Enhancement of sensitivity in capillary supercritical fluid chromatography through optimization of injection and detection techniques

The reproducibility of peak areas as a function of the technique used for sample injection was investigated in capillary supercritical fluid chromatography (SFC). An injection technique has been developed to increase the volume of sample introduced into the capillary column. Using a modified time-split injection technique, long injection duration times were successfully applied to achieve lower detection limits. Analytes were effectively focused at the head of the analytical column using a unique pressure trap program. Because this on-column focusing was performed only by pressure and temperature programming, no instrumental modifications were necessary. Up to 1.0 μL of sample solution was injected onto 50 μm i.d. columns using this technique, with no observable peak splitting. Dual detection using ultraviolet (UV) absorption and flame ionization detection (FID) was performed in series, thereby avoiding the necessity of splitting the column effluent. For the compounds investigated (five nitroaromatics and one phthalate ester), the absolute sensitivity of the UV detector was significantly greater than that of the FID.     

Large volume injection for preparative supercritical fluid chromatography

Summary A large volume injection system for preparative supercritical fluid chromatography is described. The method which is based on the solvent venting technique coupled with dilution of the sample solution consists of three steps. The first step is continuous dilution of the sample solution with liquid carbon dioxide at a controlled flow rate. The second step is solvent removal and solute trapping in a packed trap column. Combination of these two steps results in efficient solvent removal and the volume of sample which can be injected in a single injection becomes virtually unlimited. The third step is transfer and re-concentration of the solutes from the trap column on to the separation column with the pressures of both columns controlled independently; the final step is the separation. With this method, mass overloading behavior has been investigated and preparative separations performed.     

Direct injection of large sample volumes into capillary columns with packed column injector

A direct injection method for large volume samples which avoids severe tailing of the solvent peak has been developed using a packed column injector (up to 100 μl) leading into an ordinary capillary column (0.3 mm i.d.). Modifications are made to the cooler zones of the inlet port and on the carrier gas flow control system. This injection technique is based on the effective use of phase soaking and cold trapping using a retention gap. The large volume of solvent vapor is rapidly purged out of the injector with a higher flow of carrier gas while the solutes trapped at the head of the column are subsequently analyzed with another optimum flow rate. The proposed carrier gas flow regulation system is also compared with conventional split/splitless injection methods.     

Determination of cholesterol in fat and muscle of pig by HPLC and capillary gas chromatography with solvent venting injection

Two methods for determination of cholesterol in fat and muscle of pig were evaluated: extraction with chloroform:methanol (2:1, v/v) followed by saponification (method 1) and direct saponification (method 2). HPLC and GC were used to determine cholesterol concentrations. GC analysis was performed with a capillary column of 100 μm using a PTV injector in the modes of cold split and solvent venting. Cholesterol was analyzed without derivatization. Both methods of extraction did not present significant differences (p > 0.01). Sample analysis by GC with solvent venting injection and HPLC showed the lowest % r.s.d. but GC in the cold split mode allowed to obtain a shorter analysis time. Cholesterol concentrations obtained by HPLC were not statistically different from the results obtained by GC with solvent venting injection and were slightly lower than those previously reported. Cholesterol concentrations in fat and muscle tissues respectively ranged from 52 to 77 mg/100 g and from 55 to 65 mg/100 g.     

Analytical characteristics of combined gas chromatographic systems: Packed precolumn-open tubular column

The peculiarities of the flow splitting technique for sample injection into a capillary column have been discussed. A comparative investigation of reproducibility of the analytical results obtained for two gas chromatographic systems using flow splitting [1) sample injector-packed predolumn-splitter-open tubular capillary column and (2) sample injector-splitter-open tubular capillary column] has been carried out. It was experimentally shown that the first system ensures considerably better reproducibility for the results concerning the composition of the analyzed mixtures. Other advantages of the first system are also discussed.     

Transfer-volume effects in two-dimensional chromatography: adsorption-phenomena in second-dimension size-exclusion chromatography

Gradient-elution LC × LC is a valuable technique for the characterization of complex biological samples as well as for synthetic polymers. Breakthrough and viscous fingering may yield misleading information on the sample characteristics or deteriorate separation. In LC × SEC another phenomenon may jeopardize the separation. If the analytes adsorb on the SEC column under the injection-plug conditions, peak splitting may occur. In LC × LC the effluent from the first column is the sample solvent for the analytes injected into the second dimension. If a gradient-elution LC × SEC setup is used (i.e. if reversed-phase gradient-elution LC is coupled to organic SEC and if normal-phase gradient-elution LC is coupled to SEC with a polar solvent), the percentage of weak solvent may be significant, especially at short analysis times. In this case adsorption in the first-dimension-effluent zone on the second-dimension SEC column can become an issue and two peaks--the first eluting in size-exclusion mode and the second undergoing adsorption--can be obtained. The work presented in this paper documents peak splitting in LC × SEC of polymers. The adsorption of the polymer on the size-exclusion column was proven in one-dimensional isocratic runs. The observed effects were modeled and visualized through simulation. Studies on the influence of the transfer volume were carried out. Keeping the transfer volume as small as possible helped to minimize peak splitting due to adsorption.     

Rapid gas chromatographic analysis of less abundant compounds in distilled spirits by direct injection with ethanol–water venting and mass spectrometric data deconvolution

The principal trace secondary compounds common to fermentation-derived distilled spirits can be rapidly quantified by directly injecting 5 μL of spirit without sample preparation to a narrow-bore 0.15 mm internal diameter capillary column. The ethanol–water is removed in an initial solvent venting step using a programmed temperature vapourization injector, followed by splitless transfer of the target analytes to the column. The larger injection facilitates trace analysis and ethanol–water removal extends column lifetime. Problems of coelution between analytes or with sample matrix were surmounted by using mass spectral deconvolution software for quantification. All operations in the analysis from injection with solvent venting to data reduction are fully automated for unattended sequential sample analysis. The synergy of the various contributory steps combines to offer an effective novel solution for this analysis. Applications include quantification of low ppm amounts of acids and esters and sub-ppm profiling of trace compounds from both the raw material malt and the ageing in wood barrels.     

The use of precolumns for solvent focusing in capillary column gas chromatography

In trace analysis by capillary GC it is often desirable to use larger than normal injection volumes to obtain sufficient sensitivity. This, however, results in a wider solvent peak and tailing, and may reduce column efficiency. This paper describes the use of a short length of a capillary precolumn coated with a stationary phase of polarity similar to that of the sample solvent and a film thickness greater than that of the analytical column; provided the right combination of polarities of injection solvent and liquid phases are used, the precolumn focuses the solvent band, thereby enabling the maximum injection volume to be increased without measurably reducing efficiency. Typical precolumn dimensions are 1 m length, 0.32 mm i.d., and 0.5 μm stationary phase film thickness. Using a precolumn increases the maximum injection volume up to 8 or 10 μl, or three times that appropriate for a conventional analytical column, with little or no loss in efficiency.     

An Insight into Peak‐Splitting Phenomenon in On‐Column Concentration‐Micellar Electrokinetic Capillary Chromatography for Aqueous Sample Solution

Abstract

A detailed study was carried out to investigate the origin of the peak‐splitting phenomena in on‐column concentration‐micellar electrokinetic capillary chromatography for aqueous sample solution. The system studied was a basic phosphate and borax mixed buffer with sodium dodecyl sulfate (SDS) as micellar phase. Phenol, benzyl alcohol, phenyl ethanol, salicylic acid, and p‐hydroxy benzyl acid were selected as the analytes. Several factors that affect peak splitting were investigated. The injection time, SDS micellar concentration, hydrophobicity of the analytes, and analytes concentration were the most important factors. A hypothesis was proposed to explain the peak‐splitting phenomena. Several means to avoid peak‐splitting phenomena were proposed, such as controlling sample injection time and hydrophobicity of the analyte, decreasing SDS concentration and increasing sample concentration. However, the most practical method for avoiding peak splitting was to control the sample injection time.     

Analysis of volatile fatty acids in biological specimens by capillary gas chromatography

A method was developed to analyze and quantitate volatile fatty acids such as acetic, propionic, butyric, iso-butyric, valeric, and iso-valeric acid from samples of biological origin. A capillary column system including an automatic on-column injection device as well as a precolumn of larger internal diameter than the analytical column was elaborated for this purpose. In order to obtain well resolved and correctly quantifiable chromatographic peaks it turned out to be essential to work under acidic/aqueous conditions. To achieve a better sample transfer into the chromatographic system an organic solvent had to be used together with the aqueous milieu, thus improving wetting properties of the liquid sample plug introduced into the column. Cold on-column injection was applied in order to avoid discrimination of the various acids due to sample splitting and the automatic technique was chosen in view of the large number of samples from biological extractions which had to be analyzed.     

On-line liquid chromatography – gas chromatography for automated clean-up and analysis of polycyclic aromatic hydrocarbons

A fully automated system, comprising a liquid chromatograph (LC) coupled on-line to a gas chromatograph (GC) by means of a loop interface, has been constructed for clean-up and analysis of polycyclic aromatic hydrocarbons (PAH). An autosampler was utilized for sample injection into the LC. By the use of a back-flush technique in conjunction with an ordinary analytical aminopropylsilica column, PAH could be isolated by LC: a concurrent solvent evaporation injection technique was then used for on-line transfer of the PAH fraction to the GC, where the PAH analysis was completed. Compared with ordinary off-line LC clean-up followed by GC analysis, the sensitivity has been increased by a factor of 50–100, yielding a detection limit for individual PAH of a few nanograms per sample when using flame ionization detection. Further, irreproducible losses of low molecular weight PAH as a result of solvent evaporation steps in off-line clean-up procedures have been eliminated. Reproducibility of retention times and relative peak areas is high, facilitating automatic peak identification and calculation of concentrations, and the system can thus be used for automatic sample evaluation. The total time required for clean-up and analysis is only 1.5 hours, and the demand on personnel time for the analysis of PAH has been drastically reduced. The technique has been demonstrated with samples of urban air and of used automobile engine lubricating oil.     

Cold on-column – solvent split injection with a three-way press-fit device

In a previous paper we described the possibilities of cold on-column – sample split injection achieved by means of an inexpensive and simple three way press-fit device [1]. The same arrangement is proposed here for cold on-column – solvent split injection in which specific elimination of the solvent, without loss of any other sample components, is achieved by opening the splitting tube (or better, in this case, the early solvent vapor exit) during solvent elution, and then closing it during elution of the sample's other components. Discrimination between solvent and other sample components is achieved by means of a retention gap, a retaining precolumn, and an early vapor exit. The technique enables both selective enrichment of a sample, in order to record satisfactory mass and infrared spectra of minor components, and injection of large volumes (up to 100 μl) of dilute solutions which cannot be concentrated because of component volatility. Details of the assembly and tuning the system are given, together with some examples.     

Practical aspects of splitless injection of semivolatile compounds in fast gas chromatography

Possibilities and practical aspects of implementation of splitless injection of larger volumes for fast GC purposes utilizing narrow-bore column, hydrogen as carrier gas, fast temperature programming under programmed flow conditions and commercial instrumentation were searched. As a model sample semivolatile compounds of a broad range of volatility and polarity (7 n-alkanes and 19 pesticides) were chosen. Peak shapes, peak broadening and peak areas and its repeatability were evaluated under various experimental set-ups (liner/injection technique combinations). Various factors, such as liner design, injection technique, retention gap length, compound volatility and polarity, the solvent used, initial oven temperature influenced compound focusation and/or maximal injection volume. Combination of analytical column (CP-Sil 13 CB 25 m long, 0.15 mm i.d., film thickness 0.4 microm) with normal-bore retention gap (1 m long, 0.32 mm i.d.) allowed maximal injection volume 8 microl for 4 mm i.d. liner used without any peak distortion when solvent recondensation in the retention gap was employed.     

A new split-flow injector for preparative liquid chromatography columns. Annular injection system

The packing of large-diameter columns for liquid chromatography is still difficult and numerous publications have reported results from tests which prove the packing is heterogeneous. The slurry is more compact in the wall region and this reduces the flow of the mobile phase, leading to distortion of the sample zone in the column and generation of peak tailing. A new type of injection system for the head of the column has been developed which divides the flow of the solvent from the pump into two parts. One, without sample, is directed to a crown injector, close to the wall. By adjusting the ratio of this flow to that of the bulk flow it is possible to increase the speed of the mobile phase in this part of the stationary phase and reduce distortion of the sample zone. The other part of the solvent carries the sample to the stationary phase through a distributor. The results demonstrate the benefits of this annular injection system, which include increased efficiency and improved column stability.     

Direct injection of plasma into column liquid chromatographic systems

Summary Since the late 1970’s several techniques involving the direct injection of biological fluids, like plasma and serum have been published. Most of the systems presented utilize a precolumn for the reception of the biological fluids, trace enrichment and preliminary clean-up before the analyte fraction is transferred to the separation column. The most common solid phase for the precolumn is silanized silica, but many other types have been used. One was especially designed for the purpose of direct injections of biological fluids: the Internal Surface Reversed Phase support. The main problem with direct injections is the development of column instabilities due to the denaturation and precipitation of plasma proteins on the support. It has, however, been possible to design systems which can handle the injection of 25ml total plasma volume without the need to exchange precolumn. The most promising techniques in this respect are the so called “pre-column” venting plug technique and the use of micellar mobile phases. Losses of analytes may be a problem when protein-binding extends 95%. However, remedies are available, such as the use of more hydrophobic solid phases, the addition of competitive binders and pH adjustments of injected solutions.     

Injection of large volumes of aqueous solutions in capillary supercritical fluid chromatography and sample preconcentration by multiple injections

The possibility of using water or aqueous solutions as the solvent in capillary supercritical fluid chromatography was successfully demonstrated. Large volumes (up to 1 μl) of aqueous sample solutions were injected. Sample preconcentration was performed by means of multiple injections of aqueous sample solutions. The solutes were trapped at the beginning of the column at low density (high temperature and low pressure) and eluted using a density programme. The method can be applied to trace analysis. It proved to be linear in the range examined. Flame ionization detection was used for the studies. As this technique is not sensitive to water, no solvent peak appears, which may be an advantage for certain applications. The influence of water injections on the column performance and the reproducibility of injection was investigated.     

Rapid and Simple Technique for the Quantitation of Polyamines in Biological Samples

Abstract

A rapid and simple technique has been developed to quantify putrescine, spermidine, and spermine in biological tissue. The method, based upon several published procedures, involves protein precipitation with perchloric acid followed by dansylation with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride). After extraction on a Waters C₁₈ Sep-Pak cartridge, the samples are analyzed by high pressure liquid chromotography using a step solvent change and a 3μ C₁₈ reverse phase column. The chromotographic conditions allowed complete analysis of the three polyamines within 10 min with a total run time of 13 min (sample injection and re-equilibrium of column). Standard curves were linear up to 1 μg polyamine and the coefficient of variation for the assay ranged from 4% at l μg polyamine per sample to 11% at 50 ng polyamine per sample. The assay is therefore both rapid and simple. Moreover, unlike other available methods, the present technique does not require duel pumps, ion pairing agents, solvent extraction or a gradient control system. The concentrations of putrescine, spermidine and spermine in rat lung, liver and kidney are reported.     

Solvent venting interface for capillary gas chromatography and a microwave-induced plasma

A solvent venting system for capillary gas chromatography coupled with a microwave-induced plasma detector was constructed and evaluated. The solvent vent is based on a fluid logic system within the plasma containment tube. During solvent elution, the column effluent is routed to waste and does not pass through the plasma. During analyte elution, the fluid logic system is used to direct the analytes through the plasma for detection. Because the system has no transfer line, problems associated with interface dead volume and analyte degradation reaction due to reactive sites in the transfer line are eliminated. Switching from the vent-on to the vent-off mode is rapid and does not affect plasma stability or flow through the chromatographic column. The baseline shift during switching is negligible. The solvent venting system is simple and easily constructed. Design details are presented and chromatographic results are evaluated.     

Factors influencing chromatographic data in fast gas chromatography with on‐column injection

The use of larger volume injection with on‐column injection and fast GC commercial instrumentation was evaluated with the model mixture of n‐alkanes of a broad range of volatility (C₁₀–C₂₈). The presented configuration allows introduction of 40–80‐fold larger sample volumes without any distortion of peak shapes compared to “usual” fast GC set‐ups using narrow‐bore columns. A normal‐bore retention gap (1–5 m×0.32 mm ID) was coupled to a narrow‐bore (5 m×0.1 mm ID×0.4 μm film thickness) analytical column using a standard press‐fit connector. The connection was tight and reliable, and hence suitable for hydrogen as carrier gas. The effect of pre‐column and analytical column connector, injection volume, pre‐column length, column inlet pressure, and analyte volatility on peak shape, peak broadening, and focusing are discussed. The precision of chromatographic data measurements and peak capacity under optimised temperature programmed conditions for fast separations with large volume injection were found to be very good. The presented fast GC set‐up with on‐column injection extends the applicability of the technique to trace analysis.