Performance evaluation of evaporative light scattering detection and charged aerosol detection in reversed phase liquid chromatography

In pharmaceutical industry ultraviolet (UV) detection is often used as the preferred detection technique in HPLC analysis since most pharmaceutical compounds possess a UV-absorbing chromophore. However, in case the active pharmaceutical ingredient (API) does not have a UV-absorbing chromophore, or if some of the impurities present lack a chromophore, they will not be detected in routine HPLC analysis employing only a UV detector and alternative detection schemes have to be used. Refractive index detection or mass spectroscopy (MS) can be used but these detectors have their intrinsic weaknesses, such as lack of sensitivity or high cost. With the appearance of semi-universal techniques such as evaporative light scattering detection (ELSD), and more recent, charged aerosol detection (CAD), detection of non-UV-absorbing compounds became feasible without having to resort to such complex or costly detection methods. This paper evaluates the different performance characteristics such as sensitivity, linearity, accuracy and precision of both the ELSD and CAD detector coupled to HPLC. One disadvantage of this type of detector is the non-linear response behaviour which makes direct linear regression for making calibration curves inaccurate.     

Comparison of two aerosol-based detectors for the analysis of gabapentin in pharmaceutical formulations by hydrophilic interaction chromatography

Comparison of hydrophilic interaction chromatography (HILIC) columns coupled with an evaporative light scattering detector (ELSD) or charged aerosol detector (CAD) was done for the detection of gabapentin in pharmaceutical formulations. The chromatographic separations were achieved on four HILIC columns: ZIC HILIC, ZIC pHILIC, Luna HILIC, and Atlantis HILIC. Experimental factors such as mobile phase composition, acetonitrile content, and mobile phase pH were evaluated. Validation of method was done in terms of linearity, sensitivity, accuracy, and precision. The performance of ELSD detection method is comparable to that of CAD. The intra-day and inter-day variations were below 1.7% and 3.2% for CAD and 2.8%, and 3.4% for ELSD, respectively. In addition, detection sensitivities of ELSD, CAD, and UV detectors were also compared for HILIC and reversed phase (RP) modes and the highest sensitivities were obtained in the HILIC mode when connected with CAD and ELSD. The developed HILIC aerosol based detection methods were successfully applied to the analysis of gabapentin in commercial tablets and capsules.     

Evaluation of a condensation nucleation light scattering detector for the analysis of synthetic polymer by supercritical fluid chromatography

A condensation nucleation light scattering detector (CNLSD) was adapted for use as a detector for supercritical fluid chromatography. The performance of the CNLSD was evaluated and compared to evaporative light-scattering detector (ELSD) using a well-defined equimass mixture of uniform poly(ethylene glycol) oligomers and a certified reference material of poly(ethylene glycol) 1000. The CNLSD was able to detect a 10 times less concentrated solution of uniform oligomers compared to the ELSD. The quantitativeness of CNLSD was high enough to obtain the molecular mass distribution of poly(ethylene glycol) 1000 without any calibrations; on the other hand, the original data measured by ELSD was about 4% smaller than the certified value of poly(ethylene glycol) 1000. The CNLSD was suitable for supercritical fluid chromatography as a mass concentration detector.     

Quantitative comparison of a corona-charged aerosol detector and an evaporative light-scattering detector for the analysis of a synthetic polymer by supercritical fluid chromatography

A corona-charged aerosol detector (CAD) was developed to improve the sensitivity, reproducibility and quantitativeness of detection as compared to evaporative light-scattering detector (ELSD) for liquid chromatography. Our laboratory used the corona CAD as a detector for supercritical fluid chromatography (SFC) and evaluated its performance compared to the ELSD by using a certified reference material of poly(ethylene glycol) (PEG) and a well-defined equimass mixture of uniform PEG oligomers. The corona CAD was able to detect a 10 times more dilute solution of uniform oligomers compared to the ELSD. Although the original data of molecular mass by ELSD was 4.6% smaller than the certified value of PEG 1000, molecular mass distribution obtained by corona CAD was virtually almost the same as the certified value without any calibrations.     

Separation and determination of carbohydrates in drinks by ion chromatography with a self-regenerating suppressor and an evaporative light-scattering detector

Analysis of glucose and other carbohydrates are often performed by use of normal phase HPLC methods with acetonitrile as major eluent coupled with evaporative light-scattering detector (ELSD) or by use of anion-exchange ion chromatography (IC) methods with NaOH as eluent coupled with pulsed amperimetric electrochemical detector. In this work, a novel method for the determination of carbohydrates by IC in conjunction with a self-regenerating suppressor and an ELSD detector was investigated. Three carbohydrates (glucose, fructose, and sucrose) were separated using a KOH eluent generator to avoid the effect of carbon dioxide absorption in the alkaline eluent. Due to the use of the suppressor, non-volatile components were removed and a low salt background (K+ approximately 0.070 microg/mL) can be obtained so the suppressed eluent could directly go into an ELSD detector without obvious interference of inorganic salts. After examining the changes in retention and resolution, an optimized method was established (for IC: using 32 mM KOH as the eluent at a flow rate of 1 mL/min; for ELSD: operated at 95 degrees C, 4.0 bar nitrogen with a gas flow rate of 2.0 L/min) and the linearity, reproducibility, and the limit of detection (LOD) for the three carbohydrates were further evaluated. Regression equations revealed acceptable linearity (correlation coefficients=0.994-0.998) across the working-standard range (100-1000 microg/mL for glucose and sucrose, 150-1000 microg/mL for fructose) and LODs of glucose, fructose, and sucrose were 93, 126, and 90 microg/mL, respectively. This method has successfully been applied to the determination of the three carbohydrates in carbonated cola drinks and fruit juices. The recoveries were between 95 and 113% (n=3) for different carbohydrates.     

Polyolefin characterization in dibutoxymethane by high temperature gel permeation chromatography with a new evaporative light scattering detector

A new evaporative light scattering detector (ELSD) for the analysis of polyolefins by high temperature gel permeation chromatography (GPC) was recently introduced by Agilent Technologies. For the first time, we investigated the possibility to use this detector to measure the molecular weight distributions (MWD) of different types of polyolefins (polypropylene, linear and low-density polyethylene) in dibutoxymethane (DBM, butylal). These samples were previously characterized by GPC in trichlorobenzene (TCB) with a differential refractive index (DRI) detector in an interlaboratory study conducted by the International Union of Pure and Applied Chemistry (IUPAC) [1], and in a recent publication by GPC with the new ELSD in TCB [2]. The signal to noise of ELSD using DBM is about 10 times lower than that for TCB. However, the ELSD signal power exponent for DBM was measured as 1.35, which is much closer to unity than the value of 1.61 for TCB. After applying the required corrections to linearize the response of the ELSD signal as a function of concentration, similar average molecular weights to those measured in the interlaboratory study using DRI, were obtained for the analyzed resins.     

Analysis of triacylglycerols on porous graphitic carbon by high temperature liquid chromatography

The retention behaviour of several triacylglycerols (TAGs) and fats on Hypercarb, a porous graphitic carbon column (PGC), was investigated in liquid chromatography (LC) under isocratic elution mode with an evaporative light scattering detector (ELSD). Mixtures of chloroform/isopropanol were selected as mobile phase for a suitable retention time to study the influence of temperature. The retention was different between PGC and non-aqueous reversed phase liquid chromatography (NARP-LC) on octadecyl phase. The retention of TAGs was investigated in the interval 30-70 degrees C. Retention was greatly affected by temperature: it decreases as the column temperature increases. Selectivity of TAGs was also slightly influenced by the temperature. Moreover, this chromatographic method is compatible with a mass spectrometer (MS) detector by using atmospheric pressure chemical ionisation (APCI): same fingerprints of cocoa butter and shea butter were obtained with LC-ELSD and LC-APCI-MS. These preliminary results showed that the PGC column could be suitable to separate quickly triacylglycerols in high temperature conditions coupled with ELSD or MS detector.     

Determination of gentamicin sulfate and related compounds by high-performance liquid chromatography with evaporative light scattering detection

A rapid and simple method for the separation and quantitation of gentamicin sulfate by HPLC coupled with evaporative light scattering detection (ELSD) has been developed. Detection of the different components of gentamicin is problematic because of the lack of UV absorbing chromophore. The use of the universal ELSD avoids the need for sample derivatization or use of specific detector such as pulsed amperometry. Separation was performed on a highpurity C18 125 mm x 4 mm i.d., 3 microm, reversed phase column with 48.5 mM trifluoroacetic acid-methanol (97:3, v/v), as mobile phase at a flow rate of 0.7 ml/min. The influence of the gas nature, gas pressure and temperature of the drift tube of the detector on the detection response was investigated. Optimization was performed with the help of a specific experimental design software. This method allows the determination of the composition in components C1, C1a, C2, C2a and C2b of gentamicin sulfate samples. Mass spectrometry was employed to confirm the ELSD chromatographic profile. The method was validated using methodology described by the International Conference of Harmonization in the field of Medicinal Substances. Commercial samples of different sources were analyzed and results were in good agreement with specifications of both European and United States Pharmacopoeia.     

Calibration of an evaporative light-scattering detector as a mass detector for supercritical fluid chromatography by using uniform Poly(ethylene glycol) oligomers

The quantitativeness of an evaporative light-scattering detector (ELSD) for supercritical fluid chromatography (SFC) was evaluated by using an equimass mixture of uniform poly(ethylene glycol) (PEG) oligomers. Uniform oligomers, in which all molecules have an identical molecular mass, are useful for the accurate calibration of detectors. We calibrated the SFC-ELSD system for various concentrations and molecular masses by using an equimass mixture of PEG oligomers. ELSD not only showed a good linear response to the injected concentration over a wide concentration range, from 10(-4) to 10(-1)g/mL, but also showed a strong dependence on the molecular mass of the solute. By using chromatograms of the equimass mixture of uniform oligomers to calibrate SFC-ELSD, it was possible to determine exact values of not only the average mass but also the molecular-mass distribution for a PEG 1540 sample. The average molecular mass was shifted to a higher value by several percentage points after calibration of the ELSD.     

Micro liquid chromatography coupled with evaporative light scattering detector at ambient and high temperature: optimization of the nebulization cell geometry

The recent developments in liquid chromatography (LC) are mainly dedicated to both system miniaturization (micro-, capillary-, and nano-LC) and analysis time decrease (fast-, and ultra-fast-LC). For the latter, several strategies can be used, and high temperature liquid chromatography (HTLC) seems very promising and easy to implement, especially in miniaturized system. In LC, the evaporative light scattering detector (ELSD) is considered an attractive alternative to conventional detector such as UV-vis due to its versatility and quasi-universality. Therefore, the compatibility of ELSD with micro-LC and micro-HTLC was investigated for several pharmaceutical compounds of interest. The nebulization process appeared to be the most critical parameter for performing the coupling and maintaining an efficient separation. Therefore, appropriate modifications in the nebulization cell geometry were brought to make ELSD fully compatible with micro-LC. The impact of optimized nebulization cell on chromatographic performance was evaluated in terms of efficiency and sensitivity. Finally, highly efficient, sensitive and fast separations of pharmaceutical drugs were performed with both techniques and the customized nebulization cell design.     

Comparison of universal detectors for high-temperature micro liquid chromatography

This study compares, through micro high-temperature liquid chromatography (microHTLC), three commercial universal detectors that allow a direct detection of lipids. The detectors are: the charged aerosol detector (CAD), the evaporative light-scattering detector (ELSD) and the ion trap mass spectrometer with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) sources (APCI-MS and ESI-MS). This study shows the feasibility to use the high temperature with these detectors and hybrid behavior between concentration and mass flow rate detector in microHTLC. The detectors were compared in terms of response intensity, linearity and limit of detection for different high temperatures. The charged aerosol detector shows a linear response from 5 to 500 microg/mL and the correlation coefficients (r(2)) obtained for squalene, cholesterol and ceramide IIIB exceed 0.99.     

Use of evaporative light scattering detector in the detection and quantification of enantiomeric mixtures by HPLC

Routinely used in our laboratories at analytical scale, an evaporative light scattering detector (ELSD) has proved to be versatile in the detection of enantiomeric resolution using chiral stationary phases by HPLC. Though this kind of detector has been widely used in various domains, its application in enantiomeric resolution has not been discussed in the literature and is found to have very specific features especially in the quantitative perspective. In contrast with the UV detection, the peak area from ELSD for both enantiomers of a racemic mixture may not be the same. This complicates the assessment of the enantiomeric purity of unknown samples. This current work deals with some practical aspects in the detection of enantiomers and in accurate quantitative determination of enantiomeric purity by ELSD. Effects of analyte nature (more precisely molecular weight and volatility), peak shape and peak shape difference between enantiomers on the quantitative integration by ELSD are discussed in connection with the UV-detection results. The calibration for quantitative enantiomeric analysis and its effectiveness are demonstrated.     

Control of impurities in l-aspartic acid and l-alanine by high-performance liquid chromatography coupled with a corona charged aerosol detector

In this study a reversed phase ion-pair high-performance liquid chromatography (HPLC) method using charged aerosol detection (CAD) was developed and fully validated for the pharmaceutical quality control of l-aspartic acid (Asp). With a slight modification, the method also allows the evaluation of related substances in l-alanine (Ala). The method enables simultaneous control of related amino acids and of possibly occurring organic acids contaminants. A minimum limit of quantification of 0.03% could be achieved for all occurring related substances. Moreover, the detector sensitivity of the CAD was compared with an evaporative light scattering detector (ELSD). Depending on the analyte the CAD was found to be 3.6–42 times more sensitive than the ELSD. The HPLC method was applied to the purity testing of 8 samples of pharmaceutical grade and reagent grade Asp and of 12 samples of Ala supplied by various manufacturers. Both substances were found to be of high purity (greater than 99.8% for Asp and greater than 99.9% for Ala). Malic acid and Ala were the major impurities in Asp. Asp and glutamic acid (Glu) were the only detectable impurities in Ala.     

六味地黄丸的串联双检测器高效液相色谱分析

采用紫外检测器与蒸发光散射检测器串联的方式构建了高效液相色谱分析系统,并用于对六味地黄丸的分析研究。紫外检测器共检测到26个色谱峰;蒸发光散射检测器检测到31个色谱峰,其中15个色谱峰为可以认证的共有峰。所构建的系统适宜于中草药的指纹图谱研究。     

Packed column supercritical fluid chromatography with light-scattering detection. I. Optimization of parameters with a carbon dioxide/methanol mobile phase

Summary Evaporative light scattering detectors have, in recent years, gained acceptance in chromatography with dense mobile phases i.e. liquid and supercritical fluid chromatography. In the present work an instrument of this type has been used in packed column supercritical fluid chromatography with carbon dioxide/methanol mixtures. Detector response and signal-to-noise ratios have been determined using squalane as test compound. Nebulizer gas flow, evaporator temperature, photomultiplier sensitivity, and mobile phase composition were found to have an influence on instrument performance. With this type of detector the field of packed column SFC applications can be extended to include non-UV-absorbing substances even when mixed mobile phases or composition gradients are necessary for the separation.     

Fatty acids by high-performance liquid chromatography and evaporative light-scattering detector

A high-performance liquid chromatographic (HPLC) separation method with an evaporative light-scattering detector (ELSD) has been developed for the separation and quantitative analysis of fatty acid methyl esters (FAME) in three different oils. Reverse-phased C18 HPLC separation of 13 FAME is achieved using a methanol/water eluent mixture. The retention times (RT) reflect the elution behavior of these compounds on C18 reversed-phase HPLC. The proposed method is tested on: soybean oil (Glycine max L.) as reference sample, rice bran oil (Oryza sativa L.), pumpkin seed oil (Cucurbita pepo L.) and algal oil (Arthrospira platensis Nordst.).     

Analysis of carbohydrates in drinks by high-performance liquid chromatography with a dynamically modified amino column and evaporative light scattering detection

A high-performance liquid chromatographic method with a dynamically modified amino column and evaporative light-scattering detector (ELSD) was established for the direct analysis of the carbohydrates in some drinks. A separation column (Zorbax Rx-SIL, 250 mm x 4.6 mm I.D., 5 microm, Hewlett-Packard, USA) which was modified by ethylenediamine and a guard column (Zorbax Rx-SIL, 12.5 mm x 4.6 mm I.D., 5 microm) were used. The mobile phase was a mixture of water-acetonitrile (1:2.6, v/v) containing 0.03% (v/v) ethylenediamine. Regression equations revealed linear relationship (correlation coefficients=0.996-0.999) between the mass of carbohydrates injected and the carbohydrates peak areas detected by ELSD. The detection limits of ELSD (S/N=3) were between 0.2 and 1.2 microg for different carbohydrates. This method is simple and sensitive.     

Determination of artemisinin and artemisinic acid by capillary and packed supercritical fluid chromatography

Artemisinin (an antimalarial compound) and its bioprecursor artemisinic acid, present in the plant Atemisia annua L., were analyzed by supercritical fluid chromatography (SFS) using capillary and packed columns, coupled respectively with a flame ionization detector (FID) and an evaporative light scattering detector (ELSD). Both methods were optimized and validated with columns of different polarity in order to separate artemisinin and artemisinic acid. Analytical results were comparable, but the paced SFC-ELSD method was faster. Indeed, artemisinin and artemisinic acid were separated with an aminopropyl silica column in less than 8 minutes instead of about 25 minutes by capillary SFS. Contrary to conventional gas and liquid chromatography coupled to an UV-visible detector, SFS methods determined both compounds directly, without degradation and/or derivatization in the concentration range expected in the plant material. Results obtained on plant extracts by capillary SFS-FID and packed SFS-ELSD were confirmed by GC-MS.     

Quantification in the analysis of polyethylene glycols and their monomethyl ethers by liquid adsorption chromatography with different detectors

It is shown that the molar mass distribution of polyethylene glycols (PEGs) and their monomethyl ethers can be determined by liquid adsorption chromatography (LAC) on reversed phases using isocratic or gradient elution. In gradient LAC, the evaporative light scattering detector (ELSD) has to be used, which is, however, problematic with respect to quantification. The response factors of the individual oligomers depend strongly on the operating conditions, molar mass, and sample size. These problems do not arise with density and refractive index detection, which can, however, only be applied with isocratic elution. A comparison of the results obtained with these three detectors showed that calibration of the ELSD has to be performed very carefully.

Size exclusion chromatography with Corona charged aerosol detector for the analysis of polyethylene glycol polymer

A technique of using size exclusion chromatography (SEC) with the Corona charged aerosol detector (CAD) was developed and evaluated in comparison with refractive index (RI) and evaporative light scattering detection (ELSD) for fast screening of polyethylene glycol (PEG), a polymer used in preparing pegylated pharmaceutical compounds. These detection techniques were used in the analysis of multiple lots of PEG reagents. CAD was found to provide more accurate impurity and polydispersity profiles of PEG reagents that better differentiate their quality, while RI was not suitable for this application due to its low sensitivity and ELSD led to underestimation of the impurity and polydispersity. The accuracy of polydispersity determination by SEC-CAD was validated against a commercial reference standard of known polydispersity. The SEC-CAD technique and the observed differences between the three detectors can also be applied to polymer analysis in general.