. The precision expected will be high because preliminary results gave standard deviations of around 0.04% for the separation factors, below 0.15% for the retention times and around 1% for the column efficiency in short-term repeatability experiments performed with one column.

Analysis of lecithin in cosmetics by reversed-phase liquid chromatography/electrospray tandem mass spectrometry

A rapid, sensitive and selective method by reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for the determination of lecithin in a cosmetic raw material for quality control of the product formulation. The mixture of the phosphatidylcholine, phosphatidyl-ethanolamine, and phosphatidylinositol in lecithin was separated using aqueous methanol–acetonitrile solvent with a reversed-phase LC column, and detected as forms of pseudomolecular ions. Using this LC/ESI-MS technique, simultaneous separation and detection of phospholipid classes and molecular species within each class were achieved for the first time. Additionally informative fragmentation patterns were obtained by employing LC/ESI-MS/MS in both positive and negative ionization modes to identify these fatty acid chains and polar head groups in each molecular species of the phospholipids. © 1998 John Wiley & Sons, Ltd.

Analysis of lecithin in cosmetics by reversed‐phase liquid chromatography/electrospray tandem mass spectrometry

A rapid, sensitive and selective method by reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for the determination of lecithin in a cosmetic raw material for quality control of the product formulation. The mixture of the phosphatidylcholine, phosphatidyl-ethanolamine, and phosphatidylinositol in lecithin was separated using aqueous methanol–acetonitrile solvent with a reversed-phase LC column, and detected as forms of pseudomolecular ions. Using this LC/ESI-MS technique, simultaneous separation and detection of phospholipid classes and molecular species within each class were achieved for the first time. Additionally informative fragmentation patterns were obtained by employing LC/ESI-MS/MS in both positive and negative ionization modes to identify these fatty acid chains and polar head groups in each molecular species of the phospholipids. © 1998 John Wiley & Sons, Ltd.

Comparative study of test methods for reversed-phase columns for high-performance liquid chromatography

Column selection in reversed-phase liquid chromatography (RPLC) is still not a straightforward process. A number of tests to characterise and classify RPLC columns have been suggested. Several tests are already applied in laboratory practice, while others are under development. The results of the various tests, however, are not always qualified to describe the properties of columns for RPLC. In this study different tests for RPLC-columns are studied and compared, viz. the Engelhardt, Tanaka, Galushko and Walters tests. The column descriptors hydrophobicity and silanol activity are investigated in particular. The tests are studied using approximately 20 silica, alumina and polymer based C 8- and C 18-columns. Hydrophobicity data from the tests generally were good and interchangeable between the tests resulting in a column classification that is independent of the applied test. It appears that buffering of the eluent is mandatory for adequate testing of column silanol activity. In contrast with the high-quality hydrophobicity data, the silanol activity results of the various tests differ significantly. As a consequence column classification with respect to silanol activity depends considerably on the applied test method.

Detector cell based on plastic liquid-core waveguides suitable for aqueous solutions: one-to-two decades improved detection limits in conventional-size column liquid chromatography with absorption detection

A new type of detector cell is presented, which is designed to improve concentration detection limits in conventional-size reversed-phase column liquid chromatography (LC) with ultraviolet (UV)–visible absorption detection. It is based on a recently developed plastic liquid-core waveguide (LCW), suitable for light guidance through aqueous solutions since the refractive index of the plastic perfluorinated polymeric material (denoted as Teflon AF2400) is 1.29, i.e., smaller than that of water (1.33). An LCW of 90 cm length (280 μm I.D.) was brought into a standard UV–Vis absorption detector and a 1.0 m long quartz optical fibre was installed in the reference channel. This LCW detector cell combines an optical pathlength of about 90 cm with an internal volume of 55 μl. An LC separation of three test analytes – the pesticides atrazine, diuron and linuron, which were detected at 250 nm – provided detection limits of 0.3–0.5 μg/l. This is 30- to 50-fold better than obtained with the standard detector cell. The linear range extended up to about 1 mg/l. As an application 1 ml of tap water spiked with the test analytes at the 0.2 μg/l level was analysed; the results indicated that such a volume is sufficient to reach the 0.1 μg/l threshold value of the European Union directive without using analyte preconcentration.

Role of stationary phase and eluent composition on the determination of log P values of N-hydroxyethylamide of aryloxyalkylen and pyridine carboxylic acids by reversed-phase high-performance liquid chromatography

The partition coefficients, P, between n-octanol and water of a number of growth stimulating substances, N-hydroxyethylamide of aryloxyalkylen- and pyridine carboxylic acids were obtained from Pomona College ( C log P), and Rekker's (log P Rekker) revised fragmental constant system was used to calculate log P data sets. Both of these data sets were correlated with two different substance lipophilicity parameters, log k w and ϕ 0. Log k w was obtained by extrapolation of log retention factor ( k) to 0% organic modifier measured in reversed-phase liquid chromatography (RPLC) systems. ϕ 0 values were obtained from the slopes and intercepts of these relationships. The RPLC experiments were performed on four commercially available reversed-phase columns. Binary mixtures of methanol–water, methanol–phosphate buffer (pH 7.0), methanol–tricine buffer (pH 7.0) and acetonitrile–water were used as mobile phases for the determination of log k w values. For the methanolic eluents linear regression provided satisfactory correlations ( r>0.99) for the relationships log k vs. organic modifier content in the eluent, while for the acetonitrile-containing eluents a second-degree polynominal regression was necessary. For all four RPLC columns, by linear regression satisfactory correlations ( r>0.99) were obtained between log k w and log P data using methanolic eluents. In such eluents ϕ 0 values were shown to be the second-best lipophilicity parameters. For acetonitrile-containing eluents the use of second-degree polynominal regression was necessary and, in contrast to methanol, significant influence of the applied column on regression results was observed. For acetonitrile-containing eluents the ϕ 0-index does not provide satisfactory results for our substances. No difference in regression results between the use of buffered and non-buffered eluents was observed.

Determination of Deoxynivalenol in White Flour, Whole Wheat Flour, and Bran by Solid-Phase Extraction/Liquid Chromatography: Interlaboratory Study

A liquid chromatographic (LC) method for determining deoxynivalenol (DON) in white flour, whole wheat flour, and bran at or above the U.S. Food and Drug Administration advisory level of 1 microgram/g was evaluated by an interlaboratory study. Test samples of processed wheat (flour and bran) were extracted by blending with acetonitrile-water (84 + 16). Extracts were filtered and passed through a solid-phase extraction (SPE) column. The eluate was then chromatographed on a reversed-phase LC column with a water-methanol gradient. DON was measured at 220 nm. Naturally contaminated white flour, whole wheat flour, and bran samples and spiking solutions of DON to be added to the 3 commodities at 0.5, 1.0, and 2.0 micrograms/g were sent to 4 collaborators in Kansas, Louisiana, Missouri, and Washington states. Three collaborators completed the study. Average recoveries of DON from the 3 commodities spiked at 0.5, 1.0, and 2.0 micrograms/g were 94, 87, and 97%, respectively. Within-laboratory relative standard deviations for repeatability (RSDr) ranged from 3.1 to 21.7% and between-laboratory relative standard deviations for reproducibility (RSDR) ranged from 10.8 to 38.7%. On the basis of the results of this study, the SPE/LC method for DON in white flour, whole wheat flour, and bran was adopted as a peer-verified method by AOAC INTERNATIONAL.

Column preparation for reversed-phase high-temperature open tubular column liquid chromatography

A method for the preparation of stable reversed-phase columns for high temperature open tubular column liquid chromatography (HT-OT-LC) has been developed and the effect of covalent bonding of the stationary phase to a covalently bonded deactivation layer, as well as, crosslinking of the polysiloxane stationary phase has been investigated. It was found that the column lifetime depended primarily on the extent of crosslinking of the stationary phase film with azo- tert.-butane and a dynamic crosslinking procedure was developed for this purpose. By extensive crosslinking of the polysiloxane stationary phase, column lifetimes of more than several hundreds of hours could be obtained at 150°C. Inadequate crosslinking, on the other hand, resulted in shorter column lifetimes most likely as a result of hydrolysis of the stationary phase polymer leading to its collapse in the centre of the column. No significant difference in column lifetime was seen between columns prepared with 50% n-octylpolymethylsiloxane or 49% n-octyl-, 1% n-octenylpolymethylsiloxane stationary phases, with cyano or octyl groups in the deactivation layer, or between columns with different stationary phase film thickness (in the range between 0.25 and 0.60 μm).

98/02739 Manufacture method of blast-furnace coke: Arima, T. and Suzuki, T. Jpn. Kokai Tokkyo Koho JP 09,255,967 [97,255,967] (Cl. C10B57/04), 30 Sep 1997, Appl. 96'64,374, 21 Mar 1996, 4 pp. (In Japanese)

A reversed-phase column liquid chromatograpic method was developed for the assay of ascorbyl dipalmitate in whitening powders. The linear calibration range was 0.1–0.7 mg/ml (r = 0.9999), and recoveries were generally greater than 98.8%. The coefficient of variation was less than 0.64%. The method has been applied to whitening powders produced by four different cosmetics companies. The assay results were compared with those obtained from a sodium hydroxide titration assay of ascorbyl dipalmitate in commercial whitening powders. The method can be successfully used for routine quality control and offers advantages in speed, simplicity and reliability.

Rapid verification of disulfide linkages in recombinant human growth hormone by tandem column tryptic mapping

An automated tryptic mapping method was developed for characterization of disulfide linkages in recombinant human growth hormone (rhGH). The hormone was trypsin digested and the peptide fragments concentrated by eluting rhGH through an immobilized trypsin column and transferring the peptides directly to a reversed-phase liquid chromatography (RP-LC) column where they were collected. Reaction time was controlled by the flow-rate. Following tryptic digestion of a sample, the immobilized enzyme column was uncoupled from the flow train by a switching valve and the RP-LC column eluted with a solvent gradient ranging from 0.1% trifluoroacetic acid (TFA) with 1% acetonitrile (ACN) to ACN with 0.1% TFA and 5% water. This two-step mapping process was achieved within 2 h on both native and reduced rhGH samples. The chromatographic elution position and mass spectra matrix-assisted laser desorption ionization time-of-flight mass spectrometry of native rhGH and sulfur-containing peptides were determined with standards. Standards of the individual sulfhydryl (–SH) containing peptides and all possible disulfide linked peptides that could result from coupling the –SH peptides in disulfide linkages were obtained by synthesis and chromatographic purification. This approach allowed the chromatographic elution position of all possible mismatched disulfide containing peptides to be established and samples of rhGH to be examined for improper folding.

Determination of glutamate and aspartate in microdialysis samples by reversed-phase column liquid chromatography with fluorescence and electrochemical detection

Five different systems for fast determination of aspartate and glutamate in microdialysis samples are described: (I) a high-speed HPLC using a gradient pump with a sharp elution profile, (II) a column switching technique, (III) an isocratic pump with a low-pressure switching valve for one-step gradients, (IV) microbore chromatography using injections of acetonitrile as a wash-out step, (V) on-line connection of microdialysis and HPLC/derivatization. In all cases, automated precolumn derivatization with o-phthalaldehyde-2-mercaptoethanol reagent were used. Both fluorescence and electrochemical detection techniques were evaluated in terms of reproducibility, sensitivity, interference, maintenance and troubleshooting. The electrochemical detection method required a second derivatization step with 0.2 M iodoacetamide to remove excess of a thiol moiety and regular recalibrations after each six to ten injections. Under these conditions the correlation coefficients for electrochemical vs. fluorescence detectors were 0.918 for Asp and 0.988 for Glu for 65 microdialysis samples. Coefficients of variation for six analyses between calibrations were below 3% for both detectors. The limits of detection for both amino acids were about 0.4 pmol for electrochemical detection with a thiol scavenger step, 50 fmol for fluorescence detection using conventional columns and about 20–30 fmol for the microbore system. All systems are suitable for detecting basal levels of Asp and Glu in 5–10 μl microdialysis samples from a rat brain where typical concentrations lie around 1–10 pmol or more. It is concluded that a microbore setup with one isocratic pump and an autosampler optimized for injections of washing solvent between samples is the most practical and economical. The system allows analysis of minute sample volumes down to 1–2 μl.

High sensitivity determination of the remoxipride hydroquinone metabolite NCQ-344 in plasma by coupled column reversed-phase liquid chromatography and electrochemical detection.

An HPLC method for the determination of NCQ-344, a remoxipride metabolite with a hydroquinone structure, in human plasma is described. Special precautions for the sampling were necessary as the compound rapidly decomposes. An efficient clean-up of the plasma samples was necessary to make use of the inherent sensitivity of the electrochemical detector. This was accomplished by a fast and simple liquid-liquid extraction at pH 7.05 combined with further cleaning on-line by using a short cyanopropyl column as the first column in a column switching system. A heart-cut from the cyanopropyl column containing the NCQ-344 fraction was then injected onto the analytical octadecyl silica column and NCQ-344 was detected at an oxidation potential of 0.70 V. The absolute recovery was > 95% and concentrations down to 0.10 nM could be determined with acceptable precision. The NCQ-344 levels in a limited number of samples from patients given remoxipride were found to be between 0.10 and 1 nM. The remoxipride concentrations in the same samples were 5,000-20,000 nM.

Effect of domain size on the performance of octadecylsilylated continuous porous silica columns in reversed-phase liquid chromatography

We prepared continuous porous silica columns having double pore structures with micrometer-size co-continuous through-pores and silica skeletons which have mesopores. The through-pore volumes and the ratio of through-pore diameters to silica skeleton diameters were kept constant, and the domain sizes were varied between 2.3 and 5.9 μm. We examined the effect of the domain size on the performance of silica rod columns in reversed-phase liquid chromatography after octadecylsilylation. The rod column having a smaller domain size showed lower plate height, though not so low as expected based on their skeleton size, and a smaller effect of linear velocity of mobile phase on the plate height for alkylbenzenes. The tendency was more pronounced for a larger molecule such as insulin. It was found that flow resistance parameters and separation impedance of the rod columns were smaller than those of conventional packed columns by a factor of 5 or more and showed little dependence on the domain size. The silica monoliths with double pore structure can provide a higher efficiency at a lower back pressure than conventional particle-packed columns.

Capillary reverse-phase high-performance liquid chromatography (HPLC) with electrospray condensation particle counting detection

Protein standards are separated on a reverse-phase liquid chromatography column with 40 μm inner diameter. They are introduced to the gas phase by micro-electrospray and detected by a condensation particle counter. The micro-electrospray needle is a new design which allows for the use of a nanoliter-per-minute makeup flow instead of the conventional microliter-per-minute flow. The observed separation efficiency is very good and the sheath micro-electrospray needle performs flawlessly while the system detection limit is comparable to on-column UV absorption. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 467–471, 1998

Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides.

A two-dimensional liquid chromatography system is described here which uses size exclusion liquid chromatography (SEC) followed by reversed phase liquid chromatography (RPLC) to separate the mixture of peptides resulting from the enzymatic digestion of a protein. A novel LC/LC interface, using two RPLC columns in parallel rather than storage loops, joins the two chromatographic dimensions. This new interface design permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC, making construction and maintenance of this system very easy. The reversed phase chromatography utilizes 1.5 microns diameter, nonporous C-18 modified silica particles, which produce fast and efficient analyses. Following the high-resolution two-dimensional chromatographic separation, an electrospray mass spectrometer detects the peptide fragments. The mass spectrometer scans a 2000 m/z range to identify the analytes from their molecular weights. The analyses of tryptic digests of ovalbumin and serum albumin are each described.

Comprehensive on-line RPLC-CZE-MS of peptides

Peptide standards and tryptic digests of ribonuclease B are separated by comprehensive two-dimensional reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) and detected on-line by electrospray mass spectrometry. The RPLC column is coupled to the CZE column by a transverse flow gating interface. A new rugged microelectrospray needle is described that combines high ionization efficiency, low flow rates, and a sheath flow. The result is a system combining the separation capabilities of both RPLC and CZE with on-line mass spectrometric detection, all in about 15 min.

Preparation of continuous beds for electrochromatography and reversed-phase liquid chromatography of low-molecular-mass compounds

High-performance capillary columns for electrochromatography and reversed-phase liquid chromatography are often prepared from silica beads. However, the synthesis of the beads involves many expensive and complicated steps and the packing of reproducible, stable and uniform beds requires great experience. In this paper we propose a simpler and more cost-effective approach, not based on preformed beads but on continuous polymer beds synthesized in situ in the chromatographic tube. The continuous bed columns were prepared by polymerizing two different monomer solutions in two steps directly in the capillary, followed by effective derivatization with hydrophobic ligands (C 18). Electroendosmosis was created by embedding a long, charged polymer (dextran sulfate). The continuous beds, as synthesized previously for the separation of proteins by reversed-phase chromatography, showed low resolution for the separation of low-molecular-mass compounds owing to a relatively low ligand density. This disadvantage was overcome by a new method designed to increase the ligand density and, in addition, to achieve a more rigid gel matrix. For rapid removal of the Joule-heat generated in electrochromatography, it is mandatory to employ very narrow columns. Continuous polymer bed columns with an inner diameter of 25 μm or less are easy to prepare. The potential of this type of capillary column is demonstrated by the separation of non-charged polycyclic aromatic hydrocarbons at an efficiency of 120 000 plates/m for a retained solute using electro-driven buffer flow. The performance is thus comparable to that of electrochromatography columns of 40–50 μm I.D. packed with 3–5 μm silica beads. In accordance with theoretical considerations only a somewhat higher plate height was obtained when the same continuous bed column was eluted with a pressure-driven flow. The columns have the distinct advantage over conventional capillary columns packed with beads that air bubbles seldom form and spoil a run, partly due to the absence of a supporting frit.

Effect of skeleton size on the performance of octadecylsilylated continuous porous silica columns in reversed-phase liquid chromatography

We prepared continuous porous silica rods that had silica skeletons with sizes of 1.0–1.7 μm and through-pores of 1.5–1.8 μm, and evaluated their performance as a column in reversed-phase liquid chromatography. The mesoporous silica monoliths (mesopore size: 14 or 24 nm) were derivatized to C 18 phase by on-column reaction with octadecyldimethyl-N,N-diethylaminosilane. The C 18 silica rods gave minimum plate heights of 10–15 μm for aromatic hydrocarbons in 80% methanol and 20–30 μm for insulin in acetonitrile-water mixtures in the presence of trifluoroacetic acid. The performance of the silica rods at a high flow-rate was much better than that of conventional columns packed with 5 μm C 18 silica particles with pores of 12 or 30 nm, especially for high-molecular-mass species. Silica rods with the smaller sized silica skeletons resulted in Van Deemter plots showing a minimum plate height at the higher linear velocity of the mobile phase and a smaller dependence of plate height on the linear velocity. Separation impedance of less than 1000 was achieved with the continuous silica columns. The higher performance and lower pressure drop of silica rods at high flow-rates compared with particle-packed columns is provided by the small silica skeletons and large through-pores.

Liquid Chromatographic Determination of Fish Decomposition Indexes from Analyses of Plaice, Whiting, and Herring

Abstract A simple and rapid liquid chromatographic (LC) method was used for simultaneous determination of ammonia, cadaverine, histamine, methylamine, putrescine, spermidine, spermine, tryptamine, and tyramine. These amines were separated as dansyl chloride derivatives in 22 min on a reversed-phase LC column using a binary elution gradient (water and acetonitrile). Three species of fish (plaice, whiting, and herring) stored in ice at 0C were analyzed to determine new decomposition indexes (ratios), which are less species-dependent than measurements of total volatile basic nitrogen (TVBN), trimethylamine (TMA), and P % (TMA/TVBN).