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Tips and Sips Analysis of Acidic Constituents in the Fermentation Process of Beer Using the Cary 630 FTIR Confidentiality Label 1 October 2, 2017 What can you measure with infrared light? IR spectroscopy can be used to analyze samples from all three states of matter – it can be used to analyze gases, liquids, and solids. Infrared spectroscopy has been a cornerstone of analytical measurements for over 50 years. Routine measurement, used for qualitative and quantitative analysis IR Spectroscopy IR energy causes molecular vibrations Every different type of chemical bond that absorbs O infrared light naturally vibrates at a specific frequency. H When the frequency of IR light matches the frequency of H vibration of atoms bonded together, the light will be absorbed. O The amount of energy absorbed is proportional to the strength of a bond. H H The set of IR absorbances for a sample is referred to as an IR spectrum O H H Io I O H H Infrared Spectrum The IR spectrum of a sample is a plot of the amount of IR energy (y-axis) that is absorbed at frequencies (x-axis) in IR the region of the electromagnetic spectrum. Every sample has a unique IR spectrum; an IR spectrum can serve as a compound’s fingerprint. Absorbance 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm-1) 4000 3500 3000 2500 2000 1500 1000 Wavenumbers (cm-1) Beers Law - (I’ll drink to that…) Beer’s Law (Absorbance Law): IR absorbance is directly proportional to concentration A=abc A is Absorbance, b=thickness, c=concentration, a=absorptivity constant FT-IR measures each peak separately FT-IR can measures all compounds in a mixture at the same time (one spectrum can yield concentrations for dozens of components) Current Analysis of Liquids by FTIR For longer pathlength measurements (>30 microns - lower concentration solutes; IR transparent solutions) • Fixed pathlength transmission cells • Demountable, variable pathlength transmission cells For shorter pathlength measurements (<30 microns – higher concentration solutes; IR opaque solutions) • Internal reflection No real change in the past 30 years on how liquids are measured via mid IR Liquid Samples Anyone who has measured liquids with traditional cells will be AMAZED at how much faster and easier it is to measure liquids with the DialPath or TumblIR. This patented technology is a HUGE time saver in the lab and is available only from Agilent. Traditional liquid cell The New ‘Agilent’ Way 10 10 Minutes Seconds 7 Revolutionary technology for Liquids Analysis “Dialpath” Agilent Confidential Proprietary Transmission Technology As quick and easy to carry out longer pathlength transmission measurements as ATR makes shorter pathlength analysis • DialPath: Choice of three factory calibrated, fixed pathlengths can be selected in seconds • TumblIR: One dedicated Pathlength • Covers pathlengths that ATR cannot • No spacers, windows, or syringes needed • No fringing Sample introduction and sample cleaning is simple – takes seconds • Compatible with samples having a wide range of viscosities • Eliminates the need to heat samples to reduce viscosity Increased Sensitivity vs. ATR 75 um DialPath Cell 30 um DialPath Cell ATR Experimental Background • Proof of concept • Monitor acid ingredients in fermentation matrixes • Acids and high amounts of sugar reduce ethanol formation • Need quick spectrographic analysis • help control the fermentation process in real time • save time • augment current analytical methods (e.g., titration and chromatography) • Use Standard Addition method to spike two matrixes of beer (SB1 and SB2) with lactic acid (LA) and acetic acid (AA) Confidentiality Label 11 October 2, 2017 Confidentiality Label 12 October 2, 2017 Reference Spectra of Acetic Acid (Blue) and Lactic Acid (Red) Acetic Acid Reference 1.3 Lactic Acid Referece AA Index 1280 1.2 1.1 1.0 0.9 0.8 LA Index 1150 0.7 0.6 Absorbance 0.5 0.4 0.3 0.2 0.1 0.0 4000 3500 3000 2500 2000 1500 1000 Wavenumbers (cm-1) Confidentiality Label 13 October 2, 2017 Overlay of Spectra Taken from Different Stages in the Fermentation Process 4.5 sugars ferm cal 10 75um r1_2011-10-18t13-36-26(1) 4.0 3.5 Lactic & Acetic Acids 3.0 Absorbance 2.5 2.0 EtOH 1.5 1550 1500 1450 1400 1350 1300 1250 1200 1150 1100 1050 1000 950 Wavenumber Matrix SB1 Confidentiality Label 15 October 2, 2017 Spectra Used for Calibration Curve (From Standard Addition of LA and AA) 1.8 1.7 1.6 1280 AA 1.5 1.4 1130 LA 1.3 1.2 Absorbance 1.1 1.0 0.9 0.8 1500 1400 1300 1200 1100 1000 Wavenumbers (cm-1) Confidentiality Label 16 October 2, 2017 Calibration Curve for LA (Peak Area at 1280 cm-1) Confidentiality Label 17 October 2, 2017 Calibration Curve for AA (Peak Area at 1130 cm-1) Confidentiality Label 18 October 2, 2017 Matrix SB2 Confidentiality Label 19 October 2, 2017 Spectra Used for Calibration Curve (From Standard Addition of LA and AA) 1.55 1.50 1.45 1.40 1.35 1.30 1.25 1.20 1.15 1.10 Absorbance 1.05 1.00 1130 LA 1280 AA 0.95 0.90 0.85 0.80 0.75 1500 1400 1300 1200 1100 1000 Wavenumbers (cm-1) Confidentiality Label 20 October 2, 2017 Calibration Curve for LA (Peak Area at 1280 cm-1) Confidentiality Label 21 October 2, 2017 Calibration Curve for AA (Peak Area at 1130 cm-1) Confidentiality Label 22 October 2, 2017 Summary • IR is a well-established analytical technique • Sensitivity is improved by using transmission • Dial Path facilitates quick analysis, providing actionable results in seconds • Fermentation processes can be quickly monitored using FTI Confidentiality Label 23 October 2, 2017.
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