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International Max Planck Research School for Global Biogeochemical Cycles Analytical Techniques – Extraction and Chromatography September 28, 2016 Molecular Biogeochemistry Group

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Page 1: Analytical Techniques - IMPRS-gBGC · Analytical Techniques – ...  . transition . ... Braithwaite – Chromatographic Methods

International Max Planck Research School for

Global Biogeochemical Cycles

Analytical Techniques – Extraction and Chromatography

September 28, 2016 Molecular Biogeochemistry Group

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International Max Planck Research School for Global Biogeochemical Cycles

1. Introduction 1.2 Lecture - Content

Morning: o Introduction Afternoon: o Extraction

• Liquid-liquid extraction • Polarity of solvent • Solid-liquid extraction

o Chromatography • Column chromatography • Solid phase extraction • HPLC • GC

o Identification • FID • MS

o Quantification

Gerd

Gerd

Vanessa

Vanessa

Natalie

Vanessa

2

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International Max Planck Research School for Global Biogeochemical Cycles

2. Extraction 2.1. Liquid-liquid extraction

McMaster – HPLC A practical user’s guide

Mixture

3

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International Max Planck Research School for Global Biogeochemical Cycles

2. Extraction 2.1. Liquid-liquid extraction

from the rest

Separated

Mixture

4

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International Max Planck Research School for Global Biogeochemical Cycles

2. Extraction 2.1. Liquid-liquid extraction

McMaster – HPLC A practical user’s guide 5

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International Max Planck Research School for Global Biogeochemical Cycles Mitra– Sample Preparation Techniques

in Analytical Chemistry

2. Extraction 2.1. Liquid-liquid extraction

Selection of extraction solvents - Immiscible with the second solvent - Volatile for easy removal after extraction - Compatible with method of analysis (e.g. GC, HPLC)

6

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International Max Planck Research School for Global Biogeochemical Cycles Mitra– Sample Preparation Techniques

in Analytical Chemistry

2. Extraction 2.1. Liquid-liquid extraction

Hydrophobicity of substance: N-octanol/water partition coefficient log KOW < 1: highly hydrophilic log KOW > 3-4: highly hydrophobic

𝐾𝑂𝑂 =𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝒘𝒐𝒐𝒘𝒘

Substance log KOW T Methanol −0.82 19 °C Formic acid −0.41 25 °C Diethyl ether 0.83 20 °C p-dichlorobenzene 3.37 25 °C Hexamethyl benzene 4.61 25 °C

7

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

2. Extraction 2.1. Liquid-liquid extraction

8

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International Max Planck Research School for Global Biogeochemical Cycles

S. Karlowsky

2. Extraction 2.1. Liquid-liquid extraction

9

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International Max Planck Research School for Global Biogeochemical Cycles

2. Extraction 2.1. Liquid-liquid extraction

Iodine in methanol

Malachite green oxalate in water

Mixture of malachite green oxalate and iodine in water and methanol

Heptane

malachite green Oxalate and residues of iodine in water and methanol

Heptane and iodine

10

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

2. Extraction 2.1. Liquid-liquid extraction

Ether

Water

Compounds better soluble in organic solvent

Compounds better soluble in water

11

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

2. Extraction 2.1. Liquid-liquid extraction

Distribution coefficient KD (Partition Coefficient KP)

𝐾𝐷 =𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑠𝑐𝑠𝑠𝑐𝑐𝑐 1𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑠𝑐𝑠𝑠𝑐𝑐𝑐 2

=𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑐𝑐𝑡 𝑡𝑝𝑐𝑠𝑐

𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑏𝑐𝑐𝑐𝑐𝑏 𝑡𝑝𝑐𝑠𝑐

𝐾𝐷 = [ ] 𝑠𝑐𝑠𝑠𝑐𝑐𝑐 1 𝑠𝑐𝑠𝑠𝑐𝑐𝑐 2

= 𝟐

Most of the time water or water miscible solvents are in the bottom phase

Example

Nernst distribution law

12

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

2. Extraction 2.1. Liquid-liquid extraction

𝟐𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠 𝟏𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠

= 𝟐 = 𝑲

30 molecules

100 mL solvent 2

100 mL solvent 1

13

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

2. Extraction 2.1. Liquid-liquid extraction

𝟐𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠 𝟏𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠

= 𝟐 = 𝑲

30 molecules

100 mL solvent 2

100 mL solvent 1

𝟐𝟐𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠 𝟏𝟐𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠

= 𝟐 = 𝑲

300 molecules

100 mL solvent 2

100 mL solvent 1

14

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

2. Extraction 2.1 Liquid-liquid extraction

𝟐𝟐𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠/200𝑏𝑚 𝟔𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠/100𝑏𝑚

= 𝟐 = 𝑲

300 molecules

200 mL solvent 2

100 mL solvent 1

If you use a larger amount of extraction solvent, more solute is extracted

15

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

2. Extraction 2.1. Liquid-liquid extraction

𝟐𝟐𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠 𝟏𝟐𝟐 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠

= 𝟐 = 𝑲

300 molecules

100 mL solvent 2

100 mL solvent 1

If you extract twice with 100 mL of solvent

200 molecules in 100 mL solvent

𝟔𝟔 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠 𝟑𝟑 𝑏𝑐𝑠𝑐𝑐𝑚𝑠𝑐𝑠

= 𝟐 = 𝑲 100 molecules

in 100 mL solvent

+

100 mL solvent

67 molecules in 100 mL solvent

16

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

2. Extraction 2.1. Liquid-liquid extraction

If you extract once with 200 mL of solvent If you extract twice with 100 mL of solvent

200 molecules in 100 mL solvent

67 molecules in 100 mL solvent

+

240 molecules in 100 mL solvent

267 molecules in 200 mL solvent

=

Multiple extractions are more efficient For maximum efficiency the rule of thumb is to extract three times

17

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International Max Planck Research School for Global Biogeochemical Cycles

2. Extraction 2.2 Solid-liquid extraction

http://www.gunt.de/download/extraction_english.pdf

transition component

solvent with dissolved transition component

solid carrier phase with transition component

solvent

depleted solid carrier phase

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International Max Planck Research School for Global Biogeochemical Cycles

2. Extraction 2.2 Solid-liquid extraction

http://blog.effjot.net/en/2009/10/practical-courses-for-students-of-secondary-school/ Kenkel – Analytical Chemistry for Technicians Mitra– Sample Preparation Techniques in Analytical Chemistry

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International Max Planck Research School for Global Biogeochemical Cycles

2. Extraction 2.2 Solid-liquid extraction

Solid-liquid extraction

http://blog.effjot.net/en/2009/10/practical-courses-for-students-of-secondary-school/ Kenkel – Analytical Chemistry for Technicians Mitra– Sample Preparation Techniques in Analytical Chemistry

Automated extraction systems

Shaking

Soxhlet extraction

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http://blog.effjot.net/en/2009/10/practical-courses-for-students-of-secondary-school/ Kenkel – Analytical Chemistry for Technicians Mitra– Sample Preparation Techniques in Analytical Chemistry

International Max Planck Research School for Global Biogeochemical Cycles

2. Extraction 2.2 Solid-liquid extraction

Solid-liquid extraction

Automated extraction systems

Shaking

Soxhlet extraction

21

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International Max Planck Research School for Global Biogeochemical Cycles http://www.chemistryviews.org/details/education/2040151/Tips_and_Tricks_for_the_Lab_Column_Packing.html

Braithwaite – Chromatographic Methods

Distribution coefficient KD

𝐾𝐷 =𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑠𝑐𝑠𝑐𝑠

𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑠𝑐𝑠𝑠𝑐𝑐𝑐 Nernst distribution law

2. Extraction 2.2 Solid-liquid extraction

22

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3. Chromatography

Braithwaite – Chromatographic Methods International Max Planck Research School for

Global Biogeochemical Cycles

The International Union of Pure and Applied Chemistry (IUPAC) defines chromatography as follows [1]: ‘Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (the stationary phase), while the other (the mobile phase) moves in a definite direction. A mobile phase is described as “a fluid which percolates through or along the stationary bed in a definite direction”. It may be a liquid, a gas or a supercritical fluid, while the stationary phase may be a solid, a gel or a liquid. If a liquid, it may be distributed on a solid, which may or may not contribute to the separation process.’

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3. Chromatography 3.1 Column chromatography

Braithwaite – Chromatographic Methods

The individual components in the mobile phase are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent.

International Max Planck Research School for Global Biogeochemical Cycles

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3. Chromatography 3.1 Column chromatography

Braithwaite – Chromatographic Methods International Max Planck Research School for

Global Biogeochemical Cycles

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3. Chromatography 3.1 Column chromatography

Braithwaite – Chromatographic Methods International Max Planck Research School for

Global Biogeochemical Cycles

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3. Chromatography 3.1 Column chromatography

Braithwaite – Chromatographic Methods International Max Planck Research School for

Global Biogeochemical Cycles

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International Max Planck Research School for Global Biogeochemical Cycles

Braithwaite – Chromatographic Methods

Fundament: Liquid-solid extraction

Distribution coefficient KD

𝐾𝐷 =𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑠𝑐𝑠𝑐𝑠

𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑠𝑐𝑠𝑠𝑐𝑐𝑐 Nernst distribution law

3. Chromatography 3.1 Column chromatography

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International Max Planck Research School for Global Biogeochemical Cycles Mitra– Sample Preparation Techniques

in Analytical Chemistry

Absorption: into a 3D matrix Adsorption: onto a 2D surface Occur simultaneously Use the term “Sorption”

3. Chromatography 3.1 Column chromatography

Sorption

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International Max Planck Research School for Global Biogeochemical Cycles

3. Chromatography 3.1 Column chromatography

30 Schmidt-Traub – Preparative Chromatography

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31 Braithwaite – Chromatographic Methods International Max Planck Research School for

Global Biogeochemical Cycles

Choose solvents and sorbents dependent on analytes

3. Chromatography 3.1 Column chromatography

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International Max Planck Research School for Global Biogeochemical Cycles http://www.chemistryviews.org/details/education/2040151/Tips_and_Tricks_for_the_Lab_Column_Packing.html

Dry-pack method 1

Pro Con Creates less usage of equipment

Difficult to get a well-packed column

3. Chromatography 3.2 Column packing

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International Max Planck Research School for Global Biogeochemical Cycles http://www.chemistryviews.org/details/education/2040151/Tips_and_Tricks_for_the_Lab_Column_Packing.html

Dry-pack method 2

Pro Con Tight-packed column Requires a lot of solvent

3. Chromatography 3.2 Column packing

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International Max Planck Research School for Global Biogeochemical Cycles http://www.chemistryviews.org/details/education/2040151/Tips_and_Tricks_for_the_Lab_Column_Packing.html

Slurry method

Pro Con Quick and easy Creates the most usage

of equipment

3. Chromatography 3.2 Column packing

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International Max Planck Research School for Global Biogeochemical Cycles

Retains the analyte or the matrix selectively Advantages of solid phase extraction • Separate analytes / fractionate classes of compounds • Enriches sample components present in low concentration • Desalts samples • Exchanges solvents

3. Chromatography 3.3 Solid phase extraction

Choose solvents and sorbents dependent on analytes

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International Max Planck Research School for Global Biogeochemical Cycles

cond

ition

ing

load

ing

rinsi

ng

dryi

ng

elut

ing

3. Chromatography 3.3 Solid phase extraction

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International Max Planck Research School for Global Biogeochemical Cycles http://www.waters.com/waters/de_DE/Goals-and-Benefits-of-SPE/nav.htm?cid=10083495&locale=de_DE

3. Chromatography 3.3 Solid phase extraction

Choose solvents and sorbents according to analytes

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International Max Planck Research School for Global Biogeochemical Cycles McMaster – HPLC A practical user’s guide

By Gerd Gleixner

3. Chromatography 3.4 HPLC

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International Max Planck Research School for Global Biogeochemical Cycles

3. Chromatography 3.5 GC

By Gerd Gleixner

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International Max Planck Research School for Global Biogeochemical Cycles Schmidt-Traub – Preparative Chromatography

4. Identification 4.1 Detected Signal

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International Max Planck Research School for Global Biogeochemical Cycles

4. Identification 4.1 Detected Signal

Schmidt-Traub – Preparative Chromatography

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International Max Planck Research School for Global Biogeochemical Cycles Schmidt-Traub – Preparative Chromatography

4. Identification 4.1 Detected Signal

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International Max Planck Research School for Global Biogeochemical Cycles

• Universal GC detector • High sensitivity to all organic compounds • Little or no response to H2O, CO2, carrier

gas impurities • Stable baseline • Linear dynamic range of 107

Ionization: -CH• + O• → -CHO+ + e-

4. Identification 4.2 Flame Ionization Detector

Braithwaite – Chromatographic Methods

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International Max Planck Research School for Global Biogeochemical Cycles

4. Identification 4.3 Mass Spectrometry

A mass spectrometer’s building blocks

• Inductively coupled plasma (ICP)

• Electron ionization (EI) • Chemical ionization (CI) • Atmospheric pressure

chemical ionization (APCI) • Electrospray ionization (ESI) • Matrix-assisted laser

desorption/ionization (MALDI)

• …

• Time-of-flight (TOF) • Quadrupole mass filter • Ion trap • Orbitrap • Fourier transform ion

cyclotron resonance (FT-ICR)

• Accelerator mass spectrometry (AMS)

• …

• Faraday detector • Focal plane detector • Electron multiplier • Scintillation detector • Image Current Detection

(FT-ICR and Orbitrap) only nondestructive detection method in MS

• …

Ion source Mass analyzer Detector

Separates species according to their

mass-to-charge ratio (m/z) Converts energy into

a current signal Creates gas phase ions

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International Max Planck Research School for Global Biogeochemical Cycles

4. Identification 4.3 Mass Spectrometry

Electron ionization (EI)

Ekman – Mass Spectrometry – Instrumentation, Interpretation, and Application

Requires gas phase molecules For GC-MS

[M] [M]+•

M + e-(70 eV) → M+• + 2e-

EI spectra are reproducible for library search

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International Max Planck Research School for Global Biogeochemical Cycles

4. Identification 4.3 Mass Spectrometry

Atmospheric pressure chemical ionization (APCI)

Dass – Fundamentals of contemporary mass spectrometry

For LC-MS

• Soft ionization • Fragment spectra highly depend on instrument and experimental conditions Disadvantage for spectra libraries

[M]

[M+H]+

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International Max Planck Research School for Global Biogeochemical Cycles

4. Identification 4.4 EI vs. APCI

Ekman – Mass Spectrometry – Instrumentation, Interpretation, and Application

GC-MS/EI

LC-MS/APCI

Diazepam: C16H13ClN2O Monoisotopic mass: 284 g/mol

[M+H]+

[M-1]+

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International Max Planck Research School for Global Biogeochemical Cycles

4. Identification 3.5 RT and fragments

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International Max Planck Research School for Global Biogeochemical Cycles

Correlation: Is there a connection between x and y?

Correlation coefficient: measure of the strength of linear (Pearson product-moment correlation coefficient r) or statistical (Spearman's rank correlation coefficient ρ or rs ) dependence between two variables giving values between -1 and +1.

5. Quantification 5.1 Correlation and Regression

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International Max Planck Research School for Global Biogeochemical Cycles

Correlation: Is there a connection between x and y?

Correlation coefficient: measure of the strength of linear (Pearson product-moment correlation coefficient r) or statistical (Spearman's rank correlation coefficient ρ or rs ) dependence between two variables giving values between -1 and +1. Analytical methods are based on strict dependencies. Correlation coefficient is not relevant for calibration because: a) The interdependence of the measured values, y , and the analytical values, x, is well-

known a priori - mostly by natural laws - and is, therefore, not subject of verification as a rule1

b) The analytical values of the calibration standards, xstandard, are no random variables but

fixed ones and carefully selected1

Danzer, Klaus (2007) Analytical Chemistry – Theoretical and Metrological Fundamentals. Springer, Berlin - Heidelberg

5. Quantification 5.1 Correlation and Regression

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International Max Planck Research School for Global Biogeochemical Cycles

coefficient of determination R2: coefficient of determination; measure of the amount of variance of y explained by x, giving values between 0 and 1.

Regression: Use known dependency between two (univariate) or more (multivariate) variables and quantify it. Use known dependency between two or more variables to model a mathematical equation that describes the relationship. E.g.: y = ax + b or y = ax + bx2 + c

The goal of regression analysis is both modelling and predicting1. The values of the independent variable x are already determined before the experiment (e.g. calibration standards).

Danzer, Klaus (2007) Analytical Chemistry – Theoretical and Metrological Fundamentals. Springer, Berlin - Heidelberg

5. Quantification 5.1 Correlation and Regression

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International Max Planck Research School for Global Biogeochemical Cycles

Example of Anscombe

0

5

10

15

3 8 13

1

0

5

10

15

3 8 13

2

0

5

10

15

3 8 13

3

0

5

10

15

3 8 13 18

4

x1 y1 x2 y2 x3 y3 x4 y4 10 8.04 10 9.14 10 7.46 8 6.58 8 6.95 8 8.14 8 6.77 8 5.76 13 7.58 13 8.74 13 12.74 8 7.71 9 8.81 9 8.77 9 7.11 8 8.84 11 8.33 11 9.26 11 7.81 8 8.47 14 9.96 14 8.1 14 8.84 8 7.04 6 7.24 6 6.13 6 6.08 8 5.25 4 4.26 4 3.1 4 5.39 19 12.5 12 10.84 12 9.13 12 8.15 8 5.56 7 4.82 7 7.26 7 6.42 8 7.91 5 5.68 5 4.74 5 5.73 8 6.89

Anscombe, Francis J. (1973) Graphs in statistical analysis. American Statistician, 27, 17–21.

5. Quantification 5.1 Correlation and Regression

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Example of Anscombe

0

5

10

15

3 8 13

1

0

5

10

15

3 8 13

2

0

5

10

15

3 8 13

3

0

5

10

15

3 8 13 18

4

Anscombe, Francis J. (1973) Graphs in statistical analysis. American Statistician, 27, 17–21.

Same regression key figures for all four datasets n 11 mean of x 9 mean of y 7.5 regression equation y = 3 + 0.5 x standard deviation of slope 0.118 residual sum of square 13.75 correlation coefficient r 0.82 coefficient of determination R2 0.67

Check if linear relation is an

appropriate solution

Plot your data

Do not rely only on r or R2

5. Quantification 5.1. Correlation and Regression

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5. Quantification 5.2 Detected Signal

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5. Quantification 5.2 Detected Signal

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Response factor RF

𝑅𝑅𝑐 = 𝑆𝑐𝑆𝑐𝑐𝑠 𝑐𝑜 𝑐𝑐𝑐𝑠𝑎𝑐𝑐𝑐

𝐶𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑜 𝑐𝑐𝑐𝑠𝑎𝑐𝑐𝑐

= 𝑃𝑐𝑐𝑃 𝑐𝑐𝑐𝑐 𝑐𝑜 𝑐𝑐𝑐𝑠𝑎𝑐𝑐𝑐

𝐶𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑜 𝑐𝑐𝑐𝑠𝑎𝑐𝑐𝑐

𝐶𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑜 𝑐𝑐𝑐𝑠𝑎𝑐𝑐𝑐 = 𝑃𝑐𝑐𝑃 𝑐𝑐𝑐𝑐 𝑐𝑜 𝑐𝑐𝑐𝑠𝑎𝑐𝑐𝑐

𝑅𝑅𝑐

5. Quantification 5.3 Response Factor

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Calibration: In analytical chemistry, calibration represents a set of operations that connects quantities in the sample domain with quantities in the signal domain Generation of a mathematical model that describes the signal response in dependence of the analyte‘s concentration. Calibration function Analytical function

𝑎 = 𝑜(𝑥)

𝑥 = 𝑆(𝑎)

Danzer – Analytical Chemistry – Theoretical and Metrological Fundamentals

𝑎� = 𝑐𝑥 + 𝑏𝑥𝑥

𝑥� = 𝑐𝑦 + 𝑏𝑦𝑎

Based on regression of y onto x by minimizing the y deviations according to the Gaussian least squares criterion

5. Quantification 5.4 Calibration and Regression

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Example Chinones

5. Quantification 5.4 Calibration and Regression

Ubichinon 10 (UQ-10) Vitamin K1 (K1)

conc. in pg/µl Peak area conc. in pg/µl Peak area 1 1 381 5 5 2170

10 422 10 4419 20 1067 20 9276 51 2420 51 25374

101 4638 101 52053 152 7093 152 76984 202 10033 202 108104 505 27204 505 261335

1010 46823 1010 571785 1515 71981 1515 863203 2020 94055 2020 1183199 2525 118036 2525 1522019 3030 138779 3030 1777905

𝑅𝑅𝑈𝑈10 =

𝟔𝟏𝟕𝟕𝟏 𝑐𝑐𝑚𝑐𝑐𝑠/𝑠𝑐𝑐𝟏𝟏𝟏𝟏 𝑡𝑆/µ𝑚

= 𝟐𝟕

𝑅𝑅𝐾1 =

𝟕𝟔𝟑𝟐𝟐𝟑 𝑐𝑐𝑚𝑐𝑐𝑠/𝑠𝑐𝑐𝟏𝟏𝟏𝟏 𝑡𝑆/µ𝑚

= 𝟏𝟔𝟐

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5. Quantification 5.4 Calibration and Regression

y = 46.238x + 511.45 R² = 0.9994

y = 592.36x - 10320 R² = 0.9993

-2000000

200000400000600000800000

100000012000001400000160000018000002000000

0 500 1000 1500 2000 2500 3000 3500

peak

are

a in

cou

nts/

sec

concentration in pg*µL-1

linear regression

UQ-10K1

𝑅𝑅𝑈𝑈10 =𝟐𝟕

𝑅𝑅𝐾1 =𝟏𝟔𝟐

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5. Quantification 5.5 Practical part

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