An investigation of microbore reverse-phase columns containing 3 μm packing materials

A procedure for packing 15 cm × 1 mm id reverse-phase microbore columns with 3 μm silicas obtained from different manufacturers is described. The speed of analysis and detection limits are compared to those obtained with a 50 cm × 1 mm id column packed with 10 μm ODS. The effect of detector time constant on the system, and flow rates on column efficiency are also examined.     

Preparation,evaluation, and comparison of wide bore (320 μm) and narrow bore (50 μm) cyanosilicone-coated capillary columns for gas chromatography

The preparation of wide bore (320 μm) and narrow bore (50 μm) fused silica capillary columns is described for immobilized cyanopropyl substituted silicones containing 60 and 88% substitution. The effect of high temperature deactivation with cyanopropylcyclosiloxanes was studied with a special test mixture. Curing was achieved with dicumyl peroxide or azo-tert-butane. The columns were evaluated and compared in terms of efficiency, activity, polarity, and temperature stability. Different coating methods were compared for the narrow bore columns. The activity of the 60% cyanopropyl columns that had been immobilized with dicumyl peroxide was significantly larger than for azo-tert-butane immobilized columns. The polarity of polar columns appeared to depend greatly on column temperature and is completely different for wide and narrow bore columns.     

Open tubular capillary column of 50 μm internal diameter with a very high separation efficiency for the separation of peptides in CEC and LC

A specially designed long open tubular capillary column (50 μm internal diameter and 112 cm effective length) was prepared by fabrication of a thin three‐component co‐polymer layer on the inner surface of silica capillary. A pretreated silica capillary was reacted with 4‐(chloromethyl)phenyl isocyanate in the presence of dibutyltin dichloride as catalyst followed by sodium diethyl dithiocarbamate. Then a thin polymer layer was made on the inner surface of capillary by reversible addition‐fragmentation transfer polymerization of styrene, N‐phenylacrylamide, and methacrylic acid. A carefully adjusted formulation of reaction mixture and elaborated procedures were adopted to secure formation of the co‐polymer layer of enhanced separation performance. The co‐polymer immobilized open tubular capillary column was used for the separation of a synthetic mixture of five peptides and excellent separation efficiency (over 1.7 million per column) was obtained in the capillary electrochromatography mode. Such excellent separation efficiencies of ca. 1 m column have not been obtained in the isocratic elution mode so far. The column was also used for separation of the peptides in the liquid chromatography mode to show very good separation efficiency (average 286 700 per column).     

Electrochromatography and micro high-performance liquid chromatography with 320 μm I.D. packed columns

Electrochromatography is a chromatographic method in which the mobile phase (liquid or supercritical fluid) is “pumped” through a stationary phase in a microbore or capillary column by electroosmosis using an electric field. The technique permits separation of charged and uncharged compounds with higher resolution and superior efficiency when compared with micro-HPLC with an identical column. It is desirable to work with packed capillary columns with wide diameter in electrochromatography in order to improve detectability and column loadability. This study shows that we have moved a step forward towards this goal in spite of problems and difficulties, due to Joule heating, frit making and column packing in using wide-diameter columns. The paper demonstrates that the pressure pump of micro-HPLC with a commercially available 320 μm I.D. column can be replaced by the electroosmotic “pump” of capillary zone electrophoresis. Experiments were carried out in a chromatographic system under both electroosmosis and pressure-driven flow with 320 and 50 μm I.D. columns packed with 3- and 5-μm ODS. The advantage of electrochromatography over conventional micro-HPLC is shown.     

High speed capillary GC on 10 m × 100 μm i.d. FSOT Columns

The chromatographic performance of 10 m × 100 μm i.d. capillary columns, coated with different stationary phases, was studied. TZ-ū plots were measured at different temperature program rates. The polarity and selectivity are strongly influenced by the temperature program rate. The analysis time depends on the capacity ratio and the selectivity factor; stationary phase selection thus is an important aspect in high speed capillary GC. A number of applications is presented and the applicability of split injection is discussed.     

Large diameter open tubular columns in gas chromatographic analysis

Large diameter open tubular columns provide the packed column chromatographer with a simple route to higher resolution gas chromatography. They can be operated in a high-flow (lower resolution) mode that permits their direct substitution for a packed column, or they can be operated in a low-flow (higher resolution) mode that maximizes separation at the cost of longer analysis times. Inlet design and column installation can influence both the chromatographic results and quantiative reliability. Make-up gas is not required in the high-flow mode, and its benefits in the low-flow mode are restricted to enhanced detector sensitivity, provided the outlet end of the column resides in the detector jet (FID). The columns seem fully compatible with all common modes of detection.     

Effects of Injected Volume and Applied Voltage on Column Efficiency in Capillary Electrochromatography with Open Tubular Columns of 10 μm i. d.

The effect of some instrumental parameters on column efficiency in open tubular capillary electrochromatography (OTCEC) has been evaluated. First, it was investigated whether band broadening due to the sample injection process is within a tolerable range when an open tubular column (OTC) of about 10 μm i. d. is used. As a result of the small injection profile factor (K² = 1.3), injected volumes must be sufficiently small (less than 10 pL) to avoid a significant efficiency loss (>5%) when hydrodynamic injection by siphoning is employed. Secondly, the kinetic performance of OTCs in a CEC system was estimated from the variation of the reduced plate height (h) with the reduced linear velocity (ν) which was controlled by the voltage applied. Reasonable agreement was obtained between the theoretical h versus ν curve and the experimental values for a group of polycyclic aromatic hydrocarbons used as test compounds. Values of 0.25 for minimum h at an optimum ν of 16 are estimated, which permit separations with around 400,000 plates per meter to be obtained in less than 5 min. Finally, the possibility of estimating the diffusion coefficients of the solutes in the mobile phase from the plot of the height of a theoretical plate versus electroosmotic flow velocity is shown.     

CGC analysis of the essential oils of citrus fruits on 100 μm i.d. columns

Narrow bore columns (100 μm) have successfully been applied in the analysis of commercial lemon oils. On these columns, high resolution is obtained in a short analysis time. Different Brazillian lemon oils are characterized qualitatively and quantitatively.     

Gas chromatographic separations on packed microcolumns using various 10 μm support particles

Packed columns using 10 μm HPLC-packing materials as supports for various liquid phases have been studied. The glass columns used were 80 mm long (o.d. 3 mm and i.d. 1.5 mm), the amount of solid support in the column being approximately 0.15 g. A. wide variety of separation characteristics were obtained by combining support material of various surface areas, chemically bonded surfaces, applied liquid phases, and various liquid phase loadings. This type of column is also characterized by a very short gas hold-up time, leading to possibilities for relatively fast separations. Other advantages are high sample loading, high sample capacity, and low operating cost.     

Large sample injection method (≥ 100 μL) for capillary GC using a commercial thermodesorption unit as injection device

A commercial thermodesorption and cold trap unit can be used as a large sample volume injection device in high resolution gas chromatography. Volumes of more than 100 μl can be injected. More than 97% of solvents, such as hexane, can be removed by a Tenax TA precolumn without any loss of compounds with volatilities comparable to naphthalene. Experimental conditions, recovery rates of polycyclic aromatic hydrocarbons, and the reproducibility of the method are described in detail. The method was successfully applied for the determination of PAH in air particulate matter extracts.     

Packed-column hydrodynamic chromatography using 1-μm non-porous silica particles

150×3 mm I.D. columns, packed with 1-μm non-porous spherical silica particles, were used to separate soluble synthetic polymers by hydrodynamic chromatography. The columns exhibited a plate height of about 1.4 μm allowing very fast and efficient separations of polymers in the molecular mass range 10³−2·10⁶ g/mol. The migration behaviour of polymers could be well described by a simple theoretical model. The applicability of packed bed HDC for the fast separation of polymers was illustrated with separations of polystyrene and poly(methyl methacrylate) mixtures.     

An optimized gas chromatographic determination of priority pollutant phenols

Priority pollutant phenols are determined by gas chromatography on an SE-30 WCOT column using hydrogen carrier and flame ionization detection with dual internal standards. Linear responses over ranges ca. 12–125 ng per component are achieved and detection limits at or below 1 ng are obtained. Carrier flow and temperature program are optimized to maintain baseline resolution while affording an analysis time of less than 15 minutes.     

Preparation and evaluation of 3 m open tubular capillary columns with a zwitterionic polymeric porous layer for liquid chromatography

A 3 m zwitterionic polymeric porous layer open tubular column (3 m × 25 μm id × 375 μm od) with a polymeric porous layer thickness of 4 μm was fabricated by the copolymerization of [2‐(methacryloyloxy)ethyl] dimethyl‐(3‐sulfopropyl) ammonium hydroxide and N,N’‐methylenebis(acrylamide). The effects of the diameter of the capillary, reaction temperature, and polymerization time on the preparation of the open tubular column were investigated. Characterized by scanning electron microscopy, the zwitterionic layer was observed to be rough and throughout the fused‐silica capillary homogenously, which increased the phase ratio. The separation of neutral, basic, and acidic compounds demonstrates the strong hydrophilicity of the poly[2‐(methacryloyloxy)ethyl] dimethyl‐(3‐sulfopropyl) ammonium hydroxide coating. In addition, the poly[2‐(methacryloyloxy) ethyl] dimethyl‐(3‐sulfopropyl) ammonium hydroxide porous layer open tubular column was applied for the analysis of flavonoids from the rootstalk of licorice, revealing the potential in separating complex samples. The relative standard deviation of retention time for run‐to‐run (n = 5), day‐to‐day (n = 3), and column‐to‐column (n = 3) of toluene, N,N‐dimethylformamide, formamide, and thiourea were below 1.2%, exhibiting good repeatability.     

Fast separation of native proteins using sub‐2 µm nonporous silica particles in a chromatographic cake

A novel method for the fast separation of native proteins was investigated using sub‐2 µm nonporous silica packing inside a chromatographic cake having a diameter much larger than its thickness. Various silica‐based particles ranging from 630 nm to 1.2 µm were synthesized and chemically modified with polyethylene glycol 600. The packing material was laterally packed into a series of chromatographic cakes containing the same diameter (10 mm) and different thicknesses, ranging from 2 to 10 mm, and tested by hydrophobic interaction chromatography. The results showed that the sub‐2 µm NPS particles in a small chromatographic cake were found to have a high efficiency at a flow rate of 10 mL/min and a backpressure of <20 MPa. The effect of the thickness of the chromatographic cake on the resolution of the proteins was also investigated and it was found that too short a column length could dramatically decrease the protein resolution; the minimum column length was also qualitatively evaluated. The presented method is expected to be useful for routine analysis of native and/or intact proteins in hospitals and as a tool for the fast screening protein drugs and optimization of experimental laboratory conditions. Copyright © 2014 John Wiley & Sons, Ltd.     

Packed columns vs. wall coated open tubular columns in quantitative analyses of polychlorinated biphenyls

This article describes and critically evaluates a complete method for the quantitation of PCB in sediment samples. The extraction and clean-up procedure is described. Packed column and capillary column results are compared. Capillary column quantitation yields lower values for total PCB loading. Preferences for capillary column quantitation are discussed and explained. Capillary quantitation is based on the combination of an external Aroclor standard and an internal decachlorobiphenyl standard for normalizing data. In light of the recent report of synthesis of all 209 PCB congeners a suggestion is made to use these as absolute standards to establish a bank of primary standard Aroclors.     

Green ultra‐fast high‐performance liquid chromatographic method using a short narrow‐bore column packed with fully porous sub‐2 μm particles for the simultaneous determination of selected pharmaceuticals as surface water and wastewater pollutants

Fast separations are very desirable in laboratories that analyze large numbers of samples per day or those needing short turn‐around times. Traditional HPLC methods using conventional stationary phases and standard column dimensions require significant amounts of organic solvents and generate large volumes of waste. With growing awareness about the environment, the development of green technologies has been receiving increasing attention. In this work, a very fast green analytical method based on LC‐UV using a short narrow bore column packed with fully porous sub‐2 μm particles has been developed for simultaneous determination of nine pharmaceuticals in wastewater and surface water. The chromatographic separation was optimized in order to achieve short analysis time and good resolution for all analytes in a single run. All analytes could be separated in 1 min with good resolution. Sample preparation was executed by solid phase extraction using Oasis HLB cartridges. The method developed was validated based on parameters such as linearity, precision, accuracy, detection, and quantification limits. The recovery ranged from 70.9 to 92.5% with SDs not higher than 5.4%, except for acetaminophen and sulphanilamide. LODs ranged from 0.6–2.5 μg/L, while the LOQs were in the range 2–8 μg/L.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Considerations of speed and efficiency in capillary gas chromatography

The analysis time for a given resolution is a complex function of stationary phase selectivity, column radius, and thickness of the stationary phase film. Variation of these parameters has a large effect not only on analysis time, but also on the column inlet pressure and other instrumental requirements. The minimum amount that can be reliably detected as well as the maximum sample capacity of a column are strongly related to the selected column dimensions.     

Simultaneous determination of quinolones in fish by liquid chromatography coupled with fluorescence detection: Comparison of sub‐2 μm particles and conventional C18 columns

A simple and effective multi‐residue analysis method is presented for the extraction and determination of eleven quinolones (pipemidic acid, enoxacin, norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin, gatifloxacin, difloxacin, oxolinic acid, nalidixic acid and flumequine) in fish tissues. In this study, multi‐residue separations on four columns packed with 5 μm or sub‐2 μm particles were simultaneously developed for the purpose of comparison. Various gradients were optimized and best resolutions were achieved on each column. A short and sub‐2 μm particle‐sized HPLC column was chosen for its advantages in analysis time and column performance. Additionally, considering the matrix effect of the complex crude fish tissue, an effective extraction protocol was also established for sample pre‐treatment procedure. Good recoveries (71–98%) were obtained from samples fortified with a mix of eleven quinolones at three levels, with satisfactory relative standard deviations and limits of detection. As a result, the sub‐2 μm HPLC column and proposed analytical procedures have been evaluated and applied to the analysis of different fish tissues. Detectable residues were observed in 8 of 30 samples, at concentrations ranging from 4.74 to 23.27 μg/kg.