Volumetric Analysis

VOLUMETRIC ANALYSIS (volumentry, titrimetric analysis) encompasses those methods of quantitative chemical analysis in which the amount of substance is judged by the volume of a reagent solution of definite concentration expended to produce a reaction with the substance being analyzed. Obviously, in volumetric analysis, precise measurement of volumes, precise determination of the titer of solutions (i.e., the volumetric concentration of substances dissolved in them, from which the name of such solutions—titrated—is derived), and establishment of the endpoint of reaction between reacting solutions are of great importance. For the latter purpose, indicators are used (see). Concentrations are usually expressed in normalities (see Normal solutions). Solutions of exactly the same normality react with each other in equal volumes; for example, to neutralize 10 cm³ of HCl, exactly 10 cm³ of alkali is required; if the solutions are not of exactly the same normality, then a different amount of alkali will be required for 10 cm³ of HCl, from which it is easy to calculate a correction coefficient (factor), by multiplication of which the titration results are recalculated to a strictly normal solution. For measuring volumes in volumetric analysis, special measuring apparatus proposed by Mohr is used: measuring (volumetric) flasks, pipettes, burettes. Measuring flasks have a long narrow neck, on which is marked a line indicating the volume of liquid that the flask holds up to the line; the flask is closed with a ground-in stopper. Flasks are calibrated at a certain temperature, usually at 15°. The volume occupied by 1 kg of water, weighed in a vacuum at 4° in a flask having a temperature of 15°, is called a normal liter. Due to the impossibility of weighing water at 4° in a flask at 15°, water is weighed in the flask at 4° in air and corrections are made for the change in weight in a vacuum and for the change in volume of the flask at 15°. On normal flasks there is a mark -?; the line is applied to the bottom meniscus. The allowable error in the weight of water in a liter flask is not more than 0.2 g. According to Mohr, flasks are calibrated at 17.5°; such a flask holds 2 cm³ more than a true liter; 17.5° the mark is -~^. There are flasks calibrated to the true liter, but at 17.5° 17.5°\", "-- /15° 20°\ -^-J ; other flasks also exist (^, -^J). Pipettes are intended for taking a definite volume of solution; a pipette is a tube with an expansion (reservoir) in the middle; the lower end of the pipette is narrowed, and on the upper end there is a line marking the volume (1, 2, 3, 5, 10, 15, 20, 25, 30, 50, 75, 100 cm³); after drawing the liquid into the mouth above the line, the pipette is pinched from above with the index finger. By loosening the pressure of the finger, the liquid is released to the line (to the bottom meniscus), then, removing the finger, the liquid is released into the prepared vessel (flask, beaker), applying the narrowed end to the wall of the vessel. There are various pipettes: for free flow, for blowing out (the last drops are blown out); it is convenient to remove the last drop by pinching the upper end of the pipette with the index finger of the right hand, and the reservoir with the left hand (from the heat of which the air in the pipette expands and displaces the drop). Pipettes are checked and calibrated by weighing the water released from them at a certain temperature. The error in taking a definite volume for pipettes of different capacities is expressed as 0.1-1.0%; the smaller the pipette, the greater the percentage error. There are pipettes graduated in cubic centimeters and fractions of cm³ (0.1-0.01); with such pipettes, any volume can be taken (within the entire volume of the pipette).-Burettes are used for measuring the liquid flowing out during titration; there should be no air bubbles in the tip of the burette, as this changes the volume. Before use, the burette should be dry or rinsed with the solution with which it will be filled. Burettes are calibrated and checked by weighing the water released from them (of different volumes). The titration itself is performed as follows: a definite volume of solution is measured into a beaker or Erlenmeyer flask with a pipette, 1-2 drops of indicator are added, and the other solution is carefully poured from the burette; toward the end, the liquid is released drop by drop until the color of the indicator changes. From the figures obtained, knowing the concentration of the known solution, the concentration of the unknown solution can be calculated.-Volumetric analysis is divided into several sections: the neutralization method (acidimetry and alkalimetry—determination of acids and alkalis), oxidation methods (oxidimetry, iodometry), and the precipitation method. In the latter method, the reacting solutions form an insoluble compound. This includes, for example, the methods of Mohr and Volhard (see Urine, analysis of urine). With the precipitation method, chlorides, cyanides, silver (by conversion to AgN0₃ and direct titration with NH₄CNS), Cu (by first converting to cuprous salt and then titrating with NH₄CNS) can be determined.

I. Yaichnikov. OVARIN, a name under which various preparations from the ovaries of large animals were formerly made, without regard to their specific activity. Ovarian preparations were either dry preparations of ovaries or nearly unrefined extracts from them. The impetus for a complete revision of the question of the ovarian hormone (ovarian hormone) was provided by the work of Stockard and Papanicolaou (1917), who studied the picture of the sexual cycle (already described in general outline as early as 1892 by Lataste) in rodents and established the possibility of determining, by histological examination of vaginal discharge, the specific changes in the uterine horn of small animals (rat, mouse) characteristic of the period of estrus. Allen and Doisy (1923-24), by injecting castrated animals with ovarian extracts prepared by various methods, were able to induce estrus with certain fractions. Thus, the specific reaction of the ovarian hormone (test-reaction, Allen-test) was established. The reaction is usually conducted on rats and mice, in whose vaginal discharge periodic microscopic examinations are made. In normal animals in the resting stage (dioestrus), the smear consists predominantly of leukocytes, a small amount of mucus, and occasional impurities (bacteria, etc.). In the next period—the beginning of estrus (pro-oestrus)—the smear shows a sharply decreased number of leukocytes and a large number of epithelial cells (sometimes in entire layers) with a well-staining nucleus according to Giemsa. The presence of these cells is explained by the temporary, during the period of estrus, proliferation of the vaginal mucosa, reaching 8-10-14 layers instead of the usual 2-4, and the desquamation of this epithelium. In the period of full estrus (oestrus), the smear consists entirely of large, clumpy, anucleate cells, which represent cornified epithelial cells; in this stage, leukocytes are completely absent from the smear. It is characteristic that in small rodents, erythrocytes are not observed in the vaginal secretion throughout the entire period of estrus. In castrated animals, the smear gives a picture of the resting stage. The introduction of active ovarian preparations in castrated animals can induce a complete picture of a single estrus, the natural duration of which (several days) can be arbitrarily prolonged by daily administration of preparations. Research by a number of authors has established the content of ovarian hormone in the female body at different periods and thereby indicated the paths for obtaining specifically active preparations. It further turned out that ovarian tissue is relatively poor in hormone, while the largest amount of ovarian hormone is contained in the follicular fluid at the time of ovulation. The hormone is present in the blood of women at different times in different quantities (maximum in the intermenstrual period). During pregnancy, the ovarian hormone is present in the blood in increasing amounts, reaching a maximum at the beginning of labor. From the blood, the hormone is excreted in the urine, where it is correspondingly present in maximum amounts in the last months of pregnancy. The initial idea about the internal secretory function of the ovaries during pregnancy, and hence about the overflow of the body with unnecessary hormone at this time, which is therefore excreted in the urine, has recently given way to another view, confirmed by facts, according to which large amounts of hormone enter the body from the placenta, which produces it continuously in increasing quantities. The mature placenta contains significantly more ovarian hormones than the ovaries. The high hormone content in urine and placenta and the availability of these products have led to the fact that many firms now prepare preparations from this type of raw material. Substances specifically acting on the female reproductive sphere have also been found in the plant kingdom (in rhubarb leaves, yeast, sprouting oats, willow leaves and catkins, sunflower, etc.). To obtain specifically active preparations, the raw material (ovaries, placenta, urine after evaporation and preliminary purification) is usually extracted with some lipid-soluble substance (chloroform, acetone, alcohol, benzene, etc.); after further processing, consisting of saponification, re-extraction with ether or benzene, and some other manipulations, the preparation can be converted into an aqueous solution. Previously, unrefined preparations of ovarian hormone were used in the form of oil solutions. The ovarian hormone, without changing, withstands heating up to 250° (in oil solutions even up to 260°), pressure up to 40 atm., is not destroyed by treatment with caustic alkalis (up to 20% KOH) and acids (up to 25% H₂SO₄) even when heated with them, dialyzes through parchment and collodion and is extremely easily adsorbed. Digestive tract enzymes do not act on the ovarian hormone, yet the effect of aqueous preparations per os is many times weaker than their effect when administered subcutaneously. Active ovarian preparations are dosed in biological units, which are established by the above-mentioned biological test on castrated animals. The minimum amount of preparation capable of inducing a single estrus in castrated rats (1 rat unit) or mice (1 mouse unit) is taken as the unit of action (ED); administration is done subcutaneously in several doses, since with fractional administration the effect of the preparation is enhanced and more pronounced; the effect appears already on the 2nd-3rd day. Numerous previous dry preparations of ovaries and extracts from them in most cases contain almost no specifically active principles. The study of the ovarian hormone from a chemical point of view culminated in obtaining it in crystalline form (Doisy, Butenandt, Laqueur and others). The crystalline preparation was obtained in very small quantities by prolonged and complex processing of huge amounts of urine. The crystals of ovarian hormone are colorless, plate-like and leaf-shaped, melt at 240°, dissolve well in lipid solvents and poorly in water. The molecular formula is not finally established (according to various authors: C₁₆H₂₀O₂, C₂₃H₂₈O₃, C₁₈H₂₄O₃); chemical analysis of the crystals indicates the content of C, H, and O; N, unlike other studied hormones, is not part of the crystals. The crystalline ovarian hormone belongs neither to proteins nor to carbohydrates; by its chemical properties it should rather be classified as a sterol or bile acid. The activity of the crystalline preparation is colossal: 1 g contains up to 10,000,000 (and according to some authors even much more) mouse units or up to 3,000,000 rat units. Commercial preparations are of course far from such purity, but are still sufficiently active and contain in 1.0 cm³ from several tens to several hundred mouse units. Ovarian preparations are extremely numerous. They can be divided into a group containing the specific ovarian hormone and a group of preparations of the previous type. Among the first, the most well-known are: novoovarikrin (Institute of Experimental Endocrinology, Moscow), ovarin ('Farmakon', Leningrad), Folliculin-Men-formon ('Degewop'), Hogival (Bad. Homburg), Unden or Hormovar (Bayer Meister Lucius), Progynon (Schering-Kahlbaum, tablets from placenta) and a whole series of others. The activity of preparations of this group, in most cases intended for subcutaneous administration, varies: 1.0 cm³ contains from several tens to several hundred mouse units. To the second group belong almost all without exception dry preparations—ovarin in tablets ('Farmakon', in Leningrad and Kiev san.-bact. institute), Ovaria sicca (Merck), Oototal (Laboschin), Ovaraden (Knoll), etc., and such liquid preparations as ovarin (Ukrainian organotherapeutic institute), ovarikrin (Institute of Exper. Endocrin.), Ovarium-ampoules ('Henning'), Ovosan ('Sanabo'), etc. The preparation Ovowop ('Degewop')—tablets representing a mixture of preparations of both types.

K. Sargin. The application of preparations from the ovary, prepared in various ways and administered per os, subcutaneously, and intravenously, has a considerable history. The indications for the use of ovarian hormone have been and are at present: 1) infantilism, hypoplasia of the genitals, virilism, 2) disorders of blood formation during sexual maturity - chlorosis, anemia, 3) disorders of menstruation - amenorrhea, oligomenorrhea, hypomenorrhea, dysmenorrhea, 4) early menopause (natural, artificial), physiological (symptoms of atrophy), 5) general disorders of nutrition and metabolism (obesity), 6) skin diseases associated with sexual maturity, normal and pathological menstruation, menopause, 7) nervous diseases - epilepsy, psychoses, 8) inflammatory diseases of the ovaries, 9) infertility (not on the basis of infection), 10) diseases of the endocrine glands (Basedow's disease, acromegaly), 11) disorders during pregnancy (threatened or beginning miscarriage, toxicoses and dermatoses). The results of treatment with ovarian preparations were not always positive, and the data of various authors were contradictory. Now, when it has been established that ovarian hormone is very stable with respect to high temperatures, acids, is not broken down under the influence of gastric juice and pancreatic juice, it becomes understandable the positive therapeutic effect in individual cases when using certain dry preparations from the ovary. On the other hand, the liquid preparations used in the past, examined by the Allen-Doisy method, turned out to be almost completely devoid of ovarian hormone (Zondek). The certain success achieved by these preparations should be explained by the action of the protein substances, amino acids, etc., contained in them. At the present time, when it has become possible to prepare extracts and preparations that actually contain ovarian hormone and to obtain them in any concentration, it has become necessary to review and verify the action of ovarian hormone in various pathological conditions. In general, the established indications for the use of ovarian hormone remain in force at the present time. However, the real value of ovarian hormone as a therapeutic factor is limited to disorders of menstrual function; but even in this direction, the evaluation of ovarian hormone cannot yet be considered complete. The possibility of giving a final judgment on the therapeutic value of ovarian hormone is considerably hampered by the fact that observations until recently were conducted with preparations that varied both in the method of their preparation (follicular fluid, pregnant urine, placenta, dry preparations, aqueous solutions, etc.) and in the amount of mouse units contained in them. Not all cases treated with ovarian hormone have actual value, since only those cases deserve attention where every element of chance is excluded, where observations were conducted on as homogeneous material as possible and for a sufficiently long time. The hopes placed on the use of ovarian hormone for amenorrhea have not been fully realized despite a number of experiments noting the growth of the uterus under the influence of administered ovarian hormone and observations where after its injection to women with infantile genitals the uterus enlarged, as if pregnant (Shushania). According to modern views, some forms of amenorrhea are the result of diseases of many endocrine glands. Therefore (as noted by Zondek) in appropriate cases of amenorrhea, combined treatment with ovarian hormone preparations with extracts from the anterior pituitary gland ('prolan'), thyroid gland ('thyovarin' of the Ukrainian organotherapeutic institute) is indicated. However, it should be borne in mind that in amenorrhea there may not be a decrease, but, on the contrary, an increase in circulating ovarian hormone in the blood, its appearance in the urine (Zondek). Such a form of amenorrhea, when the administration of ovarian hormone is contraindicated, is observed in girls, in women in the premenopausal period, where after varying durations of absence of menstruation, metrorrhagia (hemorrhagic metropathy) occurs. It is quite understandable that the use of ovarian hormone is also contraindicated in those cases where amenorrhea is caused by ovarian insufficiency due to weakening of the entire organism on the basis of various general diseases, etc. The use of ovarian hormone in such cases is indicated only after the general condition of the organism has been restored by various measures, and the function of the ovary nevertheless remains reduced. The data from the clinic of Stoeckel deserve attention, where the use of ovarian hormone for amenorrhea gave success in 21%, for oligo-, hypomenorrhea - in 46% and hypermenorrhea - in 61.5%. Such increasing success apparently finds its explanation in the position expressed by Schroeder that some forms of hypermenorrhea are a manifestation of one of the mildest degrees of ovarian insufficiency. It should be noted that according to the opinion of most researchers, the success of treatment with ovarian hormone is the more successful the less the phenomena of ovarian insufficiency. Similarly, the conclusion of those authors who consider that success in treatment can be expected in those cases where on examination the pelvic organs do not have clearly expressed signs of an infantile state. A favorable factor for treatment is also the short duration of the disease (6 months - 1^1/2 years). According to the data of the Halban clinic, treatment of ovarian insufficiency with ovarian hormone gives success in 60%. According to the conclusions of the Stoeckel clinic, more effective than in the treatment of menstrual function anomalies is the use of ovarian hormone for symptoms of atrophy of functions; successful treatment was noted not only with parenteral administration of the hormone, but also with its administration per os. Here should be noted the observations of Laqueur, who established an increase in metabolism under the influence of the ovarian hormone preparation in castrated females; similar are the observations of Zondek on the increase in metabolism in castrated women. Favorable success is noted in the treatment of skin diseases during the climacteric period with ovarian hormone, in kraurosis vulvae, acne, etc. The treatment of infertility with ovarian hormone with apparently favorable results in individual cases has been little studied. In some cases during treatment, enlargement of the uterus, mammary glands, a feeling of tension in them, pulling pains in the lower abdomen; a feeling of heaviness, as observed before menstruation; in isolated cases, an increase in libido was observed. The use of ovarian hormone is contraindicated in amenorrhea on the basis of tuberculosis (exacerbation of the process); in menorrhagias, treatment should be carried out very cautiously (intensification of bleeding). The mechanism of action of the hormone introduced into a woman's body is not yet completely clear. Some authors assume only a substituting action of the hormone. Shushania, introducing menformon to infantile rabbits, observed rapid growth of the uterus and mammary glands, while the ovaries themselves remained without visible changes. Other authors admit, in addition, an activating influence of the administered ovarian hormone on the ovary directly or through the pituitary gland. The activating role of the administered ovarian hormone is indicated with some justification by the case of the rapid appearance of menstruation after the start of treatment, since ovarian hormone can in essence only cause proliferation of the uterine mucosa and its hyperemia. From this point of view, the observation of Zondek, noting changes in the uterine mucosa corresponding to the secretory phase in a castrated woman under the influence of administered folliculin, is not quite clear; the Stoeckel clinic, conducting similar observations, does not note such data. The question of the dosage of ovarian hormone for therapeutic purposes has not yet been resolved in final form. Until the present time, observations were conducted with preparations that varied both in their method of preparation and in the amount of mouse units. The Halban clinic, for example, uses 'Novomar' containing 3-5 ME in 1 cm³, administering it daily subcutaneously for 28 days. The Stoeckel clinic used various preparations: 'Horigal' 4 ME, Folliculin-Menformon 4-40 ME - in 1 cm³, in the form of injections under the skin of 2 cm³ 3 times a week until the appearance of menstruation and continues treatment until the appearance of the next menstruation. The doses used also vary within wide limits, for example in the Pankow clinic, where Folliculin-Menformon is administered from 3 to 1,200 ME (total dose 36-80,000 ME). It should be noted that, having determined the content of ovarian hormone in the entire ovary at 4-6 ME, Zondek considered it sufficient to use for therapeutic purposes doses of 3-5 ME. However, Bochkarev quite rightly points out that such an approach to establishing dosage is not correct, since the organism does not work with the amount of hormone that is in the ovary at the time of its examination, but with the continuously hormone coming from the ovary.

From this point of view, observations with the introduction of preparations containing large amounts of ovarian hormone deserve attention. These observations provided very valuable data in that large doses are well tolerated by the body, without affecting blood pressure or heart activity, but at the same time, and this is especially important, are not accompanied by a noticeably better therapeutic effect compared to preparations containing a small amount of ovarian hormone; there is an assumption that part of the ovarian hormone is excreted unused. The insufficient effect with intravenous administration of ovarian hormone is explained by its rapid excretion from the body; along with this, the clinic of Pankov notes the significant advantages of the action of the oil solution of ovarian hormone (slow absorption). Observations of the clinic of Steckel note a favorable result in cases where the ovarian hormone was used exclusively in the form of a dry preparation (Ovowop) per os, three to four times a day, for twelve weeks, with a four-week break after the first six weeks. The effect of the administered ovarian hormone (according to observations of the Steckel clinic), e.g. in climacteric phenomena, was discovered quite soon (in individual cases after 5 days). All authors unanimously recommend a long course of treatment with ovarian hormone, which it is desirable to continue for some time even after the achieved effect. Some authors (Bokharev, Siebke) recommend along with the subcutaneous administration of ovarian hormone its simultaneous administration in the form of dry preparations per os. (Bokharev proposes such treatment for three to four weeks and continues for the same amount of time if no effect is observed.) It is quite clear, however, that each individual case requires an individual approach, and it is the physician's duty, depending on the peculiarities of the case, to make certain changes in terms of the duration of the use of ovarian hormone. It remains unclear why in the case of unsuccessful treatment with one preparation, a favorable effect is achieved by using another preparation. Along with this, doubts arise regarding the equivalence of the ovarian extract and the ovarian hormone obtained from the urine of pregnant women and the placenta, in which other hormones are also contained, which have been little studied to this day. All this requires further observations and detailed processing on as homogeneous material as possible; the resolution of these questions is considerably hampered by the fact that there is no method for determining the effect of the hormone in women (analogously, for example, to what happens in the treatment of INSULIN).

D. Gudim-Levkovich*