Annex White book.pdf

ml of water and adding 20 ml of ammonia at 30%. The second dissolventis prepared by dissolving 0,4 g of potassium chloride in 50 ml of tertiarybutanol, 12 ml of propionate acid and 38 ml of water. We place 10μl of the sample extract and 10 μl of the reference solution on celluloseplates. We let them develop for approximately 45 minutes with the dissolvent1 and almost 8 hours with the dissolvent 2.Possible existing dye substances are detected by comparing the Rf of thedye substances of the witnessing solution to those of the sample extract.A4.3.2. High pressure liquid chromatography (HPLC)Since revision of the ISO 3632:1993 has begun, the efforts were focusedon creating a method that would make the detection of artificialwater-soluble acid dye substances possible. During the entire time, therewere differences between the Spanish and French standardization organizationsregarding the issue of extraction and chromatography. Theactual ISO regarding saffron contains a Technical Specification equivalent,given the fact that no agreement was ever achieved regarding theabove method, during the ISO TC34/SC7 committee meeting in Toledo(Spain) in 2002. Recently, during a second meeting that took place inthe city of Kalocsa (Hungary) between September and October 2005,an agreement was reached between Spain, France and Iran regardingthe consensus of a HPLC method based on the initial Spanish propositioncombined with an adaptation of the initial French proposition. Themethod was recently approved by the ISO authorities and the comparativeprocedure has already begun by a Spanish laboratory.The above two methods are set out here. The first applies as technicalspecification ISO/TS 3632:2003 while the second will be includedin the new ISO3632 publication.A4.3.2.1. Valid technique ISO/TS 3632:2003According to ISO 3632-2 2003, high pressure liquid chromatographyenables a quantitative as well as qualitative detection of artificialwater-soluble acid dye substances. The dye substances identified are:cynoline yellow, S – naphtol yellow, tartrazine, amaranth, A – cochinealred, azorubine, orange II, erythrocine and rocceline. The detection procedureis as follows: we place a 500 mg of sample in a centrifugation tubeand add 10 ml of water at 60 o C. The solution rests for 10 to 12 minutes.Agitation and centrifugation follows. Afterwards we add 250 μl of formicacid or 2 μl of acetic acid. The sample is then placed in a solid phase extraction(SPE) tube with polyamide 6 as a filling material. The sample iswashed successively with water, methanol, acetone until the dissolventcomes colorless out of the column. After washing with water, the pH value162