Multiplexed detection: fast comprehensive sample analysis of tobacco leaf extracts using HPLC with AFT columns

Detector-based comprehensive screening analysis of complex samples of natural origin using High Performance Liquid Chromatography (HPLC) can be a complicated and time-consuming task. There are a number of ways multidetection characterization can be achieved; however, there are limitations associated with each technique. Active Flow Technology (AFT) in Parallel Segmented Flow (PSF) mode allows for multiplexed detection HPLC analysis within a single injection, whereas maintaining chromatographic performance and allowing the use of multiple destructive detectors to achieve a comprehensive yet efficient screening of a complex sample. In this study, a comprehensive characterization analysis of tobacco leaf extract was carried out through multiplexed detection using a PSF column for the detection of biomolecules by UV-Vis detection, DPPH• for reactive-oxygen species (ROS) detection, and mass spectrometry, the latter two detection methods being sample destructive.

A hybrid FIA/HPLC system incorporating monolithic column chromatography

We have combined the generation of solvent gradients using milliGAT pumps, chromatographic separations with monolithic columns and chemiluminescence detection in an instrument manifold that approaches the automation and separation efficiency of HPLC, whilst maintaining the positive attributes of flow injection analysis (FIA), such as manifold versatility, speed of analysis and portability. As preliminary demonstrations of this hybrid FIA/HPLC system, we have determined six opiate alkaloids (morphine, pseudomorphine, codeine, oripavine, ethylmorphine and thebaine) and four biogenic amines (vanilmandelic acid, serotonin, 5-hydroxyindole-3-acetic acid and homovanillic acid) in human urine, using tris(2,2′-bipyridyl)ruthenium(III) and acidic potassium permanganate chemiluminescence detection.

Studies on the behaviour of polystyrene in reversed phase chromatography

Polystyrene behaviour in reversed phase high performance liquid chromatography was influenced mainly by the solvent system, but secondary affects were observed depending on the stationary phase. A variety of reversed phase columns were investigated using mobile phase combinations of dichlorom ethane-methanol, dichloromethane-acetonitrile, ethyl acetate-methanol and ethyl acetate-acetonitrile. Several different modes of behaviour were observed depending on the polymer solubility in the solvent system. In the dichloromethane-methanol solvent system, polymer-stationary phase interactions only occurred when the molecules had pore access. Retention of excluded polystyrene depended on the kinetics of precipitation and redissolution of the polymer. Peak splitting and band broadening occurred when the kinetics were slow and molecular weight separations were limited !o oligomers and polystyrenes lower than 5-10(4) dalton. Excellent molecular weight separations of polystyrenes were obtained using gradient elution reversed phase chromatography with a dichloromethane-acetonitrile mobile phase on C18 columns. The retention was based on polymer-stationary phase interactions regardless of the column pore size. Separations were obtained on large diameter pellicular adsorbents that were almost as good as those obtained on porous adsorbents, showing that pore access was not essential for the retention of high molecular weight polystyrenes. In the best example, the separation ranged from the monomer to 10(6) dalton in a single analysis. Very little adsorption of excluded polymers was observed on C8 or phenyl columns. Polystyrene molecular weight separations to 7-10(5) dalton were obtained in an ethyl acetate-acetonitrile solvent system on C18 columns. Adsorption was responsible for retention. When an ethyl acetate-methanol solvent system was used, no molecular weight separations were obtained because of complex peak splitting. Reversed phase chromatography was compared to size exclusion chromatography for the analysis of polydisperse polystyrenes. Similar results were obtained using both methods. However, the reversed phase method was less sensitive to concentration effects and gave better resolution.

An illustration of the physical robustness of silica monolithic rod columns

A commercial silica monolithic rod column (100 × 4.6 mm) was cut into smaller sections using a saw. Each time a section was cut from the column, the performance of the remaining intact monolith was retested. No significant change in the performance of the bed was recorded following the removal of 40 mm of the column in three separate cut sections. The work illustrates that monoliths are extremely robust and that they can be remodelled to different lengths if required, or a blocked section of the column (i.e. inlet) could be removed in much the same manner as for GC columns.

Automated methods for the location of the boundaries of chromatographic peaks

Several simple techniques are presented for the identification of the boundaries of chromatographic peaks. These methods provide a significant reduction in the time needed to perform the rapid, automatic calculation of the central peak moments and to evaluate the quality of a separation while improving the accuracy of the measurements of column efficiencies. It was found that the identification of the peak boundaries as functions of the peak widths and the examination of the slope of the signal to noise versus time plot are viable alternatives to a manual determination.

Application of power functions to chromatographic data for the enhancement of signal to noise ratios and separation resolution

Chromatographic detection responses are recorded digitally. A peak is represented ideally by a Guassian distribution. Raising a Guassian distribution to the power ‘n’ increases the height of the peak to that power, but decreases the standard deviation by √n. Hence there is an increasing disparity in detection responses as the signal moves from low level noise, with a corresponding decrease in peak width. This increases the S/N ratio and increases peak to peak resolution. The ramifications of these factors are that poor resolution in complex chromatographic data can be improved, and low signal responses embedded at near noise levels can be enhanced. The application of this data treatment process is potentially very useful in 2D-HPLC where sample dilution occurs between dimension, reducing signal response, and in the application of post-reaction detection methods, where band broadening is increased by virtue of reaction coils. In this work power functions applied to chromatographic data are discussed in the context of (a) complex separation problems, (b) 2D-HPLC separations, and (c) post-column reaction detectors.

Performance of columns packed with the new shell particles, Kinetex-C18

The performance of the new Kinetex-C₁₈ column was investigated. Packed with a new brand of porous shell particles, this column has an outstanding efficiency. Once corrected for the contribution of the instrument extra column volume, the minimum values of the reduced plate heights for a number of low molecular weight compounds (e.g., anthracene and naphtho[2,3-a]pyrene) were between 1.0 and 1.3, breaking the legendary record set 3 years ago by Halo-C₁₈ packed columns. The liquid-solid mass transfer of proteins (e.g., insulin and lyzozyme) is exceptionally fast on Kinetex-C₁₈ much faster than on the Halo-C₁₈ column. The different contributions of dispersion and mass transfer resistances to the column efficiency were determined and discussed. The possible reasons for this extremely high column efficiency are discussed.

Ammonia nitrogen removal from aqueous solution using functionalized zeolite columns

In this study, a functionalized zeolites column was developed to remove ammonia nitrogen with a low concentration (50 mg/L) from aqueous solution. The absorption properties and regeneration capacity were investigated. Through breakthrough and elution curve for dynamic adsorption, we found the wastewater with 50 mg/L ammonia nitrogen took 7 h to flow 10 g modified zeolites column with diameters of 24 to 64 meshes at a flow rate of 2 mL/min. The saturated extent of adsorption was up to 7.95 mg/g, and the saturated adsorption time was 22 h. The process of dynamic adsorption could be fitted by the Thomas Model. The regeneration ability was optimized by 0.1 M Na2CO3 as a regenerant. With excellent absorption ability for removing ammonia nitrogen with a low concentration, the functionalized zeolites could be potentially used a high-performance adsorbent for removing ammonia nitrogen.

The impact of column connection on band broadening in very high pressure liquid chromatography

A series of experiments was conducted to evaluate the degree of band broadening in very high pressure LC due to column connections. Different column manufacturers use slightly different designs for their column fittings. If the same column connections are repeatedly used to attach columns of different origins, different void volumes form between capillary tubes and column inlets. An Agilent Ultra Low Dispersion Kit (tubing id 75 μm) was installed on an Agilent Infinity 1290 ultra HPLC and used to connect successively an Agilent, a Phenomenex, and a Waters column. A series of uracil (unretained) samples were injected and eluted at a wide range of flow rates with a water/acetonitrile mixture as eluent. In order to determine the variance contribution from column connections as accurately as possible a nonretained probe compound was selected because the variance contribution from the column is the smallest for analytes, which have very low k values. Yet, this effect still has an impact on the resolution for moderately retained compounds (k > 2) for narrow-bore columns packed with fine particles, since variance contributions are additive for linear chromatographic systems. Each injection was replicated five times under the same experimental conditions. Then NanoViper column connections (tubing id 75 μm) were used and the same injections were made. This system was designed to minimize connection void volumes for any column. Band variances were calculated as the second central moment of elution peaks and used to assess the degree of band broadening due to the column connections. Band broadening may increase from 3.8 to 53.9% when conventional metal ferrules were used to join columns to connection sites. The results show that the variance contribution from improper connections can generate as much as 60.5% of the total variance observed. This demonstrates that column connections can play a larger role than the column packing with respect to band dispersion.

Investigating retention characteristics of a mixed-mode stationary phase and the enhancement of monolith selectivity for high-performance liquid chromatography

The synthesis and chromatographic behavior of an analytical size mixed-mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3-cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance π-type bonding within a single separation environment. Selectivity studies were undertaken using n-alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the π-type selectivities afforded by the two different ligands on the mixed-mode column.

Improving quantification using curtain flow chromatography columns in the analysis of labile compounds: a study on amino acids.

The performance of curtain flow chromatography column technology with MS detection was evaluated for the analysis of labile compounds. The curtain flow column design allows for separations that are faster and/or more sensitive than conventional columns, depending on how exactly the curtain flow column is configured. For example, when mass spectral detection is employed, the curtain flow column can yield separations that are 5-times faster than conventional columns when the curtain flow and the conventional columns have the same internal diameter. Or when the internal diameter of the conventional column is reduced in order to yield the same analytical through-put as the curtain flow column, the sensitivity on the curtain flow column can be as much as 66-fold higher than the conventional column. As a consequence of the higher analytical through-put less standardization is required in the analysis of labile compounds because less sample degradation is apparent. Consequently the sample integrity is preserved yielding data of a higher quality.