BIOL1177 SM1 2019 Session 3 Proforma-converted-merged

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Royal Melbourne Institute of Technology *

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Biology

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Jun 3, 2024

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CHROMATOGRAPHY I: ION EXCHANGE Session date:3/4/2019 Name: Akeel Doush Submission date:10/4/2019 Partner: Affaan Nawaz Group:13 Demonstrator: Yuh Ping Chong Aim: To determine the concentration of an unknown patient’s sample using the Ninhydrin assay to generate a standard curve at a wavelength of 570nm. using ion exchange chromatography to isolate the amino acids using the anion exchange resin amberlite IR400 that is separated by charge. Method: Refer to Biochemistry and Molecular Biology 1 Practical Course Manual: Semester 1. 2019. Session 3, (Page 37 – 44) Results: The Isolation of total amino acids in urine using Amberlite IR 400 Table 1. (Absorbance and Concentration Readings for Total AAs in Urine using Amberlite IR400 and Ninhydrin Method) Tube 1 2 3 4 5 6 Standard amino acid solution (0.25 mM), mL 0 0 0.2 0.2 0.4 0.4 H 2 O, mL 2.0 2.0 1.8 1.8 1.6 1.6 Diluted eluate, mL - - - - - - Absorbance (570 nm) 0 0 0.0665 0.0685 0.1285 0.1325 [amino acid] (μM) 0 0 25 25 50 50 Mean concentration of amino acids (μM) - - - - - - Tube 7 8 9 10 11 12 Standard amino acid solution (0.25 mM), mL 0.6 0.6 0.8 0.8 - - H 2 O, mL 1.4 1.4 1.2 1.2 - - Diluted eluate, mL - - - - 2.0 2.0 Absorbance (570 nm) 0.1915 0.2005 0.2605 0.2595 0.1705 0.1885 [amino acid] (μM) 75 75 100 100 63.25 72.25 Mean concentration of amino acids (μM) - - - - 67.75

• Plot the standard curve (include appropriate figure title and axes labels) and attach it to this proforma. • Read off the values of the concentration of amino acids for the diluted eluate and record in Table 1. Calculations: Q1. Allowing for the dilution used, calculate the concentration of amino acid present in the original 2 mL of collected eluate. Total Concentration of AA’s = Unknown mean concentration x Dilution factor = 67.75 x 100/2 = 67.75 x 50 = 3387.5 μM amino acids Q2. Calculate the amount of amino acid present in this 2 mL. n=cv n= 3387.5 x 0.002L = 6.775 μ mol Q3. Calculate the amount of amino acid present in the original 20 mL of collected eluate. Concentration of amino acids in the original 20 mL = Concentration of amino acids in 2 mL x (Original 20ml eluate / dilution volume) n= 6.775 μmol x 20ml/2ml = 67.75 μ mol Q4. Assume the urine to be part of a 24-hour sample of 1450 mL. Calculate the amino acid output for the 24 hour period. Results should be reported as mmol/24 h. Amino acid concentration for 24/hr = Amino acid in the original 2mL x (Urine sample in 24/hr / urine in column) = 67.75 μmol x (1450/10) = 9823.75 μmol /24hr =9823.75 / 1000 (μmol → mmol) = 9.82 mmol/24hr Reference range: 5 – 15 mmol/24hr

Q5. Comment on your results. In figure 1, the standard curve is linear, and all duplicates were close and touching the line which means it’s a positive standard curve. The Ninhydrin assay was reliable as the duplicates were consistent and the mean absorbance for the diluted samples was 0.1795 which falls within the range of the standard curve. The total amino acids with the 24-hour period was 9.82mmol/24hr that is within the reference range of (5- 15mmol/24hours), therefore the patient is normal and has a normal AAs count in their urine sample Conclusion: By using the Ninhydrin assay to generate a standard curve, we were able to determine the total number of amino acids in the patient’s urine sample via ion exchange chromatography under an optimal wavelength of 570nm. The standard curve formed a linear correlation without any outliers which means the results are accurate therefore the mean concentration for the patient that was determined was 67.75 μmol in figure 1. The patient’s urine sample was calculated, and it was 9.82 mmol/24hr that is within the reference range of 5 – 15 mmol/24hrs. therefore the results are reliable, and the patient is healthy and there are no clinical implications needed.

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