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.     

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.     

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.     

Polyketide analysis using mass spectrometry,evaporative light scattering,and charged aerosol detector systems

In this study, a mass spectrometer (MS), an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD) were used to analyze an erythromycin precursor (termed 6-deoxyerythronolide B). The work highlights the capabilities of each detector to analyze a representative polyketide compound that does not possess a natural chromophore, and presents the first comparison to include a charged aerosol system. Each detector was evaluated based upon limit of detection (LOD), dynamic range, and precision in the context of polyketide analysis. Due to its low LOD, wide dynamic range, and ability to provide molecular weight information, the MS was deemed the best detection option for the analysis of low-concentration, poorly identified polyketide compounds. Alternatively, both the CAD and ELSD systems studied showed better precision and accuracy. The ELSD demonstrated the best precision at 3%, but its LOD was limited to concentrations primarily greater than or equal to 1 mg/L. The Corona CAD demonstrated a LOD (0.012 mg/L) and dynamic range comparable to mass spectroscopy and therefore serves as a more cost-efficient alternative for polyketide production schemes with low titers.     

Evaluation of the quantitativeness of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an equimolar mixture of uniform poly(ethylene glycol) oligomers

Quantitativeness of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was elucidated using an equimolar mixture of uniform poly(ethylene glycol) (PEG) oligomers with no molecular weight distributions. Uniform PEG oligomers with degrees of polymerization n = 6-40 were separated from commercial PEG samples by preparative super-critical fluid chromatography. MALDI-TOF mass spectra of an equimolar mixture of the uniform PEG oligomers were recorded by adding a mixture of 2,5-dihydroxybenzoic acid as a matrix reagent and four chlorinated salts, i.e. LiCl, NaCl, KCl and RbCl. Remarkable non-quantitative effects were observed in the MALDI-TOF mass spectra in both the lower and higher molecular mass regions. At higher molecular masses greater than about 10(3), PEG oligomers with larger molecular mass yielded lower spectral intensities irrespective of the species of adduct cations and higher laser powers induced larger decreases in mass spectral intensities with the increase in their molecular masses. On the other hand, in the lower molecular mass region, less than about 10(3), the observed non-quantitative effect greatly depends on the species of adduct cations, indicating that the stability of the PEG-cation complex affects the MALDI-TOF mass spectral intensities of uniform PEG oligomers.     

Linearization of evaporative light scattering detector signal

Recent advances in nebulizer technology of the evaporative light scattering detector (ELSD) allow the evaporation of trichlorobenzene (TCB) at much lower temperatures than the previous instruments, thus avoiding the sample loss in the lower molecular weight domain. Therefore, the new ELSD opens the possibility to correctly evaluate the molecular weight distribution (MWD) of polymers by gel permeation chromatography, after linearizing the ELSD signal intensity in function of concentration. To find the correct exponent parameter for linearization, it is necessary to take into account not only the peak area, but each point of the chromatogram. The evaluation method for this exponent parameter, found to be 1.61 for the analysis in TCB of polyolefin and polystyrene samples, is presented in this study. This value was verified by the excellent correlation found between the obtained MWD of a high-density polyethylene both with the ELSD and with the traditional differential refractive index detector.     

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.     

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.     

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.     

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.

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.     

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.     

Precise measurement of the self-diffusion coefficient for poly(ethylene glycol) in aqueous solution using uniform oligomers

Uniform poly(ethylene glycol) (PEG) oligomers, with a degree of polymerization n=1-40, were separated by preparative supercritical fluid chromatography from commercial monodispersed samples. Diffusion coefficients, D, for separated uniform PEG oligomers were measured in dilute solutions of deuterium oxide (D(2)O) at 30 degrees C, using pulsed-field gradient nuclear magnetic resonance. The measured D for each molecular weight was extrapolated to infinite dilution. Diffusion coefficients obtained at infinite dilution follow the scaling behavior of Zimm-type diffusion, even in the lower molecular weight range. Molecular-dynamics simulations for PEG in H(2)O also showed this scaling behavior, and reproduced close hydrodynamic interactions between PEG and water. These findings suggest that diffusion of PEG in water is dominated by hydrodynamic interaction over a wide molecular weight range, including at low molecular weights around 1000.     

Application of size exclusion chromatography with surfactant eluent to the study of polymer–surfactant interactions: oligomeric and micellar chromatographic effects

Complexation of sodium dodecyl sulphate (SDS) with a wide range of molecular weights of poly(ethylene glycol) (PEG) and poly(ethylene oxide) (PEO) has been studied by size exclusion chromatography using aqueous SDS eluent. A multi-angle laser light scattering detector and a differential refractometer were applied to give direct measurement of the molecular weight of complexes without reference to elution volume, since the latter is not a reliable indicator of the complex size. Background light scattering from micellar eluents hampered quantitative size measurements, but was minimal in sub-micellar eluent, where saturated binding was observed for polymers larger than 1000 g mol⁻¹. Multiple peaks and voids were observed in the elution profiles of low molecular weight polymers (up to a mass of 600 g mol⁻¹) in eluent at micellar concentrations. Several sources contribute to this behavior, including micellar chromatographic separation of the PEG oligomers due to their different distribution coefficients between the micellar and water phases. Preliminary results are reported for distribution coefficients of individual oligomers in a 600 g mol⁻¹ PEG sample. Three distinct binding behaviors are observed with increasing degree of polymerization of PEG: no interaction for small glycols, equilibrium partitioning of intermediate oligomers in and out of micelles, and binding of micelles to the larger polymers.     

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.     

HPLC-electrospray mass spectrometry for the analysis of temoporfin-poly(ethylene glycol) conjugates

The photodynamic therapeutic agent temoporfin, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) conjugated with poly(ethylene glycol) 2000 (PEG), has been analysed by high performance liquid chromatography (HPLC), linked to electrospray ionisation mass spectrometry (ESI-MS). Sufficient separation of m-THPC-PEG 2000 oligomers was achieved, enabling determination of molecular mass. The use of ESI-MS alone could not achieve this, because of too great a complexity in the mass spectrum, resulting from the presence of four PEG 2000 side chains with a wide molecular mass distribution. The technique is applicable to similar PEG conjugated compounds.     

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.     

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.     

Certification and uncertainty evaluation of the certified reference materials of poly(ethylene glycol) for molecular mass fractions by using supercritical fluid chromatography

Poly(ethylene glycol) (PEG) is a useful water-soluble polymer that has attracted considerable interest in medical and biological science applications as well as in polymer physics. Through the use of a well-calibrated evaporative light-scattering detector coupled with high performance supercritical fluid chromatography, we are able to determine exactly not only the average mass but also all of the molecular mass fractions of PEG samples needed for certified reference materials issued by the National Metrology Institute of Japan. In addition, experimental uncertainty was determined in accordance with the Guide to the expression of uncertainty in measurement (GUM). This reference material can be used to calibrate measuring instruments, to control measurement precision, and to confirm the validity of measurement methods when determining molecular mass distributions and average molecular masses. Especially, it is suitable for calibration against both masses and intensities for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Figure Comparison between the molecular mass fractions of PEG 1000 before calibration (si) (○) and after calibration (wi) (⧫). The error bar shows the expanded uncertainty of k = 2 of each mass fraction