chemical analysis for the winery: practical aspects

Chemical analysis for the winery: Practical aspects

Chemical analysis for the winery: Practical aspects

Anita Oberholster

IntroductionIntroduction

What to measure? methods

When to measure?

Why measure?

What does it mean?

What to measure? methods

When to measure?

Why measure?

What does it mean?

Juice/wine analysisJuice/wine analysis

°Brix 1°Brix = 1 g of sugar per 100 g of solution Hydrometry or refractometer measures total

soluble solids (90–94% sugars) Not analytical technique, only give estimation of amount of

sugar Why measure?

Indication of fruit ripeness Potential ethanol production in wine

» Yeast vary in their efficiency to convert sugar to alcohol – could differ up to 0.8 % v/v

Follow progress of fermentation

°Brix 1°Brix = 1 g of sugar per 100 g of solution Hydrometry or refractometer measures total

soluble solids (90–94% sugars) Not analytical technique, only give estimation of amount of

sugar Why measure?

Indication of fruit ripeness Potential ethanol production in wine

» Yeast vary in their efficiency to convert sugar to alcohol – could differ up to 0.8 % v/v

Follow progress of fermentation


Reducing sugars Mainly glucose and fructose Methods

Rebelein method (titration with color change) Enzymatic analysis FOSS or “winescan”, HPLC (expensive)

Why measure? Important for regulatory and wine style reasons, especially

when aiming for specific sugar content

Reducing sugars Mainly glucose and fructose Methods

Rebelein method (titration with color change) Enzymatic analysis FOSS or “winescan”, HPLC (expensive)

Why measure? Important for regulatory and wine style reasons, especially

when aiming for specific sugar content


pH Measure free H+ ions pH = -log [H+] Why is knowing the pH important?

Microbial stability Effectiveness of SO2

» Molecular SO2 is the form effective against microorganisms

pH Measure free H+ ions pH = -log [H+] Why is knowing the pH important?

Microbial stability Effectiveness of SO2

» Molecular SO2 is the form effective against microorganisms


pH Mouth-feel (“flabby”) – wine style Color of red wine Color of red wine

pH Mouth-feel (“flabby”) – wine style Color of red wine Color of red wine


Titratable acidity (TA) Measures concentration of all available

hydrogen ions (both free and bound to dissociated acids (eg H2T) How do you measure it?

Titrating with known vol of NaOH to end point indicated by color change of indicator (pH 8.2)

» Expressed as g/L tartaric acid Foss, HPLC

Titratable acidity (TA) Measures concentration of all available

hydrogen ions (both free and bound to dissociated acids (eg H2T) How do you measure it?

Titrating with known vol of NaOH to end point indicated by color change of indicator (pH 8.2)

» Expressed as g/L tartaric acid Foss, HPLC

Juice/wine analysisJuice/wine analysis Why is knowing the TA important?

Guide to acid taste of wine, desired amounts depends on wine style

Adjust pH of wine

Why is knowing the TA important? Guide to acid taste of wine, desired amounts depends on

wine style Adjust pH of wine


Adjust wine pH Change in pH not directly related to acid

addition Depends on wines buffer capacity Rule of thumb: 1 g/L of tartaric acid, decrease pH by 0.1

Add L(+)-tartaric acid DL-tartaric acid addition

Increase calcium tartrate instability

Adjust wine pH Change in pH not directly related to acid

addition Depends on wines buffer capacity Rule of thumb: 1 g/L of tartaric acid, decrease pH by 0.1

Add L(+)-tartaric acid DL-tartaric acid addition

Increase calcium tartrate instability


Measure malic acid (and lactic acid) conc TLC, Enzymatic analysis, HPLC or FOSS Why measure?

Chances of MLF Following MLF

Measure malic acid (and lactic acid) conc TLC, Enzymatic analysis, HPLC or FOSS Why measure?

Chances of MLF Following MLF

T L M


Nitrogen (NH3, NH4+ and amino acids)

Enzymatic analysis or HPLC, FOSS Why measure?

N2 deficiency» Stuck fermentation» Utilization of sulfur containing amino acids – formation of H2S

Too much N2» Modify aroma character» Formation of ethyl carbamate

N2 needed» 100-200 mg/L needed for fermentation

Nitrogen (NH3, NH4+ and amino acids)

Enzymatic analysis or HPLC, FOSS Why measure?

N2 deficiency» Stuck fermentation» Utilization of sulfur containing amino acids – formation of H2S

Too much N2» Modify aroma character» Formation of ethyl carbamate

N2 needed» 100-200 mg/L needed for fermentation


Yeast assimilable nitrogen (YAN) Ammonia (NH3) and free alpha amino nitrogen (FAN)

How to measure? Formol titration (FAN + NH4

+)» Only need pH meter

NOPA method (primary amino acids)» Need Vis spectrophotometer» Derivatisation of primary amino acid groups with o-phthaldehyde/N-

acetyl-L-cysteine reagent» Resulting iso-indole derivative absorb at 335 nm» Quantification by using calibration curve using known iso-leucine

concentrations FOSS (FAN + NH3)

Yeast assimilable nitrogen (YAN) Ammonia (NH3) and free alpha amino nitrogen (FAN)

How to measure? Formol titration (FAN + NH4

+)» Only need pH meter

NOPA method (primary amino acids)» Need Vis spectrophotometer» Derivatisation of primary amino acid groups with o-phthaldehyde/N-

acetyl-L-cysteine reagent» Resulting iso-indole derivative absorb at 335 nm» Quantification by using calibration curve using known iso-leucine

concentrations FOSS (FAN + NH3)

Must/wine analysisMust/wine analysis

Sulfur dioxide analysis (free and bound) Why use?

Antimicrobial and antioxidant properties Why measure?

Regulatory Too little – no protection Too much – inhibit yeast, used to produce sulfides

Sulfur dioxide analysis (free and bound) Why use?

Antimicrobial and antioxidant properties Why measure?

Regulatory Too little – no protection Too much – inhibit yeast, used to produce sulfides

Must/wine analysisMust/wine analysis

Sulfur dioxide analysis (free and bound) How to measure

Aspiration method» Stream of air through acidified solution passes released SO2

through H2O2 solution» H2O2 + SO2 H2SO4» Titrated with NaOH» Boiling releases bound SO2

Ripper method (Metrohm)» Titration with iodine (redox reaction), less accurate

because phenols and sugars also oxidized by I2 Winescan SO2 (new application)

» Measure free and total SO2

Sulfur dioxide analysis (free and bound) How to measure

Aspiration method» Stream of air through acidified solution passes released SO2

through H2O2 solution» H2O2 + SO2 H2SO4» Titrated with NaOH» Boiling releases bound SO2

Ripper method (Metrohm)» Titration with iodine (redox reaction), less accurate

because phenols and sugars also oxidized by I2 Winescan SO2 (new application)

» Measure free and total SO2

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Wine analysisWine analysis

Alcohol analysis Mostly EtOH, small amount of MeOH, PrOH,

BuOH and glycerol Why measure?

Regulatory, TAX Mouth-feel, wine style

How to measure? Distillation with hydrometry or

pycnometry» Accurate (0.1% v/v)

Alcohol analysis Mostly EtOH, small amount of MeOH, PrOH,

BuOH and glycerol Why measure?

Regulatory, TAX Mouth-feel, wine style

How to measure? Distillation with hydrometry or

pycnometry» Accurate (0.1% v/v)

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Alcohol analysis How to measure?

Distillation with hydrometry or pycnometry Ebulliometry –cheap and easy but less accurate HPLC, GC – accurate more expensive Densitometry

» Uses the fact that alcohol is less dense thanwater, so the lower the density the higher the alcohol content

Alcohol analysis How to measure?

Distillation with hydrometry or pycnometry Ebulliometry –cheap and easy but less accurate HPLC, GC – accurate more expensive Densitometry

» Uses the fact that alcohol is less dense thanwater, so the lower the density the higher the alcohol content

Termometer

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Volatile acidity Volatile fatty acids (acetic acid – 90%) Why measure?

Indicator of spoilage» Normal 0.2-0.4 g/L produced during fermentation» Fault 0.7-1.1 mg/L depending on wine style

Regulatory (<1.2 g/L white wine, < 1.4 g/L red wine) How to measure?

Steam distillation followed by titration

Volatile acidity Volatile fatty acids (acetic acid – 90%) Why measure?

Indicator of spoilage» Normal 0.2-0.4 g/L produced during fermentation» Fault 0.7-1.1 mg/L depending on wine style

Regulatory (<1.2 g/L white wine, < 1.4 g/L red wine) How to measure?

Steam distillation followed by titration


Volatile acidity How to measure?

Steam distillation followed by titration (± 0.05 g/L)» Possible problems – if boil to fast, other acids also distilled» Degas or CO2 will distil as carbonic acid» Add H2O2 to bind SO2

- SO2 distilled as sulfuric acid HPLC, GC, FOSS

Volatile acidity How to measure?

Steam distillation followed by titration (± 0.05 g/L)» Possible problems – if boil to fast, other acids also distilled» Degas or CO2 will distil as carbonic acid» Add H2O2 to bind SO2

- SO2 distilled as sulfuric acid HPLC, GC, FOSS

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FOSS (FTIR) How does it work?

FOSS (FTIR) How does it work?


Phenol analyses Folin-Ciocalteu

Measures all OH groups» Interference from sugar, also ox SO2 etc.

FC reagent (yellow) oxidizes phenols and reduces product (blue-green) measured at 765 nm

[Phenol] in gallic acid equivalents (GAE) mg/L

Phenol analyses Folin-Ciocalteu

Measures all OH groups» Interference from sugar, also ox SO2 etc.

FC reagent (yellow) oxidizes phenols and reduces product (blue-green) measured at 765 nm

[Phenol] in gallic acid equivalents (GAE) mg/L


Folin-Ciocalteu Why measure?

Phenols source of browning substance Responsible for color and taste (astringency) Aging potential Useful if historic knowledge of grapes and wine style aiming

for Could adapt winemaking accordingly Range of values white table wine (40 – 1,300, av 225; red

table wine 190 – 3,800, av 1,800)

Folin-Ciocalteu Why measure?

Phenols source of browning substance Responsible for color and taste (astringency) Aging potential Useful if historic knowledge of grapes and wine style aiming

for Could adapt winemaking accordingly Range of values white table wine (40 – 1,300, av 225; red

table wine 190 – 3,800, av 1,800)


Phenol analyses Color measurements (280, 420 and 520 nm) 280 nm similar to FC – indication of total

phenolics 520 nm – red color [anthocyanin]

A = c» A = absorbance» = extinction coefficient» = pathlength (cm)» c = concentration of sample

Corr HPLC analysis, less expensive

Phenol analyses Color measurements (280, 420 and 520 nm) 280 nm similar to FC – indication of total


A = c» A = absorbance» = extinction coefficient» = pathlength (cm)» c = concentration of sample

Corr HPLC analysis, less expensivenm250 300 350 400 450 500 550 600

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Anthocyanin profile by RP-HPLC Color measurements (280, 420 and 520 nm) 280 nm similar to FC – indication of total



Anthocyanin profile by RP-HPLC Color measurements (280, 420 and 520 nm) 280 nm similar to FC – indication of total



min0 10 20 30 40 50 60 70 80

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Why measure color? Determine opt time to harvest Red color major quality indicator for red wine

» Color corr with wine quality Aging potential Historical knowledge/values of vineyard or wine style to

compare – adapt winemaking practices 280/520 ratio quality indicator

» Depends on terroir» Personally found it between 1 to 3

420/520 nm ratio in wine gives hue» Indicative of oxidation, age of wine» In young wine <0.7» Approach 1 with aging

Why measure color? Determine opt time to harvest Red color major quality indicator for red wine

» Color corr with wine quality Aging potential Historical knowledge/values of vineyard or wine style to

compare – adapt winemaking practices 280/520 ratio quality indicator

» Depends on terroir» Personally found it between 1 to 3

420/520 nm ratio in wine gives hue» Indicative of oxidation, age of wine» In young wine <0.7» Approach 1 with aging


Phenol analyses Color measurements (280, 420 and 520 nm)

280 nm similar to FC – indication of total phenolics 520 nm – red color [anthocyanin]

» Corr HPLC analysis, less expensive

Why measure? Determine opt time to harvest Red color major quality indicator for red wine Aging potential Historical knowledge/values of vineyard or wine style to

compare – adapt harvest date and winemaking approach

Phenol analyses Color measurements (280, 420 and 520 nm)

280 nm similar to FC – indication of total phenolics 520 nm – red color [anthocyanin]

» Corr HPLC analysis, less expensive

Why measure? Determine opt time to harvest Red color major quality indicator for red wine Aging potential Historical knowledge/values of vineyard or wine style to

compare – adapt harvest date and winemaking approach


Phenol analyses Adams-Harbertson assay

Measures tannin, anth, small polymeric pigments (SPP) and large polymeric pigments (LPP)

» Use combination of protein (BSA) precipitation and SO2 addition

Why measure? Similar to 280 nm and FC, except more specific to tannin Best correlation to observed astringency Also give anthocyanin and polymeric pigment conc

Phenol analyses Adams-Harbertson assay

Measures tannin, anth, small polymeric pigments (SPP) and large polymeric pigments (LPP)

» Use combination of protein (BSA) precipitation and SO2 addition

Why measure? Similar to 280 nm and FC, except more specific to tannin Best correlation to observed astringency Also give anthocyanin and polymeric pigment conc

Juice analysisJuice analysis

Laccase activity Enzymatic analysis

Activity measurement based on rate the enzyme oxidizes syringaldazine to a purple colored oxidation product

Measured at 530 nm with spectrophotometer Acitivity expressed as U/mL Unfortunately not very sensitive SO2 and ascorbic acid interfere with analysis

Ranges from 0 in sound fruit to 140 U/mL 5-10 U/mL indicate some influence of laccase >10 U/mL apparent influence likely

Laccase activity Enzymatic analysis

Activity measurement based on rate the enzyme oxidizes syringaldazine to a purple colored oxidation product

Measured at 530 nm with spectrophotometer Acitivity expressed as U/mL Unfortunately not very sensitive SO2 and ascorbic acid interfere with analysis

Ranges from 0 in sound fruit to 140 U/mL 5-10 U/mL indicate some influence of laccase >10 U/mL apparent influence likely

Juice analysisJuice analysis

Laccase activity Why measure?

If Botrytis in the vineyard – knowing the number and potential risk

Better adaptation during the winemaking process

Laccase activity Why measure?

If Botrytis in the vineyard – knowing the number and potential risk

Better adaptation during the winemaking process


Tartrate stability Wine is a supersaturated solution of KHT How to measure?

Cold test (-2ºC - 5ºC, up to 5 days) Mini-contact test (seed with 4 g/L) Conductivity test with seeding

» Short version , 30 min with 10 g/L KHT addition

Why measure? Prevent crystal formation and precipitation Consumer satisfaction Important, needs to be stabilized for conditions of storage

and export market

Tartrate stability Wine is a supersaturated solution of KHT How to measure?

Cold test (-2ºC - 5ºC, up to 5 days) Mini-contact test (seed with 4 g/L) Conductivity test with seeding

» Short version , 30 min with 10 g/L KHT addition

Why measure? Prevent crystal formation and precipitation Consumer satisfaction Important, needs to be stabilized for conditions of storage

and export market


Protein stability Many tests, based on denaturing protein by

heat, acid or alcohol Done in combination with bentonite trial to

determine opt fining addition Most commonly used –heat stability test

Why measure? Unstable proteins in wine can lead to development of a haze

or deposit. Mainly problematic in white wine In red proteins react with tannin, precipitate during

fermentation and maturation

Protein stability Many tests, based on denaturing protein by

heat, acid or alcohol Done in combination with bentonite trial to

determine opt fining addition Most commonly used –heat stability test

Why measure? Unstable proteins in wine can lead to development of a haze

or deposit. Mainly problematic in white wine In red proteins react with tannin, precipitate during

fermentation and maturation


Oxidative and color stability Not absolute test, indication Based on accelerated aging

White wine: Heat 50°C, measure 420 nm » Evaluation base ond speed of 420 nm increse0.15 unit

increase in 2 to 3 days, likely unstable Red wine: Aerate wine, compare after 3 days with control (wine + 30 mg/L SO2)

» Obvious browning, oxidized character – considered unstable» If control also oxidized then very unstable

Why measure? Determine aging potential Release date of wine, opt storage

Oxidative and color stability Not absolute test, indication Based on accelerated aging

White wine: Heat 50°C, measure 420 nm » Evaluation base ond speed of 420 nm increse0.15 unit

increase in 2 to 3 days, likely unstable Red wine: Aerate wine, compare after 3 days with control (wine + 30 mg/L SO2)

» Obvious browning, oxidized character – considered unstable» If control also oxidized then very unstable

Why measure? Determine aging potential Release date of wine, opt storage


Pinking potential Usually in white wine made under reductive

conditions How to measure?

Treat wine with H2O2 and compare with control» Visible pinking or increase 520 nm – do fining trial with PVPP,

casein or combination

Why measure? To prevent “pinking “of wine Consumer satisfaction

Pinking potential Usually in white wine made under reductive

conditions How to measure?

Treat wine with H2O2 and compare with control» Visible pinking or increase 520 nm – do fining trial with PVPP,

casein or combination

Why measure? To prevent “pinking “of wine Consumer satisfaction

Wine analysisWine analysis Musty taints What off-odours classified as musty taints?

Fungal, earthy, moldy, corky, mushroom or straight musty What causes musty taints?

Haloanisoles (TCA, TCB) Alkylmethoxypyrazine (MDMP) Carbon unsaturated aliphatic compounds with carbonyl

function (1-octen-3-one, 1-nonen-3-one) (-)-Geosmin

Musty taints What off-odours classified as musty taints?

Fungal, earthy, moldy, corky, mushroom or straight musty What causes musty taints?

Haloanisoles (TCA, TCB) Alkylmethoxypyrazine (MDMP) Carbon unsaturated aliphatic compounds with carbonyl

function (1-octen-3-one, 1-nonen-3-one) (-)-Geosmin

Wine analysis – Musty taintsWine analysis – Musty taints Why measure? To prevent fault Identify source of contamination Identify compound responsible

80-85% TCA responsible Bottle to bottle variation indicate cork taint

Possible removal from contaminate

Why measure? To prevent fault Identify source of contamination Identify compound responsible

80-85% TCA responsible Bottle to bottle variation indicate cork taint

Possible removal from contaminate

2,4,6-trichloroanisole (TCA)

Wine analysis – Musty taintsWine analysis – Musty taints How to measure?

Screening method usually based on sensory assessments of wine soaks or corks in damp environment Advantage – low cost, detect both known an unknown taints Disadvantage – variation in performance of assessors Low sensitivity and corks soaked in batches Soaked few hrs to 2 days in mostly aqueous alcohol

Instrumental analysis of TCA Identify specific taints as well precursors

GC-MS (expensive)

How to measure? Screening method usually based on sensory

assessments of wine soaks or corks in damp environment Advantage – low cost, detect both known an unknown taints Disadvantage – variation in performance of assessors Low sensitivity and corks soaked in batches Soaked few hrs to 2 days in mostly aqueous alcohol

Instrumental analysis of TCA Identify specific taints as well precursors

GC-MS (expensive)


Wine analysis – Musty taintsWine analysis – Musty taints How to measure?

No screening method reflects accurately level of taint in bottle Whole surface of cork is extracted in cork soaks Short soaking periods means only ‘rapidly released’ TCA measured Batches soaked, if one cork contaminated the average could be below

detection limit However, these measurements still help predict

bottle taint

How to measure? No screening method reflects accurately level of taint

in bottle Whole surface of cork is extracted in cork soaks Short soaking periods means only ‘rapidly released’ TCA measured Batches soaked, if one cork contaminated the average could be below

detection limit However, these measurements still help predict

bottle taint


Wine analysisWine analysis Many many more chemical analyses Identify taints/compounds of interest

Methoxypyrazines Ethyl acetate Volatile phenols Sulfur compounds........

GC-FID or GC-MS

Many many more chemical analyses Identify taints/compounds of interest

Methoxypyrazines Ethyl acetate Volatile phenols Sulfur compounds........

GC-FID or GC-MS

Wine analysisWine analysis Many many more chemical analyses Phenol compounds of interest –RP-HPLC

Catechin, epicatechin, resveratrol, quercetrin

Many many more chemical analyses Phenol compounds of interest –RP-HPLC

Catechin, epicatechin, resveratrol, quercetrin

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Wine analysisWine analysis Many many more chemical analyses Health and regulatory pruposes Pesticides

GC-MS Trace metals

<300 ppb lead, <500 ppb copper Coupled Plasma MS

Allergen labelling proposed by TTB Currently voluntary Milk, Eggs, Fish, Wheat, Tree nut, Peanuts, Soy, Shellfish

» Allergen in the proteinEnzyme linked Immunosorbent Assay – most cost effective

Many many more chemical analyses Health and regulatory pruposes Pesticides

GC-MS Trace metals

<300 ppb lead, <500 ppb copper Coupled Plasma MS

Allergen labelling proposed by TTB Currently voluntary Milk, Eggs, Fish, Wheat, Tree nut, Peanuts, Soy, Shellfish

» Allergen in the proteinEnzyme linked Immunosorbent Assay – most cost effective

ConclusionsConclusions A lot of supportive data available Some more useful or necessary than others In more difficult years

Larger need for supportive data to be able to adapt Compositional data useful in building

knowledge base regarding specific vineyards and/or wines Valuable when aiming for similar wine style Determine optimal harvest date

A lot of supportive data available Some more useful or necessary than others In more difficult years

Larger need for supportive data to be able to adapt Compositional data useful in building

knowledge base regarding specific vineyards and/or wines Valuable when aiming for similar wine style Determine optimal harvest date

Questions?Questions?

chemical analysis for the winery: practical aspects

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