Method 1
The following is a general procedure for all reducing sugars and may be used to determine the starch content in botanical articles.
Malt Extract
Use clean new barley malt of known efficacy, and grind just before use. Prepare malt extract just prior to use. For every 80 mL of malt extract needed, digest 5 g of ground malt with 100 mL of water at room temperature for 2 hours. [noteIf an electric mixer is used, stir the mixture for 20 minutes.] Filter to obtain a clear extract, filtering again, if necessary, and mix the infusion well.
Test Solution
Extract about 5 g of the finely ground test specimen with five 10-mL portions of ether, using a filter that will completely retain the smallest starch granule. Allow the ether to evaporate from the residue, and wash with 250 mL of aqueous alcohol solution (10 in 100). Carefully wash the residue from the paper into a 500-mL beaker with about 100 mL of water. Heat to about 60
(avoiding, if possible, gelatinizing starch), and allow to stand for about 1 hour, stirring frequently to effect complete solution of sugars. Transfer to a wide-mouth bottle, rinse the beaker with a little warm water, and cool. Add an equal volume of alcohol, mix, and allow to stand for not less than 1 hour.
Centrifuge until the precipitate is closely packed on the bottom of the bottle, and decant the supernatant. Wash the precipitate with successive 50-mL portions of alcohol solution (50 in 100) by centrifuging and decanting through a suitable filter until the washings are sugar-free. [noteTo test for the presence of sugar, transfer a few drops of the washings to a test tube, add 3 or 4 drops of a 20% solution of 1-naphthol in alcohol, prepared by dissolving 200 mg of 1-naphthol in 1 mL of alcohol and 2 mL of water. Shake the test tube well to allow uniform mixing, allow 2 to 4 mL of sulfuric acid to flow down the sides of the test tube, and hold the test tube upright. If sugar is present, the interface of the two liquids is colored faint to deep violet, and on shaking, the whole solution becomes blue-violet.]
Transfer the residue from the bottle and hardened filter to a beaker with about 50 mL of water. Immerse the beaker in boiling water, and stir constantly for 15 minutes or until all of the starch is gelatinized. Cool the beaker to 55
, add 20 mL of
Malt Extract, and hold at this temperature for 1 hour. Heat again to boiling for a few minutes, cool to 55
, add 20 mL of
Malt Extract, and hold at this temperature for 1 hour or until the residue when treated with
iodine TS shows no blue tinge upon microscopic examination. Cool, dilute with water to 250 mL, and filter.
General Procedure
Transfer 200 mL of the
Test Solution to a flask fitted with a reflux condenser, add 20 mL of hydrochloric acid, and heat in a boiling water bath for 2½ hours. Cool, nearly neutralize with
sodium hydroxide TS, complete neutralization with
sodium carbonate TS, dilute with water to 500 mL, mix, and filter. The volume of aliquot taken depends on the starch content of the specimen under test (see
Table 1). The aliquot should contain between 100 and 200 mg of dextrose. Transfer 50 mL of the filtrate to a 400-mL alkali-resistant glass beaker, add 50 mL of alkaline cupric tartarate TS, cover the beaker with a water glass, and heat. Adjust the flame in the burner so that the contents of the flask begin to boil in 4 minutes and continue boiling for exactly 2 minutes. Filter the hot solution at once through a sintered-glass filter. Wash the precipitate of cuprous oxide thoroughly with water at about 60
, then with 10 mL of alcohol, and finally with 10 mL of ether.
Table 1. Determination of the Optimum Aliquot
% of Expected Starch Content |
Aliquot in mL |
60 |
25 |
50 |
35 |
40 |
50 |
30 |
50 |
20 |
50 |
For solutions of reducing sugars of comparatively high purity, proceed as directed under Method 1A to determine the amount of reduced copper obtained by weighing the dried cuprous oxide. For solutions of reducing sugars containing large amounts of organic impurities, including sucrose, proceed as directed under Method 1B to determine the amount of reduced copper obtained by titration with sodium thiosulfate.
method 1a
Dry the precipitate obtained under
General Procedure for 30 minutes in an oven at 110 ± 2
, cool to room temperature in a desiccator, and weigh. Refer to
Table 2 to find the quantity of dextrose, in mg, corresponding to the weight of cuprous oxide found. Determine the percentage of dextrose and then the content of starch by the following formula:
Percentage of dextrose = |
|
wt. of dextrose in mg × 0.1 × 500 |
|
wt. of sample in g × aliquot in mL |
Content of starch = % dextrose × 0.9. |
Table 2. Calculating Dextrose (Applicable when Cu2O is weighed directly) (Expressed in mg)
Cuprous Oxide (Cu2O) |
Dextrose (d-Glucose) |
Cuprous Oxide (Cu2O) |
Dextrose (d-Glucose) |
Cuprous Oxide (Cu2O) |
Dextrose (d-Glucose) |
Cuprous Oxide (Cu2O) |
Dextrose (d-Glucose) |
Cuprous Oxide (Cu2O) |
Dextrose (d-Glucose) |
Cuprous Oxide (Cu2O) |
Dextrose (d-Glucose) |
10 |
4.0 |
90 |
38.9 |
170 |
75.1 |
250 |
112.8 |
330 |
152.2 |
410 |
193.7 |
12 |
4.9 |
92 |
39.8 |
172 |
76.0 |
252 |
113.7 |
332 |
153.2 |
412 |
194.7 |
14 |
5.7 |
94 |
40.6 |
174 |
76.9 |
254 |
114.7 |
334 |
154.2 |
414 |
195.8 |
16 |
6.6 |
96 |
41.5 |
176 |
77.8 |
256 |
115.7 |
336 |
155.2 |
416 |
196.8 |
18 |
7.5 |
98 |
42.4 |
178 |
78.8 |
258 |
116.6 |
338 |
156.3 |
418 |
197.9 |
|
|
|
|
|
|
|
|
|
|
|
|
20 |
8.3 |
100 |
43.3 |
180 |
79.7 |
260 |
117.6 |
340 |
157.3 |
420 |
199.0 |
22 |
9.2 |
102 |
44.2 |
182 |
80.6 |
262 |
118.6 |
342 |
158.3 |
422 |
200.1 |
24 |
10.0 |
104 |
45.1 |
184 |
81.5 |
264 |
119.5 |
344 |
159.3 |
424 |
201.1 |
26 |
10.9 |
106 |
46.0 |
186 |
82.5 |
266 |
120.5 |
346 |
160.3 |
426 |
202.2 |
28 |
11.8 |
108 |
46.9 |
188 |
83.4 |
268 |
121.5 |
348 |
161.4 |
428 |
203.3 |
|
|
|
|
|
|
|
|
|
|
|
|
30 |
12.6 |
110 |
47.8 |
190 |
84.3 |
270 |
122.5 |
350 |
162.4 |
430 |
204.4 |
32 |
13.5 |
112 |
48.7 |
192 |
85.3 |
272 |
123.4 |
352 |
163.4 |
432 |
205.5 |
34 |
14.3 |
114 |
49.6 |
194 |
86.2 |
274 |
124.4 |
354 |
164.4 |
434 |
206.5 |
36 |
15.2 |
116 |
50.5 |
196 |
87.1 |
276 |
125.4 |
356 |
165.4 |
436 |
207.6 |
38 |
16.1 |
118 |
51.4 |
198 |
88.1 |
278 |
126.4 |
358 |
166.5 |
438 |
208.7 |
|
|
|
|
|
|
|
|
|
|
|
|
40 |
16.9 |
120 |
52.3 |
200 |
89.0 |
280 |
127.3 |
360 |
167.5 |
440 |
209.8 |
42 |
17.8 |
122 |
53.2 |
202 |
89.9 |
282 |
128.3 |
362 |
168.5 |
442 |
210.9 |
44 |
18.7 |
124 |
54.1 |
204 |
90.9 |
284 |
129.3 |
364 |
169.6 |
444 |
212.0 |
46 |
19.6 |
126 |
55.0 |
206 |
91.8 |
286 |
130.3 |
366 |
170.6 |
446 |
213.1 |
48 |
20.4 |
128 |
55.9 |
208 |
92.8 |
288 |
131.3 |
368 |
171.6 |
448 |
214.1 |
|
|
|
|
|
|
|
|
|
|
|
|
50 |
21.3 |
130 |
56.8 |
210 |
93.7 |
290 |
132.3 |
370 |
172.7 |
450 |
215.2 |
52 |
22.2 |
132 |
57.7 |
212 |
94.6 |
292 |
133.2 |
372 |
173.7 |
452 |
216.3 |
54 |
23.0 |
134 |
58.6 |
214 |
95.6 |
294 |
134.2 |
374 |
174.7 |
454 |
217.4 |
56 |
23.9 |
136 |
59.5 |
216 |
96.5 |
296 |
135.2 |
376 |
175.8 |
456 |
218.5 |
58 |
24.8 |
138 |
60.4 |
218 |
97.5 |
298 |
136.2 |
378 |
176.8 |
458 |
219.6 |
|
|
|
|
|
|
|
|
|
|
|
|
60 |
25.6 |
140 |
61.3 |
220 |
98.4 |
300 |
137.2 |
380 |
177.9 |
460 |
220.7 |
62 |
26.5 |
142 |
62.2 |
222 |
99.4 |
302 |
138.2 |
382 |
178.9 |
462 |
221.8 |
64 |
27.4 |
144 |
63.1 |
224 |
100.3 |
304 |
139.2 |
384 |
180.0 |
464 |
222.9 |
66 |
28.3 |
146 |
64.0 |
226 |
101.3 |
306 |
140.2 |
386 |
181.0 |
466 |
224.0 |
68 |
29.2 |
148 |
65.0 |
228 |
102.2 |
308 |
141.2 |
388 |
182.0 |
468 |
225.1 |
|
|
|
|
|
|
|
|
|
|
|
|
70 |
30.0 |
150 |
65.9 |
230 |
103.2 |
310 |
142.2 |
390 |
183.1 |
470 |
226.2 |
72 |
30.9 |
152 |
66.8 |
232 |
104.1 |
312 |
143.2 |
392 |
184.1 |
472 |
227.4 |
74 |
31.8 |
154 |
67.7 |
234 |
105.1 |
314 |
144.2 |
394 |
185.2 |
474 |
228.3 |
76 |
32.7 |
156 |
68.6 |
236 |
106.0 |
316 |
145.2 |
396 |
186.2 |
476 |
229.6 |
78 |
33.6 |
158 |
69.5 |
238 |
107.0 |
318 |
146.2 |
398 |
187.3 |
478 |
230.7 |
|
|
|
|
|
|
|
|
|
|
|
|
80 |
34.4 |
160 |
70.4 |
240 |
108.0 |
320 |
147.2 |
400 |
188.4 |
480 |
231.8 |
82 |
35.3 |
162 |
71.4 |
242 |
108.9 |
322 |
148.2 |
402 |
189.4 |
482 |
232.9 |
84 |
36.2 |
164 |
72.3 |
244 |
109.9 |
324 |
149.2 |
404 |
190.5 |
484 |
234.1 |
86 |
37.1 |
166 |
73.2 |
246 |
110.8 |
326 |
150.2 |
406 |
191.5 |
486 |
235.2 |
88 |
38.0 |
168 |
74.1 |
248 |
111.8 |
328 |
151.2 |
408 |
192.6 |
488 |
236.3 |
method 1b
Sodium Thiosulfate Solution
Transfer 3.9 g of sodium thiosulfate, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Potassium Iodide Solution
Dissolve 42 g of potassium iodide in 100 mL of water.
Sodium Acetate Solution
Dissolve 5.74 g of sodium acetate in 10 mL of water.
Copper Solution
Transfer about 0.3 g of pure electrolytic copper, accurately weighed, to a 250-mL flask, add 5 mL of nitric acid to dissolve the copper, add about 25 mL of water, and boil to expel red fumes. Add about 5 mL of bromine TS, and boil until the bromine is completely removed. Cool, add 10 mL of
Sodium Acetate Solution followed by 10 mL of
Potassium Iodide Solution, and titrate with
Sodium Thiosulfate Solution to a light yellow color. Add enough starch TS to produce a marked blue color, and continue the titration. As the endpoint nears, add 2 g of potassium thiocyanate, and stir until completely dissolved. Continue titration until the precipitate is completely white. One mL of sodium thiosulfate solution is equivalent to about 10 mg of copper.
[noteIt is essential that the concentration of
Potassium Iodide Solution be carefully regulated. If the solution contains less than 320 mg of copper at the completion of titration, add 4.2 to 5 g of potassium iodide to make a total solution of 100 mL. If greater amounts of Cu are present, add
Potassium Iodide Solution slowly from buret with constant agitation in amounts proportionately greater.
]
Traps for Volatile Oil Apparatus
Procedure
Wash the precipitated cuprous oxide obtained under
General Procedure with water, cover this filter with a watch glass and dissolve the cuprous oxide with 5 mL of nitric acid directed under the watch glass with a pipet. Collect the filtrate in a 250-mL flask, wash the watch glass and the filter with water. Collect all the washings in the flask. Boil the contents of the flask to expel red fumes. Add about 5 mL of bromine TS, and boil until the bromine is completely removed. Cool, and proceed as directed under
Copper Solution beginning with add 10 mL of
Sodium Acetate Solution. From the volume of
Sodium Thiosulfate Solution consumed, obtain the weight of copper, in mg, by multiplying by 1.1259 to obtain the weight, in mg, of cuprous oxide. From
Table 2, find the quantity of dextrose, in mg, corresponding to the weight of cuprous oxide. The content of starch is equivalent to the weight, in mg, of dextrose obtained times 0.9. Conduct a blank determination, using 50 mL of
alkaline cupric tartrate TS and 50 mL of
Malt Extract. If the weight of the cuprous oxide so obtained exceeds 0.5 mg, correct the result of the determination accordingly.
[noteThe alkaline cupric tartrate TS deteriorates on standing and the quantity of cuprous oxide obtained in the blank determination increases.
]
Method 2
The following method is specific for dextrose (glucose), and because of its extreme sensitivity it may account for differences noted between values obtained from the same specimen. Duplicate determinations do not vary more than 2%.
Glucoamylase Solution
Prepare a solution of glucoamylase in water containing 30 International Units (IU) per mL. Use glucoamylase obtained preferably from Rhizopus delemar. The total glucoamylase activity of the test specimen being used should be not less than 150 IU.
Acetate Buffer Solution
Dissolve 16.4 g of sodium acetate in 100 mL of water, add 12.0 mL of glacial acetic acid, and mix. The pH of this solution is 4.8.
Phosphate Buffer
Dissolve 3.63 g of tris (hydroxymethyl) aminomethane and 5.0 g of monobasic sodium phosphate in 50.0 mL of water. At 37
, adjust with phosphoric acid to a pH of 7.0, dilute with water to 100.0 mL, and mix.
[noteThe pH of the buffer medium is sensitive to temperature and should be adjusted to the desired pH at the temperature to be used during incubation.
]
Enzyme Solution
Dissolve 30 mg of glucose oxidase (Type II from Aspergillus niger), 3 mg of peroxidase (Type I from horseradish), and 10 mg of potassium ferrocyanide in 100 mL of Phosphate Buffer. [noteThis mixture can be stored in a refrigerator for up to 10 days.]
18 N Sulfuric Acid
Add slowly, while stirring, 54 mL of sulfuric acid to 102 mL of water, allow to cool to 25
, and mix.
Standard Solutions
Dissolve an accurately weighed quantity of
USP Dextrose RS in water to obtain a solution containing 1.0 mg of
USP Dextrose RS per mL. Quantitatively dilute a known volume of this solution with water to obtain
Standard Solutions A,
B,
C,
D, and
E, having known concentrations of 10, 20, 25, 40, and 50 µg per mL of
USP Dextrose RS, respectively.
[noteAllow 4 hours for complete mutarotation before use.
]
Test Solutions
Extract about 5 g of finely ground test specimen with five 25-mL portions of 80% alcohol, and filter. Remove all the alcohol from the residue by drying in an air oven at 105
for about 8 hours.
[N
ote 1Any traces of alcohol remaining in the residue will inhibit glucoamylase.
] Cool, and transfer the flask containing the dried test specimen to a desiccator. Transfer about 1 g, accurately weighed, of the test specimen to a previously tared flask, add 25 mL of water, and adjust with phosphoric acid to a pH between 5.0 and 7.0, if necessary. Boil the suspension for about 3 minutes, transfer the flask to an autoclave, and heat to 135
for 2 hours. Remove the flask from the autoclave, maintain the temperature near 55
, and add 2.5 mL of
Acetate Buffer Solution and sufficient water to adjust the total weight of the solution to 45 ± 1 g. Immerse the flask in a water bath maintained at 55 ± 1
, and add 5 mL of
Glucoamylase Solution. Continuously swirl the flask for 2 hours to effect hydrolysis, filter through filter paper into a 250-mL volumetric flask, wash quantitatively with water, and collect all the washings in the flask. Dilute the contents of the flask with water to volume, and mix. Transfer 1 mL of an aliquot containing 20 to 60 µg of
d-glucose to each of five test tubes.
[N
ote 2In order to obtain the range of concentration of glucose in the hydrolysate, quantitatively dilute, if necessary, with water to volume.
] Add 2 mL of
Enzyme Solution to each of the five test tubes, and place the test tubes in the dark at 37 ± 1
for exactly 30 minutes to develop the color. At the end of 30 minutes, add 2 mL of
18 N Sulfuric Acid to each of the test tubes to stop the reaction, and mix.
Control Solution
Transfer an accurately weighed quantity of about 0.4 g of starch to a previously tared flask and proceed as directed under Test Solutions beginning with add 25 mL of water and, adjust the pH with phosphoric acid.
Procedure
Concomitantly determine the absorbances of the
Standard Solutions and the
Test Solutions at the wavelength of maximum absorbance at about 540 nm, with a suitable spectrophotometer, using the
Control Solution as the blank to set the instrument. Plot the absorbance values of the
Standard Solutions versus concentration, in µg per mL, of dextrose, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration,
C, in µg per mL, of dextrose in each of the
Test Solutions, calculate the average concentration, in µg per mL, of the solution under test. The percentage of starch content in the weight of the test specimen taken by the equation is calculated by the formula:
(0.9C / 106)(V1)(250 / V0)(100 / E)(100 / W) = 2.25CV1 / V0EW
in which
E is the weight, in g, of the test specimen taken;
V0 is the volume, in mL, of the aliquot taken from the 250-mL volumetric flask;
W is the percentage of dry weight of the test specimen; and
V1 is the volume, in mL, if extra dilution is done (see
Note 2 under
Test Solutions).
[noteV0 is 1.0 when no extra dilution is done.
]