Evaporative light scattering detector is a general-purpose detector, which can analyze any compound whose volatility is lower than that of mobile phase. The principle is as follows: firstly, the column eluent is atomized to form aerosol, then the solvent is evaporated in the heated drift tube, and finally the remaining nonvolatile solute particles are detected in the light scattering detection cell.
Differential detection is disturbed by the front peak of solvent, which complicates the analysis, and the baseline is very unstable and incompatible with gradient elution due to its extremely sensitive to temperature. In addition, the response of the differential detector is not as sensitive as ELSD; The low wavelength UV detector is plagued by baseline drift under the condition of sharp gradient, and the analyzed compound is required to have chromophores. ELSD is not subject to these restrictions. Different from these detectors, ELSD can obtain a stable baseline in the case of multi solvent gradient, which makes the resolution better and the separation speed faster. In addition, because the response of ELSD does not depend on the optical properties of the sample, the sample is not required to contain chromophores or fluorescent groups during ELSD detection.
The column eluent separated by HPLC enters the atomizer, where it is mixed with stable atomized gas (generally nitrogen) to form aerosol. The aerosol consists of uniformly distributed droplets whose size depends on the gas flow rate used in the analysis. The lower the gas flow rate, the larger the droplet formed, and the larger the droplet, the more scattered light, which improves the analysis sensitivity, but the larger the droplet, the harder it is to evaporate in the drift tube. Each method has an optimized gas flow rate that produces the best signal-to-noise ratio. The lower the flow rate of mobile phase, the lower the gas flow rate requiring proper atomization. Using a micro diameter column with an inner diameter of 2.1mm to replace the standard analytical column with an inner diameter of 4.6mm can greatly reduce the flow rate of mobile phase and improve the sensitivity of analysis.
The volatile components in the evaporated aerosol evaporate in a heated stainless steel drift tube. Setting the appropriate drift tube temperature for a particular application depends on the mobile phase composition and flow rate, as well as the volatility of the sample. The drift tube temperature required for evaporation of high organic content mobile phase is lower than that of high water content mobile phase. The lower the flow rate of the mobile phase, the lower the temperature of the drift tube required for evaporation. Semi volatile samples require a lower drift tube temperature to obtain the best sensitivity. The optimum temperature needs to be determined by observing the signal-to-noise ratio at each temperature.
Detection: the sample particles suspended in the mobile phase steam enter the light scattering detection cell from the drift tube. In the detection cell, the sample particles scatter the light emitted by the laser light source, while the evaporated mobile phase does not scatter. The scattered light is detected by the silicon photodiode, generates an electrical signal, transmits the analog signal output port, and is used for data acquisition of analog output.
Application advantages of ELSD
- The response value of evaporative light scattering has nothing to do with the functional groups and optical properties of the measured substances, so it can be used for the detection of various substances. The detection of macromolecular organic compounds with no characteristic UV absorption or only UV terminal absorption shows great advantages.
- The sample pretreatment process is simplified and the analysis time is shortened.
- The baseline is stable and the sample detection sensitivity is high. The evaporation of mobile phase makes ELSD compatible with gradient elution, which can improve the resolution of chromatographic separation and shorten the analysis time.
- Only one standard can quantify two or more unknown substances, so unknown compounds can be quantified by comparing with internal standard. This characteristic is particularly useful for the quantification of compounds synthesized by combinatorial technology in combinatorial chemistry.
- The selected unknown compounds have similar structures but different molecular weights, and the quantitative accuracy is higher.
- It is difficult to quantify unknown compounds by UVD. The UV absorption value of the sample is often independent of the chromatographic peak representing the sample quality. For ELSD, its response does not depend on the optical characteristics of the sample and represents the composition of the sample. The detection signal is more direct and represents the real concentration of the sample, which is directly proportional to the mass concentration of the sample. Therefore, it can be used to determine the purity of the sample or detect unknown substances.
Precautions and supplements
- The eluent needs to be atomized, so the purity and pressure of atomized gas flow will affect the signal-to-noise ratio of the detector.
- The mobile phase needs to evaporate, so non-volatile substances cannot be used to adjust the pH value of the mobile phase. The components with lower boiling point than the measured substance can be evaporated by adjusting the evaporation temperature. On the premise of not evaporating the measured substance, the higher the temperature, the more complete the evaporation of the mobile phase, the better the baseline of the chromatogram and the higher the signal ratio. The concentration is not linear with the peak area, which is linear after taking the natural logarithm respectively.