Chemical laboratories are usually looked as the starting points of modern chemical and pharmaceutical industries. The contamination of the wastewater produced by chemical laboratory is a worldwide problem. Seeking for efficient methods to reuse laboratory wastewater produced in laboratory teaching and applying the methods in chemistry laboratory courses will help students establish the philosophy of environmental protection and sustainable development ^[1-8].

The "determination of alkali hydroxide concentration" was selected as an experiment in chemistry laboratory teaching, because it is widely used in chemical and pharmaceutical industries. At Peking University Health Science Center, this experiment is included in the first year course of basic chemistry for medical undergraduate students or the second year course of analytical chemistry for pharmacy undergraduate students ^[9]. In previously laboratory teaching, the concentration of aqueous sodium hydroxide was measured using potassium hydrogen phthalate as the reference material and the ethanol solution of phenolphthalein for endpoint indication. A typically environmental consideration was collecting the wastewater produced by titration and treating it through water purification system.

Different from the previous methods, this paper describes a successful design of the teaching experiment into a green one which does not need to purify the wastewater produced by titration. The teaching reform will help students learn how to design and carry out a green chemical experiment ^[6-9].

Firstly, we select potassium hydroxide (KOH) as the analyte and an aqueous solution of oxalic acid as the titrant. The chemical reaction for determination of KOH concentration is as follows:

$\mathrm{H}_{2} \mathrm{C}_{2} \mathrm{O}_{4}+2 \mathrm{OH}^{-} \rightleftharpoons \mathrm{C}_{2} \mathrm{O}_{4}^{2-}+2 \mathrm{H}_{2} \mathrm{O}$

This is a generally used reaction for determination of acid-base neutralization. Oxalic acid is a diprotic acid (K_a1= 5.6 × 10^-2, K_a2 = 1.5 × 10^-4). Because the two-step ionization constants are close, it is difficult to distinguish two chemical endpoints when titrating the oxalic acid with potassium hydroxide. Therefore, the concentration of potassium hydroxide is determined through titration of the total quantity of hydrogen ion in oxalic acid solution.

Secondly, the aqueous solutions of potassium oxalate produced by titration is used in irrigation of plants. The oxalate, a natural product widely existing in plants, can be biodegraded in soil. Potassium cation is an indispensable nutrient for growth of plants. Hence, in this green experiment, the product of titration solution could be directly involved in natural circulation without treatment. In addition, the pigment extracted from a black tea is used to indicate the titration endpoint, because the black tea and the extractant acetic acid are also biodegradable.

To address those improvements, we have conducted three different titration methods. Their similarity is that all of them use oxalic acid as the reference material. The differences of these three methods are that one is selected the pigment extracted from black tea (BTP) as the chromogenic agent to indicate titration endpoints; another is applied potentiometric titration without addition of any color indicator; and the third one is the control experiment using phenolphthalein as the color indicator for titration endpoints.

To address the improvements, we have compared three different titration methods, using oxalic acid as the reference material. The difference of these three methods is the way of indicating the titration endpoint: one uses BTP, one uses phenolphthalein, and one uses potentiometry. Herein, we report the reformed teaching experiment and the corresponding results (Fig. 1).

The lapsang souchong black tea fragments were bought from Maliandao tea leaf market in Beijing. Acetic acid, potassium hydroxide (KOH), and phenolphthalein (PP) (A. R, Beijing Chemical Plant, China). The reference material of oxalic acid (H₂C₂O₄∙2H₂O crystal, M_w: 126.07, 99.99%–100.01% mass contents, Tianjin Jinke Fine Chemical Co., Ltd, China). The plant, Spathiphyllum kochii, was bought from the Beijing flower market.

Denver INSTRUMENT UB-7 pH/mV PH meter, was calibrated by potassium hydrogen phthalate (pH 4.01), sodium hydrogen phosphate/potassium hydrogen phosphate (pH 6.86) and borax (pH 9.18) buffer. The elemental analysis of C, H, and N was carried out with Vario EL III elemental meter. DSC-TGA determination was carried out with SDT Q600 V20.9 Build 20, (American TA Co., Ltd, United States), Sample: 3.5770 g; temperature increasing rate: 10 K∙min^-1; N₂ flow: 100.0 mL∙min^-1; reference: Al₂O₃.

Powder X-ray diffraction spectra were determined on MiniFlex 600 X-ray polycrystalline diffractor (Rigaku, Japan) with Cu K_α radiation (0.15418 nm). Fourier transform infrared (FTIR) spectra were determined on NEXUS-470 FTIR spectrometer (Nicolet) with KBr pelleting. UV-Vis spectra were determined with UV-6100 spectrophotometer (MAPADA Instrument Co., Ltd, Shanghai, China).

Mass spectrometric spectra were determined on Xevo G2 ESI Q-Tof MS meter (Waters). Sample: BTP aqueous solution; inlet: 0.2 μL; positive ionization voltage: +3 kV; negative ionization voltage: -2 kV; Vacuum: 10^-4–10^-5 Pa.

50 g of lapsang souchong black tea leaf fragments were soaked in 400 mL of 0.1 mol∙L^-1 acetic acid aqueous solution at room temperature for 72 h. The extraction solution with dark brown color was filtrated under vacuum, followed by rotation evaporation to 150 mL under 150 rpm at 50 ℃, and cooled to room temperature. The concentrated liquid was frozen at -80 ℃ for 4 h, then desiccated under 33 Pa at -78 ℃ for 48 h. A solid powder of BTP with brown red color and aromatic odor was obtained (yield 8.5%). The pH of BTP aqueous solution (1 mg∙mL^-1) was 4.8.

An aqueous solution (RS, 0.05003 mol∙L^-1) of reference material oxalic acid was prepared by the standard weighted method. A KOH solution was prepared. The concentration of KOH solution was determined by the following three methods.

The 25.00 mL of oxalic acid reference solution (RS) containing 3 drops (0.2 mL each) of chromogenic agent BTP aqueous solution (25 mg∙mL^-1) was titrated with KOH sample solution until pH 8–10 (chemical endpoint detected by pH meter). This pale brown-yellow colored solution in an Erlenmeyer flask, as the reference solution1 (RS1), was sealed to prevent absorption of CO₂ from the air. Then another 25.00 mL of oxalic acid solution (RS) containing 3 drops (0.2 mL each) of BTP chromogenic agent was titrated with KOH sample solution to a point with color was similar to that of RS1 (Fig. 3 (I)). The volume of KOH aqueous solution was recorded. Three replicated measurements were conducted.

The 25.00 mL of oxalic acid reference solution (RS) was added into a measurement cup equipped with a thermostatic bath maintained at room temperature (25.0 ± 0.1 ℃). Then the reference solution (RS) was titrated with KOH solution with stirring. Consecutive addition of about 1 mL of KOH solution was performed until pH 5.5; then consecutive addition of 0.10 mL of KOH solution was performed until pH 10.3; after that, consecutive addition of 0.20 mL of KOH solution was performed until pH 12. Three replicate measurements were conducted. For obtained the chemical endpoint, the titration curve (pH–V diagram) (Fig. 4) was plotted and the titration data were processed.

As the control experiment, the concentration of KOH sample solution was also determined with phenolphthalein as the indicator (2% mass concentration of phenolphthalein in absolute ethyl alcohol). The oxalic acid reference solution (RS) was titrated with KOH sample solution until the color of titration solution turned to pale pink-red. Three replicate measurements were conducted.

All above data were processed statistically with Microsoft EXCEL 2007.

The titration solutions produced from the three different methods were collected in three different containers and labeled, respectively. Because the final pH of the solution produced by means of potentiometric titration was 12, the product solution was neutralized with oxalic acid solution until pH 7.49. Table 1 shows the pH of the solutions used in irrigation. Some of the pH values were lower than those at the endpoint, because the solutions had absorbed CO₂ gas in air for a long time.

The different product solutions were used to irrigate Spathiphyllum kochii, a plant with white flowers and green leaves (Fig. 5). For comparison, distilled water was also used in irrigation. All the plants grew under similar sunniness, temperature and moisture. Firstly, the plants had grown through irrigation with a plant nutrient solution (80 mL each plant) for 10 days. After that, the plants were irrigated with 80 mL of three different titration product solutions and distilled water on the 1st day, 5th day, 17th day and 31th day, respectively (each plant was irrigated with 80 mL of different solution). The growth of the plant was documented at the same time with visual observation and digital camera.

Twelve undergraduate students in School of Basic Medicine, Peking University participated in the teaching research. They determined the concentration of another KOH sample solution with three methods mentioned above, observed the growth of the plants, and filled out a questionnaire about this teaching experiment reform.

The pigment extracted from the black tea (BTP) was a mixture powder with brown red color. Its elemental composition was: C 36.75% ± 0.09%; H 5.35% ± 0.02%; N 4.51% ± 0.05%.

The results of DSC-TGA, XRD, ESI Q-TOF MS, and FT-IR spectra showed that the BTP powder contained the multiple solid phases with high amount of caffeine and trace of chromogenic components of theaflavin, theaflavin 3-gallate or theafalvin 3'-gallate, theaflavin 3, 3'di-gallate, catechin or epicatechin, gallocatechin or epigallocatechin, epigallocatechin gallate, epicatechin gallate, and other components ^[10] (Fig. S1–4).

In order to demonstrate BTP suitable as a chromogenic agent, we determined UV-Vis spectra of a series of BTP aqueous solution at different pH. Fig. 2 indicates no significant changes of absorbance (380–450 nm) in the UV-Vis spectra of BTP solution at pH between 2 and 6, and the evident changes of absorbance of visible light (380-450 nm) can be observed at the pH between 8 and 10 which is the pH range of titration endpoint. The results suggest the possibility of directly observing the titration endpoint through the color depth of titration solution using BTP as the chromogenic agent in visual colorimetry.

Fig. 3 shows the results of determination of KOH concentration with visual colorimetry. The 25.00 mL of the reference solutions of oxalic acid (RS) was titrated with KOH aqueous solution until the color depth of the product solution was similar to that of RS1 (I). The endpoint was acquired by directly visualizing and comparing the color depth of sample solution with that of RS1 during titration. The results with this method were repeated easily.

Fig. 4 shows the titration curve of the oxalic acid reference solution (RS, 0.05003 mol∙L^-1, 25.00 mL) titrated with KOH sample solution by means of potentiometric titration. The concentration of KOH sample solution can be acquired after data processing. This method does not need any chemical color indicator for titration endpoints.

The results acquired with the three different methods are summarized in Table 2. The t-test data show that the results with visual colorimetry using BTP as the indicator and that with potentiometric titration are not significantly different from the result of control experiment using phenolphthalein as the indicator.

The results showed that the plant, Spathiphyllum kochii, irrigated with three different product solutions grew well (Fig. 5). Even the plant irrigated with the potassium oxalate/phenolphthalein solution also grew well, because there were little quantity of synthetic phenolphthalein and alcohol in the product solution. With the observation of growth of the plants, we had not found evident differences of the plants irrigated with these three different titration product solutions. The results indicate the plant can be directly irrigated with these solutions. The problem is that in these solutions the nutrients is not enough for growth of the plant. Therefore, on the 15th day, equal amount of the plant nutrient reagent was added to each solution for irrigation.

Table 3 shows the results of KOH concentration determined by the undergraduate students. The data include the results with BTP and phenolphthalein methods by 10 students, and those with potentiometry by 6 groups of students (2 students in each group). The results of the concentration of KOH sample showed the better repeatability. Then they collected the titration product solutions and irrigated the plant, Spathiphyllum kochii with these titration product solutions and observed the plants through the method mentioned above (Fig. 5).

The results of questionnaire showed that most of the students were interested in this teaching experiment. They considered that from the experiment, they could learn not only laboratory skills, but also how to design a chemical analysis experiment based on green consideration. Spathiphyllum kochii has a popular Chinese name that means "everything is going on smoothly". When they observed the plant irrigated with the titration product solutions, they showed the feeling of freshness. Besides, this teaching experiment is more comprehensive and has integrated the knowledge points and skill trainings of several previous teaching experiments. But the students considered that the teaching contents (prepare reference solution of oxalic acid, determine concentration of KOH solution, and collect product solution in class, irrigate the plants and take pictures in class free time) of a 4-hour laboratory course with three titration methods seemed too much for the beginners.

According to the students' proposals, we have revised the teaching material. We plan to complete the green titration course in two teaching experiments (total 8 hours for two experiments, 4 hours each).

In recent years, natural products extracted from plants such as the anthocyanidin from roselle or the juice from red cabbage have been used as the color indicators of acid-base titration teaching experiments in some middle schools and universities ^[11-15]. A paper reported the results using tea water as an indicator to determine concentrations of alkali hydroxide aqueous solution ^[15]. We found that because of the complex components in tea, it was difficult to use the mixture in tea as a usual color indicator in acid-base titration (Fig. S5). The indicator method mentioned usually is based on the visible absorption wavelength changes of solution at different pH to indicate titration endpoint. We use BTP as the chromogenic agent to indicate the titration endpoint in visual colorimetry and have gotten the repeatable results.

Different from the previously teaching experiments, we have considered the environmental problems of whole titration system and do not use potassium hydrogen phthalate, a toxicant as the reference material. Besides, the wastewater produced from titration is used in directly irrigating plants. This consideration follows the "Designing Safer Chemicals and Design for Degradation " in the twelve principles of green chemistry ^[1]. We try to give our students a new philosophy that designing an environment-friendly experiment is more superior to treating the results of a system-polluted experiment. We also hope them apply this philosophy in their future profession.

For recent years, we have completed some reforms on the classic chemistry teaching experiments ^[16]. Some of the classic teaching experiments are very important because the concepts and the skill trainings in these experiments are still useful in modern chemistry."Green" is a critical criterion for evaluation of what we should reserve or give up in a new teaching system of chemistry experiment. We consider that the classic teaching experiments in chemistry courses need a green reform. This reform includes reservation of those important knowledge points and selection of some green chemical reactions. The green consideration needs to be extended to the fates of wastes produced by chemistry teaching experiments. Besides, the conception of "green", especially the green philosophy of experiment-design, should be added into textbooks of chemistry laboratory.

We have reformed the teaching experiment "determination of alkali hydroxide concentration" into a green one. Further research needs detailed analysis of the degraded components of the titration solutions in soil and investigation on the bioeffects of these components on the plants through cooperation with agriculture laboratories. In addition, we need more cases of teaching experiments designed with the green philosophy.

No conflict of interest in this research.

Acknowledgement: We thank 12 undergraduate students, Changxian Xiong, Yifan Yao, Luhui Zhang, Weiyi Zhang, Jixuan Xu, Chen Zhang, Yundi Tang, Huiling Zhao, Shuning Sun, Meixin Zhang, Qingyun Bai, Tengrui Zhang, in School of Basic Medicine, Peking University, for their participation of this project. Thanks for Dr. Jun Li & Ms. Kening Xu in State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, and Ms. Fei Zhang in Analytical Instrumentation Center, College of Chemistry and Molecular Engineering, Peking University for their kind help of determination of elemental contents, FT-IR spectra, ESI Q-TOF MS and DTA-TG of the black tea pigment, respectively.

We also thank Prof. Dr. Ping Xu, Dr. Xihui He, Mr. Xuesheng Yan, Mr. Ji Li, Mr. Youqi Yan, Ms. Xuezhi Feng, Ms. Yan Lu, Ms. Hongyan Zhang & Mr. Xu Wang in School of Pharmaceutical Sciences, Peking University for their kind help in this research.

Supporting Information: available free of charge via the internet at http://www.dxhx.pku.edu.cn.