Basics for qnmr Peter Lankhorst, Joep van Rijn & John Gauvin September 2017
Quantitative NMR: The principle Purity determination P x A A x st n n st x MW MW x st W W st x P st P = purity A = NMR area n = number of protons MW = molecular weight A x ~ n x W = weight Page 1
3 types of qnmr applications Concentration of producs and impurities Down to ppm level is possible Purity determination of standards for other techniques: HPLC. Highest accuracy and precision required. Composition of complex mixtures. Many compound classes at the same time. E.g. metabolomics Page 2
NMR linearity Range R 2 = 0.99999 1..1300 Range 1 240000 R 2 = 0.99989 Page 3
Page 4 Diacetyl impurities Quantitative NMR Dynamic range
Page 5 Quantitative NMR Dynamic range
Page 6 Quantitative NMR Dynamic range
Quantitative NMR Dynamic range Acetaldehyde 7 ppm Page 7
Amoxicillin Purity The problem: Production batches were rejected. Purity on HPLC was too high!!! > 100.5% means out of spec Purity determination with USP standards is required Strategy: Check standards with quantitative NMR Page 8
Amoxicillin 7 6 3 5 2 dmso 1 1 acetonitril 4 DSM 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 (ppm) USP * * * * 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 (ppm) Page 9
Quote CS Marcel van Tilborg HPLC is like you girlfriend, she will tell you what you want to hear. NMR is like you mother, she will tell you the truth Page 10
Complex mixtures - wine Tartaric acid glycerol Internal standard Lactic acid Succinic acid butanediol Page 11
Complex mixtures - wine proline Galacturonic acid Vitamin C tyrosine Page 12
Complex mixtures wine Benzoic acid Page 13
Key aspects for qnmr Sample variation Keep track of your standards Accurate weighing Biological variation/sample inhomogeneity Changes over time? Simple? Accurate pipetting Relax and remember your T1 lessons When internal standard in solution is added Which pulseprogram to use? 30 or 90 pulse? Page 14
Important tips for accurate weighing 1.Use tweezers 2.Clean the table 3.Clean the weighing platform of the balance before weighing. 4.Put an empty vial on the balance a few minutes before first weighing. 5.Check if there are no magnetic objects near the balance. 6.Use vials with a narrow rim 7.Tap the vial before the weighing 8.Do not add material while the vial is on the balance!! 9.Do not attach stickers to the vial!! 10.Close the doors of the balance fully!! 11.Wait until the balance has stabilized before zeroing!! 12.Store the vials close to the balance 13.Do not use vials immediately after opening the package Page 15
Is weighing the most important source of errors? Idea: weigh more compound and standard use microbalance (0.001 mg read-out) Batch weighing Succinic acid S0503 A 99.61% avg 99.57% S0503 B 99.58% stdev 0.03% S0503 C 99.53% max 99.61% S0503 D 99.56% min 99.53% S0503 E 99.58% Page 16
Important tips for accurate weighing 1.Use tweezers 2.Clean the table 3.Clean the weighing platform of the balance before weighing. 4.Put an empty vial on the balance a few minutes before first weighing. 5.Check if there are no magnetic objects near the balance. 6.Use vials with a narrow rim 7.Tap the vial before the weighing 8.Do not add material while the vial is on the balance!! 9.Do not attach stickers to the vial!! 10.Close the doors of the balance fully!! 11.Wait until the balance has stabilized before zeroing!! 12.Store the vials close to the balance 13.Do not use vials immediately after opening the package Page 17
Requirements for NMR internal standards High purity, typically > 99% NMR signals in non-crowded area of spectrum Soluble Not hygroscopic Stable over a long period, chemically inert Simple compound, with one or two NMR signals Non volatile Page 18
NMR standard examples Amongst others: O O O O HO O - K + HO OH Potassium biftalate Maleic acid O - CH 3 N + CH 3 O O O H 3 C p-nitrotolueen dimethoxybenzeen Page 19
Doing the mathematics T1 relaxation with 90 pulse M z (τ) = M z,eq (1 e τ/t1 ) results τ = interpulse delay experiments When τ =T1 63% recovery to equilibrium Example: 99% rec - τ /T1 = ln(1 0.99)= -4.6 M z (τ) M z,eq = 1 e τ/t1 1 M z(τ) M z,eq = e τ/t1 ln(1 M z(τ) M z,eq )= - τ /T1 99% recovery > d1= 4.6*T1 99.9% recovery > d1= 6.9*T1 99.99% recovery > d1= 9.2*T1 Page 20
21 NMR sensitivity & quantification B o Z Z Z Y Y Y X X X
22 Net magnetization with 30 or 90? B o Z Z Z Y Y Y X X X
NET MZ (%) 23 How long do you need to wait? T1 relaxation 2.5s 120 @ 30 @ 90 100 80 60 40 20 0 0 5 10 15 20 25 INTERPULSE DELAY (S)
NET MZ (%) 24 How long do you need to wait? T1 relaxation 10s 120 100 @ 30 @ 90 80 60 40 20 0 0 10 20 30 40 50 60 INTERPULSE DELAY (S)
SiNo relative 25 Relax and go 90 s SiNo vs number of scans 18,0 16,0 14,0 12,0 10,0 8,0 6,0 4,0 2,0 0,0 @ 90 @ 30 0 100 200 300 400 500 600 700 800 900 1000 1100 Number of scans Sensitivity improves With 2 with a double amount of scans. ~4x as much scans are needed to achieve same SiNo.
SIGNAL INCREASE (REL) 26 Relax and go 90 s 200,00 180,00 160,00 @90 Signal increase per unit time @45 @30 @90 @45 140,00 120,00 100,00 @30 30dg, 2.5s 45dg, 2.5s 90dg, 2.5s 80,00 30dg, 10s 60,00 40,00 45dg, 10s 90dg, 10s 20,00-0 20 40 60 80 100 120 140 160 180 200 TIME SPENT (S) 30 pulse needs ~2x more measurement time for the same SiNo
Thank you! Acknowledgments DSM Peter Lankhorst Joep van Rijn Page 27