Simultaneous Analysis of Tartrazine and Its Intermediates by Reversed Phase Liquid Chromatography D. P. Wittmer,' N. 0. Nuessle, and W. G. Haney, Jr. University of Missouri-Kansas City, School of Pharmacy, 5100 Rockhill Rd., Kansas City, Mo. 64 110
Tartrazine (FD and C Yellow 5 ) , a sulfonic acid dye used extensively in the food, drug, and cosmetic industry, is synthesized by reaction of sulfanilic acid with 3-carboxy-l-(4sulfophenyl)-5-pyrazolone (Pyrazolone T). Quantities of uncombined intermediates in the dye are currently determined by a laborious and relatively imprecise (f1096) column chromatographic procedure ( I ) which would seem to be a logical candidate for replacement with a high-pressure liquid chromatographic procedure. Methods have, in fact, been developed for the analysis of uncombined intermediates in structurally related dyes (2, 3 ) . However, these procedures are slow (25 minutes), require gradient elution, and suffer from a lack of reproducibility attributable to degradation of the resin over the course of a series of analyses. High pressure liquid chromatography appears to be the method of choice for rapid analysis of most highly polar substances; however, the large retention volumes observed for sulfonic acids on conventional adsorption media have ruled out normal-phase systems as a useful means of separating these compounds. In addition, the (recommended) guides which are normally used for rational selection of solvent systems are not applicable to ionic compounds ( 4 ) . The use of reversed-phase chromatography would seem to be a logical approach to separation of ionic solutes, since the polarity of the stationary phase can, in concept, be manipulated to obtain optimum affinity of the phase for the ionic solute. However, this approach has not been generally successful, a fact due in part to the absence of sufficiently polar stationary phases. Therefore, in the procedure described here, small quantities of a quaternary or tertiary amine have been added to the mobile phase in an effort to increase affinity of the dye and its synthetic intermediates for a lipophilic stationary phase. EXPERIMENTAL Apparatus and Operating Conditions. A Model ALC 202 Liquid Chromatograph equipped with a Model 6000 pump (Waters Associates, Milford, Mass.) was used and effluents were monitored with the 280-nm detector. Areas were determined with an electronic digital integrator (Varian Model 505). The flow rate was 0.9 ml/min. Column. A 30-cm X 4-mm i.d. gBondapak CIS (Waters Associates) column was used. KBondapak CIS has a monomolecular layer of octadecyltrichlorosilane chemically bonded to Porasil beads having an average particle size of 10 microns. The number of theoretical plates, based on rn-chlorobenzoic acid, was determined to be 2,500, and ko was 1.9 ml. Reagents. Authentic samples of tartrazine, Pyrazolone T, and sulfanilic acid were supplied by Warner-Jenkinson. Tetrabutylammonium hydroxide (TBAH) was used as a 10% solution in methanol, while tetraethylammonium hydroxide (TEAH) and tridecylamine (TDA) were used as 10%solutions in water. Mobile Phase. Methanol (400 ml), deionized water (400 ml) and formic acid (1.0 ml) was used as the solvent mixture with varying amounts of the substances listed in Table I. Standard Solutions. Solutions of sulfanilic acid, Pyrazolone T, and 3-nitro salicylic acid were prepared in methanol/water (50/50) to contain 0.10 mg/ml. Solutions of rn-chlorobenzoic acid (15.0 mg/ml) and tartrazine (10.0 mg/ml) were also prepared. Present address, Waters Associates, Milford, Mass. 01757. 1422
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 , JULY 1975
Sample Solution. A sample of dye (1000.0 mg) was weighed accurately and transferred to a 100-ml volumetric flash. Water (50 ml) was added, the flask shaken until solution was achieved, and methanol was added to volume. An aliquot (1.0 ml) of this solution was mixed with 1.0 ml each of the rn-chlorobenzoic acid and 3nitro-salicylic acid solutions. An appropriate amount (2-6 microliters) of this solution was injected into the chromatographic system. Analysis of components present in the dye was performed under the conditions given under Figure 1. The concentration of constituents present in the sample was determined by peak area ratios with the areas of sulfanilic acid and Pyrazolone T measured relative to 3-m-chlorobenzoic acid, and tartrazine relative to 3-nitrosalicylic acid. Accuracy and precision of the procedure were determined with laboratory prepared samples of known composition, and the results are summarized in Table 11.
RESULTS AND DISCUSSION Initial attempts to separate constituents of the dye sample on conventional adsorption or partition media were unsuccessful. The affinity of the components for the stationary phase was either too high (as in the case of microparticulate silicic acid) or too low (as with the permanently bonded supports). It was noted, however, that use of a lipophilic packing material in combination with the addition of selected salts to the mobile phase gave sufficient selectivity to effect the separation (Table I). When small quantities of formic acid are added to the mobile phase, tridecylamine may serve in the same capacity as the quaternary ammonium salts. I t appears that the mechanism for such a separation depends on the reversible formation of ion-pairs within the chromatographic system and separation of the constituents on the basis of differences in the lipophilicity of the ionpairs so formed. Thus, the more lipophilic the cationic constituent of the salt, the longer the retention time of the sulfonic acid derivatives on the lipophilic column. The generalities of ion-pair formation have been extensively reviewed (5, 6) and ion-pair formation has in fact been used Table I. Effect of Selected Salts on the Retention Volumea of Tartrazine and Its Synthetic Intermediates Sulfanilic acid,
Pyrazolone
Tartrazinc,
Moles
ml
T, m l
n.1
TEAH (1.0 x 10-3) TBAH (3.0 x 10-3) TDA (3.0 x 10-4) TBAH + TDA (3.0 x 10-3)(3.0x TBAH + TDA (3.0 X 10'3)(1.50 x lo-') TBAH + TDA (3.0 x 10-3)(0.60 X lo-') a Flow rate of 0.9 ml/min.
2.2
2.2
3.4
2.4
3 .O
6.0
8.9
13.0
4.5
6.5
60.0
4.2
5.5
14.0
3.2
4.0
7.1
90
Table 11.Quantitative D a t a Derived from Laboratory Prepared Samples P) razolonr T
Sulfanilic a c i d
Sample
Added, mg
1 2 3 4 5 6
0.05 0.10 1.00 10.0 10.0 0.05
Founda
98.4 99.7 100.2 98.4 99.7 100.8
Added, mg
(2.14) (1.41) (0.54) (1.46) (1.26) (2.44)
Percent recovered (% s t a n d a r d d e v i a t i o n ) ,
O
2
Found'
0.05 0.10 1.oo 10 .o 0.05 10.0
101.4 100.6 100.7 101.4 101.8 98.4
Added, m g
(1.63) (0.82) (0.21) (1.04) (1.51) (1.69)
100.0 100.0 98.0 80 .O 90.0 90.0
Found a
100.3 100.1 99.7 98.7 100.8 98.2
(0.54) (0.69) (0.52) (1.50) (0.06) (1.64)
N = 10.
5
1
Tartrazme
3
I
I
I
I
2
4
6
8
TIME imml
Tartrazine, synthetic intermediates, and internal standards separated on 30 cm X 4 mm MBondapak C18 (ko = 1.9 ml) with a mobile phase of water, methanol, formic acid (400:400:1) and 3.0 X of TDA of TBAH and 0.60 X
Figure 1.
Relative concentration of analytes: (1) Sulfanilic acid (1.O%); (2) Pyrazolone T (1 0%); (4) Tartrazine (98.0%). Concentration of Internal Standards: (3) 3rnetachlorobenzoic acid (0.033 rng/rnl), (5) m-chlorobenzoic acid (5.0 rng/rnl)
previously as a tool for effecting chromatographic analysis of selected organic ions ( 7 , 8 ) .The approach has been, however, t o use stationary phases heavily loaded with salts, and, as a result, column packing material has been shortlived. The use of selected salts in the mobile phase does not suffer from this disadvantage, and columns have been used for four months with no evidence of instability. I t is interesting t,o note (Table I) that the retention times of constituents in mobile phases obtained by mixing individual solutions containing different salts generally appear to be closely related to their retention times in the individual solutions. This relationship makes selection of the optimum mobile phase for the analysis rational and rapid.
Initially, the separation was monitored a t 254 nm. However, a t this wavelength, the molar absorptivity of sulfanilic acid is much greater than that of Pyrazolone T. The 280nm monitor for which the equimolar response is approximately equivalent, was used therefore to facilitate the simultaneous determination of the two uncombined intermediates. When the ratio of tartrazine to uncombined intermediates in the sample exceeds 50/1,an increase in signal attenuation after emergence of the first internal standard is necessary to keep all peaks on scale. Since it is necessary to quantitate as little as 0.1% of the synthetic intermediates of the dye, it was deemed necessary to use one internal standard for the two minor constituents and another for the major constituent. Standard curves were prepared daily for 10 days, and the standard deviation of the slope of the curves, the average correlation coefficient, and the average y-intercept value were calculated. The respective values for sulfanilic acid are 0.54%, 0.996,and -0.008;for 3-carboxy-l-(4-sulfophenyl)-5-pyrazolone 0.32%, 0.998,and -0.003;for tartrazine 0.40%, 0.998, and -0.002. These values indicate that, in each case, the procedure is amenable to use of a single-point standard. The procedure is relatively rapid with duplicate analysis of one sample requiring approximately one hour. More than eight hundred injections have been made with no discernible change in the chromatographic characteristics of the system.
CONCLUSIONS High pressure, reversed-phase liquid chromatography is an effective method for the simultaneous analysis of tartrazine and its uncombined intermediates when selected quaternary and tertiary amines are added to the mobile phase. This general procedure would appear to offer a logical approach to the analysis of other sulfonic acid dyes and their intermediates, as well as other ionic substances. L I T E R A T U R E CITED W. B. Link, J. Assoc. Off. Agric. Chem., 44, 43 (1961). M. Singh, J. Assoc. Off. Anal. Chem., 57, 219 (1974). M. Singh, J. Assoc. Off. Anal. Chem., 57, 358 (1974). L. R. Snyder, J. Chromatogr., 63, 15 (1971). R. Modin, Acta Pharm. Suecica, 9, 157 (1972). B. A . Persson, Acta Pharm. Suecica 8, 217 (1971). B. L. Karger and B. A. Persson, J. Chromatogr. Sci.. 12, 521 (1974). ( 8 ) B. L. Karger, S. C. Su, S. Marchese, and B. A. Persson, J. Chromatogr. Sci., 12, 678 (1974).
(1) (2) (3) (4) (5) (6) (7)
RECEIVEDfor review January 6,1975.Accepted March 31, 1975.
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