Introduction and Background An isocratic RP-HPLC method with UV detection was developed to quantify ascorbic acid in app
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Introduction and Background An isocratic RP-HPLC method with UV detection was developed to quantify ascorbic acid in apple juice. It was used to quantify the content of ascorbic acid in one sample of apple juice using external standard solutions. HO OH OH OH ascorbic acid Objectives 1. Review and finalize the laboratory report (fill in required information). 2. What is the retention factor k of ascorbic acid? 3. What can you learn from the provided chromatograms? 4. Calculate the quantity of ascorbic acid in one glass of apple juice (125 ml) and report the result in mg/glass. HPLC method details 0.1 % formic acid in water-MeOH (80:20, Mobile phase: v/v) Flow rate: 950 ul min-1 Injection volume: 3.0 uL Phenomenex Gemini C18, 100 x 4.6 mm, Column: 3 um, 110 Å 1 Column temperature: 30°C Detection: UV, 245 nm Run time: 8 min
Materials • Volumetric pipettes, borosilicate glass, class A • Volumetric flaks, borosilicate glass, class A Analytical Balance • Ascorbic acid, Certified Reference Material, P/N HJXL-23B, certified purity 944 mg/g Syringe filter: Regenerated Cellulose (RC), 15 mm i.d., 0.45 um . Analytical program Preparation of the reference stock solution (RSS) 94.2 mg of ascorbic acid CRM was weighed into a 1000 mL volumetric flask. It was dissolved in approximately 30 mL of water in an ultrasonic bath (3 min), then filled up to the mark with water. Preparation of the reference solutions (RS_LIN1, RS_LIN2, RS_LIN3, RS_LIN4, and RS_LIN5) Five reference solutions with different concentrations of ascorbic acid were prepared by further diluting aliquots of RSS using volumetric pipettes and volumetric flasks (Table 1). Table 1 Preparation of linearity solutions as reference solutions Solution П Volume RSS (Volumetric pipette) [ML] 5.00 Volumetric flask (Filled up to) [ML] 250.0 Concentration of ascorbic acid [mg mL-1] ??? ??? 15.00 200.0 RS_LIN1 RS_LIN2 RS_LIN3 RS_LIN4 RS_LIN5 15.00 100.0 ??? 10.00 50.0 50.0 ??? ??? 15.00
Preparation of the test solution 25.00 mL of apple juice (batch no. XUTJ23) was transferred into a 100.0 mL volumetric flask and filled up to the volume with water. It was filtered through a syringe filter into a HPLC vial discarding the first mL of solution. Chromatographic analysis and calculations The HPLC system is setup accordingly and equilibrated. A blank, the reference solutions in increasing concentration, a blank, the test solutions, and a blank are injected in the HPLC system consecutively in an analytical sequence. Quantification of the analyte in the test solution using peak areas of the analyte in reference solutions (ESTD). Chromatograms 0.7 0.6 0.5 0.4 Detector signal [AU] 0.3 0.2 0.1 0.0 0 5 8 4 Time [min] Figure 1 Chromatogram of blank (water)
0.7 0.6 0.5 0.4 Detector signal [AU] 0.3 0.2 0.1 0.0 0 Time [min] Figure 2 Chromatogram of the reference solution (RS_LIN3) 0.7 0.6 0.5 0.4 Detector signal [AU] 0.3 0.2 0.1 0.0 0 5 6 ו 8 Time [min] Figure 3 Chromatogram of the test solution (TS)
J Results and discussion Chromatographic analysis Blank, linearity (reference) solutions and the test solution were injected in the order outlined in Table 2. Integration results of ascorbic acid are also provided in Table 2. Table 2 Sequence table and integration results Inj. # Sample name Retention time Ascorbic acid [min] n.d. Peak area Ascorbic acid [mV min] 1 n.d. Blank (water) LIN1 2 3.19 4.18 3 LIN2 3.19 16.87 LIN3 3.18 32.16 41.63 5 LIN4 3.19 LIN5 3.18 n.d. 61.93 n.d. 7 Blank (water) 8 TS (batch XUTJ23) 9 Blank (water) n.d. not detected 3.15 n.d. 18.90 n.d.
Observations Discuss the chromatograms. Anything noteworthy? Retention factor What is the retention factor k of ascorbic acid? Provide all relevant parameters, their units, and formulas using the Microsoft Word formula editor. Regression analysis Perform a linear regression analysis to obtain the response factor (slope) of ascorbic acid for this HPLC method. Add all required raw data and their units in a tabular form. Provide all relevant calculations including units of parameters using the Microsoft Word formula editor. What is the dependent variable? What is the independent variable? Add a graph of the linear regression analysis (don't forget correct labelling of the axes). Quantification Calculate the amount of ascorbic acid in apple juice batch no. XUTJ23. Report the result in mg/serving. One serving is 125 ml. Provide all relevant calculations including units of parameters using the Microsoft Word formula editor. Method details Discuss the choice of the mobile phase.