Concentration-sensitive Detector Research Articles

Enhanced Sensitivity in Comprehensive Liquid Chromatography: Overcoming the Dilution Problem in LC × LC via Temperature-Responsive Liquid Chromatography.

In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension (1D), which lead to peak broadening in the second dimension (2D). In the majority of the current LC × LC column combinations, analytes are less than optimally refocused upon transfer to the second column, which negatively affects sensitivity. Furthermore, the typical combination of 1 or 2.1 mm columns in the 1D paired with a 3 mm (or broader) column in the 2D leads to at least a 9- or 4-fold dilution and a corresponding loss of sensitivity when using concentration-sensitive detectors. This occurs due to the enhanced radial dilution of the analytes in a broader column, while the sensitivity problem is further exacerbated in LC × LC due to the high flow operated 2D. In this paper, we introduce a solution to neutralize and inverse this dilution problem through a reconcentrating solution using temperature-responsive liquid chromatography (TRLC) in the 1D, which is a purely aqueous separation mode. Full solute refocusing at the 2D column head is thereby obtained when TRLC is combined with reversed-phase liquid chromatography (RPLC). This is shown for the combination of a 2.1 mm I.D. TRLC column with decreasing RPLC column diameters (3-2.1-1 mm) operated at the same linear velocities, hence a resulting decrease in dilution, respectively. Ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection were used to determine the experimental detection limits. Sensitivity improvements with UV detection were somewhat lower than expected, but represent ∼1.5- and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the 2D, respectively. This is attributed to extra-column dispersion and the poorer performance of 1 mm I.D. columns. A major benefit of the use of 1 mm I.D. columns in the 2D is that it allows split-free coupling of 2D effluent with ESI-MS (at 450 μL/min), making the coupling robust and simple. When using ESI-MS even better, albeit more variable, sensitivity enhancements were obtained on the narrower columns. The benefits of the methodology are demonstrated for paraben test solutes and for phenolic compounds in a blueberry extract by TRLC × RPLC-UV-ESI-TOF-MS.

Characterization of Nano-Gravimetric-Detector Response and Application to Petroleum Fluids up to C34.

A nano-gravimetric detector (NGD) for gas chromatography is based on a nanoelectromechanical array of adsorbent-coated resonating double clamped beams. NGD is a concentration-sensitive detector and its sensitivity is analyte-dependent based on the affinity of the analyte with the porous layer coated on the NEMS surface. This affinity is also strongly related to the NGD temperature (NGD working temperature can be dynamically set up from 40 to 220 °C), so the sensitivity can be tuned through temperature detector control. An adsorption-desorption model was set up to characterize the NGD response on a large set of n-alkanes from C10 to C22 at different NGD temperatures. For fast identification of petroleum mixture based on chromatogram fingerprint, a general strategy for NGD temperature program design was developed leading to a constant relative response factor between 0.96 and 1.03 for all the alkanes, and then chromatograms are very similar to those obtained with a flame ionization detector (FID). The analysis of a real petroleum fluid was also performed and compared to FID results: quantitative results obtained for all the analytes were satisfactory according to precision (<5%) and accuracy (average relative error = 4.3%). Based on such temperature control strategy, NGD sensitivity and the dynamic linear range can be adjusted and detection limits at a picogram level can be easily achieved for all n-alkanes.

Is vacuum ultraviolet detector a concentration or a mass dependent detector?

The vacuum ultraviolet detector (VUV) is a very effective tool for chromatogram deconvolution and peak identification, and can also be used for quantification. To avoid quantitative issues in relation to time drift, such as variation of peak area or peak height, the detector response type has to be well defined. Due to the make-up flow and pressure regulation of make-up, the detector response (height of the peak) and peak area appeared to be dependent on experimental conditions such as inlet pressure and make-up pressure. Even if for some experimental conditions, VUV looks like mass-flow sensitive detector, it has been demonstrated that VUV is a concentration sensitive detector.

Periodate oxidation and macromolecular compaction of hyaluronan

Partial periodate oxidation of hyaluronan (HA) leads to ring opening of the 4-linked D-glucuronate (GlcA) residues, providing a particularly flexible element within otherwise semi-flexible chains. This leads to compaction of the chains as demonstrated by a pronounced decrease in the intrinsic persistence length, which was determined on the basis of the molecular weight dependence of radius of gyration (R G) and the intrinsic viscosity. These parameters were readily obtained using size-exclusion chromatography (SEC) with an online multi-angle laser light scattering (MALLS) detector, a viscosity detector, and a concentration-sensitive detector. The electrostatic contribution to the total persistence length increased with increasing degree of oxidation. Compared to alginates and chitosans, HA becomes less degraded during the oxidation, which is attributed to a protective effect of periodate-resistant N-acetyl-D-glucosamine (GlcNAc) residues adjacent to periodate-sensitive GlcA residues in HA.

There’s plenty of gloom at the bottom: the many challenges of accurate quantitation in size-based oligomeric separations

There is a variety of small-molecule species (e.g., tackifiers, plasticizers, oligosaccharides) the size-based characterization of which is of considerable scientific and industrial importance. Likewise, quantitation of the amount of oligomers in a polymer sample is crucial for the import and export of substances into the USA and European Union (EU). While the characterization of ultra-high molar mass macromolecules by size-based separation techniques is generally considered a challenge, it is this author's contention that a greater challenge is encountered when trying to perform, for quantitation purposes, separations in and of the oligomeric region. The latter thesis is expounded herein, by detailing the various obstacles encountered en route to accurate, quantitative oligomeric separations by entropically dominated techniques such as size-exclusion chromatography, hydrodynamic chromatography, and asymmetric flow field-flow fractionation, as well as by methods which are, principally, enthalpically driven such as liquid adsorption and temperature gradient interaction chromatography. These obstacles include, among others, the diminished sensitivity of static light scattering (SLS) detection at low molar masses, the non-constancy of the response of SLS and of commonly employed concentration-sensitive detectors across the oligomeric region, and the loss of oligomers through the accumulation wall membrane in asymmetric flow field-flow fractionation. The battle is not lost, however, because, with some care and given a sufficient supply of sample, the quantitation of both individual oligomeric species and of the total oligomeric region is often possible.

Comparison of the quantitative performance of constant pressure versus constant flow rate gradient elution separations using concentration-sensitive detectors

This contribution discusses the difference in chromatographic performance when switching from the customary employed constant flow rate gradient elution mode to the recently re-introduced constant pressure gradient elution mode. In this mode, the inlet pressure is maintained at a set value even when the mobile phase viscosity becomes lower than the maximum mobile phase viscosity encountered during the gradient program. This leads to a higher average flow rate compared to the constant flow rate mode and results in a shorter analysis time. When both modes carry out the same mobile phase gradient program in volumetric units, normally identical selectivities are obtained. However, small deviations in selectivity are found due to the differences in pressure and viscous heating effects. These selectivity differences are of the same type as those observed when switching from HPLC to UHPLC and are inevitable when speeding up the analysis by applying a higher pressure. It was also found that, when using concentration-sensitive detectors, the constant pressure elution mode leads to identical peak areas as the constant flow rate mode. Also the linearity is maintained. In addition, the repeatability of the peak area and retention time remains the same when switching between both elution modes.

Viscosimetric detection in size‐exclusion chromatography (SEC/GPC): The Goldwasser method and beyond

Size-exclusion chromatography (SEC or GPC) is the most widely used separation method to characterize polymers. The high level of complexity of most polymeric materials necessitates the use of not only concentration-sensitive detection but also structure-sensitive detection. Viscometry is usually used in conjunction with a concentration-sensitive detector and universal calibration to determine molecular weights of polymers. Goldwasser proposed to use a viscometer as a single detector to determine number-average molecular weights, M(n) (ACS Symposium Series, 521, 143). The method is particularly of interest when concentration-sensitive detection is not available, because the sample is isorefractive or not UV-absorbing, or because composition is not constant (copolymers). It has known very little applications so far. It actually does not only allow determining M(n), but also the number hydrodynamic volume distribution. This opens a wider range of applications for the Goldwasser method. Size-exclusion chromatography only yields inaccurate molecular weight distributions for some complex branched polymers. Hydrodynamic volume distributions have then a strong potential for comparative studies owing to their far higher accuracy. Our experimental tests highlight the fact that the method is highly sensitive to noise and careful optimization of the injection concentration is needed, but number distribution can be obtained as well as M(n).

Hydrodynamic chromatography of latex blends

The ability to characterize the size and shape distributions of broadly polydisperse analytes is a driving force in particle size analysis. Multi-detector hydrodynamic chromatography (HDC), which has previously shown promise in its ability to characterize the size and shape of monodisperse, spherical polystyrene latex standards, is applied here to include the characterization of bi-, tri-, and tetramodal latex blends and their constituents varying in size, chemistry, and compactness. The ability of multi-detector HDC to distinguish between similar-sized particles of different chemistry is realized by the coupling of a concentration-sensitive detector and two different types of light scattering detectors. The use of both multi-angle static and quasi-elastic light scattering permits for determination of two different size parameters across the elution profiles of the blends. Combining the size information obtained from both light scattering methods provides a measure of how particle compactness changes as a function of size in a latex blend. Multi-detector HDC was shown to be a rapid and precise method for characterizing particle size, shape, and their distributions of broadly polydisperse analytes.

An SEC/MALS study of alternan degradation during size-exclusion chromatographic analysis

Ultrahigh-molar-mass (M) polymers such as DNA, cellulose, and polyolefins are routinely analyzed using size-exclusion chromatography (SEC) to obtain molar mass averages, distributions, and architectural information. It has long been contended that high-M polymers can degrade during SEC analysis; if true, the inaccurate molar mass information obtained can adversely affect decisions regarding processing and end-use properties of the macromolecules. However, most evidence to the effect of degradation has been circumstantial and open to alternative interpretation. For example, the shift in SEC elution volume as a function of increased chromatographic flow rate, observed using only a concentration-sensitive detector, may be the result of degradation or of elution via a nondegradatory slalom chromatography mechanism. Here, using both concentration-sensitive and multiangle static light-scattering detection, we provide unambiguous evidence that the polysaccharide alternan actually degrades during SEC analysis. The decrease in molar mass and size of alternan with increasing flow rate, measured using light scattering, allows ruling out an SC mode of elution and can only be interpreted as due to degradation. These findings demonstrate the extreme fragility of ultrahigh-M polymers and the care that must be taken for accurate characterization.

Quantitative LC−MS of Polymers: Determining Accurate Molecular Weight Distributions by Combined Size Exclusion Chromatography and Electrospray Mass Spectrometry with Maximum Entropy Data Processing

We report on the successful application of size exclusion chromatography (SEC) combined with electrospray ionization mass spectrometry (ESI-MS) and refractive index (RI) detection for the determination of accurate molecular weight distributions of synthetic polymers, corrected for chromatographic band broadening. The presented method makes use of the ability of ESI-MS to accurately depict the peak profiles and retention volumes of individual oligomers eluting from the SEC column, whereas quantitative information on the absolute concentration of oligomers is obtained from the RI-detector only. A sophisticated computational algorithm based on the maximum entropy principle is used to process the data gained by both detectors, yielding an accurate molecular weight distribution, corrected for chromatographic band broadening. Poly(methyl methacrylate) standards with molecular weights up to 10 kDa serve as model compounds. Molecular weight distributions (MWDs) obtained by the maximum entropy procedure are compared to MWDs, which were calculated by a conventional calibration of the SEC-retention time axis with peak retention data obtained from the mass spectrometer. Comparison showed that for the employed chromatographic system, distributions below 7 kDa were only weakly influenced by chromatographic band broadening. However, the maximum entropy algorithm could successfully correct the MWD of a 10 kDa standard for band broadening effects. Molecular weight averages were between 5 and 14% lower than the manufacturer stated data obtained by classical means of calibration. The presented method demonstrates a consistent approach for analyzing data obtained by coupling mass spectrometric detectors and concentration sensitive detectors to polymer liquid chromatography.

Size-Based Characterization by the Coupling of Capillary Electrophoresis to Taylor Dispersion Analysis

In this work, a new methodology is presented for performing capillary electrophoresis (CE) coupled to Taylor dispersion analysis (TDA). The CE step allows the separation of the different compounds of the injected mixture, while the diffusion coefficient related to each sample zone can be derived from the subsequent TDA step. TDA is an absolute and straightforward nonseparative method allowing the determination of the diffusion coefficient (or hydrodynamic radius) from the peak dispersion obtained in an open tube under Poiseuille laminar flow conditions. With a mass concentration sensitive detector, the hydrodynamic radius derived from TDA is a weight average value calculated upon all the molecules present in the sample zone. Since CE can be hardly coupled to light scattering detection for technical reasons (low volumes, short detection path length), TDA represents an interesting alternative for the size characterization, without calibration, of sample mixtures using CE-based separation techniques. The coupling of CE to TDA can be implemented on a commercial CE apparatus.

Improving the universal response of evaporative light scattering detection by mobile phase compensation

Mobile phase compensation, first reported for the charged aerosol detector (CAD), was used as a suitable method to overcome problems related to the mobile phase-dependent response of the evaporative light scattering detector (ELSD). Mobile phase compensation was effectively performed both in the flow injection- and in gradient modes. Without compensation, the response factors of the ELSD for six sulfonamide drugs differed by a factor of two when varying the mobile phase composition between 10 and 90% acetonitrile. This change could be effectively eliminated using the technique of mobile phase compensation, where a secondary pump with a reversed gradient was used to provide the detector with a constant composition of the mobile phase. For identical experimental conditions, the ELSD showed a nearly constant, albeit somewhat reduced, response with compensation. This indicates that under such conditions, the ELSD behaved as a concentration-sensitive detector. The analysis of sulfonamides drugs at 0.05% level using gradient UPLC-ELSD separation with mobile phase compensation is demonstrated.

Onset of non-linearity in zonal elution separators: The concept of effective analyte concentration

When an analyte injected in a zonal separation method (chromatography, capillary zone electrophoresis, field-flow fractionation) is not highly diluted in the carrier fluid, the retention ratio, R – or ratio of the cross-sectional average migration velocity of the analyte to that of the carrier fluid – depends on the local concentration, c, of the center of mass of the analyte zone, and the zone migration occurs in non-linear conditions. Because the zone broadens as it moves along the separator, R varies continuously from the inlet to the outlet of the separator. That concentration, c eff, for which R( c eff) is equal to the length-averaged apparent retention ratio, R app, is called effective concentration, and that distance, z eff, from the separator inlet, for which c( z eff) is equal to c eff, i.e. for which R( z eff) is equal to R app, is called effective position. Assuming that near the onset the non-linear behavior, R( c), is a linear function, values of R app, c eff and z eff have been computed in a wide range of operating conditions which are typical of situations encountered in capillary zone electrophoresis, liquid chromatography, or field-flow fractionation. Computations have been performed both in presence and in absence of the dispersion arising from the concentration dependence of the analyte migration rate (called thermodynamic dispersion in chromatography or electromigration dispersion in capillary zone electrophoresis). It is found that, whatever the range of analyte concentration covered from inlet to outlet of the separator, c eff is always close to two times the analyte concentration, c out, at the outlet of the separator, and z eff between one-fourth and one-third of the separator length. As c out is easily determined from the peak recorded by a concentration-sensitive detector, a simple pragmatic expression is given for the estimation of c eff. This effective concentration is the appropriate concentration to be used for comparing predictions of theoretical models of R( c) with experimental retention data. This is of particular interest for validating such models in field-flow fractionation.

Capillary columns in liquid chromatography: between conventional columns and microchips

Liquid chromatography on columns with small internal diameters has been reviewed as the intermediate technique between conventional liquid chromatography and microchip separations. The development of micro column separations in the early years has been described, starting with the papers of Horváth and co-workers and Ishii and co-workers, continuing into the first part of the eighties, then making a leap in time to recent innovations with small-bore columns. Based on internal diameters a classification of the different analytical HPLC columns has been suggested. The advantages of small-bore columns have been discussed, with particular emphasis on the advantage of coupling to concentration sensitive detectors when the sample amount is limited. Open tubular columns are treated as a part of the historic background. The recent developments include a brief look into the current status of monolithic columns, the use of packed nano columns and micro columns with electrospray mass spectrometry, and the potential of two-dimensional comprehensive liquid chromatography. Finally, the coupling of sample preparation to analytical columns and the future applications of the novel technological improvements to the microchip separation methods have been discussed.

Analysis of cyclic nucleotides and cytokinins in minute plant samples using phase-system switching capillary electrospray-liquid chromatography-tandem mass spectrometry

Using an electrospray tandem mass spectrometer as a concentration-sensitive detector, a method has been developed to quantify femtomole amounts of plant growth regulators (i.e. isoprenoid type cytokinins, zeatin, dihydrozeatin, isopentenyladenine and their respective riboside and glucoside analogues) and the second messenger adenosine 3′:5′-cyclic monophosphate (3′:5′-cAMP). Miniaturisation of the chromatographic setup using capillary high performance liquid chromatographic (HPLC) ion spray mass spectrometry increased the sensitivity to the low femtomole region. Application of automated capillary column switching allowed the introduction of large injection volumes into the HPLC system. Aliquots (25 µL) were injected into one dimension of the HPLC set-up and stacked onto a micro pre-column. By means of mobile phase switching the pre-column was back-flushed to introduce the analytes onto the analytical column. For cytokinin analysis positive electrospray ionisation was used and resulted in 2.5–25 fmol detection limits. Cyclic nucleotides were separated under ion-pair conditions using tetrabutyl ammonium bromide as ion-pair reagent and were detected under negative electrospray ionisation conditions. Here a 25 fmol detection limit was determined. Following this approach, cytokinins and 3′:5′-cAMP extracted from only mg amounts of apical shoot meristem and chloroplasts obtained from Nicotiana tabacum cv. Petit Havana SR1 were identified and quantified. Copyright © 1999 John Wiley & Sons, Ltd.

Flow field-flow fractionation of high-molecular-mass polyacrylamide

The fractionation of high-molecular-mass polyacrylamide in aqueous solutions by size-exclusion chromatography is complicated by concerns over adsorption and shear degradation. The coupling of flow field-flow fractionation (flow FFF), multi-angle laser light scattering (MALLS) detection and a concentration-sensitive detector provided absolute measurement of molecular mass distributions in a low shear environment. Critical to the success of the method was the careful selection of the carrier solution. A number of commercial polyacrylamide standards with mean molecular masses in the range 0.35 to 9.00·10 6 were studied. The breadth of the distributions indicated the presence of entangled or agglomerated polymer. Diffusion coefficients calculated from both light scattering and flow FFF retention were comparable, as well as being consistent with extrapolations from literature values for lower-molecular-mass polyacrylamides.

Liquid chromatographic-mass spectrometric studies on the enzymatic degradation of β-endorphin by endothelial cells

An on-line HPLC-mass spectrometric procedure with an electrospray atmospheric pressure ionization (ESI-API) ion source was developed to identify the enzymatic degradation products (peptides) generated by incubation of human β-endorphin (hβE) with cultured aortic endothelial cells. The samples from the complex incubation mixture were prepurified and enriched using a small reversed-phase (RP) perfusion precolumn. Flow switching was applied to transfer the peptides from this precolumn to the analytical RP column of 2 or 0.32 mm I.D. and to separate them by gradient elution. The peptides were detected by means of an on-line coupled triple quadrupole mass spectrometer (TSQ 700) with an ESI-API ion source operated in the positive ion mode. This MS system behaves as a concentration sensitive detector at flow-rates from 5 to 150 μl/min. MS-MS experiments supported the unambiguous assignment of the peptide structures. Thus most of the peptide fractions were identified and the region 16-17-18 (-L-F-K-) of hβE was found to be primarily attacked by the enzymes of the endothelial cells.

Ion spray mass spectrometric detection for liquid chromatography: A concentration- or a mass-flow-sensitive device?

As the mass spectrometer becomes more accepted as a detector for HPLC, its characteristics should become better understood by those performing routine LC-MS experiments. In particular, the ion current response for quantitative analysis studies involving significant dynamic range in concentration for target analytes must be determined as well as other factors that affect MS response. This work describes the concentration-sensitive response for the ion spray (pneumatically-assisted electrospray) LC-MS interface from the chromatographer's perspective. A comparison of LC-MS ion current response in the isocratic mode resulting from studies of a synthetic mixture containing alkyl benzoates is presented. LC-MS total ion current chromatograms from three different column sizes (1 mm I.D., 2.1 mm I.D. and 4.6 mm I.D.) with and without a post-column split, and high-flow ion spray LC-MS without a post-column split illustrates that the former behaves as a concentration-sensitive detector whereas the latter behaves as a mass-flow-sensitive detector. The flexibility of ion spray to high-flow applications allows the use of HPLC eluent flow ranging from 0.001-2.0 ml/min. The use of solvent—buffer post-column addition also allows optimization for improved analyte ion current response.

Selective On-Line Preconcentration of Proteins by Tandem Metal Chelate Capillaries-Capillary Zone Electrophoresis

Abstract Fused-silica capillaries having surface bound iminodiacetic acid metal chelating functions were developed for the selective on-line preconcentration of dilute protein samples prior to capillary zone electrophoresis. The metal chelate capillaries with immobilized zinc proved effective in the selective accumulation of detectable amounts of proteins from dilute samples. They permitted the detection of 25 times less concentrated samples than by CZE alone with concentration sensitive detectors. Large sample volumes could be introduced without affecting separation efficiency. Besides demonstrating the effectiveness of metal chelate capillaries in the quantitative determination of proteins from dilute samples, the effects of pH. composition of electrolytes and sample feeding time on the analytical signal were examined with carbonic anhydrase, a zinc binding protein.

Use of small-diameter capillaries for increasing peptide and protein detection sensitivity in capillary electrophoresis-mass spectrometry.

The use of small ID capillaries is shown to provide a substantial increase in sensitivity for capillary electrophoresis-electrospray ionization/mass spectrometry (CE-ESI/MS). In a comparison using capillaries ranging from either 100 to 10 microns or 50 to 5 microns ID and chemically modified with aminopropylsilane, a 25- to 50-fold increase in sensitivity was observed for both peptide and protein mixtures. This enhanced solute sensitivity allowed the detection of approximately 150 attomoles of melittin (2845 Da) with selected ion monitoring and 600 attomoles of carbonic anhydrase (29,157 Da) while scanning for CE-MS with a quadrupole mass spectrometer. For the protein mixture, mass spectra of sufficient quality for precise molecular weight determination (< or = 0.05%) were obtained for 600 attomole injections using a 5 microns ID capillary. The increase in sensitivity with small capillary diameters can be primarily attributed to a reduced mass flow rate of buffer and other background constituents into the electrospray source, which allows for greater sample ionization efficiency. A model that qualitatively accounts for the results is presented, but quantitative agreement is precluded due to difficulties in accounting for contributions due to a liquid sheath flow used with the electrospray source. The model accounts for the observation that the ESI/MS appears to function as a concentration-sensitive detector under many conditions using large-diameter capillaries. A transition occurs, however, to a regime where the ESI/MS functions as a mass flow-sensitive detector for small-diameter capillaries, where the ESI current is limited by the rate of delivery to the ESI source of charge carrying species in solution. These results suggest peptide and protein analysis at low attomole and subattomole levels should be obtainable with alternative types of mass spectrometers.