Expanding Your HPLC and UHPLC Capabilities with Universal ...

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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

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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

espo

nse

Mass on Column

Maj

or

resp

onse

err

or

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

Res

pons

e ng

pA*min

Typical ELSD sigmoidal response curve. Typical Charged Aerosol Parabolic Response Curve

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• 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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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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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 ]


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


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


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• 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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• 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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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 µL 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 = 0.9997R² = 0.9999R² = 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 = 0.9995R² = 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)

Peak

Are

a (C

harg

ed A

eros

ol D

etec

tor)

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

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Expanding Your HPLC and UHPLC Capabilities with Universal ...

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