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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