13 



The plant material is extracted in a large metal Soxhlet ex- 

 tractor for 18 hours. The extract is then evaporated in vacuo 

 (700 to 740 mm.) to a small volume and made up to a definite volume, 

 e.g. 500 c.c. Of this 2 portions of 20 c.c. each are evaporated to dry- 

 ness and dried in vacuo for 18 hours at 100°C. This gives the 

 total dry matter in the extract. 440 c.c. are treated with the requisite 

 volume of basic lead acetate solution, filtered under pressure on a 

 Buchner funnel, washed and made up to a known volume, 2 litres. 

 This is called Solution A. 



300 c.c. of solution A are deleaded by means of solid Na 2 COg 

 and made up to 500 c.c. This is called Solution B. 



(1) 25 c.c. of B are used for direct reduction and polarised; * 

 the reduction is due to dextrose, laevulose, maltose, pentoses. 



(2) For Cane Sugar. Invert 50 c.c. of B : 



(a) By invertase. Make neutral to methyl orange by a 

 few drops of concentrated sulphuric acid, and add 1 — 2 c.c. auto- 

 lysed yeast and two or three drops of toluene and leave 24 

 hours at 38 — 40°C. After this period, add 5 to 10 c.c. alumina 

 cream, filter and wash to 100 c.c. Take the reducing power 

 of 50 c.c. ( = 25 c.c. B) and polarise. 



(b) By 10 per cent, citric acid. Make faintly acid to methyl 

 orange by a few drops of concentrated sulphuric acid and add 

 a weighed quantity of citric acid crystals so as to have 10 per 

 cent, of the crystalline acid (C 6 H 8 7 + H 2 0) present. Boil 

 10 minutes, cool, neutralise (to phenolphthalein) with sodium 

 hydroxide, make to 100 c.c. and determine reducing power of 

 50 c.c. ( = 25 c.c. B) ; polarise. 



Cane SUGAR is calculated from the increase of reducing 

 power or change of rotation caused by inversion. The values 

 obtained by the two methods a and b should agree closely. 



(3) For Maltose. Another 300 c.c. of Solution A is deleaded 

 by means of hydrogen sulphide and filtered, the precipitated sulphide 

 being washed until the total volume of filtrate and washings is about 

 450 c.c. Air is then sucked through this for about l\ hours to expel 

 hydrogen sulphide, a very little ferric hydroxide is added to remove 

 the last traces of the latter, and the solution is made to 500 c.c. It 

 is filtered and 



50 c.c. fermented (a) with S. marxianus 

 ,, ,, {b) ,, S. anomalus 

 (c) „ S. exiguus 

 and two lots d and e of 50 c.c. are fermented with baker's yeast. 

 It is generally necessary in order to ensure good growth of the yeast 

 to reduce the acidity by adding 2 to 5 c.c. of N -sodium carbonate to 

 the 50 c.c. to be fermented ; 5 c.c. of sterilised yeast water is also 

 added, the mixture is sterilised in the usual way and inoculated in 

 the inoculating chamber with the pure culture of yeast. It is then 

 stoppered with cotton wool and the yeast allowed to incubate for 21 

 to 28 days at 25°. __J 



* The polarisation of these dilute solutions is usually small and it is therefore 

 necessary to take the reading with a long tube (at least 200 mm. in length) with 

 an instrument reading accurately to jfo°, trj e temperature being maintained con- 

 stant at 20° C within 1 J ?T . It is an easy matter, using a Lowry thermo-regulator 

 and circulating the water by means of a small pump, to keep the temperature 

 constant to y^ but differences of temperature less than -j^° hardly make a per- 

 ceptible difference in the readings with such dilute solutions as these. 



