Evaporative Light Scattering Detector Response Research Articles

Optimization of Evaporative Light Scattering Detector using Response Surface Methodology for Liquid Chromatography Analysis of Frondoside A

AbstractResponse surface methodology is employed to investigate the optimum evaporative light scattering detector (ELSD) operating conditions for high performance liquid chromatography (HPLC) analysis of frondoside A, which is a bioactive compound valuable for its anti‐cancer activity. The results suggest that ELSD response is mostly affected by nebulizing gas flow rate and evaporator temperature. The optimum response of frondoside A is achieved by adjusting ELSD evaporator and nebulizer temperature at 80 °C and 35 °C, respectively, with nebulizing gas flow rate of 0.9 SLM. The analysis of variance (ANOVA) test results demonstrated that the established model can successfully describe the relationship between the signal response and ELSD experimental parameters, and predict the optimum ELSD parameters for frondoside A analysis. Furthermore, analytical method validation performed in optimum conditions shows that the proposed method also exhibits adequate sensitivity and linearity, with acceptable precision and accuracy. The results demonstrate the suitability of HPLC‐ELSD technique for the development of routine analysis procedures of frondoside A in natural product samples.

Optimization of Detection of Native Amino Acids with Evaporative Light Scattering Detector in Chiral Supercritical Fluid Chromatography

Based on previously reported chiral separation of free amino-acids (AAs) using packed-column supercritical fluid chromatography (pSFC), it was desirable to investigate parameters influencing the response of evaporative light scattering detector (ELSD). The effect of several parameters, such as SFC conditions (outlet pressure, temperature and flow rate) and ELSD settings (drift tube temperature of nebulisation and nebulizer gas pressure) on the sensitivity (response) of ELSD have been studied. Based on optimization an outlet pressure of 170 bar, a temperature of 40 °C and a flow rate of 3 mL/min were chosen, and a drift tube temperature of 60 °C and a nebulizer gas pressure of 3.0 bar were found to be optimal. Limits of detection (LOD) and limits of quantification (LOQ) were determined for studied amino acids (AAs) and were found to be lower than values determined without optimization. The enantiomeric elution order was also determined for leucine, phenylalanine, isoleucine, valine, carnitine and histidine by the injection of the L-AAs in the above-mentioned optimized conditions. The L-enantiomers eluted first for four out of the six AAs studied. Conclusions on the influence of the screened variables on ELSD sensitivity and recommendations for AAs analytical studies in SFC are given.

Fully Automated Three‐Dimensional Column‐Switching SPE–FIA–HPLC System for the Characterization of Lipids by a Single Injection: Part I. Instrumental Design and Chemometric Approach to Assess the Effect of Experimental Settings on the Response of ELSD

AbstractThis article presents the first application of fully automated three‐dimensional (3D) column‐switching SPE–FIA–HPLC system for the characterization of lipids by a single injection. The whole system was designed and set up by modifying Agilent 1200 Series HPLC system in our laboratory. By using this system, a complete separation profile of the oil samples was achieved in a very short time period by using single injections. This approach was applied on vegetable oils which contains a large number of relatively high‐class lipid components, such as TG, FFA, sterols, tocopherols, DG, ester and MG. In this part of the study, we focused on the optimization of evaporative light scattering detector (ELSD) by using an experimental design and RSM. Three experimental parameters were chosen as an independent variables which are the flow rate of mobile phase, nebulization temperature and evaporation temperature. A multivariate five level experimental design was used to establish a quadratic model as a functional relationship between the response values and independent variables. The optimal values of parameters were found to be a flow rate of 1.25 mL min−1, nebulization temperature of 80 °C, and evaporation temperature of 40 °C. Regression analysis with an R2 values indicated as a satisfactory correlation between the experimental and predicted values. ANOVA test results were also illustrate that the models can be successfully used to predict the optimum parameters of ELSD. Thus, the proposed system is suitable for a large number of applications including research and development of new quality control and characterization methods for vegetable oils.

Determination of γ-Aminobutyric Acid (GABA) in Rambutan Fruit cv. Rongrian by HPLC-ELSD and Separation of GABA from Rambutan Fruit Using Dowex 50W-X8 Column.

A high-performance liquid chromatography method coupled with an evaporative light scattering detector (ELSD) was validated for the determination of γ-aminobutyric acid (GABA) in rambutan fruit without any sample pretreatment or derivatization. In the concentration range of 0.05-1.0 mg/mL GABA, the ELSD response was linear with a correlation coefficient (r) >0.999. Limit of detection and limit of quantitation were found to be 0.7 and 2.0 µg/mL, respectively. The method enabled the complete separation of GABA in the aqueous extract of rambutan flesh from the impurity peaks at 45.7 min. The recoveries of sample added GABA were obtained in the range of 92.0-99.3%. Intraday and interday relative standard deviations were <5.3%. Repeatability of the extraction process showed the acceptable precision. From the analysis of GABA content in rambutan flesh, 0.71 ± 0.23 mg of GABA was found in 1 g fresh weight. The recovery of GABA after passing through the Dowex 50W-X8 column was 96.65%. The analytical methodology could be potentially applied to the detection and quantification of GABA in other fruits and complex matrices when a sufficient quantity is available.

Analysis of non-phthalates plasticizers on porous graphitic carbon by supercritical fluid chromatography using evaporative light scattering detection

The analysis of several plasticizers, widely used in the production of medical devices, was investigated on porous graphitic carbon (PGC) stationary phase in supercritical fluid chromatography (SFC) with an evaporative light scattering detector (ELSD). Due to strong interaction of compounds with the PGC support, solvents of strong eluotropic strength were added to the CO2 supercritical fluid. The effect of alkyl chain (pentane, hexane, heptane) and chlorinated (CH2Cl2, CHCl3, CCl4) solvents was studied on the retention and on the ELSD detection of plasticizers. A co-solvent mixture composed of CHCl3/heptane, eluted under gradient mode, allowed a significant improvement of the ELSD response compared to the use of each solvent individually. Then, a central composite design (CCD) was implemented to optimize both the separation and the detection of plasticizers. The parameters involved were the outlet pressure, the gradient slope, the co-solvent composition and the drift tube temperature of the ELSD. After optimization, baseline separation of plasticizers was achieved in 7min and best signal-to-noise ratios were obtained with outlet pressure and drift tube temperature of ELSD set at 200bar and 31°C, respectively. The co-solvent mixture was also composed of CHCl3/heptane (35/65 v/v) and a gradient from 15 to 60% of co-solvent in 2.2min was employed. The results demonstrated that CCD is a powerful tool for the optimization of SFC/ELSD method and the response surface model analysis can provide statistical understandings of the significant factors required to achieve optimal separation and ELSD sensitivity.

HPLC/ELSD analysis of amidated bile acids: An effective and rapid way to assist continuous flow chemistry processes

The employment of the flow N-acyl amidation of natural bile acids (BAs) required the in-line connection with suitable analytical tools enabling the determination of reaction yields as well as of the purity grade of the synthesized glyco- and tauro-conjugated derivatives. In this framework, a unique HPLC method was successfully established and validated for ursodeoxycholic (UDCA), chenodeoxycholic (CDCA), deoxycholic (DCA) and cholic (CA) acids, as well as the corresponding glyco- and tauro-conjugated forms. Because of the shared absence of relevant chromophoric moieties in the sample structure, an evaporative light scattering detector (ELSD) was profitably utilized for the analysis of such steroidal species. For each of the investigated compounds, all the runs were contemporarily carried out on the acidic free and the two relative conjugated variants. The different ELSD response of the free and the corresponding conjugated BAs, imposed to build-up separate calibration curves. In all the cases, very good precision (RSD% values ranging from 1.04 to 6.40% in the long-period) and accuracy (Recovery% values ranging from 96.03 to 111.14% in the long-period) values along with appreciably low LOD and LOQ values (the former being within the range 1–27ngmL−1 and the latter within the range 2–44ngmL−1) turned out.

Quantitative analysis of tris(2-carboxyethyl)phosphine by anion-exchange chromatography and evaporative light-scattering detection

Tris(2-carboxyethyl)phosphine (TCEP) belongs to the trialkylphosphine class of reducing agents that are widely used in research and industry. In this paper, we discuss a sensitive high-performance liquid chromatography (HPLC) method equipped with an evaporative light scattering detector (ELSD) for the determination of TCEP in pharmaceutical samples containing therapeutic protein and stabilizing additives. TCEP was first completely oxidized with hydrogen peroxide to form TCEP oxide (TCEPO). Proteins and salts were removed from the sample by solid phase extraction. TCEPO concentrations were determined by anion exchange chromatography coupled with ELSD. Because of the 1:1 oxidation stoichiometry for the reaction, the concentration of TCEP in the sample is directly proportional to the measured concentration of TCEPO. A good linearity fit of ELSD response versus TCEPO concentration was observed over the range of 20–2000 μM. The specificity, precision, accuracy, and robustness of the method were evaluated and suitable for the quantitation of TCEP in biological samples. Moreover, selective treatment with peroxide prior to solid-phase extraction may be used to determine the mass balance of TCEP species or track the oxidation rate in pharmaceutical samples.

Effects of Drift Tube and Nebulizing Gas Temperatures on the Response of Evaporative Light-Scattering Detector

The drift tube temperature of an evaporative light-scattering detector for inorganic anions and cations response has been evaluated. The results of the detector are independent of temperature in the range studied (40–80°C) for cations, sulfate, and phosphate, but the responses for chloride and nitrate decrease when tube temperature increases. We also studied the effect of nebulizing gas temperature on the response of the evaporative light-scattering detector and noted a 200% improvement of the intensity of the signal.

On the Issue of Characteristic Evaporative Light Scattering Detector Response

In recent years, the evaporative light scattering detector (ELSD) has increasingly been used in conjunction with liquid chromatography for detection of various analytes. Due to the specificity of the detection method, which is based on the scattering of laser light on the non-volatile analyte particles, the ELSD has been considered a universal detector. This article presents issues concerned with the response of this detector. A review of experimental results presented by researchers who used the detector in question clearly show that two major issues tend to recur. Interestingly enough, the researchers do not have a uniform opinion on those issues. First, the majority of authors treat the detector signal as a mass signal, i.e., the response is believed to depend only on the analyte mass. Second, a number of different approaches are offered to analyze how the detector signal depends on analyte mass.

Application of a xenon arc lamp as a light source for evaporative light scattering detection

The standard tungsten-halogen light source used in a commercial evaporative light scattering detector (ELSD) was replaced with a 180 W xenon arc lamp. The xenon arc lamp possesses a broader spectrum in the UV region than the halogen source. The influence of the UV transmittance of five selected solvents was studied with a size-exclusion chromatography column. This solvent parameter was not observed to influence the ELSD response between the two light source settings. With the solvents studied, better sensitivity was obtained with the xenon arc lamp than the halogen lamp. This high-energy source was applied to ceramide III analysis with an octadecyl-grafted silica column and methanol:tetrahydrofuran 97:3 as the mobile phase, and the sensitivity of the quantification of ceramide III increased 16-fold for injected amounts of 14 approximately 140 ng. The molecular species in a sample of naturally occurring ceramides was analyzed using two C18 columns at 40 degrees C and gradient elution from 100% acetonitrile to 100% isopropanol in 30 min. The increased ELSD sensitivity achieved when using the xenon arc lamp allowed both the minor and major ceramide species to be observed, in contrast to the results achieved when the halogen lamp was used, where the increased photomultiplier voltage needed to observed the signals from the minor species caused the signals from the major ceramide species to occur above the detector response window.

Microanalytical systems for separations of stratum corneum ceramides

The small amount of lipids from human skin obtained with noninvasive sampling method led us to investigate microanalytical separation techniques. The lipid class analysis was performed with a micro polyvinyl alcohol-silica (PVA-Sil) column. The gradient elution was from heptane to acetone/butanol 90:10 v/v in 4%/min at 78 microL/min. In addition an evaporative light scattering detector (ELSD) was modified for micro-LC. All solvents contained 0.1% of triethylamine and formic acid in stoichiometric amount, which increased the ELSD response. In these conditions, the cholesterol eluted before free fatty acid, and squalene and triglycerides close to the dead volume. The various ceramide classes eluted following the order of the increased number of hydroxyl groups. The LOD for ceramides was 2.2 ng. The advantages of this method are the use of a normal stationary phase more reliable due to its chemical stability, its surface homogeneity and its development in microchromatography without chlorinated solvents which offers small LOD and the whole profile of lipids present in stratum corneum (SC). A method using a narrow-bore PVA-Sil column was used to collect ceramide fraction. Then the molecular species were analysed with a porous graphitic carbon column in capillary LC using a gradient from CH3OH/CHCl3 70:30 v/v to CHCl3 at 2%/min with a flow rate at 5 microL/min. The LOD obtained for ceramide was 1 ng. Both methods were assessed with SC samples obtained by rinsing a 5.7 cm2 area of the forearm with 25 mL of ethanol.

Development and validation of a novel LC non-derivatization method for the determination of amikacin in pharmaceuticals based on evaporative light scattering detection

A novel method for the direct determination of the aminoglycoside antibiotic amikacin and its precursor component kanamycin was developed and validated, based on reversed phase LC with evaporative light scattering detector (ELSD). ELSD response to amikacin was found to be enhanced by: (a) use of ion-pairing acidic reagents of increased molecular mass, (b) increase of mobile phase volatility and (c) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity and/or ratio of organic solvent to water). Utilizing a Thermo Hypersil BetaBasic C 18 column, the selected optimized mobile phase was water–methanol (60:40, v/v), containing 3.0 ml l −1 nonafluoropentanoic acid (18.2 mM) (isocratic elution with flow rate of 1.0 ml min −1). ELSD experimental parameters were: nitrogen pressure 3.5 bar, evaporation temperature 50 °C, and gain 11. Amikacin was eluted at 8.6 min and kanamycin at 10.4 min with a resolution of 1.5. Logarithmic calibration curves were obtained from 7 to 77 μg ml −1 ( r > 0.9995) for amikacin and 8 to 105 μg ml −1 ( r > 0.998) for kanamycin, with a LOD equal to 2.2 and 2.5 μg ml −1, respectively. In amikacin sulfate pharmaceutical raw materials, the simultaneous determination of sulfate ( t R = 2.3 min, LOD = 1.8 μg ml −1, range 5–40 μg ml −1, %R.S.D. = 1.1, r > 0.9997), kanamycin and amikacin was feasible. No significant difference was found between the results of the developed LC–ELSD method and those of reference methods, while the mean recovery of kanamycin from spiked samples (0.5%, w/w) was 97.3% (%R.S.D. ≤ 2.0, n = 6). Further, the developed method was applied for the determination of amikacin in pharmaceutical formulations (injection solutions) without any interference from the matrix (recovery from spiked samples ranged from 95.6 to 103.8%).

Direct determination of kanamycin in raw materials, veterinary formulation and culture media using a novel liquid chromatography–evaporative light scattering method

A novel method for the direct determination of kanamycin A and its minor component kanamycin B was developed and validated based on reversed phase liquid chromatography with evaporative light scattering detector (ELSD). ELSD response to kanamycins was found to be enhanced by: (a) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity or ratio of organic solvent to water), (b) use of ion-pairing acidic reagents of increased molecular mass, and (c) increase of mobile phase volatility. Utilizing an Spherisorb ODS-2 C 18 column, the selected optimized mobile phase was water–acetonitrile (60:40, v/v), containing 11.6 mM heptafluorobutyric acid (isocratic elution with flow rate of 1.0 ml min −1). Kanamycin A was eluted at 3.9 min and kanamycin B at 5.0 min with a resolution of 2.7. Logarithmic calibration curves were obtained from 0.6 to 28 μg ml −1 ( r > 0.9998) for kanamycin A and 4–36 μg ml −1 ( r > 0.9994) for kanamycin B, with a LOD equal to 0.20 and 1.4 μg ml −1, respectively. In kanamycin acid sulfate pharmaceutical raw materials, the simultaneous determination of sulfate ( t R = 2.1 min, LOD = 2.3 μg ml −1, %R.S.D. = 1.7, r > 0.9998) and kanamycins was feasible. No significant difference ( t-test) was found between the results of the developed LC–ELSD method and those of reference methods, while recovery from kanamycin B spiked samples ranged from 95 to 105%. The developed method was also applied with very good accuracy for the determination of kanamycin A in veterinary formulation (%recovery 95–103, %R.S.D. < 1.4, n = 3) and for the determination of kanamycins A and B in bacteria culture media (%recovery 102 and 99, respectively).

Optimisation of the separation of four major neutral glycosphingolipids: application to a rapid and simple detection of urinary globotriaosylceramide in Fabry disease

A simple method for the separation of the four major neutral glycosphingolipids, present in all human tissue, was developed. This gradient normal phase-HPLC method utilises a polyvinyl alcohol bonded stationary phase and an evaporative light-scattering detection (ELSD). Screening pure solvents in a binary gradient elution mode allowed, in a first step, to assess the behaviour of the studied solutes and to select the solvents for further mobile phase optimisation. The proportion of the remaining solvents was defined to reach a maximal resolution. The reduction of the analysis time and the enhancement of the signal were obtained by optimising the gradient slope and the flow-rate. Optimal levels of triethylamine and formic acid (TEA-FA) for the enhancement of the evaporative light scattering detector response were established at 0.1% (v/v). Thus, the optimal conditions for the separation of the four glycosphingolipids was obtained with a gradient elution from a 100% chloroform to a 100% acetone:methanol (90:10 (v/v)) mobile phase at 0.2 ml min −1, using a 10% min −1 gradient slope. Finally, this method was applied to detect the excess of one of the neutral sphingolipids, namely globotriaosylceramide (Gb 3) in the urine of patients affected with Fabry disease. A liquid–liquid extraction of the sediments obtained from an aliquot of only ten ml of urine proved sufficient to detect the excess of Gb 3 present in both hemizygote and heterozygote patients. In all, the ability of our method to detect abnormal amounts of Gb 3 in urinary sediments could allow the diagnosis of weakly symptomatic Fabry patients in large screening programs

Mechanism of response enhancement in evaporative light scattering detection with the addition of triethylamine and formic acid.

Triethylamine with an equimolar amount of formic acid added to the mobile phase provides an enhancement of the evaporative light scattering detector (ELSD) response. After characterization of the influence of various parameters on the ELSD response, a sequential strategy was defined to elucidate this response enhancement. The response enhancement was more marked at low mobile phase flow rate, and was highly dependent on solutes and solvents. The influence of drift tube temperature on response enhancement with various solutes demonstrated that triethylamine and formic acid mainly act as mass amplifiers by the inclusion of triethylamine-formic acid clusters inside the droplets.

Advantages of Evaporative Light Scattering Detection for the Purity Control of Commercial Cyclodextrins

Using an Evaporative Light Scattering Detector (ELSD) which is a universal detection system compatible with gradient elution, allowed satisfactory analysis of the non volatile impurities in cyclodextrins. ELSD performances for cyclodextrin analysis were evaluated. The detection capabilities of ELSD are better than those of RI and Polarimetric detectors (detection thresholds obtained with the ELSD are about 80 ng). Analyses were performed on different Phenyl columns with acetonitrilewater mobile phases in isocratic or gradient mode. Potential impurities of β-CD and of TM-β-CD, which included 7 linear (glucose to maltoheptaose), 2 unbranched cyclic (α- and γ-CD) glucose polymers and partially methylated cyclodextrins, have been determined. As the ELSD response factors of these homologous compounds are highly similar, it is possible to evaluate impurity levels without previous identification. The methods presented provide good selectivity and sensitivity and can also be used to compare different sources of cyclodextrins.