Quality Control in chemical industry: Quantitative NMR Exercise - - PowerPoint PPT Presentation

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Exercise Analytical Strategy Debora Thöny, Simon Schneider

23.11.15 1

Quality Control in chemical industry: Quantitative NMR

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

§ Analysis of NMR peak intensities enables quantitative measurements § Other methods suitable for quantitative analysis

§ GC § HPLC § Absorption measurements § etc.

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Principles of qNMR

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy 23.11.15 3

Principles of qNMR

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

§ All kinds of quantitative analysis

§ Quantitative Metabolomics § Content determination § Purity determination (this presentation)

§ Quality control of drugs etc.

§ Definition of new standard materials which can be used for quantitative measurements by GC and HPLC

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Applications of qNMR

Anal Bioanal Chem (2012) 403:247–254; Accred Qual Assur (2009) 14:79–86; J Nat Prod (2012) Apr 27, 75(4), 834-85

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy 23.11.15 5

Comparison qNMR vs. GC, HPLC

Anal Bioanal Chem (2012) 403:247–254; Accred Qual Assur (2009) 14:79–86; J Nat Prod (2012) Apr 27, 75(4), 834-85

Chromatographic Methods (GC/HPLC) qNMR Costs and operation

• Choosing the appropriate experimental

conditions can be difficult (column etc.)

• Expensive, maintenance-intense

Reference

• Reference material is the same as analyte
• Reference material is different from analyte
• Provides a method for the validation of other

analytical techniques and for the certification of reference materials Sample preparation

• Mixtures are separated in the column
• Might require derivatization (GC)
• More difficult when sample is not pure (impurity

detection by H,H-COSY NMR) Calibration

• Calibration curve required
• No calibration curve required (internal calibration)
• Highly reproducible

Analytes

• Applicable for a wide range of analytes
• Recovery of analytes possible
• Only possible for 1H, 13C, 19F, 31P etc.
• Recovery of analytes possible
• Analysis of sensitive compounds is possible

(protecting gas)

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

§ Requirements for target nuclei

§ Nuclear spin ≠ 0 § Natural abundance § Gyromagnetic ratio determines NMR sensitivity

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Internal Standard for qNMR

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

§ Requirements for standard material

§ Soluble in solvent § Peak of internal standard must not interfere with analyte § Must be stable

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Internal Standard for qNMR

Anal Bioanal Chem (2012) 403:247–254

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

§ The purity of the analyte is given as

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qNMR measurement uncertainty

Accred Qual Assur (2009) 14:79–86

§ p: purity § I: Integral; area of the peak § N: number of equivalent 1H nuclei N § M: molar mass § m: mass

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

§ Every measurand in the equation contributes to the uncertainty of the purity of the analyte

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qNMR measurement uncertainty

Anal Bioanal Chem (2012) 403:247–254

| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

§ p: purity § I: Integral; area of the peak § N: number of equivalent 1H nuclei § M: molar mass § m: mass

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Influence on the measurement uncertainty

Accred Qual Assur (2009) 14:79–86

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Mass m: § balance measures Bu: buoyancy

(german: Auftrieb)

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Influence on the measurement uncertainty

Bu ¼ 1 ða=rÞ 1 ða=sÞ ¼ sðr aÞ rðs aÞ

ms ¼ Bu ws

MicroChimica Acta (2004) 148 (3-4): 133-141

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy 23.11.15 12

Influence on the measurement uncertainty

Reference weight: steel alloy with density of 8000 kg m-3 Therefore samples with lower density than steel have a lower weighing

• value. (Bu > 1)

MicroChimica Acta (2004) 148 (3-4): 133-141

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Mass m: § u(Bu): ρair calculate extreme values, ρcal, ρsample § u(ws): Repeatability, Nonlinearity, Sensitivity Tolerance, Temperature Coefficient, Eccentric Load

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Influence on the measurement uncertainty

ucðmsÞ ms ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi uðBuÞ Bu

• 2

þ uðwsÞ ws

• 2

s

MicroChimica Acta (2004) 148 (3-4): 133-141

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Mass m: § Repeatability: up to 50 g: u(REP)= 0.015 mg from 50 to 200 g: u(REP)= 0.04 mg Repeatability can be worse if the weighing isn’t done carefully or if the weighing objects are volatile or hygroscopic § Sensitivity Tolerance: Sensitivity of the balance

Note: Every listed uncertainty refers to the semi-micro electronic balance AT 201 (Mettler Toledo)

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Influence on the measurement uncertainty

MicroChimica Acta (2004) 148 (3-4): 133-141

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Mass m: § Nonlinearity: within 10 g: NLmax = 0.03 mg within 200 g: NLmax = 0.12 mg § Temperature Coefficient: temperature drift of the sensitivity § Eccentric Load: occurs if the centre of gravity of the weighing object isn’t placed vertically above the centre of the weighing pan.

Note: Every listed uncertainty refers to the semi-micro electronic balance AT 201 (Mettler Toledo)

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Influence on the measurement uncertainty

MicroChimica Acta (2004) 148 (3-4): 133-141

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Mass m: § Minimize errors by electrostatics with aluminium pans as weighing vessel and anti-static kit.

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Influence on the measurement uncertainty

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Integral: § Integral width: Using 64 times of the half width: 99 % of the signal (630 times is required for 99.9 %) Other possibility: Using the 13C satellites + 25 Hz as the integral limits (~133 times of the half width)

BUT: Always make sure that the peaks don’t overlap! è use solvent effects to separate signals

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Influence on the measurement uncertainty

Anal Bioanal Chem (2012) 403:247–254; Prog Nucl Magn Reson Sepctrosc 57(2): 229-240

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Influence on the measurement uncertainty

Integral: § Resolution: The S/N should be at least 20’000 else: increase number of scans (S/N improves only with √n) § Resolution can also sometimes be enhanced with increasing temperature

Prog Nucl Magn Reson Sepctrosc 57(2): 229-240

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Integral: § Baseline correction § Phase correction § Offset: Therefore a π/2 pulse is used to minimize the offset effect

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Influence on the measurement uncertainty

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Integral: § Acquisition time: should be at least as long as the spin-lattice relaxation time T1 § All relaxation times T1 need to be determined by an inversion recovery experiment

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Influence on the measurement uncertainty

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Molar mass M, number of nuclei N: § Isotope ratio § Accumulation

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Influence on the measurement uncertainty

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| | Quality Control in chemical industry: Quantitative NMR Spectroscopy

Standard purity: § Exactly known purity and uncertainty § No overlapping peaks § T1 similar or smaller than T1 of the analyte § No reaction or interaction with the analyte

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Influence on the measurement uncertainty

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Uncertainty budget

0.02 0.04 0.06 0.08 0.1 0.12

Integration ratio Molecular mass standard Molecular mass analyte Initial mass standard Initial mass analyte Disregarding air buoyancy Purity standard Combined uncertainty

Relative uncertainty [%]

Anal Bioanal Chem (2012) 403:247–254