Electrochemical determination of indole‑3‑acetic acid and indole‑3‑butyric acid using HPLC with carbon felt detector

 

 

Hana Dejmkova1    · Marjorie de Araújo Daniel

Introduction
Carbon felt (CF) is industrially manufactured material, con- sisting of randomly ordered carbon fibers. Its advantageous properties, concerning both mechanical and electrochemical point of view, support its applications in many fields of sci- ence, including electrochemistry. Among these properties, we can name, for example, chemical and physical stability, large surface area, high porosity, high electrical conductiv- ity, and low price [1]. The electrochemical applications use carbon felt mostly as a part of the fuel cells [2]. Neverthe- less, electroanalytical applications are also known, particu- larly those involving coulometry [3, 4] or anodic stripping voltammetry [5, 6].

 

Recently, we have introduced a novel construction of coulometric detector based on carbon felt and suitable for combination with flow injection analysis or HPLC [7]. This time, we try to extend the experience with the detector by the development of determination method of different class of compounds, indole auxins, namely indole-3-acetic acid (IAA, Fig. 1a) and indole-3-butyric acid (IBA, Fig. 1b). These compounds are phytohormones influencing forma- tion of buds and roots and supporting plant growth. Both of them are naturally produced by the plant metabolism, where IBA is a precursor of IAA. Both of them are also used in agriculture as pesticides or rooting stimulators [8, 9]. Elec- trochemical oxidizability of these compounds enables to use electrochemical methods for their determination [10]. Previ- ously, voltammetric methods were tested for this purpose [11]. Among their common determination methods, HPLC with mass spectrometric detection [12, 13] or electromigra- tion methods [14, 15] prevail; determination is often com- bined with specific extraction techniques [16, 17]. Recently, the determination methods of auxins were reviewed in [18]. Aim of the work is the application of carbon felt detec- tor for the determination of IAA and IBA, including the development of the determination method, confirmation of the determination parameters, and testing the performance of the method for the determination of analytes in gardening product.

 

Results and discussion
Method optimization

Chromatograms under various detection potentials were measured using solutions of the analytes of concentration 100 µmol dm−3 in mobile phase with pH of aqueous buffer 2.5, 4.0, and 6.0. On the basis of preliminary experiments, content of methanol as organic modifier was set to 60%, as it allowed the separation of both compounds in short time. In the resulting dependences (Fig. 2), we can see the increase of the detection potentials with decreasing pH, accompanied with the same shift of the potential window width. Electro- chemical behavior of both compounds is similar, but the response of IAA is generally higher.

In the same series of measurement, it was possible to observe the influence of pH on the retention time (tR). Peak position remained the same for pH 2.5 and pH 4.0, where tR was 1.80 min and 2.47 min for IAA and IBA, respectively, but for the pH 6.0, it decreased to 1.25 min and 1.46 min for IAA and IBA, respectively. This effect is caused by the deprotonization in the pH range above the pKA of the compounds, which reaches value around 4.75 for both com- pounds. For the further measurements, buffer of pH 4.0 and detection potential + 1.5 V was selected, because these con- ditions join the high response, low background current and higher retention of the compounds.
Under the described conditions, repeatability of the meas- urement was observed, using three series of ten injections of the mixture of the analytes, with each series performed on the new piece of carbon felt. Relative standard deviation of the measurements reached 3.1% and 2.5% for IAA and IBA, respectively, which confirms stability of the measurements both concerning the electrode passivation and felt exchange.

 

Concentration dependences

For the observation of linearity and determination limits, concentration dependences of the analytes were measured in the range of concentration from 0.4 to 100 µmol dm−3. Resulting straight lines together with the selected chromato- grams are depicted in Fig. 3 and the main parameters of the dependences are summarized in Table 1. The dependences are linear in the whole examined concentration range, as confirmed also by the values of correlation coefficients. The difference between their slopes mirrors the higher electrode response of IAA, mentioned in previous section. The inter- cepts are negligible. Determination limits (LOQ) reached  value of 1.1 µmol dm−3 and 1.8 µmol dm−3 for IAA and IBA, respectively., this value is sufficient for the auxin determi- nation in many types of samples, including plant samples.

 

Determination in rooting preparation

To confirm the applicability of the developed method, determination of IAA and IBA in liquid rooting prepara- tion Stimulax II was performed by HPLC with detection on carbon felt (HPLC–ED). The manufacturer declares the content of IAA as 0.05% and IBA as 0.06%. However, due to the presumable instability of the auxins in the solution, HPLC with UV detection was used as a reference method for IAA and IBA determination.

 

Obtained results, recalculated to percentage in the prepa- ration in agreement with the form of the manufacturer dec- laration, are shown in Table 2. Agreement of the results of both determination methods is high, as well as the preci- sion of the methods. Nevertheless, the measured values are clearly different from the declared values; they reach only approximately 20% and 70% of the said values in the case of IAA and IBA, respectively. The results are not surprising, as the instability of auxins is known, even the fact, that IAA is less stable than IBA, explains its relatively lower content in the sample. However, the suitability of the distribution of such unreliable products among the customers is doubtful.

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