Techni cal Repor t vol . 6Principles and Practical Applications of Shimadzu's ELSD-LT 2

Evaporative Light Scattering DetectorC190-E1081. I nt roduct i onIn the history of high-performance liquid chromatographs, which dates to the early 1960s, refractive index detectors (RI detectors) have often been used as general-purpose detectors. RI detectors enable the detection of components that do not possess UV absorbance and give a proportional relationship between the heights of detected peaks and the quantities of detected components. So, in comparison with absorbance detectors (UV detectors), they offer advantages such as the ability to ascertain unknown component quantities and obtain molecular weight distributions for macromolecules. On the other hand, they also have various disadvantages. For example, they cannot be used for gradient analysis, the baselines they produce are susceptible to the influence of fluctuations in the ambient temperature, their sensitivity is low compared to that of UV detectors, and they are prone to giving negative peaks, which make quantitative analysis difficult. Furthermore, with both UV and RI detectors, in cases where the solvent peaks of the analyzed samples appear at the start of the chromatogram, it is sometimes not possible to detect target substances with short elution times. Therefore, RI detectors cannot truly be described as general-purpose detectors. The principle of evaporative light scattering detectors (ELSD), which solve these problems, is extremely simple. The target components are converted to a fine spray by a nebulizer and heated so that only the mobile phase is evaporated. Light is directed at the remaining target substances and the scattered light is detected. ELSDs can detect almost all components that are less volatile than the mobile phase. These detectors first appeared in 1966, but were subsequently overshadowed by high-performance liquid chromatographs, which advanced significantly at that time. They were first commercialized in the early 1980s, and the basic technology of modern-day ELSDs was established in the mid-1980s. In addition to describing the operating principles and practical benefits of ELSDs, this article uses the ELSD-LT 2, a product that achieves greater sensitivity, speed, and convenience than conventional products by incorporating the latest technology, to present application examples that utilize ELSD characteristics.22. What I sanEvaporat i veLi ght Scat t eri ngDet ect or( ELSD) ?The target substances separated in a column are, together with the mobile phase, converted to a fine spray by a nebulizer, and this spray is carried to a drift tube. In the drift tube, heat is applied so that only the mobile phase is evaporated. The remaining target substances that were in the mobile phase are converted to minute solid particles and are carried to the detection unit. In the detection unit, the target substance particles cause the light emitted from a light source to be scattered. This scattered light is measured by a photomultiplier and the target substances are thereby detected. The intensity of the signal detected in the ELSD can be represented by the following equation:(Signal intensity) = a x (Quantity of target substance)bHere, "a" and "b" are constants that are determined by a variety of factors, such as the size of the particles, the concentration and type of the target substances, the gas flow rate, the mobile phase flow rate, and the temperature of the drift tube. In principle, ELSDs are capable of analyzing all substances that have an evaporation temperature lower than that of the mobile phase, and can attain roughly the same level of detection sensitivity for any compound. For this reason, they are well-suited to the detection of components such as sugars, fats, surfactants, synthetic macromolecules, and steroids, as these components have low light absorbance, making them difficult to detect with UV detectors. If the target substances are nonvolatile, detection is possible down to the nanogram level in nearly all cases.One of the most important considerations when performing high-sensitivity analysis with an ELSD is the set temperature of the drift tube. In particular, when analyzing semi-volatile substances with low boiling points, the intensity of the signals obtained varies greatly with this set temperature. If it is too high, semi-volatile substances may be partially or completely evaporated together with the mobile phase; consequently, and the quantity of minute solid particles that scatter light maydecrease. This may lead to a reduction in detection sensitivity or make detection completely impossible. In such cases, decreasing the set temperature of the drift tube increases the number of target substance particles present after evaporation, and makes it possible to attain a higher signal intensity. Fig.2 illustrates the differences in the peak heights that are obtained for caffeine, which is a semi-volatile substance, at different set temperatures. It can be seen that the peak height for a set temperature of 30C is roughly 10 times the peak height for a set temperature of 50C.Fig. 1 Flow of ELSD AnalysisFig. 2 Variation in Sensitivity for Different Set Temperatures1. Separation with HPLC 2. Nebulization 3. Evaporation of mobile phase 4. DetectionFl owof anal ysi sThe mobile phase is converted into a spray of minute droplets using a gas stream.The droplets are carried into a heated drift tube where the mobile phase evaporates and the target components dry and are converted into minute particles.The scattered light created by the collision of light with the minute particles that emerge from the drift tube is detected.33. ELSDsandRI Det ect orsLike RI detectors, ELSDs are classified as general-purpose detectors but they differ from RI detectors in the following ways: 1.They are 5 to 10 times more sensitive than RI detectors. 2. They support the use of the gradient elution method. 3. They are not easily affected by changes in the ambient temperature. 4. They are not affected by interference due to solvent peaks. 5. Time is not required to allow for the instrument and the baseline to stabilize.Although the gradient elution method is effective for the batch analysis of, for example, multiple components in natural products, RI detectors cannot be used for this because of fluctuations in the baseline caused by changes in the refractive index of the mobile phase.With ELSDs, baseline fluctuations do not occur in gradient elution, meaning this method can be used to perform the efficient, high-sensitivity analysis of multiple components.This feature of ELSDs is useful for the following types of analysis: 1.The analysis of compounds that cannot be detected with UV detectors:Carbohydrates (sugars), sugar alcohols, alcohols, terpenoids, surfactants, natural macromolecules, and synthetic macromolecules 2.The batch analysis of compounds for which the gradient elution method is difficult to use because absorbance occurs only in the short wavelength region:Fats, phospholipids, glycerides, fatty acids, natural macromolecules, and synthetic macromoleculesAlso, because ELSDs can be applied to all aspects of the methods used for LC/MS, including the mobile phases, they can be used as substitute detectors in place of expensive LC/MS instruments in the screening of compounds.Fig. 3 Variation in Sensitivity for Different DetectorsTable 1 Analytical ConditionsSampleColumnMobile PhaseFlow RateDetection(ELSD)Previous ELSDRI detectorFructose, GlucoseSucrose, MaltoseAsahipak NH2P-50 (250mmL. x 4.6mm i.d.)Acetonitrile/ Water = 7/3 (v/v)1mL/minTemperature: 35CGas Pressure: 350kPa44. Feat uresof ELSD- LT24- 1. Hi gh- Sensi t i vi t yDet ect i onof Semi - Vol at i l eSubst ances Achi evedwi t hLow- Temperat ureEvaporat i onof Mobi l ePhaseWith an ELSD, the larger the nebulized droplets, the higher the evaporation temperature must be set in order to evaporate them. If analysis is performed at a low temperature, the larger droplets that are not evaporated create scattered light that gives rise to a high level of noise. The ELSD-LT2 solves this problem by incorporating a glass cell with a unique structure. Minute droplets that leave the ELSD-LT2 nebulizer are carried by the nebulizer gas stream through the glass cell and into the drift tube. Larger droplets, however, are not carried by the nebulizer gas stream and adhere to the inside surface of the glass tube, where they change to liquid form. This liquid accumulates in a siphon tube and is subsequently discharged. There is always waste liquid in the siphon section, so all of the nebulizer gas flows into the drift tube (siphon split method). In this way, the larger droplets that cause noise are separated selectively, and the smaller droplets are efficiently carried into the drift tube. This technology makes it possible for the ELSD-LT2 to suppress noise, even at low evaporation temperatures. Because mobile phases can be evaporated at low drift tube set temperatures in the range of 35C (organic solvent mobile phases) to 40C (aqueous mobile phases), efficient, high-sensitivity analysis is possible for nearly all compounds. Also, with the ELSD-LT2, assisting gas is projected in a cylindrical shape centering on the drift tube outlet. This increases the concentration of the target substances that reach the detection unit from the drift tube and, consequently, increases sensitivity.In this way, with both a reduction in noise achieved through low-temperature evaporation technology and an increase in peak intensity achieved through superior detection technology, the ELSD-LT2 realizes high-sensitivity detection.Fig. 4 Structure of Nebulizer and Glass Cell (Siphon Split Method)The assisting gas that merges with the flow of sample particles just before they reach the detection unit causes the particles to converge on the focal point of the light source, en high-sensitivity detection. Also, because the particles are not dispersed in the detection unit, there is little contamination of the measurement system, and frequent maintenance is not required.Glass cellNebulizerNebulizer gasMobile phaseTo the drainage outletPhotomultiplierAssisting gas Assisting gasLight sourceTo the drift tubeTo the drift tubeQuantity of dropletsTo the drainage outletSmall Size of droplets LargeFig. 5 Assisting Gas Focusing (Detection Unit)4- 2. MoreUserFri endl ySetting the drift tube temperature and gain is the only preparation required to perform analysis with the ELDS-LT2. It is also possible to turn off the lamp and stop the nebulizer after the completion of analysis using the auto power-down function. The use of a long-life LED lamp and the auto power-down function enable reductions in the frequency of lamp replacement and the consumption of nebulizer gas, and thereby making it possible to reduce running costs. Furthermore, an automatic cleaning function for the drift tube helps make maintenance easier.55. Hi gh- SpeedAnal ysi sUsi ngt heELSD- LT2The ELSD-LT2 supports ultrahigh-speed analysis, which has received much attention in recent years, using the technology described below. Backed by the detecting versatility that is characteristic of ELSDs, it enables efficient execution of the batch analysis of multiple components in natural products, something that calls for ultrahigh-speed analysis. 1. High-speed sampling rate that helps produce sharp peaks (20 msec) 2. Suppression of peak spreading by reduction of the drift tube's innert diameter 3. Peak focusing using assisting gasFurthermore, the siphon split method, which ensures that only a fine mist enters the drift tube, and the assisting gas focusing function ensure that contamination of the detection unit is kept to an absolute minimum, and that there is no loss of sensitivity, even in the continuous analysis of multiple samples. Using the ELSD-LT2 in combination with the Prominence UFLC series and the Shim-pack XR-ODS high-speed separation column ensures optimal performance.Fig. 6 Ultrahigh-Speed Analysis Based on Four Semi-Volatile Substances (Alkyl Parabens)Table 2 Analytical ConditionsColumnMobile PhaseFlow RateColumn Temp.Shim-pak XR-ODS (75mmL. x 3mm i.d.)Acetonitrile/ Water =6/4 (v/v)1mL/min30C6. Exampl esof Anal ysi sUsi ngt heELSD- LT2Analysis of Polyethylene GlycolPolyethylene glycol has little UV absorbance and is therefore difficult to analyze with a UV detector. In general, an RI detector is used for the analysis of such compounds. However, when separating low-molecular-weight polyethylene glycol according to the polymerization degree using the reverse-phase mode, the separation achieved with isocratic elution using an RI detector is limited, and a relatively long time is required for analysis. Fig. 7 shows an example of the results obtained by subjecting polyethylene glycol 1000 to gradient elution using the reverse-phase mode, and performing detection using an ELSD. This example shows how using an ELSD makes it possible to separate polyethylene glycol according to the polymerization degree with a stable baseline using the gradient elution method.Fig. 7 Example of Analysis of Polyethylene GlycolTable 3 Analytical ConditionsPolyethylene Glycol1000Shim-pack VP-ODS (250mmL. x 4.6mm i.d.)A) Water B) MethanolB Conc.40% (0min)60% (15min)1.0mL/min 40CTemperature : 40CGas Pressure : 350kPaSampleColumnMobile PhaseTime ProgramFlow RateColumn Temp.Detection (ELSD)The contents of this brochure are subject to change without notice.JQA-0376SHIMADZUCORPORATION.InternationalMarketingDivision3.Kanda-Nishikicho1-chome,Chiyoda-ku,Tokyo101-8448,JapanPhone: 81(3)3219-5641Fax. 81(3)3219-5710URL http://www.shimadzu.comFounded in 1875, Shimadzu Corporation, a leader in the development of advanced technologies, has a distinguished history of innovation built on the foundation of contributing to society through science and technology. We maintain a global network of sales, service, technical support and applications centers on six continents, and have established long-term relationships with a host of highly trained distributors located in over 100 countries. For information about Shimadzu, and to contact your local office, please visit our Web site at www.shimadzu.comAnalysis of OligosaccharidesIn the separation of oligosaccharides according to the glucose polymerization degree, although the normal-phase mode (HILIC mode) demonstrates superior selectivity, the time required for elution increases greatly as the number of glucose units increases. With an RI detector, gradient elution cannot be used, so analysis must be performed repeatedly using mobile phase compositions that are suitable for the separation of each polymerization degree. Fig. 9 shows an example of the results obtained by subjecting maltooligosaccharides to gradient elution using the normal-phase mode, and performing detection using an ELSD. Using an ELSD makes it possible to identify maltooligosaccharides with glucose polymerization degrees of up to around 20.Analysis of TriglyceridesTriglycerides, which are the main components of cooking oil, exist in many different forms that vary according to the acyl group, and the separation of these components is required for purposes such as quality control. Although detection is possible for triglycerides based on the absorbance of the acyl group in the range of 200 to 210 nm (UV), with systems that use acetone as the mobile phase, there is a high level of background absorbance, and UV detectors cannot be used. Fig. 8 shows an example of the results obtained by subjecting sesame oil to gradient elution using an acetone-based mobile phase in the reverse-phase mode, and performing detection using an ELSD. This example shows how an ELSD can be an effective detection tool even in cases where the mobile phase has UV absorbance.Fig. 9 Analysis of MaltooligosaccharidesTable 5 Analytical ConditionsMaltooligosaccharidesAsahipak NH2P-50(250mL. x 4.6mm i.d.)A ) Acetonitrile/Water=7/3 (v/v)B ) Acetonitrile/Water=4/6 (v/v)B Conc.0% (0min)100% (25min)1.0mL/min40CTemperature : 40CSampleColumnMobile PhaseTime ProgramFlow RateColumn Temp.Detection (ELSD)Gas Pressure : 350kPaFig. 8 Analysis of Sesame OilTable 4 Analytical ConditionsColumnMobile PhaseTime Program(40min)Flow RateColumn Temp.Detection (ELSD)Shim-pack VP-ODS (250mmL. x 4.6mm i.d.)A ) AcetonitrileB ) AcetoneB Conc. 50% (10min)70% (40min)1.0mL/min30CTemperature : 35CGAIN : 5Nebulizer Gas : AirGas Pressure : 350KPaPrinted in Japan 3295-08705-30ANS