Part 33

The material in which the starch is to be determined is dried and extracted with ether. From two to four grams of the extracted material, according to the amount of starch which it contains, are boiled for half an hour with 100 cubic centimeters of water, cooled to 65°, treated with ten cubic centimeters of malt extract and kept at the temperature named for half an hour. It is then again boiled for fifteen minutes, cooled to the temperature mentioned and again treated with malt extract as above. Two treatments with malt extract are usually sufficient to bring all the starch into solution. Finally it is again boiled and the volume completed to 250 cubic centimeters and thrown upon a filter. Two hundred cubic centimeters of the filtrate are converted into dextrose by boiling with hydrochloric acid, and the rest of the analysis is conducted in the usual manner. The dextrose value of the quantity of malt extract used must be determined upon a separate portion thereof, and the quantity of dextrose found deducted from the total amount obtained in the analysis.

=181. Separation by Hydrolysis with Water at High Temperatures.=—Instead of dissolving the starch with diastase, it may be brought into solution by heating with water under pressure. The former method employed of heating in sealed flasks has been entirely superceded by heating in an autoclave. The materials are best held in metal beakers furnished with a cover which prevents loss from boiling if the pressure should be removed too rapidly after the completion of the operation. The autoclave is a strong metal vessel capable of resisting the pressure of several atmospheres. It is furnished with a pressure gauge C and a safety valve D, as shown in the figure. The top is securely screwed on by means of a wrench, shown at the right hand side. In the figure a portion of the case is represented cut away to show the arrangement of the metal beakers inside.

In the method of Reinke, as practiced at the Halle station, and in this laboratory, about three grams of the starchy substance are placed in each of the metal beakers with twenty-five cubic centimeters of a one per cent lactic acid solution and thirty cubic centimeters of water. The contents of the beaker are thoroughly mixed and they are then heated for two and a half hours in the autoclave, at a pressure of three and a half atmospheres. The addition of the lactic acid is for the purpose of protecting any sugar which may be present from decomposition at the high pressure and temperature employed. After the completion of the heating, the autoclave is allowed to cool, the cover is removed and the beakers taken out and their contents washed with hot water into quarter liter flasks. After cooling, the volume is completed with cold water, and after standing for half an hour, with frequent shaking, the contents of the flasks are filtered and 200 cubic centimeters of the filtrate in each case converted into dextrose with hydrochloric acid in the usual way. In order to obtain agreeing results, it is highly necessary that the substance before treatment should be ground to a fine powder. The addition of the lactic acid, as practiced in the reinke method, tends to give somewhat high results, due probably to the hydrolytic action of the acid on the fiber present. When starchy bodies are heated in the autoclave for the determination of their starch by polarimetric methods, or for ordinary determinations, the use of lactic acid should be omitted.

Example.—The following data indicate the methods of calculation to be followed in the determination of the percentage of starch in the material by diastatic hydrolysis: Three grams of a barley were inverted by diastase, as directed above, the volume of the solution made a quarter of a liter, filtered, 200 cubic centimeters of the filtrate converted into dextrose by hydrochloric acid, the volume completed to half a liter with water and fifty cubic centimeters thereof oxidized by the alkaline copper solution in the usual way. The amount of copper obtained was 331 milligrams, corresponding to 174 milligrams of dextrose. The amount of malt extract used in hydrolyzing the barley mentioned above, was ten cubic centimeters. The diastatic solution inverted with hydrochloric acid and treated as indicated above, yielded 191 milligrams of copper, corresponding to ninety-eight milligrams of dextrose in ten cubic centimeters of the malt extract. The quantity of malt extract represented in the final determination of copper, however, was only one and six-tenths cubic centimeters. We then have:

Total dextrose 174 milligrams Dextrose in one and six-tenths cubic centimeters malt extract 16 milligrams Dextrose corresponding to 240 milligrams of barley 158 milligrams

Calculated on the proportion that dextrose is to starch, as ten is to nine, this is equivalent to 142 milligrams of starch. The percentage of starch in the original substance, therefore, was equivalent to 142 multiplied by 100, divided by 240, viz., 59.17.

=182. Principles of the Methods of Determination.=—In the approximately pure state in which starch exists in the trade, it may be determined by conversion into dextrose and estimating the latter by one of the methods given. It is probable that there is no known method by which starch can be entirely converted into dextrose, and all the methods of hydrolysis, when used for quantitive purposes, must be standardized, not by the theoretical quantity of dextrose which a given weight of pure starch should yield, but by the actual quantity obtained. Starch is not largely converted into dextrose by any of the diastatic ferments which produce principally maltose and dextrins. Recourse must therefore be had to strong acids. In practice, hydrochloric is the one usually employed. By the action of a hot mineral acid, not only is starch converted into dextrose, but also the dextrose found is subjected to changes. In such cases an opposing action seems to be exerted by the hydrolytic agent, a part of the dextrose formed suffering a partial condensation, and thus assuming a state of higher molecular weight, approaching the constitution of the dextrins. Another part of the dextrose may also suffer oxidation and thus disappear entirely in respect of the further steps in starch analysis.

In such cases, the best the analyst can do is to conduct the hydrolysis in as nearly as possible constant conditions, and to assume that the percentage of dextrose present at a given time bears a constant ratio to the quantity of starch hydrolyzed. In reality almost all the starch appears finally as dextrose, and by proceeding on the assumption noted above a fairly satisfactory accounting may be made of the remainder.

Starch being insoluble, it cannot be determined directly by its rotatory power. When heated for a few hours in contact with water at a high pressure, starch becomes soluble, and in this state has a fairly constant gyrodynat, viz., [a]_{D} = 197°.

Starch is also rendered soluble by rubbing it in a mortar for about ten minutes with an excess of strong hydrochloric acid, and in this way a quick approximate idea may be obtained of the percentage present. Starch prepared in this manner, however, has a strong reducing power on metallic salts, showing that a part of it has already, even in so short a time, assumed the state of maltose or dextrose. The gyrodynat of pure anhydrous starch in such conditions varies from [a]{D} = 197° to [a]{D} = 194°. Starch is also rendered soluble by boiling with salicylic acid, whereby a solution is obtained having a gyrodynat of [a]_{D} = 200°(circa). The methods of procedure for the analysis of starch will be set forth in detail in the following paragraphs.

=183. Estimation of Water.=—In prepared or commercial starches the water may be determined by heating in a partial vacuum. The temperature at first should be low, not exceeding 60°. After drying for an hour at that heat the temperature may be gradually increased. The last traces of water come off from starch with difficulty, and the final temperature may be carried a little beyond 100° without danger of decomposition.

Ost recommends the use of an atmosphere of hydrogen or illuminating gas. One and a half grams of the finely powdered sample are placed in the drying tube described in paragraph =23=, and heated in a stream of dry hydrogen. The temperature at first is kept at about 60° for several hours and is then gradually increased to 120°. Ost states that even at 150° the sample preserves its pure white color, but so high a temperature is not necessary. Maercker, at the Halle station, makes use of the same process, but employs illuminating gas instead of hydrogen. The importance of beginning the desiccation at a low temperature arises from the fact that at a higher temperature, before the greater part of the water is driven off, the starch will suffer a partial fusion and form a paste which is very difficult to dry. The dried sample must be kept in a stoppered vessel to prevent the absorption of hygroscopic moisture.

=184. Estimation of Ash.=—When the drying is accomplished in a flat platinum dish, the same sample may serve for incineration. Otherwise the incineration may be accomplished in another portion of the sample by following directions already given.

=185. Nitrogen.=—Even very pure samples of starch may contain a little nitrogen which is most conveniently determined by moist combustion.

As a rule, in commercial starches of good quality, the quantity of pure starch may be considered to be the remainder after subtracting the sum of the weights of water, ash and nitrogen multiplied by 6.25, from the original weight of the sample taken.

Example:—

Per cent of moisture found 12.85 ” ” ” ash found 0.08 ” ” ” nitrogen × 6.25 0.27 ----- Sum 13.20 Per cent of pure starch in sample 86.80

Samples of starch usually contain also traces of fat and fiber, and these when present in weighable quantities, should be determined and proper deductions made.

=186. Hydrolysis with Acids.=—The acids commonly chosen for hydrolyzing starch are sulfuric and hydrochloric. The former has the advantage of being more easily removed from the finished product but the latter performs the work with less damage to the sugars formed. For commercial purposes sulfuric and for analytical practice hydrochloric acids are commonly employed.

The best process for analytical purposes is the one proposed by Sachsse. In this method the starch is heated with the hydrolyzing mixture in the proportion of three grams to 200 cubic centimeters of water and twenty of hydrochloric acid of 1.125 specific gravity, containing five and six-tenths grams of the pure gas. The heating is continued for three hours on a steam-bath. Maercker recommends, instead of the above procedure, heating for two hours at gentle ebullition in an oil-bath. In this method three grams of the starch are reduced to paste with 200 cubic centimeters of water, and then boiled for two hours with fifteen cubic centimeters of hydrochloric acid of 1.125 specific gravity. The erlenmeyers in which the hydrolysis takes place are heated in an oil-bath and are provided with reflux condensers made of long glass tubes on which some bulbs have been blown, as shown in the accompanying figure. In all cases after hydrolysis the solution is neutralized, made to a standard volume and an aliquot part, after filtration, diluted to contain an amount of dextrose suited to the use of the table by Allihn for calculating the percentage of sugar. In diluting the solution preparatory to the estimation of dextrose, it is well to remember that nine parts of starch will furnish theoretically ten parts of dextrose. Since three grams of the sample are used, containing approximately eighty-five per cent of starch, the quantity of dextrose present is a little less than three grams. The solution should therefore contain not less than 300 cubic centimeters.

=187. Factor for Calculating Starch from the Dextrose Obtained.=—If all the starch could be converted into dextrose without loss, the quantity of it could be easily calculated theoretically on the supposition that the formula of starch is (C₆H₁₀O₅)ₙ. The factor by this assumption is, starch = dextrose × 0.90. If the starch have the formula assigned to it by Nägeli, viz., C₃₆H₆₂O₃₁ the formula becomes, starch = dextrose × 0.918.

Ost prefers to work by Sachsse’s method and to use the factor 0.925 to convert the dextrose into starch.

In view of all the facts in the case it appears that the analyst will reach nearly correct results by converting the starch into dextrose by heating for three hours at 100° with hydrochloric acid or for two hours at gentle ebullition as directed above, determining the resultant dextrose and multiplying the weight thereof by 0.92.

=188. Polarization of Starch.=—Starch may be prepared for polarization by dissolving it in cold hydrochloric acid. The process as carried out by Effront is as follows. Five grams of starch are rubbed with twenty cubic centimeters of cold concentrated hydrochloric acid for nearly ten minutes or until the solution is quite clear. The volume is completed to 200 cubic centimeters with water and the solution polarized. By this process there is always produced a notable quantity of reducing sugars, and for this reason it must be admitted that a portion of the starch has suffered complete hydrolysis. Ost therefore recommends the use of an acid of 1.17 specific gravity, and the gyrodynat of the soluble starch thus produced is found to vary from [a]{D} = 196°.3 to 196°.7. When acid of 1.20 specific gravity is employed the gyrodynat falls to [a]{D} = 194.2. For approximately correct work the solution with the weaker hydrochloric acid and subsequent polarization is to be recommended as the most rapid method for starch determination.

It will be of interest to add the observation that the gyrodynat of maltose has lately been redetermined by Ost, who finds it to be [a]_{D}²⁰ ° = 137°.04 ± 0.19.

=189. Solutions of Starch at High Pressure.=—Starch may also be brought into a condition suited to polarization by dissolving in water at a high temperature and pressure. The solution is accomplished in an autoclave as described in =181=.

From two to three grams of starch are used and from eighty to ninety cubic centimeters of water. The starch is first reduced to a pasty state by heating with the water and, when evenly distributed throughout the flask, is rendered soluble by heating from three to five hours in an autoclave at from two to three atmospheres. The material is entirely without action on an alkaline copper solution. After heating, the volume of the solution is completed to 100 cubic centimeters and it is then polarized. The gyrodynat of starch dissolved in this way varies from [a]_{D} = 196°.5 to 197°.

Starch is prepared by Baudry for polarization by boiling with salicylic acid. The gyrodynat of starch dissolved in this way is [a]_{D} = 200°.25.