Transcript ELSD in the Pharmaceutical Lab ()

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
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
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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:
ELSD
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,
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gas=1.0 L/min)
UV @ 220nm
Advantages of Evaporative Light Scattering
Detection: Uniform Response
ELSD allows for a more uniform response than UV detection
Column:
Eluent A:
PLRP-S 100Å 5µm, 150x4.6mm
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
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Advantages of Evaporative Light Scattering
Detection
• 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)
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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
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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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Ideal Complement to LC-MS
Sample mixture of 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)
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ELSD application areas
Analytical Techniques
• HPLC analytical,
microbore, preparative
• SFC
• LC-MS
• High Throughput
Screening
• GPC/SEC
• High temperature GPC
• GPEC
• 2D Chromatography
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Application Areas
• Pharmaceuticals
• Nutraceuticals
• Combinatorial libraries
• Carbohydrates
• Lipids
• Phospholipids
• Triglycerides
• Fatty acids
• Amino acids
• Polymers
• Surfactants
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 JouleThompson 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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Complex Eluent Systems:
Phospholipid Separation
Column:
Lichrospher DIOL 5µm,150x2.1mm
IPA/Hexane/Water/Ammonia Hydroxide 57.8/40/2/0.2
Eluent B: IPA/Hexane/Water/Ammonia Hydroxide 51.8/40/8/0.2
Eluent A:
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
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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
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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
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Inj Vol:
20µl
Detector:
PL-ELS 2100
(neb=25°C evap & gas flow varied)
PL-ELS 2100 DMSO Transparency:
Ideal for High Throughput Screening
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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Nutraceutical Application: St John’s Wort
Column:
Eluent A:
Eluent B:
Gradient:
Flow Rate:
Detector:
Samples :
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Aquasil C18 5µm, 150 x 4.6mm
Water + 0.1% TFA
ACN + 0.1% TFA
50-95% B in 10 mins, hold 5 mins
1.0mL/min
PL-ELS 2100 (neb=30°C, gas=1.6 L/min)
Commercial Tablet
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
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Combichem Application: DMSO Stability
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Nutraceutical : Vitamins
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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
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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
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PL-ELS 2100 Specification
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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