Determination L-ascorbic acid in some tropical fruits by High - PDF document

30/07/2012 HPLC Basic instrument Determination L-ascorbic acid in some tropical fruits by High performance liquid chromatography. By RIBE Team HCMC - July 2012 History: Typical HPLC Stack (Agilent 1100) • Discovered by Russian botanist Mikhail Tswett at the turn of the century. He separated various plant pigments by passing solutions through a glass column containing calcium carbonate. • Chroma meaning “color” + graphein meaning “to write” Chromatography Principle Liquid Chromatography (HPLC) High Pressure Chromatography High Performance Chromatography • In chromatography process, separations are based on differences in migration rates among the sample Suitable for separation of “small” organic compounds, but also proteins components because they have differences interactive and peptides. Especially those with UV-Vis absorption or forces with stationary phase. fluorescence. Typical applications Gas chromatography Liquid chromatography  Vitamins Pesticides  Drugs Mycotoxins Mobile phase Carrier gas: N2, He, Polar and non-polar solvent such  Steroids Flavours Ar… as: MeOH, acetonitril, water…  Organic acids Carbohydrates Solid: film layer in Solid or liquid covering particles Stationary Advantages capillary column in packed column phase  Speed and improved resolution Samples easily Samples easily degrade by heat or Analyzed  Reusable columns evaporate (<300 o C) or has molecule mass > 3000 u. objects durable with heat.  Sample recovery  Low temperature separation  No need for derivatization 1

30/07/2012 Identification Vegetable, Fruit and Products Concerned What is component A? (96/23/EC) Grapes Apple Banana Pineapple Litchi Potato Cabbage Cauli Flower Determination Results obtained by HPLC What is the concentration of component A? Chromatogram In HPLC • Stationary Phase: Typical surface coatings: Normal phase (-Si-OH, -NH 2 ) Reverse phase (C 8 , C 18 , Phenyl) + ) Anion exchange (-NH 4 Cation exchange (-COO - ) • Typical Solvents - hexane, methylene chloride, chloroform, methanol, acetonitrile (normal phase) - methanol, acetonitrile, water (reverse phase) 2

30/07/2012 HPLC - Column Separation Stainless steel tubing with low volume end fittings. Solvent Length 3 - 30 cm, becoming shorter A+B Inner diameter usually 3 – 4.6 mm Packing material B Packed A – Microparticular 3, 5 or 10 µ column – Irregular or spherical shape – Different types, silica based Guard column – Short column of same packing material before separation column detector – Adsorb impurities and particles t 0 t 1 – Can be replaced Signal Time, t HPLC - Detectors Separation Solvent A+B B Packed A B column A detector t 0 t 1 t 2 Signal Time, t Separation Separation Solvent Solvent A+B A+B B Packed Packed A B column column A B detector detector A t 0 t 0 t 1 t 2 t 3 Signal Signal A Time, t Time, t 3

30/07/2012 Preparation of ascorbic acid standard curve Separation Solvent • Stock solution: dissolve 10 mg of ascorbic acid in 10 ml of extracting solution. Carry to 10 ml using dark volumetric flask. A+B • Prepare ascorbic acid standard solutions of ascorbic acid with B concentrations of 5,10, 20, 30, 40 mg/l. Packed A B column - Mix 2.5 ml stock solution with extracting solvent, fill up to 50 ml in volumetric flask-  solutions of ascorbic acid with A B concentrations of 50 mg/l. - Mix 1 ml stock solution with extracting solvent, fill up to 25 detector A B ml in volumetric flask-  solutions of ascorbic acid with t 0 t 1 t 2 t 3 t 4 concentrations of 40 mg/l. Signal - Mix 1.5 ml stock solution with extracting solvent, fill up to A B 50 ml in volumetric flask-  solutions of ascorbic acid with Time, t concentrations of 30 mg/l. Factors Affecting Chromatography Preparation of ascorbic acid standard curve t’ R(B) - Mix 0.5 ml stock solution with extracting solvent, fill up to 25 ml in volumetric flask-  solutions of ascorbic acid with t’ R(A) k (A) = t’ R(A) / t m concentrations of 20 mg/l. Retention time t R(A) - Mix 0.5 ml stock solution with extracting solvent, fill up to t m 50 ml in volumetric flask-  solutions of ascorbic acid with Detector response concentrations of 10 mg/l. Peak Area - Mix 1 ml solution of ascorbic acid with concentrations of 50 Peak height mg/l with extracting solvent, fill up to 10 ml in volumetric Injection flask  solutions of ascorbic acid with concentrations of 5 mg/l. Baseline t 0 Solvent Solute A Solute B time “peak” Determination L-ascorbic acid (AA) Procedure of determination AA (reference to Agilent application) • AA is a water soluble organic compound that participates • Weigh 2-5 g sample into centrifugal bottle. • Add 50 ml acidic solvent pH 2.1 (extracting solvent). in many biological processes. • Mix thoroughly to obtain homogenous slurry by using Ultra- • In samples, vitamin C concentration is total content of Turax in 3 minutes. ascorbic acid and dehydroascorbic acid; dehydroascorbic • Centrifuge at 4000 rpm, 4 o C in 20 minutes. acid did not account for more than 10% of total vitamin C • Give extract in 100 volumetric glass flask. in any of the analysed fruits as has been described by Lee • Wash centrifugal bottle with 10 ml acidic solvent pH 2.1 and Kader (2000). • Combine washing solvent in volumetric glass flask and add • In our lab, we extract AA in matrices by using acid acidic solvent up to 100 ml. solvent which has pH 2.1 (titrate by H 2 SO 4 ) and quantify • Filter extract through 0.2 µm membrane. its concentration by using HPLC with RP-column and UV • Inject 20 µl filtrate into HPLC system. detector. 4

30/07/2012 Practical steps: Standard curve Inject sample into HPLC Analytical conditions Reference • Agilent 1100 • M.A. Romero R., M.L. Vazquez O., J. Lopez H., J. Simal • Column: Agilent 10 cm x 4.6 cm x 3 µm L.Determination of vitamin C and organic acids in various fruits by HPLC (1992). Journal of Chromatographic Science , • Flow: isocratic elution 0.5 ml/minute. 30(11):433-437 • UV Detector: 254 nm. • HPLC for food analysis (Agilent’s application) • Mobile phase: 100 % acidic solvent pH 2.1 Calculation: Cm x V (ml) Vitamin C conc. = ------------------ (mg/Kg or mg/L ) m (g or ml) Cm: Vitamin C conc. is calculated base on standard curve. Vm: 100 ml (the final volume of extract). m: weight of sample (g). Example HPLC Separation 5