OIV-MA-AS321-01 Total bromide

Type IV method

 

1. Principle

The wine is ashed at 525 oC in presence of an excess of soda lime. A solution of the residue (at pH 4.65) is treated with chloramine T to liberate bromide. The bromide is reacted with phenolsulfonephthalein to form phenoltetra-bromophthalein-3’-3’’-disulfonic acid, which is determined by spectrophotometer at 590 nm.

2. Apparatus

2.1.   Boiling water‑bath 100C

2.2.   Temperature‑controlled electric furnace

2.3.   Spectrophotometer capable of measuring absorbance at wavelengths between 300 and 700 nm

3. Reagents

 

3.1.   Sodium hydroxide solution, 50% (m/m)

3.2.   Calcium hydroxide suspension containing 120 g of CaO per liter

3.3.   Phenolsulfonephthalein solution:

0.24 g of phenolsulfonephthalein (phenol red) are dissolved in 24 mL sodium hydroxide solution, 0.1 M, and made up to the liter with distilled water.

3.4.   pH 4.65 buffer solution:

Acetic acid, 2 M	500 mL
Sodium hydroxide, 2 M	250 mL
Distilled water to	1 L

3.5.   Oxidizing solution:

Chloramine T	2 g
Distilled water to	1 L

Prepare this solution 48 hours before use

Storage: two weeks at 4 oC

3.6.   Reducing solution:

Sodium thiosulfate	25 g/L
Distilled water to	1 L

3.9.   Potassium bromide solution corresponding to 1 g of bromide per liter.  1.489g of potassium bromide, KBr, is dissolved in distilled water and made up to one liter.

4. Procedure

 

4.1.   How to obtain ash and ash solution

Place 50 mL of wine in a silica dish of 7 cm diameter, add 0.5 mL 50% sodium hydroxide solution, (3.1), and 1 mL calcium hydroxide suspension (3.2). Check that the pH is at least pH 10. Leave the dish covered with a watch glass for 24 hours. Evaporate the liquid until dry on a boiling water bath. To accelerate the evaporation, a hot air current can be used in the final stages.

Ash as follows: place the dish 30 minutes in a furnace (2.2) at 525°C. After cooling, mix the residue with a little distilled water.  Evaporate on the boiling water-bath. Ash again at 525C. Repeat the operation until the ash is gray/white.

Mix the residue with 5 mL boiling distilled water. Add using a burette: first 10% sulfuric acid (3.7), then sufficient 1% sulfuric acid (3.8) to bring the pH to between 4 and 5 as measured by indicator paper. Let X mL = the volume added of sulfuric acid (3.7 & 3.8). Add 10.2 - (X+5) mL of distilled water. Crush the precipitated calcium sulfate with a glass rod.  Transfer the content of the dish to a centrifugation tube.  Centrifuge for 10 min.  Place 8 to 9 mL of the clear supernatant into a test tube.

4.2.   Qualitative test

This test is performed to determine if the bromide content of the wine is between 0 and 1 mg/L, which would enable the determination to be performed on the undiluted ash solution.

Place in a small test tube:

• 1 mL of ash solution
• 1 drop of pH 4.65 buffer solution
• 1 drop of phenolsulfonephthalein solution
• 1 drop of chloramine T solution

After exactly 1 minute, stop the reaction by adding 1 drop of sodium thiosulfate solution.

If the coloration obtained is yellow, brownish yellow or greenish yellow, the ash solution can be used undiluted.

If the obtained coloration is blue, purple or violet, the wine contains more than 1 mg of bromide per liter and the ash solution must be diluted 1/12 or 1/5 until the coloration obtained corresponds to the conditions above.

4.3.   Quantitative method

Place in a test tube:

• 5 mL of ash solution, diluted or undiluted, add:
• 0.25 mL of pH 4.65 buffer solution
• 0.25 mL of phenolsulfonephthalein solution
• 0.25 mL T chloramine solution

Wait exactly 1 minute and add:

• 0.25 mL of sodium thiosulfate

Measure using a spectrophotometer set at 590 nm with a 1 cm cell, the difference in absorbance between the sample and the blank obtained by adding the same quantities of reagents to 5 mL of distilled water.

Note: When the bromide content is low (yellow coloration, slightly greenish) determine the absorbance in a cell of 2 cm optical path.

4.4.   Preparation of the calibration curve

At the time of use, prepare a solution containing 10 mg of bromine per liter by making 2 successive dilutions (1/10) of standard potassium bromide solution, 1 g/L.

In a set of 8 test tubes, place 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 2.00 and 2.50 mL respectively of bromide standard, 1g/L (3.9) and make up to 5 mL with distilled water. (The solutions are equivalent to 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80 and 1 mg of bromine per liter of wine without dilution of the ash solution).  Continue as in 4.3 using the calibration solutions instead of the ash solution.  Determine the absorbance of these solutions and a blank, as in 4.3, using 5 mL of distilled water in the blank solution. The absorbance obtained corresponding to the bromide concentration is plotted on a line that curves slightly towards the origin.

5. Expression of results

5.1.   Calculations

The bromide content in wine is obtained by plotting on the calibration curve, the net absorbance of the ash solution (taking into account the thickness of the cell used and any dilution of the ash solution) and interpolating the bromide concentration. The total bromide content is expressed in milligrams per liter (mg/L) to two decimal places.

Bibliography

• DAMIENS A., Bull. Sci. Pharmacologiques, 1920, 27, 609; Ibid, 1921, 28, 37, 85 et 105.
• BALANTRE P., J. Pharm. Chem., 1936, 24, 409.
• PERRONET M., ROCQUES Mme S., Ann. Fals. Fraudes, 1952, 45, 347.
• CABANIS J.‑C., Le brome dans les vins, Thèse doct. Pharm., Montpellier, 1962.
• JAULMES P., BRUN Mme S., Cabanis J.‑C., Chim anal., 1962, 327.
• STELLA C., Riv. Viticolt. Enol., Conegliano, 1967, 5.

OIV-MA-AS321-03 Determination of fluoride content in wine using a fluoride selective ion electrode and a standard addition method

Type II method

 

1. Scope

This method is applicable to the analysis of fluoride in all wines. With proper dilution, the range of detection is 0.1 mg/l to 10.0 mg/l.

2. Principle

The concentration of fluoride in the sample is measured after addition of a buffer, using a fluoride ion selective electrode. The buffer provides a high, constant background ionic strength; complexes iron and aluminium (which would otherwise complex with fluoride); and adjusts the pH to a level that minimises the formation of a HF•HF complex. The matrix effects are then minimised using standard addition.

 

3. Reagents

 

3.1.  Deionized or distilled water

3.2.  Sodium chloride 99.0% purity

3.3.  Trisodic citrate 99.0% purity

3.4.  CDTA (1,2-diaminocyclohexane-N,N,N’,N’- tetracetic hydrate acid) 98.0% purity3.5 Sodium hydroxide to 98.0% purity

3.5.  Sodium hydroxide solution 32% (w/v) made from 3.5

3.6.  Glacial acetic acid 99.0% purity

3.7.  Sodium fluoride 99.0% purity

3.8.  Commercial Total Ionic Strength Adjustment Buffer (TISAB) (i.e. III-Orion Research Inc. Cat. # 940911) or equivalent (See 4.2).

3.9.  Alternative TISAB:

3.9.1.      To ca. 700 ml water (3.1) in a 1 l beaker (4.3), add 58.0 g ± 0.1 g sodium chloride (3.2) and 29.4 g ± 0.1 g of tri-sodium citrate (3.3).

3.9.2.      Dissolve 10.0 g ± 0.1 g of CDTA (1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid) (3.4) and 6 ml of 32% (w/v) sodium hydroxide (3.6) in approximately 50 ml of distilled water. (3.1)

3.9.3.      Mix the two solutions together then add 57 ml of glacial acetic acid (3.7) and adjust pH to 5.5 with 32% (m/v) sodium hydroxide (3.6). Cool to room temperature, transfer to 1 l volumetric flask (4.10), and dilute to volume with water (3.1).

3.10.        Fluoride standard solutions

3.10.1. Fluoride stock standard solution (100 mg/l):

Weigh 221 mg 1 mg of sodium fluoride (3.8) (dried at 105°C for 4 hours) into a 1 l polyethylene volumetric flask (4.10) and make to volume with water. (3.1)

3.10.2. Fluoride calibration standards at 1.0 mg/l, 2.0 mg/l and 5.0 mg/l : make 1.0 mg/l, 2.0 mg/l, and 5.0 mg/l calibration standards by pipetting 1 ml, 2 ml, and 5 ml of the 100 mg/l stock standard (3.11.1) into three polyethylene 100 ml volumetric flasks (4.10) respectively and diluting to volume with water (3.1).

3.11.        Wine blank : a wine known to be fluoride free is used as a matrix blank

3.12.        1 mg/l spiked wine standard  - Place 10 ml (4.11) of 100 mg/l fluoride stock standard solution(3.11.1)into a 1 l volumetric flask (4.10) and bring to volume with fluoride free wine (3.12).

4. Apparatus

 

4.1.  pH/ion analyser with standard addition capability (e.g. Corning pH/ion Analyser 455, Cat. # 475344) or pH/ion analyser with extended mV range.

4.2.  Fluoride ion selective electrode and single junction reference electrode or combination electrode (e.g., Corning Fluoride Electrode Cat. # 34108-490).

4.3.  Beakers - 150 ml, 1 l, polyethylene

4.4.  Cylinder - 50 ml graduated, polyethylene,.pouring.

4.5.  Magnetic stirrer

4.6.  Magnetic stir bars, PTFE coated.

4.7.  Plastic bottles with caps, 125 ml (Nalgene or equivalent)

4.8.  Precision pipette, 500 µl

4.9.  Ultrasonic bath

4.10.        Volumetric flasks, Class A, 50 ml, 100 ml, and 1 l

4.11.        Volumetric pipettes, Class A, 1 ml, 2ml, 5 ml, 10 ml, 20 ml, and 25 ml

5. Preparation of calibration standards

 

5.1.  Place 25 ml (4.11) of 1.0 1.0 mg/l, 2.0 mg/l, and 5.0 mg/l standard solutions (3.11.2) respectively into three 150 ml beakers (4.3), add 20 ml (4.11) of water (3.1) and (4.11) 5 ml of commercial TISAB (3.9) to each. Mix with a magnetic stirring. (4.5 and 4.6).

5.2.  If using alternative TISAB reagent (3.10) : place 25 ml (4.11) of each standard solution (3.11.2) into three 150 ml beakers (4.3) and add 25 ml (4.11) of alternative TISAB reagent (3.10) to each. Mix with a magnetic stirrer. (4.5 and 4.6)

6. Preparation of the test samples

 

Mix the wine sample thoroughly before sampling. Sparkling wines should be degassed before sampling by transferring to a clean beaker and placing in an ultrasonic bath (4.9) until gas no longer evolves.

6.1.  If using reagent (3.9), commercial TISAB : place 25 ml (4.11) of wine sample into a 150 ml beaker (4.3) with 20 ml (4.11) of water (3.1) and add 5 ml (4.11) of ommercial TISAB (3.9) solution. Mix with a magnetic stirrer (4.5 and 4.6). Dilution factor (DF) = 1.

6.2.  If using alternative TISAB reagent (3.10) : place 25 ml (4.11) of wine sample  in a 150 ml beaker (4.3) and add 25 ml (4.11) of alternative TISAB reagent (3.10). Mix with a magnetic stirrer (4.5 and 4.6). Dilution factor (DF) = 1.

 

7. Procedure

 

Measurement (all standard and wine sample solutions must be at the same temperature).

7.1.  Calibration standards

Measure the potential of each of the calibration solutions, using the meter (4.1), fluoride selective electrode (4.2), and reference electrode (4.2). The final reading must be taken when the readings have stabilised (stability is obtained when the potential varies by not more than 0.2 to 0.3 mV/ 3 minutes). Record the readings for each of the calibration standards.

The of each of the standard concentrations versus the millivolt reading measured for each standard concentration is plotted on graph paper in order to determine the slope of the electrode.

7.2.  Wine samples

Measure and record the potential expressed in mV (E1) of the sample (6.1 or 6.2) after the readings have stabilised. Add 500 µl (4.8) of 100 mg/l fluoride standard (3.11.1) to the sample (6.1 or 6.2). After the readings have stabilised, read and record the  potential expressed in mV (E2) of the wine solution.

The final concentration must be at least double the fluoride concentration in the sample solution. To make sure, if the fluoride concentration in the test sample is above 2 mg/l on the first determination, a second determination must be made after dilution of the sample as follows (7.2.1 or 7.2.2).

7.2.1.      When using the commercial TISAB buffer (3.9): pipette (4.11) 25 ml of wine sample in a 50 ml volumetric flask (4.10) and bring to volume with water. Take 25 ml (4.11) of this diluted wine in a 150 ml cylindrical beaker (4.3) and add 25 ml of commercial TISAB (3.9). Mix with a magnetic stirrer (4.5 and 4.6) and then proceed with measurement as in 7.02. Dilution factor (DF) = 2.

7.2.2.      When using the alternative TISAB buffer (3.9): pipette (4.11) 25 ml of wine sample in a 50 ml volumetric flask (4.10) and bring to volume with water.  Pour 25 ml (4.11) of this diluted wine in a 150 ml cylindrical beaker (4.3) and add 25 ml of alternative TISAB buffer (3.10). Mix with a magnetic stirrer (4.5 and 4.6) and then proceed with measurement as in 7.2. Dilution factor: (DF) = 2.

8. Calculation

The fluoride content of the sample solution expressed in mg/l is obtained by using the following formula:

If the added standard solution V _std is ‹ 1%  of the volume of the solution after the addition, so =   and

  = fluoride concentration of the sample solution (mg/l)

DF = dilution factor. If it is necessary to dilute the sample as in (7.2.1) or in (7.2.2), use the identical values for the dilution and the sample. That is to say, DF = 2 for a diluted sample (7.2.1) and (7.2.2) or DF = 1 if it is not as in (6.1) or (6.2)

= initial volume of the sample solution before standard addition (ml)

= volume of the solution after standard addition (ml)

= difference between potentials E1 and E2 obtained in (7.2) in mV.

S = slope of the calibration curve of the electrode.

where

₌ concentration (in mg/l) of fluoride added to the sample volume ( ) obtained by multiplying  the standard volume (3.11.1) added to the solution  (V_std) by the concentration () of standard (3.11.1) and divided by the sample volume (25 ml) using (6.1) or (6.2)

= volume added standard (3.11.1) (0.5 ml)

= sample volume used in (6.1) or (6.2), = 25 ml

= standard concentration (3.11.1)

Calculation example:

 

(1) for a sample prepared as in (6.2) and measured as in (7.2)

DF = 1

= 19.6 mV

S = -58.342

=  1.71 mg / l of fluoride

(2) for a sample prepared as in (7.2.2), and measured as in (7.2)

DF = 2

 

= 20.4 mV

S = -55.937

 

= 2 x  2 mg / l x  0.760 = 3.04 mg / l of fluoride

 

9. Precision

 

The details of inter laboratory study are given in Annex B. the Horrat () ranges from 0.30 to 0.97 and indicates a very good reproducibility among participants.

The results of the statistical calculations are given in Annex B table 2.

The standard deviation of repeatability () ranges from 1.94% to 4.88%. The standard deviation of reproducibility () ranges from 4.15% to 18.40%. Average % recovery ranged between 99.8% and 100.3% of the mean target.

10. Quality assurance and management

10.1.        Analyse a standard solution from 1.0 mg/l (3.11.2) at the beginning and end of each series of measurement. The results must be 1.0 0.1 mg/l.

10.2.        Before each measurement series analyse a blank sample (3.12) and for the  internal quality control (CQI) a overloaded wine (3.13). The blank sample must not be over 0.0 mg/l 0.1 mg/l. and the CQI must not be over 1.0 mg/l 0.2 mg/l.

 

Annex A

 

References

• AOAC International, AOAC Official Methods Program, Associate Referee’s Manual On Development, Study, Review, and Approval Process,1997
• Postel, W.; Prasch, E., Wein-Wissenschoft, (1975) 30 (6), 320-326
• Office International de la Vigne et du Vin, Compendium of International Methods of Wine Analysis, 255–257
• Gil Armentia, J. M.; Arranz, J. F.; Barrio, R. J.; Arranz, A., Anales de Bromatologia, (1988) 40 (1) 71-77
• Gran, G; Analyst (1952) 77, 661
• Corning fluoride ion selective electrode – Instruction Manual, 1994
• Corning Instruction Manual pH/ion analyzer 455, 109121-1 Rev. A, 11/96
• Horwitz,W.; Albert, R.; Journal of the Association of Official Analytical Chemists, (1991) 74 (5) 718

 

Annex B Inter laboratory Study

 

Validation of a fluoride ion selective electrode, standard addition method for the measurement of fluoride in wine

 

B.1 Introduction

 

The validation by collaborative trial of a fluoride selective ion electrode, standard addition method for the determination of fluoride in wine is described. The collaborative trial involved a total of twelve participants, six European and six Americans, who took part in the study. The collaborative study was performed using the AOAC, Youden protocol⁽¹⁾.

B2 Participants

The twelve participants of this validation consisted of laboratories from Austria, France, Germany, Spain, and the United States and comprised of the following: BATF Alcohol and Tobacco Laboratory—Alcohol Section, SF, Walnut Creek, CA., United States; BATF, National Laboratory Ctr., Rockville, MD, United States; Bundesinstitut für Gesundheitlichen Verbraucherschutz, Berlin, Germany; Canandaigua Winery, Madera, CA, United States; CIVC, Epernay, France; E. & J. Gallo Winery-Analytical Services Laboratory, Modesto, CA, United States; E. & J. Gallo Winery-Technical Analytical Services Laboratory, Modesto, CA, United States; ETS Labs, St. Helena, CA, United States; Höhere Bundeslehranstalt & Bundesamt für Wein und Obstbau, Klosterneuburg, Austria; Institut Catala de la Vinya i el Vi, Vilafranca del Penedes (Barcelona),Spain; Laboratorio Arbitral Agroalimentario, Madrid, Spain; and Sutter Home Winery, St. Helena, CA., United States.

B3 Samples used in the trial

 

The samples used in the trial are given in Appendix I. They were distributed as twelve wine samples (six Youden pairs of samples comprised of three red wines and three white wines).

Sample - Sample description

 

1 White wine with no fortification (total of 0.6 mg/l F^-)

2 White wine fortified with 0.3 mg /l (total of 0.9 mg/l F^-)

3 White wine fortified with 0.9 mg /l (total de 1,5 mg/l F^-)

4 White wine fortified with 1.2 mg /l (total de 1,8 mg/l F^-)

5 White wine fortified with 1.4 mg /l (total de 2,0 mg/l F^-)

6 White wine fortified with 1.7 mg /l (total de 2,3 mg/l F^-)

7 Red wine with no fortification (total de 0,2 mg/l F^-)

8 Red wine fortified with 0.3 mg /l (total de 0,5 mg/l F^-)

9 Red wine fortified with 0.8 mg /l (total de 1,0 mg/l F^-)

10 Red wine fortified with 1.1 mg /l (total de 1,3 mg/l F^-)

11 Red wine fortified with 2.5 mg /l (total de 2,7 mg/l F^-)

12 Red wine fortified with 2.8 mg /l (total de 3,0 mg/l F^-)

 

B.4 Results

A summary of the results obtained by the twelve participants is given in Table I. None of the laboratories reported any difficulties with the analysis. One Youden pair from one laboratory was determined to be an outlier, using the Cochran’s test. These results are noted^(c) in Table I, and were not used in the statistical analysis.

 

Table 1

Collaborative data for the determination of fluoride in wine by fluoride selective electrode, standard addition^[a]

Lab	Pair 1[b]		White Wine Pair 2b			Pair 3b		Pair 4b		Red Wine Pair 5b			Pair 6b
Number	1	2		3	4	5	6	7	8	9	10	11		12
1	0.55	0.80		1.33	1.56	1.86	2.24	0.19	0.45	0.89	1.17	2.54		2.77
2	0.52	0.81		1.39	1.64	1.86	2.31	0.19	0.46	0.92	1.20	2.58		2.77
3	0.52	0.81		1.40	1.70	1.92	2.25	0.14	0.42	0.96	1.22	2.64		2.95
4	0.62	0.98		1.48	1.64	1.85	2.14	0.28	0.56	1.00	1.32	2.64		2.72
5	0.48	0.78		1.34	1.64	1.84	2.11	0.12	0.39	0.88	1.16	2.56		2.82
6	0.53	0.84		1.45	1.74	1.97	2.30	0.13	0.43	0.92	1.21	2.66		2.93
7	0.53	0.76		1.27	1.64	1.89	2.06	0.14	0.40	0.88	1.12	2.44		2.83
8	0.57	0.88		1.51	1.85	2.11	2.33	0.48[c]	0.48[c]	1.01	1.32	2.64		3.08
9	0.51	0.81		1.40	1.71	1.90	2.20	0.13	0.42	0.90	1.19	2.60		2.86
10	0.54	0.84		1.43	1.71	1.93	2.22	0.18	0.44	0.96	1.23	2.66		2.87
11	0.60	0.93		1.48	1.75	1.98	2.32	0.25	0.57	1.06	1.31	2.68		2.82
12	0.65	0.94		1.54	1.79	2.05	2.32	0.21	0.52	1.03	1.24	2.81		3.07
N of cases	12	12		12	12	12	12	11	11	12	12	12		12
Minimum	0.48	0.76		1.27	1.56	1.84	2.06	0.12	0.39	0.88	1.12	2.44		2.72
Maximum	0.65	0.98		1.54	1.85	2.11	2.33	0.28	0.57	1.06	1.32	2.81		3.08
Range	0.17	0.22		0.27	0.29	0.27	0.27	0.16	0.18	0.18	0.20	0.37		0.36
Mean	0.55	0.85		1.42	1.70	1.93	2.23	0.18	0.46	0.95	1.22	2.62		2.87
Median	0.54	0.83		1.42	1.71	1.91	2.25	0.18	0.44	0.94	1.22	2.64		2.85
Std Dev	0.050	0.069		0.079	0.079	0.084	0.091	0.052	0.063	0.061	0.065	0.090		0.114

Table 2

 

Statistical data from the collaborative study on the analysis of fluoride in wine by fluoride selective ion electrode, standard addition method

STATISTIC	Pair 1	White Wine   Pair 2	Pair 3	Pair 4	Red Wine   Pair 5	Pair 6
Total # of Labs	12	12	12	11[d]	12	12
Number of "replicates” per lab	2	2	2	2	2	2
Mean (split levels)	0.55 0.85	1.42 1.70	1.93 2.23	0.18 0.46	0.95 1.22	2.62 2.87
Repeatability variance	0.0006	0.0015	0.0026	0.0002	0.0005	0.0049
Repeatability Standard Deviation	0.0235	0.0382	0.5106	0.0156	0.0211	0.0703
Relative standard deviation RSDr, repeatability	3.35 %	2.45 %	2.45 %	4.88 %	1.94 %	2.55 %
Reproducibility variance	0.0039	0.0070	0.0089	0.0034	0.0042	0.0130
Reproducibility standard deviation	0.0625	0.0835	0.0945	0.0587	0.0647	0.1141
Relative standard deviation RSDR, reproducibility	8.92 %	5.36 %	4.54 %	18.39 %	5.95 %	4.15 %
Horwitz Equation Applied (as RSDR)	16.88	14.97	14.33	19.00	15.80	13.74
HORRAT Value HoR (RSDR (measured)/RSDR (Horwitz))	0.53	0.36	0.32	0.97	0.38	0.30
Average % recovery	93.1	94.6	96.7	91.0	94.4	96.4

^[a] Units are mg fluoride/L. 

^[b] Youden pairs

^[c] Value was deleted from data set by Cochran’s Test and was not included in the statistical analysis

^[c] 

^[d] One lab pair was deleted from data set by Cochran’s Test

OIV-MA-AS321-04 Total phosporus

Type IV method

 

1. Principle

After nitric oxidation and ashing, and dissolution in hydrochloric acid, phosphoric acid is determined colorimetrically as the yellow phospho-vanadomolybdate complex.

2. Apparatus

2.1.  Boiling water‑bath 100C

2.2.  Hot plate

2.3.  Temperature‑controlled electric furnace.

2.4.  Spectrophotometer measuring absorbance at wavelengths between 300 and 700 nm

3. Reagents

Solution A: dissolve 40 g of ammonium molybdate, , in 400 mL water.

Solution B: dissolve 1 g of ammonium vanadate, NH₄VO₃, in 300 mL water and 200 mL nitric acid (ρ₂₀ = 1.39g/L) (3.1). Leave to cool.

Vanadomolybdate reagent: place first solution B then solution A into a 1 liter flask, and make up to the mark with water. Reagent to be used within 8 days of preparation.

3.4.  solution, 0.1 g/L.

Prepare a solution 1 g/L by dissolving 2.454 g of di-potassium hydrogen phosphate, , in a liter of water. Dilute 10% (v/v).

4. Procedure

4.1.  Ashing

Place 5 mL^[*] wine or must in a platinum or silica dish and evaporate on a boiling water‑bath (2.1). When the residue is nearly dry add 1 mL nitric acid (3.1), place the dish on a hot plate (2.2) for 1 hour then in a furnace (2.3) at 600‑650 oC until the ash is white.

4.2.  Determination

Add 5 mL of hydrochloric acid, approximately 3 M (3.2) to the ash and transfer the solution to a 100 mL volumetric flask.  Rinse the dish with 50 mL distilled water and pour the washings into the flask. Add exactly 25 mL of vanadomolybdate reagent, stir and leave for 15 to 20 min to allow the color to develop. Determine the absorbance at 400 nm.

Simultaneously, prepare standard solutions. Place in five 100 mL volumetric flasks, 5, 10, 15, 20 and 25 mL respectively of solution, 0.1 g/L (3.4). Make up to 50 mL with distilled water and add 25 mL vanadomolybdate reagent. Leave for the exact same time as the samples, to allow the color to develop. Make up to the mark with water and measure the absorbance at 400 nm.

In order to remain in the best absorbance zone do not reset to zero with distilled water, but set the deviation of the spectrophotometer galvanometer on a given absorbance for a determined concentration.

5. Expression of results

5.1.  Calculation

The total phosphorous content expressed in milligrams per liter of phosphoric anhydride, , is obtained by entering the absorbance of the wine sample on the calibration graph and interpolating the total phosphorus concentration.

The total phosphorous content is expressed in milligrams per liter to the nearest whole number.

Bibliography

• A.F.N.O.R., Norme U, 42‑246, Tour Europe, Paris.
• Sudraud P., Bull. O.I.V., 1969, 462‑463, 933.

^[*] A 5 mL sample volume is suitable for P₂O₅ content, of between 100 and 500 mg/L.  Outside these concentration limits, increase or decrease the sample volume.