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
