伏安极谱仪应用报告——维生素C及其化合物的测量

No. 98/3 e Application Bulletin Of interest to: General analytical laboratories; Food; B C G 1, 3, 4, 7, 8 Pharmaceutical industry; Biology Determination of ascorbic acid (vitamin C) and its compounds Summary Ascorbic acid, as well as its salts and esters, can be determined by titration with 2,6-dichlorophenol indophenol (DPIP) or by means of polarography. The determina- tion is based on the oxidation of ascorbic acid to dehydroascorbic acid: C6H8O6 – 2 H+ – 2 e– C6H6O6 ascorbic acid dehydroascorbic acid Using 2,6-dichlorophenol indophenol as titrant, this will be reduced by the ascorbic acid: C12H7Cl2NO2 + 2 H+ + 2 e– C12H9Cl2NO2 For the titrimetric determination bi-voltametric or photometric endpoint indication can be used. Be aware, though, that only the bi-voltametric indication is independ- ent of inherent coloration of the sample. The polarographic method is the most selective of the described methods, since other reducing or oxidizing substances do not interfere. Instruments and accessories For titration: • 702 SET/MET Titrino, 716 DMS Titrino, 736 GP Titrino, 751 GPD Titrino or 785 DMP Titrino or 726 or 796 Titroprocessor with 700 Dosino or 685 Dosimat • 2.728.0040 Magnetic Stirrer • 6.3014.213 or 6.3014.223 Exchange Unit • 6.0309.100 double Pt sheet electrode with 6.2104.020 electrode cable (bi-volt- ametry) or • 6.5501.01X Spectrode 610 nm (photometry) For polarography: • 757 VA Computrace or • 746 VA Trace Analyzer with 747 VA Stand Application Bulletin No. 98/3 e Determination of ascorbic acid (vitamin C) and its compounds Page 2 Reagents • Oxalic acid solution: 1 g oxalic acid or 1.4 g oxalic acid dihydrate is dissolved in dist. water and made up to 1 L. • Sodium acetate solution, w(CH3COONa) = 10% in dist. water • Acetate buffer pH = 4.64: 8.2 g sodium acetate is dissolved in dist. water. Add 6 mL glacial acetic acid and make up to 100 mL with dist. water. • Vitamin C standard, ρ(vitamin C) = 0.5 g/L: 50.0 mg vitamin C is dissolved in oxalic acid solution and made up to 100 mL. This solution should be freshly prepared every day. 1 mL standard corresponds to 0.5 mg vitamin C • Titrant: c(DPIP) ≈ 0.001 mol/L: Approx. 330 mg 2,6-dichlorophenol indophenol sodium salt dihydrate (e.g. Merck no. 103028) is dissolved in dist. water. Add approx. 100 mg NaHCO3 and make up to 1 L with dist. water. Sample preparation • Drinks, fruit and vegetable juices can be analyzed directly. • For tablets and other vitamin preparations, a diluted solution in de-aerated dist. water is first prepared, an aliquot of which is then used for the titrimetric or pola- rographic determination. • Foods, stimulants and animal feeds are extracted using the appropriate proce- dures. Colored substances present in the samples need not be extracted with ether, since these do not interfere with the determination of vitamin C. Analysis 1. Titrimetric determination Place 10 mL dist. water, 15 mL oxalic acid solution and 1 mL sodium acetate solu- tion in the titration vessel and purge with nitrogen for 3 ... 5 min. Add the sample or standard solution (containing 0.05 … 0.5 mg – for the photometric titration at least 0.2 mg – vitamin C), then titrate with c(DPIP) ≈ 0.001 mol/L using, e.g., the MET mode. Calculation 1 mL c(DPIP) = 0.001 mol/L corresponds to 0.176 mg vitamin C Application Bulletin No. 98/3 e Determination of ascorbic acid (vitamin C) and its compounds Page 3 Figures 'pa 785 DMP Titrino 02287 785.0010 user date 1999-05-04 time 10:26 22 MET Ipol ******** parameters >titration parameters V step 0.10 ml dos.rate max. ml/min signal drift OFF mV/min equilibr.time 8 s start V: OFF pause 0 s I(pol) 1 µA electrode test: OFF temperature 25.0 °C >stop conditions stop V: abs. stop V 5 ml stop U OFF mV stop EP 9 filling rate max. ml/min >statistics status: OFF >evaluation EPC 5 mV EP recognition: greatest fix EP1 at U OFF mV >preselections req.ident: OFF req.smpl size: OFF limit smpl size: OFF activate pulse: OFF ------------ 'fr 785 DMP Titrino 02287 785.0010 user date 1999-05-04 time 10:16 21 U(init) 141 mV MET Ipol ******** smpl size 2 ml EP1 4.179 ml 28 mV RS1 0.368 g/l stop V reached ============ 'cu 785 DMP Titrino 02287 785.0010 user date 1999-05-04 time 10:16 21 start V 0.000 ml MET Ipol ******** 2.0 ml/div dU=50.0 mV/div Fig. 1: Parameter settings on the 785 DMP Titrino for the titration of vitamin C with bi-voltametric indication. Fig. 2: Result block and titration curve for the determination of vitamin C in orange juice (bi-voltametric indication). Application Bulletin No. 98/3 e Determination of ascorbic acid (vitamin C) and its compounds Page 4 'pa 785 DMP Titrino 02287 785.0010 user date 1999-05-04 time 08:28 9 DET U ******** parameters >titration parameters meas.pt.density 4 min.incr. 100 µl dos.rate max. ml/min signal drift 30 mV/min equilibr.time 32 s start V: OFF pause 0 s meas.input: 1 temperature 25.0 °C >stop conditions stop V: abs. stop V 15 ml stop U OFF mV stop EP 9 filling rate max. ml/min >statistics status: OFF >evaluation EPC 5 EP recognition: greatest fix EP1 at U OFF mV pK/HNP: OFF >preselections req.ident: OFF req.smpl size: OFF limit smpl size: OFF activate pulse: OFF ============ 'fr 785 DMP Titrino 02287 785.0010 user date 1999-05-04 time 08:45 11 U(init) 164 mV DET U ******** smpl size 1.3 ml EP1 3.820 ml 106 mV Vit C 0.52 g/l manual stop ============ 'cu 785 DMP Titrino 02287 785.0010 user date 1999-05-04 time 08:45 11 start V 0.000 ml DET U ******** 2.0 ml/div dU=50.0 mV/div Fig. 3: Parameter settings on the 785 DMP Titrino for the titration of vitamin C with photometric indication. Fig. 4: Result block and titration curve for the determination of vitamin C in the stan- dard (photometric indication). Application Bulletin No. 98/3 e Determination of ascorbic acid (vitamin C) and its compounds Page 5 2. Polarographic determination Place 10 mL dist. water and 1 mL acetate buffer pH = 4.64 in the polarographic vessel and purge with nitrogen. Add 1 mL sample solution, mix and purge briefly with nitrogen once again. Afterwards record the DP polarogram using the following parameters: working electrode DME stirrer speed 2000 rpm mode DP purge time 300 s equilibration time 5 s pulse amplitude 50 mV start potential –50 mV end potential +200 mV voltage step 6 mV measure time 20 ms pulse time 40 ms voltage step time 0.6 s sweep rate 10 mV/s peak potential +100 mV The concentration is determined by standard addition. Figures ================== METROHM 746 VA TRACE ANALYZER (5.746.0101) ================== Method: AB98 .mth OPERATION SEQUENCE Title : Determination of Vitamin C. AB98 -------------------------------------------------------------------------------- Instructions t/s Main parameters Auxiliary parameters ------------- ----- ------------------------- ------------------------- 1 SMPL/M V.fraction mL V.total L 2 DOS/M V.added 11.000 mL 3 PURGE 4 STIR 300.0 Rot.speed 2000 /min 5 (ADD 6 PURGE 7 STIR 10.0 Rot.speed 2000 /min 8 SEGMENT Segm.name pol 9 ADD>M Soln.name AscStd V.add 0.100 mL 10 ADD)3 11 END Method: AB98 SEGMENT pol -------------------------------------------------------------------------------- Instructions t/s Main parameters Auxiliary parameters ------------- ----- ------------------------- ------------------------- 1 0PURGE 2 0STIR 5.0 3 (REP 4 DME 5 DPMODE U.ampl 50 mV t.meas 20.0 ms t.step 0.40 s t.pulse 40.0 ms 6 SWEEP 18.0 U.start -50 mV U.step 6 mV U.end 200 mV Sweep rate 15 mV/s 7 REP)1 8 PURGE 9 STIR Rot.speed 2000 /min 10 0MEAS U.standby mV 11 END Fig. 5: Method for the determination of vitamin C with the 746 VA Trace Analyzer. Application Bulletin No. 98/3 e Determination of ascorbic acid (vitamin C) and its compounds Page 6 Method: AB98 SUBSTANCES Ascorb - pol -------------------------------------------------------------------------------- Recognition Display / Plot ------------------------- --------------------------- U.verify 100 mV I.scale auto U.tol (+/-) 50 mV U.div 50.00 mV/cm U.width min 10 mV U.begin 0 mV U.width max 200 mV U.end 200 mV I.threshold 200 pA Baseline Evaluation ------------------------- --------------------------- Type linear Mode VA Scope whole Quantity I.peak dU.front auto Sign. digits 4 S.front auto dU.rear auto S.rear auto Calibration 2000-08-22 17:24:39 Coefficients --------------------------------- ------------------------- Technique std.add. Y.reg 4.103e-07 Curve type linear Slope 3.836e-05 Nonlin. Mean dev. 9.826e-09 Additions ------------------------------------------------------------ Soln.name AscStd ---------- Mass conc. 1 g/L g/L g/L g/L Range min g/L g/L g/L g/L Range max g/L g/L g/L g/L M.conc./cm g/L g/L g/L g/L Method: AB98 CALCULATION max. 15 lines -------------------------------------------------------------------------------- Quantity Formula (R##, C##, A##) Res.unit Sig.dig. ------------ ---------------------------------------- -------- -------- Ascorb R1000=MC:Ascorb #g/L 5 Fig. 6: Method (continued). Application Bulletin No. 98/3 e Determination of ascorbic acid (vitamin C) and its compounds Page 7 ================== METROHM 746 VA TRACE ANALYZER (5.746.0101) ================== Determ. : 08221608 User: Date: 2000-08-22 Modified : no Run : 0 Time: 16:08:59 Sample table: - -------------------------------------------------------------------------------- Pos. Ident.1/S1 Ident.2/S2 Ident.3/S3 Method.call Sample size/S0 O_Saft 0.5 mL -------------------------------------------------------------------------------- Method : AB98 Title : Determination of Vitamin C. AB98 Remark1 : 10 ml water + 1 ml buffer Remark2 : + 0.5 ml sample -------------------------------------------------------------------------------- Substance : Ascorb Comments Mass conc.: 246.1 mg/L Mass : 123 ug ------------------ MC.dev. : 5.15 mg/L (2.09%) Add.mass : 100 ug Cal.dev. : - V0.sample: 500 uL VR U/mV I/uA I.mean Std.dev. I.delta Comments --- ----- ------ ------ ------- ------- ------------------ 00 103 0.4100 0.4096 0.0006 01 103 0.4091 10 104 0.7410 0.7419 0.0013 0.3323 11 104 0.7428 20 104 1.039 1.056 0.0243 0.3139 21 105 1.073 30 105 1.348 1.373 0.0359 0.3176 31 106 1.399 Substance Techn. Y.reg/offset Slope Nonlin. Mean deviat. --------- -------- ------------ ---------- ---------- ------------ Ascorb std.add. 4.103e-07 3.836e-05 9.826e-09 Final results +/- Res.dev. % Comments --------------------------------- ----------------- ------------------ Ascorb = 246.06 mg/L 5.15 2.09 Substance: Ascorb VR(**) U (mV) I ( µA ) 25 50 75 100 125 150 175 0.2 0.4 0.6 0.8 1 1.2 1.4 Standard addition curve: Ascorb rho(eff) (mg/L) I ( µA ) 5 10 15 20 25 30 35 0.2 0.4 0.6 0.8 1 1.2 1.4 Fig. 7: Results, polarograms and standard addition curve for the determination of vitamin C in orange juice. Application Bulletin No. 98/3 e Determination of ascorbic acid (vitamin C) and its compounds Page 8 Remarks • Instead of 2,6-dichlorophenol indophenol, other oxidizing titrants can be used as well, e.g. iodine, iodide/iodate, chloramine-T, Fe(III). Here, however, the danger is greater that apart from ascorbic acid other oxidizable compounds present in the sample will also be determined. • Larger quantities of chloride ions (e.g. in sauerkraut) interfere with the polaro- graphic determination of vitamin C. They are removed from the sample solution by precipitation with silver nitrate and subsequent filtration. Literature • Metrohm Application Note T-30 Ascorbic acid (vitamin C) in fruit juices Metrohm Ltd., Herisau. • Metrohm Application Note T-54 Vitamin C (ascorbic acid) by photometric titration Metrohm Ltd., Herisau. • L. Erdey, G. Svehla Ascorbinometric Titrations Verlag Akademiai Kiado, Budapest, 1973. • G. Pongracz Neue potentiometrische Bestimmungsmethoden für Ascorbinsäure und dessen Verbindungen Fresenius Z. Anal. Chem. 253 (1971) 271–274. • K. K. Verma, A. K. Gulati Determination of vitamin C with chloramine T Anal. Chem. 52 (1980) 2336–2338. • D. Amin Application of differential pulse polarography to the assay of ascorbic acid Microchem. J. 28 (1983) 174–179. • S. Kozar, A. Bujak, J. Eder-Trifunovic, G. Kniewald Determination of L-ascorbic acid in fresh and processed fruit and vegetables by differential pulse polarography Fresenius Z. Anal. Chem. 329 (1988) 760–763.