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The Use of Evaporative Light Scattering Detection in Pharmaceutical Analysis

The Use of Evaporative Light Scattering Detection in Pharmaceutical Analysis. 1. Introduction. Traditional HPLC detectors such as UV and RI have limited capabilities RI detection is not gradient compatible RI detection is susceptible to thermal instability and long equilibration times

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The Use of Evaporative Light Scattering Detection in Pharmaceutical Analysis

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  1. The Use of Evaporative Light Scattering Detection in Pharmaceutical Analysis 1

  2. Introduction • Traditional HPLC detectors such as UV and RI have limited capabilities • RI detection is not gradient compatible • RI detection is susceptible to thermal instability and long equilibration times • Different analytes produce different UV responses depending on their extinction coefficient • UV and RI are not compatible with all solvents • Important information can be missed with an RI and a UV detector • ELSD better alternative than UV detection at low wavelengths

  3. Advantages of Evaporative Light Scattering Detection • Universal detection : responds to all compounds in the mobile phase • Detects compounds that do not possess a UV chromophore e.g. polymers, sugars • Not dependent on the spectroscopic properties of an analyte • Produces more uniform detection sensitivity for analytes • Not susceptible to baseline drift during gradient elution, temperature or solvent pump fluctuations • ELSD compatible with a much wider range of solvents compared to refractive index

  4. 3 5 1 2 4 0 min 30 Advantages of Evaporative Light Scattering Detection: Compounds With No Chromophore Column: PL Hi-Plex Ca, 250x4.0mm Eluent: Water Flow Rate: 0.6ml/min Inj Vol: 10µl Temp: 85°C Detector: PL-ELS 2100 (neb=30°C, evap=90°C, gas=1.6 L/min) Samples: 1. Fructose, 2. Glucose, 3. Sucrose I, 4. Lactose, 5. Stachyose 4

  5. Advantages of Evaporative Light Scattering Detection: Pharmaceutical Mixture Detects all components in a single run Samples : 1. α-cyclodextrin 2. ß-cyclodextrin 3. Ibuprofen Column: Aquasil C18 5µm, 150x4.6mm Eluent A: Water Eluent B: Acetonitrile Gradient: 50-95% B in 5 mins Flow Rate: 1.0 ml/min Inj Vol: 20µl Detector: PL-ELS 2100 (neb=30°C , evap=50°C, gas=1.0 L/min) ELSD UV @ 220nm 5

  6. Advantages of Evaporative Light Scattering Detection: Uniform Response ELSD allows for a more uniform response than UV detection Column: PLRP-S 100Å 5µm, 150x4.6mm Eluent A: 50% 0.1% TFA in Water : 50% 0.1% TFA in ACN Flow Rate: 1.0ml/min Inj Vol: 10µl Detector: PL-ELS 2100 (neb=30°C, evap=30°C, gas=1.4 L/min) UV-VIS @ 280nm 6

  7. Removal of mobile phase eluent allows rapid HPLC gradients • Fast setup and equilibration • Flow rates up to 5ml/min can be achieved with no effect on baseline stability • No interference from solvent front peaks • Ideal for High Throughput Screening • Removes the need for derivatization steps • (e.g. for amino acids, toxins) Advantages of Evaporative Light Scattering Detection 7

  8. min 0 30 Underivatized Amino Acids ELSD removes the need for derivatization for applications such as amino acids 1. Serine, 2. Glutamic Acid, 3. Arginine, 4. Proline, 5. Valine, 6. Methionine, 7. Isoleucine, 8.Leucine, 9. Phenylanine, 10. Tryptophan 8

  9. Advantages of Evaporative Light Scattering Detection: Ideal Complement to LC-MS • Similar operating principles to LC-MS • Volatile buffers • Favors lower flow rates (i.e. 0.2-0.5ml/min) • Can develop LC methods on ELSD then transfer to • LC-MS • ELSD can provide supporting information when used in tandem with LC-MS 9

  10. Ideal Complement to LC-MS Sample mixtureof known 1:1 ratio LC-MS results show ratio to be 3:1 UV-Vis result show ratio to be 10:1 PL-ELS 2100 results show ratio to be 1:1 (Response independent of optical properties) 10

  11. ELSD application areas • Application Areas • Pharmaceuticals • Nutraceuticals • Combinatorial libraries • Carbohydrates • Lipids • Phospholipids • Triglycerides • Fatty acids • Amino acids • Polymers • Surfactants • Analytical Techniques • HPLC analytical, microbore, preparative • SFC • LC-MS • High Throughput Screening • GPC/SEC • High temperature GPC • GPEC • 2D Chromatography 11

  12. Supercritical Fluid Chromatography: Steroid Separation ELSD capable of handling supercritical CO2 Properties of supercritical CO2 are complementary to the nebulization step in ELSD Low baseline noise & improved S/N with SFC–ELSD Nebulizer must be heated to counter the Joule-Thompson cooling effect; prevents blockage of the nebulizer ELSD provides an alternative detection method to FID and UV for SFC separations 12

  13. Supercritical Fluid Chromatography: Steroid Separation Sample: PEG 1000 Column: Berger SFC Diol Column 60Å 6µm, 250x4.6mm Eluent: Carbon Dioxide with 5% Methanol Flow Rate: 2ml/min, Pressure: Carbon Dioxide was pressure programmed from 100bar to 250bar at 10bar/min. Inj Vol: 10µl Detector: PL-ELS 2100 (neb=40°C, evap=30°C, gas=0.9 L/min) 13

  14. Supercritical Fluid Chromatography: Steroid Separation Minimal band broadening allows high resolution SFC separations to be performed PEG 1000 14

  15. Complex Eluent Systems: Phospholipid Separation Column: Lichrospher DIOL 5µm,150x2.1mm Eluent A: IPA/Hexane/Water/Ammonia Hydroxide 57.8/40/2/0.2 Eluent B: IPA/Hexane/Water/Ammonia Hydroxide 51.8/40/8/0.2 Gradient:0-100% B in 7 min, 8 min hold; 100-0% B in 5 min, 10 min hold Flow Rate: 0.3ml/min Detector: PL-ELS 2100: (neb=30°C, evap=80°C, gas=1.0 L/min) Samples: 1: Cholesterol, 2: Phosphatidylethanolamine, 3: Phosphatidylcholine, 4: Sphingomyelin, 5: Lysophosphatidylcholine Stable baseline for complex mobile phase systems 15

  16. PL-ELS 2100: DMSO Transparency • Combinatorial libraries are commonly stored in DMSO • DMSO peak can mask fast eluting compounds • DMSO peak can be removed at elevated evaporator temperatures (e.g. >50°C) • Loss of volatile species occurs at higher evaporator temperatures • Eliminates DMSO removal step in sample preparation 16

  17. PL-ELS 2100: DMSO Transparency Sample: Pharmaceutical mixture in DMSO Column: C8 5µm, 50x4.6mm Eluent A: Water Eluent B: Acetonitrile Gradient: 5-100% B in 5 mins Flow Rate: 1.0ml/min Inj Vol: 20µl Detector: PL-ELS 2100 (neb=25°C evap & gas flow varied) 17

  18. PL-ELS 2100 DMSO Transparency:Ideal for High Throughput Screening Loss of volatile species occurs as temperature is increased 18

  19. PL-ELS 2100 DMSO Transparency:Ideal for High Throughput Screening The unique use of evaporation gas in the PL-ELS 2100 makes it transparent to DMSO at 25-30°C PL-ELS 2100: neb=25°C, evap=30°C 19

  20. Nutraceutical Application: St John’s Wort Column: Aquasil C18 5µm, 150 x 4.6mm Eluent A: Water + 0.1% TFA Eluent B: ACN + 0.1% TFA Gradient: 50-95% B in 10 mins, hold 5 mins Flow Rate: 1.0mL/min Detector: PL-ELS 2100 (neb=30°C, gas=1.6 L/min) Samples : Commercial Tablet 20

  21. Fast Centrifugal Partition Chromatography: St John’s Wort • PL-ELS 2100 is compatible with Centrifugal Partition Chromatography, for prep scale fractionation of compounds • The LED intensity can be reduced for the high loading levels • The evaporator gas can be used to control baseline noise, which can be a problem with this technique • UV detection is noisy with this technique, because the mobile phase can form cloudy emulsions 21

  22. Fast Centrifugal Partition Chromatography: St John’s Wort • FCPC-ELSD was used to isolate Hypericin from a 3g sample of raw plant product • Flow Rate 20ml/min • Rotation speed 1000rpm • Solvent: Heptane/MeOH/ Water/Ethyl Acetate 22

  23. Fast Centrifugal Partition Chromatography: St John’s Wort 23

  24. Nutraceutical Application: Black Cohosh 24

  25. Combichem Application: DMSO Stability 25

  26. Nutraceutical : Vitamins 26

  27. Features of the PL-ELS 2100 • The PL-ELS 2100 offers the general benefits of evaporative light scattering detection which are well documented: • Universal detector, does not rely on the optical properties of the solute • Excellent baseline stability, compatible with gradient elution • High sensitivity • Removes the need for derivatization 27

  28. Additional Benefits of the PL-ELS 2100 • Low temperature operation, even for 100% water • Improved detection of volatile compounds • Extremely low dispersion for high resolution separations • High eluent flow rates, up to 5ml/min • Wide application range • Extremely small footprint, stackable • Ergonomic design • Easy to use 28

  29. PL-ELS 2100 Specification Light source: LED 480nm Detector: Photomultiplier tube Nebulizer temperature: Ambient to 90°C in 1°C increments Evaporator temperature: Ambient to 120°C in 1°C increments Gas flow rate: Up to 3.25 L/min Gas pressure: Typically 60-100psi, maximum 100psi Eluent flow rate: Up to 5ml/min Analog output: 0-1V FSD Communications: Serial I/O (RS232), contact closure, TTL Power requirements: 90/120V AC or 220/250V AC 50/60 Hz Dimensions: 200x450x415mm (wxdxh) Weight: 11kg 29

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