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Analysis of Acidic

Constituents in the

Fermentation Process of

Beer Using the Cary 630

FTIR

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

Io I

H

O

H

H

O

H

H

O

H

H

O

H

IR Spectroscopy

IR energy causes molecular vibrations

Every different type of chemical bond that absorbs

infrared light naturally vibrates at a specific frequency.

When the frequency of IR light matches the frequency of

vibration of atoms bonded together, the light will be

absorbed.

The amount of energy absorbed is proportional to the

strength of a bond.

The set of IR absorbances for a sample is referred to as

an IR 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.

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

Ab

so

rban

ce

1500200025003000 35004000

Wavenumbers (cm-1)

1000

Every sample has a unique IR spectrum;

an IR spectrum can serve as a compound’s fingerprint.

Infrared Spectrum

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 (

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

Minutes10

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

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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)

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October 2, 2017

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AA Index 1280

LA Index 1150

Acetic Acid Reference

Lactic Acid Referece

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Abs

orba

nce

1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Reference Spectra of Acetic Acid (Blue) and Lactic Acid (Red)

Overlay of Spectra Taken from Different Stages in the

Fermentation Process

ferm cal 10 75um r1_2011-10-18t13-36-26(1)

1550 1500 1450 1400 1350 1300 1250 1200 1150 1100 1050 1000 950

4.5

4.0

3.5

3.0

2.5

2.0

1.5

Wavenumber

Absorb

ance

sugars

EtOH

Lactic & Acetic Acids

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Matrix SB1

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1280 AA

1130 LA

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

Absorb

ance

1000 1100 1200 1300 1400 1500

Wavenumbers (cm-1)

Spectra Used for Calibration Curve (From Standard

Addition of LA and AA)

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Calibration Curve for LA (Peak Area at 1280 cm-1)

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Calibration Curve for AA (Peak Area at 1130 cm-1)

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Matrix SB2

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1280 AA 1130 LA

0.75

0.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

1.40

1.45

1.50

1.55

Absorb

ance

1000 1100 1200 1300 1400 1500

Wavenumbers (cm-1)

Spectra Used for Calibration Curve (From

Standard Addition of LA and AA)

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Calibration Curve for LA (Peak Area at 1280 cm-1)

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Calibration Curve for AA (Peak Area at 1130 cm-1)

Summary

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• 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