Selecting Detectors for Compounds with No Optical Absorbance

C146-E114 Selecting Detectors for Compounds with No Optical Absorbance Technical Report vol.15 1. Features of Absorbance Detectors In HPLC, the detector is selected in accordance with the analyte. Many of the detectors used are based on optical absorbance, such as UV-Vis absorbance detectors and photodiode array (PDA) detectors. Absorbance detectors have the following features: The detector sensitivity and selectivity can be adjusted by setting an appropriate detector wavelength. The sensitivity is relatively high (although it depends on the molar extinction coefficient). The relationship between the elution concentration and the detection response is linear and covers a wide range (Lambert-Beer law). The gradient elution method can be used. The stability is high. The difference between instruments is small. They perform nondestructive detection and can therefore be used for fractionation. It is partly because of this large number of features and the consequent high level of versatility that absorbance detectors are used so widely. 2. Principle of Absorbance Detectors Fig. 1 illustrates the principle of absorbance detectors. When light of a certain wavelength is directed at a flow cell, the substance inside the flow cell absorbs the light. As a result, the intensity of the light that leaves the flow cell is less than that of the light that enters it. An absorbance detector measures the extent to which the light intensity decreases (i.e., the absorbance). During analysis, the mobile phase and sample continuously flow through the flow cell, and the data obtained by monitoring this absorbance in real time is represented as a chromatogram. As the absorbance varies in proportion to the compound concentration, the areas of the peaks in the chromatogram can be used to ascertain the concentrations of the target components. Ein Flow cell L Eout C: Concentration Absorbance Absorbance = C L = -log (Eout / Ein ) Time Fig. 1 Principle of Absorbance Detectors UV-Vis Absorbance Detector SPD-20A/20AV Photodiode Array Detector SPD-M20A Fig. 2 Absorbance Detectors

3. Limitations of Absorbance Detectors Although absorbance detectors are highly versatile and afford a high level of selectivity, they are not capable of handling every type of application. If the analyte (target compound) does not absorb UV light, then due to its operating principle, an absorbance detector cannot generate a peak for this compound. Techniques that can be used to analyze compounds that do not absorb UV light include the following: (1) Introduce chromophores to the target compound to derivatize it. (2) Use a detector other than an absorbance detector. Techniques that, like (1), use absorbance detectors together with derivatization have many advantages but they are not appropriate for situations where it is desirable or essential that the target compound is analyzed in its original state (i.e., intact). Also, the necessity of derivatization requires extra pretreatment before analysis. For this reason, in the analysis of target compounds with no absorbance, it is common to employ method (2), namely, use a detector based on a different detection principle. 4. What Kind of Detectors Complement Absorbance Detectors? If there were detectors that were as versatile as, and could compensate for the deficiencies of, absorbance detectors, then it would be possible to handle all types of compounds, including those with no absorbance. The following types of detectors are possible candidates for this role: (1) Refractive index detector Example: Shimadzu RID-10A Refractive Index Detector (2) Evaporative light scattering detector Example: Shimadzu ELSD-LT2 Evaporative Light Scattering Detector (3) Charged particle detector Example: ESA Biosciences Corona CAD * 1 (Charged Aerosol Detector) (4) Mass spectrometer (LCMS) Example: Shimadzu LCMS-2010EV Liquid Chromatograph Mass Spectrometer These detectors all have their own features and so, in order to ensure the successful analysis of target compounds, selection of the optimum detector should be based on a thorough understanding of the detectors' operating principles and features. Let us look, then, at the operation principle of each type of detector. Refractive Index Detector RID-10A Evaporative Light Scattering Detector ELSD-LT2 Charged Aerosol Detector Corona CAD Liquid Chromatograph Mass Spectrometer LCMS-2010EV Fig. 3: Detectors That Complement Absorbance Detectors 4-1. Principle of Refractive Index Detectors (RID) Fig. 4 illustrates the principle of RID. As shown in the left part of the figure, the "refractive index" is a measure of the extent to which the direction of light changes at the interface between two media when it passes from one medium into another. A RID uses difference in refractive index measured when the medium on the far side of the interface consists of mobile phase without eluted sample and mobile phase with eluted sample. As shown in the right part of the figure, the flow cell in a RID consists of two neighboring independent cells called the "reference cell" and the "sample cell". The reference cell contains only mobile phase, while the sample cell is part of the analysis flow line, so therefore contains both mobile phase and eluted sample. As the sample compounds separated in the column pass through the sample cell, the refractive index continuously changes. The amount of change is measured by the RID. As the refractive index varies in proportion to the compound concentration, the areas of the peaks in the chromatogram obtained can be used to ascertain the concentration of compounds. Incidence angle Air Refraction angle Water Sample cell Light-detection point Fig. 4: Principle of RID Reference cell Detection cell Light-receiving surface 4-2. Principle of Evaporative Light Scattering Detectors (ELSD) Fig. 5 illustrates the principle of ELSD. The column eluate that reaches the glass tube is nebulized by nitrogen gas that is simultaneously introduced into the tube, and then enters the drift tube. As the droplets flow through the drift tube, which is heated at a constant temperature, the mobile phase solvent evaporates, and as a result, the nonvolatile sample that remains takes the form of dust-like particles. In this state, the sample proceeds to the detection section. When the sample is exposed to light emitted from a light source, light is scattered, and the intensity of the scattered light is measured. Column eluate Fine droplets Glass tube Nitrogen gas Light-receiving unit Drift tube Nitrogen gas Minute particles of dried sample Fig. 5: Principle of ELSD 2

Thus, ELSD are based on the following principles: (1) The column eluate is nebulized by nitrogen gas, and converted to fine droplets. (2) When the fine droplets are heated, the mobile phase evaporates and minute particles of dried sample remain. (3) In the detection section, light is directed at the minute sample particles, and the intensity of the scattered light created by the collision between the particles and the light is measured. Light-receiving unit From drift tube Fig. 6: Detection Section in ELSD 4-3. Principle of Corona Charged Aerosol Detection Fig. 7 illustrates the principle of Corona CAD. Both Corona CAD and the ELSD described above use nebulization to form droplets. In a Corona CAD, nitrogen carrier gas is used to evaporate mobile phase solvent and convey the nonvolatile sample particles to a mixing chamber. A second stream of nitrogen gas acquires charge by passing over a corona discharge and collides with these analyte particles to form charged particles. These charged particles travel to the detection section, where the intensity of the current that they create is measured. In other words, the Corona CAD is based on the following principles: (1) The column eluate is nebulized by nitrogen gas, and converted to fine droplets. (2) When passing through a drying tube, the mobile phase evaporates and minute particles of dried sample remain. (3) The minute sample particles collide with charged nitrogen gas created by corona discharge and thereby become charged particles. (4) In the detection section, the intensity of the charged particles is measured. HPLC column eluate Gas inlet Large droplets to waste Nebulizer and impactor Drying tube Electrometer Charge Is measured by a sensitive electrometer Fig. 7: Principle of Corona CAD Signal out Signal is directly proportional to quantity of analyte in sample Collector Analyte particles transfer their charge Ion trap Negatively charged ion trap removes high-mobility particles Secondary gas stream positively charged Positive charge transferred by a high-voltage platinum Corona wire to analyte particles by charged opposing secondary gas stream 4-4. Principle of Mass Spectrometers (LCMS) As with the Corona CAD described above, mass spectrometers* 3 can also use Corona discharge to perform ionization for certain types of analyses. Mass spectrometers differ greatly from Corona CAD, however, in that the process starting with the mass separation performed after ionization and culminating with the final ion detection is carried out under vacuum conditions, and that separation is performed using the masses of the sample molecule ions mass to charge ratio (m/z). There are many different ionization methods that are used in mass spectrometry. Fig. 8 and Fig. 9 illustrate the APCI (atmospheric pressure chemical ionization) method, which is based on a principle similar to that used in Corona CAD. In a mass spectrometer, after the column eluate that reaches the ion source is heated and evaporated by a heater, it is nebulized with nitrogen gas. There is a Corona needle positioned at the outlet of the nebulizer, and the Corona discharge emitted from the tip of the needle causes the nitrogen gas and solvent molecules in the immediate vicinity to be ionized. Due to the exchange of protons between these reactant ions and the sample molecules, the sample molecules are ionized. The sample molecule ions then enter the ion focusing section, which is kept under vacuum, and the range of the direction of travel is narrowed. After the molecules are led into the mass separation section and separated according to mass, they travel to the detector, where their intensities are measured. In other words, mass spectrometers are based on the following principles: (1) After the column eluate is evaporated by heating, it is nebulized by nitrogen gas. (2) The sample molecules collide with reactant ions that are charged by Corona discharge, and are thereby ionized. (3) As the sample molecule ions pass through the mass separation section, they are separated according to mass. (4) In the detection section, the intensities of the sample molecule ions are measured. Ion source Ion focusing section Heater Vacuum Mass separation section Fig. 8: Principle of Mass Spectrometers Corona needle Fig. 9: Ion Source Used with APCI Method Detection section To mass separation section 3

5. Comparison of Detector Features In order to facilitate an understanding of the characteristics of the different detectors, such as mass spectrometers, Table 1 compares their properties, such as quantitative performance and selectivity, and other important points relevant to analysis, such as mobile phase restrictions. The evaluations presented here are general in nature, while in practice, the suitability of a given detector varies with the analyte. Table 1: Comparison of Detectors Detection Principle Versatility Polarity of Target Compound Sensitivity to Compounds of Medium Polarity Quantitative performance Dynamic Range Linearity Absorbance Detector Refractive Index Detector Absorbance intensity Refractive index Not applicable Evaporative Light Scattering Detector Scattered light intensity Medium polarity Charged Aerosol Detector Ion intensity Medium polarity Mass Spectrometer Ion intensity Medium/high polarity Stability Calibration Curve Selectivity Substances of Medium Polarity Ionization Ion Mass Log-log calibration curve required. Restrictions on Mobile Phase Nonvolatile Buffer Solutions Solvents with Absorbance Ultrapure Water Applicable Analyses Isocratic Analysis Gradient Analysis Yes Required No Yes Essential Multicomponent Simultaneous Analysis High-Throughput Analysis Ease of Use Stability Reproducibility Utilities (Nitrogen Gas) Not required Required 6. Important Points Concerning Detector Selection As can be seen from Table 1, there are various features that distinguish the different detectors, and in order to ensure that the analysis objective is attained, the optimum detector must be selected. Let us look at some of the points that must be considered when selecting a detector. 6-1. RID RID are very versatile because they can detect all compounds that have a different refractive index from the mobile phase. With GPC (gel permeation chromatography), for example, this characteristic is used effectively. However, because RID perform detection using the refractive index, they are subject to some restrictions. For example, it is difficult to improve the sensitivity and, because the refractive index changes if the relative proportion of the mobile phase changes, the gradient elution method cannot be used. Also, because these detectors are easily influenced by changes in temperature and pressure, various considerations must be addressed in order to obtain stable results. With Shimadzu's RID-10A refractive index detector, temperature fluctuation is suppressed with the adoption of a double temperature control mechanism. This means that, compared to other RID, the time required for stabilization with the RID-10A is relatively short and, because it is not easily influenced by temperature changes, stable data can be obtained. Optical system Heater Aluminum block Heat insulation material Fig. 10: Double Temperature Control Mechanism in RID-10A Optical System 4

6-2. ELSD, Corona CAD, and Mass Spectrometers Selection of an ELSD, a Corona CAD or mass spectrometer is appropriate if the sensitivity or stability provided with a RID is inadequate, or if gradient analysis is required. Because these three types of detectors use nebulization, their application is restricted to the use of mobile phases that do not contain nonvolatile salts or reagents. With HPLC analysis, because ph adjustment is extremely important for separation, phosphate and citrate buffer solutions are often used. If, however, these types of nonvolatile buffer solutions are used with a nebulization-type detector, the efficiency of the generation and desolvation of fine droplets by nebulization decreases significantly. Therefore, the following reagents are usually used with nebulization-type detectors: Acids: Acetic acid, formic acid, trifluoroacetic acid Bases: Ammonia (water), diethylamine, triethylamine Salts: Ammonium acetate, ammonium formate Ion-pair reagents: Fluorinated alkyl carboxylic acids such as pentafluorobutyric acid, alkylammoniums such as dibutylammonium acetate In addition, unless high purity water is used with these detectors, a high level of noise may be expected. Therefore, it is advisable to use water produced with an ultrapure water purification system. Furthermore, although there is no problem using organic solvents such as acetonitrile and methanol, as long as they are of HPLC grade, care is required when using solvents such as THF (tetrahydrofuran) because they may contain nonvolatile stabilizers. Nitrogen gas Glass tube Fig. 11: Nebulization Method Used in ELSD To drift tube 6-3. ELSD In addition to the points described above, ELSD also have the restriction of not being able to analyze volatile or sublimable compounds. This is because ELSD are only capable of detecting scattered light for compounds that are not removed by evaporation. Shimadzu's ELSD-LT 2* 4 evaporative light scattering detector can evaporate mobile phases at low temperatures and so it can be used for a wide range of mobile phases and target compounds. If the above restriction is overcome, ELSD have the following advantages over refractive index detectors: The sensitivity is 5 to 10 times higher. The gradient elution method can be used. They are not easily influenced by changes in temperature and pressure. These characteristics are utilized in the analysis of sugars, surfactants, lipids, and pharmaceutical products that have no chromophores. Fig. 12: Prominence ELSD-2 System 6-4. Corona CAD The Corona CAD detector is based on nebulization as in ELSD, and therefore is used in similar applications. Whereas ELSD optically measure scattered light, the Corona CAD detector performs electrical measurements for charged particles and so, in comparison to ELSD, attains higher sensitivity and a larger dynamic range. 6-5. Corona CAD and Mass Spectrometers Both the Corona CAD detector and mass spectrometers use corona discharge. A large amount of nitrogen is present in the area where corona discharge takes place and so reactive nitrogen ions (N2 + ) are generated. However the fundamental use of this phenomenon differs in the two techniques. Furthermore in mass spectrometers, if a protic solvent is present, ions such as H3O + and CH3OH2 + are generated. These ions become reactant ions, and the components separated in the column become ionized by a charge transfer process. However, not all compounds are ionized. Only compounds strong enough to take protons from the H3O + and CH3OH2 + ions (i.e., compounds with a high proton affinity) are ionized. The mass spectrometers have the restriction, then, that they cannot detect compounds that are not ionized. Because the Corona CAD detector does not operate in the same way as a mass spectrometer it does not have this restriction. Although mass spectrometers and Corona CAD have similarities, the process starting with the nebulization and culminating in detection differs greatly between the two types of detectors. With a Corona CAD, the total volume of the charged particles is used for the detection response, whereas with a mass spectrometer, the generated ions are detected after separation according to the mass-to-charge ratio (m/z). This is a fundamental difference and is extremely significant in terms of selectivity. 5

6-6. Mass Spectrometers Here we will look at important consideration in the selection of a mass spectrometer as a detector. (1) Application to Analysis of Impurities With other detectors, the chromatogram retention times are the only qualitative criteria available and so in the analysis of impurities, for example, impurities cannot be identified just by the retention time. With a mass spectrometer, however, estimation and identification are possible using information about the masses. Impurities can be estimated with the LCMS-2010EV single-stage quadrupole mass spectrometer or LC-MS/MS, and structual analysis is possible with the LCMS-IT-TOF ion-trap/time-of-flight hybrid mass spectrometer, which can perform accurate mass measurement and MS n analysis* 5. This is because mass spectrometry is a technique in which sample molecules are ionized, and after the generated sample molecule ions are separated according to mass-to-charge ratio (m/z), the ion intensities are measured, and so information about the masses of the sample compounds can be used. In other words, in situations where a high qualitative capability is required of analysis data, such as the analysis of impurities, it is important to select a mass spectrometer as the detector. Also, as they have a much higher detection sensitivity than other detectors, mass spectrometers could be described as ideal for the analysis of impurities. (2) Application to High-Throughput Analysis In applications such as high-throughput analysis and multicomponent simultaneous analysis, it is unlikely that complete separation is achieved for all compounds simply with column separation. In this kind of situation, the separation capacity of a mass spectrometer is an extremely effective tool for analysis requiring a higher level of selectivity. (3) Application to Thermally Labile Samples Mass spectrometers, the removal of the mobile phase (i.e., desolvation) is performed by heating. In the analysis of thermally labile compounds, however, it may not be possible to use this kind of heating. With mass spectrometers, replacing the ion source makes it possible to apply ESI (electrospray ionization), which does not involve heating, to thermally labile compounds. This means that mass spectrometers can be used for a wider range of applications than ELSD and Corona CAD. Drying gas Fig. 13: Ion Source Used in ESI Fig. 14: LCMS-2010EV System To mass separator *1: ESA, Corona, CAD, arec registered trademark of ESA Biosciences, Inc. All other trade names, trademarks, and registered trademarks are the property of their respective holders. *2: Source: P. Garnache, R. McCarthy, S. Freeto, D. Asa, D.M. Woodcock, K. Laws, and R. Cole: HPLC Analysis of Nonvolatile Analytes Using Charged Aerosol Detection. LCGC North America 23(2), 516-520 (2005) *3: In this report, "mass spectrometry" refers to mass spectrometry based on APCI unless stated otherwise. *4: Refer to Technical Report vol. 6, "Principles and Practical Applications of Shimadzu s ELSD-LT2 Evaporative Light Scattering Detector". *5: Refer to Technical Report vol. 3, "Structural Prediction of Impurities in Drugs using MS n Data". Or refer to Technical Report vol.16, "Structural Analysis by In-Depth Impurity Search Using MetID Solution and High Accuracy MS/MS". The information contained in this report is protected by copyright by the publisher, Shimadzu Corporation ( Shimadzu ). The sale, use, reproduction or alteration of this information for any purpose is forbidden without Shimadzu s express written consent, which may be granted or withheld in Shimadzu s sole discretion. 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