Experiment1_SimoneDo_14186997_250822

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FOUNDATION EXPERIMENT 1: EXTERNAL CALIBRATION -HPLC Name: Simone Do Student Number: 14186997 Date of Submission:25/08/2022 Date of Practical Session:11/08/2022 Experimental unknown Code: U2006 HPLC column:C18 5 μm 3.9x150mm Column Mobile Phase:65% Water 35% Methanol Injection volume:5.0 mL Mobile phase flow rate:1.000 mL/min Detector: Diode Array Detector Detection Wavelength:273nm and 254nm Column Temperature:30.0°C Data file directory: 20220811_AC2 Results Part 1: Standards Table 1: Concentration standards. Volumetric Flask Standard Concentration (ppm) Volume of 1000-ppm stock to be added (μL) 1 2 20 2 4 40 3 6 60 4 8 80 5 10 100 6 12 120

Part 2 : Standard blank Figure 1: Chromatogram of instrument blank.

Part 3: Identification of peaks Figure 2: Chromatogram of Spike T (theophylline) indicating both peak 1 and 2. Figure 3: Chromatogram of Spike C (caffeine) indicating both peaks 1 and 2.

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Part 5. Construction of calibration curve and analysis of unknown Figure 4: Chromatogram of blank standard. Figure 5: Chromatogram of the unknown indicating 3 different peaks. Part 7: Data processing and reporting Table 2: HPLC results 20ppm-120ppm. Sample Identity Peak 1 Tr (m in ) Peak 1 area ( mAU* min ) Peak 1 height ( mAU ) Peak 2 Tr ( Min ) Peak 2 area ( mAU* min ) Peak 2 height ( mAU ) Run 1

20 ppm 2.600 2.380 2.502 3.243 0.205 1.824 40 ppm 2.600 0.520 5.477 3.243 0.419 3.710 60 ppm 2.603 0.727 8.089 3.247 0.582 5.473 80 ppm 2.603 0.965 10.675 3.247 0.722 7.143 100 ppm 2.603 1.044 11.258 3.247 0.855 7.702 120 ppm 2.603 1.302 14.099 3.247 1.116 10.136 Run 2 20 ppm 2.600 0.236 2.499 3.243 0.206 1.814 40 ppm 2.597 0.493 5.286 3.237 0.395 3.579 60 ppm 2.603 0.726 8.131 3.247 0.581 5.463 80 ppm 2.603 0.959 10.458 3.247 0.763 6.984 100 ppm 2.603 1.057 11.355 3.247 0.865 7.796 120 ppm 2.597 1.333 14.099 3.240 1.141 10.247 Calibration curve Figure 6: Calibration curve of theophylline (purple) and caffeine (orange) in peak height. y = 1.1239x + 0.8083 R² = 0.98 y = 0.7967x + 0.4121 R² = 0.9856 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 Peak Height (mAU) Concentration (ppm) Peak Height vs Concentration

Figure 7: Calibration curve of theophylline (green) and caffeine (blue) in peak area. Determination of unknown C1=c2v2/v1 C1=? V1=1mL C2=calibration curve conc V2=10mL Therefore, C1=(1.897mg/mL *10mL)/1mL =18.97mg/mL Theophylline C1=(1.797mg/mL*10ml)/1mL = 17.97mg/mL Theophylline C1= (7.22mg/mL*10mL)/1mL =72.2mg/mL Caffeine C1=(7.04mg/mL*10mL)1mL =70.4mg/mL Caffeine.

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Discussion Questions 1) Comment on any peaks present in the standard blank. At 1.50 min there is a peak that reaches an absorbance of between 1.00-.1.20 absorbance. However, the other peaks are just the multiple noise from the instrument. 2) Why did your perform a standard blank? The purpose of the standard blank is to measure the possibility of background contamina- tion or interferences. 3) Comment on the quality of your calibration curve, e.g., is it linear, does it pass though the origin, were there any outliers, etc. If the calibration curve is inadequate, provide possible explanations. The overall quality of the calibration curve of the peak height is adequate as it shows a linear curve, however, does not pass through the origin. Most of the points were within with line furthermore, there was no outliers. Peak area was also like height, showed an adequate calibration curve, indicating a linear line with the points within the range. 4) Is there a difference in your unknown concentration using the two methods? Which method is superior, peak height or peak area? Both peak area and height was quite similar as the r2 value was both around 0.98. The peak height calibration has a wider gap in comparison to the peak area which indicates the peak area is more superior. Furthermore, using the calibration curve concentration, caffeine was around 72.2mg/mL which was closer to the literature value of 75 but also within the range of 30-100ppm. Whereas the theophylline (17.97mg/mL) was below the range. 5) Comment on how you would improve this practical. To improve the quality of the experiment, the sample for the theophylline date would be completed again to achieve a closer result to the ideal number within the range. For better accuracy, assign one person to measure and dilute to keep the sample consistent instead of having three different people doing the same task.