In this work, a green and simpler method for photometric determination of sulfite based on a flow injection-gas diffusion (FI-GD) system using a natural reagent extracted from roselle (
Sulfite is widely used in food and beverage industries as a preservative to inhibit microbial activity and to control enzymatic reactions during the fermentation and storage stages. Although sulfite is often used as food additives, contents above the permissible limit may lead to adverse effects to the consumers, such as nasal congestion, coughing, breathing difficulties, asthma, itching, and other skin rashes [
There are several analytical methods for sulfite determination in food and beverages, including the Monier-Williams method [
Bleaching reactions of anthocyanins by sulfites have been well known for many years. The addition of sulfite in the C-2 or C-4 position of flavilic ring and blocking of electronic delocalization in anthocyanin molecule results in color fading of the solution to become colorless [
A flow injection analysis can provide rapid, high precision, high accuracy, lower amount of reagents and solvents, and automated method for sulfite determination [
In this work, we proposed the environmentally friendly method for the determination of sulfite by exploiting roselle extract as a natural reagent in a simple FI-GD photometric system. A crude extract of spray-dried roselle powder was used as a reagent in an acceptor solution of the FI-GD system. Sulfur dioxide that is converted from sulfite in an acidic donor stream diffuses through a PTFE membrane of the GD unit to dissolve in an acceptor stream. It reacts with anthocyanin contained in the natural reagent, leading to the bleaching of the reagent color, which is monitored by a home-made photometer. The conditions for operating the developed system were optimized. The proposed method was applied for the determination of sulfite in various types of wine samples.
All chemicals used were of analytical reagent grade. Deionized water (obtained from a water purification system of Elgstat Option 3A, Elga, England) was used throughout. A stock standard solution (1000 mg·L−1) was prepared by dissolving 0.1575 g of Na2SO3 (Sigma Aldrich, USA) in 1.0% (v/v) ethanol (RCI Labscan, Thailand) and filling the volume up to 100 mL with this ethanol solution. Working standard solutions were daily made by dilutions of the stock one in 1.0% (v/v) ethanol. Acetate buffer solution pH 3 (0.1 mol L−1) was prepared by mixing 982.3 mL of 0.1 mol·L−1 acetic acid (Merck, Germany) with 17.7 mL of 0.1 mol·L−1 CH3COONa∙3H2O (BDH, England). The solutions used for the Ripper method were prepared according to the reference method [
The roselle reagent was daily prepared by extracting 1.4 g of the spray-dried roselle powder (purchased from Thiptipa Company Ltd., Thailand) with 100 mL of acetate buffer solution pH 3.0. The suspension was then centrifuged at 4,000 rpm for 10 minutes to obtain a clear solution, filtered through a Whatman #1 filter paper, and the volume was then made up to 100 mL. To confirm the extraction reproducibility of different batches of roselle reagent, the absorbance of the obtained solution was measured before use. The acceptable absorbance of the extracted solution was 1.525 ± 0.025 at 510 nm. The content of total anthocyanin in the roselle solution determined by pH differential method [
The FI-GD photometric analyzer (Figure
Manifold of FI-GD photometric flow system for determination of sulfite; P = peristatic pump, V = six-port valve, S = standard/sample, C1 and C2 = mixing coils, and PC = personal computer.
The GD unit was made of two acrylic blocks (160 mm long, 50 mm wide, and 15 mm thick), engraved by a CO2 laser cutting machine (CNC Bro, China) for donor and acceptor channels (each 300 mm long, 1.5 mm wide, and 0.5 mm deep), as depicted in Figure
A standard/sample solution was injected manually into a 0.3 mol·L−1 HCl donor stream and flowed through a mixing coil (C1) to a GD unit, where any sulfite present was converted to sulfur dioxide and diffused through a PTFE membrane into an acceptor stream of the roselle reagent. Sulfur dioxide reduced the color intensity of the reagent, which was monitored by measuring a fading of the color at the flow-through cell. The output signal from the detector was recorded as a peak on a personal computer. Peak height was directly proportional to the concentration of sulfite in the injected solution. An analytical curve was constructed by plotting peak height versus sulfite concentration.
An accurate sample volume of 10.00 mL was transferred to an Erlenmeyer flask; an aliquot of 5.00 mL of 1% w/v starch indicator and a pinch of sodium hydrogen carbonate were added. After that, 5.00 mL of 33% (v/v) sulfuric acid was added, and the solution was immediately titrated with an 0.25 mmol·L−1 iodine solution to a blue endpoint (color stable for 20 seconds) [
White, red, and sparkling wines were purchased from local markets in Bangkok, Thailand. The samples were poured into a beaker and degassed for 20–30 minutes before analysis by the Ripper method and the proposed system.
Anthocyanin undergoes structure transformations with a change in the pH, which has a dramatic effect on its color. The bleaching reaction of anthocyanin by sulfite was investigated under different pH values (1.0 to 14.0), adjusting by NaOH or HCl solutions. Solutions containing 100 mg·L−1 sulfite and 1.0% (w/v) of roselle extract in different pH media were scanned for absorption spectra, using correspondent reagent blanks at each pH as references. The difference of absorbances (at 510.0 nm) of roselle extract without and with the added standard solution is shown in Figure
Influence of pH on ΔAbs. of roselle extract. Inset: spectra of roselle extract in various pH.
The optimization of the FI-GD analyzer was performed by using the following conditions: 0.3 mol·L−1 HCl as a carrier stream for donor solution, 30 cm mixing coil length for mixing between sample/standard and the HCl solution, and 100 cm mixing coil length for mixing between the diffused SO2 and roselle extract reagent. Effects of roselle extract concentration, types of PTFE plumber tape, the effect of the flow rate of acceptor and donor stream, and effect of the sample volume were investigated.
Various roselle extract concentrations were studied in the range of 0.6–2.6% (w/v). Sulfite standard solutions in the concentration range of 5–100 mg·L−1 were injected into the system to construct an analytical curve for each reagent concentration. The analytical curve was made by plotting peak height (difference between signals without and with analyte presence) obtained versus sulfite concentration. It was found that the sensitivity (slope of graph) decreased with the increase of reagent concentrations (Figure
Effect of reagent concentration (% w/v) on sensitivity of sulfite determination.
Six commercial PTFE plumber tapes (represented by M1 to M6, Figure
Influence of the membrane types on peak height of 100 mg·L−1 sulfite when using HCl or DI water as a donor stream.
Influence of the acceptor flow rates was studied in the range of 0.5–2.0 mL·min−1 by fixing the flow rate of the donor stream at 2.0 mL·min−1. The result was presented in Table
The effect of donor flow was also examined in the range of 0.5–2.0 mL·min−1, which was related to the acceptor flow rate. From Table
Sample volume was optimized in the range of 50–300
Using the optimal conditions, roselle extract 1.4% (w/v) in acetate buffer pH 3 as an acceptor stream and 0.3 mol·L−1 HCl as a carrier stream for donor solution, flow rate of each stream of 1.0 mL·min−1, the length of mixing coil 1 and mixing coil 2 of 30 and 100 cm, respectively, and a sample volume of 100
The analytical performances of spectrometric determination of sulfite using various natural reagents.
Crude extract | Reagent | Method | Linear range (mg L−1) | LOD (mg L−1) | Sample throughput (h−1) | Samples | Ref. |
---|---|---|---|---|---|---|---|
Sweet potato root ( | Polyphenol oxidase | FI | 3.2–48 | 0.18 | 26 | White wine, white vinegar, juice | [ |
Anthocyanin | Batch | 2–10 | — | — | White wine | [ | |
Roselle ( | Anthocyanin | FI-GD | 5–100 | 2 | 21 | Sparkling wine, white wine, red wine | This work |
The effects of different interfering compounds on the analytical signal of 10, 50, and 100 mg·L−1 standard solution were investigated. The tolerance limit of the interfering compounds was evaluated based on the recovery percentages within the range of 95–105%. All of the interfering compounds did not affect the percentage recovery except ethanol and ascorbic acid (Table
Effect of some potential interfering species.
Potential interferences | Tested concentrations | Results |
---|---|---|
Ethanol | 5, 10, 15, 20, 30, 50% | Interfere at 30% |
Ascorbic acid | 10, 50, 100, 500, 1000 mg·L−1 | Interfere at 500 mg·L−1 |
Glucose | 10, 50, 100, 500, 1000 mg·L−1 | Do not interfere at 1000 mg·L−1 |
Fructose | 10, 50, 100, 500, 1000 mg·L−1 | |
Sucrose | 10, 50, 100, 500, 1000 mg·L−1 | |
Tartaric acid | 10, 50, 100, 500, 1000 mg·L−1 | |
Citric acid | 10, 50, 100, 500, 1000 mg·L−1 | |
Acetic acid | 10, 50, 100, 500, 1000 mg·L−1 | |
Lactic acid | 10, 50, 100, 500, 1000 mg·L−1 | |
Tannin | 10, 50, 100, 500, 1000 mg·L−1 |
The determination of free sulfite was carried out in the various wine samples. Wine samples were prepared as described in Section
Recorder output of a routine run. Number of replicates: 3; left: standard solutions, right: sample; inset: plot of the analytical curve.
Sulfite contents in wines as determined by the proposed method and the Ripper method.
Sample number | Sample type | Sulfite concentration (mg L−1)∗ | |
---|---|---|---|
Ripper method | Proposed method | ||
1 | Sparkling wine 1 | 24.1 ± 0.2 | 23.9 ± 0.4 |
2 | Sparkling wine 2 | 20.3 ± 1.9 | 19.9 ± 0.2 |
3 | White wine 1 | 12.9 ± 0.9 | 12.6 ± 0.7 |
4 | White wine 2 | 20.6 ± 0.2 | 20.4 ± 0.5 |
5 | White wine 3 | 34.7 ± 0.7 | 34.9 ± 0.3 |
6 | White wine 4 | 21.5 ± 0.9 | 22.1 ± 0.2 |
7 | White wine 5 | 35.5 ± 0.9 | 34.5 ± 0.3 |
8 | White wine 6 | 31.7 ± 0.8 | 30.5 ± 0.1 |
9 | White wine 7 | 27.3 ± 1.3 | 23.1 ± 0.3 |
10 | Red wine 1 | 3.5 ± 0.2 | 2.28 ± 0.2 |
11 | Red wine 2 | 8.3 ± 0.1 | 7.72 ± 0.8 |
12 | Red wine 3 | 10.6 ± 0.8 | 10.9 ± 0.2 |
∗Mean of triplicate results.
The simple flow injection-gas diffusion photometric system was developed using roselle extract, a natural reagent, for the determination of sulfite. The developed method is an environmentally friendly and green analytical method and offered high selectivity by using a commercial PTFE plumber tape as a gas-permeable membrane in the GD unit. Moreover, a low-cost laboratory-made photometer provided good sensitivity and high sample throughput for the quantitation of sulfite in real samples. The results obtained from the proposed method and the Ripper method are in good agreement as compared by paired
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest.
The authors gratefully acknowledge the Department of Chemistry, Faculty of Science and Technology, Thammasat University. Partial support from Chiang Mai University is also acknowledged. The authors would like to thank Dr. Duangduan Chaiyaveij (Thammasat University) for her useful comments.
Figure S1: A schematic drawing of a home-made gas diffusion unit for determination of sulfite. Illustration of the zig-zag channel on acrylic block. A photograph of a home-made gas diffusion unit for determination of sulfite. Figure S2: Influence of the sample volume on the response sensitivity. Table S1: Influence of roselle extract concentration on the response sensitivity. Table S2: Influence of the acceptor flow rate on the response sensitivity. Table S3: Influence of the donor flow rate on the response sensitivity.