USP 36 Chemical Tests / 〈541〉 Titrimetry 213

is used to correct the fluorometer at frequent intervals for variation in sensitivity from reading to reading within an as- 〈541〉 TITRIMETRY say. Prepare this solution fresh on the day of use. Standard Thiamine Hydrochloride Stock Solution— Transfer about 25 mg of USP Thiamine Hydrochloride RS, Direct Titrations—Direct titration is the treatment of a accurately weighed, to a 1000-mL volumetric flask. Dissolve soluble substance, contained in solution in a suitable vessel the weighed Standard in about 300 mL of dilute alcohol so- (the titrate), with an appropriate standardized solution (the lution (1 in 5) adjusted with 3 N hydrochloric acid to a pH titrant), the endpoint being determined instrumentally or of 4.0, and add the acidified, dilute alcohol to volume. Store visually with the aid of a suitable indicator. in a light-resistant container, in a refrigerator. Prepare this The titrant is added from a suitable buret and is so cho- stock solution fresh each month. sen, with respect to its strength (normality), that the volume Standard Preparation—Dilute a portion of Standard Thi- added is between 30% and 100% of the rated capacity of amine Hydrochloride Stock Solution quantitatively and step- the buret. [NOTE—Where less than 10 mL of titrant is re- wise with 0.2 N hydrochloric acid to obtain the Standard quired, a suitable microburet is to be used.] The endpoint is Preparation, each mL of which represents 0.2 µg of USP Thi- approached directly but cautiously, and finally the titrant is amine Hydrochloride RS. added dropwise from the buret in order that the final drop Assay Preparation—Place in a suitable volumetric flask added will not overrun the endpoint. The quantity of the sufficient amount of the material to be assayed, accurately substance being titrated may be calculated from the volume weighed or measured by volume as directed, such that and the normality or molarity factor of the titrant and the when diluted to volume with 0.2 N hydrochloric acid, the equivalence factor for the substance given in the individual resulting solution will contain about 100 µg of thiamine hy- monograph. drochloride (or mononitrate) per mL. If the sample is diffi- Residual Titrations—Some Pharmacopeial assays require cultly soluble, the solution may be heated on a steam bath, the addition of a measured volume of a volumetric solution, and then cooled and diluted with the acid to volume. Dilute in excess of the amount actually needed to react with the 5 mL of this solution, quantitatively and stepwise, using substance being assayed, the excess of this solution then 0.2 N hydrochloric acid, to an estimated concentration of being titrated with a second volumetric solution. This consti- 0.2 µg of thiamine hydrochloride (or mononitrate) per mL. tutes a residual titration and is known also as a “back titra- Procedure—Into each of three or more tubes (or other tion.” The quantity of the substance being titrated may be suitable vessels), of about 40-mL capacity, pipet 5 mL of calculated from the difference between the volume of the Standard Preparation. To each of two of these tubes add volumetric solution originally added, corrected by means of rapidly (within 1 to 2 seconds), with mixing, 3.0 mL of Oxi- a blank titration, and that consumed by the titrant in the dizing Reagent, and within 30 seconds add 20.0 mL of back titration, due allowance being made for the respective isobutyl alcohol, then mix vigorously for 90 seconds by normality or molarity factors of the two solutions, and the shaking the capped tubes manually, or by bubbling a equivalence factor for the substance given in the individual stream of air through the mixture. Prepare a blank in the monograph. remaining tube of the standard by substituting for the Oxi- Complexometric Titrations—Successful complexometric dizing Reagent an equal volume of 3.5 N sodium hydroxide titrations depend on several factors. The equilibrium con- and proceeding in the same manner. stant for formation of the titrant-analyte complex must be Into each of three or more similar tubes pipet 5 mL of the sufficiently large that, at the endpoint, very close to 100% Assay Preparation. Treat these tubes in the same manner as of the analyte has been complexed. The final complex must directed for the tubes containing the Standard Preparation. be formed rapidly enough that the analysis time is practical. Into each of the six tubes pipet 2 mL of dehydrated alco- When the analytical reaction is not rapid, a residual titration hol, swirl for a few seconds, allow the phases to separate, may sometimes be successful. and decant or draw off about 10 mL of the clear, superna- In general, complexometric indicators are themselves tant isobutyl alcohol solution into standardized cells, then complexing agents. The reaction between metal ion and in- measure the fluorescence in a suitable fluorometer, having dicator must be rapid and reversible. The equilibrium con- an input filter of narrow transmittance range with a maxi- stant for formation of the metal-indicator complex should mum at about 365 nm and an output filter of narrow trans- be large enough to produce a sharp color change but must mittance range with a maximum at about 435 nm. be less than that for the metal-titrant complex. Indicator Calculation—The number of µg of C12H17ClN4OS · HCl in choice is also restricted by the pH range within which the each 5 mL of the Assay Preparation is given by the formula: complexation reaction must be carried out and by interfer- ence of other ions arising from the sample or the buffer. (A − b)/(S − d) Interfering ions may often be masked or “screened” via ad- dition of another complexing agent. (The masking tech- in which A and S are the average fluorometer readings of nique is also applicable to redox titrations.) the portions of the Assay Preparation and the Standard Prep- Oxidation-Reduction (Redox) Titrations—Determina- aration treated with Oxidizing Reagent, respectively, and b tions may often be carried out conveniently by the use of a and d are the readings for the blanks of the Assay Prepara- reagent that brings about oxidation or reduction of the tion and the Standard Preparation, respectively. analyte. Many redox titration curves are not symmetric Calculate the quantity, in mg, of thiamine hydrochloride about the equivalence point, and thus graphical determina- (C12H17ClN4OS · HCl) in the assay material on the basis of tion of the endpoint is not possible; but indicators are avail- the aliquots taken. Where indicated, the quantity, in mg, of able for many determinations, and a redox reagent can of- thiamine mononitrate (C12H17N5O4S) may be calculated by ten serve as its own indicator. As in any type of titration, the multiplying the quantity of C12H17ClN4OS · HCl found by ideal indicator changes color at an endpoint that is as close 0.9706. as possible to the equivalence point. Accordingly, when the titrant serves as its own indicator, the difference between the endpoint and the equivalence point is determined only by the analyst’s ability to detect the color change. A com- mon example is the use of permanganate ion as an oxidiz- ing titrant since a slight excess can easily be detected by its pink color. Other titrants that may serve as their own indica- tors are iodine, cerium (IV) salts, and potassium dichromate. 214 〈541〉 Titrimetry / Chemical Tests USP 36

In most cases, however, the use of an appropriate redox For the titration of an acidic compound, two classes of indicator will yield a much sharper endpoint. titrant are available: the alkali metal alkoxides and the tetra- It may be necessary to adjust the oxidation state of the alkylammonium hydroxides. A volumetric solution of sodium analyte prior to titration through use of an appropriate oxi- methoxide in a mixture of methanol and toluene is used dizing or reducing agent; the excess reagent must then be frequently, although lithium methoxide in methanol-ben- removed, e.g., through precipitation. This is nearly always zene solvent is used for those compounds yielding a gelati- the practice in the determination of oxidizing agents since nous precipitate on titration with sodium methoxide. most volumetric solutions of reducing agents are slowly oxi- The alkali error limits the use of the glass electrode as an dized by atmospheric oxygen. indicating electrode in conjunction with alkali metal alkoxide Titrations in Nonaqueous Solvents—Acids and bases titrants, particularly in basic solvents. Thus, the antimony- have long been defined as substances that furnish, when indicating electrode, though somewhat erratic, is used in dissolved in water, hydrogen and hydroxyl ions, respec- such titrations. The use of quaternary ammonium hydroxide tively. This definition, introduced by Arrhenius, fails to rec- compounds, e.g., tetra-n-butylammonium hydroxide and ognize the fact that properties characteristic of acids or trimethylhexadecylammonium hydroxide (in benzene-meth- bases may be developed also in other solvents. A more gen- anol or isopropyl alcohol), has two advantages over the eralized definition is that of Br¨onsted, who defined an acid other titrants in that (a) the tetraalkylammonium salt of the as a substance that furnishes protons, and a base as a sub- titrated acid is soluble in the titration medium, and (b) the stance that combines with protons. Even broader is the defi- convenient and well-behaved calomel-glass electrode pair nition of Lewis, who defined an acid as any material that may be used to conduct potentiometric titrations. will accept an electron pair, a base as any material that will Because of interference by carbon dioxide, solvents for donate an electron pair, and neutralization as the formation acidic compounds need to be protected from excessive ex- of a coordination bond between an acid and a base. posure to the atmosphere by a suitable cover or by an inert The apparent strength of an acid or a base is determined atmosphere during the titration. Absorption of carbon diox- by the extent of its reaction with a solvent. In water solution ide may be determined by performing a blank titration. The all strong acids appear equally strong because they react blank should not exceed 0.01 mL of 0.1 N sodium methox- with the solvent to undergo almost complete conversion to ide VS per mL of solvent. oxonium ion and the acid anion (leveling effect). In a The endpoint may be determined visually by color weakly protophilic solvent such as acetic acid the extent of change, or potentiometrically, as indicated in the individual formation of the acetate acidium ion shows that the order monograph. If the calomel reference electrode is used, it is of decreasing strength for acids is perchloric, hydrobromic, advantageous to replace the aqueous potassium chloride sulfuric, hydrochloric, and nitric (differentiating effect). salt bridge with 0.1 N lithium perchlorate in glacial acetic Acetic acid reacts incompletely with water to form ox- acid for titrations in acidic solvents or potassium chloride in onium ion and is, therefore, a weak acid. In contrast, it dis- methanol for titrations in basic solvents. solves in a base such as ethylenediamine, and reacts so Where these or other mixtures are specified in individual completely with the solvent that it behaves as a strong acid. monographs, the calomel reference electrode is modified by The same holds for perchloric acid. first removing the aqueous potassium chloride solution and This leveling effect is observed also for bases. In sulfuric residual potassium chloride, if any, by rinsing with water, acid almost all bases appear to be of the same strength. As then eliminating residual water by rinsing with the required the acid properties of the solvent decrease in the series sul- nonaqueous solvent, and finally filling the electrode with the furic acid, acetic acid, phenol, water, pyridine, and butyl- designated nonaqueous mixture. amine, the bases become progressively weaker until all but In nearly all cases, except those where silver ion might the strongest have lost their basic properties. In order of interfere, a silver-silver chloride reference electrode may be decreasing strength, the strong bases are sodium 2-ami- substituted for the calomel electrode. The silver-silver chlo- noethoxide, potassium methoxide, sodium methoxide, and ride electrode is more rugged, and its use helps to eliminate lithium methoxide. toxic mercury salts from the laboratory. Generally, a salt Many water-insoluble compounds acquire enhanced bridge may be used to circumvent interference by silver ion. acidic or basic properties when dissolved in organic solvents. The more useful systems for titration in nonaqueous sol- Thus the choice of the appropriate solvent permits the de- vents are listed in Table 1. termination of a variety of such materials by nonaqueous Indicator and Potentiometric Endpoint Detection—The titration. Furthermore, depending upon which part of a simplest and most convenient method by which the equiva- compound is the physiologically active moiety, it is often lence point, i.e., the point at which the stoichiometric ana- possible to titrate that part by proper selection of solvent lytical reaction is complete, may be determined is with the and titrant. Pure compounds can be titrated directly, but it use of indicators. These chemical substances, usually is often necessary to isolate the active ingredient in pharma- colored, respond to changes in solution conditions before ceutical preparations from interfering excipients and carriers. and after the equivalence point by exhibiting color changes The types of compounds that may be titrated as acids that may be taken visually as the endpoint, a reliable esti- include acid halides, acid anhydrides, carboxylic acids, mate of the equivalence point. amino acids, enols such as barbiturates and xanthines, A useful method of endpoint determination results from imides, phenols, pyrroles, and sulfonamides. The types of the use of electrochemical measurements. If an indicator compounds that may be titrated as bases include amines, electrode, sensitive to the concentration of the species un- nitrogen-containing heterocyclic compounds, oxazolines, dergoing titrimetric reaction, and a reference electrode, quaternary ammonium compounds, alkali salts of organic whose potential is insensitive to any dissolved species, are acids, alkali salts of weak inorganic acids, and some salts of immersed in the titrate to form a galvanic cell, the potential amines. Many salts of halogen acids may be titrated in ace- difference between the electrodes may be sensed by a pH tic acid or acetic anhydride after the addition of mercuric meter and used to follow the course of the reaction. Where acetate, which removes halide ion as the unionized mercuric such a series of measurements is plotted correctly (i.e., for halide complex and introduces the acetate ion. an acid-base titration, pH versus mL of titrant added; for a For the titration of a basic compound, a volumetric solu- precipitimetric, complexometric, or oxidation-reduction ti- tion of perchloric acid in glacial acetic acid is preferred, al- tration, mV versus mL of titrant added), a sigmoid curve though perchloric acid in dioxane is used in special cases. results with a rapidly changing portion (the “break”) in the The calomel-glass electrode system is useful in this case. In vicinity of the equivalence point. The midpoint of this linear acetic acid solvent, this electrode system functions as pre- vertical portion or the inflection point may be taken as the dicted by theory. USP 36 Chemical Tests / 〈541〉 Titrimetry 215

Table 1. Systems for Nonaqueous Titrations Acidic (for titration Relatively Neutral Relatively Neutral of bases and their (for differential Basic (for titration (for differential Type of Solvent salts) titration of bases) of acids) titration of acids) Solvent1 Glacial Acetic Acid Acetonitrile Dimethylformamide Acetone Acetic Anhydride Alcohols n-Butylamine Acetonitrile Formic Acid Chloroform Pyridine Methyl Ethyl Ketone Propionic Acid Benzene Ethylenediamine Methyl Isobutyl Ketone Sulfuryl Chloride Toluene Morpholine tert-Butyl Alcohol Chlorobenzene Ethyl Acetate Dioxane Indicator Crystal Violet Methyl Red Thymol Blue Azo Violet Quinaldine Red Methyl Orange Thymolphthalein Bromothylmol Blue p-Naphtholbenzein p-Naphtholbenzein Azo Violet p-Hydroxyazobenzene Alphezurine 2-G o-Nitroaniline Thymol Blue Malachite Green p-Hydroxyazobenzene Electrodes Glass–calomel Glass–calomel Antimony–calomel Antimony–calomel Glass–silver–silver chloride Calomel–silver–silver chlo- Antimony–glass Glass–calomel Mercury–mercuric acetate ride Antimony–antimony2 Glass–platinum2 Platinum–calomel Glass–calomel 1Relatively neutral solvents of low dielectric constant such as benzene, toluene, chloroform, or dioxane may be used in conjunction with any acidic or basic solvent in order to increase the sensitivity of the titration end-points. 2In titrant. endpoint. The equivalence point may also be determined ically and records the electrode potential differences during mathematically without plotting a curve. However, it should the course of titration as the expected sigmoid curve. In the be noted that in asymmetrical reactions, which are reactions second type, titrant addition is performed automatically un- in which the number of anions reacting is not the same as til a preset potential or pH, representing the endpoint, is the number of cations reacting, the endpoint as defined by reached, at which point the titrant addition ceases. the inflection of the titration curve does not occur exactly at Several acceptable electrode systems for potentiometric ti- the stoichiometric equivalence point. Thus, potentiometric trations are summarized in Table 2. endpoint detection by this method is not suitable in the Blank Corrections—As previously noted, the endpoint case of asymmetric reactions, examples of which are the determined in a titrimetric assay is an estimate of the reac- precipitation reaction, tion equivalence point. The validity of this estimate depends upon, among other factors, the nature of the titrate constit- + –2 2Ag + CrO4 uents and the concentration of the titrant. An appropriate blank correction is employed in titrimetric assays to enhance and the oxidation-reduction reaction, the reliability of the endpoint determination. Such a blank

+2 – correction is usually obtained by means of a residual blank 5Fe + MnO4 . titration, wherein the required procedure is repeated in every detail except that the substance being assayed is All acid-base reactions, however, are symmetrical. Thus, omitted. In such instances, the actual volume of titrant potentiometric endpoint detection may be employed in equivalent to the substance being assayed is the difference acid-base titrations and in other titrations involving symmet- between the volume consumed in the residual blank titra- rical reversible reactions where an indicator is specified, un- tion and that consumed in the titration with the substance less otherwise directed in the individual monograph. Two types of automatic electrometric titrators are availa- ble. The first is one that carries out titrant addition automat-

Table 2. Potentiometric Titration Electrode Systems Titration Indicating Electrode Equation1 Reference Electrode Applicability2 Acid-base Glass E = k + 0.0591 pH Calomel or silver–silver Titration of acids and bases chloride Precipitimetric (silver) Silver E = E° + 0.0591 log [Ag +] Calomel (with potassium Titration with or of silver in- nitrate salt bridge) volving halides or thiocya- nate Complexometric Mercury–mercury(II) E = E° + 0.0296(log k′ − Calomel Titration of various metals pM) (M), e.g., Mg+2, Ca+2 Al+3, Bi+3, with EDTA Oxidation–reduction Platinum E = E° + (0.0591/n) × log Calomel or silver–silver Titrations with arsenite, [ox]/[red] chloride bromine, cerate, dichro- mate, exacyonoferrate(III), iodate, nitrite, permanga- nate, thiosulfate 1Appropriate form of Nernst equation describing the indicating electrode system: k = glass electrode constant; k′ = constant derived from Hg–Hg(II)–EDTA equilibrium; M = any metal undergoing EDTA titration; [ox] and [red] from the equation, ox + ne red. 2Listing is representative but not exhaustive. 216 〈541〉 Titrimetry / Chemical Tests USP 36 present. The corrected volume so obtained is used in calcu- Calculate the alpha tocopherol content, in mg, in the as- lating the quantity of the substance being titrated, in the say specimen taken by the formula: same manner as prescribed under Residual Titrations. Where potentiometric endpoint detection is employed, the blank 30.2 AD/(LCD) correction is usually negligible. in which AD is the corrected absorbance; L is the length, in cm, of the absorption cell; and CD is the content of the assay specimen in the alcohol solution employed for the measurement of absorbance, expressed as g, capsules, or tablets per 100 mL. 〈551〉 ALPHA TOCOPHEROL ASSAY

The following procedure is provided for the determination 〈561〉 ARTICLES OF BOTANICAL of tocopherol as an ingredient. ORIGIN Hydrogenator—A suitable device for low-pressure hydro- genation may be assembled as follows. Arrange in a rack or in clamps two conical centrifuge tubes, connected in series by means of glass and inert plastic tubing and suitable stop- pers of glass, polymer, or cork (avoiding all use of rubber). SAMPLING Use one tube for the blank and the other for the assay spec- imen. Arrange a gas-dispersion tube so that the hydrogen In order to reduce the effect of sampling bias in qualita- issues as bubbles at the bottom of each tube. Pass the hy- tive and quantitative results, it is necessary to ensure that drogen first through the blank tube and then through the the composition of the sample used be representative of the specimen tube. batch of drugs being examined. The following sampling Procedure—Pipet into a suitable vessel 25 mL of the final procedures are the minimum considered applicable to vege- washed ether solution of the unsaponifiable fraction ob- table drugs. Some articles, or some tests, may require more tained as directed for When Tocopherol Is Present under Pro- rigorous procedures involving more containers being sam- cedure in the Vitamin A Assay 〈571〉, and evaporate to about pled or more samples per container. 5 mL. Without applying heat, remove the remaining ether in a stream of inert gas or by vacuum. Dissolve the residue in sufficient alcohol to give an expected concentration of Gross Sample about 0.15 mg of alpha tocopherol per mL. Pipet 15 mL into a 50-mL centrifuge tube, add about 200 mg of palla- Where external examination of containers, markings, and dium catalyst, stir with a glass rod, and hydrogenate for labels indicates that the batch can be considered to be ho- 10 minutes in the Hydrogenator, using hydrogen that has mogeneous, take individual samples from the number of been passed through alcohol in a blank tube. Add about randomly selected containers indicated below. Where the 300 mg of chromatographic siliceous earth, stir with a glass batch cannot be considered to be homogeneous, divide it rod, and immediately centrifuge until the solution is clear. into sub-batches that are as homogeneous as possible, then Test a 1-mL aliquot of the solution by removing the sol- sample each one as a homogeneous batch. It is recom- vent by evaporation, dissolving the residue in 1 mL of chlo- mended to include samples from the first, middle, and last roform, and adding 10 mL of antimony trichloride TS: no containers where the No. of Containers in Batch (N) is 11 or more and each container in the batch is numbered or let- detectable blue color appears. [NOTE—If a blue color ap- pears, repeat the hydrogenation for a longer time period, or tered in order. with a new lot of catalyst.] Pipet 2 mL of the supernatant into a glass-stoppered, No. of Containers No. of Containers opaque flask, add 1.0 mL of a 1 in 500 solution of ferric in Batch (N) to be Sampled (n) chloride in dehydrated alcohol,* and begin timing the reac- 1 to 10 all tion, preferably with a stop watch. Add immediately 1.0 mL 11 to 19 11 of a 1 in 200 solution of 2,2′-bipyridine in dehydrated alco- >19 n = 10 + (N/10) hol, mix with swirling, add 21.0 mL of dehydrated alcohol, close the tube, and shake vigorously to ensure complete (Round calculated “n” to next highest whole number.) 1 mixing. When about 9 /2 minutes have elapsed from the Samples are taken from the upper, middle, and lower sec- beginning of the reaction, transfer part of the mixture to tions of each container. If the crude material consists of one of a pair of matched 1-cm spectrophotometer cells. Af- component parts which are 1 cm or less in any dimension, ter 10 minutes, accurately timed, following the addition of and in the case of all powdered or ground materials, with- the ferric chloride-dehydrated alcohol solution, determine draw the sample by means of a sampling device that the absorbance at 520 nm, with a suitable spectrophotome- removes a core from the top to the bottom of the con- ter, using dehydrated alcohol as the blank. Perform a blank tainer, not less than two cores being taken from different determination with the same quantities of the same re- angles. For materials with component parts over 1 cm in agents and in the same manner, but using 2 mL of dehy- any dimension, withdraw samples by hand. In the case of drated alcohol in place of the 2 mL of the hydrogenated large bales or packs, samples should be taken from a depth solution. Subtract the absorbance determined for the blank of 10 cm because the moisture content of the surface layer from that determined for the assay specimen, and designate may be different from that of the inner layers. the difference as AD. Prepare the gross sample by combining and mixing the *NOTE—The absorbance of the blank may be reduced, and the precision of individual samples taken from each opened container, tak- the determination thereby improved, by purification of the dehydrated alco- ing care not to increase the degree of fragmentation or sig- hol that is used throughout the assay. Purification may be accomplished by nificantly affect the moisture content. the addition of a few crystals (about 0.02%) of potassium permanganate and of a few pellets of potassium hydroxide to the dehydrated alcohol, and sub- For articles in containers holding less than 1 kg, mix the sequent redistillation. contents, and withdraw a quantity sufficient for the tests.