LAB ANALYSES for BEGINNERS

to INTERMEDIATES

Barry H. Gump, Ph. D.

Professor of Beverage Management

Chaplin School of Hospitality & Tourism

Management

Florida International University

North Miami, FL

bgump@fiu.edu

What’s Important?

1. Fruit Maturity -- Soluble solids, titratable acidity, tartaric/malic ratio,

& pH

2. Harvest/pre-fermentation -- Soluble solids/sugar per berry, pH and

Titratable acidity, & Nitrogen

3. During fermentation – Brix

4. End of primary fermentation – Brix, Volatile acidity, Malolactic

fermentation, Ethanol, & Nitrogen

5. Post-fermentation -- Residual Sugar, pH/Sulfur dioxide, Heat

stability, Cold Stability

pH, Titratable Acidity, and YAN

What are Acids?

Acids dissociate to produce protons (hydrogen ions) in solution

Acids found in juices and wines are termed “weak acids” – they only partially dissociate

Tartaric, malic, and small amounts of citric found in grapes

Tartaric, malic/lactic, succinic acids are primary in wines

What is pH?

• pH is a concentration term for free (dissociated) protons in solution

• pH = - log[H+], the logarithmic concentration of free protons with the sign changed (to make pH values positive numbers)

• On the pH scale values below pH 7 denote acidic solutions, values above 7 denote alkaline or basic solutions

Consult operator's manual for standardization using two buffer solutions.

Rinse the beaker with sample. Place enough fresh sample in beaker to cover electrode junctions. Allow to come to defined temperature.

Place electrode(s) in the sample.

Allow meter reading to stabilize and record value.

pH Measurements

Titratable Acidity (TA) refers to the total concentration of free protons and

undissociated acids in a solution that can react with a strong base and be

neutralized

Typical concentrations of free protons in a juice or wine range from ~ 0.1 to

1 mg/L, whereas TA values might be 4 to 8 g/L

A Titratable Acidity (TA) titration will generally use the strong base, NaOH,

and either a chemical indicator or pH meter to signal when equivalent

amounts of base have been metered into the sample

The concentration of sodium hydroxide used is typically 0.1 N

(same as 0.1 M) or less

What is Titratable Acidity?

Two units used to express the concentration of an analytical reagent

Molarity denotes the concentration in moles of reagent per liter, eg. Mol NaOH/L

Normality denotes the concentration in moles of reacting unit per liter, eg. Mol OH-/L

For solutions of NaOH the Normality equals the Molarity

Normality vs. Molarity

Two burets with NaOH (0.100 N and 0.0100 N)

Titration

Accurately pipette a 10 mL juice or wine

sample into the beaker and note volume

reading on buret (V1)

Add 0.1 N NaOH from buret to pH 8.2 endpoint

and note volume reading (V2). VNaOH = V2 – V1

TA ANALYSIS

Pipettes

H2T + HT- + T= +H++OH- H2O + T=

T= is a base, so at the end of this titration the solution

will be alkaline

TA (g/L H2T) = VNaOH x MNaOHx ½ x 0.150 x 1000/10

TA (g/L H2T) = VNaOH x 0.75 if NaOH is 0.1 M

TA MEASUREMENTS

What Is Importance of YAN• YAN – Yeast Assimilable Nitrogen

• The chemical and physical environment of grape juice fermentation,

coupled with competition from indigenous yeast and bacteria, can present

significant challenges to the growth of Saccharomyces cerevisiae.

• Nitrogen compounds in grapes play important roles as nutrients for

microorganisms involved in winemaking and wine spoilage and as aroma

substances and precursors.

• The nitrogenous components of grapes and juice which are metabolically

available to yeasts are present as ammonium salts (NH4+) and primary or

“free alpha-amino acids” (FAN).

Measuring YAN

Nitrogen

Using 1 M NaOH, pre-titrate formaldehyde to pH 8.2

Select buret with 0.01 M NaOH

Add 2 mL formaldehyde (eyedropper) to juice/wine sample and titrate with 0.01 N NaOH from buret to return to pH 8.2 endpoint

Note beginning volume reading and endpoint volume reading (V3 and V4)

VNaOH = V4 – V3

0.1 MNaOH0.01 MNaOH

MNaOH

Nitrogen

N (mg/L) = VNaOH x NNaOHx 14 x 1000/10

N (mg/L) = VNaOH x 14 using 0.01 N NaOH

FORMOL NITROGEN ANALYSIS

SULFUR DIOXIDE

AO & Ripper Methods

SO2 is an anhydride of sulfurous acid

SO2 + H2O H2SO3

Sulfurous acid – although technically a weak acid --can dissociate in

wine

H2SO3 H+ + HSO3-

HSO3- H+ + SO3

2-

Wine pH affects concentration of sulfite species

pH vs. % Sulfite Species

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11

pH

%S

ulfit

e S

peci

es

H2SO3 HSO3

-SO3

=

pH vs. % Sulfite Species

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11

pH

%S

ulfit

e S

peci

es

H2SO3 HSO3

-SO3

=

Free SO2 Needed to Obtain 0.5 or 0.8 mg/L (molecular) at Various pH

Levels

0.0

20.0

40.0

60.0

80.0

100.0

120.0

2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0

pH

Fre

e S

O2 (

mg

/L)

0.8 mg/L

0.5 mg/L

Aspiration Method

Sodium Hydroxide (0.01 M): Easiest to take known standard NaOH of ~ 0.1 M and do a 1+9 (1:10) dilution.

Hydrogen Peroxide (~ 3%): Purchase at drugstore – use 10 mL

Phosphoric Acid (1 + 3): Using o-phosphoric acid (85% stock) carefully prepare an approximate 1 + 3 solution with deionized water – use 10 mL

Indicator Solution is available commercially –mixture of Methyl Red and Methylene Blue –violet (acid) – colorless – green (> pH 6) –use 1-3 drops

Distillation of Sample Remove sample bottle from refrigerator and bring to

room temperature. Pipette exactly 20.0 mL sample to the

round bottom flask

Add ~10 mL phosphoric acid to the same flask.

Replace stopper in round bottom flask and begin

aspirating vigorously (1.5 liters/minute)

Continue aspirating sample for exactly 10 min

Titration

At the end of the 10 min period turn off aspiration.

Remove flat-bottomed receiver and rinse the inside of the

vacuum adapter, and the outside of the Pasteur pipette

connected to it, into the receiver.

Titrate contents of flask with 0.01 N NaOH to the end

point. Read the titration volume to the nearest 0.01 mL

(V2)

Chemistries & Calculations

3H+ + H2SO3 + HSO3-

+ SO3= 3H2SO3 3SO2 + 3H2O

H2O2 + SO2 H2O+ SO3 H2SO4

H2SO4 + 2OH- 2H2O + SO4

=

Calculate SO2 as follows: VNaOH = V2 – V1

SO2 (mg/L) = VNaOH x MNaOH x 64/2 x 1,000 / 20 (20 mL sample size)

SO2 (mg/L) = VNaOH x 16

SULFUR DIOXIDE: RIPPER

TITRAMETRIC METHOD

USING IODINE

• standard iodine is used to titrate free or total sulfur

dioxide. The end point of the titration is traditionally

monitored using starch indicator solution

• Free sulfur dioxide is determined directly. Total sulfur

dioxide can be determined by first treating the sample

with sodium hydroxide to release bound sulfur dioxide

EQUIPMENT

250-mL Erlenmeyer flask (preferably wide-mouth)

10-mL burette

25-mL volumetric pipette

High-intensity light source

REAGENTS

Working Iodine Solutions – 0.01M (0.02N)

I2 + SO2 + H2O 2HI + SO3

(It may be necessary to standardize working solutions against

primary standard sodium thiosulfate)

Sulfuric Acid (1 + 3) - Carefully dilute 1 vol of concentrated

acid into 3 vol of deionized water.

Starch Indicator (1%): Mix 10 g of soluble starch and 1 L of

deionized water in a beaker. Heat solution to incipient boiling,

and then cool.

FREE SULFUR DIOXIDE

Volumetrically transfer 25 mL of wine or must to a clean 250-mL Erlenmeyer flask.

Add approximately 5 mL of starch indicator

Add 5 mL of the (I + 3) H2SO4.

Rapidly titrate with standard iodine solution to a blue end point that is stable for approximately 20 sec.

CALCULATION OF ANSWER

Calculate the free SO2 concentration (in mg/L):

SO2 (mg/L) = (mL iodine) (Miodine) (64) (1,000)/ 25

mL wine sample

SO2 (mg/L) = (mL iodine) x 25.6 for 25 mL sample

Calculation of Answer

If you make your own iodine solutions – an iodine

concentration of 0.0078 M causes the VI2x (factor) to equal

VI2x 20

SO2 (mg/L) = (mL iodine) x 20 for 25 mL sample

Comparison of chemistries &

Calculations

Working Iodine Solutions – 0.01M (0.02N)

I2 + SO2 + H2O 2HI + SO3

SO2 (mg/L) = (mL iodine) (Miodine) (64) (1,000)/ 25 mL wine

sample

H2SO4 + 2OH- 2H2O + SO4

=

SO2 (mg/L) = VNaOH x MNaOH x 64/2 x 1,000 / 20 (20 mL sample

size)

Comparison of Ripper and A/O Sulfur Dioxide Values for Three

Wines by Six Student Groups

WINE

Free SO2

mg/L

Total SO2

mg/L

Group #

N

Iodine Ripper

A/

O Ripper A/O

Valdiquie

Average

Values 0.0205 9.2 2.8 27.8 31.9

0.0003 1.1 0.6 6.5 6.2

Carignan

Average

Values 17.1 4.5 55.8 56.3

3.5 0.0 1.9 1.1

CA

Sunshine

Average

Values 9.8 7.6 71.5 60.3

2.0 1.9 8.3 6.6

Alternative Reagent

IO3

- + 5I- + 6H+ 3I

2+ 3H

2O

Potassium Iodate – primary standard 214.001 g/mol

2.140 g/L 0.03MI2

0.7133 g/L 0.01 MI2

0.5564 g/L 0.0078 MI2

Reagents for Electrochemical Endpoint

Potassium iodate – 1.1128 g/L

Potassium iodide – 10 g/L KI + 50 mL of (1+3) sulfuric

acid

SO2 (mg/L) = VI2(mL) x 0.0078 mmol I2/mL x

1mmol SO2/1mmol I2 x 64mg SO2/1mmol SO2 x

1000 (mL/L)/25 mL = VI2(mL) x 20

MLF - easily monitored by paper or thin-layer

chromatographic separation

absence of a malic acid spot

Visual resolution for malic acid is presumed to limit at

approximately 100 mg/L

most winemakers prefer 15 to 30 mg/L malic acid to

consider their wine “safe”

HO

HOOC - CH – CH2 – COOH (Malic Acid)

HO

HOOC - CH – CH3 + CO2 (Lactic Acid)

Tracking a Malo – Lactic Fermentation

Whatman No.1 chromatography paper

Chromatography developing tank

Micropipettes (20 mL)

PAPER CHROMATOGRAPHY

Wine acid standards (0.3%) purchased as part of kit

Chromatography solvent – purchased as part of kit

100 mL n-butanol

100 mL de-ionized water

10.7 mL stock formic acid

15 mL indicator solution prepared by dissolving 1 g of water-soluble bromocresol green in 100 mL of de-ionized water.

PAPER CHROMATOGRAPHY

Taking care to handle chromatography paper only by the edges, cut a piece of appropriate size to fit into developing tank.

Using a pencil, draw a line parallel to, and approximately 2.5 cm from the bottom edge of the paper.

Using micropipettes, spot standard acids and wine samples at equal intervals along baseline. Spots should be of as small a diameter as possible (less than 1 cm). Re-spot at least twice in order to achieve this goal.

Each spot should be at least 2.5-3.0 cm apart. A hair dryer can be used to assist in drying the spots between applications.

PAPER CHROMATOGRAPHY

Transfer solvent to developing tank, allowing at least 30 min for vapor saturation to occur (shake tank).

A minimum depth of 0.75 cm of solvent is required for adequate development.

Immerse baseline side of paper into tank, taking care that solvent moves uniformly up the paper.

When the solvent has ascended to near the upper edge of paper, chromatogram may be removed and allowed to dry.

When dry, results may be interpreted by noting the positions of yellow spots (acids) on blue background. Identification of various wine acids may be made by comparison to standard acids

PAPER CHROMATOGRAPHY

Results

Limitations on paper chromatographic method ~ 100 mg/L

Available kits

Production of NADH

Small sample volumes

Need to run standards

Enzymatic Malic Acid

ENZYMATIC MALIC ACID

L-Malate + NAD Malate dehydrogenase oxaloacetate + NADH

NADH + color reagent (oxidized) Diaphorase

NAD + color agent (reduced)

ENZYMATIC MALIC ACIDSamples:

• Wine used as is

• Juice/must diluted 1:20 (if > 500 mg/L)

• Samples do not need to be filtered or treated with color removing substance

Procedure:

• Squeeze bulb and dip tip into wine/juice/ must and aspirate sample

• Transfer sample to rectangular absorbent layer on back of test strip

(squeeze bulb)

• Allow sample to absorb into absorbent layer

• Wait four to six minutes for color development

ENZYMATIC MALIC ACID

Procedure:

• Compare developed color on strip to color chart

• Read Malic Acid level in mg/L from color chart

• Correct answer for any sample dilution

• Best to read using incandescent or natural light

Physical Methods

Ebulliometry

Distillation plus Refractometry

Distillation plus Hydrometry

Gas Chromatography

Chemical Methods

Enzymatic Analysis

Dichromate Oxidation

Determination of Alcohol Content

Ethanol Boiling Point Diagram

Ebulliometer

Determine boiling point of water

Add approximately 30 mL of deionized water to boiling

chamber "A." There is no need to add cold tap water to

condenser "D" at this time.

Insert thermometer "C." Position instrument over flame.

When thermometer reaches a stable point, allow 15-30 sec

for minor fluctuations to occur. At this time, take boiling point

reading and set inner scale opposite 0.0% alcohol on the

"Degres Alcoholique Du Vin" outer scale.

EBULLIOMETRIC ETHANOL

Determine boiling point of wine

Rinse boiling chamber several times with a few milliliters of wine to be analyzed and drain (This prevents dilution of sample).

Dilute 50 mL of wine to 100 mL in a volumetric flask. Place approximately 50 mL of wine in boiling chamber.

Fill condenser with cold tap water

Insert thermometer such that it is partially immersed in the liquid, and place instrument over heat source.

EBULLIOMETRIC ETHANOL

Determine boiling point of wine

When thermometer reaches a stable level, allow 15-30 sec

for changes and take reading.

Locate the boiling point of wine on the inner "Degres du

Thermometre" scale and record the corresponding alcohol

content (% vol/vol) on the outer scale.

EBULLIOMETRIC ETHANOL

Ebulliometric Temperature Difference Readings vs. % Ethanol

%Ethanol Delta T %Ethanol Delta T %Ethanol Delta T %Ethanol Delta T %Ethanol Delta T %Ethanol Delta T%Ethan

olDelta

T

0.00 0.00 2.10 1.96 4.60 4.01 7.10 5.79 9.60 7.32 12.10 8.71 14.60 9.91

0.01 0.05 2.20 2.05 4.70 4.09 7.20 5.87 9.70 7.39 12.20 8.77 14.70 9.95

0.10 0.10 2.30 2.13 4.80 4.16 7.30 5.91 9.80 7.44 12.30 8.82 14.80 10.01

0.15 0.14 2.40 2.22 4.90 4.22 7.40 5.99 9.90 7.51 12.40 8.87 14.90 10.04

0.20 0.20 2.50 2.31 5.00 4.31 7.50 6.05 10.00 7.57 12.50 8.92 15.00 10.09

0.25 0.24 2.60 2.41 5.10 4.39 7.60 6.11 10.10 7.61 12.60 8.97 15.10 10.12

0.30 0.30 2.70 2.50 5.20 4.45 7.70 6.18 10.20 7.68 12.70 9.02 15.20 10.17

0.35 0.34 2.80 2.59 5.30 4.52 7.80 6.24 10.30 7.73 12.80 9.07 15.30 10.21

0.40 0.39 2.90 2.68 5.40 4.59 7.90 6.30 10.40 7.79 12.90 9.12 15.40 10.26

0.50 0.49 3.00 2.76 5.50 4.68 8.00 6.37 10.50 7.85 13.00 9.18 15.50 10.30

0.60 0.58 3.10 2.83 5.60 4.73 8.10 6.41 10.60 7.91 13.10 9.21 15.60 10.33

0.70 0.67 3.20 2.91 5.70 4.81 8.20 6.49 10.70 7.97 13.20 9.26 15.70 10.38

0.80 0.77 3.30 3.01 5.80 4.89 8.30 6.54 10.80 8.01 13.30 9.31 15.80 10.42

0.90 0.85 3.40 3.09 5.90 4.96 8.40 6.60 10.90 8.08 13.40 9.36 15.90 10.47

1.00 0.94 3.50 3.18 6.00 5.02 8.50 6.66 11.00 8.12 13.50 9.41 16.00 10.50

1.10 1.03 3.60 3.25 6.10 5.10 8.60 6.72 11.10 8.19 13.60 9.46 16.10 10.53

1.20 1.12 3.70 3.32 6.20 5.18 8.70 6.79 11.20 8.23 13.70 9.51 16.20 10.59

1.30 1.22 3.80 3.41 6.30 5.23 8.80 6.84 11.30 8.29 13.80 9.56 16.30 10.63

1.40 1.31 3.90 3.49 6.40 5.31 8.90 6.91 11.40 8.35 13.90 9.61 16.40 10.67

1.50 1.41 4.00 3.58 6.50 5.39 9.00 6.97 11.50 8.40 14.00 9.64 16.50 10.70

1.60 1.50 4.10 3.64 6.60 5.46 9.10 7.02 11.60 8.45 14.10 9.69 16.60 10.74

1.70 1.59 4.20 3.71 6.70 5.52 9.20 7.09 11.70 8.51 14.20 9.73 16.70 10.79

1.80 1.69 4.30 3.80 6.80 5.59 9.30 7.14 11.80 8.57 14.30 9.79 16.80 10.82

1.90 1.79 4.40 3.87 6.90 5.66 9.40 7.21 11.90 8.61 14.40 9.82 16.90 10.87

2.00 1.88 4.50 3.93 7.00 5.72 9.50 7.28 12.00 8.67 14.50 9.88 17.00 10.90

Lab Glassware Ebulliometer

VOLATILE ACIDITY

Defined as those steam-distillable acids present in the

wine sample. Mainly acetic acid, which can be

produces during/by

Normal fermentation

Malo-lactic bacteria

Spoilage yeasts

Acetic acid bacteria

Steam Distillation

Enzymatic analysis

HPLC and GC procedures

METHODS OF ANALYSIS

Turn on condenser cooling water

Through funnel fill boiling chamber with deionized water to the

approximate level indicated in Figure

Reposition stopcock so that sample is delivered to the inner chamber

Volumetrically transfer 10 mL of wine to funnel E. Rinse the sample into

inner chamber with deionized water. Add 3-5 drops of 3% H2O2.

Turn heater on and bring water in chamber to moderate boiling. Carbon

dioxide present in water is vented through funnel for 10-15 sec before

closure of stopcock.

Collect 100 mL of distillate into receiving flask.

PROCEDURE

Immediately upon completion, turn the heater unit off

Open stopcock to water aspirator and remove the sample from

the inner chamber.

Add 1-2 drops of phenolphthalein indicator to distillate and

titrate, using 0.1 N NaOH, to end point lasting 15-20 sec.

Record the volume of NaOH used in titration and calculate the

volatile acidity (VA in g/L)

CH3COOH + OH- H20 + CH3COO

-

VA (g/L) = (mL NaOH) (N NaOH) (0.060) (1,000) / mL wine =

VNAOH X 0.6

PROCEDURE

SOLUBLE SOLIDS MEASUREMERNTS (BRIX)

and SUGAR PER BERRY

Refractometry - used in determinations of soluble solids

The refractive index varies as a function of composition, wavelength, and temperature

monochromatic sodium light at 589 nm and 20°C used as reference values

standardize with de-ionized water so that the sugar concentration of 0.0 B.

Dry the prism with the lens paper or just flush the prism with several drops of juice and read

If refractometer is not temperature corrected, note the temperature of the reading

Caution: particulate matter in the sample may scratch the prisms.

SUGAR PER BERRY: DETERMINATION

BY REFRACTOMETRY

Sugar per berry utilizes the same initial Brix measurement as made above on a representative sample of grapes.

Sugar per berry takes into account the actual weight of the sample.

Sugar per berry allows one to observe changes in the amount of sugar in the berries due to maturation, even though the Brix reading of the sample does not change.

Changes in

Sugar/Berry

Changes in Berry Weight

Decreases No Change Increases

Increases Maturation &

dehydration

Maturation (a) Major increase: maturation and

dilution

(b) Minor increase: maturation

No change Dehydration No change Dilution

Decreases Dehydration &

sugar export

Sugar export Sugar export & dilution

Source: Long 1984

Sugar per Berry

Degrees Brix – g soluble solids/100g juice

Weigh 80 berries (or any number – count them)

Crush in baggie and determine Brix

Example: 80 berries weigh 102 g, Brix is 22

22/100 = X/102 X = 22.4 g per 80 berries

Sol solids per berry = 22.4 g/80 = 0.281 g/berry

Analysis for Reducing

Sugars (residual sugars)

• glucose & fructose (hexoses)

• pentoses

Analytical Methods

Lane-Eynon

Rebelein (Gold Coast)

Clinitest

Enzymatic

HPLC

FTIR

GC

Residual Sugar Comparisonresults in g/L

Wine Scan Rebelein Clinitest

Nouveau Blanc 22 20.3 27

Tail Gate Red 2.2 4.9 2.9

Sherry 106.3 83.8 143

% Rs 0.0% 0.05% 0.10% 0.20% 0.40% 0.60% 1.0%

Color Gn/Blk Dk Olive Olive Olv/Ong Mud Bn Dk Sand Orange

ENZYMATIC RESIDUAL

SUGAR

fructose glucose invertase glucose

Glucose + O2 + H2O glucose oxidase glucono-

δ-lactone + H2O2

H2O2 + color agent (oxidized) peroxidase 4 H2O +

color agent

(reduced)

ENZYMATIC RESIDUAL

SUGAR

Samples:

• Wine used as is

• Juice/must diluted 1:20

• Samples do not need to be filtered or treated with

color removing substances

ENZYMATIC RESIDUAL

SUGAR

Procedure:

• Squeeze bulb and dip tip into wine/juice/ must and

aspirate sample

• Transfer sample to rectangular absorbent layer on

back of test strip (squeeze bulb)

• Allow sample to absorb into absorbent layer

• Wait two minutes for color development

• Read residual sugar level in mg/L from color chart

HEAT & COLD STABILITY

TESTING

•Protein precipitation – haze formation

•Bentonite treatment for removal of proteins, and other

insoluble materials

Evaluation of Protein Stability via Heat

Testing

Various temperature vs. time protocols – low temperature/long

time vs. high temperature/short time

Typical procedures

49 oC for 24 hours

80 oC for 6 hours

80 oC for 2 hours

Precautions

Effect of ---

Malo-lactic fermentation

Blending

Acid additions

Spirits additions

Heat

Heat stability testing done following all winery operations

Cold Stability

Tartrate crystalline deposit formation

Potassium hydrogen tartrate (KHT)

Calcium tartrate (CaT)

Natural phenomenon during aging

Crystal formation in bottle

Tartrate Stability Tests

Freeze tests

Electrical conductivity

Precautions - stability testing done following all winery

operations