LAB ANALYSES for BEGINNERS to INTERMEDIATES
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1 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
2 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
3 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
4 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
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9 ph Measurements 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.
10 What is Titratable Acidity? 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
11 Normality vs. Molarity 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
12 TA ANALYSIS Two burets with NaOH (0.100 N and N) Titration Accurately pipette a 10 ml juice or wine sample into the beaker and note volume reading on buret (V 1 ) Add 0.1 N NaOH from buret to ph 8.2 endpoint and note volume reading (V 2 ). V NaOH = V 2 V 1
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17 TA MEASUREMENTS H 2 T + HT - + T = +H + +OH - H 2 O + T = T = is a base, so at the end of this titration the solution will be alkaline TA (g/l H 2 T) = V NaOH x M NaOH x ½ x x 1000/10 TA (g/l H 2 T) = V NaOH x 0.75 if NaOH is 0.1 M
18 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 (NH 4+ ) and primary or free alpha-amino acids (FAN).
19 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 (V 3 and V 4 ) V NaOH = V 4 V 3
20 0.1 M NaOH 0.01 M NaOH M NaOH
21 FORMOL NITROGEN ANALYSIS Nitrogen N (mg/l) = V NaOH x N NaOH x 14 x 1000/10 N (mg/l) = V NaOH x 14 using 0.01 N NaOH
22 SULFUR DIOXIDE AO & Ripper Methods SO 2 is an anhydride of sulfurous acid SO 2 + H 2 O H 2 SO 3 Sulfurous acid although technically a weak acid --can dissociate in wine H 2 SO 3 H + + HSO 3 - HSO 3 - H + + SO 3 2- Wine ph affects concentration of sulfite species
23 %Sulfite Species %Sulfite Species ph vs. % Sulfite Species H 2 SO HSO3 = SO ph
24 Free SO 2 (mg/l) Free SO 2 Needed to Obtain 0.5 or 0.8 mg/l (molecular) at Various ph Levels mg/l mg/l ph
25 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 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
26 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
27 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)
28 Chemistries & Calculations 3H + + H 2 SO 3 + HSO SO3 = 3H2 SO 3 3SO 2 + 3H 2 O Calculate SO 2 as follows: H 2 O 2 + SO 2 H 2 O+ SO 3 H 2 SO 4 H 2 SO 4 + 2OH - 2H 2 O + SO 4 = V NaOH = V2 V1 SO 2 (mg/l) = V NaOH x M NaOH x 64/2 x 1,000 / 20 (20 ml sample size) SO 2 (mg/l) = V NaOH x 16
29 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
30 EQUIPMENT 250-mL Erlenmeyer flask (preferably wide-mouth) 10-mL burette 25-mL volumetric pipette High-intensity light source
31 REAGENTS Working Iodine Solutions 0.01M (0.02N) I 2 + SO 2 + H 2 O 2HI + SO 3 (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.
32 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) H 2 SO 4. Rapidly titrate with standard iodine solution to a blue end point that is stable for approximately 20 sec.
33 CALCULATION OF ANSWER Calculate the free SO 2 concentration (in mg/l): SO 2 (mg/l) = (ml iodine) (M iodine ) (64) (1,000)/ 25 ml wine sample SO 2 (mg/l) = (ml iodine) x 25.6 for 25 ml sample
34 Calculation of Answer If you make your own iodine solutions an iodine concentration of M causes the V I2 x (factor) to equal V I2 x 20 SO 2 (mg/l) = (ml iodine) x 20 for 25 ml sample
35 Comparison of chemistries & Calculations Working Iodine Solutions 0.01M (0.02N) I 2 + SO 2 + H 2 O 2HI + SO 3 SO 2 (mg/l) = (ml iodine) (M iodine ) (64) (1,000)/ 25 ml wine sample H 2 SO 4 + 2OH - 2H2O + SO 4 = SO 2 (mg/l) = V NaOH x M NaOH x 64/2 x 1,000 / 20 (20 ml sample size)
36 Comparison of Ripper and A/O Sulfur Dioxide Values for Three Wines by Six Student Groups WINE Valdiquie Group # N Iodine Free SO2 mg/l Ripper Total SO2 mg/l A/ O Ripper A/O Average Values Carignan CA Sunshine Average Values Average Values
37 Alternative Reagent IO I - + 6H + 3I 2 + 3H 2 O Potassium Iodate primary standard g/mol g/l 0.03M I g/l 0.01 M I g/l M I2
38 Reagents for Electrochemical Endpoint Potassium iodate g/l Potassium iodide 10 g/l KI + 50 ml of (1+3) sulfuric acid SO 2 (mg/l) = V I2 (ml) x mmol I 2 /ml x 1mmol SO 2 /1mmol I 2 x 64mg SO 2 /1mmol SO 2 x 1000 (ml/l)/25 ml = V I (ml) x 20 2
39 Tracking a Malo Lactic Fermentation 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 CH 2 COOH (Malic Acid) HO HOOC - CH CH 3 + CO 2 (Lactic Acid)
40 PAPER CHROMATOGRAPHY Whatman No.1 chromatography paper Chromatography developing tank Micropipettes (20 ml)
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42 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 deionized water.
43 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 cm apart. A hair dryer can be used to assist in drying the spots between applications.
44 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
45 Results
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47 Enzymatic Malic Acid Limitations on paper chromatographic method ~ 100 mg/l Available kits Production of NADH Small sample volumes Need to run standards
48 ENZYMATIC MALIC ACID L-Malate + NAD Malate dehydrogenase oxaloacetate + NADH NADH + color reagent (oxidized) Diaphorase NAD + color agent (reduced)
49 ENZYMATIC MALIC ACID Samples: 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
50 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
51 Determination of Alcohol Content Physical Methods Ebulliometry Distillation plus Refractometry Distillation plus Hydrometry Gas Chromatography Chemical Methods Enzymatic Analysis Dichromate Oxidation
52 Ethanol Boiling Point Diagram
53 Ebulliometer
54 EBULLIOMETRIC ETHANOL 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 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.
55 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.
56 EBULLIOMETRIC ETHANOL Determine boiling point of wine When thermometer reaches a stable level, allow 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.
57 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 ol Delta T
58 Lab Glassware Ebulliometer
59 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
60 METHODS OF ANALYSIS Steam Distillation Enzymatic analysis HPLC and GC procedures
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63 PROCEDURE 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% H 2 O 2. Turn heater on and bring water in chamber to moderate boiling. Carbon dioxide present in water is vented through funnel for sec before closure of stopcock. Collect 100 ml of distillate into receiving flask.
64 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 sec. Record the volume of NaOH used in titration and calculate the volatile acidity (VA in g/l) CH 3 COOH + OH - H CH 3 COO - VA (g/l) = (ml NaOH) (N NaOH) (0.060) (1,000) / ml wine = V NAOH X 0.6
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66 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.
67 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.
68 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
69 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 = g/berry
70 Analysis for Reducing Sugars (residual sugars) glucose & fructose (hexoses) pentoses
71 Analytical Methods Lane-Eynon Rebelein (Gold Coast) Clinitest Enzymatic HPLC FTIR GC
72 Residual Sugar Comparison results in g/l Wine Scan Rebelein Clinitest Nouveau Blanc Tail Gate Red Sherry
73 % 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
74 ENZYMATIC RESIDUAL SUGAR fructose glucose invertase glucose Glucose + O 2 + H 2 O glucose oxidase gluconoδ-lactone + H 2 O 2 H 2 O 2 + color agent (oxidized) peroxidase 4 H 2 O + color agent (reduced)
75 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
76 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
77 HEAT & COLD STABILITY TESTING Protein precipitation haze formation Bentonite treatment for removal of proteins, and other insoluble materials
78 Evaluation of Protein Stability via Heat Testing Various temperature vs. time protocols low temperature/long time vs. high temperature/short time Typical procedures 49 o C for 24 hours 80 o C for 6 hours 80 o C for 2 hours
79 Precautions Effect of --- Malo-lactic fermentation Blending Acid additions Spirits additions Heat Heat stability testing done following all winery operations
80 Cold Stability Tartrate crystalline deposit formation Potassium hydrogen tartrate (KHT) Calcium tartrate (CaT) Natural phenomenon during aging Crystal formation in bottle
81 Tartrate Stability Tests Freeze tests Electrical conductivity Precautions - stability testing done following all winery operations
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