152 MISC. PUBLICATION 5 40, XL S. DEPT. OF AGRICULTURE 



Procedure: Five grams of finely divided sample material is weighed, placed 

 upon a Biichner funnel and washed with warm water (50° C.) under gravity 

 flow (about 200 ml. total). The filter paper and the softened washed sample 

 are transferred quantitatively to a blender and disintegrated in the presence of 

 200 ml. of 90-percent acetone. 11 Five grams of Filter-Cel is added during the last 

 30 seconds of desintegration and the suspension is filtered under reduced pressure 

 directly into a 500-ml. volumetric flask through a sintered-glass funnel. The cake 

 formed upon the filter by means of a tamper and the application of vacuum is 

 successively washed with a 50-ml. portion of 90 percent acetone, resuspended in 

 an equal portion of acetone, reformed, washed, resuspended, etc., until the residue 

 and filtrate are colorless. The filtrate is then made to volume (500 ml.) with 

 90-percent acetone. 



In the analysis of fresh samples, 25 gm. of thinly sliced plant material (50 

 gm. if weakly pigmented) is disintegrated in a blender in the presence of 200 ml. 

 of 90-percent acetone. 15 Subsequent steps are identical with those given above 

 for dehydrated materials. 



A 100-ml. aliquot of the 90-percent acetone extract is mixed with 75 ml. of 

 diethyl ether in a 500-ml. separatory funnel and the acetone removed by the con- 

 tinuous washing method of Hubert (22) and LeRosen (26). In this method water 

 is continuously introduced below the ether layer in the separatory funnel through 

 a glass tube bent upward at the end. The tube is held in a rubber stopper which 

 closes the upper opening of the funnel. The stream of water introduced through 

 the tube and emitted from the lower stopcock washes the acetone from the ether 

 hyperphase. Traces of water remaining in the hyperphase are forced out with 

 the addition of 20 ml. of petroleum ether and the water drawn off through 

 the lower stopcock. 



The hyperphase is then made to volume in a 100-ml. volumetric flask with petro- 

 leum ether. Triplicate 25-ml. aliquots of this solution are placed in 50-ml. 

 round-bottom flasks, and the solvents are evaporated off under reduced pressure 

 with a small stream of nitrogen in a constant-temperature bath held at 40° C. 

 Each of these samples is redissolved in a few cubic centimeters of petroleum ether 

 and transferred quantitatively to a calcium phosphate (CaHPCM chromato- 

 graphic column as described by Moore (32) . Carotene is washed through the col- 

 umn with petroleum ether and caught in a 100-ml. volumetric flask. Other pig- 

 ments are retained on the column. 



After dilution to volume, the carotene is determined spectrophotometrically by 

 the use of wave length 436 [i (a=199) as suggested by Beadle and Zscheile (6) 

 or colorimetrically with the use of a blue filter. In the latter method, the colorim- 

 eter can be calibrated by the use of solutions of known concentration of pure 

 crystalline beta-carotene dissolved in petroleum ether. 



ASCORBIC ACID 



The following procedure for the determination of ascorbic acid (27) 

 has been found particularly useful in handling fresh, frozen, and de- 

 hydrated food materials. 



Apparatus : Photoelectric colorimeter of the direct-reading type with a filter 

 in the 520-millimicron range, blender, calibrated pipettes of 1-ml. and 9-ml. 

 capacities. 



Reagents: Metaphosphoric acid, 1 percent (freshly prepared); indophenol 

 dye solution, approximately 13 mg. per liter. If metaphosphoric acid is unavail- 

 able, oxalic acid (0.25 percent) can be substituted, provided the extracts are 

 tested promptly. 



Procedure : Blend 25 to 50 gm. of fresh fruit or vegetable tissue with 500 ml. 

 of 1-percent metaphosphoric acid in a blending machine operated for 5 minutes 

 at high speed. If the material is of high ascorbic acid content, such as leafy 

 vegetables, raspberries, strawberries, or asparagus, use the smaller quantity. 

 Fifty grams is used with foods containing less ascorbic acid, such as potatoes, 

 carrots, peaches, plums, and apricots. If a dehydrated fruit or vegetable is 



14 This washing process is unnecessary for many dehydrated vegetables, such as carrots, 

 spinach, chard, and others. In these cases 25 ml. of hot water (50° C.) is added to the 

 "> -in. sample in the weighing bottle and allowed to stand 1 hour. The sample and excess 

 wator are then transferred quantitatively to a blender with 175 ml. of acetone, and the 

 sample is disintegrated and extracted as described. 



15 Some difficulty has been experienced in obtaining complete extraction of raw carrots 

 by the procedure described. Steam blanching for 2 to 5 minutes prior to disintegration 

 lias been found to facilitate the extraction. Since carotene is stable in steam blanching of 

 this duration, the inclusion of this step is recommended with raw carrots. 



