(Gibson, Peacock, Seligman & Sack, 1946), the radioactive phosphorus (Hevesy,

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1 170 J. Physiol. (I949) I09, I70-I76 6I2.II6:667.2I2 A MEBTHOD FOR TESTING THE PURITY OF COMMERCIAL SAMPLES OF TI824 WITH OBSERVATIONS ON THE AMOUNTS OF COLOURED IMPURITIES PRESENT AND THE ERRORS IN PLASMA VOLUME ESTIMATION CAUSED BY THEM BY D. LEESON AND E. B. REEVE* From The Clinical Research Unit, Guy's Hospital, London (Received 23 November 1948) It has been shown by a number of workers that the estimate of total red cell volume given by the dye (T 1824)-haematocrit method is greater than the estimate given by reliable marked red cell methods, such as the radioactive iron (Gibson, Peacock, Seligman & Sack, 1946), the radioactive phosphorus (Hevesy, Koster, Sorensen, Warburg & Zerahn, 1944; Reeve & Veall, 1949) and the Ashby methods (Barnes, Loutit & Reeve, 1948a). A review of the data will be found in Reeve (1948). A possible cause of the dye-haematocrit overestimate is the presence of coloured impurities in the samples of T 1824 used (Barnes, Loutit & Reeve, 1948b). To determine the plasma volume a known quantity (N) of dye is injected into the circulation, and when it is distributed through the plasma the quantity (n) in unit volume of plasma is determined. Plasma volume (V) is given by the equation V = N/n. N and n are estimated in terms of the amount of red light absorbed by dyed plasma solutions, measurement being made with a spectrophotometer or photoelectric photometer. N is estimated from the concentration of a standard dyed plasma solution, prepared by diluting a small quantity of the dye solution with undyed plasma. This solution will therefore contain T 1824 and any coloured impurities present. From the dye contents of plasma samples drawn at intervals of some minutes after the injection of dye, n is determined. If the coloured impurities present absorb significant amounts of the red light used for the photometric estimation of the dye, then their differential removal from the circulating plasma could result in erroneous estimates of plasma volume, and hence of the dye-haematocrit estimate of total red cell volume. It has been shown that samples of T 1824 may contain coloured impurities * Work undertaken on behalf of the Medical Research Council.

COLOURED IMPURITIES IN T1824 171 (Gregersen & Gibson, 1937). These workers used the spectrophotometer, an instrument neither easy to use nor generally available.

2 COLOURED IMPURITIES IN T (Gregersen & Gibson, 1937). These workers used the spectrophotometer, an instrument neither easy to use nor generally available. As a rough test of purity Gregersen & ]Rawson (1943) have used the 'capillary' filter-paper test. This method may, or may not, detect coloured impurities (see, e.g., Reeve, 1948). We now describe a simple chromatographic method which may be used for the detection and rough estimation of the amounts of coloured impurities present, and a rather more complex adaptation of this method by which the amounts of the coloured impurities may be estimated accurately and their significance assessed. We report observations with both methods on five commercial samples of T1824 supplied by pharmaceutical firms, two British and three American. The simple chromatographic method should be of use in the routine testing of new samples of T The tests we report below have been made on watery solutions of dyes autoclaved for 30 min. at 15 lb. pressure, since solutions used for plasma volume estimations are treated thus. METHODS Simple chromatographic method. Samples -of T 1824 can be adsorbed on a column of alumina. Coloured impurities and the dye itself can then be separated by elution with ammoniated aqueous ethanol mixtures of appropriate concentrations. Apparatu8. Glass tubing, diameter about 1-5 cm., length 20 cm., drawn out to a jet at one end which is lightly plugged with a small piece of cotton-wool. Pressure bottle consisting of a stout 1000 ml. bottle connected by pressure tubing (a) with a sphygmomanometer bulb, (b) with a rubber bung fitting the head of the above glass tubing. Reagents. Aluminium oxide, A1203, 'for chromatographic adsorption analysis according to Brockmann'; Savory and Moore Ltd., L9ndon. Glass wool, ground fine in a mortar. Acetic acid (1-5%); 1-5 ml. of glacial acetic acid and 98-5 ml. water. Ammonia solution (1%); 1 ml. of NH40H (sp.gr. 0-88) added to 100 ml. distilled water. Because of the volatility of concentrated ammonia solutions the 1 % ammonia solution was roughly titrated against HCI. 5 ml. of 1% NH40H was equivalent to about 8 ml.- of N110 HCI. Absolute ethanol.' Eluting reagent A: 80 ml. of absolute ethanol and 20 ml. 1% NH40H. Eluting reagent B: 20 ml. of absolute ethanol and 80 ml. 1% NH4OH. Preparation of column. A portion of a well-stirred mixture of alumina and distilled water is poured into the plugged glass tube. The alumina is allowed to settle and then pressed down firmly with a cork on the end of a glass rod. More of the mixture is added and the process is repeated until a uniform column of alumina about 6 cm. high is obtained. To protect the top surface a layer 3 mm. thick of ground glass-wool (added to the column as a watery suspension) is allowed to settle on the alumina. The column is washed three times with about 5 ml. of water, care being taken not to disturb the surface of the column nor to let the column run dry. The water and all succeeding solutions, unless otherwise stated, pass through the column at a steady rate of about 2 ml./min. They are forced through by air pressure applied at the head of the column by the pressure bottle. This method minimizes the escape of ammonia from the solution. Adsorption of dye. The column is washed with 10 ml. of 1-5% acetic acid. If this is omitted some samples of alumina fail to adsorb all the added dye. About 10 mg. of dye in 10 ml. distilled water are now added carefully and the water in the solutions allowed to pass through the column slowly under atmospheric pressure. The dye is adsorbed as a narrow blue band on the top of the column and is washed with 10 ml. of distilled water.

$172 D. LEESON AND E. B. REEVE Elution of fractions. Eluting reagent A is now added and forced through the column by air pressure.$

3 172 D. LEESON AND E. B. REEVE Elution of fractions. Eluting reagent A is now added and forced through the column by air pressure. In all samples tested this separates from the dye adsorbed at the top of the column a red impurity which rapidly passes down the column. The width of band and depth of colour of the red impurity are noted. The red impurity in 10 mg. can be completely eluted and collected in about 40 ml. of solution. When the red impurity has been removed eluting reagent B is added. This solution very rapidly brings the T 1824 fraction off the column. The T 1824 content of 10 mg. of commercial samples can be collected in about 20 ml. of reagent. In all samples tested a small amount of a violet compound was left at the head of the column. The width of the band and intensity of its colour are noted. This compound is partly decolorized on standing. We have been unable to elute it. It is possible that it consists of more than one compound, as on occasion we have separated it into two bands. RESULTS Tests with the simple chromatographic method on commercial samples oft Samples of T 1824 were obtained from British Drug Houses, Ltd., and Imperial Chemical Industries, Ltd. of Great Britain, and from the Eastman Kodak Co., the National Aniline Division of the Allied Chemical, and Dye Corporation, and the Warner Institute of the United States. Portions of these dissolved in distilled water were autoclaved at 15 lb. pressure for 30 min., and then fractionated on alumina columns as described above. The amounts of impurities were roughly measured from the width and intensity of the coloured bands. The results are summarized in Table 1. It can be seen that all samples of TABLE 1 Samples of T 1824 supplied by Red impurity Violet impurity I.C.I B.D.H E.K Warner N.A.D , indicates a trace; + +, shows an easily visible quantity; + + +, a rather greater quantity than + +; , a fair amount. dye tested contained appreciable amounts of the red and traces of the violet impurity. The sample supplied by the Warner Institute showed the least quantity of the red impurity and that supplied by Imperial Chemical Industries the greatest. The sample supplied by the British Drug Houses showed the greatest quantity of violet impurity. The T 1824 content of plasma samples is estimated from the optical densities measured in the red in the region of 620 m,u. with the spectrophotometer or photoelectric photometer. Red impurities transmit a high proportion of red light, but violet impurities may absorb a high proportion. Appreciable amounts of red impurity are therefore unlikely to cause much error, but much smaller amounts of a violet impurity might cause error. On the basis of these rough tests samples of dye for plasma volume estimation would be selected which

COLOURED IMPURITIES IN T1824.173 3 show a minimal quantity of violet impurity, and to be on the safe side no more than a moderate amount of the red impurity.

4 COLOURED IMPURITIES IN T show a minimal quantity of violet impurity, and to be on the safe side no more than a moderate amount of the red impurity. From the results shown in Table 1 the sample supplied by British Drug Houses would be taken as insufficiently pure because of the greater proportion of the violet impurity. The quantitative tests now to be described show, however, that the amounts of impurity present in this sample are permissible. Quantitative estimation of effects of impurities. The proportion of the total red light absorbed by the coloured impurities may be determined by comparing the optical density in the red of a known concentration of the unpurified commercial sample of dye with that of a purified sample having the same T 1824 concentration. By the chromatographic method described the fraction containing T 1824 can be separated from the red and violet impurities. This fraction, washed off the column with eluting reagent B, must be freed from alcohol and ammonia before its spectral properties can be compared with those of the original sample of dye. The following method was used. The small quantity of alumina particles present was removed by ifitering with suction through a sintered glass filter of porosity 4. Any dye adsorbed to the particles was freed by washing them with eluent B. The filtrate and washings were quantitatively transferred to a distillation flask. 20 mg. of sodium chloride were added to minimize adsorption of T 1824 to the glass surface of the flask. The ammonia, alcohol and some of the water were then distilled off under reduced pressure. Occasionally, in spite of the addition of sodium chloride, small quantities of T 1824 became adsorbed to the distillation flask walls and were lost. By measurement it was found that the amount so adsorbed was always less than 2% of the total. Comparison of the optical densities in the red of the unpurified and purified samples. The solvent, ph, salt content and protein content influence the absorption spectrum of solutions of T Therefore these variables must be standardized. Ideally they should approximate to the conditions in serum. Two solvents have been used: (a) Sorensen phosphate buffer of ph 7*3 and (b) diluted human serum. The latter if appreciably cloudy, but not otherwise, was extracted with about one-fifth of its volume of ether to remove fat. One volume of serum was diluted with one volume of 0X9 % sodium chloride solution. Purified and unpurified dye samples were prepared for measurement as follows. The unpurified dye solution consisted of 10 mg. of the commercial sample dissolved in 100 ml. of water. The purified dye solution consisted of the T 1824 fraction separated, as described above, from 10 mg. of the commercial sample and dissolved in 100 ml. of water: (1) to 5 ml. each of purified and unpurified dye solutions were added 5 ml. of Sorensen phosphate buffer of ph 7-3; (2) to 1 ml. each of purified and unpurified dye solutions were added 5 ml. of dilute serum. The optical densities of 5 mm. thicknesses of the dye solutions in phosphate buffer, and 1 cm. thicknesses of the dye solutions in serum were determined with the photoelectric photometer and red colour filters, cutting off a narrow band with its centre at 620 mgu., as described by Gibson & Evelyn

5 174 D. LEESON AND E. B. REEVE (1938). The percentage of the light absorption of the impure samples caused by the coloured impurities was then calculated from D impure sample - D pure sample 100 D impure sample where D=optical density. The results of a number of observations expressed thus are shown in Table 2. It will be noted that the samples supplied by the Warner Institute and Eastman Kodak have the least coloured impurities causing red light absorption, and that no sample as judged by the phosphate buffered solutions has more than 4% of its total red light absorption caused by coloured impurities. In dilute serum solution, though one sample showed an average of 6% of its total red light absorption to be due to coloured impurities, all the others showed 4% 6r less. On the whole, the agreement shown by the estimates in phosphaite and dilute serum solutions is good. Estimations on the dye contents of the dilute serum solutions are probably rather less precise, since slight variations in the light absorptions of the protein solutions themselves may influence the results. Hence results in phosphate buffer are to be preferred. As a further check on the light absorption of the coloured impurities, the red impurity was separated from a measured amount of the Imperial Chemical Industries' sample and its red light absorption compared with that of the same amount of unpurified sample. The method used was exactly as above, except that the red impurity was quantitatively washed off the column with eluent A, collected, then purified and concentrated by vacuum distillation. The results' are shown in Table 2, where it can be seen that its red light absorption in TABLE 2 Mean percentages of total red light absorption of five commercial samples of T 1824 due to the presence of coloured impurities. (a) Measured in phosphate buffer of ph 7-3; (b) measured in diluted human serum (a) Mean % red (b) Mean % red Samples of light absorption light absorption T1824 No. of of impurities in of impurities in supplied by analyses phosphate buffer Range dilute plasma Range.0I.C.I *6-6-8 B.D.H. 2 3*1 1* Warner *2-2*8 E.K "4-2 N.A.D Mean percentage of total red light absorption of the sample of T 1824 supplied by I.C.I. due to its contained red impurity, measured in phosphate buffer of ph phosphate buffer of ph 7-3 is about 1 % of the total red light absorption of the anpurified sample. Hand spectroscopic examination of the watery solutions of the red impurity showed its main light absorption to lie between 600 and

COLOURED IMPURITIES IN T 1824 175 520 m,u., that is in the orange and green.

6 COLOURED IMPURITIES IN T m,u., that is in the orange and green. The light absorptions of the red impurity and the unpurified dye sample were therefore measured in the green with the photoelectric photometer with a colour filter transmitting a band between 520 and 560 m,. In this region, in both phosphate buffer and in plasma, the red impurity has a light absorption of about 10% of the total of that of the unpurified dye. One observation was made on the Imperial Chemical Industries' sample to determine if prolonged autoclaving altered the relative amounts of T 1824 and its impurities. Portions of the same solutions in distilled water were autoclaved for 30 and 90 min. at'15 lb. pressure. The percentages of total red light absorbed by impurities were respectively 3-8 and 3-5 %. Thus prolonged autoclaving does not increase the amounts of coloured impurities. DISCUSSION The results presented show that in the commercial samples of T 1824 tested between 2 and 4% of the total light absorption in the red was due to coloured substances other than T If 1 % be allowed for the average losses in the method of purification then no sample contained impurities absorbing more than 3% of its total red light absorption. It is possible, but unlikely, that the fraction containing T 1824 contained other coloured impurities. This we have not completely'excluded, but in many -experiments with a variety of eluents, when we were searching for the best co"nditions for fractionating the dye samples on the column, we found no evidence that the T 1824 fraction contained other coloured compounds. The colour of the red impurity is rather similar to that of the alkaline form of T 1824, but in acid solution the two show quite different colours. The amounts of coloured impurities found in the samples tested are too small, even if after injection the impurities are very rapidly removed from the circulation, to cause significant errors in plasma volume estimates and hence significant overestimates of total red cell volume. With less pure samples, however, error might be caused. If the purity of a sample is in doubt it is wise to test it by the simple method described. If much red, violet or other coloured impurity is found, the sample should either be discarded for a better or purified. For purifying, the impure sample may be fractionated on an alumina column as described here, the T 1824 collected, filtered and either concentrated by vacuum distillation, or precipitated by salting out. Care should be taken in the choice of colour filters for the measurement of T 1824 concentration. Our observations show that the red impurity has a greater absorption in the green and orange than in the red. Filters and spectrophotometer slits should be used transmitting narrow bands centred at about 620 ma. If orange or green filters are used rapid removal of the red impurity might cause significant errors.

7 176 D. LEESON AND E. B. REEVE SUMMARY 1. Tests with a simple chromatographic method, by which the presence of coloured impurities may be detected and their amounts roughly estimated, are described on five commercial samples of T A method is described by which the' T1824 fraction and a fraction containing a red impurity can be quantitatively separated from impure commercial samples and concentrated. 3. It is shown that the coloured impurities in the five commercial samples of T 1824 tested absorb at most 4% of the total red light absorption of the sample. Hence the coloured impurities present cannot be responsible for the overestimate of total red cell volume given by the dye-haematocrit method. Our thanks are due to R. W. R. Baker, Esq. and Dr A. Shore for chemical advice and help, Dr J. Magladery for obtaining for us samples of T 1824, and Imperial Chemical Industries and the Warner Institute for gifts of T1824. REFERENCES Barnes, D. W. H., Loutit, J. F. & Reeve, E. B. (1948a). Clin. Sci. 7, 135. Barnes, D. W. H., Loutit, J. F. & Reeve, E. B. (1948b). Clin. Sci. 7, 155. Gibson, J. G. 2nd, & Evelyn, K. A. (1938). J. din. Invest. 17, 153. Gibson, J. G. 2nd, Peacock, W. C., Seligman, A. M. & Sack, T. (1946). J. din. Inve,st. 25, 838. Gregersen, M. I. & Gibson, J. G., 2nd (1937). Amer. J. Phy8iol. 120, 494. Gregersen, M. I. & Rawson, R. A. (1943). Amer. J. Phy8iol. 138, 698. Hevesy, G., Koster, K. H., Sorensen, G., Warburg, E. & Zerahn, K. (1944). Acta med. Scand. 116, 561. Reeve, E. B. (1948). Nuir. Abstr. Rev. 17, 811. Reeve, E. B. & Veall, N. (1949). J. Phy8iol. 108, 12.

EXPERIMENT 1. AIM: To prepare benzilic acid from benzyl using Green approach.

EXPERIMENT 1. AIM: To prepare benzilic acid from benzyl using Green approach. EXPERIMENT 1 AIM: To prepare benzilic acid from benzyl using Green approach. Requirements: 100 ml conical flask, 200ml beakers, filter paper, oven,0.5 g benzil,20% alc KH, dil.sulfuric acid, benzene for