eas 104-1999_alcoholic beverages - sampling & tests

EAS 104:2000 ICS 67.160.10

HS 2203 HS 2204 HS 2205 HS 2206 HS 2207 HS 2208

© EAC 2008 First Edition 2008

EAST AFRICAN STANDARD Alcoholic beverages — Methods of sampling and test

DKS AS 4044: 2004

EAST AFRICAN COMMUNITY

EAS 104:2000

ii © EAC 2008 — All rights reserved

Foreword Development of the East African Standards has been necessitated by the need for harmonizing requirements governing quality of products and services in East Africa. It is envisaged that through harmonized standardization, trade barriers which are encountered when goods and services are exchanged within the Community will be removed. In order to meet the above objectives, the EAC Partner States have enacted an East African Standardization, Quality Assurance, Metrology and Test Act, 2006 (EAC SQMT Act, 2006) to make provisions for ensuring standardization, quality assurance, metrology and testing of products produced or originating in a third country and traded in the Community in order to facilitate industrial development and trade as well as helping to protect the health and safety of society and the environment in the Community. East African Standards are formulated in accordance with the procedures established by the East African Standards Committee. The East African Standards Committee is established under the provisions of Article 4 of the EAC SQMT Act, 2006. The Committee is composed of representatives of the National Standards Bodies in Partner States, together with the representatives from the private sectors and consumer organizations. Draft East African Standards are circulated to stakeholders through the National Standards Bodies in the Partner States. The comments received are discussed and incorporated before finalization of standards, in accordance with the procedures of the Community. Article 15(1) of the EAC SQMT Act, 2006 provides that “Within six months of the declaration of an East African Standard, the Partner States shall adopt, without deviation from the approved text of the standard, the East African Standard as a national standard and withdraw any existing national standard with similar scope and purpose”.

East African Standards are subject to review, to keep pace with technological advances. Users of the East African Standards are therefore expected to ensure that they always have the latest versions of the standards they are implementing.

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© 2008 EAC — All rights of exploitation in any form and by any means reserved worldwide for EAC Partner States’ NSBs.

EAS 104:2000

© EAC 2008 — All rights reserved iii

Contents 1 Scope ......................................................................................................................................1 2 Normative references...............................................................................................................1 3 Terms and definitions ..............................................................................................................1 4 Sampling requirements ............................................................................................................1 5 Quality of reagents...................................................................................................................3 6 Determination of ethyl alcohol..................................................................................................3 7 Determination of specific gravity ..............................................................................................9 8 Determination of volatile acidity ............................................................................................. 10 9 Determination of fixed acidity................................................................................................. 10 10 Determination of total acidity.................................................................................................. 11 11 Determination of esters as ethyl acetate ................................................................................ 11 12 Determination of aldehydes as acetaldehydes....................................................................... 12 13 Determination of furfural ........................................................................................................ 12 14 Determination of methanol..................................................................................................... 13 15 Determination of fusel oils, methanol and dimethyl sulphide in alcoholic beverages by headspace gas chromatography — Reference method...................................................................... 15 16 Detection of acetones, other ketones, isopropanol and tertiary butylalcohol .......................... 22 17 Determination of ash.............................................................................................................. 22 18 Determination of suspended solids ........................................................................................ 23 19 Determination of dissolved solids........................................................................................... 23 20 Determination of total solids................................................................................................... 24 21 Determination of higher alcohol as amyl alcohol .................................................................... 24 22 Test for miscibility with water ................................................................................................. 28 23 Test for alkalinity.................................................................................................................... 28 24 Test for permanganate reaction time ..................................................................................... 28 25 Determination of copper......................................................................................................... 29 26 Determination of lead............................................................................................................. 30 27 Determination of sulphur dioxide in wines.............................................................................. 32 28 Determination of sorbic acid .................................................................................................. 33 29 Wine analysis......................................................................................................................... 34 30 Determination of atmospheric pressure in sparkling wines .................................................... 35

EAS 104:2000

iv © EAC 2008 — All rights reserved

Introduction Alcoholic beverages are consumed by many people and hence their effects on people in terms of health, income and social status is quite significant. The production and consumption of alcoholic beverages is likely to increase in the very near future, hence in order to protect the consumer the control of product quality become very necessary, through the application of acceptable sampling and testing procedures for the evaluation of the various inherent characteristics. This standard is intended for introduction of uniform method of sampling and tests for alcoholic beverages. In the preparation of this standard considerable assistance was derived from IS 3753:1967, Method of sampling for alcoholic drinks published by the Indian Standards Institution; IS 3762:1967, Methods of test for alcoholic drinks published by Indian Standards Institution and IS 3506:1967, Tables for alcoholometry by pyknometer method published by the Indian Standards Institution. In reporting the result of a test or analysis made in accordance with this standard, if the final value observed or calculated is to be rounded off, it shall be done in accordance with EAS 124.

EAS 104:2000

© EAC 2008 — All rights reserved 1

Alcoholic beverages — Methods of sampling and test 1 Scope This East African Standard prescribes methods of sampling and tests for alcoholic beverages.

2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. OIML R22, International alcoholometric tables EAS 123, Distilled water — Specification EAS 124, Rounding off numerical values 3 Terms and definitions

For the purposes of this standard, the following terms and definitions shall apply.

3.1 observed specific gravity the specific gravity obtained by the pyknometer method at t °C with respect to that of water at 15 °C 3.2 single bulk method when a measured volume, say 100 ml, of a spiritous preparation is diluted and distilled to collect the same volume of distillate, that is 100 ml 3.3 double bulk method when a measured volume say 50 ml, of a spiritous preparation is diluted and distilled to collect distillate twice the original volume of sample, that is 100 ml 3.4 treble bulk method when a measured volume, say 25 ml, of a spiritous preparation is diluted and distilled to collect thrice the volume of sample, that is 75 ml 3.5 quadruple bulk method when a measured volume say 25 ml, of a spiritous preparation is diluted and distilled to collect four times the volume of sample, that is 100 ml. 4 Sampling requirements 4.1 General precautions in sampling In drawing, preparing, storing and handling samples, the following precautions and directions shall be observed as far as possible: a) samples shall be taken in a protected place not exposed to damp air, dust or soot; b) the sampling instruments shall be clean and dry;

EAST AFRICAN STANDARD

EAS 104:2000

2 © EAC 2008 — All rights reserved

c) to draw representative sample, the contents of each container selected for sampling shall be

mixed as thoroughly as possible by suitable means; d) precautions shall be taken to protect the samples, the material being sampled, the sampling

instrument and the container for samples from extraneous contamination;

e) samples shall be placed in suitable clean, dry and air tight glass containers;

f) the sample containers shall be of such size that sufficient head space to allow for expansion of the liquid is left after pouring in the samples;

g) each sample container shall be sealed air tight with a suitable stopper after filling and marked with full details of sampling, the date of sampling and the year of manufacture of the materials and

h) samples shall be stored in cool, dark and dry place. 4.2 Sampling instrument Appropriate sampling cans and tubes in term of size and volume shall be used. 4.3 Scale of inspection 4.3.1 Lot — Lot shall mean the quantity of packed alcohol of the same type from the same batch of manufacture at one time. 4.3.2 The samples shall be selected and tested for each lot separately for ascertaining their conformity to the requirements of relevant specifications. 4.3.3 The number of bottles to be selected from a lot shall as far as practicable be as given in the table.

Table — Scale of Sampling

Number of bottles in the lot Number of bottles to be selected up to 12000 9

1201 to 3600 12 3601 to 10800 15

10801 and above 21 4.3.4 The bottles form the lot shall be chosen at random. As a first step, 2 % of the cases or bags shall be chosen at random. All the cases thus selected shall be opened and the bottles examined visually for conditions of packing, the external appearance and the fill. The lot shall be considered satisfactory if the bottles in cases or bags opened are found conforming to the requirements of the characteristics. In case any defectives are found, 5 % of the cases shall be opened and bottles examined for similar characteristics. .

From each of the cases or bags opened, an approximately equal number of bottles shall be picked up from its different parts of layers so as to obtain the required number of bottles in the sample (see the table). 4.3.5 The sample bottles selected as in 4.3.4 shall be divided at random into three equal sets and labelled with all the particulars of sampling. One of these sets of sample bottles shall be for the purchaser, another for the vendor and the third for the reference. 4.4 Number of tests

Tests for ethyl alcohol content shall be made on the individual sample bottle. Organoleptic tests shall also be carried. Tests for other requirements of the relevant specification shall be made on the composite sample prepared by thorough mixing equal quantities from individual sample bottles.

EAS 104:2000


4.5 Criteria for conformity 4.5.1 For those characteristics where a composite sample has been tested from a lot, only one test result shall be available and the result shall satisfy the requirements of the specifications. 4.5.2 When three or more sample bottles have been analyzed individually from a lot for any particular characteristic, the following procedure shall be followed for judging the conformity to the requirements of that characteristic: 4.5.2.1 For the different test results obtained by analyzing different individual samples, the average (X) and the range (R) shall be calculated as follows:

( )results testofNumber

results test theofsum TheAverage =X

Range (R) = The difference between the maximum and the minimum values of the test results. 4.5.2.2 If the specification limit for the characteristic is given as a minimum, then the value of the expression ( X - 0.6R) shall be calculated from the relevant test results. If the value so obtained is greater than or equal to the minimum limit, the lot shall be declared as conforming to the requirement of that characteristic. 4.5.2.3 If the specification limit for the characteristic is given as a maximum, then the value of expression ( X + 0.6R) shall be calculated from the relevant test results. If the value so obtained is less than or equal to the maximum limit, the lot shall be declared as conforming to the requirement of that characteristic. 4.5.2.4 If the characteristic has two-sided specification limits then the value of the expression ( X – 0.6R) and ( X + 0.6R) shall be calculated from the relevant test results. If the values so obtained lie between the two specification limits, that lot shall be declared as conforming to the requirements of that characteristic. 5 Quality of reagents Unless otherwise specified, analytical grade reagents and distilled water in accordance with EAS 123 shall be used in all tests. NOTE Pure chemicals, should mean chemicals that do not contain impurities which affect the results of analysis (chemicals of analytical grade). 6 Determination of ethyl alcohol 6.1 General Ethanol content of a liquor preparation can be quantitatively determined by specific gravity determination. However, prior to this determination ethanol contained in the liquor as a rule, has to be obtained practically free from all other dissolved and undissolved substances except water. Simple direct distillation suffices where the admixed or dissolved ingredients are not volatile with steam. When volatile bodies are present, it is necessary either to render them incapable of distillation or to remove them. All liquor preparations containing volatile acids or ammonia (or amines) are neutralized by an alkali or acid (sodium hydroxide or sulfuric acid). Free iodine, if present, may first be converted into sodium iodide by treatment with sodium thiosulfate. Volatile oils, solvents etc. are removed by adopting the single, double, treble or quadruple bulk method. Through use of any of these methods a definite volume of the distillate is collected and its specific gravity determined by pyknometer method. Through suitable use of Table 1 or Table 2, in Annex A, ethanol content of liquor preparation is determined.

EAS 104:2000


NOTE On account of high vapour pressure of ethyl alcohol and its affinity for water, distillation of a concentrated liquor preparation into its own volume of water (single bulk method) so as to obtain an accurate determination of the amount of alcohol present is practically impossible with the apparatus and method commonly used. Consequently double, treble and quadruple bulks are generally used. As regards the extent of dilution, it has been found that alcohol in 25 ml of concentrated liqour preparation when diluted with water even to the extent of 500 ml and the solution saturated with salt may be entirely removed in the first 100 ml of the distillate. In the case of very low concentration liquor (under 20 % alcohol) the single bulk method is normally employed. 6.2 Apparatus 6.2.1 Distillation assembly — The apparatus are assembled as in Figure 1. The distillate end of the condenser is attached to a 50 ml pipette suitably shortened at the upper end and attached to the condenser nozzle by means of rubber tubing. The lower part of this pipette is suitably curved so as to reach the bottom of the receiver where it is slipped into the minimum quantity of distilled water. 6.2.2 Receiver, 100 ml capacity round-bottomed flask. 6.2.3 Pyknometer, as shown in Figure 2, 50 ml capacity. 6.2.4 Thermometer, with the range of 0 °C to 50 °C with sub-divisions at every 0,5 °C shall be used. 6.3 Reagents 6.3.1 Common salt, of the highest purity. 6.3.2 Petroleum hydrocarbon solvent, 60/80. 6.4 Procedure a) Where the liquor contains admixed or dissolved ingredients, which are not volatile, direct

distillation using single, double, treble or quadruple methods shall be used. b) Where volatile constituents are present prior to distillation, the volatile constituents shall be

rendered incapable of distillation. All liquor containing volatile acids or ammonia (or amines), shall be neutralized by alkali or acids (sodium hydroxide or sulfuric acid as the case may be) prior to distillation.

If free iodine is present, convert it into sodium iodide by treatment with sodium thiosulfate. c) Where volatile oils, solvents etc, are present, the following procedure shall be followed:

Take 25 ml. (or 50 ml or 100 ml) depending on whether single, double or quadruple method is to be used, of the sample measured at air temperature to t °C. Wash the sample into a separator and make up to the volume of 100 ml; add enough common salt to saturate the liquid and shake vigorously for about 5 min with 50 ml to 80 ml of petroleum hydrocarbon solvent. Allow standing for 15 min to 20 min after which the petroleum hydrocarbon solvent shall be completely separated carrying with it the only matter. Draw off the lower saline alcohol layer into the distillation assembly. Wash petroleum layer twice with saturated sodium chloride solution (about 10 ml) and transfer the saline washings also to the distillation flask. Neutralize if necessary, and distill to collect 100 ml of the distillate at t °C.

d) Determination of specific gravity — When several tests are carried out, rinse the interior of the

pyknometer after each test so that the previous liquid, which has wetted the sides of the pyknometer, may not alter the density of the liquid under test. Fill the pyknometer with the alcoholic liquid. Dip the thermometer into the liquid and note the exact temperature of the liquid in the pyknometer before the perforated stopper is inserted. Then weigh the pyknometer with the alcoholic liquid thus filled at t °C. Calculate the net mass in grams of the alcoholic liquid at t °C in the pyknometer by subtracting the mass of the empty pyknometer. Divide the mass so obtained by the water equivalent; that is, the mass in air of 50 ml of water content in the pyknometer at 15 °C. This gives the specific gravity of the alcoholic liquid in air at t °C/15 °C. Record temperature t °C to the nearest 0.5 °C. Determine the ethanol content of the spirit through use of Tables 1 and 2 given in OIML R22.

EAS 104:2000


Figure 1 — Distillation assembly

Figure 2 – Pyknometer capillary stoppered

6.5 Tables 6.5.1 Table 1 — Specific gravity conversion table from t °C/15 °C to 15 °C/15 °C. This table gives specific gravity in vacuum at various temperatures (10 °C to 40 °C at intervals of every 0.5 °C) with respect to that of water at 15 °C for various percentages of ethanol (by volume at 15 °C).

EAS 104:2000


6.5.2 With the help of this table, the specific gravity at a particular temperature t °C/ 15 °C found by the pyknometer method can be converted into the corresponding specific gravity at 15 °C/ 15 °C. 6.5.3 Table 2 — Conversion table for specific gravity data into ethanol content at specified temperature. This table gives data for specific gravity in vacuum and specific gravity in air at 15 °C for every 0.1 % by volume of ethanol content at 15 °C/15 °C. 6.6 Use of tables 6.6.1 The steps involved for the determination of ethanol content of a liquor preparation shall be as follows: a) Dilution of 25 ml of the sample with fixed amount of water (depending on whether double, treble

or quadruple bulk is desired) and distillation of a fixed quantity of the distillate. b) Determination of specific gravity in air at ambient temperature (t °C/15 °C). c) Conversion of specific gravity in air at ambient temperature WC / 15°C to specific gravity in air at

15°C / 15°C by the use of Table 1. d) Conversion of specific gravity in air at 15 °C/15 °C to the specific gravity in vacuum at 15 °C/15

°C by the use of Table 2. e) Conversion of specific gravity in vacuum at 15 °C/15 °C to ethanol content at 15 °C by the use of

Table 2. f) Conversion of ethanol content as obtained under (e) to ethanol content at 15 °C of the distillate at

t °C by the use of Table 1 of OIML R22. 6.6.2 Table 1. The first vertical line of this table gives the specific gravities at various temperatures for a given percentage of ethanol (by volume) at 15 °C. The horizontal lines indicate the various specific gravities at a particular temperature for varying percentage of ethanol (by volume) at 15 °C/15 °C. For instance; Specific gravity for 76.9 % ethanol at 26 °C/15 °C = 0.86360 (see Table 1). Then look for the value in the first horizontal line corresponding to 76.9 % ethanol, which is 0.87260 in this case. Therefore, if the observed specific gravity of 76.9 % ethanol at 26 °C/15 °C is 0.86360, the corresponding specific gravity at 15 °C/15 °C is 0.87260. 6.6.3 Table 2. The first column of Table 2 gives the ethanol content, percent by volume, at 15 °C. The second column of Table 2 gives the specific gravity of alcoholic liquids at 15 °C/15 °C in vacuum. For instance; specific gravity in vacuum for 50.0 % of ethanol at 15 °C/15 °C = 0.93437 (see Table 2 ) which is the ratio of the respective densities of alcoholic liquid at 15 °C in vacuum. The third column of Table 2 gives the specific gravity in air at 15 °C, that is, the ratio of densities of alcoholic liquid in air at 15 °C and that of water at 15 °C in vacuum. NOTE The values of specific gravity into air at 15 °C are calculated by converting the specific gravities at t °C/15 °C in air found by the pyknometer method (which is standardized in vacuum at 15 °C) with the use of Table 1. For instance; specific gravity at 15 °C/15 °C for 78 % of ethanol = 0,86857 (see Table 2) which gives the ratio between the densities of alcoholic liquids in air at 15 °C and water in vacuum at 15 °C. 6.6.4 The corresponding specific gravities in vacuum are indicated in column 2 of Table 2 and are calculated by multiplying with 0.999 126 which is the density of water in vacuum at 15 °C. This gives the density of the alcoholic liquid in air at 15 °C which is then converted into the specific gravity of the alcoholic liquid in vacuum at 15 °C by using the alcoholometric Table 2 of OIML R22. Similarly, any specification in vacuum at 15 °C can be converted into corresponding specific gravity in air at 15 °C by first finding out from the alcoholometric Table 2 of OIML R22. The corresponding density in air 15 °C which is then divided by 0.999 126 (the density of water in vacuum at 15 °C) gives the specific

EAS 104:2000


gravities in vacuum and specific gravities in air at 15 °C for every 0.1 % by volume of ethanol content at 15 °C/15 °C. For instance; specific gravity in air at 15 °C /15 °C for 77.6 % ethanol content as determined by the pyknometer method = 0.86965 (see Table 2). The specific gravity value 0.869 65 in air at 15 °C when multiplied by 0.999 126, the density of water in vacuum at 15 °C gives 0.868 89, the density of the alcoholic liquid in air at 15 °C. From the alcoholometric Table 2, corresponding to 0.868 89, the density of alcoholic liquid in air at 15 °C is thus obtained. The figure 0.870 73 is therefore, the specific gravity in vacuum at 15 °C corresponding to 0.869 65, the figure for specific gravity in air at 15 °C obtained by the pyknometer method. 6.6.5 The specific gravities in air at 15 °C are also independently converted into specific gravities in vacuum at 15 °C using the following formula:

( )( ) ( ) ( ) kam

arramr

aramM ×−=

−−=×

−−

=

where M is the density in air; m is the density in vacuum; a is the density of air at 15 °C and 760 mm pressure (which is equal to 0.001 225) and r is the average density of cast iron weights, which has been taken as 7.3.

Density in air at 15 °C = specific gravity in air at 15 °C × 0.999 126 Density in vacuum at 15 °C = specific gravity in vacuum × 0.999 126

arr−

is a constant k which is equal to

67900110012255037

37 ...

.=

−

Hence M = (m - a) x 1.0001679

91671.0005)2250.0011260.999vacuumingravity(Specific1260.999airingravitySpecific ×−×=×

72939990.72250.0011260.999airingravityspecific

1269990.91671.00091671.0002250.0011260.999airingravityspecific

vacuumingravitySpecific

+×=

××+×

=

In other words the specific gravity in air

1260.99972250.00172930.999vacuumingravityspecific −×

=

Using the above formula, any specific gravity in air as found by the pyknometer method can be converted into the corresponding specific gravity in vacuum and vice versa. For instance: specific gravity in air at 15 °C for 78.2 % ethanol content as determined by the pyknometer method = 0.868 03 (see Table 2).

11869072930.999

2250,0011260.999038680vacuumingravitySpecific .

.=

+×= (the same value as given in

col.2 of Table 2). Thus the various specific gravities in vacuum at 15 °C are also arrived at from the specific gravities in air at 15 °C by using the same formula. 6.6.6 Examples and calculations. For guidance, a few typical examples are given below.

EAS 104:2000


Example 1 Observed specific gravity at 22 °C/15 °C for 76.5 % ethanol content = 0.868 02. This specific gravity is determined by the pyknometer method in air. To convert the specific gravity in air to specific gravity in vacuum, use Table 2 of this standard and obtain the value 0.869 11 as the specific gravity in vacuum corresponding to the specific gravity in air 0.868 02. In Table 1, follow the horizontal line indicated by 0.869 11 under the vertical temperature column of 22 °C until the vertical line headed by 15 °C is encountered. At this point, the figure 0.874 74 is found. The figure 0.874 74 is the specific gravity in vacuum at 15 °C/15 °C corresponding to the specific gravity of 0.868 03 in air at 22 °C/15 °C. By referring to Table 2, it is found that the specific gravity (in vacuum) 0.874 74 corresponds to 76.1 % by volume of ethanol content at 15 °C. Then using Table 1 of OIML R22 following the horizontal line indicated by 22 °C (the observed temperature) until the vertical column indicated under 76.1 is encountered. At this point, the figure 73.40 is found which is the percentage by volume of ethanol at 15 °C corresponding to the specific gravity in air of the alcoholic liquid at 22 °C/15 °C. This means then that an observed volume of 100 litres of the alcoholic liquid at 22 °C contains 73.40 litres of alcohol by volume at 15 °C. NOTE 1 If the pyknometer is not standardized at 15 °C in vacuum, the specific gravity at t °C/t °C should be reduced to t °C/15 °C knowing the density of water at t °C at 15 °C. NOTE 2 For routine type of work, the conversion of specific gravities in air at 15 °C, to the specific gravities in vacuum at 15 °C, correction factors as indicated below may be added.

Specific gravity in air Correction factor to be added

1.000 to 0.9600

0.9601 to 0.9000

0.9001 to 0.8200

below 0.8200

0.00106

0.00107

0.00108

0.00109 Example 2 Specific gravity at 28 °C /15 °C = 0.983 18. This specific gravity is in air. From Table 2, the specific gravity in vacuum corresponding to specific gravity 0.983 18 in air is 0.984 24 (for routine type of work, the correction of 0.000 106 may be added to get the specific gravity in vacuum). Thus the specific gravity at 28 °C /15 °C in vacuum is 0.983 18 + 0.001 06 = 0.984 24. In Table 1, follow the vertical line indicated by 0.984 24 under the horizontal temperature column of 28 °C until the horizontal line headed by 15 °C is encountered. At this point the figure 0.98711 is found which is the specific gravity in vacuum at 15 °C/15 °C corresponding to the specific gravity 0.98318 in air at 28 °C/15 °C. From Table 2, it is found that the specific gravity (in vacuum) figure 0.98711 corresponds to 9.5 % by volume of ethanol content at 15 °C. Then using Table 1 of OIML R22 follow the horizontal line indicated by 28 °C (the observed temperature) until the vertical column indicated under 9.5 is encountered. At this point, the figure 7.25 is found which is the percentage by volume of ethanol at 15 °C corresponding to the specific gravity in air of 0.983 18 at 28 °C/15 °C. This means that an observed volume of 100 litres of alcoholic liquid at 28 °C contains 7.25 litres of ethanol by volume at 15 °C. 6.7 Double bulk, treble bulk and quadruple bulk If double bulk, treble bulk and quadruple bulk methods are employed in the determination of specific gravity by the pyknometer method for want of sufficient amount of the liquid, the percentage of the ethanol by volume at 15 °C is found as detailed under 6.5, from the specific gravity obtained by pyknometer method. Then, the actual percentage of ethanol at 15 °C is calculated by multiplying it by 2, 3 or 4 depending upon whether double bulk, treble bulk or quadruple bulk method is employed.

EAS 104:2000


7 Determination of specific gravity 7.1 Definition For the purpose of this standard, the specific gravity of a sample shall mean the ratio of the mass of a given volume of the sample at the specified temperature to the mass of an equal volume of distilled water at the same temperature.

7.2 Method The specific gravity may be determined by means of a pycnometer (specific gravity bottle) or a special hydrometer for example the international organization of Legal Metrology (OIML) hydrometer. For accurate work the method using the specific gravity bottle shall be adopted in conjunction with the OIML R22.

7.3 Apparatus For determination of specific gravity, two alternate specific gravity bottles showing Figure 2A and 2B are prescribed. In case of dispute the vacuum jacketed specific gravity bottle shown as Figure 2A shall be used.

Figure 2A — Vacuum jacketed specific gravity bottle

Figure 2B — Renault’s specific gravity bottle

7.4 Procedure 7.4.1 Weigh empty and dry pyknometer and record its weight. Cool the test sample of the material to 20 °C and fill into the pyknometer cooled to 20°C. Fill the pycnometer to overflowing, taking care to avoid any head space and close with stopper. Clean and thoroughly dry the pycnometer before recording the sample temperature. Weigh immediately.

EAS 104:2000


7.4.2 Calculation Specific gravity at 20°C/20°C

BCBA

−−

=

where, A = mass in grams of the specific gravity bottle with the sample at 20 °C B = mass, in grams of the specific gravity bottle and C = mass, in grams, of the specific gravity bottle with water at 20 °C. 8 Determination of volatile acidity 8.1 Reagents 8.1.1 Standard sodium hydroxide, 0.05 M. 8.1.2 Phenolphthalein indicator. 8.2 Procedure Take 50 ml of the distillate obtained from the determination of ethyl alcohol (6.1) and titrate against standard alkali using phenolphthalein as indicator (1 ml of standard sodium hydroxide is equivalent to 0.003 g of acetic acid). 8.3 Calculation Volatile acids expressed as acetic acid, grams per 100 litres of absolute alcohol

1

20030100V

V ×××=

.

where V is the volume in ml of alkali used for titration; and V1 is the alcohol, % by volume. 9 Determination of fixed acidity 9.1 Reagents 9.1.1 Standard sodium hydroxide, 0.05 M. 9.1.2 Phenolphthalein indicator, 10g/l, prepared by putting 2.5 of phenolphthalein in 250 ml of rectified spirit. 9.1.3 Distilled water, distilled and boiled to remove carbon dioxide and neutralized. 9.2 Procedure Take 50 ml of the sample and evaporate to near dryness. Dilute with neutralized distilled water to 100 ml and again evaporate to dryness. Dilute and titrate against standard sodium hydroxide solution using phenolphthalein as indicator. (One millilitre of the standard sodium hydroxide is equivalent to 0.003 75 g of tartaric acid).

EAS 104:2000


9.3 Calculation Fixed acidity expressed as tartaric acid, grams %, 100 litres of absolute alcohol

1

21000100750030V

V ××××=

.

where V is the volume in ml of the standard sodium hydroxide used in the titration; and V1 is the alcohol, % by volume. 10 Determination of total acidity 10.1 Reagents 10.1.1 0.1 N Sodium Hydroxide 10.1.2 Phenolphthalein indicator 10.2 Procedure Take 50 mL of the sample and add about 200 mL of neutralized, distilled water. Titrate against sodium hydroxide solution using phenolphthalein as indicator. 1 mL of 0.1 N sodium hydroxide solution is equivalent to 0.003 75 g of tartaric acid. 10.3 Calculation Total acidity, expressed as tartaric acid, grams per 100 litres of absolute alcohol.

1V2 000 1 100 V 0.00375 ××××

=

where, V = Volume, in mL of 0.1 N sodium hydroxide solution used for titrations; and V1 = Absolute alcohol, per cent by volume. The total acidity as both acetic and tartaric acid shall be determined by calculating the sum of both volatile acidity (8.3) and fixed acidity (9.3). 11 Determination of esters as ethyl acetate 11.1 Reagents 11.1.1 Standard sodium hydroxide, 0.1 M. 11.1.2 Standard sulfuric acid, 0.1 M. 11.2 Procedure 11.2.1 To the neutralized distillate from the volatile acidity determination (8.2) add 10 ml of the standard alkali and reflux it on a steam bath for an hour. Cool and back titrate the excess alkali with standard sulfuric acid. (1 ml of standard alkali is equivalent to 0.008 8 g of ethyl acetate. 11.2.2 Simultaneously run a blank taking 50 ml of distilled water in place of the distillate of the sample in the same way. The difference in titration value in millilitres of the standard acid solution gives the equivalent ester.

EAS 104:2000


11.3 Calculation Esters expressed as ethyl acetate, grams per 100 litres of absolute alcohol

1

280080100V

V ×××=

.

where V is the difference in ml of standard acid used for blank and experiment, and V1 is the alcohol, % by volume. 12 Determination of aldehydes as acetaldehydes 12.1 Apparatus Iodine flask, of 250 ml capacity 12.2 Reagents 12.2.1 Sodium bisulfate solution, approximately 0.05 M. 12.2.2 Standard iodine solution, 0.05 M 12.2.3 Standard sodium thiosulfate solution, 0.05 M. 12.2.4 Starch, as indicator 12.3 Procedure Take 50 ml of the distillate obtained during ethanol determination, in 250 ml iodine flask and add 10 ml of bisulfate solution. Keep the flask in a dark place for 30 min with occasional shaking. Add 25 ml of the standard iodine solution and back titrate excess iodine against standard sodium thiosulfate solution using starch as indicator. Run a blank taking 50 ml of distilled water in place of the distillate of the liquor in the same way. The difference in titration value in millilitres sodium thiosulfate solution gives the equivalent aldehydes. (1 ml of the standard sodium thiosulfate solution is equal to 0.001 1 g of the acetaldehydes). 12.4 Calculation Aldehydes expressed as acetaldehydes, grams per 100 litres of absolute alcohol

1

1000100210010VV ××××

=.

where V is the difference in ml of standard sodium thiosulfate solution used for blank and experiment and V1 is the alcohol, % by volume 13 Determination of furfural 13.1 Apparatus 13.1.1 Nessler tubes, with flat bottom tubes of thin colourless 25 mm in diameter, about 150 mm in length and graduated at 50 ml. The depth measured internally from the graduation mark to the bottom shall not vary by more than 2 mm.

EAS 104:2000


13.1.2 Nessler comparator 13.2 Reagents 13.2.1 Aniline, distilled and colourless. 13.2.2 Hydrochloric acid, sp. gr. 1.125. 13.2.3 Furfural free alcohol. Let alcohol containing 5 g of m-phenylenediamine hydrochloride stand at least for 24 h with frequent shaking (previous treatment with potassium hydroxide is not necessary). Reflux for at least 8 h, longer if necessary. Let stand overnight and distill, rejecting the first 100 ml and the last 200 ml of the distillate. If this gives a coloration with aniline hydrochloride, repeat the treatment. 13.2.4 Standard furfural solution. Dissolve 1 g of redistilled, colourless furfural in 100 ml of the furfural free alcohol. Prepare standard furfural solution by diluting 1 ml of this solution to 100 ml with 50 % furfural free alcohol. 1 ml of this diluted solution contains 0.1 mg of furfural (strong furfural solution shall retain its strength but the diluted standard solution should be prepared afresh every time). 13.3 Procedure 13.3.1 Take 5 ml of the distillate obtained for ethanol determination, add 1 ml of the colourless aniline and 0.5 ml of the hydrochloric acid, and keep for 15 min. Red colour indicates the presence of furfural. Proceed for quantitative estimation if colour develops. 13.3.2 Dilute measured portion of the distillate with 50 % furfural free alcohol to 50 ml. First add 2 ml of the colourless aniline and then 0.5 ml of hydrochloric acid. Mix and keep at 15 °C for 15 min. Compare the colour developed with standard furfural solution by using a Nessler comparator. 13.4 Calculation Furfural, grams per 100 litres of absolute alcohol

21

1000001VV

W×

××=

where W is the weight in grams of the furfural present in volume used for matching the experimental

solution; V1 is the volume in ml of experimental solution used for estimation; and V2 is the alcohol, % by volume 14 Determination of methanol 14.1 Apparatus 14.1.1 Separating funnel 14.1.2 Spectrophometer 14.2 Reagents 14.2.1 Potassium permanganate solution. 3.0 g KMnO4 and 15.0 ml H3PO4 shall be dissolved in 100 ml water. The solution shall be prepared monthly.

EAS 104:2000


14.2.2 Sodium salt of chromotropic acid (sodium 1,8-dihydroxynaphthalene - 3,6 disulfonate) 5 % aqueous solution. If not clear, the sodium salt chromotropic acid shall be filtered. It shall be prepared weekly. 14.3 Procedure 14.3.1 Purification of chromotropic acid. If absorbance of blank is greater than 0.05, the reagent shall be purified as follows: 10 g chromotropic acid or its salt shall be dissolved in 25 ml water. 2 ml H2SO4 shall be added to the aqueous solution of the salt to convert it to free acid. 50 ml methanol shall be added to precipitate free chromotropic acid. More isopropyl alcohol may be added to increase yield of purified acid. 14.3.2 The sample of spirits shall be prepared as follows: a) The sample shall be diluted or adjusted to total alcoholic concentration of 5 % to 6 %. Using 50

ml sample distillation shall be carried out through a still apparatus, collecting 40 ml of the distillate and diluting it to 50 ml with water. If alcohol has been determined previously distillate may be adjusted to 5 % to 6 % alcoholic concentration and used for determination.

b) If more than 0.05 % methanol by volume is present dilute to approximately that concentration

with 5.5 % alcohol content. For samples containing less than 0.05 % methanol by volume, 200 ml shall be measured into an efficient fractionating still and the system placed under total reflux for 15 min, and then slowly distilled at rate of reflux (at least 20:1). 10 ml distillate shall be collected and diluted to 10 ml with water.

14.3.3 A portion of the sample prepared under 14.3.2 shall be used. 14.4 Determination 14.4.1 2 ml KMnO4 solution shall be pipetted into a 50 ml volumetric flask. The solution shall then be chilled in an ice bath, add 1 ml chilled diluted sample, and allow to stand for 30 min in ice bath. Decolourizing with little dry NaHSO3 shall be carried out and 1 ml chromotropic acid solution added. 15 ml H2SO4 shall be added slowly with swirling and then placed in hot water bath at 60 °C to 75 °C for 15 min. The contents of the flask shall then be cooled, enough water added to bring to approximately 50 ml mark, mixed and diluted to volume with water at laboratory temperature. 14.4.2 The absorbance of the test portion shall be read at 575 nm, using reagent blank of 5.5 % alcohol treated similarly as reference. Standard methanol solution containing 0.025 % by volume of methanol shall be treated in 5.5 % alcohol simultaneously in the same manner and absorbance read (see note below). 14.5 Expression of results. The methanol content, in the test portion shall be calculated as follows:

1

0250A

FAMethanol

××=

.%,

where A is the absorbance of the test portion; A1 is the absorbance of the standard methanol; and F is the dilution factor of the test portion. NOTE The difference in temperature between the standard and the test portion should be within 1 °C since temperature affects depth of colour. If the colour of test portion is too intense, it should be diluted with H2SO4 alcohol blank according to 14.4. Note more than three-fold dilution should be permitted.

EAS 104:2000


15 Determination of fusel oils, methanol and dimethyl sulphide in alcoholic

beverages by headspace gas chromatography — Reference method 15.1 Scope and field of application This method separates and quantifies acetaldehyde, ethyl acetate, Iso-butyl acetate, n-propanol, Iso-butanol, Iso-amyl acetate, n-buttanol , Iso-amyl alcohol, ethyl hexanoate and dimethyl sulphide in alcoholic beverages. The method has been shown to be linear up the following concentrations for each compound: acetaldehyde (16 mg/litre). Ethyl acetate (44 mg/litre), Iso buryl acetate (1.7 mg/litre), n-propanol (64 mg/litre), Iso butanol (76 mg/litre), Iso amyl acetate (7 mg/litre), Iso amyl alcohol (188 mg/litre), ethyl hexanoate (0.86 mg/litre), and dimethyl sulphide (280 mg/litre). This method has also been adapted to determine methanol levels in alcoholic beverages and has successfully shown to give linearity up to 60 mg/litre. 15.2 Principle 15.2.1 An aliquot of the sample is mixed with an excess of ammonium sulphate, in a vial at 35 ºC, until an equilibrium has been established between the sample and the headspace. 15.2.2 A sample of the headspace is injected into a gas chromatograph fitted with a flame ionization detector. 15.2.3 The fusel oils, methanol and dimethyl sulphide are separated and their respective concentrations calculated from their peak area ratios to the internal standards with respect to the calibration standards. 15.3 Reagents All analytical reagents are labelled Good Laboratory Practice (GLP) Regulations. The precautions recommended under the GLP scheme and in the laboratory safety manual must be adhered to at all times. 15.3.1 Acetaldehyde, puriss grade, >99.5 per cent anhydrous 15.3.2 Ethylacetate, puriss grade, > 99.5 per cent 15.3.3 Ethyl caproate (Ethyl hexanoate), purum grade 99 per cent 15.3.4 Iso butanol, puriss grade, > 99.5 per cent 15.3.5 Iso butyl acetate, puriss, grade, 99 per cent 15.3.6 Iso pentyl acetate (isoamyl acetate), purum grade, > 97 per cent 15.3.7 Methyl acetate, purum grade 15.3.8 2-Methyl-l-butanol, purum grade, > 98 per cent 15.3.9 3-Methyl-1-butanol, puriss grade, >99.0 per cent 15.3.10 Methyl sulphide (Dimethyl sulphide), puriss, grade, > 99 per cent 15.3.11 1-Pentanol, puriss grade, > 99.0 per cent 15.3.12 Propanol, Aristar. 15.3.13 Absolute Alcohol, AR.

EAS 104:2000


15.3.14 Ammonium sulphate, Analar. 15.3.15 Methyl Alcohol, A.R, 99.85 per cent w/w min. 15.4 Apparatus 15.4.1 Gas chromatography fitted with a flame ionization detector. CAUTION! Hydrogen is used as the carrier gas and is explosive. Ensure the column oven is continuously purged with nitrogen and that a gas leak detector is located in the same room as the gas chromatography. 15.4.2 Automated Headspace Sampler 15.4.3 Computerised Data Handling System 15.4.4 Adjustable pipettes, 250 µl, 1 mL, 5 mL and 10 mL 15.4.5 ‘A’ grade pipettes, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 20 mL and 25 mL 15.4.6 ‘A’ grade volumetric flasks, 100 mL and 1 Litre 15.4.7 50 mL stoppered conical flask 15.4.8 50 mL measuring cylinder 15.4.9 Electronic analytical balance to weigh to 4 decimal places 15.4.10 10 mL, 20 mL and 150 mL crimp-top vials, aluminium crimp caps and PTFE-faced seals 15.4.11 Crimper and decaper 15.4.12 Rotary shaker, Gerhardt Minischuttler MS 15.4.13 Incubator, held at 35 ºC ± 1.0 ºC, Gallen Kamp 15.4.14 Thermometer for 27.4.11. 15.4.15 Calculator, capable of performing linear regression analysis 15.5 Calibration procedure Unless otherwise stated, only use reagents and apparatus listed above, and deionized water when preparing calibration solutions. All solutions are stable for 6 months if kept refrigerated and must be clearly labelled. 15.5.1 Stock solutions To prepare the stock solutions FO A, FO B, FO C and FO D weigh the volume of each compound given, to 4 decimal places, into a 100 ml volumeric flask, approximately half-full with absolute alcohol. Make up to the mark with absolute alcohol. 15.5.1.1 Stock solutions, FO A

0.32 g ± 0.05 g propanol by pipetting 0.4 mL 0.47 g ± 0.05 g 2 methyl-l-butanol by pipetting 0.6 mL 0.46 g ± 0.05 g 3-methyl-l-butanol by pipetting 0.6 mL

EAS 104:2000


0.39 g ± 0.05 g iso butanol by pipetting 0.5 mL 15.5.1.2 Sock solution FO B

2.2g ± 0.05g ethyl acetate by pipetting 2.5 mL 7g ± 0.05g acetadehyde by pipetting 1 mL 0.85g ± 0.005g iso butyl acetate by pipetting 0.1 mL 0.35g ± 0.05g iso amyl acetate by pipetting 0.4 mL

15.5.1.3 Stock solution, FO C

0.08 g ± 0.005 g ethyl hexanoate by pipetting 0.1 mL 15.5.1.4 Stock solution FO D

0.075g ± 0.005g dimethyl sulphide by pipetting 0.1 mL 15.5.2 Working Stock Solution FO E Pipette 20 mL of FO A, 2mL of FO B, 1 mL of FO C and 0.4 mL of FO D into a-100 mL volumetric flask and make up to the mark with water. 15.5.3 Standard Solution FO 0, FO 1, FO 2, FO 3, FO 4, FO 5 and FO 6 15.5.3.1 Pipette 4 mL of absolute alcohol into a 100 mL volumetric flask, make up to the mark with water and label FO 0. 15.5.3.2 Pipette the following volumes of FO E and absolute alcohol into separate 100 mL volumetric flasks, approximately half full with deionized water.

1 mL FO E, 3.76 mL absolute alcohol 2 mL FO E, 3.52 mL absolute alcohol 3 mL FO E, 3.28 mL absolute alcohol 4 mLFO E, 3.04 mL absolute alcohol 5 mL FO E, 2.80 mL absolute alcohol 6 mL FO E, 2.56 absolute alcohol

Make up to the mark with water and label FO 1, FO 2, FO 3, FO 4, FO 5, FO 6 respectively. 15.5.4 Internal Standard Solution FO IS 15.5.4.1 Pipette 1 mL of methyl acetate and 2 mL of pentanol into a 1 litre volumeric flask approximately half-full with water. Using a measuring cylinder, add 40 ml of absolute alcohol to the flask and make up to the mark with water. 15.5.4.2 Decant the solution into 150 mL crimp top glass vials until full, crimp on the caps and label each vial FO IS. 15.5.4.3 On the day of analysis, transfer the solution FO IS from a vial to a stoppered 50 ML conical flask. Keep the flask in an ice bath. NOTE This solution is stable for one week if kept refrigerated.

EAS 104:2000


15.5.5 Standard calibration procedure FOCC 15.5.51 Pipette 14 mL of absolute alcohol into a 500 mL volumetric flask 15.5.5.2 Pipette 25 mL of FO E into the flask, make up to the mark with water and label FO CC 15.5.5.3 Pipette 10 mL of FO CC into separate 10 mL crimp top glass vials, crimp on the caps and label each vial FO CC. 15.5.6 Methanol calibration 15.5.6.1 Pipette 4 mL of absolute alcohol into a 100 mL volumetric flask, make up to the mark with water and label ME O. 15.5.6.2 Pipette 4 mL of absolute alcohol into six separate 100 mL volumetric flasks labelled ME 10, ME 20, ME 30, ME 40, ME 50, and ME 60. 15.5.6.3 Pipette the following volumes of methanol into the six respective 100 mL flasks and make up to the mark with deionized water.

1.0 g ± 0.05 g methanol by pipetting 1.3 mL 2.0 g ± 0.05 g methanol by pipetting 2.5 mL 3.0 g ± 0.05 g methanol by pipetting 3.8 mL 4.0 g ± 0.05 g methanol by pipetting 5.1 mL 5.0g ± 0.05g methanol by pipetting 6.3 mL 6.0 g ± 0.05 g methanol by pipetting 7.6 mL

15.6 Sample and control procedure All samples and controls must be kept in an ice bath for at least 1 hour prior to preparation. Controls are interspaced amongst samples at a rate of approximately 1 control to every 8 samples. 15.6.1 Weigh 4.7 g ± 0.25 g of ammonium sulphate into a 20 mL crimp top glass vial. 15.6.2 Cool the vial to approximately — 20 ºC. 15.6.3 Pipette 5 mL of sample (or control) and 250 µL of internal standard solution, FO IS, into the vial and crimp on the cap. 15.6.4 Transfer the vial to the rotary shaker inside the incubator. Allow to shake for 30 to 35 minutes. 15.6.5 Record the vial number alongside the name of the sample (or control) in the Fusel Oil Workbook. 15.7 Analytical procedure 15.7.1 Set up the gas chromatograph according to the instructions given in the Operators Manual. Set the following parameters: 15.7.1.1 Column — 60m × 0.25mm i.e. CP Wax 57 – CB (WOOT) fused silica column, film thickness 0.41µm

EAS 104:2000


15.7.1.2 Temperature — Injector block 150 °C

Detector block 200 ºC Column temperature program: Initial temp. 30 ºC Initial time 3.0 min 1st temp ramp rate 4 ºC/min 1st upper temp 90 ºC

1st upper time 0 min 2nd Ramp 25 ºC/min 2nd upper temp 160 ºC 2nd upper time 0 min

15.7.1.3 Gas Flow Rates — Carrier gas (H2)2 mL/min

split vent 13 mL/min 15.7.1.4 Detector Range 1 15.7.1.2 Allow the gas chromatograph to stabilise for approximately 30 minutes. NOTE It may be necessary to alter the conditions slightly in order to optimise the separation and quantification of the components. Alterations to the conditions listed above are recorded in the Fusel Oils Workbook. 15.7.3 Set up the headspace autosampler according to the instructions given in the Operators Manual. 15.7.4 Set up the Computerised Data Handling System according to the instructions given in the Users Manual. 15.7.5 Injection procedure Place the vials into the autosampler. Press the “Run/stop” button on the autosampler to initiate the injection procedure. The components are eluted in the following order.

Acetaldehyde Dimethl sulphide Ethyl acetate (methanol) methyl acetate Iso butyl acetate Propanol Iso butanol Iso amyl acetate Iso amyl alcohol ethyl hexanoate pentanol

The two isomers, 2 methyl-butanol and 3 methyl-1-butanol co-elute and are referred to as iso amyl alcohol. 15.7.6 Linear calibration — Perform the linear calibration at intervals not exceeding three months.

EAS 104:2000


15.7.6.1 Prepare the standards FO 0, FO 1, FO 2, FO 3, FO 4, FO 5 and FO 6 (27.5.3.1 and 27.3.2) or ME 0, ME 10, ME 20, ME 30, ME 40, ME 50 and ME 60 (27.5.6.1 to 27.5.6.3), in triplicate, by following steps 6.1 to 6.5 inclusive. 15.7.6.2 Set up the components list and enter the values of each standard as described in the operators instruction manual. 15.7.6.3 In the sequence file enter the first of each triplicate as “CAL Replace” and the other two as “CAL Average”. Inject each standard in turn and obtain chromatograms. 15.7.6.4 Go to the “Fit Line” page and carry out the calibration procedure according to the operators instruction manual. 15.7.6.5 Ensure that the r2 value of each line is no greater than 0.98. If not then return to 15.7.6.1 15.7.6.6 Record calibration details on the form entitled “Fusel Oils Calibration Record”. 15.7.6.7 Sample (and control) determination. 15.7.6.8 Inject each sample (or control) in turn and obtain the chromatogram. 15.7.6.9 Calibration Check — Check the linear calibration by analysing the standard calibration check solution (FO CC) daily at the beginning of each run. 15.7.6.10 Treat FO CC as a sample following the sample and control procedure (section 6), the anlaytical procedure (15.7.7 and calculation procedure (8.1 to 8.2 inclusive). 15.8 Expression of results 15.8.1 Set the “Set up” file to print both chromatogram and results. 15.8.2 The concentrations of the fusel oil and dimethyl sulphide in each sample (or control) are calculated by the computerised data handling system. 15.8.3 Record the results in the fusel oils workbook. 15.8.4 Any results that are less than the detection limits below should be reported as detection limit:

Detection limit

Acetaldehyde 0.5 mg/litre

Ethyl Acetate 0.25 mg/litre

Iso Butyl Acetate 0.04 mg/litre

Iso Amyl Acetate 0.15 mg/litre

Ethyl Hexanoate 0.01 mg/litre

Iso Amyl Alcohol 2.5 mg/litre

Dimethyl Sulphide 8µg/litre 15.8.5 Any results which exceed the linear range of the method as detailed in section 1, are reported as ‘>x’ where x is the upper limit of the linear range for any one compound. 15.8.6 Report the concentrations of each fusel oil to the nearest whole number for concentrations greater than 100 mg/litre to 1 decimal place for concentrations greater than 1 mg/litre, to 2 decimal places for concentrations less than 1 mg/litre. Report the concentration of dimethyl sulphide to the nearest whole number in µg/litre.

EAS 104:2000


15.9 Performance characteristics 15.9.1 Quality control procedures are followed in order to ensure the reliability of results. Control samples are analysed and their results plotted on a control sheer. 15.9.2 The difference, d, between the results of one sample analysed in duplicate should not exceed the value(s) calculated from the following equations: Acetaldehyde d = 0.64 + 0.061 x mg/litre Ethyl Acetate d = 0.38 + 0.095 x mg/litre Iso butyl acetate d = 0.014 + 0.14 x mg/litre n- Propanol d = 0.49 + 0.083 x mg/litre Iso Butanol d = 0.85 + 0.047 x mg/litre ISO Amyl Acetate d = 0.039 + 0.11 x mg/litre n-Butanol d = 0.113 + 0.22 x mg/litre Ethyl Hexanoate d = 0.004 + 0.22 x mg/litre Iso Amyl Alcohol d = 2.37 + 0.091 x mg/litre Dimethyl Sulphide d = 6.00 + 0.14 x mg/litre Where X = The mean concentration of the two duplicates. 15.9.3 The IOB published F25 and R25 values for some of the compounds are given below:

Range R95 R95 Acetaldehyde Ethyl Acetate n-Propanol Iso Butanol Iso Amyl Acetate Iso Amyl Alcohol Ethyl Hexanoate

4.9 – 8.1 9.9 – 54 8.7 – 2.3 5.4– 2.2 0.8 – 4.7 45 - 105 0.1 – 0.4

0.208 m 3 1 0.251 m 0.56 + 0.085 m 0.36 0.14 m 0.073

0.60 m 2.1 + 0.29 2.5 - 0.57 m 1.7 + 0.15 m 0.2 + 0.58 m 0.22 m 0.1 + 0.91 m

15.9.4 The results of the standard calibration check solution (FO SCC) are treated as a control sample (see 9.1). If the results fall outside the stated limits the appropriate action is taken. 15.10 Test results 15.10.11 For the procedure for reporting results and layout of report forms. 15.11 Safety 15.11.1 Take all necessary precautions as recommended in the COSHH regulations and the laboratory safety manual. 15.11.2 Take note of the hazards associated with using the gas chromatography and the automated headspace sampler by referring to their respective operators manuals.

EAS 104:2000


16 Detection of acetones, other ketones, isopropanol and tertiary

butylalcohol 16.1 Apparatus 16.1.1 Kohlrausch flask. 16.1.2 Water bath. 16.2 Reagents Mercuric sulfate solution is prepared as follows: 5 g of mercuric oxide (HgO) shall be mixed with 40 ml of water and 20 ml H2SO4 and 40 ml of water added by stirring. Stirring shall be continued until the substances added are completely dissolved. 16.3 Procedure 16.3.1 To 2 ml distillate of the sample add 3 ml of water and 10 ml of HgSO4 solution. Heat on boiling water bath for 3 min. White or yellow precipitate forming within 3 min shall indicate the presence of acetone, other ketones or tertiary butyl alcohol. Any precipitate forming after 3 min on the boiling water bath shall be disregarded. 16.3.2 If no precipitate forms, testing for isopropanol shall be conducted as follows: 8 g of CrO3 shall be placed in 100 ml Kohlrausch flask and 15 ml of water and 2 ml of H2SO4 added. The flask shall be connected to a reflux condenser and 5 ml of the test portion added very slowly through the condenser. Boiling under reflux condenser shall be carried out for 30 min then cooled and 2 ml distilled off, collecting distillate in the cylinder. 3 ml of water and 10 ml of the HgSO4 solution shall be added and the operation specified in 15.3.1 carried out. 17 Determination of ash 17.1 Apparatus 17.1.1 Muffle furnace 17.1.2 Desiccator 17.1.3 Water bath 17.1.4 Dish, 50 ml capacity, silica or platinum. 17.2 Procedure Shake the contents of the container. Evaporate 100 ml of the sample in a dried, tared dish on a water bath. Place the dish with the residue in the muffle furnace maintained at 450 °C to 500 °C for about an hour. Cool the dish in a desiccator and weigh. 17.3 Calculation

( )V

WW100volumebymass%Ash,

12 −=

where W1 weight of the empty dish; W2 weight of the dish with ash; and V volume in ml of liquor taken for ash determination.

EAS 104:2000


18 Determination of suspended solids 18.1 Apparatus 18.1.1 Hot air-oven 18.1.2 Filter Paper — Whatman no 42 or equivalent of about 9 cm diameter. 18.1.3 Dessicator 18.2 Procedure 18.2.1 Dry a filter paper at 105 °C for 3 hours in an oven. Cool in the dessicator and weigh immediately. Mix the contents of the container thoroughly by shaking. Take 250 ML and filter through the filter paper. Dry the filter paper in air-oven at 105 °C for 3 hours. Cool in dessicator and weigh. Express the results to four decimal places. 18.3 Calculation — Suspended solids, per cent (mass/volume)

( )V

MM 12100 −=

where,

M2 = Mass of the filter paper with dry material; M1 = Mass of the filter paper, and V = Volume, In mL, of sample taken.

19 Determination of dissolved solids 19.1 Apparatus 19.1.1 Hot air-oven 19.1.2 Water-bath 19.1.3 Volumetric Flask — 200 mL capacity. 19.1.4 Dish – 50 mL capacity, silica or platinum. 19.1.5 Dessicator 19.2 Procedure Evaporate 200 mL of filtrate (see 18.2) in a dried, tarred dish on a water-bath. Dry the dish in an air-oven at 105 °C for 3 hours, cool in a dessicator and then weigh the dish. Calculate the dissolved solids as per cent mass by volume. Express the result to four decimal places. 19.3 Calculation — Dissolved solids, per cent mass / volume

( )V

MM 12 100 −=

where, M2 = Mass of the, dish with dry material,

EAS 104:2000


M1 = Mass of the dish, V = Volume in ml of sample taken.

20 Determination of total solids 20.1 Determine the percentage of total solids by adding the suspended solids (18.3) and dissolved solids (19.3). The total solids may also be determined by the method given in 20.2. 20.2 Procedure Place 200 mL of the sample in a dried, tared dish into an air-oven. Bring the temperature to 105°C and hold for at least 3 hours or until the liquid is completely evaporated. Cool in a dessicator and reweigh the dish. Express the result to four decimal places. 20.3 Calculation Total solids, per cent (mass / volume)

( )V

MM 12100 −=

where M2 , M1 and V represent the same parameters as in 19.3 21 Determination of higher alcohol as amyl alcohol 21.1 General Three methods have been prescribed. Method(i) may be used to determine approximately the quantity of higher alcohol in process control. Method (ii) shall be employed for accurate determination while – Method (iii) shall be used as a reference method.

EAS 104:2000


Figure 3 — Apparatus for the determination of higher alcohol

21.2 Method (I) (Komarowski Method). 21.2.1 Reagents 21.2.1.1 Salicycllic Aldehyde — Dissolve 1 % salicylic aldehyde in 95 per cent alcohol, free of higher alcohol.

EAS 104:2000


21.2.1.2 Sulphuric acid – AR 21.2.2 Procedure 21.2.2.1 Take a clean glass-stoppered bottle and wash it twice with the product to be tested. Similarly wash small graduated cylinder or a 100-mL pipette. 21.2.2.2 Take 10 mL of sample in the bottle and add 1 ml of 1 per cent salicylic aldehyde and 20 mL of concentrated sulphuric acid. Allow to stand at room temperature for over 12 hours. Note the colour changes. For quick routine analysis, the colour change may be noted after a shorter interval. 14.2.2.3 The colours developed after the reaction indicate the amount of higher alcohol as follows:

Colour — Amount of higher alcohol Light yellow — Only traces Yellow to brownish — About 0.01 per cent (V/V) Brown — 0.02 to 0.03 per cent (V/V) Red — 0.05 to 0.1 per cent (V/V) Dark red to black — Above 0.1 per cent (V/V)

21.3 Method (ii) — Titrimetric Method 21.3.1 Apparatus — As shown in Figure 3. It consists of a 800 mL Kjeldahl flask fitted with splash head connected to distillation head. The other head of the distillation is connected to a liebig condenser, which opens into a receiver having capacity of 250 mL. All joints are made of ground glass. 21.3.2 Reagents 21.3.2.1 Oxidizing Mixture — Dissolve 100 g of potassium dichromate in 100 mL of sulphuric acid (AR) and make the volume up to 1 litre. 21.3.2.2 Standard Sodium Hydroxide — 0.1 N 21.3.2.3 Carbon Tetrachloride — AR 21.3.2.4 Sodium Chloride 21.3.2.5 Sodium Sulphuric 21.3.2.6 Phenolphthalein Indicator 21.3.3 Procedure 21.3.3.1 Add 50 mL of water to the solution resulting from the determination of esters (see 21.2.1). Saturate it with sodium chloride, extract the saturated solution four times with carbon tetrachloride using 40, 30, 20 and 10 mL respectively. Combine the extracts and wash three times with saturated sodium chloride solution and twice with saturated sodium sulphate solution.

EAS 104:2000


21.3.3.2 Filter carbon tetrachloride and add 50 mL of oxidizing mixture. Reflux for 2 hours. Cool, wash the reflux condenser with some water and transfer it to the distillation flask using about 50 mL water. Distil till 50 mL is left over in the flask. Avoid charring. 21.3.3.3 Titrate the distillate against standard alkali with phenolphthalein indicator. 1 mL of standard alkali is equivalent to 0.0088 grams of amyl alcohol. 21.3.3.4 Run a blank in the same way taking some of distilled water in place of the distillate of the liquor. 21.3.4 Calculation — Higher alcohol as amyl alcohol, grams per 100 litres of absolute alcohol.

21 V V100 100 1000 0.0088 V

×××××

=

where, V = Difference, in mL, of standard alkali used for blank and experiment; V1 = Volume of sample taken; and V2 = Alcohol, per cent by volume. 21.4 Method (iii) 21.4.1 Apparatus Spectrophotometer 21.4.2 Reagent 21.4.2.1 P-Dimethylaminobenzaldehyde solution — In 100- mL volumetric flask dissolve 1 g. P-dimethylaminobenzaldehyde in mixture of 5 mL sulphuric acid and 90 mL water and dilute to mark with water. 21.4.2.2 Isobutyl alcohol 21.4.2.3 Isoamyl alcohol 21.4.2.4 Ethyl alcohol — Redistilled middle 50 per cent fraction. 21.4.2.5 Synthetic standard higher alcohol Weigh 2 g isobutyl alcohol and 8 g isoamyl alcohol into 1 litre volumetric flask and dilute to mark with water. Pipette two 10 mL portions into 100-mL volumetric flasks and dilute to mark, one with water and other with alcohol. Prepare working standards for products in range 0 to 170 proof containing 1.0 to 6.0 g synthetic higher alcohol per 100 litres by diluting 1.0 to 6.0 mL aliquots of aqueous standard solution to 100 mL with alcoholic solution of proof expected for dilute sample when pipetted analysis tube. Prepare similar working standard for products in range 170 to 190 proof by diluting 1.0 to 6.0 aliquots of standard solution of proof of sample or its dilution. 21.4.3 Procedure 21.4.3.1 Preparation of sample

EAS 104:2000


Take 200 mL of sample liquor in 500 mL erlenmeyer flask, add about 35 mL water and a few pieces of anti-bumping granules.Distil slowly into 500 ml volumetric flask until distillate is nearly at mark. Dilute to mark and mix. 21.4.3.2 Pipette 2 mL aliquots of sample or dilute sample, 2 mL water (for reagent blank) and 2 mL aliquots of standards into 15 × 150 mm stoppered or covered test tubes stopper or cover tubes, and place in rack then in ice-bath. Pipette 1 mL P-dimethylaminobenzaldehyde solution into each tube. Shake tubes individually and replace in ice-bath for 3 minutes. Transfer rack of tubes from ice-bath to boiling water bath and boil for 20 minutes. Transfer tubes to ice-bath for 3 to 5 minutes then to room temperature bath. Read percentage transmittance at 530 to 535 mm against reagent black or reference (Use same wavelength for both standards and unknown). Plot a higher alcohol per 100 litres on linear scale as abscissa against percentage T as ordinate on log scale of semi log paper. Convert percentage transmittance of samples to g higher alcohol per 100 litres from standard curve. If dilution was used, multiply g higher alcohol to obtain g higher alcohol per 100 litres (found by dilution factor to obtain g higher alcohol per 100 litres) in original sample. Analyse 2 levels of standard with each series of unknowns. 21.4.4.4 Precision expected Whiskies and Brandies ± 5 per cent; Rum ± 8 per cent, in Vodka Spirits ± 0 per 100 litres. 22 Test for miscibility with water 22.1 Mix 10 mL of the sample with 90 mL distilled water in a suitable glass vessel and allow to stand at room temperature for one hour. Compare the clarity of the mixture with that of an equal volume of water. 22.2 The material shall be taken to comply with the specified requirement if there is no noticeable difference in clarity between the mixture and water. 23 Test for alkalinity 23.1 Reagents 23.2 Procedure Place 100 mL of water and a few pieces of Porous pot in 500 mL conical, flask. Boil gently for 5 minutes to eliminate carbon dioxide then cool slightly and add 100 mL of the sample. Boil gently for a further period of 5 minutes. At the end of this period, close the neck of the flask with a stopper carrying a soda-lime guard tube, and allow to cool to room temperature. Remove the stopper, add 0.5 mL of phenolphthalein indicator and examine for alkalinity. 24 Test for permanganate reaction time 24.1 Apparatus Graduated measuring cylinder — stoppered, 100 mL capacity. 24.2 Reagents Standard Potassium Permanganate solution — 0.01N 24.3 Procedure

EAS 104:2000


24.3.1 Clean thoroughly the graduated measuring cylinder first with concentrated hydrochloric acid, then with water and finally with the sample to be tested. Place 20 mL of the sample in the cylinder, bring the temperature to 20 ºC placing the cylinder in cold water, add 1.0 mL of standard permanganate solution by means of a 1 mL pipette noting the exact time as soon as addition is over. Mix the contents at once and keep the cylinder at 20 ºC and away from bright light. 24.3.2 The sample shall be taken to have satisfied the test if the pink colour does not disappear for 30 minutes.

Figure 4 — Pressure gauge for testing atmospheric pressure in sparkling wines 25 Determination of copper 25.1. Apparatus 25.1.1 Spectrophotometer 25.2. Reagents 25.2.1 Citric acid — solid 25.2.2 Ammonium Hydroxide solution — sp. gr. 0.90

EAS 104:2000


25.2.3 Sodium Diethyldithiocarbamate solution — 0.1 per cent (v/v), aqueous 25.2.4 Carbon Tetrachloride — Redistilled 25.2.4 Sodium Sulphate — Anhydrous 25.2.5 Dilute Nitric Acid — Concentrated nitric acid of Sp. Gr. 1.42 diluted with an equal volume of water. 25.2.6 Standard Copper Solution — Weigh accurately 0.100g of pure copper turnings, carefully dissolve in the minimum amount of nitric acid, cool and dilute to one litre in a graduated flask. Pipette 10 mL of this solution into 100 mL graduated flask and dilute to the mark. This solution contains 10 micrograms of copper per litre. 25.3 Procedure 25.3.1 Transfer a 10 mL aliquot of the test solution to a separating funnel. Add one gram of citric acid and dissolve it by shaking. Make the solution alkaline to litmus by adding ammonium hydroxide solution in small quantities. Add 5 mL of the sodium diethyl — dithiocarbamate solution, shake thoroughly and extract with 5 mL portions of carbon tetrachloride until the final extract is colourless. Dry the combined extracts by shaking thoroughly with anhydrous sodium sulphate. Filter the dry extract and wash the filter paper with carbon tetrachloride. Make up the volume of filtrate to 25 mL with carbon tetrachloride and measure the absorption at 437 mm by means of the spectrophotometer. Simultaneously, carry out blank determinations on the water and the reagents. 25.3.2 Prepare a series of standards by treating aliquots of the standard copper solution (22.2.7) in the same manner as the test solution. From the absorption of the standard solution, prepare a standard curve plotting absorption values against concentrations. From the curve, obtain the weight of copper present in the test solution. 26 Determination of lead 26.1 Principle The sample is either wet digested as described in 23.4.1 or dry-ashed and the lead is extractedas its APDC complex for a buffered acid solution, the PH of which is adjusted withammonia, into a water-immiscible ketone which is aspirated into the acetylene/air flame or an Atomic Absorption Spectrophotometer (AAS). The precision of this method is ± 0.1 ppm (mg/kg). 26.2 Reagents 26.2.1 Ammonia solution 25 per cent v/v — Dilute 25 mL 0.880 ammonia solution to 100 mL with water. 26.2.2 Sulphuric Acid — sp.gr. 1.84 26.2.3 Nitric Acid — sp. Gr. 1.42 26.2.4 Perchloric Acid — 60 per cent 26.2.5 Sodium Acetate solution, saturated — This should be ‘cleaned’ before use, i.e. to 100 mL of the saturated solution add 2 mL of 2 per cent APDC extract with successive 10 mL quantities of MIBK until the solvent layer is colourless. 26.2.6 Methyl orange solution, 0.5 per cent w/v aqueous. 26.2.7 Ammonium pyrrolidine Dithiocarbamate (APDC), a 2 per cent w/v aqueous solution.

EAS 104:2000


26.2.8 Methyl 150 Butyl Ketone (MIBK), (i.e. 4 methyl – pentan – 2 – one) 26.2.9 Stock standard solution — Dissolve 1.6000g lead nitrate in about 100 mL water in a one litre graduated flask, add about 10 mL nitric acid, cool, dilute to volume with water. 26.2.10 Working standard lead solution – Dilute 10 mL of 23.2.9 to 100 mL with water immediately before the test [1 mL = 0.1 mg pb]. 26.3 Apparatus All glass ware must be soaked in 10 per cent nitric acid, rinsed in de-ionized water and even dried. 26.3.1 Top-pan balance — weighing to ± 0.01 g 26.3.2 Kjeldahl flasks (150 mL). 26.3.3 Hot plate or appropriate heating device. 26.3.4 Glass beads. 26.3.5 Separating Funnels — 150 mL capacity, marked 100 mL. 26.3.6 Filter paper — Whatman No. 541. 26.3.7 Filter funnels. 26.3.8 Graduated Flasks — 10 mL capacity 26.3.9 Atomic Absorption Spectrophotometer (AAS) 26.4 Procedure 26.4.1 Preparation of test solution Weigh 10 g sample using a top balance and place the sample into a Kjeldahl flask containing two glass beads. Wet oxidize the sample by adding 5 mL sulphuric acid and 10 ml nitric acid and warm gently until reaction commences. Remove the flask from the heat and add further 10 mL aliquots of nitric acid and then continue heating. When the digestate is colourless or light yellow/brown colour, cool and add 10 mL water. Add 2 mL perchloric acid and boil to sulphuric fumes, cool and add 10 mL water. 26.4.2 Transfer the acid digest from wet oxidation to a separating funnel and add 10 ml sodium acetate solution. Add 5 drops of methyl orange solution only if wet oxidation method is used and titrate with ammonia solution until the indicator changes from pink to yellow. Dilute solution to 100 mL with water, and add 2 mL of the APDC solution. Mix by swirling, and allow to stand for at least 2 minutes. NOTE It is important to proceed from here with no intervals. Pipett 10 mL MIBK into the funnel and shake vigorously for 30 seconds. Allow the phases to separate (about 1 min), Reject the aqueous (lower) layer phase, remove the organic phase by pouring gently through the top of the funnel and filtering through a dry 541 filter into a 10 mL graduated flask. NOTE This must NOT be diluted to volume. Stopper the flask until the sample is aspirated into the Atomic Absorption Spectrophotometer. 26.4.3 Aspirate MIBK through AAS followed by the reagent blank, standards and samples. NOTE 1 A reagent blank must be carried through the analysis and it is important that for every 10 mL in the wet oxidation, blank is similarly treated.

EAS 104:2000


NOTE 2 The calibration curve is constructed by taking 0.5 mL and 1.0 mL of the working standard solution through the procedure commencing at 23.4.2. 27 Determination of sulphur dioxide in wines 27.1 Free sulphur dioxide 27.1.1 Ripper method 27.1.1.1 Reagents a) Iodine solution — 0.02 mole/L standardized. b) Sodium bicarbonate. c) Starch indicator solution — Titrate 5g starch and 0.01g of mercuric iodide with 5g starch water

and slowly pour it with stirring into a litre of boiling water. Boil for three minutes. Allow to cool and decant off the supernatant clear liquid.

d) Sulphuric acid — 1:3 27.1.1.2 Procedure Pipette 50 mL of wine into a 250 mL Erlenmeyer flask. Add 5 mL of sulphuric acid and a pinch of sodium bicarbonate to expel air. Rapidly titrate the free sulphurous acid using iodine solution. The end-point shall be the first darkening of solution to bluish colour persisting for 1-2 minutes. 27.1.1.3 Calculation

Sulphur dioxide mg/litre 2

1000321

MMM ×××

=

where,

M1 = mL of Iodine used, M = concentration of iodine, and M2 = mass of sample

27.2 Total sulphur dioxide 27.2.1 Distillation method 27.2.1.1 Apparatus illustrated as Figure 2 shall be well fitting; all with ground grass joint. 27.2.1.2 Reagents

a) Hydrogen peroxide – 3 per cent. b) Concentrated hydrochloric acid. c) Sodium hydroxide – 0.05 mole/L.

27.2.1.3 Procedure Circulate cold water through the condenser. Add from a graduated cylinder 20 mL of 3 per cent hydrogen peroxide to the 250 mL conical flask. Measure about 50 ml wine of the gas inlet tube and adding 300 mL of water. Replace gas inlet tube immediately, making sure all connections are well greased and again remove the gas inlet tube and slowly add 20 mL of concentrated hydrochloric acid.

EAS 104:2000


Replace the tube and see if bubbles of CO2 or nitrogen enter the receiving flasks. If not, check the joints leaks. Adjust CO2 (passed through a gas washing bottle fined with sodium carbonated solution) or nitrogen (99.9% pure) to give a flow 15 to 20 bubbles per minute through the tube. Connect to a 500 watt heater and turn to high position. In about 5 minutes, when the solution starts to boil, adjust the heater to give a slow boil. Boil for 30 minutes, wash the hydrogen peroxide solution from the trap into the conical flask. Rinse the trap with water. Add 3 drops of bromophenol blue indicator and titrate with 0.05 mole/l NaOH solution to a pale sky blue and point, using a ml microburette. (For samples with very high in SO2 use a 50 ml burette and 0.1 mole/l (NaOH). Run a blank titration on 20 mL hydrogen peroxide and correct the results accordingly. 27.2.1.4 Calculation 1 mL of 0.1 mol/L NaOH = 3.2 mg of SO2

SO2 mg/L = sample of W

1000 32 NaOH ofion Concentrat Titre 1

×××

27.2.2 Ripper Method a) Sodium hydroxide — 1 moL/L 27.2.2.1 Procedure To 20 mL of wine sample in a flask, add 25 mL of the sodium hydroxide. Cool immediately and let it stand for 10 minutes for hydrolysis to take place. Add a pinch of sodium bicarbonate, 5 ml starch indicator – and 10 Ml sulphuric acid. Titrate rapidly with standard (0,012 5 mole/L iodine to a faint blue colour. 1 mL of 0.0, 12 5 mole/L iodine is equivalent to 0.4 mg of total SO2. 27.2.2.2 Bound SO2 shall be the difference between the total sulphur dioxide and free sulphur dioxide. 28 Determination of sorbic acid 28.1 Principle 28.1.1 This analysis involves steam distillation of the free sorbic acid oxidation to malonaldehlyde using dichromate and reaction with thiobarbituric acid to form a red complex. This determined spectrophotometrically at 532 runs. 28.2 Apparatus 28.2.1 Steam distillation apparatus. 28.2.2 Volumetric flasks, 50mL, 100mL, 500mL, 1litre. 28.2.3 Boiling water bath. 28.2.4 Spectrophotometer. 28.3 Reagents 28.3.1 Magnesium Sulphate Heptahydrate. 28.3.2 Sulphuric acid solution, 0.5 m and 0.005 m. 28.3.3 Sodium hydroxide solution, 1 m Dichromate solution. 28.3.4 Mix equal volumes of 0.15 m sulphuric acid and a solution of 0.5 g potassium dichromate (K2Cr2O7) in one litre of water, prepare fresh as needed.

EAS 104:2000


28.3.5 Thiobarbituric acid (TBA) solution: dissolve 0.5 g TBA in 25 mL water + 20 ml 0.5 M sodium hydroxide, add 11 mL 1 m hydrochloric acid and dilute, to 100 mL with water. 28.4 Procedure Weigh 50g sample to a litre steam distillation flask. Add 100 magnesium sulphate (Mg SO4.7H2O) and 100 mL 0.5 m sulphuric acid. Place 10 mL 1m sodium hydroxide in the steam distillation apparatus receiver. Steam distil rapidly. NOTE Do not heat the flask containing the sample during distillation, otherwise a coloured distillate may arise. Collect about 450 mL distillate in 30 min. Cool and transfer the distillate to a 500 mL volumetric flask. Add 15 mL 0.5M sulphuric acid and make up to volume with water then mix. Pipette 2 mL of distillate into a test tube and add 2mL of freshly prepared dichromate solution. Heat in a boiling water bath (at 100ºC) for 5 min. Cool and then add 2 mL thiobarbituric acid solution and heat in a boiling water bath for 10 min. Cool rapidly and transfer to a 50 mL volumetric flask with water. Make to volume with water. Measure the absorbance of the solution at 532 mm using a 1cm cuvette, water as the reference. Prepare a standard curve as follows: Dissolve 1.0 g of sorbic acid in a small volume of 1 M NAOH and dilute to 1 litre with water. This is the stock solution (1mg/ml). Prepare a blank and four working standard solutions by first pipetting 25.0 mL of the stock solution to a 500 ML volumetric flask (50 µg/mL) and diluting to volume with water. Next, pipette 0.0, 10.0, 20.0, 50.0 and 80.0 of this solution into five 100 ml volumetric flasks and dilute to volume with water (range 0.5, 10.25 and 40 µg/ml). Pipette 2 mLof each of the working standards and blank into five test tubes and continue as in the above procedure, starting at addition of dichromate solution. Plot absorbance versus µg Sorbic acid for a standard curve. (µg Sorbic = 0, 10, 20, 50, 80 in the 2 mL aliquots). 28.5 Calculation

Sorbic acid ppm 2

500 ×=SA

where, A = µg sorbic acid corresponding to the sample absorbance, taken from the standard curve. S = Sample weight in g.

29 Wine analysis 29.1 The determination of the total acidity 29.1.1 Reagents — N/3 Sodium hydroxide (NaOH) 1% Phenolphthalein Indicator 29.1.2 Apparatus i) 25 mL pipette

ii) 950 mL burette iii) 150 mL beaker iv) Glass rod

29.1.3 Procedure Pipette 25 mL wine into a beaker. Titrate with N/3 Sodium Hydroxide to neutral, using phenolphthalein indicator. 29.1.4 Calculation 1 mL N/3 NaOH = 1 gm/L acidity calculated as tartaric acid (COOH (CHOH)2COOH).

EAS 104:2000


29.2 The determination of the volatile acid content of wines 29.2.1 Reagents (i) N/12 Sodium hydroxide (NaOH)

(ii) 1% phenolphthalein indicator 29.2.2 Apparatus Steam distillation unit including:

500 mL flask 50 mL burette 50 mL pipette 500 mL graduated flask Thermometer 0 – 100 ºC

29.2.3 Procedure Transfer 50 mL of wine to the distillation flask and team distil for approximately 30 min. Note that approximately 200 mls of the distillate is to be collected and that the residue should have an upper limit of 25 mL and a lower one of 15 mL, which is corrected by adjustment of the two burners. Heat distillated to 80ºC to remove excess of carbon dioxide and titrate with the N/12 NaOH solution, using phenolphthalein as indicator. 29.2.4 Calculation — 1 mL N/12 NaOH = 0.1 gm/L acetic acid (CH3COOH) 30 Determination of atmospheric pressure in sparkling wines 30.1 Apparatus 30.1.1 Aphrometer The apparatus enabling the excess pressure in bottles of sparkling and semi-sparkling wine is called an aphrometer. It takes the form shown to in which the bottle is stoppered (metallic top, cap, cork or plastic stopper, see Figure 4) They are graduated in pascals (Pa) (12), although it is more practical to use 105 Pa or the kilopascal (kPa) as the unit. They fall into various classes. The class of a manometer is the precision of a reading in relation to a full scale reading expressed as a percentage (e.g. 1 000 Kpa manometer class I means that the maximum pressure of 1 000 kPa can be read to within ± 10 kPa). A class I instrument is recommended for accurate measurement. Aphrometers must be checked regularly (at least once a year). 30.2 Procedure Measurements must be carried out on bottles whose temperature has been stabilized for at least 24 hours. After having pierced the cap, the cork or the sopper, the bottle must then be shaken vigorously until the pressure is constant in order to take the reading. Figure 4: Aphrometer for corks and stoppers. Expression of results The excess pressure at 20 °C (Paph 20) is expressed in pascals (Pa) or in kilopascals (kPa). It must be quoted in a form consistent with the precision of the manometer (e.g. 6,3x105 Pa or 630 kPa and not 6,33 x 105 Pa or 633 Kpa for a class I manometer with a full scale reading of 1 000 kPa).

EAS 104:2000


If the temperature at which the measurement is carried out is different from 20 °C, a correction should be made by multiplying the measured pressure by the coefficientgiven in Table 1. This relates the result to 20 °C. 30.3 Relationship between the Pressure and the Quantity of Carbon Dioxide contained in a Semi-Sparkling Wine (13) From the excess pressure at 20 °C (Paph 20), the absolute pressure at 20 °C (Pabs 20) is calculated using the formula: Pabs 20 = Patm + Paph 20 Where the Patm is the atmospheric pressure expressed in bars. The quantity of carbon dioxide contained in a wine is given by the following relationships:- in litres of CO2 per litre of wine: 0.987 × 10-5 Pabs 20 (0,86 0,01A) (1 0,00144S), - in grams of CO2 per litre of wine: 1.951 × 10-5 Pabs 20 (0,86 0,01A) (1 0,00144S), where A is the alcoholic strength of the wine at 20 °C, S is the sugar content of the wine in grams per litre.

eas 104-1999_alcoholic beverages - sampling & tests

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