Evaporative Light Scattering Detection Response Research Articles

Effects of selected parameters on the response of the evaporative light scattering detector in supercritical fluid chromatography

Due to the renewal of the use of supercritical fluid chromatography (SFC), the coupling of SFC with evaporative light scattering detection (ELSD) needs to be revisited. Indeed, SFC and ELSD apparatus have evolved, while understanding of the nebulisation process has improved. This detector, first developed for polymer analyses can be used for a lot of compounds that do not display sufficient UV absorption. Because the response of ELSD is related to numerous parameters, such as mobile phase composition, the velocity difference between the mobile phase and the nebulisation gas, or the nebulisation temperature, many additional studies are necessary to understand its behaviour in SFC as compared to HPLC. The effects of the nature of co-solvent added to carbon dioxide (methanol, ethanol and acetonitrile), the injected volume and the flow rate on the ELSD response were studied. Certain parameters induce great changes on the nebulisation recovery (the proportion of mobile phase entering the drift tube), allowing for a dramatic improvement in peak area. Moreover, effects of the particle size in the aerosol were also observed, related, as expected, to the modifier nature, but strangely also to the injected volume. Specific and positive behaviour of the ELSD detection on the apparent peak efficiency was confirmed in SFC, for large injected volumes. Finally, a flow rate increase reduces peak area, showing that the use of high flow rates, although attractive to shorten analysis duration, is not favourable to ELSD response. Numerous parameters acting on detector response are modified by the flow rate change, such as the mobile phase velocity. Moreover, measurement of the nebulisation chamber temperature shows the strong decrease of temperature for high flow rates, due to the supercritical fluid depressurisation.

Evaporative Light Scattering Detection Based HPLC Method for the Determination of Polysorbate 80 in Therapeutic Protein Formulations

An evaporative light scattering detection (ELSD) based high-performance liquid chromatography (HPLC) method is developed for the determination of polysorbate 80 (tween 80) in therapeutic protein formulations. The method is simple and overcomes the difficulties associated with specificity and sensitivity. The method is suitable for the quantitation of polysorbate 80 in the usual formulation range (0.01-0.1%) as well as in trace amounts ≥13 µg/mL. The analysis is based on the removal of protein first by solid-phase extraction using Oasis HLB cartridges followed by HPLC analysis using Inertsil ODS-3 C 18 column (4.6×150 mm, 5 µm) using reversed-phase conditions. The detector response changes exponentially with an increase in polysorbate concentration. A very good linear fit of log ELSD response against log polysorbate 80 concentration is observed. The specificity, sensitivity, precision, and accuracy of the method are suitable for the quantitation of polysorbate 80 in protein formulations.

Identification and quantification of polyethylene glycol types in polyethylene glycol methyl ether and polyethylene glycol vinyl ether

Quantitative determination of polyethylene glycol (PEG) impurities in two monofunctional polyglycol types, PEG methyl ether (M-PEG) and PEG vinyl ether (V-PEG), has been carried out by reversed-phase liquid chromatography with evaporative light scattering detection (ELSD). In addition to optimizing the resolution between PEG and monofunctional PEG peaks, the major focus has been to determine the molecular weights of PEG impurities in M-PEG and V-PEG of diverse molecular weights. The latter is achieved by examining liquid chromatography–mass spectrometry (LC–MS) mass spectra of both monofunctional PEG and PEG in several cases, and matching peak retention times with those of available PEG standards for all M-PEG and V-PEG sample types. This information is helpful in selecting the appropriate PEG standard to determine PEG content in each sample type. ELSD response factors for various PEG standards have also been compared. It has been found that PEG standards with molecular weights from 1000 Da to 8000 Da show responses that are within 10% of each other. However, a low molecular weight PEG such as PEG 400, provides approximately 30% less response compared to its higher molecular weight counterparts.

Determination of artesunate using reversed‐phase HPLC at increased temperature and ELSD detection

Artesunate (ART) determination can be performed by evaporative light scattering detection with mobile phase composed of CH(3)CN/HCOOH 0.01 M (40:60 v/v; pH 2.85). Evaporative light scattering detection instead of UV detection allowed to improve the sensitivity and the LOD. However, the evaporative light scattering detection response of dihydro-artemisinin appears weaker than for ART, whereas with UV detection the response of ART and dihydroartemisinin seemed similar. Constant analysis time was obtained on using the mobile phase with a flow rate of 0.5 mL/min and column temperature at 60 degrees C instead of 0.7 mL/min at room temperature. This led to less solvent consumption. Moreover, decrease in the flow rate and increase in the column temperature were advantageous for higher sensitivity with both evaporative light scattering detection and UV detection. ART determination in rectal gel and suppositories were compared with these different detection modes and similar results were obtained.

Evaluation of evaporative light-scattering detection for metabolite quantification without authentic analytical standards or radiolabel

Development of a sensitive and specific technique for the quantitation of drug metabolites without the use of synthetic analytical standards or radiolabel would represent a major advance in preliminary route of metabolism screening in drug discovery. In this study, the ability of evaporative light-scattering detection (ELSD) to quantify metabolites of 7-ethoxycoumarin (EC) was evaluated. Because ELSD operates as a mass detector, the complex nature of in vitro-derived samples from hepatocyte incubations resulted in an inability to detect the analytes of interest in this matrix using ELSD. Additionally, the gradient nature of the analysis required to temporally separate ethoxycoumarin from its metabolites and matrix components interfered with the ELSD response. Furthermore, using less-complex contrived mixtures, ELSD demonstrated insufficient sensitivity (limit of detection of 1000–10,000 ng/mL) and an inconsistent inter-analyte response. Together, the limitations outlined in these experiments demonstrate that ELSD is at present an inadequate technique for generating semi-quantitative data on metabolites in drug discovery.

Enhancement of evaporative light scattering detection in high-performance liquid chromatographic determination of neomycin based on highly volatile mobile phase, high-molecular-mass ion-pairing reagents and controlled peak shape

In the frame of the development of a novel HPLC–ELSD (evaporative light scattering detection) method for the determination of the aminoglycoside antibiotic neomycin sulfate, the influence of mobile phase composition and peak broadening on ELSD response was evaluated. ELSD response was enhanced by: (a) increase of mobile phase volatility (solvents examined: water, acetonitrile, methanol and acetone), (b) increase of molecular mass of ion-pairing species [acidic reagents tested: formic, acetic, trifluoroacetic, trichloroacetic and heptafluorobutyric acid (HFBA)], and (c) decrease of peak width and asymmetry obtained by controlling the concentration of the ion-pairing acidic reagent (HFBA). Utilizing a Waters ODS-2 C 18 Spherisorb column, evaporation temperature of 45 °C and nitrogen pressure of 3.5 bar, the optimized mobile phase was water–acetone (50:50), containing 11.6 mM HFBA, in an isocratic mode at a rate of 1.0 ml/min. Neomycin was eluted at 4.9 min, with asymmetry factor 1.3. Logarithmic calibration curve was obtained from 2 to 50 μg/ml ( r > 0.9997). Limit of detection (LOD) was 0.6 μg/ml and R.S.D. = 1.7% ( n = 3, 3.3 μg/ml). In raw materials, the simultaneous determination of sulfate (LOD = 3 μg/ml, R.S.D. = 1.7%, r > 0.9998) and of minor impurities was feasible. The developed method was also applied for the determination of neomycin in pharmaceutical formulations (powder, aerosol and cream) without any interference from excipients (recovery from spiked samples ranged from 99 to 102%) and a %R.S.D. of <2.1 ( n = 3). The HPLC–ELSD method was also found applicable in the determination of neomycin in animal feeds (LOQ = 0.2%) without any interference from the feed matrices.

An Investigation into Detector Limitations Using Evaporative Light-Scattering Detectors For Pharmaceutical Applications

Evaporative light-scattering detection (ELSD) high-performance liquid chromatography (HPLC) is an alternative technology to low-wavelength UV analysis that is often employed when compounds lack sufficient absorptivity. Although ELSD provides an additional detector option for liquid chromatographers, studies in our laboratory indicate analyte properties may adversely affect the ability to detect certain molecules. In this investigation, a series of low-molecular-weight compounds of pharmaceutical interest are evaluated with two commercially available ELSDs. It is observed that melting point is a useful analyte property to consider in optimizing ELSD detectors. The melting point of the analyte should be significantly higher than what the compound will experience in the nebulizer/evaporator chambers to achieve the best analyte response. It is found that some analytes could not be distinguished from the evaporated mobile phase background when ELSD temperatures exceed the melting point of the compound. Though useful for many applications and of particular interest for compounds that are weak chromophores, ELSD falls short of being a "universal detector" technology. In addition to boiling points of mobile phase components, scientists should also consider the melting point and volatility of the analyte(s) when optimizing ELSD response.

Adaptation of an evaporative light-scattering detector to micro and capillary liquid chromatography and response assessment

A commercially available evaporative light-scattering detection (ELSD) system was adapted for micro and capillary LC. Therefore the various parameters involved in the droplet formation during the nebulization step in the ELSD system were studied. It was shown that the velocity term in the Nukiyama Tanasawa equation remains constant, leading to droplets of the same order of magnitude for narrow bore and capillary columns. Consequently, the ELSD modification was performed by decreasing the internal diameter of the effluent capillary tube in the nebulizer nozzle and by keeping its external diameter constant. Next, response curves for a conventional and the developed micro and capillary LC were compared as to investigate why a linear ELSD response is often obtained when used in micro or capillary LC. By splitting the flow rate post column, we showed that the nebulization process was not at the origin of the phenomenon. For ceramide III and tripalmitin, the response curves were found to be non-linear. However the curvature was less significant when the columns internal diameter decreased. Calculated particle size profiles for micro or capillary LC suggest that the particle entering the detection chamber are bigger than under conventional LC conditions. Last, triethylamine and formic acid were used to increase the response of the detector. The response enhancement, expected from previous studies, was established for the two lipids involved in this study.

Post-column addition as a method of controlling triacylglycerol response coefficient of an evaporative light scattering detector in liquid chromatography–evaporative light-scattering detection

The non-linear response is generally the main limitation to the general quantitative use of evaporative light-scattering detection (ELSD). In the particular case of triacylglycerol (TG) analysis, we present a preliminary paper dealing with the use of post-column additives as a means of monitoring the response of such a detector. As TG can form molecular association complexes (ligand–ligate associations) with either cholesterol, urea or silver nitrate, we report the influence of the concentration of each of these chemical compounds in the liquid phase directed towards the ELSD system. The results show that the response coefficient b of the calibration curve either decreases from 1.25–1.30 to 0.51 or increases from 1.25–1.30 to 1.78 according to the nature and concentration of post-column additive. The use of cholesterol as additive, at a discrete concentration, may lead to a linear response curve ( b=1), i.e. to the direct proportionality of ELSD response versus the TG concentration, making quantitative analysis of such solutes easier. On the other hand, to improve sensitivity, the addition of silver nitrate may be chosen for an increase in b value.

Identification and Quantification of the Molecular Species of Acylglycerols in Castor Oil by HPLC Using ELSD

Sixteen molecular species of acylglycerols (AG) in castor oil have been identified and quantified. Evaporative light scattering detection (ELSD) responses of different amounts of the standards of molecular species of AG were nearly linear and similar. In general, the addition of a double bond and a hydroxyl group on the acyl chain and the shortening of the acyl chain decreased the ELSD response of AG slightly. The quantification of molecular species of AG was based on the percentage peak area in the HPLC chromatogram. Triricinolein (RRR) constituted about 71% of castor oil. The contents of the molecular species of diricinoleoyltriacylglycerol (RR‐TAG) were, in total, about 18% and were in the order of RRO (8.8%), RRL (6.6%), RRS (1.1%), RR‐lesqueroleate (0.67%), RRP (0.47%) and RRLn (0.15%). The level of R‐TAG was about 0.3%. The level of TAG containing no ricinoleate was less than 0.1%. Diricinoleoylglycerol, a diacylglycerol, was also identified and quantified (0.14%) in castor oil.

SFC with evaporative light-scattering detection and atmospheric-pressure chemical-ionisation mass spectrometry for methylated glucoses and cyclodextrins analysis

Packed-column supercritical-fluid chromatography (pSFC) with a CO2-polar modifier mobilephase was used for the analysis of methylated glucoses and methylated-β-cyclodextrins. The interface with evaporative light-scattering detection (ELSD) was studied with regards to the effect of the position of the restrictor capillary (either at the column outlet or at the ELSD interface) and to the influence of the internal diameter of the nebulization capillary both on peak shape and on ELSD response. An unmodified LC interface of ELSD can be successful and it led to an increase in signal-to-noise ratios of methylated glucoses by a factor from 10 to 30. Likewise, an unmodified LC interface of atmospheric-pressure chemical-ionisation mass spectrometry (APCI-MS) can successfully be used for pSFC-APCI-MS analysis of methylated-β-cyclodextrin mixtures. pSFC-ELSD is a good and inexpensive approach for pSFC-APCI-MS method development.

Determination of nonvolatile components of heated soybean oils separated with high‐efficiency mixed‐bed polystyrene/divinylbenzene columns

AbstractWhole heated soybean oils and their polar fractions were analyzed for nonvolatile components by high‐performance size‐exclusion chromatography (HPSEC) with evaporative light scattering detection (ELSD). High molecular‐weight (MW) polymer compounds with MW ≥ trimer were efficiently separated with new 3‐µm mixed‐bed styrene/divinylbenzene copolymer columns. Peaks of high MW polymer components in the new column system appeared to be sharper and more symmetrical than those obtained with other columns. In the model systems studied, continuous addition of water to partially simulate frying conditions resulted in a significant increase (up to 30%) in the polar lipid content of the heated oils evaluated. Due to relatively high concentrations of monomeric triglycerides (84.6–93.5%) present in the whole unfractionated oils, small but erratic variations in the compositional distribution of components were observed in oils containing different amounts of added water. On the other hand, HPSEC‐ELSD analyses of the polar fractions (monomeric triglycerides, 25.4–62.6%) showed significant changes in the content and composition of nonvolatile components with the amount of water added. In general, prolonged heating with increasing amounts of water accelerated hydrolysis and polymerization of heated soybean oils. Discrepancies in total polymerization of heated soybean oils. Discrepancies in total polymeric materials obtained from HPSEC composition data for whole oils and polar fractions are discussed in terms of nonuniformity in sample matrices, detection limitations for minor components, and a nonlinear ELSD response rationale.

Separation of acetylated polypropylene glycol di- and triamines by gradient reversed-phase high-performance liquid chromatography and evaporative light scattering detection

Polypropylene glycol di- and triamines, the so-called Jeffamines, were reacted with a mixture of equal volumes of acetic anhydride and pyridine to give the corresponding acetamides prior to chromatographic separation by reversed-phase high-performance liquid chromatography using a linear binary solvent gradient and evaporative light scattering detection (ELSD). The procedure was applied to decrease silanophilic interactions of the solutes with the column matrix in order either to improve the peak resolution R s or to decrease peak tailing. Removal of excess of derivatizing agent prior to sample injection is not required owing to the volatility of pyridine, which does not yield any ELSD response. Therefore, the signals of oligomers with lower retention that are hidden by the broad and strongly tailing pyridine peak when measured by UV detection, are now clearly detectable. Separation was performed on C 18, C 8, C 6, C 4 and C 1 stationary phases with either acetonitrile or methanol as organic modifiers. With acetonitrile either complete elution or excellent separation of the low-molecular-mass samples ( M r ⋍ 400) is achieved on the C 18, C 8, C 6 and C 4 matrices. However, R s decreases with increasing M r but the recoveries of high- M r samples ( M r ⋍ 2000–5000) increase markedly with decreasing hydrophobicity of the stationary phase in the order C 18 < C 8 < C 6 < C 4. Complete elution of the whole family of investigated polyether amines was accomplished with either acetonitrile on a C 1 column or methanol on all sorbents used in the study. The optimum peak resolution was obtained on a C 4 column with either acetonitrile or methanol. Complete elution of all samples in particular on matrices with either high or intermediate hydrophobicity with methanol compared with acetonitrile is presumably attributable to a better solvation of the polypropylene glycol backbone by hydrogen bonding between the ether oxygens and the hydroxyl group of the protic solvent.