Supplementary information 1. INSTRUCTIONS FOR STUDENTS

1 2 Supplementary information 1. INSTRUCTIONS FOR STUDENTS 1.- SAFETY AND WASTE DISPOSAL Reactant CAS Pictogram * Hazards L-ascorbic acid 50-81-7 - Not a dangerous substance Hexadimethrine bromide 28728-55-4 Acute toxicity, Oral Hydroxide sodium 1310-73-2 Skin corrosion Phosphoric acid 7664-38-2 Skin corrosion Potassium phthalate 877-24-7 - Not a dangerous substance Sodium tetraborate 1303-96-4 Reproductive toxicity 3 4 5 6 7 8 9 10 11 * Pictogram source: Globally Harmonized System of Classification and Labelling of Chemicals (GHS) supported by United Nations Economic Commission for Europe (UNECE) - Wear lab coats, protective gloves, and eye protection. - Dispense wastes in containers, following the guidelines of laboratory hazardous waste management. 2.- EQUIPMENT Reagents Distilled water Filtered solution of phthalate (1000 mg/l) Filtered solution of phosphoric acid at 2% and 5% Filtered solution of L-ascorbic acid (1000 mg/l) Filtered solution of borate buffer (400 mm, ph =8 and hexadimethrine bromide 0.01%) Tomato samples Materials Graduated pipets of 2 ml and propipets Volumetric flask of 50 ml Centrifuge for Eppendorf vials Rack for Eppendorf vials Pasteur pipet Ultrasonic bath Membrane filters 0.22/0.45 µm and syringes Automatic pipets of 1000 µl Electrophoresis vials and taps Vortex shaker Instruments UV/Vis spectrophotometer EC instruments equipped with uncoated fused-silica capillaries (27 cm total length, 20 cm effective length, 50 mm i.d.) 1

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 3.- EXPERIMENTAL PROTOCOLS 3.1. UV measurement - Prepare a solution of phthalate at 20 mg/l and a solution of ascorbate at 10 mg/l in borate buffer (V = 50 ml). - Register the UV spectra of both solutions using UV/Vis spectrophotometer in the region of 200-300 nm. Note: Blank of instrument is registered by measuring borate buffer solution. - Propose the best wavelength for determination of ascorbate by CE technique. 3.2. Standard solutions for EC calibration - Prepare six standard solutions in electrophoresis vials (V = 1800 µl). The concentrations are phthalate at 200 mg/l, phosphoric acid at 1 %, and L-ascorbic acid ranged between 0 mg/l and 250 mg/l. Note: Don t forget shake the solutions. Degas all solutions in an ultrasonic bath. 3.3. Programming CE protocol - Following the instructions of CE instrument, create the following protocol. Step Initial Rinse Injection Separation Final Rinse Detection Conditions Solution: buffer Mode: Pressure 20 psi Time: 1 min Mode: Hydrodynamic at 1 psi Time: 5 s Voltage: -20 kv (reverse polarity) Temperature: 25 ºC Time: 5 min Solution: buffer Mode: Pressure 30 psi Time: 2 min Acquisition: single or multi-wavelength Wavelength: See results of section - Following the instructions of CE instrument, create a sequence for the injection of standards and other for tomato samples (three replicates). - The literature values for the acidity constants and molecular weight of ascorbic acid are pk a1 = 4.10; pk a2 = 11.80; MW = 176 g/mol. The values of phthalic acid are pk a1 = 2.76; pk a2 = 5.41; MW = 166 g/mol. Propose the expected electropherogram. - Discuss about the effect of experimental variables in the proposed electropherogram. Draw the hypothetical electropherogram for each experimental situation. 3.4. Separation of standards - Load the electrophoresis vials of standards into the autosampler and start the injection sequence of standards. - After finishing electrophoretic separations, process the registered electropherogram, following the instructions of CE instrument. - Identify the peaks based on migration times and UV spectra, according to the results of sections 3.1 and 3.3. Register the migration times of ascorbate and phthalate - Obtain peak height and peak area of each peak, following the recommendations of the instructor. 2

50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 - Discuss about the selection of peak height and peak area as analytical variable for calibration. - Obtain the calibration curves using direct calibration and corrected calibration by the internal standard response. For that, fitted data to linear equation Signal = a + b [ascorbate]. - Discuss about the advantages and limitations of using internal standard in routine analysis 3.5. Sample pretreatment - For tomato fruits, blend 5 g of pulp in presence of 5 ml of phosphoric acid solution at 2%. Perform three replicates. - For tomato juice or soups, mix 1.5 ml of sample with 1.5 ml of phosphoric acid solution at 2%. Perform three replicates. - Centrifuge the homogenates at 8000 rpm for 2 min and 12000 rpm for 5 min. - Filter the supernatant through 0.45 µm and 0.22 µm nylon filter, consecutively. - Transfer 1440 µl of extract to electrophoresis vials containing 360 µl of phtalate standard solution. Note: Don t forget shake the solutions. Degas all solutions in an ultrasonic bath. 3.6. Sample separation - Load the electrophoresis vials of samples into the autosampler and start the injection sequence of samples. - After finishing electrophoretic separations, assign the peaks of ascorbate and phthalate, according to the results of section 3.4. - Process the registered electropherogram. For that, obtain peak height and peak area of each peak, as section 3.4. - Calculate the ascorbate concentration of the extracts, interpolating the calibration curves of section 3.4. - Calculate the ascorbate concentration (average ± error) in tomato samples, according to the experimental steps of section 3.5. 3

81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 Supplementary information 2. INSTRUCTIONS AND NOTES FOR INSTRUCTORS 1.- SAFETY AND WASTE DISPOSAL - Ensure compliance of safety rules by students - Prepare containers for waste disposal 2.- EQUIPMENT Reagents L-ascorbic acid (CAS 50-81-7), sodium tetraborate (CAS 1303-96-4), phosphoric acid (CAS 7664-38-2), hydroxide sodium (CAS 1310-73-2) were obtained from Scharlau (puriss. p.a, Barcelona, Spain). Potassium phthalate (CAS 877-24-7) and hexadimethrine bromide (CAS 28728-55-4) were purchased from Sigma-Aldrich (puriss. p.a., Steinheim, Germany). All solutions were prepared in deionized water from a laboratory water station. Stock solutions were stored in the refrigerator. The samples were constituted by foods containing fruits of the genus Lycopersicon. Instruments UV/Vis spectrophotometer: Jenway 6715 spectrophotometer (Bibby Scientific Limited, Staffordshire, UK). EC instruments: P/ACE 5000 CE and MDQ instruments (Beckman Instruments, Fullerton, CA). Uncoated fused-silica capillaries (27 cm total length, 20 cm effective length, 50 mm i.d.) were also obtained from Beckman. 3.- EXPERIMENTAL PROTOCOLS 3.0. Experimental set-up - Before injection in the capillary, all solutions must be degassed in an ultrasonic bath and forced through a 0.2 mm membrane filter (Whatman, Maidstone, UK). - Prior to use, new capillaries were rinsed at 50 C as follows: 5 min with 1 M NaOH, 5 min with 0.1 M NaOH, and 10 min with water. - At the beginning of each working session the capillary was rinsed for 30 min with the separation buffer at 25 C. 3.1. UV measurement - Check the correct preparation of standard solutions. The volumes of reagents are: Standard phthalate Standard ascorbate Phthalate 2 ml - L-ascorbic acid - 1 ml - Ensure the correct use of UV/Vis spectrophotometer. - Assist in the selection of best wavelength for CE determination of ascorbate. - Discuss the effect of ph in UV spectra of ascorbate. 4

125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 3.2. Standard solutions for EC calibration - Check the correct preparation of standard solutions. The volumes of reagents are Standard Standard Standard Standard Standard Standard 0 1 2 3 4 5 Phosphoric acid (5%) 720 µl 680 µl 650 µl 610 µl 580 µl 540 µl Phthalate 360 µl 360 µl 360 µl 360 µl 360 µl 360 µl L-ascorbic acid 0 µl 90 µl 180 µl 270 µl 360 µl 450 µl Water 720 µl 670 µl 610 µl 560 µl 500 µl 450 µl 3.3. Programming CE protocol. - Prepare a short manual of CE instrument. - Explain the operation of CE instrument. - Ensure the correct creation of protocols. - Supervise the theoretical electropherograms deduced by students. - Moderate the discussion about the effect of experimental variables in the proposed electropherogram. 3.5. Separation of standards The injection of standards was performed by hydrodynamic mode (1 psi during 5 s). Nevertheless, good results in set-up experiments were also obtained by electrokinetic injection (10 kv during 5 s). - Supervise the correct use of CE instruments by students. - Guide the data analysis. - Explain the effect of integration parameters (threshold, minimum peak width, etc). - Assist in the correct peak identification and the calculation of peak height and peak area - Moderate the discussion about the analytical variable for calibration. - Moderate the discussion about the direct calibration and corrected calibration by the internal standard response. 3.5. Sample pretreatment - Supervise the correct extraction, centrifugation, and filtration of samples. - Highlight the importance of sample pretreatment in the final analytical result. 3.6. Sample separation - Supervise the correct use of CE instruments by students. - Evaluate the results of sample analysis. 5

160 161 162 163 164 165 166 167 168 169 170 171 Supplementary information 3. STUDENT OPINION SURVEY Determination of L-Ascorbic Acid in Tomato by Capillary Electrophoresis" Duration time: 5 minutes Question 1. Likert-scale question Complete the following survey about the laboratory experiment The experiment helped them to learn the CE technique An adequate time distribution was chosen for the experiment performance The difficulty level was appropriate The teaching-learning strategy was dynamic Very disagree Disagree Neutral Agree Question 2. Open-ended question Indicate the advantages and the drawbacks of the experiment in your learning Very agree 6