The Use of Evaporative Light Scattering Detection in Pharmaceutical Analysis 1.
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Transcript of The Use of Evaporative Light Scattering Detection in Pharmaceutical Analysis 1.
The Use of Evaporative Light Scattering Detection in
Pharmaceutical Analysis
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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
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
Column: PL Hi-Plex Ca, 250x4.0mmEluent: WaterFlow Rate: 0.6ml/minInj Vol: 10µlTemp: 85°CDetector: 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
300 min
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Advantages of Evaporative Light Scattering Detection: Compounds With No Chromophore
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)
Detects all components in a single run
ELSD
UV @ 220nm
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Advantages of Evaporative Light Scattering Detection: Pharmaceutical Mixture
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
ELSD allows for a more uniform response than UV detection
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Advantages of Evaporative Light Scattering Detection: Uniform Response
• Ideal for High Throughput Screening
• 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
• Removes the need for derivatization steps
(e.g. for amino acids, toxins)
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Advantages of Evaporative Light Scattering Detection
0 30min
1. Serine, 2. Glutamic Acid, 3. Arginine, 4. Proline, 5. Valine, 6. Methionine, 7. Isoleucine, 8.Leucine, 9. Phenylanine, 10. Tryptophan
ELSD removes the need for derivatization for applications such as amino acids
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Underivatized Amino Acids
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
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Sample mixture of known 1:1 ratio
LC-MS results show ratio to be3:1
UV-Vis result show ratio to be10:1
PL-ELS 2100 results show ratio to be1:1
(Response independent of optical properties)
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Ideal Complement to LC-MS
Analytical Techniques• HPLC analytical,
microbore, preparative• SFC• LC-MS• High Throughput
Screening• GPC/SEC• High temperature GPC• GPEC• 2D Chromatography
Application Areas• Pharmaceuticals• Nutraceuticals• Combinatorial libraries• Carbohydrates• Lipids• Phospholipids• Triglycerides• Fatty acids• Amino acids• Polymers• Surfactants11
ELSD application areas
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
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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)
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Supercritical Fluid Chromatography: Steroid Separation
Minimal band broadening allows high resolution SFC separations to be performed
PEG 1000
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Supercritical Fluid Chromatography: Steroid Separation
Column: Lichrospher DIOL 5µm,150x2.1mm
Eluent A: IPA/Hexane/Water/Ammonia Hydroxide 57.8/40/2/0.2Eluent 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 holdFlow Rate: 0.3ml/minDetector: 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
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Complex Eluent Systems: Phospholipid Separation
• 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
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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)
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PL-ELS 2100: DMSO Transparency
Loss of volatile species occurs as temperature is increased
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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
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PL-ELS 2100 DMSO Transparency:Ideal for High Throughput Screening
Column: Aquasil C18 5µm, 150 x 4.6mmEluent A: Water + 0.1% TFAEluent B: ACN + 0.1% TFAGradient: 50-95% B in 10 mins, hold 5 minsFlow Rate: 1.0mL/minDetector: PL-ELS 2100 (neb=30°C, gas=1.6 L/min)Samples : Commercial Tablet
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Nutraceutical Application: St John’s Wort
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
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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
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Fast Centrifugal Partition Chromatography: St John’s Wort
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Nutraceutical Application: Black Cohosh
Combichem Application: DMSO Stability
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Nutraceutical : Vitamins
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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
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Features 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
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Additional Benefits of the PL-ELS 2100
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
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