Introduction to Modern Liquid Chromatography, Third Edition part 23 ppt

176 DETECTION 123456 5 7 1234567 1 2 3 4 6 (a) (b) Figure 4.19 Response of chemiluminescent nitrogen detector (CLND) for different com- pound types. (a) Response of 50 ng nitrogen-equivalent of 1, N,N-dimethyl aniline; 2, nitrobenzene; 3, miconazole nitrate; 4, nicotinamide; 5, 4-acetamidophenol; 6,glycine;7, caffeine. (b) Response of 1, 1 ng nitrogen-equivalent of standard to 1 mg injected solvents: 2, acetone; 3, ethyl acetate; 4, hexane; 5, isopropanol; 6, methanol; 7, water. Adapted from [29]. both the concentration of the chiral compound and its molecular structure. Optical rotary dispersion (ORD) detectors operate on a similar principle to polarimeters, but use lower wavelengths (e.g., the 365-nm mercury emission line), which in theory should give stronger signals. Circular dichroism (CD) detectors are based on measuring the difference in absorption of right and left circularly polarized light when an analyte passes through the flow cell. For strong CD signals, it is desired that the analyte have a chromophore with absorption in the 200 to 420 nm range. In the example of Figure 4.21 [31], the response of CD, ORD, and UV detection is compared for the chiral chromatographic separation of ibuprofen enantiomers. The CD detector generates peaks with signal to noise (S/N) about 5-fold larger than the ORD detector, but only half that of the UV detector. Note that the two chiral detectors produce both negative and positive peaks. Another study [32] compared the response of PL, ORD, and CD detection for 6 pharmaceutical compounds. For naproxen, CD was about 6-fold more sensitive than PL, and 24-fold more than 4.11 REFRACTIVE INDEX DETECTORS 177 Hyp Asp 010203040 010203040 0 50 100 150 200 250 0 200 400 600 800 1000 Response (mV) Response (mV) Time (min) Pro Pro Ala Ala Thr Thr Glu Glu Cys Gln Gly Gly Ser Ser Asn Asn Hyp Asp (b) (a) Figure 4.20 Response of chemiluminescent nitrogen detector (CLND) for amino acids. (a)10μL injection of a 0.1 mM standard solution of 13 underivatized amino acids; (b)10μL injection of wine filtered through a 1000 Da filter. Adapted from data of [30]. ORD. The relative response for the various test compounds varied, but CD was superior in all cases. 4.11 REFRACTIVE INDEX DETECTORS The differential refractometer, or refractive index (RI) detector, responds to a difference in the refractive index of the column effluent as it passes through the detector flow cell. The RI detector is a bulk-property detector that responds to all solutes, if the refractive index of the solute is sufficiently different from that of the mobile phase. The most popular RI detector design is the deflection refractometer illustrated in Figure 4.22a. Light from the source lamp (typically tungsten) is directed through a pair of wedge-shaped flow cells. One cell is the reference cell, typically containing a trapped (static) sample of mobile phase; column effluent is directed through the sample cell. As the light passes through the detector cells, 178 DETECTION 05 Time (min) 2000 –2000 0 –500 –1500 –1000 0 400 800 1200 CD (230 nm) ORD UV (265 nm) Rotation (micro-degrees) Absorbance (mAU) 10 2 0 15 0 (a) (b) (c) Figure 4.21 Comparison of response of circular dichroism (CD), optical rotation (ORD), and UV detectors for a 10 μg injection of ibuprofen. (a) CD at 230 nm, S/N = 49.6; (b)ORD, S/N = 10.9; (c) UV at 265 nm, S/N = 113.4. Adapted from [31]. lamp slit reference cell sample cell dual photodiode detector output (a) photodiode arra y (b) Figure 4.22 Schematic of a deflection refractive index (RI) detector. (a) Dual-photodiode detector; (b) photodiode array detector (lamp and flow cell not shown). Dashed lines show optical path. it is refracted differently, depending on the instantaneous conditions in the cell. A pair of photodiodes measures the change in refraction (position of the beam) of the light passing through the flow cell and converts this to an output voltage. The conventional RI detector uses two photodiodes, as shown in Figure 4.22a.As the refractive index of the sample solution changes, the light is deflected so that the amount of light reaching each photodiode changes. More recent application 4.11 REFRACTIVE INDEX DETECTORS 179 Table 4.7 Characteristics of Refractive Index Detectors Excellent versatility; all solutes can be detected Moderate sensitivity Generally not useful for trace analyses Not useful for gradient elution Efficient heat-exchanger required Sensitive to temperature changes Reliable, fairly easy to operate Nondestructive of photodiode-array technology to the RI detector allows multiple photodiodes to be used for detection, as shown in Figure 4.22b. This configuration is claimed to improve the dynamic range of the RI detector and increase detector sensitivity. Table 4.7 summarizes the characteristics of RI detectors. Because they respond to all solutes, these devices have excellent versatility if the mobile phase is properly selected. For maximum RI detector sensitivity, the mobile phase should have a refractive index as different from the solute as possible (Table I.3 of Appendix I). However, even under optimum conditions RI detectors possess only modest sensi- tivity. Although this detector generally is not useful for trace analysis, it is possible under optimum conditions to quantify peaks at the 0.1% concentration level. A severe limitation of RI detectors is that they are unsuitable for use with gradient elution, since it would be exceedingly difficult to match the refractive indexes of the reference and sample streams (see exception in the discussion of Fig. 4.25a, Section 4.12.1). Even isocratic mobile-phase composition changes that are insignificant with UV detection can show up as baseline noise or ripple. For best results hand-mixed mobile phases will give quieter baselines than those prepared by on-line mixing. Despite the sensitivity limitation and impracticality in gradient elution, the differ- ential refractometer is widely used, particularly in size-exclusion chromatography, where sensitivity is not as critical. The sensitivity of RI detectors to temperature change also represents a severe limitation. Current models of RI detectors have been carefully designed to minimize temperature fluctuations through the use of constant-temperature detection envi- ronments and efficient heat exchangers to thermally equilibrate the mobile phase stream with the detector. For best performance, the RI detector should be turned on at all times, or allowed to warm up for at least two hours prior to use. Another good tip is to insulate the tubing connecting the column to the detector so as to minimize temperature fluctuations. Refractometers are convenient and reliable, although generally not as trouble free and easy to operate as UV detectors. RI baseline drift can result when changing from one bottle of ‘‘pure’’ solvent or ‘‘identical’’ hand-mixed mobile phase to another. Baseline drift can be severe when different solvents are involved, until the first solvent is completely flushed from the HPLC equipment and column. To maintain a homogeneous composition of the mobile phase during a series of runs, a sufficiently large volume of mobile 180 DETECTION 510 Time (min) 0 0 10 20 30 40 Refractive index units (×10 –3 ) treosulfan barbital (I.S.) Figure 4.23 Refractive index detector response for 560 μg/mL treosulfan 1 hr after onset of intravenous infusion; barbital is used as an internal standard. Adapted from data of [33]. phase should be formulated, with continuous stirring within the reservoir. For acceptable baseline stability, any change in the solvent composition (due to degassing, evaporation, water vapor pickup, etc.) normally should be avoided. In the past RI detectors based on a Fresnel design or interferometric detection were available, but the deflection refractometer is most popular today. For many years, the RI detector was the only option for ‘‘universal’’ detection with HPLC. Today, light-scattering detectors (Section 4.12) are replacing RI detectors for many applications. Low-wavelength UV detection (<210 nm) also provides better sensi- tivity than RI for many compounds that have very weak UV absorbance at higher wavelengths (see Fig. 4.25 for some comparisons of UV, RI, and ELSD responses). The sensitivity of RI detection usually precludes its use in routine drug monitoring, but in some cases it has proved useful for the determination of drug concentrations in biological samples, for example, when high drug concentrations are present, and other detection techniques have failed. In the example of Figure 4.23 the RI detector is used to measure treosulfan ( L-threitol-1,4-methanesulfonate) levels in pediatric plasma [33]. Treosulfan is an antitumor drug that is toxic to stem cells, and is administered intravenously prior to a stem cell transplant to kill all the native stem cells. Figure 4.23 shows a chromatogram for 560-μg/mL treosulfan in pediatric plasma following infusion of the drug; adjusting for sample preparation, this is equivalent to an injection of 83 μLofplasma. 4.12 LIGHT-SCATTERING DETECTORS In recent years improvements in light-scattering detectors have led to their replacing the refractive index (RI) detectors for many applications. One reason for this trans- formation (which has also boosted the practicality of mass spectrometric detectors) 4.12 LIGHT-SCATTERING DETECTORS 181 Table 4.8 Comparison of Refractive Index and Light-Scattering Detectors Property RI a ELSD a CNLSD a LLSD a Universal response ++ + + Sensitivity − 0 + na Gradient compatibility −+ + + Volatile mobile phase required No Yes Yes No Temperature sensitivity −+ + + Provides qualitative data to assist structural determination −− − + Note: +,good;0,intermediate;−, very poor; na, does not apply (detector designed for qualitative infor- mation, not sensitivity). a RI, refractive index; ELSD, evaporative light-scattering detector; CNLSD, condensation nucleation light-scattering detector; LLSD, laser light-scattering detector. is the ability of the light-scattering detector to efficiently nebulize the column effluent and evaporate the mobile phase. The most popular is the evaporative light-scattering detector (ELSD). The condensation nucleation light-scattering detector (CNLSD) is a modification of the ELSD that can provide increased performance. On the high-end of the price range are laser light-scattering detectors (LLSD), which occupy more of a specialty application niche than the ELSD and CNLSD. A comparison of some of the properties of the refractive index and light scattering detectors is presented in Table 4.8. 4.12.1 Evaporative Light-Scattering Detector (ELSD) Evaporative light-scattering detectors (ELSD) are based on evaporation of the mobile phase, followed by measurement of light scattered by particles of nonvolatile analyte. The ELSD principle is illustrated in Figure 4.24. Column effluent is nebulized in a stream of nitrogen or air and evaporated in a heated drift tube, leaving nonvolatile particles suspended in the carrier gas stream. Light scattered by the particles is detected by a photodetector mounted at a fixed angle from the incident beam. The ELSD should respond to most compounds that are analyzed by HPLC, but sensitivity may decrease for more volatile analytes. Detector response is related to the absolute quantity of analyte present, not its spectral properties. The ELSD, like the refractive index (RI) detector, is considered universal, so it has potential to be used for ‘‘any’’ sample. ELSD has the advantage over RI of having a response independent of the solvent, so it can be used with gradient elution and is insensitive to temperature or flow-rate fluctuations. The selection of the mobile phase for ELSD has similar restrictions as mass spectral detectors (Section 4.14) in that the mobile phase must be volatile and free of nonvolatile additives (e.g., phosphate buffer). Once the ELSD is adjusted (e.g., carrier flow rate, drift-tube temperature) for the mobile-phase conditions, it should provide acceptably stable operation. Linearity is somewhat limited (10- to 100-fold), but with the selection of appropriate calibration standard concentrations, ELSD can be useful for quantitative work over a wider range in analyte concentration. 182 DETECTION nebulizer gas light-scattering cell photocell lamp waste analyte nebulizer from column heated drift tube Figure 4.24 Schematic of an evaporative light scattering detector (ELSD). In general, ELSD provides a 10- to 100-fold improvement in sensitivity over the RI detector, with detection limits of 1- to 100-ng on-column. For some samples the sensitivity gain can be much greater, as is seen in Figure 4.25a for the separation of a triglyceride sample with detection by ELSD, whereas the UV detector at 205 nm and the RI detector do not respond to the triglycerides. Note that this separation is via gradient elution in the nonaqueous reversed-phase (NARP) mode (Section 6.5). Whereas water/organic gradients are not suitable for RI detection, acetonitrile and dichloromethane are sufficiently similar in refractive index that a changing mixture can be tolerated by the RI detector. The chromatograms of Figure 4.25b illustrate the superiority of the ELSD over the RI detector for a polyethylene sample analyzed by high-temperature (160 ◦ C) GPC. 4.12.2 Condensation Nucleation Light-Scattering Detector (CNLSD) The condensation nucleation light-scattering detector (CNLSD) is an enhancement of the standard ELSD for improved sensitivity and linear range. Following evaporation of the mobile phase, a saturated stream of solvent is added to the particles in the carrier gas. The particles act as condensation nuclei and the solvent condenses onto the particles, causing them to grow to a size where they are more easily detected by light-scattering detection [34]. Early work in this field [34] used butanol vapor, but current instrumentation uses water as the condensing solvent. The applications of the CNLSD are the same as those for the ELSD. In general, the CNLSD gives 10- to 100-fold improvement in sensitivity over the classic ELSD configuration. Manufacturer’s applications literature [35] shows detection of inorganic ions (Li + , Na + ,K + ) at 0.5-ng on-column, linearity for sucrose of three orders of magnitude, and five orders of magnitude of dynamic range. 4.12 LIGHT-SCATTERING DETECTORS 183 1 2 3 4 5 Figure 4.25 Comparison of ELSD detector response. (a) ELSD versus refractive index (RI) and UV at 205 nm for triglyceride sample. Shimadzu Premier C18 column; acetoni- trile/dichloromethane gradient; 1 mL/min; 30 ◦ C. (b) ELSD versus RI for the analysis of polystyrene standards by high-temperature (160 ◦ C) GPC; 200 μgsampleonPL-GelMixed B column. Sample molecular weights: 1, 2,560,000 Da; 2, 320,000 Da; 3, 59,500 Da; 4, 10,850 Da; 5, 580 Da. (a) Courtesy of Shimadzu Corporation; (b) courtesy of Varian Polymer Laboratories. 4.12.3 Laser Light-Scattering Detectors (LLSD) Laser light-scattering detectors (LLSD; also called multi-angle light-scattering, MALS) generally refer to HPLC detectors that make light-scattering measure- ments in solution, as opposed to the ELSD or CNLSD systems that measure light scattered by particles suspended in a gas. LLSD use a laser light source directed on the flow cell as the sample passes through in the mobile phase. Scattered light is measured at multiple angles (e.g., 3–18 different angles) and can be used, with the proper mathematical transformations, to determine the mass of the analyte in the absence of reference standards. These detectors are useful in conjunction with size-exclusion chromatography (see Chapter 13) for the determination of molec- ular weights of synthetic polymers and biological molecules in the range of 10 3 to 10 6 Da. Figure 4.26 shows superimposed UV chromatograms (280 nm) for a protein kinase fragment and three protein standards (ADH trimer, BSA and ADH monomer). Also shown are the LLSD-determined molecular weights (y-axis; 3 sep- arate runs). The kinase has a theoretical mass of 53.5 kDa, whereas the molecular weight of the kinase peak by LLSD is about 108,000, indicating that this is a dimer peak. The expected molecular weights of the standards are 141,000 (ADH), 67,000 184 DETECTION LLSD molecular wt. (×10 –3 ) Elution volume ( mL ) LLSD UV Kinase 250 200 150 100 50 ADH ADH sub-unit BSA dimer BSA 20 22 24 26 28 30 Figure 4.26 Size-exclusion separation of several proteins, with detection by laser (multi-angle) light-scattering detector (LLSD) and UV at 280 nm. Molecular weights by LLSD areplottedonthey-axis. Kinase, BSA, and ADH each run separately. Adapted from Wyatt Technology Corporation. (BSA), and 35,000 Da (ADH sub-unit), which closely match values by LLSD in Figure 4.26. The BSA dimer (135,000 Da) is observed to elute earlier (23.3 mL) than ADH (24.7 mL) despite its lower molecular weight. This demonstrates the greater accuracy of LLSD for molecular-weight determinations, compared to values from size-exclusion measurements (Sections 13.8, 13.10.3.1). 4.13 CORONA-DISCHARGE DETECTOR (CAD) The corona-discharge detector, also called the charged-aerosol detector (CAD) is classified as a universal HPLC detector because it responds to most analytes. The function of the CAD is illustrated in the schematic diagram of Figure 4.27. Column effluent is nebulized and the mobile phase is evaporated, the same as by the evaporative light-scattering detector (Section 4.12.1) or the mass spectrometer (Section 4.14). Analytes in the gas phase are then mixed with a stream of nitrogen gas that has been positively charged by a corona-discharge device. The charge is transferred to the analyte particles, and high-mobility charged species are removed in an ion trap to improve signal quality. The remaining charged analyte ions generate a signal that is read by an electrometer. The CAD is sensitive to nearly any compound that is sufficiently less volatile than the mobile phase so that remains in the gas phase after the mobile phase is evaporated. As with other evaporative detectors, the mobile phase is restricted to volatile components (e.g., no phosphate buffer); it also requires particles that can be charged in the detector. CAD has been applied to sugars and other carbohydrates as an alternative detector to RI or ELSD, with detection limits (S/N = 3) for oligosaccharides of 5-ng on-column and a dynamic range of > 10 4 [36]. The example of Figure 4.28 shows that the CAD can be applied to impurities analysis at 4.14 MASS SPECTRAL DETECTORS (MS) 185 gas in nebulizer analyte vaporizes charge transfer corona needle from column ion trap electrometer Figure 4.27 Schematic of the corona discharge detector. Figure 4.28 Response of corona-discharge detector to 10 μg on-column of sulfadimethoxine (6) plus 5 ng on-column each of related substances: 1, sulfaguanidine; 2, sulfamerazine; 3,sul- famethazine; 4, sulfamethizole; 5, sulfamethoxazole; and 6, sulfadimethoxin. Adapted from data of [39]. the 0.05% level relative to the active pharmaceutical ingredient (API) [37]. In this case 5-ng on-column of 5 related sulfonamide drugs (Fig. 4.28, peaks 1–5) are easily detected in the presence of 10-μg on-column of sulfadimethoxine (6). 4.14 MASS SPECTRAL DETECTORS (MS) Hyphenated HPLC detectors refer to the coupling of an independent analytical instrument (e.g., MS, NMR, FTIR) to the HPLC system to provide detection. The . cell dual photodiode detector output (a) photodiode arra y (b) Figure 4.22 Schematic of a deflection refractive index (RI) detector. (a) Dual-photodiode detector; (b) photodiode array detector (lamp and flow. heat-exchanger required Sensitive to temperature changes Reliable, fairly easy to operate Nondestructive of photodiode-array technology to the RI detector allows multiple photodiodes to be used for detection,. stream of solvent is added to the particles in the carrier gas. The particles act as condensation nuclei and the solvent condenses onto the particles, causing them to grow to a size where they are