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

Quality Control in chemical industry: Quantitative NMR Exercise Analytical Strategy Debora Thöny, Simon Schneider Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 1

Principles of qNMR § Analysis of NMR peak intensities enables quantitative measurements § Other methods suitable for quantitative analysis § GC § HPLC § Absorption measurements § etc. Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 2

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

Applications of qNMR § 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 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 | 4

Comparison qNMR vs. GC, HPLC Chromatographic Methods (GC/HPLC) qNMR Costs and operation - Choosing the appropriate experimental - Expensive, maintenance-intense conditions can be difficult (column etc.) 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 - More difficult when sample is not pure (impurity - Might require derivatization (GC) 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 - Only possible for 1 H, 13 C, 19 F, 31 P etc. - Recovery of analytes possible - Recovery of analytes possible - Analysis of sensitive compounds is possible (protecting gas) 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

Internal Standard for qNMR § Requirements for target nuclei § Nuclear spin ≠ 0 § Natural abundance § Gyromagnetic ratio determines NMR sensitivity Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 6

Internal Standard for qNMR § Requirements for standard material § Soluble in solvent § Peak of internal standard must not interfere with analyte § Must be stable Anal Bioanal Chem (2012) 403:247–254 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 7

qNMR measurement uncertainty § The purity of the analyte is given as § p: purity § I: Integral; area of the peak ! ! = ! ! ! !"# ! ! ! !"# § N: number of equivalent 1 H nuclei N ! !"# ! !"# ! ! ! !"# ! ! § M: molar mass § m: mass ! ! ! ! Accred Qual Assur (2009) 14:79–86 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 8

qNMR measurement uncertainty § Every measurand in the equation contributes to the uncertainty of the purity of the analyte Anal Bioanal Chem (2012) 403:247–254 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 9

Influence on the measurement uncertainty § p: purity § I: Integral; area of the peak ! ! = ! ! ! !"# ! ! ! !"# ! !"# § N: number of equivalent 1 H nuclei ! !"# ! ! ! !"# ! ! § M: molar mass § m: mass ! ! ! ! Accred Qual Assur (2009) 14:79–86 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 10

! ! ! ! Influence on the measurement uncertainty ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! !"# ! ! ! !"# ! ! Mass m: ! ! ! ! § balance measures m s ¼ Bu � w s Bu ¼ 1 � ð � a = � r Þ 1 � ð � a = � s Þ ¼ � s ð � r � � a Þ Bu: buoyancy � r ð � s � � a Þ (german: Auftrieb) MicroChimica Acta (2004) 148 (3-4): 133-141 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 11

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

! ! ! ! Influence on the measurement uncertainty ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! !"# ! ! ! !"# ! ! Mass m: ! ! ! ! s ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi � 2 � 2 u c ð m s Þ � u ð Bu Þ þ u ð w s Þ � ¼ m s Bu w s § u(Bu): ρ air calculate extreme values, ρ cal , ρ sample § u(w s ): Repeatability, Nonlinearity, Sensitivity Tolerance, Temperature Coefficient, Eccentric Load MicroChimica Acta (2004) 148 (3-4): 133-141 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 13

! ! ! ! Influence on the measurement uncertainty ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! !"# ! ! ! !"# ! ! 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) MicroChimica Acta (2004) 148 (3-4): 133-141 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 14

! ! ! ! Influence on the measurement uncertainty ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! !"# ! ! ! !"# ! ! Mass m: ! ! ! ! § Nonlinearity: within 10 g: NL max = 0.03 mg within 200 g: NL max = 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) MicroChimica Acta (2004) 148 (3-4): 133-141 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 15

! ! ! ! Influence on the measurement uncertainty ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! !"# ! ! ! !"# ! ! Mass m: ! ! ! ! § Minimize errors by electrostatics with aluminium pans as weighing vessel and anti-static kit. Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 16

Influence on the measurement uncertainty 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 13 C 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 Anal Bioanal Chem (2012) 403:247–254; Prog Nucl Magn Reson Sepctrosc 57(2): 229-240 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 17

! ! ! ! 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 Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 18

! ! ! ! Influence on the measurement uncertainty ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! !"# ! ! ! !"# ! ! ! ! = ! ! ! ! ! ! !"# Integral: !"# ! !"# ! ! ! ! § Baseline correction § Phase correction § Offset: Therefore a π /2 pulse is used to minimize the offset effect Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 19

! ! ! ! Influence on the measurement uncertainty ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! !"# ! ! ! !"# ! ! Integral: ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! ! ! ! § Acquisition time: should be at least as long as the spin-lattice relaxation time T 1 § All relaxation times T 1 need to be determined by an inversion recovery experiment Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 20

! ! = ! !"# ! ! ! !"# ! ! ! !"# Influence on the measurement uncertainty ! ! = ! ! ! ! ! ! !"# !"# ! !"# ! !"# ! ! ! !"# ! ! Molar mass M, number of nuclei N: ! = ! ! ! ! ! ! !"# !"# ! ! ! ! ! § Isotope ratio § Accumulation Quality Control in chemical industry: Quantitative NMR Spectroscopy | 23.11.15 | 21