Slide 1: Fatty Acid and FAME Analysis Using State-of-the-Art Gas Chromatography
Fatty Acid and FAME Analysis Using State-of-the-Art Gas Chromatography Gustavo Serrano, Ph.D. Global Product Manager - GC Columns Agilent Technologies
Slide 2: Outline
Outline • Analysis of Fats and Oils and Column Selection • DB-FATWAX Ultra Inert • DB-FastFAME • DB-HeavyWAX • Conclusions
Slide 3: The importance of fatty acid analysis
The importance of fatty acid analysis Quality Labeling/Regulatory Adulteration Process Monitoring/Product EMA: Economically Motivated Adulteration and assuring economics This Photo by Unknown Author is licensed under CC BY-SA
Slide 4: Fatty acids commonly found in food samples
Fatty acids commonly found in food samples • Milk fat (C4-C24 saturated/unsaturated FAMEs) • Vegetable oil (hydrogenated and nonhydrogenated) • Palm oil, rapeseed oil, olive oil, soybean oil • Cooking oils • Fish oil and meat fat
Slide 5: Fat and Oil Analysis - Triglycerides
Fat and Oil Analysis - Triglycerides • Used to identify composition and percentage of each TG present in the sample • Very easy to prepare and analyze, typically, only a dilution of the oil is required • Cold on-column injection is required due to the low volatility of the triglycerides • A typical
Slide 6: Triglycerides analysis of butter fat
Triglycerides analysis of butter fat Column CP-TAP CB, 25m, 0.25mm, 0.10μm (p/n CP7483) Inlet on-column CP-TAP CB for Triglycerides Carrier Hydrogen, 100kPa (1 bar, 15 psi) Oven 280 ⁰C (1 min) to 355 ⁰C at 3 ⁰C/min Detector FID Injection 0.2 uL P: Palmitic acid, (hexadecanoic acid) C16:0 Sample 0.05
Slide 7: Fat and Oil Analysis – Free Fatty Acids
Fat and Oil Analysis – Free Fatty Acids • Faster and easier sample preparation • Traditionally small-chain or volatile fatty acids are analyzed in free form • Usually difficult to analyze due to the carboxylic acid • Typically requires specialized column such as acid-modified wax phases Fat Hydrolys
Slide 8: Analysis of free fatty acids
Analysis of free fatty acids Column DB-FFAP, 30m, 0.25mm, 0.25μm (p/n 122-3232) Carrier Helium at 40 cm/sec, measured at 100 °C Oven 100 ⁰C (5 min) to 250 ⁰C (12 min) at 10 ⁰C/min Detector FID, 300 °C DB-FFAP Nitrogen makeup gas at 30 mL/min Injector Split 1:50, 250 °C Chromatogram C077 Peak No 1. A
Slide 9: Fat and Oil Analysis - FAME
Fat and Oil Analysis - FAME • Transesterification of free fatty acids is the most preferred method for fat analysis • Allows to distinguish slight differences among unsaturated fatty acids (e.g. positional geometric isomers) • Generally, sample preparation requires a ester group few complex steps Fa
Slide 10: Traditional FAME analysis on a CP-Sil 88
Traditional FAME analysis on a CP-Sil 88 • Most common method, suggested by AOAC 996.06 and AOCS Ce 1j-07 CP-Sil 88 • 100-m cyanopropyl columns are optimized for positional isomer Peak No separation of critical cis/trans FAMEs 1. C4:0 19. C18:2 (t9, t12) • Analysis time typically in the 65-76 min ra
Slide 11: Detailed Analysis of cis/trans FAMEs C18:1 positional isomers
Detailed Analysis of cis/trans FAMEs C18:1 positional isomers Column Select FAME, 200-m, 0.25 mm i.d. (P/N CP7421) Inlet 250°C, split mode, split ratio 1:20 Select-FAME Carrier Helium, 520 kPa Oven 185°C C18:1 cis group FID 250°C Injection 0.5 μL Peak No 1. C18:0 2. C18:1 7 trans 3. C18:1 8 trans C1
Slide 12: Comprehensive Portfolio for Fatty Acids, FAME and Oils Analysis
Comprehensive Portfolio for Fatty Acids, FAME and Oils Analysis
Slide 13: DB-FATWAX Ultra Inert
DB-FATWAX Ultra Inert For the analysis of: - FAMEs including Omega 3 and Omega 6 - Small organic acids and free fatty acids - Applications in fish oil and animal fat analysis
Slide 14: 37-FAME mix analysis with DB-FATWAX Ultra Inert
37-FAME mix analysis with DB-FATWAX Ultra Inert • Best option for saturated and polyunsaturated FAMEs, including Omega 3 and Omega 6 • WAX-type selectivity not ideal for cis/trans separation GC System Agilent 7890B Column DB-FATWAX UI, 30m, 0.25mm i.d., 0.25μm (G3903-63007) C10:0 Inlet 250°C, split/
Slide 15: Comparison study of the C24:0/C22:6n3 (DHA) critical pair
Comparison study of the C24:0/C22:6n3 (DHA) critical pair • DB-FATWAX UI fully resolves the C24:0/DHA critical pair • It remains stable with no change in selectivity or retention time even after 40 h at MAOT. DB-FATWAX UI Competitor A Competitor B C24:0 C24:0/DHA C24:0 C24:1 C24:1 C24:1 DHA DHA Afte
Slide 16: Analysis of real samples
Analysis of real samples Column DB-FATWAX UI, 30m, 0.25mm i.d., 0.25μm (G3903-63007) Inlet 250°C, split/splitless mode, split ratio 100:1 Carrier Helium, constant flow, 30cm/s@180°C Oven 180°C(2min), 2°C/min to 210°C(35min) 1. C14:0 FID 280°C, Hydrogen:40mL/min, Air:400mL/min, make-up gas:25mL/min 2
Slide 17: Analysis of real samples
Analysis of real samples Column DB-FATWAX UI, 20m*0.18mm I.D, 0.18um Inlet 250°C, split/splitless mode, split ratio 100:1 Carrier Hydrogen, constant flow, 2mL/min Oven 140°C,20°C/min to 190°C(3min), 5°C/min to 220°C(15min) FID 250°C, Hydrogen:40mL/min, Air:400mL/min, make-up gas:25mL/min Injection 1
Slide 18: DB-FATWAX UI provides inertness and thermal stability for organic acids
DB-FATWAX UI provides the desire inertness and thermal stability to separate challenge organic acids and fatty acids • Great need in the food, forensic and cosmetic industries to monitor the content of free and natural occurring fatty acids. • Analysis of underivatized organic acids and free acids i
Slide 19: Separation of short-chain volatile organic acids in water
Separation of Short-chain volatile organic acids in water using a competitor WAX column and DB-FATWAX Ultra Inert • Standard WAX columns don’t have the inertness to separate most organic acids 1. Acetic acid Competitor WAX column 2. Propionic acid 30m*0.25mm, 0.25um 3. Isobutyric acid 4. Butyric aci
Slide 20: Comparison between acid-modified WAX and DB-FATWAX UI after 50h at 250 ºC
Comparison between acid-modified WAX and DB-FATWAX UI after 50h at 250 ºC DB-FATWAX Ultra Inert shows superior inertness and thermal stability than acid-modified WAX pA Compound I.D. 60 1. 2-Nonanone 7. Methyl Dodecanoate 1 8. 2-Chlorophenol Standard acid-modified WAX 2 Decanal 50 3. Propionic Acid
Slide 21: Analysis of Fatty acids (FA)
Analysis of Fatty acids (FA) • Analysis of most FA and FAMEs without the need of an acid-modified WAX phase 1. Acetone 9. Hexanoic acid Column: Agilent DB-FATWAX UI, 30m, 0.25mm, 0.25μm (G3903-63007) 2. Acetic acid 10. Heptanoic acid Carrier gas: Helium, 40cm/s @ 100°C 3. Propionic acid 11. Octanoic
Slide 22: Fatty acids and fatty acid ethyl esters in alcoholic beverages
Fatty acids and naturally ocurring fatty acid ethyl esters analysis in alcoholic beverages Peak No Compounds Chinese liquor sample 1. 2. Acetaldehyde Acetone 3. Ethyl acetate 62% aqueous sample 4. Acetal (acetaldehyde diethyl acetal) Good resolution and peak shape 5. Methanol pA 6. Ethanol 6 18 7. 2
Slide 23: DB-FastFAME
DB-FastFAME For the analysis of: - Ultra fast FAME analysis - < 10 min analysis time for complex FAME mixtures 23 June 20, 2019 Agilent Confidential
Slide 24: DB-FastFAME (30 m x 0.25 mm x 0.25 μm)
DB-FastFAME (30 m x 0.25 mm x 0.25 μm) Technical note 5991-8706EN C16:0 Cis Trans C12:0 C10:0 C14:0 Stronger interaction of cis-isomers with the cyano-dipole causes trans-isomers to elute before the cis-isomers C8:0 C18:1 cis C6:0 C18:0 C20:0 C22:0 C4:0 C11:0 C13:0 C18:1 trans C24:0 C14:1 C15:0 C15:
Slide 25: DB-FastFAME 20m x 0.18mm x 0.20μm
Strong interaction between cis DB-FastFAME isomers and the dipoles of the 20m x 0.18mm x 0.20μm cyano propyl ligands. That allows the trans to elute after the cis isomers. Cis Trans Column Agilent J&W DB-FastFAME, 20 m x 0.18 mm, 0.20 µm Gas Hydrogen, 28 psi, constant pressure mode RS ≥ 1.95 for cis
Slide 26: Agilent Intuvo 9000
Agilent Intuvo 9000 • New design • New column connection • Guard chip • New column heating • Application to FAME analysis Intelligent, Intuitive, Innovative. Intuvo. 26 June 20, 2019 Agilent Confidential
Slide 27: Innovating a New Path to GC Productivity
Innovating a New Path to GC Productivity Modular Intuvo flow chips Disposable Guard chip Ferrule-free click-and-run connections No-trim column Direct heating 27 June 20, 2019 Agilent Confidential
Slide 28: Fast Analysis of a 36-FAME mix with Intuvo 20-m DB-FastFAME
Fast Analysis of a 36-FAME mix (AOAC 2012.13) With the Intuvo 20-m DB-FastFAME GC System: Agilent Intuvo 9000 GC system Column DB-FastFAME, 20m*0.18mm I.D, 0.20um, Intuvo module Inlet 250°C, split/splitless mode, split ratio 100:1 Intuvo Guard chip 200°C Carrier Hydrogen, constant pressure, 28psi Ov
Slide 29: How about more complex FAME samples like PHVO?
How about more complex FAME samples like PHVO? New DB-FastFAME GC column dimensions coming soon… 30 June 20, 2019 Title Confidentiality label Regulatory statement (if applicable)
Slide 30: Analysis of 57-FAMEs using a 90m DB-FastFAME
Analysis of 57-FAMEs using a 90m DB-FastFAME GC System: Agilent 7890B Column DB-FastFAME, 90m*0.25mm I.D, 0.25um Inlet 260°C, split/splitless mode, split ratio 50:1 Carrier Helium, constant pressure, 46psi Oven 70°C(1min), 40°C/min to 200°C(5 min), 2°C/min to 205°C(11.5 min); 15°C/min to 245°C(15 mi
Slide 31: Expanded view of 57-FAME Chromatogram 90-m DB-FastFAME
Expanded view of 57-FAME Chromatogram 90-m DB-FastFAME 26 min analysis time! FID2 B, Back Signal (C:\CHEM32\1\DATA\002-90M-DB23-7890B--62M-SA.D) C18:1 C18:2 C18:3 pA 1. C18:1 6t 7. C18:2 9t,12t 12. C18:3 9t,12t,15t 160 2. C18:1 9t 8. C18:2 9t,12c 13. C18:3 9t,12t,15c 3. C18:1 11t 9. C18:2 9c,12t 14.
Slide 32: Fast Analysis of a 54-FAME mix with Intuvo 60-m DB-FastFAME
Fast Analysis of a 54-FAME mix With the Intuvo 60-m DB-FastFAME (coming soon!) GC System: Agilent intuvo GC/FID Column DB-FastFAME Intuvo GC column, 60m*0.25mm I.D, 0.25um Inlet 260°C, split/splitless mode, split ratio 100:1 Guard chip 200°C Carrier Helium, constant pressure, 30psi Oven 70°C(1min),
Slide 33: Expanded view of C18:1 cis/trans isomers
Expanded view of C18:1 cis/tran isomers (from GC/FID Chromatogram of 54-FAMEs ) pA 45 40 35 30 25 20 15 10 5 16 16.2 16.4 16.6 16.8 17 17.2 min
Slide 34: DB-HeavyWAX
DB-HeavyWAX For the analysis of: - High temp applications for polar analytes - Essential oils and flavor & fragrances - Other high temp. requirements (GCxGC)
Slide 35: Introducing DB-HeavyWAX
Introducing DB-HeavyWAX • WAX column with increased MAOT compared to existing columns on the market • 280°C isothermal and 290°C programmed • Provides increased thermal stability • Has a low bleed level • Advantages General GC GC/MS GC×GC • Shorter runtimes when late • Desire for “zero” bleed • Exte
Slide 36: Bleed Summary at 280°C Over 100 Hours
Bleed Summary at 280°C Over 100 Hours pA 90 80 WAX A at 100 Hours 70 60 WAX B at 100 Hours 50 WAX B at 0 Hours 40 30 WAX A at 0 Hours 20 DB-HeavyWAX at 100 hours 10 DB-HeavyWAX at 0 hours 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Response Units (%) vs. Acq
Slide 37: Bleed reductions for FAMEs in Biodiesel using a DB-HeavyWAX
Bleed reductions for FAMEs in Biodiesel (IP-585) using a DB-HeavyWAX, 60m x 0.25mm x 0.50 µm x10 2 1.2 Peak FAME 1 C16:0 Agilent J&W DB-HeavyWAX 1.1 1 2 C17:0-d33 (IS) 0.9 3 C17:0 5 6 7 0.8 4 4 C18:0 0.7 5 C18:1 0.6 3 0.5 6 C18:2 2 0.4 7 C18:3 1 Bleed 3.1 pA 0.3 0.2 0.1 0 x10 2 Competitor A 1.2 1.1
Slide 38: Retention time stability on a DB-HeavyWAX
Retention time stability on a DB-HeavyWAX GC Conditions IP-585: FAMEs in aviation fuel Column 60m x 0.25 mm x 0.5 mm ➢ Less bleeds reduces noise level Carrier Helium, constant flow, 1.2 mL/min ➢ Enhance sensitivity without changing the detector ➢ Increase thermal stability→ Better stability in the S
Slide 39: Conclusion
Conclusion • New column technologies for faster & high performance FAME analysis, complements our comprehensive portfolio of GC columns for Fat & Oil analysis – DB-FATWAX UI is our new GC Column specially tested for FAMEs in Fish Oil and Animal Fat. Ultra Inert technology makes it possible to analyz
Slide 40: Thank you!
Thank you! 41 June 20, 2019 Title Confidentiality label Regulatory statement (if applicable)