CHAPTER V METHODS OF ANALYSES
D. Grain Size Apparatus
Extraction Flask Vacuum Flask Sieve Shaker
Tyler # 14, 20 and 28 Mesh Sieves Reagents
Ethyl Ether Anhydrous Methanol Procedure
1. The flask containing the sample previously collected for grain size test is swirled vigorously for two minutes so that the sugar is well mixed with the solvent.
2. Drain the solvent from the flask. After the solvent has drained, break vacuum, shake the extraction flask and place back over the vacuum flask.
Repeat two or three times.
3. After draining, return flask to an upright position, add 50 ml. of anhydrous methanol and repeat swirling and draining procedure.
4. Repeat swirling and draining procedure twice, using a 50 ml. portion of ethyl ether time. (Caution: Do not use near open flame.)
5. Place drained sugar on absorbent filter paper and allow to air dry. No lumping or caking should occur on drying. Soft conglomerates if any, should be broken by gentle hand pressure. If lumping is observed after drying, discard the sample. Begin again, starting with the affination procedure.
6. Weigh (to ± 0.1 gm.) the entire amount of affined raw sugar which has been washed with solvent and dried.
7. Assemble the screens with a 14 mesh tyler as the top screen, followed by the tyler 20 and tyler 28 mesh screens. A pan, and additional screens if neces- sary, are added to make up a set of screens that will fit on a mechanical shaker.
8. Place the weighed amount of the 14 mesh tyler screen.
9. Place the set of screens on a mechanical shaker and run for five minutes.
10. The grain size, is reported as the percentage of sample passing through the 28 mesh tyler screen, determined as follows:
weight through 28 mesh screen x 100 = % grain size starting weight
E. Moisture
It is important for this routine determination that the drying agent in the desiccator be changed regularly and always kept in a highly active state.
Oven Method Apparatus Analytical balance Desiccator Oven
Alurninium dishes (with close fitting cover)
N.B. - Dishes should be dried at least one hour at 100°C (212°F) prior to use.
Procedure
1. Transfer the moisture dish and cover to the desiccator and cool.
2. Weigh dish with cover.
3. Weigh 5 gm of well mixed sample and transfer to dish and replace cover.
4. Immediately place in oven pre-heated to 105°C (221°F) for three hours (cover must be in oven but not covering sugar).
5. Remove dish from oven, replace cover, and transfer to desiccator at once.
6. Cool to room temperature and weigh.
Calculation
Weight of dish - = 14.3689 gm Weight of dish + sugar = 19.4387 gm Weight of sugar = 5.0698 gm Weight of dish + dried sugar = 19.4245 gm Loss of weight (19.4387 - 19.4245) = 0.0142 gm
% moisture = 0.0142 x 100 = 0.28%
5.0698
•
F.Pol Apparatus Analytical balance
100 ml Kohlrausch flask (calibrated at 27.5°C) Polariscope 200 mm tube
Whatman No. 1 18.5 cm or equivalent filter paper Reagent - Home's dry lead
Procedure
1. Weigh 26.00 gm of sugar into nickel dish (to the nearest 10 mg).
2. Wash into 100 ml Kohlrausch flask, being careful to wash all sugar from nickel dish and neck of flask.
Use just enough water to half fill the flask. (i.e. 45-50 ml).
3. Dissolve sugar completely by gently swirling flask either by hand or suitable machine.
4. Make up volume to 90 ml with distilled water, and rotate flask gently to mix contents completely.
5. Fill to mark with distilled water. Carefully remove any drops of water in the neck of the flask by absorption on filter paper.
6. Clarify by using the minimum quantity of Home's dry lead (not to exceed 1.0 gm).
7. Shake flask thoroughly end over end, allow to stand for 5 mins, shake again and pour entire contents into filter paper and funnel of suitable size. Cover filter paper with watch glass.
8. When 25-30 mls of filtrate has collected, use to rinse inside of filtrate beaker and discard.
Note: Filtrate can only be returned to the funnel if it is cloudy and if funnel is covered with a watch glass during the filtering operation.
9. After rinsing filtrate jar, collect sufficient filtrate to carry out three washings of 200 mm Polariscope tube before filling.
10. Fill 200 mm Polariscope tube and take average of five readings.
Prior to use, the polariscope tube must be tested as follows:-
Fill with distilled water and place in Polariscope trough. Reading must be the same as when tube is empty. This precaution is necessary after cleaning, changing or tightening the cover glasses.
In order to avoid changes in temperature of solution, during the reading the tube (filled with solution) must be left in the trough of polariscope for 10 mins. During this period the tubulure of the tube is covered with a small watch glass.
G. Dextran Apparatus
Oven or water-bath maintained at 55°C.
100 cm3 stoppered measuring cylinders.
Membrane filtration apparatus and 0.45 micron Millipore membranes and pre-filters.
25 cm3 volumetric flasks.
Spectrophotometer capable of operating at 720 nm wavelength, with matched pairs of 40 mm and 10 mm cells.
Reagents
—Amylase enzyme Ion exchange resins
Equal weights of Amberlite resins IR-45 (OH form) and IR-120 (H form) are mixed. Both resins should be dry, 14—52 mesh size and of Analytical grade.
The mixture is washed with at least twice its weight of distilled water and drained dry. It is then treated with acetone for no longer than 2 minutes by just covering the resin twice and gently sucking dry. Finally it is dried a+
30°C.
Trichloracetic Acid, 10 g/100 cm3. Denatured anhydrous alcohol.
Test Procedure
1. 23.5 grams of whole raw sugar are weighed and dissolved in 35 cm3 of dis
tilled water in a suitable beaker or flask.
2. 0.05 g of —amylase starch-removing enzyme is added to the solution which is incubated at 55°C. for 1 hour in an oven or water bath with agitation every 15 minutes.
3. 10 g of the mixture of ion-exchange resins are added and stirred for 30 minutes. At the end of this period the resin is removed by passing the mixture through a coarse filter, filterate and washings being collected in a 100 cm measuring cylinder.
4. The volume in the cylinder is made up to 100 cm3 with distilled water, and then 10 cm3 of trichloroacetic acid added (to remove protein). The cylinder is stoppered and shaken.
5. The contents of the cylinder are filtered through a 0.45 μm Millipore mem
brane with a pre-filter pad, using the first runnings to rinse the funnel and flask. At least 30 cm3 of filtrate are collected.
6. 12.5 cm3 of filtered solution are pipetted into each of two 25 cm3 volume
tric flasks, a "sample" flask (designated A) and a "control" flask (designated B).
7. To the sample flask A is added anhydrous alcohol dropwise from a burette while swirling the flask until the 25 cm3 mark is reached. The stoppered flask is inverted gently several times and allowed to stand at room tempera- ture for 60 ± 2 minutes while the haze develops.
8. To the control flask B distilled water is added with swirling to the 25 cm3
mark.The stoppered flask is shaken.
9. One of the matched pair of 40 mm cells is filled with distilled water and the other with the control solution. After zeroing the spectrophotometer at 720 nm with the cell containing distilled water, the absorbance of the control solution is measured (B). The cell is then emptied and cleaned.
10. At the expiration of the 60 minute period, the cell is filled with the sample solution and its absorbance measured (A) after zeroing the spectrophoto- meter against the distilled water cell.
11. If the absorbance of the sample exceeds 0.7 in value, both the sample and control solutions should be re-read in 10 mm cells without delay, after zeroing the instrument with a 10 mm cell.
5. Calculation
The results are expressed as absorbance due to dextran haze in a 50 mm light path. Thus if 40 mm cells are employed
Dextran = (A-B) x 1.25 x 1000 milli-Absorbency Units (M.A.U.) With 10 mm cell
Dextran = (A-B) x 5 1000 M.A.U.
H. INSOLUBLE MATTER