Nonchromophoric compounds pose a challenge when it comes to analysis using common ultraviolet-visible (UV-vis) detectors because these analytes do not contain any functionality that leads to absorption and emission of UV or visible light. Evaporative light scattering (ELS), on the other hand, is an ideal technique for the detection of these difficult samples, including carbohydrates, triglycerides, lipids, phospolipids, underivatized fatty and amino acids, and modified peptides as well as small-molecule drugs.
“Because ELS can be used as a universal detector for both chromophoric and non-chromophoric compounds and can be coupled with conventional high-performance liquid chromatography (HPLC) and even ultra high-pressure liquid chromatography (UHPLC), its popularity has been steadily increasing,” notes Jim Anderson, R&D director with Grace Discovery Sciences.
Under optimized conditions, ELS enables rapid and reliable measurement of both quantity and purity, according to Anderson. In addition, ELS detectors respond in a mass-dependent manner that is independent of chemical structure. “For these reasons, we are seeing growing use of ELS used in conjunction with UV-vis and mass spectrometry as a complementary analytical method,” Anderson notes.
The problem with nonchromophoric compounds
Because nonchromophoric compounds do not absorb light in the ultraviolet and visible region of the electromagnetic spectrum, they are not detected by UV-vis systems and are thus not seen in HPLC chromatograms. As a result, researchers are not aware of their presence, and they cannot be identified or quantified. If these compounds co-elute with a desired, chromophoric compound, they could remain undetected as a contaminant in the product.
ELS offers universal detection
Three key steps are involved in the evaporative light-scattering detection process. First, the column effluent is nebulized by passing it through a needle and mixing it with nitrogen gas to form a dispersion of droplets similar to that in an aerosol. The droplets are then passed through a heated “drift tube,” where the mobile phase is evaporated, leaving a fine mist of dried-sample particles in solvent vapor. The sample particles are then passed through a cell and illuminated with light. The light is scattered, and the signal is collected by a detector placed at a 90° angle to ensure that only light scattered by the particles is analyzed.
The results produced by an ELS detector are similar to those generated by a UV-vis detector. The chromatogram has peaks on a baseline, and the peak size is proportional to the amount of material in the sample. The relationship isn’t linear, however, so for quantitative analysis, the preparation of a calibration curve is necessary for any given sample, according to Anderson.
ELS detectors can operate at a temperature range from ambient to about 200 °C, and the optimum temperature program is chosen based on the boiling points of the mobile phase and the analytes. “The only major restriction with ELS is that the mobile phase must be more volatile than the analytes so that it will evaporate and the analyte will not,” Anderson observes.
The biggest advantage of evaporative light scattering is that it is independent of the sample’s optical characteristics, so that any nonvolatile sample, chromophoric or nonchromophoric, can be detected. “As a result, researchers can have more confidence that they are detecting all of the compounds in a sample and that they are estimating the purity of samples more accurately,” Anderson says. In addition, the detection limits are in the nanogram range for very high sensitivity due to low noise and excellent baseline stability. Furthermore, stable baselines can be maintained even when using gradients unlike with refractive index detectors. ELS provides a more accurate representation of sample mass than UV-vis, making it effective for the analysis of impurities. “Many researches find that maximum structural and concentration information can be obtained when they use ELS, MS, and UV-Vis detectors in parallel,” comments Anderson. Recently, low-dispersion ELS detectors have been introduced that can be used effectively with UHPLC systems.
Solution for the analysis of many troublesome biologic samples
Many carbohydrates, peptides, and glycolized peptides are nonchromophoric compounds that can be difficult to detect without the use of ELS. Often, according to Anderson, ELS detectors are employed for the analysis of modified peptides, such as pegylated biotherapeutics because both the desired product and any excess, unreacted polyethylene glycol can both be detected.