Film Developing Baths. BiruteË PranaityteË, ZËydru¯nas Daunoravicius, and Audrius PadarauskasÐ. Department of Analytical and Environmental Chemistry, ...
Microchim. Acta 149, 49–54 (2005) DOI 10.1007/s00604-004-0307-2
Original Paper Micellar Electrokinetic Chromatography of X-Ray Film Developing Baths Birute˙ Pranaityte˙, Zˇydru¯nas Daunoravicˇius, and Audrius Padarauskas Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, LT-2006 Vilnius, Lithuania Received June 28, 2004; accepted September 3, 2004; published online January 5, 2005 # Springer-Verlag 2005
Abstract. A simple and fast micellar electrokinetic chromatographic (MEKC) method for the determination of five main compounds (hydroquinone, metol, phenidone, 2-hydroxy-5-methylaminobenzenesulphonate and 2,5-dihydroxybenzenesulphonate) in X-ray film developing solutions was developed. The optimal conditions for the separation were established by varying the concentration of sodium dodecyl sulfate (SDS), the electrolyte pH and the temperature. Successful results were obtained with a 25 mmol L1 Tris-phosphate buffer at pH 9.0 in the presence of 50 mmol L1 SDS using direct UV detection at 214 nm. Under these conditions, baseline separation of the five compounds was achieved in less than 8 min. The method was validated in terms of precision, linearity, accuracy, and successfully applied to the analysis of X-ray film developing baths. Key words: Micellar electrokinetic chromatography; developers.
Black and white photographic processes are extensively carried out by hospitals and many private medical companies to obtain X-ray plates. Black and white developers usually contain three main ingredients: the developing agents, the preservative (sulphite) and the alkali (carbonate, tetraborate, hydroxide etc.) [1]. The two most frequently encountered developing Author for correspondence. E-mail: audrius.padarauskas@ chf.vu.lt
agents are hydroquinone (1,4-dihydroxybenzene) and metol (4-methylaminophenol sulphate). Hydroquinone is an excellent electron donor but a slow developing agent because it is not hydrophobic. Developing agents with good surfactant properties, such as metol, transfer electrons efficiently, but oxidized forms then take time to move aside for unused molecules. When hydroquinone and metol are combined, the result is a fast acting developer that develops to a fairly high contrast. This co-operation between two developing agents of different surfactant properties is known as super-additive development. Phenidone (1-phenyl-3-pyrazolidone) is often used as a hypo-allergenic substitute for metol. Phenidone not only acts the same as metol, that is, the combination of the two is super-additive, but the phenidone possesses the property of being regenerated by the hydroquinone. This results in a developing solution that is not only active from the start, but one that retains its activity longer. To prevent the autocatalytic oxidation of the developing agent, a chemical compound called ‘preservative’ is added to the developer, the choice being almost universally sodium sulphite. Quinone formed during a developing process reacts with sulphite, forming 2,5-dihydroxybenzenesulphonate (DHBS). Similarly, metol yields the 2-hydroxy5-methylaminobenzenesulphonate (HMABS). This action prevents the auto-oxidation of the electron donor but also keeps the developing solution clear and colourless.
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The analysis of these baths is important in determining the quality and effectiveness of the developing process [2]. Moreover, in order to minimize unwanted environmental contamination, the effluents from used baths should be collected, and the harmful compounds should be converted to alternate forms (e.g. CO2, N2, H2O etc.). In the last decade various oxidation techniques have been investigated for this purpose [3–5]. The main problem of such investigations is rapid and simple monitoring of the contaminants before and during their decomposition. Since common X-ray photographic developing baths usually contain more than one developing agent with very similar chemical properties, analytical methods allowing simple, rapid and sensitive determination of all compounds in a single analysis are required. In the last few years, capillary electrophoresis (CE) has been successfully introduced in the analysis of various photographic solutions [6–12]. However, since most of the developing agents are neutral compounds, micellar electrokinetic chromatography (MEKC) should be used for their separation. MEKC is a capillary electrophoretic technique for the separation of uncharged compounds and was first reported by Terabe et al. [13]. In MEKC the separation of neutral analytes is based on analyte partition to micelles formed by addition of surfactants to the electrolyte. Of the four common classes of surfactants (anionic, cationic, nonionic, zwitterionic), anionic surfactants, especially sodium dodecyl sulfate (SDS), have seen the most use in MEKC. Although many excellent applications have been performed by the MEKC technique [14, 15], to our knowledge, there are no reports concerning MEKC analysis of developing solutions. The main aim of this study was to evaluate the MEKC technique for a rapid and simple analysis of common X-ray film developing solutions. Experimental Separations were performed on a P=ACE 2100 apparatus (Beckman Instruments Inc., Fullerton, CA, USA) equipped with a UV detector with wavelength filters (200, 214, 230 and 254 nm). Fused silica capillaries (Polymicro Technology, Phoenix, AZ, USA) of 75 mm i.d. and 57 cm total length (50 cm to the detector) were used. Samples were injected in the hydrodynamic mode by overpressure (3.43 103 Pa). System Gold software (Beckman Instruments Inc.) was used for data acquisition. UV detection was employed at 214 nm. All experiments were conducted at 25 C using a liquid thermostated capillary cartridge. The HPLC instrumentation consisted of a Varian Model 5060 high-pressure pump, an injection valve equipped with a sample loop of 20-mL and a Waters 990 variable wavelength UV detector set to
B. Pranaityt_e et al. absorb at 254 nm. The results and data were collected and plotted on a plotter=integrator SP 4290 (Spectrophysics, San Jose, CA, U.S.A.). Isocratic HPLC separations were performed on a 10 mm Separon TM SGX CN (150 3 mm i.d.) column (TESSEK Ltd., Prague). The mobile phase flow rate was 0.3 mL min1 . All electrolyte and standard solutions were prepared using doubly distilled helium degassed water. Sodium dodecyl sulfate (SDS) was purchased from Merck (Darmstadt, Germany). All other reagents were of analytical-reagent grade obtained from Aldrich (Milwaukee, WI, USA). Since the 2-hydroxy-5-methylamino-benzenesulphonate was not commercially available, this compound was kindly provided by Dr. R. Ragauskas (Institute of Chemistry, Vilnius) and was used without further purification. The stock analyte solutions (0.005 mol L1 ) were prepared daily by dissolving in oxygen-free water. All working solutions were prepared before use by suitable dilution. Carrier electrolytes were prepared by neutralization of 25 mmol L1 H3PO4 and 50 mmol L1 SDS solution with NaOH or tris-(hydroxymethyl)-aminomethane (Tris) to desired pH. The mobile phase system for HPLC analysis was 30% CH3OH and 70% 0.05 mol L1 KH2PO4. All electrolyte and sample solutions were filtered through a 0.45 mm membrane filter. Each new fused-silica capillary was flushed with 1 mol L1 NaOH for 30 min and then with deionised water for 15 min. Finally, the capillary was flushed with the carrier electrolyte for 20 min. Between all electrophoretic separations, the capillary was rinsed with carrier electrolyte for 2 min.
Results and Discussion Method Development Being strong reducing agents, all developers are very sensitive to oxygen, especially in alkaline solutions. For this reason initial MEKC separations were performed using phosphate electrolyte at pH 6.5. The concentration of SDS is a key parameter in MEKC, and its effect on the separation was studied in the range of 0–50 mmol L1 (Fig. 1). As expected, the increase in SDS concentration does not significantly affect the migration behaviour of both sulphonates. Their effective mobilities decrease approximately from 2.82104 and 3.05104 cm2 V1 s1 at zero SDS concentration to 2.57104 and 2.85104 cm2 V1 s1 at 50 mmol L1 SDS for HMABS and DHBS, respectively. A gradual decrease in the mobilities for these anions is caused mainly by the increase in electrolyte ionic strength and=or electrolyte viscosity, indicating that the charge repulsive interaction between the sulphonates with anionic SDS is very strong and prevents the analytes from partitioning into SDS micelles. Both sulphonates migrated against electroosmotic flow at a different velocity based on their charge-to-size ratio. In contrast, the migration behaviour of the neutral and cationic analytes depends primarily on the attractive interactions of each analyte with the SDS micelles and the
Micellar Electrokinetic Chromatography of X-Ray Film Developing Baths
Fig. 1. Effect of SDS concentration on the migration times of the analytes and EOF. Electrolyte: 25 mmol L1 NaH2PO4 pH 6.5. Conditions: fused silica capillary, 75 mm i.d., 57 cm total length, 50 cm to the detector; injection, pressure (3.43 103 Pa) for 8 s; voltage, 25 kV; temperature, 25 C; UV detection at 214 nm. (1) EOF (thiourea); (2) hydroquinone; (3) metol; (4) phenidone; (5) HMABS; (6) DHBS
partition of an analyte into SDS micelles is favoured with increasing SDS concentration. The results presented in Fig. 1 show that the migration time of metol increases much more than that of hydroquinone and phenidone, indicating that the positively charged metol, compared with uncharged hydroquinone and phenidone, is most strongly attracted to the micelles. It is attributed to the electrostatic interaction between metol and micelles as well as the formation of ion pairs. However, at SDS concentrations higher than 20 mmol L1 the peak of the metol begins to broaden and tail. Another important parameter affecting the separation selectivity is the pH of the carrier electrolyte. The effect of pH was measured in the pH range of 6.0–9.0 keeping the SDS concentration in the electrolyte constant (50 mmol L1 ). At each pH point, the capillary was rinsed with a fresh electrolyte for 10 min. The results obtained from this experiment are summarized in Fig. 2. At higher pH values it can be observed that the increased EOF causes a decrease in the migration times of all compounds. Only a slight increase (10–12%) in the effective mobilities for the neutral (hydroquinone and phenidone) and anionic (both sulphonates) analytes was observed in the pH range studied. By contrast, in the case of metol, its net positive charge decreases with pH due to the deprotonation. This deprotonation process resulted in reducing the electrostatic interaction of metol with anionic
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Fig. 2. Effect of electrolyte pH on the migration times of the analytes and EOF. Electrolyte: 25 mmol L1 NaH2PO4, 50 mmol L1 SDS. Other conditions as in Fig. 1. (1) EOF (thiourea); (2) hydroquinone; (3) metol; (4) phenidone; (5) HMABS; (6) DHBS
micelles (its effective mobility decreased from 3.08104 cm2 V1 s1 at pH 6.5 to 1.02 104 cm2 V1 s1 at pH 9) and in a significant improvement in peak efficiency. Based on these results, pH 9.0 was selected for further separations because it provided the shortest separation time with acceptable resolution. To avoid any adverse effects from electrolytic degradation of non-buffered electrolyte, Tris was chosen instead of sodium hydroxide for the neutralization of H3PO4 solution to pH 9.0. The effect of temperature on the separation of developers was also briefly studied. In this study, the temperature was varied from 25 to 50 C. As the temperature was increased, the migration times decreased due to a decrease in electrolyte viscosity. Separation at 50 C was complete in about 65% of the time required for the same separation at 25 C. Although increasing the temperature resulted in higher efficiencies, a subsequent loss in resolution between hydroquinone and metol took place. For this reason all further separations were performed at 25 C. The electropherogram obtained under optimum conditions for a standard solution is shown in Fig. 3. As can be seen, excellent separation of five compounds was obtained in less than 8 min. Validation Using the optimal experimental parameters described above (a carrier electrolyte of 50 mmol L1 SDS in
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B. Pranaityt_e et al.
Fig. 3. Electropherogram obtained for a standard solution of the analytes. Electrolyte: 25 mmol L1 Tris-phosphate, 50 mmol L1 SDS, pH 9.0. Other conditions as in Fig. 1. Peaks: (1) hydroquinone; (2) metol; (3) phenidone; (4) HMABS; (5) DHBS
25 mmol L1 Tris-phosphate, pH 9.0, and applied voltage of 25 kV), several analytical performance characteristics important for quantitative analysis were measured. Validation was carried out in a way similar to that generally adopted for HPLC and now employed to validate CE methods [16]. Preliminary validation of the method included assessment of stability of the solutions, precision, linearity, detection and quantification limits and accuracy. Although the stability test is often considered an essential part of the procedure, it should be carried out at the beginning of the procedure validation because it conditions the validity of the data of the other tests. As expected, the aqueous solutions of both sulphonates were found to be unchanged for at least two weeks. The stability of the aqueous solutions (1103 mol L1 ) of hydroquinone, metol and phenidone depends strongly upon the pH. These solutions were stable in the dark, at room temperature, and pH
values lower than 7 for at least 20 hours. At basic pH values the concentrations of these developers gradually decreased. For example, at pH 9, the hydroquinone concentration decreased to about 64% after 10 h. A similar behaviour was also observed for phenidone and metol. On the other hand, the same standards prepared in helium degassed water at pH 9 were stable for at least 5 h, indicating that oxidation with dissolved oxygen was the cause of instability. Consequently, all standard solutions were prepared daily just before use in oxygen-free water. The method precision was determined by measuring the repeatability and intermediate precision (between-day precision). In order to achieve acceptable precision levels, an internal standard was found to be required. The internal standard used was methyl4-hydroxybenzoate with more than 99% purity. Although the stability of the internal standard was not examined in detail during method development or validation, no significant changes in the internal standard peak area were observed in the course of these experiments or during sample analysis. In order to determine the repeatability of the method, eight replicate injections of a mixed standard solution of analytes and internal standard (1104 mol L1 each) were carried out. The intermediate precision was evaluated over 3 days by performing eight successive injections daily. The obtained relative standard deviations (RSD) of the relative migration time and relative peak area are summarized in Table 1. Detector response linearities were assessed for each analyte with seven mixed standard solutions of analytes containing the internal standard at its nominal concentration. Since an accurate standard for the HMABS was not available, the quantitative characteristics for this compound were not measured. The tested concentration range was from 1105 to 1103 mol L1 . Each concentration level was injected three times. Regression curves were obtained by plotting relative peak areas versus concentration
Table 1. Precision of the MEKC method expressed as RSD (%) values for relative migration time and relative peak area (n ¼ 8) Analyte
Hydroquinone Metol Phenidone HMABS DHBS
Repeatability
Intermediate precision
Relative migration time
Relative peak area
Relative migration time
Relative migration time
0.31 0.18 0.26 0.25 0.20
2.48 1.84 2.45 2.30 2.56
0.36 0.28 0.35 0.41 0.27
3.12 2.21 3.06 3.35 2.91
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Micellar Electrokinetic Chromatography of X-Ray Film Developing Baths Table 2. Linearity data and LOD values Parameter
Hydroquinone
Metol
Phenidone
DHBS
Linear range (mol L1 ) Slope CI (95%) Intercept CI (95%) r2 LOD (mol L1 )
5 105 –1 103 1501 148 0.018 0.026 0.9987 1.5 105
2 105 –1 103 3127 73 0.014 0.017 0.9998 5.0 106
2 105 –1 103 2728 152 0.006 0.042 0.9996 7.0 106
1 105 –1 103 3962 265 0.019 0.078 0.9993 3.2 106
using the least squares method. For determination of the limit of detection (LOD), solutions with low concentrations of analytes were injected in order to find the concentration corresponding to a signal-to-noise ratio of 3. The data obtained is summarized in Table 2. This data supports the suitability of the proposed MEKC method for its application to real samples. Although the detection limits achieved were relatively high, such sensitivity was sufficient to monitor commercial processing solutions. The accuracy of the method was evaluated by adding known amounts of each component at three concentration levels (from 5105 to 5104 mol L1 ) to X-ray film developing solutions and analysing the spiked samples with the proposed MEKC method. Comparing the responses from sample and spiked sample solutions, the following recovery data was obtained: 91–106% for hydroquinone, 98–102% for metol, 96–104% for phenidone and 96–102% for DHBS. These results suggest that interferences by the other matrix components are not significant and the MEKC conditions are suitable to obtain adequate method accuracy. Sample Analysis To evaluate the proposed MEKC system for real samples, it was applied to the analysis of commercial hydroquinone-metol and hydroquinone-phenidone X-ray film developing solutions. The only sample pre-treatment stage involves filtration of the sample through a 0.45 mm pore filter, addition of an internal standard and appropriate dilution. Figure 4 shows the electropherograms obtained for 1:50 diluted hydroquinone-metol developer samples collected at different developing process run times. Two samples were analysed by the proposed MEKC method and by the HPLC technique [17]. Since the calibration curves were found to be linear and go through or close to the origin, all determinations were performed using external calibration with one calibration point. Alternative results for anionic
Fig. 4. Electropherograms of 1:50 diluted (a) fresh and (b) spent hydroquinone-metol developer samples. Conditions as in Fig. 3. Peaks: (1) hydroquinone; (2) metol; (3) HMABS; (4) methyl-4hydroxybenzoate (internal standard); (5) DHBS
Table 3. Results of the determination of developing agents (g L1 ) in X-ray developer solutions (n ¼ 3) Developing bath
Analyte
MEKC
HPLC a
Hydroquinonemetol
Hydroquinone Metol DHBS
0.62 0.04 1.18 0.05 3.25 0.17
0.60 0.03 1.23 0.04 3.17 0.20b
Hydroquinonephenidone
Hydroquinone Phenidone DHBS
8.16 0.43 2.35 0.11 6.06 0.25
8.04 0.28 2.28 0.08 6.14 0.28b
a
Average concentration value CI (95%). mined by CE technique.
b
DHBS was deter-
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Micellar Electrokinetic Chromatography of X-Ray Film Developing Baths
DHBS were obtained by conventional CE technique using 50 mmol L1 ammonium acetate electrolyte at pH 4.5. The results are compared in Table 3. As can be seen, the MEKC method shows good agreement with the data obtained from alternative methods. The comparison of means using a t-test has shown that there is no statistically significant difference between them at a confidence level of 0.05. Conclusions MEKC was found to be a useful technique for the analysis of common developing agents and their main oxidation products in commercial X-ray film developing baths. Good and acceptable method performance was observed for all validation points. In comparison with HPLC, the MEKC method requires minimal setup time, lower costs and reagent consumption and gives better separation efficiencies in a shorter analysis time. Furthermore, the possibility of simultaneous quantification of the developing agents and their oxidation products is very important not only in determining the quality and effectiveness of the developing process but also in the rapid monitoring of the effluents from used baths during their decomposition.
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