Analytical Methods NMR Spectroscopy, a Rapid Method for Any Lipid Analysis ASAGA World Congress, Rosario Argentina, November2015 Bernd W.K. Diehl, Yulia Monakhova, Elina Zailer Spectral Service 2015 1
Analytical Methods Holistic control of edible oils based on NMR spectroscopy NMR for fast quality control of edible oils, 10 minutes measuring time, no reverence material Easy and fast sample preparation o o o o o o o o o o POZ Peroxide AV Aldehyde AV FFA 1,2 and 1,3-DAG 1- and 2-MAG Omega-3 Tocopherol Polyphenol Sterole Additives Identity test using statistical methods o PCA (Principle Component Analysis) o DA (Discriminant analyses) o PLS (Partial Least Squares) Contamination or adulteration Provenience of Olive Oil Spectral Service 2015 2
Alternative: qnmr of Lipids Quality parameter Peroxides (PV) Aldehydes (AV) TOTOX Free Fatty Acids (TAV) Saturated FA Unsaturated FA Omega-3 Iodine Value (JV) 1,2-Diglycerides 1,3-Diglycerides Sterols Cis/Trans-conjugation Tocopherols Spectral Service 2015 3
Internal Reference! CH 3 at chain end = 9H per MOL 1.0 ppm Spectral Service 2015 4
Internal Reference! Glycerol CH 2 = 4H per MOL 4.2 ppm Spectral Service 2015 5
Internal Reference! α CH 2 = 6H per MOL 2.3 ppm Spectral Service 2015 6
Internal Reference! Glycerol CH = 1H per MOL 5.20 ppm 10,00 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 Internal Reference TAG/FA y = 0,9964x R² = 1 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Spectral Service 2015 7
Jodine Value % DOBI vs Jodine Value JV 350 300 250 y = 1,1489x R² = 1 Trilinolenat 5.3 ppm 200 150 100 50 Trioleat Trilinoleat 0 Tristearat 0 50 100 150 200 250 300 Spectral Service 2015 8
Jodine Value JV 5.3 ppm 350 300 250 200 150 100 50 0 y = 1,1489x R² = 1 Butter % DOBI vs Jodine Value Sesame Oil Olive Oil Walnut Oil 0 50 100 150 200 250 300 Spectral Service 2015 9
Jodine Value Saturated Unsaturated JV unsat. 5.3 ppm 79.5 % unsat 20.5 % sat. 2.1 ppm Spectral Service 2015 10
W-3, 18:2, 18:3 18:3 18:2 ω-3 2.8 ppm 1.0 ppm Spectral Service 2015 11
Peroxide Value POV ppm Spectral Service 2015 12
Anisidine Value AV 9.8 ppm Spectral Service 2015 13
TOTOX POV ppm + AV 9.8 ppm + FA ppm Spectral Service 2015 14
1,2 DAG 1,3 DAG 1,2 DAG 1,3 DAG Ratio of 1,2 to 1,3 DAG Is essential for Olive oil quality 4.9 ppm 3.6 ppm Spectral Service 2015 15
Free Fatty Acids FFA 2.20 ppm Spectral Service 2015 16
Secondary Compound α Toc. 7.4 ppm Squalene 5.1 ppm Sterols 0.70 ppm Spectral Service 2015 17
Complete Analysis JV FFA Sterols 5.3 ppm 2.20 ppm 0.70 ppm unsat. POV 1,3 DAG 1,2 DAG 18:3 18:2 2.1 ppm ppm 4.9 ppm 3.6 ppm 2.8 ppm ω-3 AV 9.8 ppm α Toc. 7.4 ppm Squalene 5.1 ppm 1.0 ppm Spectral Service 2015 18
NMR of Olive Oils Quality parameter By 1 H NMR in 5 min Peroxides (PV) Aldehydes (AV) Free Fatty Acids (TAV) Iodine Value (JV) Saturated Unsaturated FA Omega-3 1,2- and1,3-diglycerides Sterols, Tocopherols, Polyphenols Origin and Provenience By 1 H NMR in 5 min Identity and origin of Olive oil by targeted Analysis of the Polyphenol and Sterol composition By 13 C NMR in 30 min Regioselective FA distribution Spectral Service 2015 19
Internal Calibration Integral CH=CH = Iodine Number 2 CH 2 = 4 CH = 1 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 ppm Spectral Service 2015 20
Fatty Acid Composition 18:2 α-ch 2 = 6 H Allylic = unsat. 18:3 18:3 β-ch 2 = 6 H 2.8 ppm 0.98 0.96 ppm 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 ppm Spectral Service 2015 21
NMR vs GC Spectral Service 2015 22
NMR vs GC Spectral Service 2015 23
Validation Validation data are published: POV C. Skiera, B.W.K. Diehl et al: J Am Oil Chem Soc. 2012 AV C. Skiera, B.W.K. Diehl, et al 1. Euro Food Res Tech, 2012 TOTOX B.W.K. Diehl, EJLST, sub 2014 IV Y. Miyake et al, JOFI, 12/1998 FFA C. Skiera, B.W.K. Diehl, et al, JPBA, 2013 Tocopherol Spectral Service intern Vitamins B.W.K. Diehl, Y. Monakhova, J. Eiff, JAFC, 2015 DAG MAG Spectral Service intern Fa Composition Spectral Service vs. GC/MS and HPLC/MS intern Egg liquor M. Hohmann, B.W.K. Diehl,et al, JAFC, 2015 Spectral Service 2015 24
Statistical Test A by-product Spectral Service 2015 25
Statistical Test Spectral Service 2015 26
Lipid Oxidation Hydroperoxides of FA FFA Hydroperoxides of Squalene Hexanal 11.8 11.6 11.4 11.2 11.0 10.8 10.6 10.4 10.2 10.0 ppm Spectral Service 2015 27
60 Spectroscopy 1 H NMR, Oxidation of vegetable Oils PV [meq/kg] 50 40 30 20 10 0 0 5 10 15 20 NMR [mmol/kg] sunflower oil thisle oil nut oils rapeseed oil corn oil olive oil black seed oil pumpkin seed oil further oils Spectral Service 2015 28
1,2 vs 1,3 DAG Test of Aging Squalene * 64 1,2 DAG 1,3 DAG 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 ppm Spectral Service 2015 29
1,2 vs 1,3 DAG Test of Aging Glycerol Carbons TAG sn2 TAG sn1/3 1,2 DAG sn2 1,3 DAG sn1/3 1,2 DAG sn1 1,2 DAG sn3 Sterol 1,3 DAG sn 2 Sterol 73 72 71 70 69 68 67 66 65 64 63 62 61 60 59 58 57 ppm Spectral Service 2015 30
1,2 vs 1,3 DAG Test of Aging Carbonyl 1,3 DAG 1,2 DAG 18:1 TAG sn1/3 sn1 sn 2 sat 18:1 sn1/3 sn 2 18:2 20:1 18:2 174.4 174.2 174.0 173.8 173.6 173.4 173.2 ppm Spectral Service 2015 31
1,2 vs 1,3 DAG Test of Aging 1,3 DAG 1,2 DAG sn1/3 sn1 TAG sn1/3 174.4 174.3 ppm 174.2 174.1 ppm 173.7 ppm Spectral Service 2015 32
13 C NMR Regiospecivity 18:2 1/2 hour additional measuring time on 600 MHz cqnp 18:3 18:3 Squalene 128.2 128.0 ppm 128.5 128.0 127.5 127.0 126.5 126.0 125.5 125.0 124.5 ppm Spectral Service 2015 33
13 C NMR Regiospecivity Walnut 128.3 128.2 128.1 128.0 127.9 127.8 127.7 ppm Spectral Service 2015 34
13 C NMR Regiospecivity 18:2 18:2 Hazelnut 18:3 18:3 128.3 128.2 128.1 128.0 127.9 127.8 127.7 ppm Spectral Service 2015 35
13 C NMR Regiospecivity 18:2 18:2 Linseed Oil 18:3 18:3 sn2 sn1/3 128.3 128.2 128.1 128.0 127.9 127.8 127.7 ppm Spectral Service 2015 36
Phenols Dialdehydic, hemiaacetal and aldehydic forms of oleuropein, ligstroside and elenoic acid. Spectral Service 2015 37
Polyphenols Spectral Service 2015 38
Sterols Spectral Service 2015 39
Sterols Spectral Service 2015 40
Statistical Methods Spectral Service 2015 41
Chia Seed Program V.Y. Ixtaina, M.L. Martínez,, V. Spotorno, C.M. Mateo,, D.M. Maestri, B.W.K. Diehl, S.M. Nolasco, M.C. Tomás, Characterization of chia seed oils obtained by pressing and solvent extraction. Journal of Food Composition and Analysis, volume 24, issue 2, year 2011, pp. 166 174 Muchas Gracias Spectral Service 2015 42