Comparison and characterization of reversed phases

Summary A pragmatic test procedure for comparison and evaluation of reversed phases is described. The differences in retention between RP8 and RP18 can be compensated for by adjustment of the eluent composition. A 10% increase in the water content doubles the k values, as does the exchange of an RP8 with an RP18 column. It is possible to differentiate between the two columns with the pair ethylbenzoate and toluene. Under stand-ard conditions (methanol-water, 55–45, v/v; 49–51, w/w) with RP8 ethylbenzoate is always eluted together or after toluene, whereas with RP18 it is always eluted in front of toluene.With the same eluent composition the suitability of stationary phases for the separation of basic solutes can be evaluated. With good phases—symmetrical peaks for basic solutes—aniline is always eluted before phenol and the peak asymmetry relationship of the aniline and phenol peak is less than 1.3. With such good stationary phases the retention of bases is independent of sample size if the linear capacity of 0.1 mg sample/g stationary phase is not exceeded. The test can also be used to study column stability towards hydrolysis.     

Trennung von Zuckern an chemisch modifizierten Kieselgelen

Zusammenfassung Die Zuckertrennung mit chemisch gebundenen Aminophasen beruht auf einer Verteilung der Proben zwischen einer stehenden wäßrigen Phase und dem bewegten Eluenten, einem Acetonitril-Wasser-Gemisch. Die Aufspaltung des Eluenten in zwei Phasen ist auf die gebundenen Aminogruppen zurückzuführen, die sonst nicht an der Trennung beteiligt sind. Mit der Aminophase erhält man so eine Belegung von 0,15 g/g stationärer Phase. Die Belegung steigt mit zunehmendem Stickstoffgehalt der gebundenen Phase. Bei der Triamiphase erhält man eine Belegung von 0,25 g/g. Die k'-Werte der Proben steigen — bei gleicher Eluentenzusammensetzung — mit der Wasserbelegung an. Daher kann zur Reduzierung der Analysenzeit, bei identischer Auflösung, der Wassergehalt im Eluenten bis auf das Doppelte-verglichen mit der Aminphase-erhöht werden. Dies erleichtert die Zuckeranalyse, da die Löslichkeit der Proben im Eluenten steigt.

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Separation of sugars on chemically modified silica gel

Summary The speration of sugars with chemically bonded amino groups depends on the partition of the solutes between a stagnant aqueous liquid phase and a moving acetonitrile-water mixture. The bonded amino groups cause demixing of the aqueous acetonitrile eluent, but do not otherwise participate in the separation process. With amino groups a liquid loading of 0.15 g/g is achieved. The loading increases with increasing nitrogen content of the bonded phase. With triamino phases liquid loadings of 0.25 g/g are obtained. At constant eluent composition the k' values increase with increasing water loading. With the triamino phase the water concentration in the eluent can be doubled compared to that with the standard amino phase to achieve identical resolution and analysistime. This facilitates the sugar analysis owing to the increased solubility of the solute in the eluent.

Teil der Dissertation von Peter Orth, Saarbrücken 1979.     

Preparation of open tubular molecule imprinted polymer capillary columns with various templates by a generalized procedure and their chiral and non‐chiral separation performance in CEC

A generalized preparation procedure of open tubular (OT) molecule imprinted polymer (MIP) columns is proposed for a number of templates with acidic functionality such as profen drugs and others. The template (S‐enantiomer) was mixed with methacrylic acid, ethylene glycol dimethacrylate and 4‐styrenesulfonic acid, dissolved in a porogen mixture of ACN/2‐propanol (9/1), and incubated in a pretreated and silanized fused silica capillary by the thermal non‐covalent polymerization procedure. The whole preparation procedure was exactly the same for all the MIP capillaries except for the selection of template. Nevertheless, the morphologies of the MIP layers were markedly variant depending upon the choice of template. The separation efficiency of each OT‐MIP column for chiral separation of R‐ and S‐enantiomers was examined and tuned to obtain the best separation efficiency by changing the chromatographic parameters such as eluent composition and pH. Different optimized conditions were obtained for different OT‐MIP columns. Nevertheless, a unified eluent could be used to obtain still quite satisfactory results. Non‐chiral separation of the MIP columns were also examined in the unified eluent with two sets of test mixtures, that is, a mixture of alkylbenzenes and a mixture of small polar solutes. The chiral and non‐chiral separation of this study resulted in very good separation efficiencies. This work is the very first study for the generalization of preparation of OT‐MIP columns for a number of templates.     

Ternary eluent compositions in supercritical fluid chromatography improved fingerprinting of therapeutic peptides

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide's fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2-PIC columns (each 100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed.     

Influence of intermolecular interaction with the eluent on the retention and selectivity in liquid chromatography

Summary The influence of substance — eluent (water-isopropanol mixture) intermolecular interaction on the retention and selectivity of separation in liquid chromatography on silica gel with silanized surface has been investigated for benzene and toluene derivatives. The interaction greatly depends on the nature of the polar functional groups, their position in the benzene ring and the existence of intramolecular interaction.     

Comparison of the retention behaviour of phenol derivatives on porous graphitized and octadecylsilica columns

Summary The retention of 22 ring-substituted phenol derivatives on porous graphitized carbon (PGC) (eluents: acetonitrile — water and methanol — water mixtures) and on octadecylsilica (ODS) (eluents: methanol — 0.025 M KH₂PO₄ mixtures) was determined, and the relationship between retention and physicochemical parameters were evaluated by principal component analysis followed by two-dimensional nonlinear mapping and by cluster analysis as well as by canonical correlation analysis. Calculations proved that marked differences can be detected between the retention characteristics of PGC and ODS columns, and the electronic parameters of phenol derivatives have the highest impact on their retention. The comparison of various multivariate mathematical-statistical methods indicated that principal component analysis followed by two dimensional non-linear mapping is the most appropriate method for the evaluation of large data matrices in RP-HPLC.     

Comparison of the relationships between the retention of dansyl derivatives of aliphatic amines and eluent composition for normal-and reversed-phase liquid-solid TLC systems

Summary Dansyl derivatives of four long-chain primary aliphatic amines and five secondary aliphatic amines were chromatographed in two adsorption systems: heptane+diisopropyl ether — silica and water+methanol — octadecyl cilica. In the normal-phase system R_M (log k) values were approximately linearly dependent on the log concentration of diisopropyl ether in the eluent; the separation selectivity of dansyl C₈–C₁₂ primary amines was poor. For the reversed-phase system the derivatives of both primary and secondary amines were well separated, the log k values being linearly dependent on per cent concentration of methanol in the eluent.     

Two-dimensional ion chromatography using tandem ion-exchange columns with gradient-pulse column switching

A two-dimensional ion chromatography (2D-IC) approach has been developed which provides greater resolution of complex samples than is possible currently using a single column. Two columns containing different stationary phases are connected via a tee-piece, which enables an additional eluent flow and independent control of eluent concentration on each column. The resultant mixed eluent flow at the tee-piece can be varied to produce a different eluent concentration on the second column. This allows analytes strongly retained on the first column to be separated rapidly on the second column, whilst maintaining a highly efficient, well resolved separation of analytes retained weakly on the first column. A group of 18 inorganic anions has been separated to demonstrate the utility of this approach and the proposed 2D-IC method provided separation of this mixture with resolution of all analytes greater than 1.3. Careful optimisation of the eluent profiles on both columns resulted in run times of less than 28 min, including re-equilibration. Separations were performed using isocratic or gradient elution on the first column, with an isocratic separation being used on the second column. Switching of the analytes onto the second column was performed using a gradient pulse of concentrated eluent to quickly elute strongly retained analytes from the first column onto the second column. The separations were highly repeatable (RSD of 0.01–0.12% for retention times and 0.08–2.9% for peak areas) and efficient (typically 8000–260,000 plates). Detection limits were 3–80 ppb.     

Comparative Chiral Separation of Thalidomide Class of Drugs Using Polysaccharide-Type Stationary Phases with Emphasis on Elution Order and Hysteresis in Polar Organic Mode

The enantioseparation of four phthalimide derivatives (thalidomide, pomalidomide, lenalidomide and apremilast) was investigated on five different polysaccharide-type stationary phases (Chiralpak AD, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) using neat methanol (MeOH), ethanol (EtOH), 1-propanol (PROP), 2-propanol (IPA) and acetonitrile (ACN) as polar organic mobile phases and also in combination. Along with the separation capacity of the applied systems, our study also focuses on the elution sequences, the effect of mobile phase mixtures and the hysteresis of retention and selectivity. Although on several cases extremely high resolutions (R_s > 10) were observed for certain compounds, among the tested conditions only Chiralcel OJ-H column with MeOH was successful for baseline-separation of all investigated drugs. Chiral selector- and mobile-phase-dependent reversals of elution order were observed. Reversal of elution order and hysteresis of retention and enantioselectivity were further investigated using different eluent mixtures on Chiralpak AD, Chiralcel OD and Lux Amylose-2 column. In an IPA/MeOH mixture, enantiomer elution-order reversal was observed depending on the eluent composition. Furthermore, in eluent mixtures, enantioselectivity depends on the direction from which the composition of the eluent is approached, regardless of the eluent pair used on amylose-based columns. Using a mixture of polar alcohols not only the selectivities but the enantiomer elution order can also be fine-tuned on Chiralpak AD column, which opens up the possibility of a new type of chiral screening strategy.     

Determination of trace inorganic anions in seawater samples by ion chromatography using silica columns modified with cetyltrimethylammonium ion

Conventional silica columns dynamically modified with cetyltrimethylammonium ions were evaluated for the determination of UV-absorbing bromide, nitrate, and nitrite in seawater samples. Cetyltrimethylammonium, which is a quaternary ammonium ion, was dynamically introduced onto silica surfaces. The first layer of the modifier was introduced by electrostatic interaction, whereas the second layer was introduced by hydrophobic interaction. The latter layer worked as the anion-exchange sites. The modified conventional silica columns could be used for separation of inorganic anions. Separation of authentic mixture of five anions was achieved within 17 min. The addition of 0.1 mM cetyltrimethylammonium ion to the eluent improved the repeatability of the retention time. Seawater samples could be directly injected onto the prepared conventional silica columns, and bromide, nitrate, and nitrite levels were determined to be 69, 0.13, and 0.016 ppm, respectively.     

Reduced sample recovery in liquid chromatography at critical adsorption point of high molar mass polystyrene

Low sample recovery may represent an important drawback in liquid chromatography at the critical adsorption point (LC-CAP) if the critical eluent is not carefully fitted to the system. So far, this problem was often overlooked and only few experimental examples can be found in literature. We showed that in the case of polystyrene (PS) in a tetrahydrofuran (THF)/n-hexane critical mixed eluent, PS with molar masses higher than 100 kg mol⁻¹ were not eluted from a tandem of two columns packed by bare silica gels with 30 nm and 100 nm pore size, respectively. The polymer trapped within the columns was well recovered after injection of a small volume of pure THF as demonstrated using 2D chromatography. We studied PS conformations by means of small angle neutron scattering and found that the THF/n-hexane critical eluent is in fact a theta solvent for PS. By replacing it by a CH₂Cl₂/n-hexane critical mixture, which is a good solvent for PS, the limits of reduced sample recovery was displaced towards far higher molar masses. Thus, thermodynamic quality of eluent - theta or good solvent - plays an important role on the phenomenon of sample recovery.     

Rapid determination of bromide in seawater samples by capillary ion chromatography using monolithic silica columns modified with cetyltrimethylammonium ion

Monolithic silica capillary columns dynamically modified with quaternary ammonium ions were evaluated for the determination of bromide in seawater samples. A quaternary ammonium ion such as cetyltrimethylammonium ion was dynamically introduced onto monolithic silica surfaces. The first layer of the modifier was introduced by electrostatic interaction, whereas the second layer was introduced by hydrophobic interaction. The latter layer worked as the anion-exchange sites. The modified monolithic silica capillary columns could be used for rapid separation of inorganic anions. Separation of authentic mixture of five anions was achieved within a few minutes. The addition of small amount of the modifier in the eluent improved the repeatability of the retention time. Seawater samples could be directly injected onto the prepared capillary columns, and bromide could be determined to be 63 mg/L.     

Tunable separation of anions and cations by column switching in ion chromatography

A convenient ion chromatography method has been proposed for the routine and simple determination of anions (Cl⁻, SO₄²⁻ and NO₃⁻) and/or cations (Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺) using a single pump, a single eluent and a single detector. The present system used cation-exchange and anion-exchange columns connected in series via two 6-port switching valves or a single 10-port valve. The connection order of the ion-exchange columns could be varied by switching the valve(s). The present system therefore allowed the separation of either cations or anions in a single chromatographic run. While one ion-exchange column is being operated, the other ion-exchange column is being conditioned, i.e., the columns are always ready for analysis at any time. When 2.4 mM 5-sulfosalicylic acid was used as the eluent, the three anions and the five cations could be separated on the anion-exchange column and cation-exchange column, respectively. In order to obtain the separations of the target ions, the injection valve was placed between the two columns. Complete separations of the above anions or cations were demonstrated within 10 min each. The detection limits at S/N = 3 were 19-50 ppb (μg/l) for cations and 10-14 ppb for anions. The relative standard deviations of the analyte ions were less than 1.1, 2.9 and 2.8% for retention time, peak area and peak height, respectively. This proposed technique was applied to the determination of common anions and cations in river water samples.     

Peak parking technique for the simultaneous determination of anions and cations

An ion chromatography method is described for the simultaneous determination of anions (Cl^–, NO₃^–, and SO₄^2–) and cations (Na⁺, NH₄⁺, K⁺, Mg²⁺, and Ca²⁺) using a single pump, a single eluent and a single detector. An anion-exchange column modified with chondroitin sulfate C facilitated the elution of the above three anions using 5 mM tartaric acid as the eluent in isocratic mode, whereas the same eluent facilitated the separation of the above five cations on a commercially-available cation-exchange column. The separation columns were connected in series via two six-port switching valves, so the required cation-exchange or anion-exchange separation could be carried out by selecting the appropriate positions for the switching valves. The separations were completed in 30 min.     

The static electric permittivity of solutions of organic molecules in dipolar solvents

The permittivity of solutions of pyrazine and of 1,4-diazabicyclo-[2,2,2]-octane (TED) in various dipolar solvents has been measured at 10 MHz by means of a sensitive bridge method. The solvents used were water, methanol, formamide, N-methylformamide, N,N-dimethylformamide, and dimethylsulfoxide. The results of the measurements are compared with those of theoretical mixture formulas. It is shown that — depending on structural properties — the permittivity attributed to the solvation shells around the organic solute molecules is changed with respect to the pure solvent value.     

Quantitative determination of the alkaloids ofAnabasis aphylla by thin-layer chromatography

Summary A chromatographic method has been developed for the quantitative determination of the alkaloids ofAnabasis aphylla — anabasine, aphylline, lupinine, aphyllidine, and anabasamine — and also pachycarpine in the mixture of reduction products of the high-boiling fraction of alkaloids, in a thin layer of alumina.V. I. Lenin Tashkent State University. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 772–776, November–December, 1976.     

Separation of potential flavoring compounds by high-performance liquid chromatography

High-performance liquid chromatography separated successively and quantitatively the food flavoring agents pyrimidines, purines and nucleosides, followed by nucleotides, then by polyphenols and finally by pyrazines with a reversed-phase octadecylsilica (μBondapak C₁₈) column and various proportions of methanol, water, acetic acid and tetrabutylammonium phosphate (PIC A). The polar solvent (solvent A) was water—acetic acid—PIC A (97.5:1.5:1.0) and the relatively non-polar solvent (solvent B) was methanol—acetic acid—PIC A (97.5:1.5:1.0). Purines, pyrimidines, and nucleosides were eluted with solvent A. Nucleotides were eluted with a mixture of solvents A and B (9:1). Polyphenols were separated with a gradient starting at 10% solvent B and finishing at 25% solvent B, and finally the pyrazines were removed successively from the column with a gradient starting at 25% solvent B and finishing at 45% solvent B. The resolution and reproducibility were excellent for more than 50 compounds. By this method beverages could be analyzed directly, without solvent extraction, for flavoring compounds.     

Characterization and kinetic performance of 2.1 × 100 mm production columns packed with new 1.6 μm superficially porous particles

The overall kinetic performance of three production columns (2.1 mm × 100 mm format) packed with 1.6 μm superficially porous CORTECS‐C₁₈+ particles was assessed on a low‐dispersive I‐class ACQUITY instrument. The values of their minimum intrinsic reduced plate heights (h_min = 1.42, 1.57, and 1.75) were measured at room temperature (295 K) for a small molecule (naphthalene) with an acetonitrile/water eluent mixture (75:25, v/v). These narrow‐bore columns provide an average intrinsic efficiency of 395 000 plates per meter. The gradient separation of 14 small molecules shows that these columns have a peak capacity about 25% larger than similar ones packed with fully porous BEH‐C₁₈ particles (1.7 μm) or shorter (50 mm) columns packed with smaller core–shell particles (1.3 μm) operated under very high pressure (>1000 bar) for steep gradient elution (analysis time 80 s). In contrast, because their permeabilities are lower than those of columns packed with larger core–shell particles, their peak capacities are 25% smaller than those of narrow‐bore columns packed with standard 2.7 μm core–shell particles.     

Study of biologically relevant physical-chemical properties of bile salts by reverse-phase liquid chromatography

Summary The study of chromatographic behaviour of bile salts with reverse-phase HPLC columns (C18 and C8) and buffered water — methanol eluent systems indicated that the octanol-water partition coefficient and the micellar cholesterol-solubilizing capacity are linearly correlated with the chromatographic mobilities if eluents with pH>7 and 0.154 M saline concentration are used (correlation coefficients 0.98 and 0.86 respectively). The critical micellar concentration was poorly correlated with the chromatographic mobility under the various conditions tested. The pKa values of bile salts were determined by the changes of mobility with the pH of eluent. Values in the range 4.92–5.06 and 4.27–4.47 were found respectively for unconjugated and glycineconjugated bile salt, in agreement with previous estimates. Values in the range 0.74–1.02 represent the first direct evaluation for taurine-conjugates.     

Determination of ultra trace amounts of cobalt and nickel in water samples by inductively coupled plasma-optical emission spectrometry after preconcentration on modified C18-silica extraction disks

A highly sensitive and accurate method for preconcentration and determination of ultra trace amounts of cobalt and nickel ions in water samples is proposed. The preconcentration is achieved using C₁₈-silica extraction disks modified with 5-(6-methoxy-2-benzothiazoleazo)-8-aminoquinoline (MBTAQ). The retained ions on the prepared solid phase was eluted with 10 ml of 0.01 M nitric acid and measured by inductively coupled plasma-optical emission spectrometry (ICP-OES). The influence of the type and amount of eluent used, pH, sample and eluent flow rates, amount of MBTAQ and the effect of other ions on extraction efficiency were investigated. The limits of detection of the method were 0.08 and 0.06 μg l^− 1 for cobalt and nickel, respectively, and provide an enrichment factor of 100. The results obtained on 10 successive extractions and elution cycles revealed relative standard deviations of 1.5 and 1.0% for cobalt and nickel, respectively. The proposed method has been applied to the determination of ultra trace amounts of cobalt and nickel ions in natural and synthetic water samples with satisfactory results.