chromatograms and the calibration curve with the aid of simple additional calculations. These include the finding of the size of W i = hi/Zh i the given proportion of the fraction of the MW in question and AWi/AM i the proportion of the fraction up to a single MW which may readily be obtained from the data of Table I. As an example the integral and differen- tial curves of the MWD of samples of polyglucin and rheopolyglucin are shown in Fig. 2. Proceeding from these curves it is possible to determine the proportion of the fractions above and below a definite MW which is important for the assessment of the quality of the blood substitute.

Together with calculation of the MWD the chromatographic method may be used for check- ing a dynamic change of MW of dextran by direct comparison of the normalized chromatograms. This made it possible to check the process of hydrolysis of dextran and to follow its frac- tionation, purification, and its behavior in the blood stream.

Consequently the developed method of determining the molecular weight characteristics of dextra__n pr_eparations using HPEC made it possible to determine rapidly and reliably the size of M w, Mn, and D and also to follow dynamic changes of dextran MW.

LITERATURE CITED

i. M. A. Chlenov, L. I. Kudryashov, and A. S. Reshetov, in: Molecular Liquid Chromatography [in Russian], Chernogolovka (1979), pp. 63-64.

2. M. A. Chlenov, L. I. Kudryashov, and E. V. Titova, in: Liquid Chromatography [in Rus- sian], Chernogolovka (1982), pp. 37-38.

3. P. E. Barker, B. W. Hart, and S. R. Holding, J. Chromatog., 174, 143-151 (1979). 4. H. G. Barth, J. Chromatog. Sci., 18, 409-430 (1980). 5. H. Pitz and D. Le-Kim, Chromatographia, 12, 57-62 (1979). 6. A. C. Wu, W. A. Bough, E. C. Conrad, et al., J. Chromatog., 128, 87-99 (1976). 7. W. W. Yau, J. J. Kirkland, and D. D. Bly, Modern Size-Exclusion Liquid Chromatography,

New York (1979), pp. 274-322. 8. B. Yu. Zaslavsky, L. M. Miheeva, M. A. Chlenov, et al., J. Chromatog., 202, 63-73 (1980).

INFLUENCE OF AN INERT CARRIER ON THE DESTRUCTION OF CERTAIN UNSATURATED

KETONES IN ANALYSIS BY THE METHOD OF GAS-LIQUID CHROMATOGRAPHY

N. K. Falaleeva, M. Ts. Yanotovskii, UDC 615.356.011.17.07 and M. G. Romanyuk

Unsaturated polyenic ketones, 6,10-dimethylundeca-3,5,9-trlen-2-one (I) and 6, i0,14- trlmethylpentadeca-3,5-dien-2-one (II) are the main intermediates in the production of vitamins A and E; they contain conjugated double bonds, which are responsible for their high boiling points and thermal stability.

These circumstances, in conjunction with the presence of cis- and trans- isomerism, hinder their analysis, whereas in the synthesis of I and II and their use at subsequent stages, a complete quantitative and qualitative analytical monitoring of the content both of the main substance and of accompanying impurities is essential.

The method of gas-liquid chromatography (GLC) is the most convenient for performing such a task. However, its use, based on separation of the components in the vapor state, requires rather high temperatures in the analysis, which promotes destruction of the sub- stances to be analyzed under the action of various catalytic factors, for example, the inert carrier and the material of the chromatographic columns.

"Vitamin" Scientiflc-Industrial Association, Moscow. Translated from Khimiko-farmats- evticheskii Zhurnal, Vol ~ 19, No. 7, pp. 881-884, July, 1985. Original article submitted May 22, 1984.

0091-150X/85/1907-0499509.50 �9 1986 Plenum Publishing Corporation 499

2 z

~.

J d t v' a b c

Fig. i. Chromatograms of mixture X (a, c) and mixture Y (b, d), obtained from a metallic column with chromaton NAW, 0.20-0.25 ~n (a, b) and on a glass capillary column (c, d). a (Temperature of chromatography 140~ i) III; 2) cls-l; 3) trans-l; b (temperature of chromatography 150~ i) IV~ 2) cls-ll; 3) trans-ll; c (temperature of chromatography 150~ i) III; 2) cls-l; 3) trans-l; d (temperature of chromatography 170~ I) IV; 2) cls-ll; 3) trans-ll. Here and in Figs. 2 and 3 the shading denotes the area of destruc- tion S x.

The purpose of the present work was to determine the factors responsible for the de- composition of substances during chromatography, in order to determine the conditions for reliable analysis in industry.

At the first stage of the investigations we studied the influence of the column mater- ial. The stationary phase, carbowax 6000 (5% by weight on inerton AW-HMDS, 0.16-0.20 mm, silanized HMDS), was placed in metallic and glass columns of the same length (2.5 m), and a quantitative determination of I and llwas performed. It was established that the results of the quantitative determination do not depend on the column material; therefore in sub- sequent investigations we used chiefly metallic columns.

It is known that one of the factors promoting decomposition of a substance is the care- lyrical action of the inert carrier [2], In view of this we studied the influence of the inert carrier on the destruction cf I and II during their determination by the GLC method. The inert carriers most used in practice -- chromaton NAW, 0.20-0.25 mm, and inerton AW-HMDS, 0.16-0.20 mm, silanized HMDS -- were investigated; their influence was studied at various temperatures and times of stay of the substances to be analyzed in the column (time of con- tactwith inert carrier). The latter conditions was fulfilled using columns of different lengths.

In preliminary experiments on the enumerated carriers, various kinds of chromatograms were observed (Fig. la, b), confirming the destruction of I and II.

In view of the fact that the degree of destruction, expressed by the area Sx, may vary within wlde limits depending on the conditions of chromatography, it was necessary to select a method permitting monitoring of the change in the quantitative composition of I and II with a sufficient degree of accuracy.

A method was proposed in which thecontent of the investigated compounds is related to the content of substances present in the mixture in a constant amount, not subject to de- struction. For this purpose we used 6,10-dimethylundecanone-2 (hexahydropseudoionone, III) and the saturated ketone 6,10,14-trimethylpentadecanone-2 (IV).

As can be seen from a comparison of the formulas, these compounds have a carbon skele- ton analogous to I and II and the same functional group, in the chromatography of which they are removed from I and II and pass into the mixtures that must be analyzed in industrial practice.

500

J

b

3

C

/

3

t d

Fig. 2. Chromatograma of mixture X, obtained on matallic columns with a length of 1.28 m (a, b) and 2.5 m (c, d) with an inert carrier chromaton NAW, 0.20-0.25 mm. Temperature of chromato- graphy: a, c) 140~ b) 160~ d) 150~ i) III; 2) cis-l; 3) trans-l.

2

a b

3 2

C

a 2 !

d

Fig. 3. Chromatograms of mixture Y, obtained on metallic columns with a length of 1.28 m (a, b) and 2.5 m (c, d) with the inert carrier chromaton NAW, 0.20-0.25 ram. Temperature of chromato- graphy: a, c) 150~ b, d) 160~ i) IV; 2) cis-ll; 3) trans-ll.

EXPERIMENTAL

A Khrom-5 chromatograph (Czechoslovakia) with a flame-ionization detector was used in the work.

The following inert carriers were investigated: chromaton NAW, 0.20-0.25 nun (washed with acid) and inerton AW-HMDS, 0.16-0.20 mm, silanized HMDS.

In the first case carbowax 20 M in an amount of 5% of the weight of the carrier was used as the stationary phase, and in the second case carbowax 6000 in the same amounts.

Stainless steel columns with inner diameter 3 mm, length 2.5 and 1.28 m were used for analysis; carrier gas (nitrogen) velocity 40 ml/min, hydrogen velocity 60 ml/min, air velocity 300 ml/min.

The investigations were conducted on artificial mixtures of the following composition: mixture X 0.0396 g I + 0.0011 g III; mixture Y 0.2748 g II + 0.0868 g IV.

The solvent was acetone.

The amounts of I and II relative to the substances III and IV introduced were calculated according to the formula:

$1~ S~ Q ~ Ss t .100 %,

where S~, $2, and Sst are the areas of the peaks of the cis-isomer of I or II, the trans- isomer of I or II, and compound III or IV on the chromatogram in mixture X or Y, respect- ively, mm 2 .

The ratios of the areas of the peaks of I and III and of II and IV, calculated from the chromatograms obtained on glass capillary columns with a length of 54 m and inner diameter 0.27 mm and with the stationary phase PEG-40M (Fig. Ic, d), were taken as the initial quan- titative indices. These same chromatograms served as an example of maximum separation of the components of the mixture.

501

O.2

o,I

S/

, I I I I /20 /30 140 /80 /60 ; ~

Fig. ~. Dependence of the relative value of the decompo- sition of cis- and trans-lsomers of $-ionone I and the un- saturated ketone II on the temperature on the inert car- rier chromaton NAW, 0.20-0.25 mm. 1) Mixture X, column length 1.28 m; 2) mixture X, column length 2.5 m; 3) mix- ture Y, column length 1.28 m; along x-axis: temperature (in eC); along y-axls: ratio of the areas of the peaks of the unsaturated ketone investigated (I and II, respectively) and the standard (III and IV, respectively).

rl I I q~ kW ~ k~ O O-~ O O

0~"~ 0,-.4 3 Z O,w

a b c d

Fig. 5. Chromatograms of mixtures X (a, b) and Y (c, d) on metal- lic columns with a length of 1.28 m (a, c) and 2.5 m (b, d) with the carrier inerton AW-HMDS, 0.16-0.20 mm. a, b) (Temperature of chromatography 140@C): i) III; 2) cis-l; 3) trans-l; c (temperature of chromatography 180~ i) IV; 2) cis-ll; 3) trans-ll; d (temp s erature of chromatography 150~ i) IV; 2) cis-ll; 3) trans-ll.

The chromatograms were obtained on a Bichrome-i chromatograph. The pressure of the carrier gas (nitrogen) at the inlet was 1.8 kg-f/cm =, the hydrogen pressure 0.5 kg-f/cm 2, air pressure 0.8 kg-f/cm ~. For I and III the temperature of the thermostat was 150~ temperature of the evaporator 200~ for III and IV the temperature of the thermostat was 170~ temperature of the evaporator 250~

The effectiveness of the separation for mixture X was 7056 theoretical plates (for the cis-isomer) and for mixture Y 6528 theoretical plates (for the cis-isomer).

The use of capillary chromatography was also justified by the fact that the investigated substances do not interact with the inert carrier [I].

RESULTS AND DISCUSSION

Figures 2 and 3 present chromatograms of the investigated mixtures, obtained on an in- ert ca=rler chromaton NAW, 0.20-0.25 mm (washed with acid).

As can be seen from the chromatograms, depending on the temperature in the thermostat and the length of the column, destruction of I and II is observed (peaks 2 and 3).

The dependence of the destruction on the conditions of chromatography are illustrated in Fig. 4; on the graph, on account of the impossibility of correct determination of the area of peak 3 (see the chromatogram in Fig. la), the above-mentloned dependence for mixture Y on a column with a length of 2.5 m is not plotted.

5 0 2

Under the most unfavorable conditions (temperature 160~ column 2.5 m), compound I breaks down to a degree of 43%; at a column length of 1.28 m it breaks down to an extent of only 36%.

In view of the fact that the area of destruction S x was situated between the peaks of the cis- and trans-isomers, we can assume that during passage of the isomers through the chromatographic column, isomerization occurs.

Thus, it was shown that the inert carrier chromaton NAW, 0.20-0.25 mm, promotes destruc- tion of the unsaturated ketones I and II; the degree of destruction depends on the conditions of chromatography -- the temperature in the thermostat and the length of the column-- and does not depend on the column material.

To avoid obtaining unreliable results, the analysis of compounds I and II should not be performed on the inert carrier chromaton NAW washed with acid.

It has also been shown that on the inert carrier inerton AW-HMDS, 0.16-0.20 mm, no de- composition of I and II is observed (Fig. 5).

LITERATURE CITED

i. B. A. Rudenko, Capillary Chromatography [in Russian], Moscow (1978). 2. V. Supina, Packed Columns in Gas Chromatography [in Russian], Moscow (1977), p. 52.

DIFFERENTIAL SPECTROPHOTOMETRIC DETERMINATION OF BIGUANIDE DERIVATIVES

M. S. Goizman, S. O. Sarkisyan, UDC 615.31:547.495.9].074:543.42.062 A. A. Sarkisyan, and I. V. Persianova

The present work is devoted to the creation of a specific method of quantitative deter- mination of drugs that are biguanide derivatives, used as hypoglycemlc agents (glybutld, metformin, phenformin), antibacterial and antiparasitic agents (chlorhexldlne, blgunal) [3, 4].

The quantitative determination of biguanide derivatives in substances is usually per- formed by their acidimetric titration as diacidic bases in acetic anhydride medium (TU 42- 1572-80, glybutid) or in acetic acid medium [5, 7]. In the latter case the presence of mercury acetate is necessary.

The methods of acidimetric titration are not always suitable in sensitivity and speci- ficity for the quantitative determination of the content of the active ingredient in drug forms, especially at low doses of the preparation.

A number of methods have been proposed for the determination of diguanide derivatives in drug form; however, they do not possess sufficient specificity, are methodologically complex, and do not correspond in reproducibility to the level of the modern requirements for pharmacopeia analysis. For example, it has been proposed that argentometric titration according to the chloride anion [I], gravimetric determination in the form of the copper complex, and colorimetric determination at A 480 nm after treatment with sodlumhypobromite and cetrimide [5] be used for the determination of bigunal in tablets. Phenformin in drug forms is determined colorimetrically at I 520 nm after treatment with nitroprusside reagent [5] or spectrophotometrically at A 233 nm after two extractions of the preparation from al- kaline solution, followed by evaporation of the extract, dissolution of the residue in buf- fer solution with pH 7.0, and centrifugation [7].

To increase the sensitivity and selectivity of thequantitative determination of bi- guanide derivatives in drug forms and to simplify the analytical procedures, it seemed ad- visable to us to investigate the possibility of using the method of differential spectro- photometry, employing solutions of the preparation to be determined with various acidities.

cow. 1985.

S. Ordzhonikidze All-Union Chemical Pharmaceutical Scientific-Research Institute, Mos- Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 19, No. 7, pp. 884-889, July, Original article submitted September 5, 1984.

0091-150X/85/1907-0503509.50 �9 1986 Plenum Publishing Corporation 503