a. Using acid inversion (modified Jackson & GUI is method IV) i. T h e solution to be analysed should be clarified with dry
subacetate of lead as described u n d e r 4, a a b o v e . If desired, a n d this practice is r e c o m m e n d e d for molasses solutions, the clarified filtrate is deleaded by a d d i n g pulverised a n h y d r o u s potassium oxalate a n d filtering. T w o 50 ml p o r t i o n s of the filtrate ( b o t h p o r t i o n s should be t a k e n either from the clarified or from the deleaded filtrate, n o t one p o r t i o n from the clarified a n d the o t h e r from the deleaded filtrate) a r e pipetted i n t o two 100 ml sugar flasks. To the first flask a d d 10 ml of sodium chloride solution containing 231.5 g per litre. If after addition of the sodium chloride solution there develops a cloudiness which c a n n o t be removed by filtration, clearing by the a d d i t i o n of a few d r o p s of acetic acid is often practised. M a k e up to 100 ml with distilled water a n d shake t h o r o u g h l y .
ii. To the flask containing the second 50 ml p o r t i o n a d d 20 ml water. H e a t in a water b a t h to 65°C a n d a d d immediately 10 ml hydrochloric acid = 1 . 1 0 2 9 . Mix by r o t a t i n g a n d set aside for at least 30 minutes. C o o l to r o o m t e m p e r a t u r e a n d m a k e to the m a r k with distilled water, mixing the contents by rotating j u s t before the liquid reaches the level of the neck.
Shake thoroughly.
iii. Fill two jacketed tubes with these t w o solutions. Insert an
" i n v e r s i o n " type of t h e r m o m e t e r into each t u b e , place the tubes on a r a c k a n d leave t h e m for 30 minutes in t h e sac- charimeter r o o m before polarizing. This p r o c e d u r e will avoid a possible error due to m u t a r o t a t i o n a n d will ensure a c o n s t a n t t e m p e r a t u r e which should be similar in b o t h tubes. T h e t e m p e r a t u r e of the solution at the time of t a k i n g t h e invert reading, should be recorded to the nearest 0.1 °C.
T h e t e m p e r a t u r e of t h e two solutions should n o t differ by m o r e t h a n 0.2°C. T h e reading of the first solution is t e r m e d the direct saccharimeter reading (Dr) a n d t h a t of the second solution the invert saccharimeter reading (Ir). T h e m e t h o d for calculating the percentage of sucrose in t h e solution being analysed is given in detail u n d e r M i x e d juice, chapter VIII, 5, c, iii, a n d Final molasses, chapter V I I I , 10, c, i.
Laboratory Manual for S. Afr. Sugar Factories 1962 — 49
b. Using invertase
i. T h e invertase m e t h o d is the only m e t h o d which can be depended u p o n to give correct sucrose values for cane p r o d u c t s which may contain fructose, reversion p r o d u c t s , a n d a m i n o c o m p o u n d s , provided t h a t the solution used for the direct reading a n d the inverted solution have approximately the same p H . A l t h o u g h invertase may be prepared in the labora- tory from yeast, it is recommended that the commercial p r o d u c t be purchased. In b o t h cases a check on the activity of the invertase solution is desirable.
ii. C H E C K O N A C T I V I T Y O F I N V E R T A S E S O L U T I O N : Instead of determining the k-value of the solution (which should be approximately 0.1) it is usually a d e q u a t e for practical purposes to carry out the following test:
Dilute 1 ml of the invertase p r e p a r a t i o n to 200 ml.
Transfer 10 g of p u r e sucrose (no. 1 refined) to a sugar flask graduated at 100 and 110 ml, dissolve in 75 ml of water, a d d 2 drops of glacial acetic acid, a n d dilute to the 100 ml m a r k . M a k e to the 110 ml m a r k with the diluted invertase solution a n d mix thoroughly a n d rapidly, noting the exact time at which the solutions are mixed. At the termination of exactly 60 minutes m a k e a p o r t i o n of the solution j u s t alkaline to litmus p a p e r with small a m o u n t s of a n h y d r o u s sodium carbonate a n d polarize in a 200 mm t u b e at 20°C. If the invertase solution is sufficiently active, the alkaline solution will polarize approximately 31°S without correcting for the dilution to 110 ml and the optical activity of the invertase solution.
Example: 10 grams of sucrose in 110 ml a q u e o u s solution show a reading in a 200 mm tube of 34.96°S a n d after complete inversion the reading will be — 1 1 . 2 2 ° S . If the invertase solution is the correct strength, the reading in the above test should be 31°S, corresponding to a d r o p of
(34.96—31.00) X 100 = 8.58 %
46.18
A reading lower t h a n 31°S indicates t h a t the invertase con- centrate is stronger t h a n usual. If for example the reading is 26°S instead of 31°S which corresponds to a d r o p in pol of 19.4%, the activity of the invertase is
19.40
--- = 2.26 so a correspondingly smaller volume of the 8.58
invertase solution shall be required for the analysis, as des- cribed in 5, b, iii, below a n d in chapter VIII, 5, d a n d 10, c, ii.
Laboratory Manual for S. Afr. Sugar Factories 19.40
8.58
(34.96—31.00) 46.18
X 100
= 8.58%
50 — 1962
iii. T H E D E T E R M I N A T I O N : T o determine sucrose using i n - vertase, the clarified solution 4, a, above, m u s t be deleaded with the m i n i m u m of a n h y d r o u s p o t a s s i u m oxalate a n d filtered again. Three 50 ml p o r t i o n s are pipetted into three 100 ml flasks. T h e first flask is m a d e up to the m a r k and t h o r o u g h l y mixed. T h e second p o r t i o n is used to determine the quantity of glacial acetic acid required to reduce the pH to 4.7 ± 0.2.
T h e volume of acetic acid required is noted a n d this volume is added to the third flask. T h e n a d d the requisite quantity of the invertase solution (10 ml of the invertase concentrate is adequate), a n d mix thoroughly. Place the flask in a water- b a t h a n d allow it to m a i n t a i n a t e m p e r a t u r e of 57—58°C for fifteen minutes with occasional shaking. Cool a n d add carefully with constant shaking a concentrated solution of sodium c a r b o n a t e until distinctly alkaline to a minute piece of litmus p a p e r in the flask. Dilute to the m a r k a n d mix thoroughly. Fill two jacketed tubes with these two solutions.
Insert an inversion type of t h e r m o m e t e r into each tube, place the tubes on a rack a n d leave t h e m for 30 minutes in the saccharimeter r o o m before polarizing. T h e reading of the first solution is termed the direct saccharimeter reading (Dr) a n d the second reading, which m u s t be corrected for the r o t a t i o n of the invertase present, the invert saccharimeter reading (Ir). T h e t e m p e r a t u r e of the solution at the time of taking the invert reading should be noted to the nearest 0.1°C. T h e t e m p e r a t u r e of the two solutions should n o t differ by m o r e t h a n 0.2°C.
T h e saccharimeter readings are converted into the sucrose percentage of the analysed solution as described in detail under Mixed juice, chapter VIII, 5, d, iii and Final molasses, chapter VIII, 10, c, ii.
iv. W h e n very accurate results are required it is preferable to carry out the inversion at r o o m t e m p e r a t u r e by allowing the solution, after addition of invertase, to stand overnight before taking the invert reading. A further reading is t a k e n several h o u r s later a n d if there is no change from the previous reading, the inversion is complete.
c. The " c h e m i c a l " method: Sucrose m a y be estimated chemically by the determination of reducing sugars, b o t h before a n d after in- version, either by acid or invertase. If the difference between the percentages of reducing sugars o b t a i n e d is multiplied by 0.95, the result will represent the weight of sucrose before hydrolysis. This m e t h o d of sucrose d e t e r m i n a t i o n is r e c o m m e n d e d in the case of molasses where solutions are often t o o d a r k to o b t a i n accurate optical readings (see chapter V I I I , 10, c, ii).
Laboratory Manual for S. Afr. Sugar Factories 1963 — 51