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International Max Planck Research School for
Global Biogeochemical Cycles
Analytical Techniques – Extraction and Chromatography
September 28, 2016 Molecular Biogeochemistry Group
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
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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
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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
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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
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)
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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
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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
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International Max Planck Research School for Global Biogeochemical Cycles
S. Karlowsky
2. Extraction 2.1. Liquid-liquid extraction
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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
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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
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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
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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
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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
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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
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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
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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
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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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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
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
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Braithwaite – Chromatographic Methods
Distribution coefficient KD
𝐾𝐷 =𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑠𝑐𝑠𝑐𝑠
𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑠𝑐𝑠𝑠𝑐𝑐𝑐 Nernst distribution law
2. Extraction 2.2 Solid-liquid extraction
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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.
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3. Chromatography 3.1 Column chromatography
Braithwaite – Chromatographic Methods International Max Planck Research School for
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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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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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3. Chromatography 3.1 Column chromatography
30 Schmidt-Traub – Preparative Chromatography
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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Slurry method
Pro Con Quick and easy Creates the most usage
of equipment
3. Chromatography 3.2 Column packing
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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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cond
ition
ing
load
ing
rinsi
ng
dryi
ng
elut
ing
3. Chromatography 3.3 Solid phase extraction
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3. Chromatography 3.3 Solid phase extraction
Choose solvents and sorbents according to analytes
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By Gerd Gleixner
3. Chromatography 3.4 HPLC
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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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• 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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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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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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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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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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4. Identification 3.5 RT and fragments
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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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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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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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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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International Max Planck Research School for Global Biogeochemical Cycles Schmidt-Traub – Preparative Chromatography
5. Quantification 5.2 Detected Signal
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International Max Planck Research School for Global Biogeochemical Cycles Schmidt-Traub – Preparative Chromatography
5. Quantification 5.2 Detected Signal
International Max Planck Research School for Global Biogeochemical Cycles
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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