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The world leader in serving science
Bruce Bailey, Ph.D.
Thermo Fisher Scientific, Chelmsford, MA
Pittcon™ Conference & Expo 2014
March 2-6, 2014
Expanding Your HPLC and UHPLC Capabilities with Universal Detection: Shedding Light on Compounds That Lack a Chromophore
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Outline
• Introduction to Charged Aerosol Detection
• How Charged Aerosol Technology Works
• Comparison with Evaporative Light Scattering Detectors
(ELSD)
• Examples of Applications
• Inverse Gradient Solution for Uniform Response
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Introduction to Charged Aerosol Detection
Comparison of Charged Aerosol
Detection to UV and MS
• Used to quantitate any non-volatile and
many semi-volatile analytes with LC
• Provides consistent analyte response
independent of chemical structure and
molecule size
• Neither a chromophore, nor the ability to
ionize, is required for detection
• Dynamic range of over four orders of
magnitude from a single injection (sub-ng
to µg quantities on column)
• Mass sensitive detection – provides
relative quantification without the need
for reference standards
• Compatible with gradient conditions for
HPLC, UHPLC, and micro LC
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The liquid eluent from the LC
column enters the detector (1)
where it undergoes nebulization
by combining with a concentric
stream of nitrogen gas or air (2).
The fine droplets are carried by
bulk gas flow to the heated
evaporation sector (3) where
desolvation occurs to form
particles, while any larger
droplets are drained to waste (4).
The dry particles exit from
evaporation (5) and are
combined with another gas
stream that first passes over a
high voltage Corona charger (6).
The charged gas then mixes
with the dry particles, where
excess charge transfers to the
particle’s surface (7).
Charged Aerosol Detection – How It Works
Any high mobility species are removed by an ion trap (8) while
the remaining charged particles pass to a collector where the
passing particles charges are measured with a very sensitive
electrometer (9). The resulting signal is then conveyed to a
chromatographic data software for quantitation.
Signal is directly
proportional to the
analyte quantity
1
2
3
4
5 6
7
8
9
5
Particle Charging for Charged Aerosol Detection
Mixing Chamber
• Particle size proportional
to mass of analyte +
background residue
• Charge per particle
proportional to particle
size
• Charged particles are
measured, not gas phase
ions as in MS
Charged particle
Dried particle
Charged gas ion
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Corona ultra RS vs. Corona Veo RS Detectors
Coaxial N2 flow
Capillary Inlet
Aerosol
FocusJet™ Concentric Nebulizer Tip
Thermo Scientific™ Dionex™ Corona™ Veo™ RS
Charged Aerosol Detector Thermo Scientific™ Dionex™ Corona™ ultra™ RS
Charged Aerosol Detector
Cross-flow Nebulizer
Impactor
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Corona Veo Detector – What's New?
• Radically new concentric
nebulization system improves
sensitivity and precision
• All new evaporation scheme
widens the scope of
applications to include low flow
capabilities for micro LC, as
well as UHPLC
• Usability and serviceability are
enhanced by countless
improvements, many of which
came from our customers
This entirely new detector incorporates many design and
performance improvements:
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Comparison Between
Corona Charged Aerosol Detection
vs. ELSD
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Comparisons Charged Aerosol vs. ELS Detectors
ELSD measures light scattered by the aerosol
ELSD Corona Veo Detection
Charged Aerosol Detection measures the
aggregate charge of the aerosol
Evaporating
chamber
Siphon
Heated
Nebulizer
Light
source
Detection
chamber
ELSD
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Detector Response Characteristics R
esp
on
se
Mass on Column
Ma
jor
resp
on
se e
rro
r
0 1000 2000 3000 4000 5000 6000
Mass on Column
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
Resp
on
se
ng
pA*min
Typical ELSD sigmoidal response curve. Typical Charged Aerosol Parabolic Response Curve
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Comparisons Charged Aerosol vs. ELS Detectors
• A major consequence of ELSD sigmoidal response is that the
dynamic range is relatively small and analyte signal rapidly
decreases and completely disappears as the amount of
analyte decreases.
• Unlike ELSD, Charged Aerosol Detector response does not
simply disappear for the same lower levels of analytes.
Subsequently charged aerosol detection performs better for
measurement of lower analyte levels and is generally more
sensitive and provides a wider dynamic range than ELSD.
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Calibration of the Charged Aerosol Detector
• Over short ranges, the Charged Aerosol Detector is linear.
• Over wider ranges it is parabolic in behavior. To deal with
this, several approaches are available. Which is the most
appropriate will depend upon the data.
Selection includes:
• Log-Log
• Quadratic
• Power function
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Working with Non-Linear Data
• Limits of Detection (LoD) data by extrapolation from Signal /
Noise data is only practical when working with a linear
response.
• Both charged aerosol and ELS detector are non-linear. LoDs
cannot be extrapolated from the response of high levels of
analyte and can only be determined through the generation
of calibration curves.
• Extrapolation of non-linear data produces major errors and
should be avoided.
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Comparisons Charged Aerosol vs. ELS Detectors
Corona Veo
Sedex ELSD LT90
0.00 1.00 2.00 3.00 Time [min]
-2.00
-1.00
0.00
1.00
2.00
C u r r
e n t [ p
A ]
Theophylline
Caffeine
min
pA mV
-1.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
R e s p o n s e [ m
V ]
Theophylline and Caffeine, 2 -31 ng on column
Charged Aerosol
Detector
ELSD
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Comparisons Charged Aerosol vs. ELS Detectors
Theophylline and Caffeine, 8 ng on column
8 ng injected
0.00 1.00 2.00 3.00 4.00
Time [min]
-1.00
0.00
1.00
C u
r r e
n t [ p
A ]
theophylline S/N = 238
caffeine S/N = 23
theophylline S/N = 2
-10.0
-5.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
R e
s p
o n
s e
[ m V
]
Charged Aerosol
Detector
ELSD
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Avoid Extrapolation of Non-Linear Data
Medium Level Standard
Avg. SNR for analytes
• Evaporative Light Scattering Detector - 1283
• Charged Aerosol Detector - 230
10-fold Dilution of Medium Level Standard
Avg. SNR for analytes
• Evaporative Light Scattering Detector - 8.5
• Charged Aerosol Detector - 30
0,21 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 5,50 6,00 6,50 7,00 7,50
-0,6
5,0
10,0
15,0
20,0
25,0
29,4
min
pA
1 - CAD PF1,5 #5 [manipulated] RPmix 1/10 CAD_1 -768,00
-767,00
-766,00
-765,00
-764,00
-763,00
-762,00
-761,00
min
mV
2 - ELSD #3 [manipulated] RPmix 1/10 ELSD -10,0
-766,75
-766,50
-766,25
-766,00
-765,80 mV
ELSD
Charged Aerosol
Detector
0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 5,50 6,00 6,50 7,00 7,50 7,69
-0,50
0,00
1,00
2,00
3,00
4,00
4,50
min
pA
1 - CAD PF1,5 #6 [manipulated] RPmix 1/100 CAD_1 -767,60
-767,50
-767,25
-767,00
min
2 - ELSD #4 [manipulated] RPmix 1/100 ELSD
ELSD
Charged Aerosol
Detector
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Working with Non-Linear Data
• Charged aerosol detectors performs better for the
measurement of low levels of analytes, and have a wide
dynamic range of four orders of magnitude. The analyte’s
physicochemical properties affect the detector much less
than ELSD.
• Charged aerosol detectors uses a single nebulizer to address
a wide flow rate range. ELSD requires multiple nebulizers
adding to expense and downtime.
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Working with Non-Linear Data
• The only way to estimate the LoD when response is
non-linear is to construct a calibration curve.
• Comparisons are completely meaningless when the
response of a non-linear detector to a high concentration of
standard is used to imply that the performance of one
detector is superior to the other.
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Comparisons Charged Aerosol vs. ELS Detectors
Charged Aerosol Detector ELSD
Response Curvilinear Sigmoidal
Dynamic Range >4 orders 2–3 orders
LoQ and LoD LoQ and LoD often lower (better)
than that estimated by SNR
LoQ and LoD often higher (worse)
than that estimated by SNR
Sensitivity (LoD) <1 ng >10 ng
Semivolatility Range Similar Similar
Analyte Response Independent of structure Variable - dependent on compound
Ease of Operation Simple Can be complex
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Charged Aerosol Applications:
Shedding Light on Compounds
That Lack a Chromophore
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Determination of Adjuvants
Column: Thermo Scientific™ Hypersil GOLD™
PFP 1.9 um, 2.1 × 100 mm
Mobile Phase A: 0.1% Formic acid in water
Mobile Phase B: 0.1% Formic acid in 10:90
acetonitrile:reagent alcohol
Gradient: 35% B to 83% B in 6 min to
90% B in 10 min
Flow Rate: 0.5 mL/min
Inj. Volume: 2 μL
Col. Temp: 45 ºC
Evap. Temp: 50 ºC
Analysis of Plant Saponins
UV @ 210 nm
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Glycan Analysis for Bovine Fetuin
Column: Thermo Scientific™ GlycanPac AXH-1™,
1.9 μm, 2.1 × 150 mm
Mobile Phase A: 80% Acetonitrile
Mobile Phase B: 80 mM Ammonium formate, pH 4.4
Gradient: 2.5% B to 25% B from 1 to 40 min
Flow Rate: 0.4 mL/min
Inj. Volume: 5 μL
Col.Temp: 30 ºC
Evap. Temp: 50 ºC
Separation of Oligosaccharide Alditols
Native Glycans
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Determination of Carbohydrates in Juice
Column: Amino, 3 μm, 3 × 250 mm
Mobile Phase: Acetonitrile:water (92:8)
Flow Rate: 0.8 mL/min
Inj. Volume: 2 μL
Col. Temp: 60 ºC
Post-column Temp: 25 ºC
Evap. Temp: 75 ºC
Sample Preparation: Add 20 mL of 85% acetonitrile
to 1 gram juice
Analysis of Simple Sugars
Simplified sample preparation
“Dilute-and-shoot” method
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0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
Time [min]
-1.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
C u
r r e
n t
[ p A
]
Isosteviol
Steviol
Rubusoside
Dulcoside A
Stevioside
Steviolbioside
Rabaudioside C
Rabaudioside F
Rebaudioside B
Rebaudioside A
Sodium
Rebaudioside D
Mixture Containing 11 Stevia Glycoside Standards
(n=3)
Column: Thermo Scientific™ Acclaim™ Trinity ™ P1, 3 µm, 2.1 × 150 mm
Mobile Phase: 88:12 (v/v) Acetonitrile:10 mM ammonium formate, pH 3.1
Flow Rate: 0.8 mL/min
Inj. Volume: 2 L
Col. Temp: 30 ⁰C
Detection: Corona Veo RS
Veo Settings: 2 Hz, 5 second filter, PF 1.0, Evap. Temp 35 ⁰C
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Characterization of Algae-based Biofuels
Column: Thermo Scientific™ Accucore™ C18,
2.6 μm, 3.0 ×150 mm
Mobile Phase A: Methanol:water:acetic acid (600:400:4)
Mobile Phase B: Tetrahydrofuran:acetonitrile (50:950)
Mobile Phase C: Acetone:acetonitrile (900:100)
Gradient: Time FlowRate %A %B %C
(min) (mL/min)
-10.0 1. 00 90 10 0
-0.1 1. 00 90 10 0
0. 0 0. 25 90 10 0
20.0 0. 50 15 85 0
35.0 0. 50 2 78 20
60.0 0. 50 2 3 95
65.0 0. 50 90 10 0
Flow Rate: 1.0 mL/min
Inj. Volume: 2 μL
Col. Temp: 40 ⁰C
Evap. Temp: 40 ⁰C
Analysis of Algal Oils
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Active Ingredient Composition
Analysis of Gentamicin Standard
(200 μg/mL)
Column: Acclaim RSLC PolarAdvantage II,
2.2 μm, 2.1 × 100 mm
Mobile Phase A: 0.025:95:5 HFBA:water:acetonitrile
Mobile Phase B: 0.3:95:5 TFA:water:acetonitrile
Gradient: 0 to 1.5min,1 to 10%B
1.5 to 7min,10 to 100% B
7 to 10min,100% B
4 min. pre-injection equilibration
Flow Rate: 0.45 mL/min
Inj. Volume: 1 μL
Col. Temp: 15 ⁰C
Evap. Temp: 80 ⁰C
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Formulation Testing
Column: Acclaim Trinity P1, 3 μm, 3.0 × 50 mm
Mobile Phase A: 75% Acetonitrile
Mobile Phase B: 25% 200 mM Ammonium acetate pH 4
Flow Rate: 0.8 mL/min
Inj. Volume: 5 μL
Col. Temp: 30 ⁰C
Evap. Temp: 60 ⁰C
Measurement of
Chloride Impurity
Analysis of Diclofenac-Sodium Salt
(1 mg/mL)
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Conventional Gradient Elution
Inverse Gradient Compensation
Inverse Gradient Solution for Uniform Response
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Solution for Uniform Response with Gradients
• Dual-gradient pump is the heart of this
exclusive solution
• Inverse gradient fingertight fitting kits are
supplied for LC systems
• Furnished with unique eWorkflows Dual Gradient Pump
Inverse Gradient Setup
Thermo Scientific™ Dionex™
Viper™ Fingertight Fitting
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Effects of Gradient and Mass on Calibration
R² = 0.9997
R² = 0.9999
R² = 1
0
1
2
3
4
5
6
7
0 500 1000 1500 2000 2500
Mass on Column (ng)
Sulfanilamide
Famotidine
Perphanzine
Inverse gradient extends the consistency of response
R² = 0.9999
R² = 0.9995
R² = 0.9998
0
1
2
3
4
5
6
7
0 500 1000 1500 2000 2500
Mass on Column (ng)
Sulfanilamide
Famotidine
Perphanzine
Standard Gradient (Single Pump)
Pe
ak
Are
a (
Ch
arg
ed
Ae
ros
ol
Dete
cto
r)
Inverse Gradient (Dual Pump)
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Determination of Drug Discovery Mass Balance
Charged Aerosol
UV
Column: Acclaim 300 C18, 3 μm,
4.6 ×150 mm
Mobile Phase A: 20 mM Ammonium
acetate, pH 4.5
Mobile Phase B: Acetonitrile
Gradient: 2% B to 98% B in 30 min,
Inverse Gradient
Flow Rate: 0.8 mL/min
Inj. Volume: 2 μL
Col. Temp: 30 ⁰C
Evap. Temp: 35 ⁰C
Corona offers a more uniform response than UV
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Thank You for Your Attention
For a Cleaner, Healthier, Safer World
OT70993_E