The following method employs 100 ml of sample. In cases where it is necessary or would be desirable to use a smaller sample, the method of McCullough et al. [J. Chem. Ed. 47, 57 (1970)], which employs only 50 Ál of sample, may be used.
Distillation range: The difference between the temperature observed at the start of a distillation and that observed at which a specified volume has distilled, or at which the dry point is reached.
Initial boiling point: The temperature indicated by the distillation thermometer at the instant the first drop of condensate leaves the end of the condenser tube.
Dry point: The temperature indicated at the instant the last drop of liquid evaporates from the lowest point in the distillation flask, disregarding any liquid on the side of the flask.
Distillation flask: A 200-ml round-bottomed distillation flask of heat-resistant glass is preferred when sufficient sample (in excess of 100 ml) is available for the test. If a sample of less than 100 ml must be used, a smaller flask having the capacity of at least double the volume of the liquid taken may be employed. The 200-ml flask has a total length of 17.9 cm, and the inside diameter of the neck is 2.1 cm. Attached about midway in the neck, approximately 12 cm from the bottom of the flask, is a side arm 12.7 cm long and 5 mm in internal diameter, which is inclined downward at an angle of 75° from the vertical.
Condenser: Use a straight glass condenser of heat-resistant tubing, 56 to 60 cm long and equipped with a water jacket so that about 40 cm of the tubing is in contact with the cooling medium. The lower end of the condenser may be bent to provide a delivery tube or it may be connected to a bent adapter, which serves as the delivery tube.
Note: All-glass apparatus with standard-taper ground joints may be used alternatively if the assembly employed provides results equal to those obtained with the flask and condenser described above.
Receiver: The receiver is a 100-ml cylinder, which is graduated in 1-ml sub-divisions and calibrated "to contain". It is used for measuring the sample as well as for receiving the distillate.
Thermometer: A partial-immersion thermometer, calibrated for accuracy, having the smallest practical sub-divisions (not greater than 0.2°) is recommended in order to avoid the necessity for an emergent-stem correction.
Source of heat: A Bunsen burner is the preferred source of heat. An electric heater may be used, however, if it is shown to give results comparable to those obtained with the gas burner.
Shield: The entire burner and flask assembly should be protected from external air currents. Any efficient shield may be employed for this purpose.
Flask support: An asbestos board, 6.5 mm in thickness and having a 10 cm circular hole, is placed on a suitable ring or platform support and fitted loosely inside the shield to ensure that hot gases from the source of heat do not come in contact with the sides of neck of the flask. A second 6.5 mm asbestos board, at least 225 square cm and provided with a 30 mm circular hole, is placed on top of the first board. This board is used to hold the 200 ml distillation flask, which should be fitted firmly on the board so that direct heat is applied to the flask only through the opening in the board.
Note: This procedure is to be used for liquids that distil above 50° in which case the sample can be measured and received, and the condenser water used, at room temperature (20-30°). For materials boiling below 50°, cool the liquid to below 10° before sampling, receive the distillate in a water bath cooled to below 10° and use water cooled to below 10° in the condenser.
Measure 100 ± 0.5 ml of the liquid in the 100-ml graduated cylinder and transfer the sample together with an efficient anti-bumping device to the distillation flask. Do not use a funnel in the transfer, or allow any of the sample to enter the side arm of the flask. Place the flask on the asbestos boards, which are supported on a ring or platform, and place in position the shield for the flask and burner. Connect the flask and condenser, place the graduated cylinder under the outlet of the condenser tube and insert the thermometer. The thermometer should be located in the centre of the neck end so that the top of the bulb (when present, auxiliary bulb) is just below the bottom of the outlet to the side arm. Regulate the heating so that the first drop of liquid is collected within 5 to 10 min. Read the thermometer at the instant the first drop of distillate falls from the end of the condenser tube and record as the initial boiling point. Continue the distillation at the rate of 4 or 5 ml of distillate per min, noting the temperature as soon as the last drop of liquid evaporates from the bottom of the flask (dry point) or when the specified percentage has distilled over.
Correct the observed temperature readings for any variation in the barometric pressure from the normal (760 mm Hg) by allowing 0.1° for each 2.7 mm of variation, adding the correction if the pressure is lower, or subtracting if higher than 760 mm Hg. When a total immersion thermometer is used correct for the temperature of the emergent-stem by the formula 0.00015 x N(T - t), in which N represents the number of degrees of emergent-stem from the bottom of the stopper, T the observed temperature of distillation, and t the temperature registered by an auxiliary thermometer the bulb of which is placed midway of the emergent-stem, adding the correction to the observed readings of the main thermometer.
Alternatively, the following simplified correction formula may be applied:
in which to is the boiling point at 760 mm, b is the observed pressure in mm Hg and k is the correction factor for each 1 -mm difference with normal pressure.
The factor k depends on the substance under study; it is given in handbooks and varies between 0.033 and 0.057.
The pH of an aqueous solution may be determined accurately by potentiometry using a pH meter. The practical definition of pH in water may be given by the equation:
where pH is the value for the solution being measured, pHo is the value for a standard buffer, E is the potential value for the solution being measured, Eo is the potential value for the standard buffer, and 0.0591 is the value at 25° of the Nernstian constant. The equation does not apply to solvents other than water, or to mixed solvents that include water. However, the pH meter gives reproducible readings in other solvent systems, on the basis of calibration with aqueous buffers, and while the pH readings lack thermodynamic significance they are useful in setting specifications.
The measurement of pH using a pH meter is a matter of comparing the meter reading of an unknown solution with the meter readings of standard buffers whose pH values are accurately known. Standard buffer solutions are described in compendia, such as the Merck Index. Routine measurement uses only one buffer and an approximation of the electrode slope, usually made by a temperature compensator, pH measurement accurate to ± 0.05 pH unit or better requires the use of two buffers that bracket, if possible, the expected pH range. All samples and buffer should be at the same temperature.
The choice and care of glass and reference electrodes must be carefully considered. The ordinary glass electrode begins to be sensitive to alkali metal cations at pH values above about 9, leading to the so-called alkaline error. Electrodes with a greatly reduced alkaline error should be used for readings in the alkaline range. Store the electrodes in distilled water when not in use, in order to avoid dehydration. "Flow-type" electrodes may be used if evidence of validity of pH measurement with the electrode is demonstrated.
The measurement of the pH of "highly buffered solutions" (distilled water or solutions of non-ionic organic compounds in distilled water) is a particularly difficult measurement. The addition of 0.3 ml of a saturated solution of potassium chloride per 100 ml of distilled water helps by providing a small amount of electrolyte. However, it will usually be necessary to protect the solution being measured from the carbon dioxide in air by use of a blanket of nitrogen during the measurement. Measure the pH of successive portions of the distilled water or test solutions, with vigorous agitation, until the observed results for two successive portions agree within 0.1 pH unit.
Use a suitable pH meter and follow the manufacturer's instructions. Each time the electrodes are used, rinse them with distilled or deionised water and carefully blot them dry with clean absorbent tissue. Form a fresh reference electrode liquid junction. Rinse the sample vessel three times with each new solution to be introduced.
Choose two standard buffers (standard buffer solutions are described in compendia, such as the Merck Index) to bracket, if possible, the anticipated pH of the unknown. Warm or cool these standards as necessary to match within 2° the temperature of the unknown, and initially set the temperature compensator to that temperature. Immerse the electrodes in a portion of the first standard buffer, and following the manufacturer's instructions adjust the appropriate standardization control (knob, switch, or button) until the pH reading is that of the buffer. Repeat this procedure with fresh portions of the first standard buffer until two successive readings are within ± 0.02 pH unit without an adjustment of the standardization control.
Rinse the electrodes, blot dry, and immerse them in a portion of the second standard buffer of lower pH. Do not change the setting of the standardization control. Following the manufacturer's instructions, adjust the slope control (thumbwheel switch, knob, or temperature compensator) until the exact buffer pH is displayed.
Repeat the sequence of standardization with both buffers until the pH readings are within ± 0.02 pH unit for both buffers without any adjustment of either control (the amount of sample to be used in sample preparation is given where applicable in the individual specification.). The pH of the unknown solution may then be measured (The difference between the results of two pH determinations when carried out simultaneously on in rapid succession by the same analyst, under the same conditions, should not exceed 0.05 pH unit.).
Always re-standardize the instrument after even a short period during which the amplifier is turned off.
The melting point of a pure substance is the temperature at which the substance changes state from solid to liquid. A substance containing impurities will not melt at one specific temperature, but will melt over a range.
Before determining the melting range of a substance, the sample should be dried under the conditions specified for Loss on Drying in the individual monograph. If a temperature is not specified in the monograph, the sample should be dried for 24 h in a desiccator.
Transfer a quantity of the dried powder to a dry capillary-tube about 10 cm long and sealed at one end (thickness of the wall, 0.10-0.15 mm; i.d. 0.9-1.1 mm) and pack the powder by tapping the tube on a hard surface so as to form a tightly-packed column 2-4 mm in height.
Attach the capillary-tube and its contents to a standard thermometer so that the closed end is at the level of the middle of the bulb, and heat in a suitable apparatus containing an appropriate liquid (liquid paraffin or silicone oil) and fitted with a stirring device and an auxiliary thermometer. Regulate the rise in temperature during the first period to 3° per min. When the temperature has risen to 5° below the lowest figure of the range for the substance being tested, heat more slowly: if no other directions are given, the rate of rise in temperature should be 1 -2° per min.
Unless otherwise directed, read the temperature at which the substance is observed to form droplets against the side of the tube and the temperature at which it is completely melted, as indicated by the formation of a definitive meniscus.
To the temperature readings, apply the emergent-stem correction determined as follows:
Before starting the determination of the melting range, adjust the auxiliary thermometer so that the bulb touches the standard thermometer at a point midway between the graduation for the expected melting range and the surface of the heating material. When the substance has melted, read the temperature on the auxiliary thermometer. Calculate the correction to be added to the temperature reading of the standard thermometer from the following formula:
in which T is the temperature reading of the standard thermometer, t is the temperature reading of the auxiliary thermometer and N is the number of degrees of the scale of the standard thermometer between the surface of the heating material and the level of the mercury.
The statement "melting range, ao - b°" means that the corrected temperature at which the material is observed to form droplets must be at least ao, and that the material must be completely melted at the corrected temperature b°.
The refractive index of a transparent substance is the ratio of the velocity of light in air to its velocity in that material under like conditions. It is equal to the ratio of the sine of the angle of incidence made by a ray in air to the sine of the angle of refraction made by the ray in the material being tested. The refractive index values specified are for the D line of sodium (589 nm) unless otherwise specified.
The determination should be made at the temperature specified in the individual monograph or at 25° (20░ for flavouring agents) if no temperature is specified. This physical constant is used as a means for identification of, and detection of impurities in, volatile oils and other liquid substances. The Abbé refractometer, or other refractometers of equal or greater accuracy, may be employed at the discretion of the operator.
This method is designed to determine the solidification point of food grade chemicals having appreciable heats of fusion. It is applicable to chemicals having solidification points between -20° and +150°.
Solidification point is an empirical constant defined as the temperature at which the liquid phase of a substance is in approximate equilibrium with a relatively small portion of the solid phase. It is measured by noting the maximum temperature reached during a controlled cooling cycle after the appearance of a solid phase.
Solidification point is distinguished from freezing point in that the latter term applies to the temperature of equilibrium between the solid and liquid state of pure compounds. Some chemical compounds have two temperatures at which there may be a temperature equilibrium between solid and liquid state depending upon the crystal form of the solid that is present.
The apparatus is illustrated below and consists of the components described as follows:
Sample container: Use a standard glass 25 x 150 mm test-tube with lip, fitted with a stopper bored to hold the thermometer in place and to allow stirring with stirrer.
Thermometer: A thermometer having a range not exceeding 30° graduated in 0.1° divisions, and calibrated for accuracy at 76 mm immersion, should be employed. A thermometer should be so chosen that the stopper of the sample container does not obscure the solidification point.
Stirrer: The stirrer consists of a 1 mm diameter (B & S gauge 18) corrosion-resistant wire bent in a series of 3 loops about 25 mm apart. It should be made so that it will move freely in the space between the thermometer and the inner wall of the sample container. The shaft of the stirrer should be of a convenient length designed to pass loosely through a hole in the stopper holding the thermometer. Stirring may be hand-operated or mechanically activated at 20 to 30 strokes per min.
for determining Solidification Point
for Solidification Point determination
Air jacket: Use a standard glass 38 x 200 mm test-tube with lip, fitted with a stopper bored with a hole into which the sample container can easily be inserted up to the lip.
Cooling bath: Use a 2-L beaker or similar suitable container as a cooling bath. Fill it with an appropriate cooling medium such as glycerine, mineral oil, water, water and ice or alcohol-dry ice.
Assembly: Assemble the apparatus in such a way that the cooling bath can be heated or cooled to control the desired temperature ranges. Clamp the air jacket so that it is held rigidly just below the lip and immerse it in the cooling bath to a depth of 160 mm.
Preparation of sample: The solidification point is usually determined on chemicals as they are received. Some may be hygroscopic, however, and require special drying. Where this is necessary it will be noted in the monograph. Products which are normally solid at room temperature must be carefully melted at a temperature about 10° above the expected solidification point. Care should be observed to avoid heating in such a way as to decompose or distil any portion of the sample.
Adjust the temperature of the cooling bath to about 5° below the expected solidification point. Fit the thermometer and stirrer with a stopper so that the thermometer is centred and the bulb is about 20 mm from the bottom of the sample container. Transfer a sufficient amount of the sample, previously melted if necessary, into the sample container to fill it to a depth of about 90 mm when in molten state. Place the thermometer and stirrer in the sample container and adjust the thermometer so that the immersion line will be at the surface of the liquid and the end of the bulb 20 ± 4 mm from the bottom of the sample container. When the temperature of the sample is about 5° above the expected solidification point, place the assembled sample tube in the air jacket.
Allow the sample to cool while stirring at the rate of 20 to 30 strokes per min, in such a manner that the stirrer does not touch the thermometer. Stir the sample continuously during the remainder of the test.
The temperature at first will gradually fall, then become constant as crystallization starts and continues under equilibrium conditions, and finally will start to drop again. Some chemicals may super-cool slightly below (0.5°) the solidification point; as crystallization begins the temperature will rise and remain constant as equilibrium conditions are established. Other products may cool more than 0.5° and cause deviation from the normal pattern of temperature changes. If the temperature rise exceeds 0.5° after the initial crystallization begins, repeat the test and seed the melted compound with small crystals of the sample at 0.5° intervals as the temperature approaches the expected solidification point. Crystals for seeding may be obtained by freezing a small sample in a test-tube directly in the cooling bath. It is preferable that seeds of the stable phase be used from a previous determination.
Observe and record the temperature readings at regular intervals until the temperature rises from a minimum, due to super-cooling, to a maximum and then finally drops. The maximum temperature reading is the solidification point. Readings 10 sec apart should be taken in order to establish that the temperature is at the maximum level and continues until the drop in temperature is established.
Approximate solubilities, as specified in the Identification Tests, are to be interpreted according to the following descriptive terms:
Parts of solvent required for 1 part of solute
|Very soluble||Less than 1|
|Freely soluble||From||1||to||Less than 10|
|Soluble||From||10||to||Less than 30|
|Sparingly soluble||From||30||to||Less than 100|
Less than 1,000
|Very slightly soluble||From||1,000||to||Less than 10,000|
|Practically insoluble or in soluble||More than 10,000|
Procedure: Unless otherwise specified, transfer a known amount of the sample into a flask containing known amount of the specified solvent, shake for no less than 30 sec and no more than 5 min.
Specific gravity is defined as the ratio of the mass of the sample to the mass of an equal volume of the standard material. The specific gravity (dtt) means the ratio of the weight of the sample at t'° to that of an equal volume of water at t°. Unless otherwise specified, specific gravity is noted as D2020 (D2525 for flavouring agents). Specific gravity is determined by one of the following methods, unless otherwise specified.
Measurement by Pycnometer
A pycnometer is a vessel made of glass with a capacity of usually 10 to 100 ml. It has a ground-glass stopper fitted with a thermometer, and has a side tube with a mark and a ground-glass cap. Weigh a pycnometer previously cleaned and dried, and note the weight W. Remove the stopper and the cap, fill the pycnometer with a sample, keep at the temperature of about 1° to 3° lower than that specified, and stopper, taking care not to leave bubbles. Raise the temperature gradually until the thermometer shows the specified temperature. Remove the sample above the mark from the side tube, replace the cap, and wipe the outside thoroughly. Weigh, and note the weight W1. Using the same pycnometer, perform the similar determination with water. Weigh the pycnometer containing water at the specified temperature, and note the weight W2. Calculate the specific gravity of the sample by the following formula.
Measurement by Mohr-Westphal Balance
Keep the balance horizontal; attach the glass tube in which a thermometer is enclosed by a wire onto the end of the arm. Immerse the glass tube in water in a cylinder, place the largest rider on the arm at the mark 10, and adjust the balance by moving the nut at the specified temperature.
After that, immerse the glass tube in the sample, adjust the balance by hanging riders on the arm, and read the specific gravity at the marks at which riders are placed. It is necessary to make the length of the part of wire that is immersed in a sample equal to that immersed in water by changing the height of the sample in the cylinder.
Measurement by Hydrometer
Use a hydrometer with a precision intended for use at the specified temperature. Clean the hydrometer with alcohol. Shake the sample well, and place in the hydrometer after bubbles have disappeared. At the specified temperature, when the hydrometer has settled, read the specific gravity at the upper rim of the meniscus. In case of any hydrometer, however, for which special directions are given, follow the directions.
Measurement by Sprengel-Ostwald Pycnometer
A Sprengel-Ostwald pycnometer (see figure) is a vessel made of glass with a capacity of usually 1 to 10 ml. As shown in the figure, both the ends are thick-walled fine tubes one of which has a mark on it. A platinum or an aluminium wire is attached to hang on the arm of a chemical balance.
Weigh the pycnometer, previously cleaned and dried (W). Immerse the curved tube in the sample kept at a temperature 3° to 5° lower than the specified temperature, attach a rubber tube at the end of the straight tube, and suck the sample gently until it comes up above the mark, taking care to prevent formation of bubbles. Immerse the pycnometer in a water bath kept at the specified temperature for about 15 min, and by attaching a piece of filter paper at the end of the curved tube, adjust the end of the sample to the mark. Remove the pycnometer from the water bath, and wipe the outside well. Weigh and note the weight W1. By using the same pycnometer, perform the same determination with water. Weigh the pycnometer containing water at the specified temperature, and note the weight W2. Calculate the specific gravity by the following formula:
Optical rotation of chemicals is generally expressed in degrees, as either "angular rotation" (observed) or "specific rotation" (calculated with reference to the specific concentration of 1 g of solute in 1 ml of solution, measured under stated conditions).
Specific rotation usually is expressed by the term [α]tx, in which t represents, in degrees centigrade, the temperature at which the rotation is determined, and x represents the characteristic spectral line or wavelength of the light used. Spectral lines most frequently employed are the D line of sodium (doublet at 589.0 and 589.6 nm and the yellow-green line of mercury at 546.1 nm). The specific gravity and the rotatory power vary appreciably with the temperature.
The accuracy and precision of optical rotation measurements will be increased if they are carried out with due regard for the following general considerations.
The source of illumination should be supplemented by a filtering system capable of transmitting light of a sufficiently monochromatic nature. Precision polarimeters generally are designed to accommodate interchangeable disks to isolate the D line from sodium light or the 546.1 nm line from the mercury spectrum. With polarimeters not thus designed, cells containing suitably coloured liquids may be employed as filters [see "Technique of Organic Chemistry", A. Weissberger. Vol. I, Part II, 3rd ed. (1960), Interscience Publishers, Inc., New York, N.Y.].
Special attention should be paid to temperature control of the solution and of the polarimeter. Observations should be accurate and reproducible to the extent that differences between replicates, or between observed and true values of rotation (the latter value having been established by calibration of the polarimeter scale with suitable standards), calculated in terms of either specific rotation or angular rotation, whichever is appropriate, shall not exceed one-fourth of the range given in the individual monograph for the rotation of the article being tested. Generally, a polarimeter accurate to 0.05° of angular rotation, and capable of being read with the same precision, suffices. In some cases, a polarimeter accurate to 0.01° or less, of angular rotation, and read with comparable precision, may be required.
Polarimeter tubes should be filled in such a way as to avoid creating or leaving air bubbles, which interfere with the passage of the beam of light. Interference from bubbles is minimized with tubes in which the bore is expanded at one end. However, with tubes of uniform bore, such as semimicro-or micro-tubes, care is required for proper filling. At the time of filling, the tubes and the liquid or solution should be at a temperature not higher than that specified for the determination, to guard against the formation of a bubble upon cooling and contraction of the contents.
In closing tubes having removable end-plates fitted with gaskets and caps, the latter should be tightened only enough to ensure a leak-proof seal between the end-plate and the body of the tube. Excessive pressure on the end-plate may set up strains that result in interference with the measurements. In determining the specific rotation of a substance of low rotatory power, it is desirable to loosen the caps and tighten them again between successive readings in the measurement of both the rotation and the zero-point. Differences arising from end-plate strain thus generally will be revealed and appropriate adjustments to eliminate the cause may be made.
In the case of a solid, dissolve the substance in a suitable solvent, reserving a separate portion of the latter for a blank determination. Make at least five readings of the rotation of the solution, or of the substance itself if liquid, at 25° or the temperature specified in the individual monograph. Replace the solution with the reserved portion of the solvent (or, in the case of a liquid, use the empty tube), make the same number of readings, and use the average as the blank value. Subtract the blank value from the average observed rotation if the two figures are of the same sign, or add if opposite in sign, to obtain the corrected angular rotation.
Calculate the specific rotation of a liquid substance, or of a solid in solution, by application of one of the following formulas:
in which a is the corrected angular rotation, in degrees, at temperature t; l is the length of the polarimeter tube in decimeters; d is the specific gravity of the liquid or solution at the temperature of observation; p is the concentration of the solution expressed as the number of g of substance in 100 g of solution; and c is the concentration of the solution expressed as the number of g of substance in 100 ml of solution.
The concentrations p and c should be calculated on the dried or anhydrous basis, unless otherwise specified.
The following are chemical tests to identify specific inorganic ions or organic moieties. Test solutions (TS) used are defined under the section on Media, Reagents and Solutions.
Solutions of aluminum salts yield with ammonia TS a white, gelatinous precipitate which is insoluble in an excess of ammonia TS. A similar precipitate is produced by sodium hydroxide TS or sodium sulfide TS, but it dissolves in an excess of either reagent.
Sodium hydroxide TS decomposes ammonium salts with the evolution of ammonia, recognizable by its odour and its alkaline effect upon moistened red litmus paper. The decomposition is accelerated by warming.
Solutions of bromates acidified with nitric acid (1 in 20), yield a white, crystalline precipitate with the addition of 2 or 3 drops of silver nitrate TS, which dissolves by heating. A pale yellow precipitate is produced with the addition of 1 drop of sodium nitrite TS.
Solutions of bromates acidified with nitric acid (1 in 20), produce a yellow to reddish brown colour with the addition of 5 or 6 drops of sodium nitrite TS. With the addition of 1 ml of chloroform and stirring, the chloroform layer becomes a yellow to reddish brown colour.
Free bromine is liberated from solutions of bromides upon the addition of chlorine TS, dropwise. When shaken with chloroform, the bromine dissolves, colouring the chloroform red to reddish brown. A yellowish white precipitate, which is insoluble in nitric acid and slightly soluble in ammonia TS, is produced when solutions of bromides are treated with silver nitrate TS.
Insoluble oxalate salts are formed when solutions of calcium salts are treated in the following manner: using 2 drops of methyl red TS as indicator, neutralize a solution of a calcium salt (1 in 20) with ammonia TS. A white precipitate of calcium oxalate forms upon the addition of ammonium oxalate TS. This precipitate is insoluble in acetic acid but dissolves in hydrochloric acid.
Calcium salts moistened with hydrochloric acid impart a transient yellowish red colour to a non-luminous flame.
Carbonates and bicarbonates effervesce with acids, yielding a colourless gas (carbon dioxide) which produces a white precipitate immediately when passed into calcium hydroxide TS. Cold solutions of soluble carbonates are coloured red by phenolphthalein TS, whereas solutions of bicarbonates remain unchanged or are slightly changed.
Solutions of chlorides yield with silver nitrate TS a white, curdy precipitate which is insoluble in nitric acid but soluble in a slight excess of ammonia TS. Chlorine, recognizable by its distinctive odour, is evolved when solutions of chloride are warmed with potassium permanganate and dilute sulfuric acid TS.
When solutions of cupric compounds are acidified with hydrochloric acid, a red film of metallic copper is deposited upon a bright untarnished surface of metallic iron. An excess of ammonia TS, added to a solution of a cupric salt, produces first a bluish precipitate and then a deep blue-coloured solution. Solutions of cupric salts yield with potassium ferrocyanide TS a reddish brown precipitate, insoluble in dilute acids.
Potassium ferrocyanide TS produces a dark blue precipitate in acid solutions of ferric salts. With an excess of sodium hydroxide TS, a reddish brown precipitate is formed. Solutions of ferric salts produce with ammonium thiocyanate TS a deep red colour which is not destroyed by dilute mineral acids.
To 10 ml of a 1% solution of the sample add 1 ml of ferric chloride TS. A dark blue precipitate is formed.
Potassium ferricyanide TS produces a dark blue precipitate in solutions of ferrous salts. This precipitate, which is insoluble in dilute hydrochloric acid, is decomposed by sodium hydroxide TS. Solutions of ferrous salts yield with sodium hydroxide TS a greenish white precipitate, the colour rapidly changing to green and then to brown when shaken.
Solutions of iodides, upon the addition of chlorine TS, dropwise, liberate iodine which colours the solution yellow to red. Chloroform is coloured violet when shaken with this solution. The iodine thus liberated gives a blue colour with starch TS. Silver nitrate TS produces in solutions of iodides a yellow, curdy precipitate which is insoluble in nitric acid and in ammonia TS.
Solutions of ferrous and ferric compounds yield a black precipitate with ammonium sulfide TS. This precipitate is dissolved by cold dilute hydrochloric acid TS with evolution of hydrogen sulfide.
Solutions of magnesium salts in the presence of ammonium chloride yield no precipitate with ammonium carbonate TS, but a white, crystalline precipitate, which is insoluble in ammonia TS, is formed upon the subsequent addition of sodium phosphate TS.
Solutions of manganese salts yield with ammonium sulfide TS a salmon-coloured precipitate which dissolves in acetic acid.
When a solution of a nitrate is mixed with an equal volume of sulfuric acid, the mixture cooled, and a solution of ferrous sulfate superimposed, a brown colour is produced at the junction of the two liquids. Brownish red fumes are evolved when a nitrate is heated with sulfuric acid and metallic copper. Nitrates do not decolourize acidified potassium permanganate TS (distinction from nitrites).
Nitrites yield brownish red fumes when treated with dilute mineral acids or acetic acid. A few drops of potassium iodide TS and a few drops of dilute sulfuric acid TS added to a solution of a nitrite liberate iodine which colours starch TS blue.
Solutions of peroxides slightly acidified with sulfuric acid yield a deep blue colour upon the addition of potassium dichromate TS. On shaking the mixture with an equal volume of ether and allowing the liquids to separate, the blue colour is transferred to the ether layer.
Neutral solutions of orthophosphates yield with silver nitrate TS a yellow precipitate, which is soluble in dilute nitric acid TS or in ammonia TS. With ammonium molybdate TS, a yellow precipitate, which is soluble in ammonia TS, is formed.
Potassium compounds impart a violet colour to a non-luminous flame if not masked by the presence of small quantities of sodium. In neutral, concentrated or moderately concentrated solutions of potassium salts, sodium bitartrate TS slowly produces a white, crystalline precipitate which is soluble in ammonia TS and in solutions of alkali hydroxides or carbonates. The precipitation may be accelerated by stirring or rubbing the inside of the test tube with a glass rod or by the addition of a small amount of glacial acetic acid or ethanol.
Sodium compounds, after conversion to chloride or nitrate, yield with cobalt-uranyl acetate TS a golden-yellow precipitate, which forms after several min agitation. Sodium compounds impart an intense yellow colour to a non-luminous flame.
Solutions of sulfates yield with barium chloride TS a white precipitate which is insoluble in hydrochloric and nitric acids. Sulfates yield a white precipitate with lead acetate TS, which is soluble in ammonium acetate solution. Hydrochloric acid produces no precipitate when added to solutions of sulfates (distinction from thiosulfates).
When treated with dilute hydrochloric acid TS, sulfites and bisulfites yield sulfur dioxide, recognizable by its characteristic odour. This gas blackens filter paper moistened with mercurous nitrate TS.
Solutions of thiosulfates yield with hydrochloric acid a white precipitate which soon turns yellow, liberating sulfur dioxide, recognizable by its odour. The addition of ferric chloride TS to solutions of thiosulfates produces a dark violet colour which quickly disappears.
Zinc salts, in the presence of sodium acetate, yield a white precipitate with hydrogen sulfide. This precipitate, which is insoluble in acetic acid, is dissolved by dilute hydrochloric acid TS. A similar precipitate is produced by ammonium sulfide TS in neutral or alkaline solutions. Solutions of zinc salts yield with potassium ferrocyanide TS a white precipitate which is insoluble in dilute hydrochloric acid TS.
Acetic acid or acetates, when warmed with sulfuric acid and alcohol, form ethyl acetate, recognizable by its characteristic odour. With neutral solutions of acetates, ferric chloride TS produces a deep red colour which is destroyed by the addition of a mineral acid.
Dissolve as completely as possible 0.01 g of the sample by shaking with 0.15 ml of 0.1 N sodium hydroxide and add 1 ml of acid ferric sulfate TS. Within 5 min, a cherry-red colour develops that finally becomes deep purple.
To 2 ml of a 2% solution of the sample in water add 2 ml of water, 0.1 g of sodium bicarbonate and about 0.02 g of ferrous sulfate. Shake and allow to stand. A deep violet colour is produced, which disappears on the addition of 5 ml of dilute sulfuric acid TS.
Neutral solutions of benzoates yield a salmon-coloured precipitate with ferric chloride TS. From moderately concentrated solutions of benzoate, dilute sulfuric acid TS precipitates free benzoic acid, which is readily soluble in ether.
When a few mg of a citrate are added to a mixture of 15 ml of pyridine and 5 ml of acetic anhydride, a carmine red colour is produced.
Dissolve a quantity of the sample in water to obtain a solution containing 10 mg/ml, heating in a water bath at 60°, if necessary. Similarly, prepare a standard solution of potassium gluconate in water containing 10 mg/ml.
Apply separate 5-µl portions of the test solution and the standard solution on a suitable thin-layer chromatographic plate coated with 0.25-mm layer of chromatographic silica gel, and allow to dry. Develop the chromatogram in a solvent system consisting of a mixture of ethanol, water, ammonium hydroxide, and ethyl acetate (50:30:10:10) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, and dry at 110° for 20 min. Allow to cool, and spray with a reagent, prepared as follows: Dissolve 2.5 g of ammonium molybdate in about 50 ml of 2 N sulfuric acid in a 100-ml volumetric flask, add 1.0 g of ceric sulfate, swirl to dissolve, dilute with 2 N sulfuric acid to volume, and mix. Heat the plate at 110° for about 10 min. The principal spot obtained from the test solution corresponds in colour, size, and retention to that obtained from the standard solution.
Proceed as directed under Thin Layer Chromatography (see Analytical Techniques) using the following conditions:
Sample: 1 µl of 1 in 100 solution of the sample. Add a few drops of ammonium hydroxide TS if required to dissolve.
Reference: 1 µl of a 1 in 100 solution of monosodium L-glutamate
Solvent: A mixture of 2 volumes of n-butanol, 1 volume of glacial acetic acid and 1 volume of water
Adsorbent: Silica gel
Stop the development when the solvent front has advanced about 10 cm from the point of application. Dry the plate at 80° for 30 min. Spray ninhydrin TS on the plate, heat at 80° for 10 min and observe the plate under natural light. The Rf value of the sample and that of the reference standard are identical.
Heat a few drops of the sample in a test tube with about 0.5 g of potassium bisulfate; pungent vapours of acrolein are evolved.
When solutions of lactates are acidified with sulfuric acid, and potassium permanganate TS is added and the mixture heated, acetaldehyde, recognizable by its distinctive odour, is evolved.
Transfer the solution described in the individual monograph into a porcelain dish and add 10 mg of sulfanilic acid. Heat the solution on a water bath for a few min, add 5 ml of a 1 in 5 solution of sodium nitrite and heat slightly. Make alkaline with sodium hydroxide TS. A red colour is produced.
To the solution given in the monograph add 2 ml of magnesia mixture TS. No precipitate is formed. Add 5 ml of nitric acid, boil for 10 min, neutralize with strong ammonia TS, add water to make to 100 ml, add ammonium molybdate TS, and warm. A yellow precipitate is formed, which dissolves in sodium hydroxide TS or ammonia TS.
To 3 ml of a 3 in 10,000 solution of the sample in water, add 0.2 ml of a 1 in 10 solution of orcinol in ethanol and subsequently 3 ml of a 1 in 1,000 hydrochloric acid solution of ferric ammonium sulfate. Heat in a water bath for 10 min. A green colour is produced.
When a few mg of a tartrate are added to a mixture of 15 ml of pyridine and 5 ml of acetic anhydride, an emerald green colour is produced.
Transfer 2 g of the sample, accurately weighed, into a 250-ml beaker containing 150 ml of water and 1.5 ml of sulfuric acid TS. Cover the beaker with a watch glass and heat the mixture on a steam bath for 6 h rubbing down the wall of the beaker frequently with a rubber-tipped stirring rod and replacing any water lost by evaporation. Weigh 500 mg of a suitable acid washed filter aid, pre-dried at 105° for 1 h, to the nearest 0.1 mg, add this to the sample solution and filter through a tared Gooch crucible provided with an asbestos pad. Wash the residue several times with hot water, dry the crucible and its contents at 105° for 3 h, cool in a desiccator and weigh. The difference between the total weight and the weight of the filter aid plus crucible and pad is the weight of the Acid-insoluble matter. Calculate as percentage.
Note1: Method I referenced in older specifications has been deleted. The colourimetric procedure described in Method II may be used. However, it is recommended that, whenever possible, that the determination of arsenic be carried out using AAS-hydride technique/ICP method.
Note 2: Metals or salts of metals such as chromium, cobalt, copper, mercury, molybdenum, nickel, palladium, and silver may interfere with the evolution of arsine (Method II). Antimony, which forms stibine, is the only metal likely to produce a positive interference. Stibine forms a red coloured complex with silver diethyldithiocarbamate reagent which has a maximum absorbance at 510 nm. But at 535 - 540 nm the absorbance of the antimony complex is so diminished that the results of arsenic would not be affected significantly.
Note 3: All reagents used in the limit test for arsenic should be very low in arsenic content.
Method II (Colourimetric Procedure)
The general apparatus is shown in Figure 1. It consists of a 125-ml arsine generator flask with a 24/40 standard-taper joint fitted with a scrubber unit and an absorber tube connected by a capillary of inside diameter 2 mm and outside diameter 8 mm via a ball-and-socket joint, secured with a No. 12 clamp, connecting the units. Alternatively, an apparatus embodying the principle of the general assembly described and illustrated may be used.
Figure 1. Apparatus for Arsenic Limit Test - Method II
Silver Diethyldithiocarbamate Solution
Dissolve 1 g of recrystallized silver diethyldithiocarbamate, (C2H5)2NCSSAg, in 200 ml of reagent grade pyridine in a fume hood. Store this solution in a light-resistant container and use within 1 month.
Silver diethyldithiocarbamate is available commercially or may be prepared as follows. Dissolve 1.7 g of reagent grade silver nitrate in 100 ml of water. In a separate container, dissolve 2.3 g of sodium diethyldithiocarbamate, (C2H5)2NCSSNa-3H2O, in 100 ml of water, and filter. Cool both solutions to about 15°, mix the two solutions, while stirring, collect the yellow precipitate in a medium-porosity sintered-glass crucible or funnel, and wash with about 200 ml of cold water.
Recrystallize the reagent, whether prepared as directed above or obtained commercially, as follows: Dissolve in freshly distilled pyridine, using about 100 ml of solvent for each g of reagent, and filter. Add an equal volume of cold water to the pyridine solution, while stirring. Filter off the precipitate, using suction, wash with cold water, and dry in vacuum at room temperature for 2 to 3 h. The dry salt is pure yellow in colour and should show no change in character after 1 month when stored in a light-resistant container. Discard any material that changes in colour or develops a strong odour.
Standard Arsenic Solution
Weigh accurately 132.0 mg of arsenic trioxide that has been finely pulverized and dried for 24 h over a suitable desiccant, and dissolve it in 5 ml of sodium hydroxide solution (1 in 5). Neutralize the solution with diluted sulfuric acid TS, add 10 ml in excess, and dilute to 1,000.0 ml with recently boiled water, and mix. Transfer 10.0 ml of this solution into a 1,000-ml volumetric flask, add 10 ml of diluted sulfuric acid TS, dilute to volume with recently boiled water and mix.
Use this final solution, which contains 1 μg of arsenic (As) in each ml, within 3 days.
Stannous Chloride Solution
Dissolve 40 g of reagent grade stannous chloride dihydrate, SnCl2.2H2O, in 100 ml of hydrochloric acid. Store the solution in a glass container and use within 3 months.
Lead Acetate-Impregnated Cotton
Soak cotton in a saturated solution of reagent grade lead acetate, squeeze out the excess solution, and dry in a vacuum at room temperature.
Note: When preparing and using the cotton, take great care to avoid lead contamination.
Preparation of the Sample Solution
The solution obtained by treating the sample as directed in an individual monograph is used directly as the Sample Solution in the Procedure. Sample solutions of organic compounds are prepared in the generator flask (Figure 3), unless otherwise directed, according to the following general procedure:
Caution: Some substances may react unexpectedly with explosive violence when digested with hydrogen peroxide. Appropriate safety precautions must be employed at all times.
Note: If halogen-containing compounds are present, use a lower temperature while heating the sample with sulfuric acid, do not boil the mixture, and add the peroxide, with caution, before charring begins, to prevent loss of trivalent arsenic.
Transfer 1.0 g of the sample into the generator flask, add 5 ml of sulfuric acid and a few glass beads, and digest at a temperature not exceeding 120° on a hot plate in a fume hood until charring begins. (Additional sulfuric acid may be necessary to completely wet some samples, but the total volume added should not exceed about 10 ml.) After the sample has been initially decomposed by the acid, add with caution, dropwise, 30% hydrogen peroxide, allowing the reaction to subside and reheating between drops. The first few drops must be added very slowly with sufficient mixing to prevent a rapid reaction, and heating should be discontinued if foaming becomes excessive. Swirl the solution in the flask to prevent unreacted substance from caking on the walls or bottom of the flask during digestion. Maintain oxidizing conditions at all times during the digestion by adding small quantities of the peroxide whenever the mixture turns brown or darkens. Continue the digestion until the organic matter is destroyed, gradually raising the temperature of the hot plate to 250° - 300° until fumes of sulfuric acid are copiously evolved, and the solution becomes colourless, or retains only a slight straw colour.
Cool, add cautiously 10 ml of water, again evaporate (fumes of sulfuric acid evolved), and cool. Add cautiously 10 ml of water, mix, wash the sides of the flask with a few ml of water, and dilute to 35 ml.
If the sample solution was not prepared in the generator flask, transfer to the flask a volume of the solution, prepared as directed, equivalent to 1.0 g of the substance being tested and add water to make 35 ml.
Add 20 ml of dilute sulfuric acid (1 in 5), 2 ml of potassium iodide TS, and 0.5 ml of Stannous Chloride Solution, and mix. Allow the mixture to stand for 30 min at room temperature. Pack the scrubber tube with two plugs of Lead Acetate-Impregnated Cotton, leaving a small air space between the two plugs, lubricate the ground-glass joints with stopcock grease, if necessary, and connect the scrubber unit with the absorber tube. Transfer 3.0 ml of silver diethyldithiocarbamate solution to the absorber tube, add 3.0 g of granular zinc (20-mesh) to the mixture in the flask, and immediately insert the standard-taper joint in the flask. Allow the evolution of arsine and colour development to proceed at room temperature (25 ± 3°) for 45 min, swirling the flask gently at 10-min intervals. (The addition of a small amount of isopropanol to the generator flask may improve the uniformity of the rate of gas evolution.) Disconnect the absorber tube from the generator and scrubber units, and transfer the Silver diethyldithiocarbamate solution to a 1 -cm absorption cell. Determine the absorbance at the wavelength of maximum absorption between 535 nm and 540 nm with a suitable spectrophotometer or colorimeter, using Silver diethyldithiocarbamate solution as the blank. The absorbance due to any red colour from the solution of the sample does not exceed that produced by 3.0 ml of standard arsenic solution (3 μg As) when treated in the same manner and under the same conditions as the sample. The room temperature during the generation of arsine from the standard should be held to within ± 2° of that observed during the determination of the sample.
Accurately weigh a known quantity of sample (depending on the ash content such that about 20 mg of ash is obtained) in a tared crucible, ignite at a low temperature (about 550°), not to exceed a very dull redness, until free from carbon, cool in a desiccator, and weigh. If a carbon-free ash is not obtained, wet the charred mass with hot water, spread the residue using a glass rod, dry it in an air oven and reignite. If a carbon-free ash is still not obtained, cool the crucible, add 15 ml of ethanol, break up the ash with a glass rod, then burn off the ethanol, again heat the whole to dull redness, cool in a desiccator, and weigh.
Note: If difficulty with oxidizing organic material is found, the use of an ash aid such as ammonium nitrate may prove to be more satisfactory. Addition of a few drops of hydrogen peroxide facilitate oxidation of organic matter.
Boil the ash obtained as directed under Ash (Total) above, with 25 ml of dilute hydrochloric acid TS for 5 min, collect the insoluble matter on a suitable ash-less filter, wash with hot water, ignite at 800 ± 25°, cool, and weigh. Calculate the percentage of acid-insoluble ash from the weight of the sample taken.
Method I (for solids)
Transfer the quantity of the sample directed in the individual monograph to a tared 50- to 100-ml platinum dish or other suitable container. Add sufficient diluted sulfuric acid TS to moisten the entire sample. Heat gently, using a hot plate, an Argand burner, or an infrared heat lamp, until the sample is dry and thoroughly charred, then continue heating until all of the sample has been volatilized or nearly all of the carbon has been oxidized, and cool. Moisten the residue with 0.5 ml of sulfuric acid TS, and heat in the same manner until the remainder of the sample and any excess sulfuric acid have been volatilized. Finally ignite in a muffle furnace at 800 ± 25° for 15 min or longer, if necessary, to complete ignition, cool in a desiccator, and weigh.
(Note: In order to promote volatilization of sulfuric acid, it is advisable to add a few pieces of ammonium carbonate just before completing ignition.)
Method II (for liquids)
Unless otherwise directed, transfer the required weight of the sample to a suitable tared container, add 10 ml of diluted sulfuric acid TS, and mix thoroughly. Evaporate the sample completely by heating gently without boiling, and cool. Finally, ignite in a muffle furnace at 800 ± 25° for 15 min or longer, cool in a desiccator, and weigh.
Unless otherwise specified, place the prescribed quantity of the sample in a Nessler tube, dissolve it in about 30 ml of water, and neutralize with dilute nitric acid TS if the solution is alkaline. Add 6 ml of dilute nitric acid TS and dilute to 50 ml with water. If the use of a sample solution is prescribed, transfer the sample solution into a Nessler tube and dilute to 50 ml with water. Transfer the prescribed volume of 0.01 N hydrochloric acid into another Nessler tube to serve as the standard, add 6 ml of dilute nitric acid TS, and dilute to 50 ml with water.
If the solution containing the sample is not clear, filter both solutions under the same conditions. Add 1 ml of silver nitrate TS to each solution, mix thoroughly, and allow to stand for 5 min protected from direct sunlight. Compare the turbidity of the two solutions by observing the Nessler tubes from the sides and the tops against a black background. The turbidity of the sample solution does not exceed that of the standard.
Note: The limit test described below is designed to show whether the sample contains more or less than 20 mg/kg of chromium. It is recommended to use an appropriate AAS/ICP method in the place of the limit test, if possible, for quantitative determination.
Weigh 1.0 g of the sample into a quartz dish. Char the material, raising the temperature slowly. Allow to cool, add 10 ml of a 25% magnesium nitrate solution; evaporate, heating slowly until no more nitrous vapour evolves. Heat the material in an oven at 600° until all black particles have disappeared (1 h).
Dissolve the residue by adding 10 ml of 4 N sulfuric acid and 20 ml of water. Heat on a water bath for about 5 min.
Add 0.5 ml of 0.1 N potassium permanganate. Add more permanganate if the solution decolourizes. Cover with a watch glass and heat on a water bath for about 20 min. Add 5% sodium azide solution, one drop every 10 sec, until the excess potassium permanganate has been removed (avoid excess of sodium azide; 2 drops are usually sufficient). Cool the solution in running water, and filter if manganese dioxide (black precipitate) is evident. Transfer the solution to a 50-ml volumetric flask. Add 2.5 ml of 5 M sodium dihydrogenphosphate, add 2 ml of diphenyl carbazide TS and fill to the mark with water. Measure the absorbance at 540 nm 30 min after adding the diphenyl carbazide TS. A blank with the latter two reagents should show no colour or only a slight purple colour.
At the same time run a parallel test with 1.00 ml of standard chromate TS (1 ml = 20 µg Cr) and a few ml of saccharose placed into a second quartz dish. Treat the mixture exactly as the sample and measure the extinction at the same wavelength.
Calculate the chromium content of the sample from the two extinction values observed.
Note: The method uses perchloric acid in one of the reagents. Special care shall be taken while handling it and all operations shall be conducted in a perchloric acid fume cup board.
The method is based on two-dimensional paper chromatography, in which the development is first carried out in one direction using a basic solvent. The paper is then turned through 90° and chromatographed using an acidic solvent. Spots are revealed by spraying with a perchloric acid/molybdate reagent, and are identified and qualitatively assembled by reference to chromatograms of standard phosphates. Quantitative estimation is effected by cutting out the 'spots', washing the paper with ammonia, subsequent determination of the phosphorus content by colourimetry of the molybdenum blue complex and calculation of cyclic phosphate content as % NaPO3.
Solvent A (basic): Mix together: 400 ml isopropanol, 200 ml isobutanol, 300 ml deionised water and 10 ml 0.880 sp.gr. ammonia solution.
Solvent B (acidic): Mix together: 750 ml isopropanol and 250 ml deionised water. Add: 50 g trichloro-acetic acid and 2.5 ml 0.880 sp.gr. ammonia solution.
Spray reagent: To 50 ml deionised water, add: 5 ml 60% perchloric acid, 1 ml conc. HC1 (1.18 sp.gr.) and 1 g ammonium molybdate. Make up to 100 ml with deionised water
Standard Phosphate Solutions: Prepare standard solutions of sodium tri, tetra, hexa, and octameta phosphates containing 2 μg/μl (0.2% w/v).
Draw faint pencil lines 2.5 cm from the bottom edge and 2.5 cm from the right-hand side of a 23 x 23 cm square piece of the chromatography paper. Apply 1 µl of a 10% w/v solution of the sample at the intersection of the two pencil lines. Allow the paper to dry, curve it into a cylinder, and secure with plastic clips. Stand the cylinder in the tank containing the basic solvent (Solution A), the immersion depth being about 6 mm and allow the solvent front to rise to a height of 20 cm. Remove the paper from the tank and mark the position of the solvent front. Dry the paper in an air oven at 50° and cut off the excess paper above the solvent front.
Develop the paper in acid solvent (Solution B), with the previous right-hand edge to the bottom of the cylinder, until the solvent front has travelled 20 cm. Remove and dry the paper and spray with the acid ammonium molybdate solution. Develop the spots produced by placing the paper under the U.V. lamp at 250 nm for a few minutes.
Mark out a separate piece of chromatography paper as described above. At the intersection of the pencil lines apply 1 μl of each of the meta phosphate standard solutions in turn, drying the paper after each application. Treat this standard paper in a similar manner to that described for the sample. Both tests must be run concurrently using the same solvents, tanks and spray.
Compare the sample and standard chromatograms, and identify the 'spots' with the aid of the Rf values given in the Table below.
|Phosphate||Rf basic||Rf acidic|
values for ortho-, pyro- and cyclic phosphates
(Values should be taken as a guide only).
If a spot of particular interest is too weak, the chromatogram should be repeated using 2 or 5 µl sample solution instead of 1 µl. About 2 µl of each of the various phosphates should be visible.
An approximation of the quantities of each component in the sample will be gained by a visual comparison of the two chromatograms. For a more accurate measurement, cut out each spot and analyze for total phosphorus by the following method.:
Soak each cut out area of chromatography paper in 25.00- ml of 0.1 N ammonium hydroxide solution for at least 1 h. Pipet a 20.00- ml aliquot of the resulting solution into a 50 -ml volumetric flask, add 5 ml of 10 N sulfuric acid and heat in a boiling water bath for 30 min to hydrolyse the cyclic phosphates to orthophosphate. Cool to room temperature, add 1 ml of 12.5% ammonium molybdate solution, shake the flask and then add 1 ml of 0.6% hydrazine hydrochloride. Make up to volume with water and place the flask in a boiling water bath for exactly 10 min. Cool rapidly in a cold water bath and measure the absorbance of the solution in a spectrophotometer at 830 nm using distilled water as the reference solution. Perform a blank determination using an equal area of chromatography paper known not to include any phosphate spots and subtract the blank value from the test values. Determine the amount of phosphorus present by reference to a calibration curve of absorbance at 830 nm obtained using samples of standard amounts of potassium dihydrogen orthophosphate.
Where a spot is ill-defined, compare with the standard chromatogram and cut out the zone where the spot should appear. Cut out the area occupied by all the metaphosphates to obtain total cyclic content.
If x = is µl of a 10% solution of sample put on paper and y is= µg P obtained by attached method, then:
%P in sample = y / x
% cyclic phosphates expressed as NaPO3= (102 /31)(y / x)
This method should be used unless otherwise directed in the individual monograph.
Caution: When applying this test to organic compounds, the temperature at which the distillation is conducted must be rigidly controlled at all times to the recommended range of 135° to 140° to avoid the possibility of explosion.
Note: To minimize the distillation blank resulting from fluoride leached from the glassware, the distillation apparatus should be treated as follows:
Treat the glassware with hot 10% sodium hydroxide solution, followed by flushing with tap water and rinsing with distilled water. At least once daily, treat in addition by boiling down 15 to 20 ml of dilute sulfuric acid (1 in 2) until the still is filled with fumes; cool, pour off the acid, treat again with 10% sodium hydroxide solution, and rinse thoroughly. For further details, see sections 25.050 and 25.054 in Official Methods of Analysis of the AOAC, Thirteenth Edition, 1980.
Unless otherwise directed, place a 5.0 g sample and 30 ml of water in a 125 ml distillation flask having a side arm and trap. The flask is connected with a condenser and carries a thermometer and a capillary tube, both of which must extend into the liquid. Slowly add, with continuous stirring, 10 ml of perchloric acid, and then add 2 or 3 drops of silver nitrate solution (1 in 2) and a few glass beads. Connect a small dropping funnel or a steam generator to the capillary tube. Support the flask on an asbestos mat with a hole that exposes about one third of the flask to the flame. Distil until the temperature reaches 135°. Add water from the funnel or introduce steam through the capillary, maintaining the temperature between 135° and 140° at all times. Continue the distillation until 100 ml of distillate has been collected. After the 100 ml portion (Distillate A) is collected, collect an additional 50 ml portion of distillate (Distillate B) to ensure that all of the fluorine has been volatilized.
Place 50 ml of Distillate A in a 50 ml Nessler tube. In another similar Nessler tube place 50 ml of water distilled through the apparatus as a control. Add to each tube 0.1 ml of a filtered solution of sodium alizarinsulfonate (1 in 1,000) and 1 ml of freshly prepared hydroxylamine hydrochloride solution (1 in 4,000), and mix well. Add, dropwise and with stirring, either 1 N or 0.05 N sodium hydroxide, depending upon the expected volume of volatile acid distilling over, to the tube containing the distillate until its colour just matches that of the control, which is faintly pink. Then add to each tube 1.0 ml of 0.1 N hydrochloric acid, and mix well. From a burette, graduated in 0.05 ml, add slowly to the tube containing the distillate enough thorium nitrate solution (1 in 4,000) so that, after mixing, the colour of the liquid just changes to a faint pink. Note the volume of the solution added, then add exactly the same volume to the control, and mix. Now add to the control solution, sodium fluoride TS (10 µg F per ml) from a burette to make the colours of the two tubes match after dilution to the same volume. Mix well, and allow all air bubbles to escape before making the final colour comparison. Check the endpoint by adding 1 or 2 drops of sodium fluoride TS to the control. A distinct change in colour should take place. Note the volume of sodium fluoride TS added.
Dilute Distillate B to 100 ml, and mix well. Place 50 ml of this solution in a 50 ml Nessler tube, and follow the procedure used above for Distillate A. The total volume of sodium fluoride TS required for the solutions from both Distillate A and Distillate B should not exceed 2.5 ml.
Buffer Solution: Dissolve 36 g of cyclohexylenedinitrilotetraacetic acid (CDTA) in sufficient 1 M sodium hydroxide to make 200 ml. Transfer 20 ml of this solution (equivalent to 4 g of disodium CDTA) into a 1,000-ml beaker containing 500 ml of water, 57 ml of glacial acetic acid, and 58 g of sodium chloride, and stir to dissolve. Adjust the pH of the solution to between 5.0 and 5.5 by the addition of 5 M sodium hydroxide, then cool to room temperature, dilute to 1,000 ml with water, and mix.
Unless otherwise directed in the individual monograph, transfer 8.0 g of sample and 20 ml of water to a 250-ml distilling flask, cautiously add 20 ml of perchloric acid, and then add 2 or 3 drops of silver nitrate solution (1 in 2) and a few glass beads. Following the directions, and observing the Caution and Note, as given under Method I, distil the solution until 200 ml of distillate has been collected.
Treat the glassware with hot 10% sodium hydroxide solution, followed by flushing with tap water and rinsing with distilled water. At least once daily, treat in addition by boiling down 15 to 20 ml of dilute sulfuric acid (1 in 2) until the still is filled with fumes; cool, pour off the acid, treat again with 10% sodium hydroxide solution, and rinse thoroughly. For further details, see sections 25.050 and 25.054 in Official Methods of Analysis of the AOAC, Thirteenth Edition, 1980.
Transfer a 25.0-ml aliquot of the distillate into a 250-ml plastic beaker, and dilute to 100 ml with the Buffer Solution. Place the fluoride ion and reference electrodes (or a combination fluoride electrode) of a suitable ion-selective electrode apparatus in the solution. Adjust the calibration control until the indicator needle points to the center of the logarithmic concentration scale, allowing sufficient time for equilibration (about 20 min) and stirring constantly during the equilibration period and throughout the remainder of the procedure. Pipet 1.0 ml of a solution containing 100 μg of fluoride (F) ion per ml (prepared by dissolving 22.2 mg of sodium fluoride, previously dried at 200° for 4 h, in sufficient water to make 100.0 ml) into the beaker, allow the electrode to come to equilibrium, and record the final reading on the logarithmic concentration scale. (Note: Follow the instrument manufacturer's instructions regarding precautions and interferences, electrode filling and check, temperature compensation, and calibration.)
Calculate the fluoride content, in mg/kg of the sample taken by the formula
in which I is the initial scale reading before the addition of the sodium fluoride solution; A is the concentration, in μg per ml, of fluoride in the sodium fluoride solution added to the sample solution; R is the final scale reading, after addition of the sodium fluoride solution; and W is the original weight of the sample in g.
Sodium Fluoride Solution (5 µg F per ml): Transfer 2.210 g of sodium fluoride, previously dried at 200° for 4 h and accurately weighed, into a 400-ml plastic beaker, add 200 ml of water, and stir until dissolved. Quantitatively transfer this solution into a 1,000-ml volumetric flask with the aid of water, dilute to volume with water, and mix. Store this stock solution in a plastic bottle. On the day of use, transfer 5.0 ml of the stock solution into a 1,000-ml volumetric flask, dilute to volume with water, and mix.
Standard Curve: Transfer into separate 250-ml plastic beakers 1.0, 2.0, 3.0, 5.0, 10.0, and 15.0 ml of the Sodium Fluoride Solution, add 50 ml of water, 5 ml of 1 N hydrochloric acid, 10 ml of 1 M sodium citrate, and 10 ml of 0.2 M disodium EDTA to each beaker, and mix. Transfer each solution into separate 100-ml volumetric flasks, dilute to volume with water, and mix. Transfer a 50-ml portion of each solution into separate 125-ml plastic beakers, and measure the potential of each solution with a suitable ion-selective electrode, using a suitable reference electrode. Plot the standard curve on two-cycle semi-logarithmic paper with µg F per 100 ml solution on the logarithmic scale.
Transfer 1.00 g of the sample into a 150-ml glass beaker, add 10 ml of water, and while stirring continuously, add 20 ml of 1 N hydrochloric acid slowly to dissolve the sample. Boil rapidly for 1 min, then transfer into a 250-ml plastic beaker, and cool rapidly in ice water. Add 15 ml of 1 M sodium citrate and 10 ml of 0.2 M disodium EDTA, and mix. Adjust the pH to 5.5 ±0.1 with 1 N hydrochloric acid or 1 M sodium hydroxide, if necessary, then transfer into a 100-ml volumetric flask, dilute to volume with water, and mix. Transfer a 50-ml portion of this solution into a 125-ml plastic beaker and measure the potential of the solution with the apparatus described under Standard Curve. Determine the fluoride content, in μg, of the sample from the Standard Curve.
Buffer Solution: Dissolve 150 g of sodium citrate dehydrate and 10.3 g of disodium EDTA dihydrate in 800 ml of water, adjust the pH to 8.0 with 50% sodium hydroxide solution, and dilute to 1000 ml with water.
Fluoride Standard Solutions
Fluoride Limit Solutions (for a 1-g sample)
Fluoride Limit Solutions (for a 2-g sample)
Note: Store all standard and limit solutions in plastic containers.
Sample Preparation: Accurately weigh the amount of sample specified in the monograph, transfer it into a 100-ml volumetric flask, and dissolve it in a minimal amount of water. Add 50.0 ml of the Buffer Solution, dilute to volume with water, and mix.
Electrode Calibration: Pipet 50 ml of the Buffer Solution into a plastic beaker. Place the fluoride ion and reference electrodes (or a combination fluoride electrode) into the plastic beaker and stir. At 5-min intervals, add 100 µl and 1000 µl of the 1000 mg/kg Fluoride Standard and read the potential, in millivolts, after each addition. The difference between the two readings is the slope of the fluoride electrode and should typically be in the range of 54 to 60 mV at 25°. If the difference in potential is not within this range, check, and, if necessary, replace the electrode, instrument, or solutions.
Transfer the entire sample into a plastic beaker. Place the electrode into the beaker, allow the solution to equilibrate for 5 min with stirring, and read the potential, in millivolts. Remove and rinse the electrode(s) with water. In another beaker, using a pipet, add 50 ml of the Buffer Solution followed by 50 ml of the Fluoride Limit Solution that best reflects the fluoride limit of the sample. Place the electrode in the beaker, equilibrate for 3 min, and read the potential in millivolts. If the potential of the Fluoride Limit Solution is less than that of the sample, the sample passes the test criterion for maximum acceptable fluoride level limit.
Note: It is recommended to use an appropriate AAS/ICP method in the place of the limit test, if possible, for quantitative determination of iron.
To 0.5 g of the sample, weighed to the nearest mg, add 2 ml of hydrochloric acid, and evaporate to dryness on a steam bath. Dissolve the residue in 2 ml of hydrochloric acid and 20 ml of water, and add a few drops of bromine TS. Boil the solution in a fume hood to remove the bromine, cool, dilute with water to 25 ml, and then add 50 mg of ammonium persulfate and 5 ml of ammonium thiocyanate TS. Any red colour produced should not exceed that of a control solution made the same way as the test solution but containing instead of the sample the amount of Iron Standard TS prescribed in the individual monograph.
Loss on drying is the amount of volatile matter expelled under the conditions specified in the monograph. Because the volatile matter may include materials other than water, this test is designed for compounds in which the loss on drying may not definitely be attributable to water alone. The water content may be determined by a method such as Karl Fischer titration method
Note: Suitable precautionary steps should be taken when weighing hygroscopic or deliquescent samples to ensure that they do not absorb moisture.
Unless otherwise directed in the individual monograph, conduct the determination on 1 to 2 g of the substance, previously well mixed and accurately weighed. Reduce the sample to a fine powder when it occurs as crystals. Tare a glass-stoppered, shallow weighing bottle that has been dried for 30 min under the same conditions as will be employed in the determination. Transfer the sample into the bottle, replace the cover, and weigh the bottle and the sample. Distribute the sample as evenly as practicable to a depth of about 5 mm, and not over 10 mm in the case of bulky materials. Place the bottle with its contents in the drying chamber, removing the stopper and leaving it also in the chamber, and dry the sample at the temperature and for the time specified in the monograph. Upon opening the chamber, close the bottle promptly and allow it to come to room temperature in a desiccator before weighing.
If the substance melts at a lower temperature than that specified for the determination of Loss on Drying, prepare the sample as described above, then place it in a vacuum desiccator containing sulfuric acid. Evacuate the desiccator to 130 Pa (1 mm of mercury), maintain this vacuum for 24 h, and then weigh the dried sample.
Proceed as directed for Loss on Drying. However, unless otherwise directed, ignite the sample at a temperature of 450 - 550° and use a platinum, quartz or porcelain dish instead of the weighing bottle.
All the procedures for trace metals commence with dissolution of the sample and, if applicable, with destruction of organic matter in the sample. The trace metal content may then be determined by instrumental or chemical methods.
Atomic spectroscopy (atomic absorption as well as atomic emission) combines speed with accuracy and is widely used for the determination of metallic impurities.
Chemical methods depend on the formation of coloured compounds (complexes) with metal impurities under controlled conditions. The colour intensities of sample and standards are then compared visually or by using a spectrophotometer. Some of these methods lack in specificity and are subject to interferences from other trace elements.
The samples are dissolved in acid or digested in a mixture of sulfuric, nitric and, in some cases perchloric acids. Metals (barium, cadmium, lead, copper, chromium, and zinc) in solution are determined by suitable atomic absorption spectrophotometry (AAS) or inductively coupled plasma (ICP) methods. The choice of flame/furnace AAS or ICP methods depend on the concentration of the analyte in the prepared sample solution (its concentration in the sample and limitations associated with the sample preparation). Furnace technique, offers better sensitivity, may be preferred over flame technique, when dealing with low levels of impurities in complex matrices. Antimony and arsenic may be determined by using a hydride generation AAS or ICP. Alternatively, antimony may be determined by flame atomic absorption but the hydride generation technique is more sensitive.
Because of the minute amounts of metals involved special care must be taken to reduce the reagent blanks to as low a value as possible. Contamination in the laboratory is a major concern in trace metal analysis. All apparatus should be thoroughly cleaned with a mixture of hot dilute acids (1 part hydrochloric acid, 1 part concentrated nitric acid, and 3 parts water) followed by thorough washing with water immediately before use. All operations involving acids shall be carried out in the specified fume cupboards. Note: Special care must be taken while using perchloric acid.
Kjeldahl flasks, of silica or borosilicate glass (nominal capacity 100 ml) fitted with an extension to the neck by means of a B24 ground joint, as shown in Figure 2. The extension serves to condense the fumes and carries a tap funnel through which the reagents are introduced.
Figure 2. Modified Kjeldahl Flask (open type)
Atomic absorption spectrophotometer: Any commercial instrument operating in the absorption mode may be used providing it has required accessories (furnace and vapour generation) and facilities for the selection of the required oxidant/fuel combination from a choice of air, argon, nitrous oxide, hydrogen and acetylene and has a wavelength range from 180 to 600 nm.
All automated instruments have the facility of instrument control (selection of lamp, pre- warm up of lamp, wavelength and slit width and optimization) data acquisition and processing through a suitable software in a data station. However, with classical instruments, these need to be set manually and for operations in emission mode and measurements of absorption involving the generation of a gaseous hydride, a potentiometric recorder is necessary, preferably a multi-range type covering the range 1 -20 mV.
Inductively coupled plasma-atomic emission spectrophotometer: Any commercial instrument, sequential or simultaneous system, operating in axial or radial mode may be used.
Reagents shall be of an order of purity higher than accepted analytical reagent grade quality, preferably atomic spectroscopy grade. Metal-free water (Distilled water may be re-distilled from an all-glass apparatus or may be passed down a column of cation exchange resin, e.g., Amberlite IR 120(H), shall be used throughout. Deionized water (water subjected to reversed osmosis followed by passing ultra high quality deionisers) may also be used.
Preparations of atomic absorption standard solutions from pure metals or salts in the laboratory is cumbersome and subject to errors as large numbers of dilutions are involved. Single as well as mixed standard solutions in different concentration ranges are commercially available. Certified standards are also available for reference purpose. It is recommended to use commercially available standard solutions. The analyst must exercise proper care when diluting the stock standard solutions to not exceed a dilution factor of 20 in each step while diluting the concentrated solutions, in order to minimize dilution errors. Dilute the single standard stock solutions with 1% nitric acid to get the following solutions:
Preparation of test solutions
Method I is applicable to substances soluble in dilute acids or mixtures of acids. Method II is used for other substances. The choice of method for the pre-treatment of a substance can also follow that given in the individual monograph.
Accurately weigh about 2.5 g of the sample and dissolve in a mixture of 4 ml of sulfuric acid and 5 ml of hydrochloric acid. Transfer the solution to a 50-ml volumetric flask. If barium is to be measured from the solution, add 0.0954 g of potassium chloride. Dilute to the mark with water. Mark this as Solution A.
Note: Special care shall be taken while handling perchloric acid. All operations shall be carried out in a perchloric acid fume cupboard.
Accurately weigh a known quantity of sample (about 2.5 g or based on the expected concentration of metal(s) in solution when made up to the volume, such that the concentration in solution will be higher than the first standard of the standard curve) into a 100 - 150-ml Kjeldahl flask, and add 5 ml of the dilute nitric acid. As soon as any initial reaction subsides, heat gently until further vigorous reactions cease and then cool. Add gradually 4 ml of sulfuric acid TS at such a rate as not to cause excessive frothing on heating (5-10 min is usually required) and then heat until the liquid darkens appreciably in colour, i.e., begins to char.
Add concentrated nitric acid slowly in small portions, heating between additions until darkening again takes place. Do not heat so strongly that charring is excessive, or loss of arsenic may occur; a small but not excessive amount of free nitric acid should be present throughout. Continue this treatment until the solution is only pale yellow in colour and fails to darken in colour on prolonged heating. If the solution is still coloured add 0.5 ml of the perchloric acid solution and a little concentrated nitric acid and heat for about 15 min, then add a further 0.5 ml of the perchloric acid solution and heat for a few minutes longer. Note the total amount of concentrated nitric acid used. Allow to cool somewhat and dilute with 10 ml of water. The solution should be quite colourless (if much iron is present it may be faintly yellow). Boil down gently, taking care to avoid bumping, until white fumes appear. Allow to cool, add a further 5 ml of water and again boil down gently to fuming. Finally, cool, add 10 ml 5 N hydrochloric acid and boil gently for a few minutes. Cool and transfer the solution to a 50-ml volumetric flask washing out the Kjeldahl flask with small portions of water. Add the washings to the graduated flask and dilute to the mark with water. If barium is to be measured from the solution, add 0.0954 g of potassium chloride before dilution, as an ionizing buffer to prevent ionization of barium. Mark this as Solution A.
Prepare a reagent blank using the same quantities of reagents as used in the sample preparation.
Preparation of standard curve solutions
To a series of 100-ml volumetric flasks pipette 0, 1, 2, 3, 4 and 5 ml of the appropriate standard solution [standards (a) to (f) and (h)] and dilute to about 50 ml. Add 8 ml concentrated sulfuric acid and 10 ml concentrated hydrochloric acid. In the case of barium [standard (f)], add 0.191 g of potassium chloride as an ionization buffer and shake to dissolve. Dilute to the mark with metal free water.
These solutions then contain 0, 1.0, 2.0, 3.0, 4.0 and 5.0 µg/ml for lead; 0, 2.0, 4.0, 6.0, 8.0 and 10.0 µg/ml for barium and antimony; , 0, 0.1, 0.2, 0.3, 0.4 and 0.5 µg/ml of cadmium and zinc or 0, 0.50, 1.0, 1.5, 2.0, 2.5 µg/ml for copper and chromium.
Select the wavelengths and gases to be used for the particular element under consideration from the table below.
|Element||Wave length (nm)||
The recommended settings for the various instrumental parameters vary from model to model, and certain parameters require optimization at the time of use to obtain the best results. Instruments should therefore be adjusted as described in the manufacturer's instructions using the type of flame and wavelength settings specified above.
Set the atomic absorption spectrophotometer to the appropriate conditions. Aspirate the strongest standard containing the element to be determined and optimize the instrument settings to give full-scale or maximum deflection on the chart recorder. Measure the absorbances of the other standards and plot a graph showing the net absorbance against the concentration of the element in the standard solutions. Aspirate the solution A obtained from dissolution or the wet oxidation of the sample and the corresponding blank solution and determine the net absorbance. If the concentration of the element in the solution is beyond the standard curve, dilute the solution as required and read the absorbance again. Using the graph prepared above, determine the concentration of the element in the sample solution.
Element in the sample [mg/kg] =
[Concentration of element (μg/ml) x 50] / [Weight of sample taken (g)]
For certain elements flame atomic absorption method will not reach the required determination limits (e.g. a monograph specification limit for Pb of 1.0 mg/kg: A maximum of 5.0 grams of sample digested and made up to 50 ml will give a concentration of 0.1 µg/ml in solution which cannot be read by flame technique). The analyst may choose to use an electro-thermal atomization method under such circumstances.
Preparation of standard curve solutions
The standard curve solutions given below are nominal in nature. The concentration of standard curve solutions differ based upon the operation mode of the torch (axial or radial) of the ICP instrument. The analyst may alternatively prepare appropriate standard curve solutions following the instrument operation manual.
To a series of 100-ml volumetric flasks pipette 0, 1, 2, 3, 4 and 5 ml of the appropriate standard solution [standards (a) to (h)] and dilute to about 50 ml. Add 8 ml concentrated sulfuric acid and 10 ml concentrated hydrochloric acid. Dilute to the mark with metal free water. These solutions then contain 0, 1.0, 2.0, 3.0, 4.0 and 5.0 μg/ml for lead; 0, 2.0, 4.0, 6.0, 8.0 and 10.0 μg/ml for barium and antimony; , 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg/ml of cadmium and zinc or 0, 0.50, 1.0, 1.5, 2.0, 2.5 µg/ml for copper and chromium.
Select appropriate emission wavelengths to be used with each element under consideration. The recommended settings for the various instrumental parameters vary from model to model, and certain parameters require optimization at the time of use to obtain the best results. Instruments should therefore be adjusted as described in the manufacturer's instructions.
Set the ICP instrument as stated in the operation manual. Activate the method and key in the standards data into the data station of the ICP. Aspirate the blank solution and set the instrument to zero, aspirate the standards and determine a standard curve for each element with emission intensity plotted against the concentration of the element in the standard solutions. Aspirate the solution A obtained from dissolution or the wet oxidation of the sample. If the concentration of the element in the solution is beyond the standard curve, dilute the solution as required and read it again. Using the standard curve, determine the element in the sample.
Element in the sample [mg/kg] =
[Concentration of element (μg/ml) x 50] / [Weight of sample taken (g)]
Chemical modifier solutions:
Use of chemical modifier solutions in the furnace atomization allows use of higher ashing temperatures to reduce the background absorbance. These solutions must be of very high purity and are available commercially. One or more of the following modifiers may be used for the determination of lead and cadmium in different food additives.
Preparation of standard curve solutions
In a 100-ml volumetric flask, pipette 25 ml of lead and 10 ml cadmium standards (e and h) and dilute to the mark with water (standard solution A, 1 ml = 25 µg of pb and 1.0 μg of cd). Dilute 10 ml of A to 100 ml with water (standard solution B, 1 ml = 2.5 μg of pb and 0.1 μg Cd). Dilute 10 ml of B to 100 ml with water (standard solution C, 1 ml = 250 ng of pb and 10 ng Cd). Dilute 10 ml of C to 100 ml with water (working standard solution D, 1 ml = 25 ng of pb and 1 ng Cd).
General instrumental conditions are provided in the table below. The recommended settings for the various instrumental parameters vary from model to model, and certain parameters require optimization at the time of use to obtain the best results. Instruments should therefore be adjusted as described in the manufacturer's instructions.
Wave length (nm)
Maximum Ashing Temperature
Place blank (1% nitric acid), working standard solution (solution D), a suitable modifier solution (if required) and sample solutions in the appropriate locations provided in the furnace auto sampler. Set up the furnace parameters following the instruction provided by the manufacturer to carry out triplicate injections. Clean the graphite tube and inject blank. Program the auto sampler to inject 5, 10, 15, 20 µl of standard (5 µl of modifier and remaining blank solution so as the total volume is 25 µl). Construct the standard curve from the absorbance either from peak area or height. Inject 10 µl of sample solution and calculate the concentration in the samples as follows:
|Injection volume of sample to furnace:||10 µl|
|Volume made up :||50 ml|
|Instrument reading (ng) :||R|
|Weight of sample, g :||W|
|Concentration in sample (mg/kg) = (Rx 5)/W|
Arsenic and antimony are determined after preparation of their volatile hydrides which are collected either in the generation vessel itself or, in some designs, in a rubber balloon attached to the vessel. The gases are then expelled with Argon into a hydrogen flame.
Preparation of standard curve solution
Into a series of 100-ml volumetric flasks add from a burette, 0, 1, 2, 3, 4 and 5 ml of standard arsenic or antimony solution [Standards (g) and (d)] and dilute to about 50 ml with distilled water. Add 8 ml 98% sulfuric acid TS and 10 ml hydrochloric acid [1.18 specific gravity]. Shake to dissolve, and when solution is complete, dilute to the mark with distilled water.
Using the atomic absorption spectrophotometer with the appropriate hollow cathode or electrode-less discharge lamp, select the wavelength for either arsenic (193.7 nm) or antimony (217.6 nm).
Measure 5.0 ml of the strongest standard into the generation vessel, add 25 ml of water and 2 ml 5 N hydrochloric acid. Stopper the vessel and expel any air as described in the maker's instructions, filling the apparatus with Argon. Isolate the vessel from the atomizer using the by-pass valve. Remove the atomizer and then quickly add 1 pellet (about 0.2 g) of sodium borohydride and replace the stopper. Ensure that all the joints are secure.
When the reaction slows (20 - 30 sec) open the appropriate taps to allow the Argon to drive the generated hydride into the flame. When the hydride has all been expelled as shown by the recorder trace, return the taps to their original position and empty the vessel.
Optimize the instrument settings to give full scale deflection for the strongest standard. Measure the other standards, the sample and the blank solution using the same procedure.
Plot a graph relating peak height on the recorder to concentration of the arsenic or antimony in the standards. Using the net absorbance of the sample, read the concentration of arsenic or antimony in the solution from the graph .
Arsenic or antimony in the sample (mg/kg)
[Concentration of arsenic or antimony (μg/ml) x 50] / [Weight of sample taken (g)]
The sample is digested under closed conditions by heating under reflux with sulfuric and nitric acids. The oxidation is completed by addition of potassium permanganate solution. After successive additions of hydroxylamine hydrochloride solution and stannous chloride solution, the mercury content is measured by cold vapour atomic absorption spectrometry. Alternatively, closed vessel microwave digestion system may be used for the digestion of samples.
Dilute commercially available mercury standard solution (e.g. 10 µg/ml) following a serial dilution technique (dilution factor in each dilution not to exceed 20) to get 0.02 µg Hg/ml
All the glassware must be cleaned with nitric acid (10% v/v) and washed thoroughly with water before use.
Mineralization apparatus fitted with reflux condenser (see figure 3).
Bubblers, with a ground glass stopper fitted with two tubes to permit entrainment of the mercury vapour and with a calibration mark at the required volume for measurement. The capacity of the bubbler and position of the mark depend on the atomic absorption spectrophotometer used. Clean the bubbler successively with chromic acid mixture (dissolve 4.0 g of potassium dichromate in 300 ml of 3.5 M sulfuric acid and make up to 1 litre with water), tap water and double distilled water before use.
(Alternatively, use the vapour generation accessory and follow operation instructions for its use)
Water vapour absorption apparatus, containing magnesium perchlorate.
Atomic absorption spectrophotometer suitable for the cold vapour determination of mercury in open or closed circuit, with a data station or recorder.
Figure 3. Mineralization Apparatus
Digestion of sample
Weigh out, to the nearest 2 mg, approximately 0.5 g sample containing not more than 0.5 μg total mercury. Introduce the sample into the receiver flask (M), and add a few glass beads. Connect the receiver flask to the condensate reservoir (D) and close the stopcock (R).
Introduce into the reservoir 25 ml of nitric acid (sp.gr. 1.40) followed by 10 ml sulfuric acid (sp.gr. 1.84). Mount and turn on the condenser (A). Open the stopcock carefully and allow small portions of the mixture of acids to run into the receiver flask. Interrupt the flow of acids if the reaction becomes too vigorous.
Empty the reservoir into the receiver flask, mix the contents of the latter well by careful shaking and leave the stopcock open.
Heat the receiver flask carefully. As soon as foaming has ceased, close the stopcock (R), continue heating and let the condensate collect in the reservoir.
Discontinue heating when the contents of the receiver flask begin to char. Allow a small portion of the condensate to run into the receiver flask, close the stopcock again and resume heating the receiver flask. Repeat this procedure for as long as the contents display charring when heated.
When charring has ceased, heat and add condensate as soon as white fumes appear. Continue alternately heating and adding condensate for one hour. Finally, heat the contents of the flask to white fumes.
Stop heating and allow to cool to approximately 40°. Open the stopcock and allow all the condensate to run into the receiver flask. Wash the apparatus out from the top of the condenser with 5 - 10 ml of water, collect the washings in the receiver flask and disconnect it from the reservoir.
Treatment of the Solution
Introduce the potassium permanganate solution (50.0 g/l) dropwise into the receiver flask, with agitation, until a pink colouration persists. Note the volume of permanganate solution used. (If this quantity exceeds 10 ml, repeat the procedure "Ashing" as above.)
Heat gently to boiling, then allow to cool.
Pour the contents of the receiver flask into a bubbler, wash the receiver flask with water and add the washings to the contents of the bubbler.
Measure the mercury content (see below) the same day as the treatment of the solution.
Measurement of Mercury Content
Introduce 5 ml of hydroxylamine hydrochloride (100 g/l) into the bubbler and make up to the mark either with double distilled water or with sulfuric acid (3.5 M solution) in the case of standard solutions. Add 5 ml of stannous chloride solution [prepare by dissolving 25.0 g of stannous chloride (SnCl2.2H2O) in 50 ml hydrochloric acid (sp. gr. 1.18), make up to 250 ml with water and bubble nitrogen through the solution. Store over a few granules of metallic tin], assemble the bubbler, connect it to the water vapour absorption apparatus and to the atomic absorption spectrophotometer. Set the latter in operation.
Mix the contents of the bubbler well by gentle shaking, pass air or nitrogen through, measure and record. Carry out measurements as quickly as possible after the addition of stannous chloride. If an open-circuit system is used, wait 30 sec before passing air or nitrogen.
Introduce respectively 2-, 5-, 10-, 15- and 25-ml aliquots of the standard mercury solution (0.02 µg Hg/ml) into bubblers and 25 ml sulfuric acid (3.5 M) into a sixth bubbler. Add potassium permanganate solution dropwise, with agitation, to each bubbler until a colouration persists.
Measure the mercury content as described above.
Plot the standard curve with the measured absorption values as ordinates and the corresponding mercury contents in micrograms as abscissae. The working standards contain 0, 0.04, 0.10, 0.20, 0.30 and 0.50 µg of mercury, respectively.
Method of Addition
The method of addition may be used if an open-circuit system is used.
Place one of the working standard solutions in a bubbler and add an aliquot portion of the sample solution obtained after treatment. The quantity of mercury in the bubbler must lie in the range in which the photometer gives a linear response. Measure the mercury content as described above. If necessary, carry out several such determinations, using different working standard solutions.
Carry out all the operations, from ashing to measurement, except for introduction of the sample. When treating the solution, add a quantity of potassium permanganate solution equal to that used for the experimental sample.
Read off from the standard curve the quantities, in μg, of mercury corresponding to the measured absorption values.
Subtract the quantity of mercury found in the blank from that found in the sample.
Net weight of mercury (μg) / sample weight (g) = mg/kg Hg in the sample
Note: This method is also applicable for determination of nickel in polydextroses.
Use a suitable atomic absorption spectrometer equipped with a nickel hollow cathode lamp and an air–acetylene flame to measure the absorbance of the Blank solution, the Standard solutions, and the Sample solution as directed under Procedure(below).
Dissolve 20.0 g of the sample in a mixture of equal volumes of dilute acetic acid TS and water and dilute to 100 ml with the same mixture of solvents. Add 2.0 ml of a 1% w/v solution of ammonium pyrrolidinedithiocarbamate and 10 ml of methyl isobutyl ketone. Mix and allow the layers to separate and use the methylisobutyl ketone layer.
Prepare in the same manner as the Sample solution, but omit the sample.
Prepare three Standard solutions in the same manner as the Sample solution but adding 0.5 ml, 1.0 ml, and 1.5 ml, respectively, of a standard nickel solution containing 10 mg/kg Ni, in addition to the 20.0 g of the sample.
Zero the instrument with the Blank solution. Then determine the absorbances at 232.0 nm of each of the Standard solutions and of the Sample solutionat least three times each, and record the average of the steady readings for each. Between each measurement, aspirate the Blank solution, and ascertain that the reading returns to its initial blank value.
Prepare a standard curve by plotting the mean absorbances vs concentration for the Standard solutions. Extrapolate the line joining the points on the graph until it meets the concentration axis. Read the concentration of nickel in the Sample solution at the intersection of the standard curve with the concentration axis.
Note: It is recommended to use an appropriate AAS/ICP method in the place of the limit test, if possible, for quantitative determination of nickel.
Dissolve 10 g of sample in sufficient water to produce 20 ml, add 3 ml bromine TS and 2 ml of a 20% w/v solution of citric acid, mix and add 10 ml of ammonia TS and 1 ml of dimethylglyoxime TS. Mix, dilute to 50 ml with water and allow to stand for 5 min; any colour produced is not more intense than that produced by similarly treating, at the same time, 1 ml of nickel standard solution [10 mg/kg Ni prepared by diluting 1.0 ml of a 0.401% w/v solution of nickel chloride (NiCl2.6H2O analytical reagent grade) with water to 100.0 ml] diluted to 20 ml with water (0.5 mg/kg Ni).
Caution: Provide adequate ventilation in the laboratory and do not permit accumulation of exposed mercury.
Note 1: The analyst may use commercially available automated Kjeldahl digestion and determination equipment for the determination of Kjeldahl nitrogen.
Note 2: All reagents should be nitrogen-free, where available, or otherwise very low in nitrogen content.
This method should be used unless otherwise directed in the individual monograph. It is not applicable for certain nitrogen-containing compounds that do not yield their entire nitrogen content upon digestion with sulfuric acid.
Nitrites and Nitrates Absent
Unless otherwise directed, transfer about 1 g of the substance, accurately weighed, to a 500-ml Kjeldahl flask of hard glass, wrapping the sample, if solid or semi-solid, in nitrogen-free filter paper to facilitate the transfer if desired. To the flask add 10 g of powdered potassium sulfate or anhydrous sodium sulfate, 500 mg of powdered cupric sulfate, and 20 ml of sulfuric acid. Gently heat the mixture, keeping the flask inclined at about a 45° angle, and after frothing has ceased, boil briskly until the solution has remained clear green in colour or almost colourless for 30 min. Cool, add 150 ml of water, mix, and cool again. Cautiously pour 100 ml of sodium hydroxide solution (2 in 5) down the inside of the flask so that it forms a layer under the acid solution, then add a few pieces of granulated zinc. Connect the flask to a distillation apparatus consisting of a Kjeldahl connecting bulb and a condenser, the delivery tube from which extends well beneath the surface of 50 ml of boric acid solution (1 in 25) contained in a 500-ml flask or bottle. Gently rotate the contents of the Kjeldahl flask to mix, and distil until about two-thirds of the solution has been collected in the receiving flask. To the receiving flask add methyl red/methylene blue TS, and titrate with 0.5 N sulfuric acid. Perform a blank determination substituting 2 g of sucrose for the sample and make the necessary corrections. Each ml of 0.5 N acid is equivalent to 7.003 mg of nitrogen.
Note: If it is known that the substance to be determined has low nitrogen content, 0.1 N acid may be used in place of the 0.5 N solution, in which case each ml of 0.1 N acid is equivalent to 1.401 mg of nitrogen.
Nitrites and Nitrates Present
Transfer to a 500-ml Kjeldahl flask of hard glass a quantity of the sample, accurately weighed, representing about 150 mg of nitrogen, add 25 ml of sulfuric acid in which 1 g of salicylic acid has been dissolved, mix, and allow to stand for 30 min, shaking frequently. Add 5 g of powdered sodium thiosulfate, mix, then add 500 mg of powdered cupric sulfate or mercuric oxide, and continue as directed as above, beginning with "Gently heat the mixture...". Prior to the digestion of substances known to have a nitrogen content exceeding 10%, add 500 mg to 1 g of benzoic acid to facilitate decomposition.
Transfer an accurately weighed or measured quantity of the sample equivalent to about 2 or 3 mg of nitrogen, to the digestion flask of a semi-micro Kjeldahl apparatus. Add 1 g of a powdered mixture of potassium sulfate and cupric sulfate (10 to 1), using a fine jet of water to wash down any material adhering to the neck of the flask, then pour 7 ml of sulfuric acid down the inside wall of the flask to rinse it. Add cautiously, down the inside of the flask, 1 ml of 30% hydrogen peroxide, swirling the flask during the addition (Caution: Do not add any peroxide during the digestion.)
Heat over a free flame or an electric heater until the solution has attained a clear blue colour and the walls of the flask are free from carbonized material. Cautiously add 20 ml of water, cool, then add through a funnel 30 ml of sodium hydroxide solution (2 in 5), and rinse the funnel with 10 ml of water. Connect the flask to a steam distillation apparatus and immediately begin the distillation with steam. Collect the distillate in 15 ml of boric acid solution (1 in 25) to which has been added 3 drops of methyl red/methylene blue TS and enough water to cover the end of the condensing tube. Continue passing the steam until 80 to 100 ml of distillate has been collected, then remove the absorption flask, rinse the end of the condenser tube with a small quantity of water, and titrate with 0.01 N sulfuric acid. Each ml of 0.01 N acid is equivalent to 0.140 mg (140 μg) of nitrogen.
When more than 2 to 3 mg of nitrogen is present in the measured quantity of the substance to be determined, 0.02 or 0.1 N sulfuric acid may be used in the titration if at least 15 ml of titrant is required. If the total dry weight of the material taken is greater than 100 mg, increase proportionately the quantities of sulfuric acid and sodium hydroxide added before distillation.
Unless otherwise indicated, transfer 100 ml of the sample into a tared 125-ml platinum evaporating dish, previously heated at 105° to constant weight, and evaporate the sample to dryness on a steam bath. Heat the dish at 105° for 30 min or to constant weight, cool in a desiccator, and weigh.
Weigh accurately about 200 - 300 mg of sample, dissolve in 25 ml of water and 10 ml of diluted nitric acid TS and boil for 30 min. Filter if necessary, and wash any precipitate, then dissolve the precipitate by the addition of 1 ml diluted nitric acid TS. Adjust the temperature to about 50°, add 75 ml of ammonium molybdate TS, and maintain the temperature at about 50° for 30 min, stirring occasionally. Allow to stand for 16 h or overnight at room temperature. Decant the supernatant, through a filter paper, wash the precipitate once or twice with water by decantation using 30 to 40 ml each time, and pour the washings through the same filter. Transfer the precipitate to the same filter, and wash with potassium nitrate solution (1 in 100) until the filtrate is no longer acid to litmus paper. Transfer the precipitate with filter paper to the original precipitation vessel, add 50.0 ml of 1 N sodium hydroxide, agitate and stir until the precipitate is dissolved, add 3 drops of phenolphthalein TS and titrate the excess alkali with 1 N sulfuric acid. Each ml of 1 N sodium hydroxide consumed is equivalent to 3.088 mg of P2O5.
Weigh accurately Transfer about 1.5 g of the sample, transfer into accurately weighed, into a 500-ml volumetric flask beaker, add 100 ml of water and 25 ml of nitric acid, and boil for 10 min on a hot plate. Cool, quantitatively transfer into a 500- ml volumetric flask, dilute to volume with water and mix. Pipet 20.0 ml of this solution into a 500- ml Erlenmeyer flask, add 100 ml of water and heat just to boiling. Add with stirring 50 ml of quimociac TS, then cover with a watch glass and boil for 1 min in a well ventilated hood. Cool to room temperature, swirling occasionally while cooling, then filter through a tared crucible (or fritted glass crucible of medium porosity), and wash with five 25-ml portions of water. Dry at about 225° for 30 min, cool and weigh. Each mg of precipitate thus obtained is equivalent to 32.074 µg of P205.
2,3-Diaminonaphthalene Solution: On the day of use, dissolve 100 mg of 2,3- diaminonaphthalene (C10H10N2) and 500 mg of hydroxylamine hydrochloride (NH2OHHC1) in sufficient 0.1 N hydrochloric acid to make 100 ml.
Selenium Standard Solution: Transfer 120.0 mg of powdered metallic selenium into a 1,000-ml volumetric flask, and dissolve in 100 ml of dilute nitric acid (1 in 2), warming gently on a steam bath to effect solution. Cool, dilute to volume with water, and mix. Transfer 5.0 ml of this solution into a 200-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 3 μg of selenium (Se).
Alternatively, commercially available selenium standard solution may suitably be diluted to obtain a 3 µg/ml solution.
Note: Method I referenced in older specifications is deleted — The colourimetric procedure described in Method II may be used, but it is recommended that, whenever possible, the determination of selenium is carried out using atomic absorption methods.
Preparation of Standard: Transfer 2.0 ml of the Selenium standard solution into a 150-ml beaker, add 50 ml of 2 N hydrochloric acid, and mix.
Sample Preparation: Transfer into a 150-ml beaker the amount of sample specified in the individual monograph, dissolve in 25 ml of 4 N hydrochloric acid, swirling if necessary to effect solution, heat gently to boiling, and digest on a steam bath for 15 min. Remove from heat, add 25 ml of water, and allow to cool to room temperature.
Place the beakers containing the standard preparation and the sample preparation in a fume hood. Cautiously add 5 ml of ammonium hydroxide to each beaker and to a third beaker containing 50 ml of 2 N hydrochloric acid to serve as the blank. Allow the solutions to cool, and then adjust the pH of each solution to 2.0 ± 0.2 with dilute ammonium hydroxide (1 in 2).
Add 200 mg of hydroxylamine hydrochloride to each beaker, swirl gently to dissolve, then without delay add 5 ml of 2.3-diaminonaphthalene solution to each solution, and mix. Cover each beaker with a watch glass, and allow to stand at room temperature for 100 min. Transfer the solutions into separate separators with the aid of about 10 ml of water, extract each solution with 5.0 ml of cyclohexane, shaking each separator vigorously for 2 min, and allow the layers to separate. Discard the aqueous phases, and centrifuge the cyclohexane extracts to remove any traces of water. Determine the absorbance of each extract in a 1-cm cell at the maximum at about 380 nm, with a suitable spectrophotometer, using the blank to set the instrument. The absorbance of the extract from the sample preparation is not greater than that from the standard solution when a 200-mg sample is tested, or not greater than one-half the absorbance of the extract from the standard solution when a 100-mg sample is tested.
Unless otherwise specified, place the prescribed quantity of the sample in a Nessler tube, dissolve it in about 30 ml of water, and neutralize with dilute hydrochloric acid TS if the solution is alkaline. Add 1 ml of dilute hydrochloric acid TS and dilute to 50 ml with water. If the use of a sample solution is prescribed, transfer the sample solution into a Nessler tube and dilute to 50 ml with water. Transfer the prescribed volume of 0.01 N sulfuric acid into another Nessler tube to serve as the standard, add 1 ml of dilute hydrochloric acid TS, and dilute to 50 ml with water.
If the solution containing the sample is not clear, filter both solutions under the same conditions. Add 2 ml of barium chloride TS to each solution, mix thoroughly, and allow to stand for 10 min. Compare the turbidity of the two solutions by observing the Nessler tubes from the sides and the tops against a black background. The turbidity of the sample does not exceed that of the standard.
Treat 10 g of sample, accurately weighed, with 100 ml of hot water and filter through a tared filtering crucible. Wash the insoluble residue with hot water, dry at 105° for 2 h, cool and weigh.
Note: Determine the water content by the method below, unless otherwise specified in the individual monograph.
The Karl Fischer titrimetric determination of water is based on the quantitative reaction of water with an anhydrous solution of sulfur dioxide and iodine in the presence of a buffer that reacts with hydrogen ions. In the original titrimetric solution, known as Karl Fischer Reagent, the sulfur dioxide and iodine are dissolved in pyridine and methanol. Pyridine-free reagents are more commonly used now and are to be preferred in order to avoid the use of pyridine (a hazardous reagent).
The test specimen is titrated with the Karl Fischer Reagent directly. The stoichiometry of the reaction is not exact, and the reproducibility of the determination depends on such factors as the relative concentrations of the Karl Fischer Reagent ingredients, the nature of the inert solvent used to dissolve the test specimen, the apparent pH of the final mixture, and the technique used in the particular determination. Therefore, an empirically standardized technique is used to achieve the desired accuracy. Precision in the method is governed largely by the extent to which atmospheric moisture is excluded from the system.
The titration of water is usually carried out with the use of anhydrous methanol as the solvent for the test specimen; however, other suitable solvents may be used for special or unusual test specimens. Substances that may interfere with the test results are ferric ion, chlorine, and similar oxidizing agents, as well as significant amounts of strong acids or bases, phosgene, or anything that will reduce iodide to iodine, poison the reagent, and show the sample to be bone dry when water may be present (false negative). 8-Hydroxyquinoline may be added to the vessel to eliminate interference from ferric ion. Chlorine interference can be eliminated with SO2 or unsaturated hydrocarbon. Excess pyridine or other amines may be added to the vessel to eliminate the interference of strong acids. Excess acetic acid or other carboxylic acids can be added to reduce the interference of strong bases. Aldehydes and ketones may react with the solution, showing the sample to be wet while the detector never reaches an endpoint (false positive).
Any apparatus may be used that provides for adequate exclusion of atmospheric moisture and for determination of the endpoint. In the case of a colorless solution that is titrated directly, the endpoint may be observed visually as a change in color from canary yellow to amber. More commonly, however, the endpoint is determined electrometrically with an apparatus employing a simple electrical circuit that serves to impress about 200 mV of applied potential between a pair of platinum electrodes (about 5 mm2 in area and about 2.5 cm apart) immersed in the solution to be titrated. At the endpoint of the titration, a slight excess of the reagent increases the flow of current to between 50 and 150 microamperes for 30 s to 30 min, depending on the solution being titrated. The time is shortest for substances that dissolve in the Reagent. The longer times are required for solid materials that do not readily go into solution in the Karl Fischer Reagent. With some automatic titrators, the abrupt change in current or potential at the endpoint serves to close a solenoid-operated valve that controls the burette delivering the titrant. A commercially available apparatus generally comprises a closed system consisting of one or two automatic burettes and a tightly covered titration vessel fitted with the necessary electrodes and a magnetic stirrer. The air in the system is kept dry with a suitable desiccant such as phosphorus pentoxide, and the titration vessel may be purged by means of a stream of dry nitrogen or a current of dry air.
A commercially available, stabilized solution of a Karl Fischer-type reagent may be used. Commercially available reagents containing solvents or bases other than pyridine and/or alcohols other than methanol also may be used. These may be single solutions or reagents formed in situ by combining the components of the reagents present in two discrete solutions. The diluted Karl Fischer Reagent called for in some monographs should be diluted as directed by the manufacturer. Either methanol, or another suitable solvent such as ethylene glycol monomethyl ether, may be used as the diluent.
Note: If commercial solution is not available, prepare Karl Fischer Reagent as follows:
Caution: Pyridine is hazardous in nature. The analyst shall take proper care and all operations shall be carried out in fume cupboard.
Add 125 g of iodine to a solution containing 670 ml of methanol and 170 ml of pyridine, and cool. Place 100 ml of pyridine in a 250-ml graduated cylinder, and keeping the pyridine cold in an ice bath, pass in dry sulfur dioxide until the volume reaches 200 ml. Slowly add this solution, with shaking, to the cooled iodine mixture. Shake to dissolve the iodine, transfer the solution to the apparatus, and allow the solution to stand overnight before standardizing. One milliliter of this solution, when freshly prepared, is equivalent to approximately 5 mg of water, but it deteriorates gradually; therefore, standardize it within 1 h before use, or daily in continual use. Protect the solution from light while in use. Store any bulk stock of the solution in a suitably sealed, glass-stoppered container, fully protected from light and under refrigeration.
Unless otherwise specified in the individual monograph, use an accurately weighed or measured amount of the specimen under test estimated to contain 10 to 250 mg of water. Where the monograph specifies that the specimen under test is hygroscopic, accurately weigh a sample of the specimen into a suitable container. Use a dry syringe to inject an appropriate volume of methanol, or other suitable solvent, accurately measured, into the container and shake to dissolve the specimen. Dry the syringe, and use it to remove the solution from the container and transfer it to a titration vessel prepared as directed under Procedure. Repeat the procedure with a second portion of methanol, or other suitable solvent, accurately measured; add this washing to the titration vessel; and immediately titrate. Determine the water content, in milligrams, of a portion of solvent of the same total volume as that used to dissolve the specimen and to wash the container and syringe, and subtract this value from the water content, in milligrams, obtained in the titration of the specimen under test.
Standardization of the Reagent
Place enough methanol or other suitable solvent in the titration vessel to cover the electrodes, and add sufficient Karl Fischer Reagent to give the characteristic color or 100 ± 50 microamperes of direct current at about 200 mV of applied potential. Pure methanol can make the detector overly sensitive, particularly at low mg/kg levels of water, causing it to deflect to dryness and slowly recover with each addition of reagent. This slows down the titration and may allow the system to actually pick up ambient moisture during the resulting long titration. Adding chloroform or a similar non-conducting solvent will retard this sensitivity and can improve the analysis. For determination of trace amounts of water (less than 1%), quickly add 25 µl (25 mg) of pure water, using a 25- or 50-µl syringe, and titrate to the endpoint.
The water equivalence factor F, in milligrams of water per milliliter of reagent, is given by the formula 25/V, in which V is the volume, in milliliters, of the Karl Fischer Reagent consumed in the second titration. For the precise determination of significant amounts of water (more than 1%), quickly add between 25 and 250 mg (25 to 250 µl) of pure water, accurately weighed by difference from a weighing pipet or from a precalibrated syringe or micropipet, the amount of water used being governed by the reagent strength and the burette size, as referred to under Volumetric Apparatus. Titrate to the endpoint.
Calculate the water equivalence factor, F, in milligrams of water per milliliter of reagent by the formula W/V, in which W is the weight, in milligrams, of the water, and V is the volume, in milliliters, of the Karl Fischer Reagent required.
Unless otherwise specified, transfer 35 to 40 ml of methanol or other suitable solvent to the titration vessel, and titrate with the Karl Fischer Reagent to the electrometric or visual endpoint to consume any moisture that may be present. (Disregard the volume consumed because it does not enter into the calculations.) Quickly add the test preparation, mix, and again titrate with the Karl Fischer Reagent to the electrometric or visual endpoint.
Calculate the water content of the specimen, in milligrams, by the formula SxF, in which S is the volume, in milliliters, of the Karl Fischer Reagent consumed in the second titration, and F is the water equivalence factor of the Karl Fischer Reagent.
Weigh 0.25 g of the sample to the nearest mg, and dissolve in 10 ml of water. Acidify with nitric acid and filter off the precipitate. Mix the precipitate with 0.5 g of calcium carbonate, dry the mixture and then ignite. Take up the ignition residue in 20 ml of dilute nitric acid TS and filter. Mix the filtrate with 0.5 ml of 0.1 N silver nitrate. The turbidity should not be greater than that obtained by adding 0.5 ml of 0.1 N silver nitrate to a similar volume of dilute nitric acid TS containing the amount of 0.01 N hydrochloric acid prescribed in the individual monograph.
Methyl orange-boric acid solution: Dissolve 200 mg of methyl orange and 3.5 g of boric acid in 100 ml of water, heating on a steam bath to effect solution. Allow to stand for at least 24 h, and filter before use.
Standard solution: Weigh accurately about 100 mg of cyclohexylamine in a 100-ml volumetric flask, dissolve in 50 ml of water and 0.5 ml of hydrochloric acid TS, dilute to volume with water, and mix. Transfer 5 ml of the solution into a second 100-ml volumetric flask, dilute to volume with water, and mix. Transfer 5 ml of the solution into a third 100-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 2.5 μg of cyclohexylamine.
Test preparation: Prepare the test preparation as directed in the individual monograph.
Transfer 10 ml each of the Standard solution and of the Test preparation into two separate 50-ml glass-stoppered centrifuge tubes, and transfer 10 ml of water to a third tube to serve as a blank. To each tube add 3.0 ml of disodium ethylenediaminetetraacetate solution (prepared by dissolving 10 g of disodium ethylenediaminetetraacetate and 3.4 g of sodium hydroxide in 100 ml of water) and 15 ml of a 20:1 mixture of chloroform and n-butanol, shake the tubes for 2 min, and centrifuge. Remove and discard the aqueous layer in each tube, and then transfer 10 ml of the chloroform solution from each tube into separate centrifuge tubes. To each tube add 2 ml of Methyl orange-boric acid solution, shake the tubes for 2 min, and centrifuge. Remove and discard the aqueous layer in each tube, then add to each tube 1 g of anhydrous sodium sulfate, shake well, and allow to settle. Transfer 5 ml of each clear chloroform solution into separate test tubes, add 0.5 ml of 50:1 mixture of methanol and sulfuric acid TS, and mix. Successively determine the absorbance of the solutions in 1 cm cells at 520 nm with a suitable spectrophotometer, using the blank to set the instrument at zero. The absorbance of the solution from the Test preparation does not exceed that from the Standard preparation.
Note: The procedure uses a packed column GC. In the absence of a packed column GC, capillary GC in the splitless mode, using an equivalent capillary column, may be used. GC conditions need to be established.
Standard solutions: Weigh accurately about 100 mg of dicyclohexylamine (C12H23N, Refractive index (25, D): 1.480-1.488, specific gravity: d (25, 25): 0.905-0.915, boiling point: 254-256°) in a 100-ml volumetric flask, dissolve in chloroform, dilute to volume with chloroform and mix (standard A, 1.0 mg/ml). Transfer 10 ml standard A into a 100-ml volumetric flask, dilute to volume with chloroform and mix (standard B, 100 μg/ml). Into a series of 10 ml volumetric flasks, Transfer 0.0, 1.0, 2.0, 4.0, 6.0 and 8.0 ml of standard B solution. Dilute to volume with chloroform, and mix. The working standard solutions contain 10.0, 20.0, 40, 60 and 80.0 μg/ml, respectively.
Dissolve 50 g of the sample in 300 ml of water, add 3 ml of sodium hydroxide TS, and extract with 50 ml and 30 ml of chloroform. Combine the extracts, add 2 g of anhydrous potassium carbonate and filter. Wash the container and the residue on the filter paper several times with 5 ml chloroform, combine the washings to the filtrate and concentrate in a rotary evaporator at 30° under vacuum, to about 0.5 ml, quantitatively transfer into a 2 ml volumetric flask, evaporate the solvent in the volumetric flask to about 0.5 ml under a stream of nitrogen, add 1 ml of nitrobenzene standard solution (100 mg in 500 ml chloroform) as an internal standard and make up to the mark with chloroform.
Gas Chromatographic Conditions
Column: Stainless steel, 1.5 m x 3-4 mm i.d., packed with 60-80 mesh diatomaceous earth (gas chromatographic grade) in a solution of methanolic potassium hydroxide. The final potassium hydroxide concentration should be about 3% of the diatomaceous support. Evaporate off the methanol, add a chloroform solution of polyethyleneglycol 6000, and evaporate the chloroform. The content of polyethyleneglycol 6000 should be about 10% of the diatomaceous support.
Carrier gas: Nitrogen or helium, flow rate should be set so that the retention time of nitrobenzene is about 7 min
Injection port, column, and detector temperatures: 225°, 130 -140°, and 250°, respectively.
Standard curve: Prepare a standard curve by mixing 1 ml of each of working standard solution with 1 ml of internal standard solution and analyze by gas chromatography using a flame ionization detector under the conditions described below. Prepare a standard curve by plotting concentration of dicyclohexylamine (in μg per ml), vs. the ratio of the dicyclohexylamine peak area to that of internal standard. Inject the sample solution and calculate the concentration of dicyclohexylamine (in μg per ml,) in the sample solution from the standard curve and calculate the dicyclohexylamine in the sample as follows.
Dicyclohexylamine (mg/kg) = Conc. in sample (μg/ml) x 0.02
Determine by headspace gas chromatography using the following procedure:
Stripped sample: Place 3000 g of the sample into a 5000-ml, 4-neck, round-bottom flask equipped with a stirrer, a thermometer, a gas dispersion tube, a dry ice trap, a vacuum outlet, and a heating mantle. At room temperature, evacuate the flask carefully to a pressure of less than 1 mmHg, applying the vacuum slowly while observing for excessive foaming due to entrapped gases. After any foaming has subsided, spurge with nitrogen, allowing the pressure to raise to 10 mmHg. Heat the flask to 60° while increasing the pressure to about 60mmHg. Continue stripping for 4 h, then cool to room temperature. Shut off the vacuum pump, and bring the flask pressure back to atmospheric while maintaining nitrogen sparging. Remove the sparging tube with the gas still flowing, then turn off the gas flow. Transfer the Stripped sample to a suitable nitrogen-filled container.
Standard Preparations: (Caution: Ethylene oxide and 1,4-dioxane are toxic and flammable. Prepare these solutions in a well-ventilated fume hood.) Add a suitable quantity of 1,4-dioxane to a known weight of organic-free water in a vial that can be sealed. Determine the amount added by weight difference. Using the special handling described in the following, complete the preparation. Ethylene oxide is a gas at room temperature. It is usually stored in a lecture-type gas cylinder or small metal pressure bomb. Chill the cylinder in a refrigerator before use. Transfer about 5 ml of the liquid ethylene oxide to a 100-ml beaker chilled in wet ice. Using a gastight gas chromatographic syringe that has been chilled in a refrigerator, transfer a suitable amount of the liquid ethylene oxide into the mixture. Immediately seal the vial, and shake. Determine the amount added by weight difference. By appropriate dilution with Stripped sample, prepare four solutions, covering the range from 1 to 20 mg/kg for the two components added to the matrix (e.g., 5, 10, 15, and 20 mg/kg). Transfer 10 ml of each of these solutions to separate 22-ml pressured headspace vials, seal each with a silicone septum, star spring, and pressure-relief safety aluminium sealing cap, and crimp the cap closed with a cap-sealing tool. Shake for 2 min.
Sample Preparation: Transfer 10 ± 0.01 g of sample to a 22-mL pressure headspace vial, and seal, cap, and crimp as directed for the Standard Preparations.
Gas chromatograph equipped with a balanced pressure automatic headspace sampler and a flame-ionization detector.
|Column:||50-m x 0.32-mm fused silica capillary column, or equivalent, bonded with a 5-mm film of 5% phenyl-95% ethylsiloxane, or equivalent.|
|Column temperature:||Program the column temperature from 70° to 250° at 10°/min|
|Transfer line temperature:||140°|
|Flow rate:||app. 0.8 ml/min.|
|Performance:||On the two Calibration plots, no point digresses from its line by more than 10%.|
Calibration: Place the vials containing the Standard Preparations in the automated sampler, and start the sequence so that each vial is heated at a temperature of 50° for 30 min before a suitable portion of its headspace is injected into the chromatograph. Set the automatic sampler for a needle withdrawal time of 0.3 min, a pressurization time of 1 min, an injection time of 0.08 min, and a vial pressure of 22 psig with the vial vent off. Obtain the peak areas for ethylene oxide and 1,4-dioxane, which have relative retention times of about 1.0 and 3.1, respectively. Plot the area versus milligram per kilogram on linear graph paper, and draw the best straight line through the points.
Procedure: Place the vial containing the Sample Preparation in the automatic sampler, and chromatograph its headspace as done for the Standard Preparations. Obtain the peak areas of each of the components, and read the concentrations directly from the Calibration plots.
Determine by HPLC using the following conditions:
Mobile Phase: Filtered, degassed solution of 0.01 N sulfuric acid in water.
Note: For all reference standards, do not dry before use, and keep the containers tightly closed and protected from light. Determine the water content of Fumaric Acid Reference Standard titrimetrically before use, and make the necessary correction in preparing the Standard Preparation.
Standard Preparation: Transfer about 5 mg of Fumaric Acid Reference Standard (USP or equivalent) and about 2mg of Maleic Acid Reference Standard (USP or equivalent), both accurately weighed, into a 1000-ml volumetric flask, dilute to volume with Mobile Phase, and mix.
Sample Preparation: Transfer about 100 mg of sample, accurately weighed, into a 100-ml volumetric flask, dilute to volume with Mobile Phase, and mix.
Resolution Solution: Transfer about 1 g of sample, about 10 mg of Fumaric Acid Reference Standard, and about 4 mg of Maleic Acid Reference Standard, all accurately weighed, into a 1000-ml volumetric flask, dilute to volume with Mobile Phase, and mix.
|Column:||30-cm x 6.5-mm (i.d.) column, or equivalent , packed with a strong cation exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the hydrogen form (Polypore H from Brownlee Lab, or equivalent)|
|Column temperature:||37 ±1°.|
|Flow rate:||App. 0.6 ml/min|
Inject a portion of the Resolution Solution, and obtain the chromatogram. Record the peak responses from the chromatogram. The resolution of the maleic acid and sample peaks is not less than 2.5; the resolution of the fumaric acid and sample peaks is not less than 7.0; and the relative standard deviation of the Sample Solution peak for replicate injections is not more than 2.0%.
Separately inject about 20 µl each of the Standard Preparation and the Test Preparation into the chromatograph, record the chromatograms, and measure the peak responses. The relative retention times are approximately 0.6 for maleic acid, approximately 1.0 for malic acid, and approximately 1.5 for fumaric acid. Calculate the quantities, in milligrams, of maleic acid and fumaric acid, in the portion of the sample taken by the formula 100C x (rU/rS), in which C is the concentration, in milligrams per milliliter, of the corresponding Reference Standard in the Standard Preparation, and rU and rS are the responses of the corresponding peaks from the Test Preparation and the Standard Preparation, respectively.
Boil a mixture of 200 mg of the sample and 20 ml of 10% sulfuric acid for 3 h. Allow to cool and add excess barium carbonate, mixing with a magnetic stirrer until the solution is pH 7, and filter. Evaporate the filtrate in a rotatory evaporator at 30 - 50° under vacuum until a crystalline (or syrupy) residue is obtained. Dissolve in 10 ml of 40% methanol. This is the hydrolysate. Place 1 to 5 µl spots of hydrolysate on the starting line of two Silica Gel G thin layer plates. On the same plates apply 1 to 10 μg of the reference standards specified in the individual monograph.
Develop one plate in solvent A and one plate in solvent B:
After development spray with a solution of 1.23 g anisidine and 1.66 g phthalic acid in 100 ml ethanol and heat the plates at 100° for 10 min. A greenish yellow colour is produced with hexoses, a red colour with pentoses and a brown colour with uronic acids. Compare sample spots with those for the solutions of the reference standards and identify the constituents specified in the individual monograph.
Determine by HPLC using the following:
Norbixin (purity 99 % or higher; prepare according to the procedure in Scotter et al. (1994, 1998) as it is not currently available commercially)
Note: all solvents should be HPLC-grade
HPLC system with a suitable pump, injector, and integrator
|Column:||Stainless steel; 250 x 4.6 mm|
|Stationary phase:||Mixed C8/C18 bonded phase, 5 μm or similar|
Column temperature: 35°
|Mobile phase:||Isocratic 65 % Solution A; 35 % Solution B Solution A: acetonitrile; Solution B: 2 % acetic acid (v/v)|
|Flow rate:||1.0 ml/min|
|Run time:||40 min|
Note: The retention time of norbixin is approximately 10 min
Standard solution: Weigh accurately about 25 - 50 mg of the norbixin standard and dissolve in 5 ml of 0.1 M NaOH solution. Transfer quantitatively to a 50 ml volumetric flask and dilute to volume with methanol.
Oil-soluble samples: Weigh accurately about 25 - 50 mg of the sample and dissolve in 3 to 5 ml of dimethylformamide. Transfer quantitatively to a 50 ml volumetric flask and dilute to volume with acetonitrile.
Water-soluble samples: Weigh accurately about 25 - 50 mg of the sample and dissolve in 5 ml of 0.1 M NaOH solution. Transfer quantitatively to a 50 ml volumetric flask and dilute to volume with methanol.
|As||is the peak area of the sample solution|
|ASt||is the peak area of the standard solution|
|PSt||is the purity of the standard expressed as a proportion of Norbixin in the norbixin standard (for example, 0.99 if the standard is 99% pure).|
|WSt||is the weight of the standard (mg)|
|Ws||is the weight of the sample (mg)|
Scotter, M.J.; Wilson, L.A.; Appleton, G.P. & Castle, L. Analysis of Annatto (Bixa orellana) food colouring formulations. 1. J. Agric. Food Chem. 1998, 46, 1031-1038.
Scotter, M.J.; Thorpe, S.A.; Reynolds, S.L.; Wilson, L.A. & Strutt, P.R. Characterisation of the principal colouring components of Annatto using high performance liquid chromatography withphotodiode-array detection. FoodAddit. Contam. 1994, 11, 301-315.
Dissolve 0.5 g of sample in 4 ml of water, add 3 ml concentrated hydrochloric acid and then 1 g of granulated zinc. Heat for 1 min in a boiling water bath. Let stand for 2 min at room temperature; decant the supernatant solution into a test tube containing 0.25 ml of a 1% solution of phenylhydrazine hydrochloride. Mix, heat to boiling and cool immediately. Transfer the solution into a glass cylinder with a ground glass stopper and add an equal volume of concentrated hydrochloric acid. Add 0.25 ml of a 5% solution of potassium hexacyanoferrate (III), mix well and let stand for 30 min. The colour of the solution is not more intense than that of a standard solution prepared in the same manner and containing 4.0 ml of a solution of 0.005% oxalic acid in water.
Examine by thin layer chromatography (TLC) using silica gel as the coating substance, and using standard and test solutions described in the individual monograph.
4-Aminobenzoic acid reagent: Prepare a solution by dissolving 1 g of 4-aminobenzoic acid in a solvent mixture composed of 18 ml acetic acid, 20 ml water and 1 ml phosphoric acid. Prepare this reagent immediately before use.
Sodium periodate reagent: 0.2% w/v sodium periodate in water
Apply 2 µl of each of the standard and test solution to the bottom of the TLC plate. Develop the chromatogram over a path of 17 cm using as the mobile phase a mixture of 70 volumes of propanol, 20 volumes of ethyl acetate and 10 volumes of water. Allow the plate to dry in air and spray with a mixture of 2 volumes of 4-aminobenzoic acid reagent with 3 volumes of acetone. Heat at 100° for 15 min. Spray with the sodium periodate reagent. Heat at 100° for 15 min. The principal spot in the chromatogram obtained from the test solution corresponds in position, colour and size to the principal spot obtained from the standard solution.
Proceed as directed under thin-layer chromatography (see Analytical Techniques) as follows: Sample: 2 µl of a 0.5 in 100 solution of the sample
Reference: 2 µl of a 0.5 in 100 solution of monosodium L-glutamate containing 2.5 mg of pyrrolidone carboxylic acid
Solvent: A mixture of 2 volumes of n-butanol, 1 volume of glacial acetic acid and 1 volume of water.
Adsorbent: Silica gel
Potassium iodide-starch solution: Stir and heat 0.5 g of starch in about 50 ml of water until it gelatinizes; after cooling add 0.5 g of potassium iodide and water to make up to 100 ml.
Stop the development when the solvent front has advanced about 10 cm from the point of the application dry the plate for 30 min in air.
At the same time, prepare a similar chamber to that used for developing; place a 50-ml beaker containing about 3 g of sodium hypochlorite in the chamber; slowly add 1 ml of hydrochloric acid into the beaker to generate chlorine gas; put on the lid and allow to stand for 30 sec to fill the chamber with chlorine. Place the dried plate in this chamber, put on the lid and allow to stand for 20 min. Take out the plate, keep for 10 min in air and spray with ethanol. After drying, spray with potassium iodide-starch solution and observe the plate under natural light immediately after the standard spot has appeared.
No spot corresponding to pyrrolidone carboxylic acid standard is detected in the sample (sensitivity = 0.2%).
Unless otherwise directed, add the specified quantity of the substance, finely powdered if in solid form, in small portions to the comparison container, which is made of colourless glass resistant to the action of sulfuric acid and contains the specified volume of sulfuric acid TS.
Stir the mixture with a glass rod until solution is complete, allow the solution to stand for 15 min, unless otherwise directed, and compare the colour of the solution with that of the specified matching fluid in a comparison container which also is of colourless glass and has the same internal and cross-section dimensions. View the fluids transversely against a background of white porcelain or white glass.
When heat is directed in order to effect solution of the substance in the sulfuric acid TS, mix the sample and the acid in a test tube, heat as directed, cool, and transfer the solution to the comparison container for matching.
For purposes of comparison, a series of twenty matching fluids, each designated by a letter of the alphabet, is provided, the composition of each being as indicated in the following table. To prepare the matching fluid specified, pipet the prescribed volumes of the colorimetric test solutions (TSC) and water into one of the matching containers, and mix the solutions in the container.
|Matching Fluid||Parts of Cobaltous Chloride TSC||Parts of Ferric Chloride TSC||Parts of Cupric Sulfate TSC||Parts of Water|
Note: Solutions A-D are very light brownish-yellow.; solutions E-L are yellow through reddish-yellow; solutions M-O are greenish-yellow; and solutions P-T are light pink.
Transfer about 1 g of the sample, accurately weighed, into a 250-ml Erlenmeyer flask, dissolve in 10 ml of water, and add 25 ml of alkaline cupric citrate TS and cover the flask with a small beaker. Boil gently for exactly 5 min and cool rapidly to room temperature. Add 25 ml of 10% acetic acid solution, 10.0 ml of 0.1 N iodine, 10 ml of dilute hydrochloric acid TS and 3 ml of starch TS, and titrate with 0.1 N sodium thiosulfate to the disappearance of the blue colour. Calculate the content of reducing substances (as D-glucose) by the formula:
V1 and N1 are the volume (ml) and normality, respectively, of the iodine solution,
V2 and N2 are the volume (ml) and normality, respectively, of the sodium thiosulfate solution, and 2.7 is an empirically determined equivalence factor for D-glucose.
Dissolve 7 g of the sample in 35 ml of water in a 400-ml beaker and mix. Add 25 ml of cupric sulfate TS and 25 ml of alkaline tartrate TS. Cover the beaker with glass, heat the mixture at such a rate that it comes to a boil in approximately 4 min and boils for exactly 2 min. Filter the precipitated cuprous oxide through a tared Gooch crucible previously washed with hot water, ethanol, and ether, and dried at 100° for 30 min. Thoroughly wash the collected cuprous oxide on the filter with hot water, then with 10 ml of ethanol and finally with 10 ml of ether, and dry at 100° for 30 min. The weight of the cuprous oxide does not exceed that prescribed in the individual monograph.
Proceed as directed under "Thin-layer chromatography" (see Analytical Techniques) using a sample of the solution described under Method of Assay in the monograph. Use a mixture of 80 volumes of a saturated solution of ammonium sulfate, 18 volumes of a 13.6% w/v solution of sodium acetate and 2 volumes of isopropanol as the developing solvent. Use microcrystalline cellulose as the absorbent. Stop the development when the solvent front has advanced about 10 cm from the point of the application, dry the plate in air, and observe under ultraviolet light (about 254 nm) in a dark place. Only a spot of 5'-guanylic acid or 5'-inosinic acid is detected.
The solvents listed in the table below can be determined by this method based on headspace gas chromatography. The method may also be used for the determination of isobutyl acetate and methyl acetate. However, information on the approximate retention time for these two solvents is not available.
|Solvent||Approximate retention times (min)||Solvent||Approximate retention times (min)|
sample with very low solvent content
Internal standard: 3-methyl-2-pentanone
Internal standard solution: Add 50.0 ml water to a 50 ml injection vial and seal. Accurately weigh and inject 15 (µl 3-methyl-2-pentanone through the septum and reweigh to within 0.01 mg.
Blank solution: Weigh accurately 0.20 g of the blank into an injection vial. Add 5.0 ml of water and 1.0 ml of the internal standard solution. Heat at 60° for 10 min and shake vigorously for 10 sec.
Samples: Weigh accurately 0.20 g sample into an injection vial. Add 5.0 ml water and add 1.0 ml of the internal standard solution. Heat at 60° for 10 min and shake vigorously for 10 sec.
Calibration solution: Weigh accurately 0.20 g of the blank into an injection vial. Add 5.0 ml of the water and 1.0 ml of the internal standard solution. Weigh the vial accurately to within 0.01 mg. Inject a known volume of the component of interest through the septum and again reweigh the vial. Heat at 60° for 10 min and shake vigorously for 10 sec.
Internal standard solution: Add 50.0 ml methanol to a 50 ml injection vial and seal. Accurately weigh and inject 15 (µl 3-methyl-2-pentanone through the septum and reweigh to within 0.01 mg.
Blank solution: Weigh accurately 0.20 g of the blank into an injection vial. Add 5.0 ml of methanol and 1.0 ml of the internal standard solution. Heat at 60° for 10 min and shake vigorously for 10 sec.
Samples: Weigh accurately 0.20 g sample into an injection vial. Add 5.0 ml methanol and add 1.0 ml of the internal standard solution. Heat at 60° for 10 min and shake vigorously for 10 sec.
Calibration solution: Solution A: Add 50.0 ml methanol to a 50 ml vial and seal. Accurately weigh, to within 0.01 mg, the vial and inject 50 (µl of the component of interest through the septum. Reweigh the vial. Mix well.
Weigh into an injection vial, a known amount of blank (0.20 g), add 4.9 ml of methanol and 1.0 ml internal standard solution. Introduce 0.1 ml of Solution A into the injection vial. Mix well and heat at 60° for 10 min and shake vigorously for 10 sec.
Place the sample, blank and calibration samples in the sample tray of the head-space gas chromatograph - FID system. Analyse using the following analytical conditions.
Column: Fused silica, length 0.8 m, i.d. 0.53mm, coated with DB-wax, film thickness 1 µm
Coupled with: Fused silica, length 30 m, i.d. 0.53 mm, coated with DB-1, film thickness 5 µm
|Flow rate:||208 kPa, 5 ml/min|
|Oven conditions:||35° for 5 min, then 5°/min to 90°, then 6 min at 90°|
|Head space sampler|
|Sample heating temperature:||60°|
|Sample heating period:||10 min|
|Syringe temperature: 70°|
|Transfer temperature: 80°|
|Sample gas injection: 1.0 ml in split mode|
A = relative peak area of the component concerned
B = mg internal standard C = calibration factor
Determ ination of calibration factors
D = mg component weighed
E = mg internal standard
F = relative peak area of component for the calibration solution
G = relative peak area of the same component for the blank solution
Determine by gas chromatography (see Analytical Techniques)
Standard and test solutions
Methylene chloride: Use a suitable chromatography grade (or pure solvent obtained by distillation in all-glass apparatus from analytical grade).
Internal standard stock solution: Weigh accurately, about 100 mg of 95% n-tricosane into a 10 ml volumetric flask, dissolve in n-heptane, dilute to volume with the same solvent and mix.
Stock standard preparation: Weigh accurately 20 mg each of reagent grade o-toluene-sulfonamide and p-toluenesulfonamide into a 10 ml volumetric flask, dissolve in methylene chloride, dilute to volume with the same solvent, and mix.
Dilute standard preparations: Pipet into five 10-ml volumetric flasks 0.1, 0.25, 1.0, 2.5 and 5 ml, respectively, of the "Stock standard preparation". Pipet 0.25 ml of the "Internal standard stock solution" into each flask, dilute each to volume with methylene chloride, and mix. These solutions contain 250 μg of n-tricosane, plus respectively, 20, 50, 200, 500 and 1000 μg per ml of each toluenesulfonamide.
Test preparation: Dissolve 2 g of the sample in 8.0 ml of sodium carbonate TS. Mix the solution thoroughly with 10 g of chromatographic siliceous earth (Celite 545 or equivalent). Transfer the mix into a 25 x 250-mm chromatographic tube having a fritted glass disk and a Teflon stopcock at the bottom, and a reservoir at the top. Pack the contents of the tube by tapping the column on a padded surface, and then by tamping firmly from the top. Place 100 ml of methylene chloride in the reservoir, and adjust the stopcock so that 50 ml of eluate is collected in 20-30 min. To the eluate add 25 μl of "Internal standard stock solution". Mix, and then concentrate the solution to a volume of 1 ml in a suitable concentrator tube fitted with a modified Snyder column, using a Kontes tube heater maintained at 90°.
Note: The procedure uses a packed column GC. In the absence of a packed column GC, capillary GC in the splitless mode, using an equivalent capillary column, may be used. GC conditions need to be established.
Inject 2.5 μl of the "Test preparation" into a suitable gas chromatograph equipped with a flame-ionization detector. The column is of glass, approximately 3 m in length and 2 mm in inside diameter, and it is packed with 3% methyl phenyl silicone in 100 to 120 mesh silanized calcined diatomaceous silica (Caution: The glass column should extend into the injector for on-column injection and into the detector base to avoid contact with metal). The carrier is helium flowing at a rate of 30 ml per min. The injection port, column, and detector are maintained at 225°, 180°, and 250°, respectively. The instrument attenuation setting should be such that 2.5 μl of the "Dilute standard preparation" containing 200 μg per ml of each toluene sulfonamide gives a response of 40-80% of full-scale deflection. Record the chromatogram, note the peaks for o-toluene sulfonamide, p-toluene sulfonamide, and the n-tricosane internal standard, and calculate the areas of each peak by suitable means. The retention times for o-toluene sulfonamide, p-toluene sulfonamide, and n-tricosane are about 5, 6, and 15 min, respectively.
In a similar manner, obtain the chromatograms for 2.5-µl portions of each of the five "Dilute standard preparations", and for each solution determine the areas of the o-toluene sulfonamide, p-toluene sulfonamide, and n-tricosane peaks. From the values thus obtained, prepare standard curves by plotting concentration of each toluene sulfonamide, in μg per ml, vs. the ratio of the respective toluene sulfonamide peak area to that of n-tricosane. From the standard curve determine the concentration, in μg per ml, of each toluene sulfonamide in the "Test preparation". Divide each value by 2 to convert the result mg/kg of the toluene sulfonamide in 2 g sample taken for analysis.
Note: If the toluene sulfonamide content of the sample is greater than about 500 mg/kg, the impurity may crystallize out of the methylene chloride concentrate (see "Test preparation"). Although this level of impurity exceeds that permitted by the specifications, the analysis may be completed by diluting the concentrate (usually 1:10 is satisfactory) with methylene chloride containing 250 μg of n-tricosane per ml, and by applying appropriate dilution factors in the calculation. Care must be taken to re-dissolve completely any crystalline toluene sulfonamide to give a homogeneous solution.
Determine by HPLC using the following:
Triphenylphosphine oxide (TPPO) (purity 99% or higher; ACROS 14043-0250 or equivalent)
Note: all solvents should be HPLC-grade
HPLC system with a suitable pump, injector, and integrator
|Column:||Stainless steel; 150 x 4.6 mm|
|Stationary phase:||Supelcosil LC-Si, 5 μm or similar|
|Mobile phase:||Isopropanol:hexane (1:24 v/v)|
|Flow rate:||1.5 ml/min|
|Run time:||10 min|
Note: The retention time of TPPO is approximately 8.1 min
Standard solution: Weigh accurately about 10 mg of the TPPO standard and dissolve in THF. Transfer quantitatively to a 1000-ml volumetric flask and dilute to volume with THF.
Sample solution: Accurately weigh about 1000 mg of the sample and dissolve in THF. Transfer quantitatively to a 100-ml volumetric flask and dilute to volume with THF.
|As||is the peak area of the sample solution|
|ASt||is the peak area of the standard solution|
|PSt||is the purity of the standard expressed as a proportion of|
|TPPO in the TPPO standard (for example, 0.99 if the standard is 99% pure).|
|WSt||is the weight of the standard (mg)|
|Ws||is the weight of the sample (mg)|
Note: All methods in this Section reference media and reagents, which are prepared as detailed in the Section entitled "Media, Reagents and Solutions ".
Equipment and materials
Media and reagents
Using separate sterile pipets, prepare decimal dilutions of 10-2, 10-3, 10-4, and others as appropriate, of sample homogenate by transferring 10 ml of previous dilution to 90 ml of diluent. Avoid sampling foam. Shake all dilutions 25 times in 30 cm (1 ft) arc within 7 sec. Pipet 1 ml of each dilution into separate, duplicate, appropriately marked petri dishes. Reshake dilution bottle 25 times in 30 cm arc within 7 sec if dilution stands more than 3 min before pipeting test portion into petri dish. Add 12-15 ml plate count agar (cooled to 44-46°) to each plate within 15 min of original dilution. Add agar immediately to petri dishes when sample diluent contains hygroscopic materials. Pour agar and dilution water control plates for each series of samples. Immediately mix sample dilutions and agar medium thoroughly and uniformly by alternate rotation and back-and-forth motion of plates on flat level surface. Let agar solidify, invert petri dishes, and incubate promptly for 48 ± 2 h at 35°.
After incubation, count duplicate plates in suitable range (25-250 colonies), using colony counter and tally register; record results per dilution plate counted. Duplicate plates of at least 1 of 3 dilutions should be in 25-250 colony range. When only 1 dilution is in appropriate range, compute average count per g for dilution and report as total plate count per g (see Table 1, Sample No. 1). When 2 dilutions are in appropriate range, determine average count per dilution before averaging 2 dilution counts to obtain total plate count per g (see Table 1, Sample No. 2). If none or only one of duplicate plates of required dilution yields 25-250 colonies, proceed as in "Guidelines", below. Round off counts to two significant figures only at time of conversion to total plate counts. When rounding off numbers, raise second digit to next higher number only when third digit from left is 5 or greater, and replace dropped digit with zero. If third digit is 4 or less, replace third digit with zero and leave second digit the same.
Guidelines for calculating and reporting total plate counts in uncommon cases
Report all total plate counts computed from duplicate plates containing less than 25 or more than 250 colonies as estimated counts. Use the following as a guide:
Table 1. Examples of computation of total plate count (2 plates/dilution poured)
|Sample No.||Colonies counted|
|1:100||1:1,000||1:1,000||Aerobic plate count/g|
*(Asterisk): estimated count
TNTC : Too numerous to count. Colony count is significantly beyond count range of 250 colonies.
Underlined numbers are used to calculate aerobic plate count.
The spiral plate count (SPLC) method for microorganisms uses a mechanical plater to inoculate a rotating agar plate with liquid sample. The sample volume dispensed decreases as the dispensing stylus moves from the center to the edge of the rotating plate. The microbial concentration is determined by counting the colonies on a part of the petri dish where they are easily countable and dividing this count by the appropriate volume. One inoculation determines microbial densities between 500 and 500,000 microorganisms/ml. Additional dilutions may be made for suspected high microbial concentrations.
Equipment and materials
Preparation of agar plates
Automatic dispenser with sterile delivery system is recommended to prepare agar plates. Agar volume dispensed into plates is reproducible and contamination rate is low compared to hand-pouring of agar in open laboratory. When possible, use laminar air flow hood along with automated dispenser. Pour same quantity of agar into all plates so that same height of agar will be presented to spiral plater stylus tip to maintain contact angle. Agar plates should be level during cooling.
The following method is suggested for pre-pouring agar plates: Use automatic dispenser or pour constant amount (about 15 ml/100 mm plate; 50 ml/150 mm plate) of sterile agar at 60-70° into each petri dish. Let agar solidify on level surface with poured plates stacked no higher than 10 dishes. Place solidified agar plates in polyethylene bags, close with ties or heat-sealer, and store inverted at 0-4.4°. Bring pre-poured plates to room temperature before inoculation.
Preparation of samples.
Samples are prepared as described under Procedures.
Description of spiral plater
Spiral plater inoculates surface of prepared agar plate to permit enumeration of microorganisms in solutions containing between 500 and 500,000 microorganisms per ml. An operator with minimum training can inoculate 50 plates per h. Within the range stated, dilution bottles or pipets and other auxiliary equipment are not required. Required bench space is minimal, and time to check instrument alignment is less than 2 min. Plater deposits decreasing amount of sample in Archimedean spiral on surface of pre-poured agar plate. Volume of sample on any portion of plate is known. After incubation, colonies appear along line of spiral. If colonies on a portion of plate are sufficiently spaced from each other, count them on special grid which associates a calibrated volume with each area. Estimate number of microorganisms in sample by dividing number of colonies in a defined area by volume contained in same area.
Check stylus tip angle daily and adjust if necessary. (Use vacuum to hold microscope cover slip against face of stylus tip; if cover slip plane is parallel at about l mm from surface of platform, tip is properly oriented). Liquids are moved through system by vacuum. Clean stylus tip by rinsing for 1 s with sodium hypochlorite solution followed by sterile dilution water for 1 s before sample introduction. This rinse procedure between processing of each sample minimizes cross-contamination. After rinsing, draw sample into tip of Teflon tubing by vacuum applied to 2-way valve. When tubing and syringe are filled with sample, close valve attached to syringe. Place agar plate on platform, place stylus tip on agar surface, and start motor. During inoculation, label petri plate lid. After agar has been inoculated, stylus lifts from agar surface and spiral plater automatically stops. Remove inoculated plate from platform and cover it. Move stylus back to starting position. Vacuum-rinse system with hypochlorite and water, and then introduce new sample. Invert plates and promptly place them in incubator for 48 ± 3 h at 35 ± 1°.
Check sterility of spiral plater for each series of samples by plating sterile dilution water.
Caution: Pre-poured plates should not be contaminated by a surface colony or be below room temperature (water can well-up from agar). They should not be excessively dry, as indicated by large wrinkles or glazed appearance. They should not have water droplets on surface of agar or differences greater than 2 mm in agar depth, and they should not be stored at 0-4.4° for longer than l month. Reduced flow rate through tubing indicates obstructions or material in system. To clear obstructions, remove valve from syringe, insert hand-held syringe with Luer fitting containing water, and apply pressure. Use alcohol rinse to remove residual material adhering to walls of system. Dissolve accumulated residue with chromic acid. Rinse well after cleaning.
Description: Use same counting grid for both 100 and 150 mm petri dishes. A mask is supplied for use with 100 mm dishes. Counting grid is divided into 8 equal wedges; each wedge is divided by four arcs labelled l, 2, 3, and 4 from outside grid edge. Other lines within these arcs are added for ease of counting. A segment is the area between two arc lines within a wedge. Number of areas counted (e.g., 3) means number of segments counted within a wedge. The spiral plater deposits sample on agar plate in the same way each time. The grid relates colonies on spiral plate to the volume in which they were contained. When colonies are counted with grid, sample volume becomes greater as counting starts at outside edge of plate and proceeds toward the center of plate.
Calibration: The volume of sample represented by various parts of the counting grid is shown in the operator's manual that accompanies a spiral plater. Grid area constants have been checked by the manufacturer and are accurate. To verify these values, prepare 11 bacterial concentrations in range of 106-103 cells/ml by making 1:1 dilutions of bacterial suspension (use a nonspreader). Plate all. Incubate both sets of plates for 48 ± 3 h at 35 ± 1°. Calculate concentrations for each dilution. Count spiral plates over grid surface, using counting rule of 20 (described below), and record number of colonies counted and grid area over which they were counted. Each spiral colony count for a particular grid area, divided by aerobic count/ml for corresponding spirally plated bacterial concentrations, indicates volume deposited on that particular grid area. Use the following formula:
Examination and reporting of spiral plate counts.
Counting rule of 20. After incubation, center spiral plate over grid by adjusting holding arms on viewer. Choose any wedge and begin counting colonies from outer edge of first segment toward center until 20 colonies have been counted. Complete by counting remaining colonies in segment where 20th colony occurs. Any count irregularities in sample composition are controlled by counting the same segments in the opposite wedge and recording results. Two segments of each wedge were counted on opposite sides of plate with 31 and 30 colonies, respectively. The sample volume contained in the darkened segments is 0.0015 ml. To estimate number of microorganisms, divide count by volume contained in all segments counted.
If 20 CFU are not within the 4 segments of the wedge, count CFU on entire plate. If the number of colonies exceeds 75 in second, third, or fourth segment, which also contains the 20th colony, the estimated number of microorganisms will generally be low because of coincidence error associated with crowding of colonies. In this case, count each circumferentially adjacent segment in all 8 wedges, counting at least 50 colonies, e.g., if the first 2 segments of a wedge contain 19 colonies and the third segment contains the 20th and 76th (or more), count colonies in all circumferentially adjacent first and second segments in all 8 wedges. Calculate contained volume in counted segments of wedges and divide into number of colonies.
When fewer than 20 colonies are counted on the total plate, report results as "less than 500 estimated SPLC per ml." If colony count exceeds 75 in first segment of wedge, report results as "greater than 500,000 estimated SPLC per ml." Do not count spiral plates with irregular distribution of colonies caused by dispensing errors. Report results of such plates as laboratory accident (LA). If spreader covers entire plate, discard plate. If spreader covers half of plate area, count only those colonies that are well distributed in spreader-free areas.
Compute SPLC unless restricted by detection of inhibitory substances in sample, excessive spreader growth, or laboratory accidents. Round off counts as described above. Report counts as SPLC or estimated SPLC per ml.
Equipment and materials
Media and reagents
Presumptive test for coliform bacteria
Aseptically weigh 10 g sample into sterile, screw-cap jar. Add 90 ml diluent and shake vigorously (50 times through 30 cm arc) to obtain 10-1 dilution. Let stand 3-5 min and shake to re-suspend (5 times through 30 cm arc) just before making serial dilutions and inoculations.
Prepare all decimal dilutions with 90 ml sterile dilution water plus 10 ml from previous dilution unless otherwise specified. The dilutions to be prepared depend on the anticipated coliform density. Shake all suspensions 25 times in 30 cm arc for 7 sec. Do not use pipets to deliver <10% of their total volume. Transfer 1 ml portions to 3 LST tubes for each dilution for 3 consecutive dilutions. Hold pipet at angle so that its lower edge rests against tube. Let pipet drain 2-3 sec. Not more than 15 min should elapse from time sample is blended until all dilutions are in appropriate media.
Incubate tubes 48 ± 2 h at 35°. Examine tubes at 24 ± 2 h for gas, i.e., displacement of medium in fermentation vial or effervescence when tubes are gently agitated. Re-incubate negative tubes for additional 24 h. Examine a second time for gas. Perform a confirmation test on all presumptive positive (gassing) tubes.
Confirmation test for coliforms
Gently agitate each gassing LST tube. Hold the LST tube at angle and insert a loop to avoid transfer of pellicle (if present). Transfer one loopful of suspension to a tube of BGLB broth. Incubate BGLB tubes 48 ± 2 h at 35°. Examine for gas production and record. Calculate most probable number (MPN) of coliforms based on proportion of confirmed gassing LST tubes for three consecutive dilutions.
Confirmation test for E. coli
Gently agitate each gassing LST tube and transfer a loopful of each suspension to tube of EC broth. Incubate EC tubes 48 ± 2 h at 45.5 ± 0.2°. Examine for gas production at 24 ± 2 h; if negative, examine again at 48 ± 2 h. Streak a loopful of suspension from each gassing tube to L-EMB agar. It is essential that 1 portion of plate exhibit well-separated colonies. Incubate 18-24 h at 35°. Examine plates for suspicious E. coli colonies, i.e., dark centered with or without metallic sheen. Pick two suspicious colonies from each L-EMB plate and transfer them to PCA agar slants for morphological and biochemical tests. Incubate PCA slants 18-24 h at 35°. If typical colonies are not present, pick 5-10 or more colonies deemed most likely to be E. coli, from every plate.
Perform gram stain. Examine all cultures appearing as gram-negative short rods or cocci for the following biochemical activities (the first four tests are collectively termed IMViC):
Interpretation. All cultures that (a) ferment lactose with production of gas within 48 h at 35°, (b) appear as Gram-negative non-sporeforming rods or cocci, and (c) give IMViC (the first four tests.) patterns of ++-- (biotype 1) or -+— (biotype 2) are considered to be E. coli.
Alternatively, MPN determination of E. coli, fecal coliforms and coliforms protocols are given below.
Note: Alternatively, instead of performing the IMViC tests, use commercially prepared biochemical strip tests. Use growth from PCA slants to perform these assays.
MPN - Presumptive test for coliforms, fecal coliforms and E. coli
Weigh 50 g into sterile high-speed blender jar. Add 450 ml of Butterfield's phosphate-buffered water and blend for 2 min. If <50 g of sample are available, weigh portion that is equivalent to half of the sample and add sufficient volume of sterile diluent to make a 1:10 dilution. The total volume in the blender jar should completely cover the blades.
Prepare decimal dilutions with sterile Butterfield's phosphate diluent. The number of dilutions to be prepared depends on anticipated coliform density. Shake all suspensions 25 times in 30 cm arc or vortex mix for 7 s. Do not use pipets to deliver <10% of their total volume. Transfer 1 ml portions to three LST tubes for each dilution for at least three consecutive dilutions. Hold pipet at angle so that its lower edge rests against the tube. Let pipet drain 2-3 s. Not more than 15 min should elapse from time the sample is blended until all dilutions are inoculated in appropriate media.
Incubate LST tubes at 35°. Examine tubes and record reactions at 24 ± 2 h for gas, i.e., displacement of medium in fermentation vial or effervescence when tubes are gently agitated. Re-incubate gas-negative tubes for an additional 24 h and examine and record reactions again at 48 ± 2 h. Perform confirmed test on all presumptive positive (gas) tubes.
MPN - Confirmed test for coliforms
From each gassing LST tube, transfer a loopful of suspension to a tube of BGLB broth, avoiding pellicle if present. Incubate BGLB tubes at 35° and examine for gas production at 48 ± 2 h. Calculate most probable number (MPN) of coliforms based on proportion of confirmed gassing LST tubes for 3 consecutive dilutions.
MPN - Confirmed test for fecal coliforms and E. coli
From each gassing LST tube from the Presumptive test, transfer a loopful of suspension to a tube of EC broth (a sterile wooden applicator stick may also be used for these transfers). Incubate EC tubes 24 ± 2 h at 45.5° and examine for gas production. If negative, re-incubate and examine again at 48 ± 2 h. Use results of this test to calculate fecal coliform MPN. To continue with E. coli analysis, follow protocol for Completed test for E. coli (below).
Note: Fecal coliform analyses are done at 45.5± 0.2°.
MPN - Completed test for E. coli.
Gently agitate each gassing EC tube and streak for isolation, a loopful to a L-EMB agar plate and incubate for 18-24 h at 35°. Examine plates for suspicious E. coli colonies, i.e., dark centered and flat, with or without metallic sheen. Transfer up to five suspicious colonies from each L-EMB plate to PCA slants incubate for 18-24 h at 35° and use for further testing.
Note: Identification of any one of the five colonies as E. coli is sufficient to regard that EC tube as positive; hence, not all five isolates may need to be tested.
Perform Gram stain. All cultures appearing as Gram-negative short rods, should be tested for the IMViC reactions above and also re-inoculated back into LST to confirm gas production.
Equipment and materials
Media and reagents
Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml sterile lactose broth and mix well, essentially preparing a 1:9 sample/broth ratio. Cap jar securely and let stand 60 ± 5 min at room temperature. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Loosen jar cap about 1/4 turn and incubate 24 ± 2 h at 35°.
In some cases, the analysis of samples may be hampered by the viscosity of thickening agents. Additional treatment may be required.
For Carob Bean gum and Guar gum. Transfer 1 ml mixture to 10 ml selenite cystine (SC) broth and another 1 ml mixture to 10 ml TT broth. Vortex. Incubate SC and TT broths 24 ± 2 h at 35°.
For all other samples. Transfer 0.1 ml mixture to 10 ml Rappaport-Vassiliadis (RV) medium and another 1 ml mixture to 10 ml tetrathionate (TT) broth. Vortex.
High microbial load. Incubate RV medium 24 ± 2 h at 42 ± 0.2° (circulating, thermostatically-controlled, water bath). Incubate TT broth 24 ± 2 h at 43 ± 0.2° (circulating, thermostatically-controlled, water bath).
Low microbial load (except carob bean gum and guar gum). Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulating, thermostatically controlled, water bath). Incubate TT broth 24 ± 2 h at 35 ± 2.0°.
Examine plates as follows:1. Typical Salmonella colony morphology
In addition to the positive control cultures (typical Salmonella), three additional Salmonella cultures are recommended to assist in the selection of atypical Salmonella colony morphology on selective agars. These cultures are a lactose-positive, H2S-positive S. diarizonae (ATCC 12325) and a lactose-negative, H2S-negative S. abortus equi (ATCC 9842); OR a lactose-positive, H2S-negative S. diarizonae (ATCC 29934). These cultures may be obtained from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.
Agar slant analysis
Three presumptive TSI agar cultures recovered from set of plates streaked from RV medium, if present, and presumptive TSI agar cultures recovered from plates streaked from tetrathionate broth, if present.
If three presumptive-positive TSI cultures are not isolated from one set of agar plates, test other presumptive-positive TSI agar cultures, if isolated, by biochemical and serological tests. Examine a minimum of six TSI cultures for each 25 g analytical unit.
Biochemical and Serological Testing for Salmonella1. Mixed cultures:
Streak TSI agar cultures that appear to be mixed on MacConkey agar, HE agar, or XLD agar. Incubate plates 24 ± 2 h at 35°. Examine plates for presence of colonies suspected to be Salmonella, as follows:
Transfer at least two colonies suspected to be Salmonella to TSI agar and LIA slants as described above, and continue as under 'Agar slant analysis'.2. Pure cultures:
3. Serological polyvalent flagellar (H) test:
Positive - agglutination in test mixture and no
agglutination in control.
Negative - no agglutination in test mixture and no agglutination in control.
Nonspecific - agglutination in both test mixture and control. Test the cultures giving such results with Spicer-Edwards antisera, below.
4. Spicer-Edwards serological test:
Use this test as an alternative to the polyvalent flagellar (H) test. It may also be used with cultures giving non-specific agglutination in polyvalent flagellar (H) test. Perform Spicer-Edwards flagellar (H) antisera test as described above. Perform additional biochemical tests (below) on cultures giving positive flagellar test results. If both formalinized broth cultures are negative, perform serological tests on four additional broth cultures (above). If possible, obtain two positive cultures for additional biochemical testing. If all urease-negative TSI cultures from sample give negative serological flagellar (H) test results, perform additional biochemical tests.
5. Testing of urease-negative cultures:
- Potassium cyanide (KCN) broth. Transfer 3 mm loopful of 24 h tryptophane broth culture to KCN broth. Heat rim of tube so that good seal is formed when tube is stoppered with wax-coated cork. Incubate 48 ± 2 h at 35° but examine after 24 h. Interpret growth (indicated by turbidity) as positive. Most Salmonella species do not grow in this medium, as indicated by lack of turbidity.
- Malonate broth. Transfer 3 mm loopful of 24 h tryptone broth culture to malonate broth. Since occasional uninoculated tubes of malonate broth turn blue (positive test) on standing, include uninoculated tube of this broth as control. Incubate 48 ± 2 h at 35°, but examine after 24 h. Most Salmonella species cultures give negative test (green or unchanged colour) in this broth.
- Indole test. Transfer 5 ml of 24 h tryptophane broth culture to empty test tube. Add 0.2-0.3 ml Kovacs' reagent. Most Salmonella cultures give negative test (lack of deep red colour at surface of broth). Record intermediate, varying shades of orange and pink as ±.
- Serological flagellar (H) tests for Salmonella. If either polyvalent flagellar (H) test (above) or the Spicer-Edwards flagellar (H) test tube test (above) has not already been performed, either test may be performed here.
- Discard as not Salmonella any culture that shows either positive indole test and negative serological flagellar (H) test, or positive KCN test and negative lysine decarboxylase test.
6. Serological somatic (O) tests for Salmonella:
Note: Pre-test all antisera to Salmonella with known cultures.
a. Polyvalent somatic (O) test.
Using wax pencil, mark off two sections about 1x2 cm each on inside of glass or plastic petri dish (15 x 100 mm). Commercially available sectioned slides may be used. Emulsify 3 mm loopful of culture from 24-48 h TSI slant or, preferably, tryptose blood agar base (without blood) with 2 ml 0.85% saline. Add 1 drop of culture suspension to upper portion of each rectangular crayon-marked section. Add 1 drop of saline solution to lower part of one section only. Add 1 drop of Salmonella polyvalent somatic (O) antiserum to other section only. With clean sterile transfer loop or needle, mix culture suspension with saline solution for one section and repeat for other section containing antiserum. Tilt mixtures in back-and-forth motion for 1 min and observe against dark background in good illumination. Consider any degree of agglutination a positive reaction. Classify polyvalent somatic (O) test results as follows:
Positive - agglutination in test mixture; no agglutination in saline control.
Negative - no agglutination in test mixture; no agglutination in saline control.
Nonspecific - agglutination in test and in control mixtures. Perform further biochemical and serological tests as described in Edwards and Ewing's Identification of Enterobacteriaceae (Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriacae, 4th ed. Elsevier, New York).
b. Somatic (O) group tests:
Test as in 6a, above, using individual group somatic (O) antisera including Vi, if available, in place of Salmonella polyvalent somatic (O) antiserum. For special treatment of cultures giving positive Vi agglutination reaction, refer to sec. 967.28B in Official Methods of Analysis (AOAC International). Record cultures that give positive agglutination with individual somatic (O) antiserum as positive for that group. Record cultures that do not react with individual somatic (O) antiserum as negative for that group.
Table 2. Summary of biochemical and serological reactions of Salmonella
Test or substrate
species reactions a
|1. Glucose (TSI)||yellow butt||red butt||+|
|2. Lysine decarboxylase (LIA)||purple butt||yellow butt||+|
|4. Urease||purple-red colour||no colour change||-|
5. Lysine decarboxylase broth
|6. Phenol red dulcitol broth||yellow colour and/or gas||no gas; no colour change||+b|
|7. KCN broth||growth||no growth||-|
|8. Malonate broth||blue colour at surface||no colour change||-c|
|9. Indole test||deep red colour at surface||
yellow colour at surface
|10. Polyvalent flagellar test||agglutination||no agglutination||+|
|11. Polyvalent somatic test||agglutination||no agglutination||+|
|12. Phenol red lactose broth||yellow colour and/or gas||no gas; no colour change||-c|
|13. Phenol red sucrose broth||yellow colour and/or gas||no gas; no colour change||-|
|14. Voges-Proskauer test||pink-to-red colour||no colour change||-|
|15. Methyl red test||diffuse red colour||diffuse yellow colour||+|
|16. Simmons citrate||growth; blue colour||no growth; no colour change||v|
a+ is 90% or more positive in 1 or 2 days; - is 90% or more negative in 1 or 2 days; v is variable.
bMajority of S. arizonae cultures are negative.
cMajority of S. arizonae cultures are positive.
Classify as Salmonella those cultures which exhibit typical Salmonella reactions for test Nos. 1-11, shown in Table 2, above. If one TSI culture from 25 g sample is classified as Salmonella, further testing of other TSI cultures from the same 25 g sample is unnecessary. Cultures that contain demonstrable Salmonella antigens as shown by positive Salmonella flagellar (H) test but do not have biochemical characteristics of Salmonella should be purified and retested.
Table 3. Criteria for discarding non-Salmonella cultures
|Test or substrate||Results|
|1. Urease||positive (purple-red colour)|
|2. Indole test and Polyvalent flagellar (H) test or Spicer-Edwards flagellar test||positive (violet colour at surface) negative (no agglutination)|
|3. Lysine decarboxylaseKCN broth||
negative (yellow colour) positive (growth)
|4. Phenol red lactose broth||
positive (yellow colour and/or gas) a,b
|5. Phenol red sucrose broth||positive (yellow colour and/or gas) b|
|6. KCN broth Voges-Proskauer test Methyl red test||positive (growth) positive (pink-to-red colour) negative (diffuse yellow colour)|
aTest malonate broth positive cultures further to determine if they are Salmonella arizonae.
bDo not discard positive broth cultures if corresponding LIA cultures give typical Salmonella reactions; test further to determine if they are Salmonella species.
Atypical Salmonella Colony Testing
Perform the following additional biochemical tests on cultures that do not give typical Salmonella reactions for test Nos. 1-11 in Table 12, above, and that consequently do not classify as Salmonella (see Table 23, also above).
a. Phenol red lactose broth or purple lactose broth
b. Phenol red sucrose broth or purple sucrose broth
Follow procedure described as directly above. Discard as not Salmonella, cultures that give positive sucrose tests, except those that give acid slants in TSI and positive reactions in LIA.
c. MR-VP broth
Inoculate medium with small amount of growth from each unclassified TSI slant suspected to contain Salmonella. Incubate 48 ± 2 h at 35°.
d. Simmons citrate agar
Inoculate this agar, using needle containing growth from unclassified TSI agar slant. Inoculate by streaking slant and stabbing butt. Incubate 96 ± 2 h at 35°C. Read results as follows:
Positive - presence of growth, usually accompanied by color change from green to blue. Most cultures of Salmonella are citrate-positive.
Negative - no growth or very little growth and no color change.
Alternative Method for Identification of Salmonella
As alternative to conventional biochemical tube system, use any of 5 commercial biochemical systems (API 20E, Enterotube II, Enterobacteriaceae II, MICRO-ID, or Vitek GNI) for presumptive generic identification of Salmonella. Choose a commercial system based on a demonstration in the analyst's own laboratory of adequate correlation between commercial system and the biochemical tube system outlined in this identification section.
Commercial biochemical kits should not be used as a substitute for serological tests.
Assemble supplies and prepare reagents required for the kit. Inoculate each unit according to Method 978.24 (API 20E, Enterotube II, and Enterobacteriaceae II), sec. 989.12 (MICRO-ID), and Method 991.13 (Vitek GNI) in Official Methods of Analysis, incubating for time and temperature specified. Add reagents, observe, and record results. For presumptive identification, classify cultures, according to Official Methods of Analysis (AOAC International) as Salmonella or not Salmonella.
For confirmation of cultures presumptively identified as Salmonella, perform the Salmonella serological somatic (O) test and the Salmonella serological flagellar (H) test or the Spicer-Edwards flagellar (H) test and classify cultures according to the following guidelines:
Treatment of cultures giving negative flagellar (H) test.
If biochemical reactions of certain flagellar (H)-negative culture strongly suggest that it is Salmonella, the negative flagellar agglutination may be the result of non-motile organisms or insufficient development of flagellar antigen. Proceed as follows: Inoculate motility test medium in petri dish, using small amount of growth from TSI slant. Inoculate by stabbing medium once about 10 mm from edge of plate to depth of 2-3 mm. Do not stab to bottom of plate or inoculate any other portion. Incubate 24 h at 35°C. If organisms have migrated 40 mm or more, retest as follows: Transfer 3 mm loopful of growth that migrated farthest to trypticase soy-tryptose broth. Repeat either polyvalent flagellar (H) or Spicer-Edwards serological tests. If cultures are not motile after the first 24 h, incubate an additional 24 h at 35°C; if still not motile, incubate up to 5 days at 25°C. Classify culture as non-motile if above tests are still negative.
Note: This method is suitable for the analysis in which more than 100 S. aureus cells/g may be expected. If the analyst suspects that the number of S. aureus cells is below this limit, then the MPN method should be used. If unknown, both procedures can be used.
Equipment and materials
Media and reagents
Under aseptic conditions, prepare serial dilutions of sample by transferring 10 ml of previous dilution to 90 ml of diluent using separate pipets. Avoid sample foam. Shake all dilutions 25 times in 30 cm (1 ft) arc within 7 seconds.
For each dilution to be plated, aseptically transfer 1 ml sample suspension to 3 plates of Baird-Parker agar, distributing 1 ml of inoculum equitably to 3 plates (e.g., 0.4 ml, 0.3 ml, and 0.3 ml). Spread inoculum over surface of agar plate, using sterile bent glass streaking rod. Retain plates in upright position until inoculum is absorbed by agar (about 10 min on properly dried plates). If inoculum is not readily adsorbed, place plates upright in incubator for about 1 h. Invert plates and incubate 45-48 h at 35°. Select plates containing 20-200 colonies, unless only plates at lower dilutions (>200 colonies) have colonies with typical appearance of S. aureus. Colonies of S. aureus are circular, smooth, convex, moist, 2-3 mm in diameter on uncrowded plates, grey to jet-black, frequently with light-coloured (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone; colonies have buttery to gummy consistency when touched with inoculating needle. Occasionally from various foods and dairy products, nonlipolytic strains of similar appearance may be encountered, except that surrounding opaque and clear zones are absent. Strains isolated from frozen or desiccated foods that have been stored for extended periods frequently develop less black coloration than typical colonies and may have rough appearance and dry texture.
Count and record colonies. If several types of colonies are observed which appear to be S. aureus on selected plates, count number of colonies of each type and record counts separately. When plates of the lowest dilution contain <20 colonies, these may be used. If plates containing >200 colonies have colonies with the typical appearance of S. aureus and typical colonies do not appear at higher dilutions, use these plates for the enumeration of S. aureus, but do not count nontypical colonies. Select > 1 colony of each type counted and test for coagulase production. Add number of colonies on triplicate plates represented by colonies giving positive coagulase test and multiply by the sample dilution factor. Report this number as number of S. aureus / g of food tested.
Identification of S. aureus
Ancillary identification tests
Some typical characteristics of two species of staphylococci and the micrococci, which may be helpful in their identification, are listed in Table 1.
|Table 1. Typical characteristics of S. aureus,S. epidermidis, and Micrococci(a)|
|S. aureus||S. epidermidis||Micrococci|
|Anaerobic utilization of|
|a+, Most (90% or more) strains are positive;
-, most (90% or more) strains are negative.
Note: The Most Probable Number (MPN) method is recommended for routine surveillance of products in which small numbers of S. aureus are expected and in foods expected to contain a large population of competing species.
Equipment and materials - Same as for Direct Plate Count Method.
Media and reagents - Same as for Direct Plate Count Method. Also required: Trypticase (tryptic) soy broth (TSB) containing 10% NaCl and 1 % sodium pyruvate.
Preparation of sample - Same as for Direct Plate Count Method.
Determination of MPN
Inoculate 3 tubes of TSB containing 10% NaCl and 1 % sodium pyruvate with 1 ml portions of decimal dilutions of each sample. Highest dilution must give negative endpoint. Incubate tubes 48 ± 2 h at 35°. Using 3 mm loop, transfer 1 loopful from each tube showing growth (turbidity) to plate of Baird-Parker medium with properly dried surface. Vortex-mix tubes before streaking if growth is visible only on bottom or sides of tubes. Streak inoculum to obtain isolated colonies. Incubate plates 48 h at 35°. From each plate showing growth, transfer at least 1 colony suspected to be S. aureus to TSB broth (see C of Direct Plate Count Method above). Continue procedure for identification and confirmation of S. aureus (see d of Direct Plate Count, above).
Equipment and materials
Media and reagents
Antibiotics are added to mycological media to inhibit bacterial growth. Chloramphenicol is the antibiotic of choice, because it is stable under autoclave conditions. Therefore, media preparation is easier and faster due to the elimination of the filtration step. The recommended concentration of this antibiotic is 100 mg/liter medium. If bacterial overgrowth is apparent, prepare media by adding 50 mg/liter chloramphenicol before autoclaving and 50 mg/liter filter-sterilized chlortetracycline when the media have been tempered, right before pouring plates.
Prepare stock solution by dissolving 0.1 g chloramphenicol in 40 ml distilled water; add this solution to 960 ml medium mixture before autoclaving. When both chloramphenicol and chlortetracycline are used, add 20 ml of the above chloramphenicol stock solution to 970 ml medium before autoclaving. Then, prepare chlortetracycline stock solution by dissolving 0.5 g antibiotic in 100 ml distilled water and filter sterilize. Use 10 ml of this solution for each 990 ml of autoclaved and tempered medium. Refrigerate in the dark and re-use remaining stock solutions for up to a month. Stock solutions should be brought to room temperature before adding to tempered medium.
Analyze 25-50 g from each subsample; generally, larger sample sizes increase reproducibility and lower variance compared with small samples. Add appropriate amount of 0.1% peptone water to the weighed sample to achieve 10-1 dilution, then homogenize in a stomacher for two min. Alternatively, blending for 30-60 sec can be used but is less effective. Make appropriate 1:10 (1+9) dilutions in 0.1% peptone water. Dilutions of 10-6 should suffice.
Plating and incubation of samples
Spread-plate method: Aseptically pipet 0.1 ml of each dilution on pre- poured, solidified DRBC agar plates and spread inoculum with a sterile, bent glass rod. DG18 is preferred when the water activity of the analyzed sample is less than 0.95. Plate each dilution in triplicate.
Pour-plate method: Use sterile cotton-plugged pipet to place 1 ml portions of sample dilutions into prelabelled 15 x 100 mm petri plates (plastic or glass), and immediately add 20-25 ml tempered DG18 agar medium. Mix contents by gently swirling plates clockwise then counter clockwise, taking care to avoid spillage on dish lid. Add agar within 1-2 min after adding dilution. Otherwise, dilution may begin to adhere to dish bottom (especially if sample is high in starch content and dishes are plastic) and may not mix uniformly. Plate each dilution in triplicate, using wide bore pipets. From preparation of first sample dilution to pouring of final plate, no more than 20 min, preferably 10 min, should elapse.
Incubate plates in dark at 25°. Do not stack plates higher than 3 and do not invert. Let plates remain undisturbed until counting.
Counting of plates
Count plates after 5 days of incubation. If there is no growth at 5 days, re-incubate for another 48 h. Do not count colonies before the end of the incubation period because handling of plates could result in secondary growth from dislodged spores, making final counts invalid. Count plates containing 10-150 colonies. If mainly yeasts are present, plates with 150 colonies are usually countable. However, if substantial amounts of mould are present, depending on the type of mold, the upper countable limit may have to be lowered at the discretion of the analyst. Report results in colony forming units (CFU)/g or CFU/ml based on average count of triplicate set. Round off counts to two significant figures. If third digit is 6 or above, round off to digit above (e.g., 456 = 460); if 4 or below, round off to digit below (e.g., 454 = 450). If third digit is 5, round off to digit below if first two digits are an even number (e.g., 445 = 440); round off to digit above if last two digits are an odd number (e.g., 455 = 460). When plates from all dilutions have no colonies, report mould and yeast counts (MYC) as less than 1 times the lowest dilution used.