The application of metallochromic indicators in colorimetry—I

Talanta.

1960,

Vol. 5, pp. 141 to 146.

Pergamon

Press Ltd.

Printed

in Northern

Ireland

THE APPLICATION OF METALLOCHROMIC IN COLORIMETRY-I

INDICATORS

THE SPECTROPHOTOMETRIC DETERMINATION OF MINUTE AMOUNTS OF COPPER WITH ANALOGUES OF GLYCINE THYMOL BLUE M. KOCH, V. SVOBODA and J. K~RBL* Department

of Chemistry, School of Agriculture, Brno and Spolana, N.E., Research Centre of the Lachema Plant, Brno, Czechoslovakia (Received 23 March 1960) Summary-The possibility of application of five different metallochromic derivatives of Thymol Blue (I-V) to the spectrophotometric determination of micro amounts of copper has been studied. The derivative of proline (I), applied in a solution buffered with hexamethylenetetramine and hydrochloric acid, has proved best for this purpose. Absorbance of its copper complex shows only insignificant changes in the pH range from 4.9 to 5.4 and it conforms to Beer’s law up to 25 ,ug of coppern at 595 mp. Larger quantities of iron”, cobalt”, nickeln, zinc”, lead”, uranium” and beryllium” interfere.

the development of chelatometry many new indicators have been recently introduced into chemical analysis, undoubtedly the most important of them being the so-called metallochromic indicators .l Their great sensitivity, favourable colour properties and, in some cases, good selectivity are very promising as to their potential application to other fields of analytical chemistry, e.g. many calorimetric methods have been developed with the use of these substances. In the group of “complexone-type” metallochromic indicators only the application of Cresolphthalein Complexone and recently also of Xylenol 0range3-5 to spectrophotometry have been studied. Other analogous compounds have not been investigated from this standpoint, although many of them are capable of meeting all the requirements necessary for calorimetric reagents. One of the most promising indicators of this type is Glycine Thymol Blue, introduced in recent years into chelatometry. The chelating properties of this substance, as compared with the analogous Methylthymol Blue,’ are decreased to such an extent by eliminating one carboxymethyl group in the side-chain, that only the reaction with copper is actually applicable; other cations react only weakly or fail to react at all. Analogous condensation products of Thymol Blue with formaldehyde and other aminocarboxylic acids, prepared in connection with the systematic study of new metallochromic indicators,8 behave similarly. By further modifying the auxiliary functional groups9 a still higher specificity was reached as compared with Glycine Thymol Blue. All of the prepared substances are acid-base indicators with the colour properties of the parent dye, i.e. Thymol Blue. In an acidic medium, where the original colour of the dye is yellow, they form blue complexes with copperu.* The sensitivity of these WITH

* Present address, Pharmaceutical and Biochemical Research Institute, Prague, Czechoslovakia. 141

142

M. KOCH, V. SVO~~DAand J. K~RBL

reactions, stability of the colour formed, and sufficient selectivity promise-according to preliminary investigations-the potential use of some of these substances as chromogenic reagents for s~ctrophotometric detestation of copper. From this viewpoint five most satisfactory reagents (I-V) have been studied; the results of this study are reported in this paper.

H”51?5, R*H,C 1

/$
No.

R

Name

COOH / (I)

(II) (ITI)

Proline Thymol Blue

-Nr ii CH,COOH N/ \CH,

Sarcosine Thymol Blue

-NHCH,COOH

Glycine Thymol Blue

CH, (TV)

-NHCCOOH I CH,

Methylalanine Thymol Blue

/COOH (V)

Serine Thymol Blue

-NHCH, \CH20H EXPERIMENTAL

AND

RESULTS

Apparatus and reagents The absorption spectra of the reagents and their copper complexes were measured by means of a quartz spectrophotometer SF 4 in cells of l-cm optical path. Other absorption measurements were made on a K&rig-Martens spectrophotometer with an entrance and eye-piece slit width of 0.2 mm. For the pH measurements an Acidimetr AK (Kovodruistvo, Prague), with a high-resistance glass electrode, was used. 0.1% solutions of reagents (I)-(V) were prepared in double-distilled water; all of these solutions were stable for several weeks. The stock solution (0.~1~) of coppef was prepared by dissolving 0.2497 g of CuS0,5HB0 (A. R. grade) in double-distilled water and diluting to 1 litre. Working solutions were prepared by appropriate diluting of the stock solution. O.lM solutions of other cations were prepared by dissolving the corresponding salts (A. R. grade). Buffer solutions for the pH range from 4.2 to 6.2 were prepared by mixing a 1M solution of hexamethylenetetramine (A. R. grade) and 6N hydrochloric acid in the appropriate proportions.

Determination

of copper with analogues of Glycine Thymol Blue

143

Qualitative test for copper and sensitivity of the reaction To a 5-ml sample solution of copper buffered with hexamethylenetetramine and hydrochloric acid, 3 drops of the reagent solution were added and the colour formed was compared, after a few set, with a blank. According to the amount of copper present, the resulting colour was either skyblue or, with smaller quantities of copper, purple or grey. The sensitivity of the reaction at the optimal pH values is given in Table I. TABLE I.-SENSITIVITY Reagent Proline Thymol Blue Sarcosine Thymol Blue Glycine Thymol Blue Methylalanine Thymol Blue Serine Thymol Blue

PH

PD

4.9 4.6 5.4 5.1 6.0

7.6 7.6 7.6 7.4 7.4

Absorption spectra The yellow coloured, slightly acid reagent solutions show an absorption maximum at about 450 rnp. The formation of a copper complex causes a bathochromic shift of about 150 rnp. Fig. 1 shows the absorption spectrum of Proline Thymol Blue (curve 1) and of its copper complex with an excess of the reagent (curve 2) at pH 5.2. Absorption curves of the copper-complexes of all of the compounds studied (I-V) measured against the reagent blanks are illustrated in Fig. 1 and 2.

mp

FIG. 1.

mP

FIG. 2.

Effect of pH and choice of the appropriate buffer The effect of pH on the sensitivity of the reaction was studied with different buffer solutions. However, their choice was restricted since some of them, e.g. citrate, mask the reaction. Hexamethylenetetramine was found to be the most convenient buffer. Acetate buffer is less suitable for it slightly decreases the sensitivity, and gives negative results in the presence of larger amounts of iron”’ or tinn. The relation between the absorption of the copper complex and pH was studied in hexamethylenetetramine buffer at different wavelengths and with an excess of the reagents. Fig. 3 shows the absorbancy plotted against pH at wavelengths of absorption maxima (see Fig. 2) in the pH range from 4.2 to 6.2. All the obtained curves have two branches: the ascending one represents the successive formation of the complex up to a maximum lying at the pH at which the reaction with copper is quantitative. A moderate slope of the second branch is caused by the commencing acid-base change of the reagent blank against which the measurements have been made. From this standpoint, Proline

144

M. KOCH,V.SVOBODA

and J. KORBL

Thymol Blue has been found to be the most convenient reagent for the spectrophotometric determination of copper, since the pa-curve of this reagent shows only insigniticant changes of absorbancy in rather broad limits around pH 5.2. Calibration curves

The validity of Beer’s law has been verified in the hexamethylenetetramine-hydrochloric acid buffer at a reagent concentration of 0.2 ml of a 0.1% solution in a total volume of 25 ml. Measurements were made at wavelengths of the maximum difference in absorbancy of the copper complex against the reagent blank (see Fig. 2), and at two different pH values. Proline Thymol Blue follows Beer’s law best of all these reagents. Calibration curves of this dye are illustrated in Fig. 4. Curves

A

-.-.-

PH

FIO. 3.

pg

Cu/25m

pH 49

L

FIG. 4.

No. 1 were obtained with 5-cm cells at pH 4.9 and 5.4, respectively, and conform to Beer’s law up to 10 pg of copper” in 25 ml. Calibration curve No. 2 was obtained at the same pH values in 2-cm cells for amounts of copper up to 25 pg in an equal volume. A slight “gooseneck” curvature occurs but even in this case Beer’s law is followed well within the limits of error of measurement. Influence of foreign ions

The effect of foreign ions was studied by absorption measurements in the solutions of copper complexes of all of the reagents with 0.2 ppm of copper” at the same wavelengths as in the preceding determination. As interfering were classified those ions causing a greater difference in absorptionin comparison with the absorption of the copper complex alone-than O+lO.5units. On the basis of the results obtained the reagents studied may be arranged in the following sequence of increasing sensitivity to foreign ions: Proline Thymol Blue, Methylalanine Thymol Blue, Sarcosine Thymol Blue, Serine Thymol Blue and Glycine Thymol Blue. From this viewpoint Proline Thymol Blue was again found to be the most convenient reagent for the application studied, which under the conditions used for the determination of copper reacts only with cobalt, zinc, nickel, lead, uranium and beryllium. Iron” interferes if present in an approximately 2*5-fold excess; however, this interference may be easily removed by oxidation to the tervalent form and by the subsequent addition of an appropriate quantity of ammonium fluoride. Upper limits for the concentrations of all of the interfering cations allowing the determination of 0.2 ppm of copper” are given in Table II. Chromium”’ interferes by virtue of its own colour and all cations precipitated under the conditions used prevent the determination of copper. Of the anions iodides, dithionites, ferro- and ferricyanides, oxalates, citrates, tartrates and aI1 substances giving even weak complexes with copper, interfere. Strong oxidants and reductants destroy the dye.

Determination of copper with analogues of Glycine Thymol Blue

145

Stability of coloration

The stability of the coloured complex of copper with Proline Thymol Blue was studied at the laboratory temperature by measuring the absorption at regular time intervals. The maximal colour developed over a period of a few min and no decrease of absorbance was then observed even after several days. TABLE II.-INTERFERING

I

Limiting cont., ppm

I

Added as

Ion

IONS

pH 4.9

I

0.5

Fez+

0.5 1000: 0.7 0.7 3 7 9 10

1ooo* 0.8

CoS0,.7H,O UO,(CH,COO),.2H,O NiS0,.7H,O BeS0,.4H,O ZnS04.7H,0 Pb(NO,),

COB+

uop Nia+ Bee+ Znz+ Pb2+

pH 5.4

1.8 7 10 15

40

* After oxidation to Fe3+ and addition of NH,F.

DISCUSSION A large number have been proposed justified. There are, should be included.

of reagents for to date, so that in our opinion, Its advantages

1. High sensitivity, expressed coefficient of 19600 at 595 rnp.

the spectrophotometric determination of copper the addition of any new ones must be properly serious reasons for which Proline Thymol Blue are:

by the value of pD = 7.6 and a molar

2. Absorption

of its copper complex

3. Favourable

properties

4. Practically

unlimited

sufficiently

independent

with regard to interferences stability

of the coloration

extinction

of pH.

from other cations.

formed.

The results presented in this paper show that only a very few of the reagents previously suggested and used for the spectrophotometric determination of copper can successfully compete with Proline Thymol Blue. By comparison with the sensitivity of other reagents, as expressed by molar extinction coefficients, it follows that only four substances (oxalyldihydrazide, Zincone, dithizone and 1 :Sdiphenylcarbohydrazide) are better-in this respect-than Proline Thymol Blue (cJ lo). However, Zincone and dithizone show very low specifity so that in applyjng these reagents a tedious and time-consuming separation of copper from other interfering cations is usually indispensable. The application of diphenylcarbohydrazide is restricted by the low stability of both the reagent solution and the colour produced; it is also necessary to maintain accurately both the pH (a change of 0.01 in pH implies a 2 % error in the determination) and the temperature. lo On the other hand, the absorption of the copper complex of Proline Thymol Blue changes but negligibly over a pH range from 4.9 to 5.4, the reagent is practically of unlimited stability, and only a few cations interfere with the determination. From this point of view Proline Thymol Blue seems to be very convenient for the

146

M. KOCH,V.SVOBODA

andJ.

K~RBL

determination of minute amounts of copper, especially in biological materials. In this case only the interference of ironn presents a certain difficulty; this difficulty can, however, be obviated by the oxidation of the iron to the tervalent form and by the addition of a small amount of ammonium fluoride. Zusanunenfassung-Die Moglichkeit der Anwendung von ftinf verschiedenen metallochromen Thymolblauderivaten /I-V/ fiir die spektrophotometrische Bestimmung von CuZ+-Mikromengen wurde untersucht. Am besten bewiihrte sich das Prolinderivat /I/ in Losungen, die mit Urotropin-Salzsaure gepuffert wurden. Die Absorption des Kupferkomplexes dieses Derivats bleibt in pH-Grenzen 4.9-5.4 praktisch konstant und folgt dem Beerschen Gesetz bis zu 25 pg Kupfer bei 595 rnp. Griissere Mengen von Fez+-, Co2+-, Ni2+-, Zn2+-, Pba+-, Be2+- und U0,2+-Ionen storen. R&.um&Les auteurs ont BtudiCla possibilite d’application de cinq differents derives metallochromiques du bleu de thymol au dosage spectrophotometrique de micro-quantites de cuivre. Le derive de la proline utilise en solution tampon&e avec l’hexamethylenetetramine et l’acide chlorhydrique s’est rev& au mieux pour ce dosage. L’absorption de son complexe avec le cuivre montre seulement des changements insigniliants dans le domaine de pH 4,9-5,4 et obtit a la loi de Beer jusqu’ii 25 ,og de Cue+ a 595 rnp. De plus grandes quantites de Fe e+, Co2+, Ni2+, Zn2+ Pbe+, UO:+ et Be2+ genent. REFERENCES 1 J. K&b1 and R. Pribil, CON. Czech. Chem. Comm., 1957, 22, 1122. * F. H. Pollard and J. V. Martin, Analyst, 1956,81, 348. a K. L. Cheng, Taluntu, 1959, 2, 61, 186, 266. 4 Idem, ibid., 1959,3, 81. 5 Idem, ibid., 1959,3, 147. B J. Korbl, E. Kraus and R. PHbil, Coil. Czech. Chem. Comm., 1958, 23, 1218. 7 J. Korbl and R. Piibil, ibid., 1958, 23, 873. 8 J. Korbl, V. Svoboda, D. Terzijska and R. Pfibil, Chem. and Znd., 1957, 1624. 9 J. Korbl and R. Pribil, Znd. Chemist, 1958, 34, 616. lo R. W. Turkington and F. M. Tracy, Analyt. Chem., 1958,30, 1699.