chemical analysis for the winery: practical aspects
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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
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
lug
kondenseerder
wyn
indikator
25% v/v H PO3 4
0.3% H O2 2
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)
wyn
piknometerflessie
distillaat
Wine analysisWine analysis
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
boonste inlaat
refluks kondenseerder
VerhittingskamerKraan
vlam
Wine analysisWine analysis
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
Wine analysisWine analysis
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
A
X
wyn
H2O in
Kondenseerder
Verhittingselement
H2O out
H2O oplossing
Wine analysisWine analysis
FOSS (FTIR) How does it work?
FOSS (FTIR) How does it work?
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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)
Juice/wine analysisJuice/wine analysis
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
phenolics 520 nm – red color [anthocyanin]
A = c» A = absorbance» = extinction coefficient» = pathlength (cm)» c = concentration of sample
Corr HPLC analysis, less expensivenm250 300 350 400 450 500 550 600
mAU
0
25
50
75
100
125
150
175
200
Juice/wine analysisJuice/wine analysis
Anthocyanin profile by RP-HPLC 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
Anthocyanin profile by RP-HPLC 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
min0 10 20 30 40 50 60 70 80
mAU
0
200
400
600
800
280 nm520 nm
Del
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Pet
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Peon
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Peo
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Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
Juice/wine analysisJuice/wine analysis
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
Wine analysisWine analysis
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
Wine analysisWine analysis
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
Wine analysisWine analysis
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
Wine analysisWine analysis
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)
2,4,6-trichloroanisole (TCA)
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
2,4,6-trichloroanisole (TCA)
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
Time (min)
Abs
orba
nce
Abs
orba
nce
Gal
lic a
cid
Cat
echi
n
Que
rcet
in-3
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cosi
de
Caf
taric
ac
id
Pol
ymer
ic p
heno
ls
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?
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