Week 10 UV and Fluorescence Analysis PDF

Instrumental Analysis  Indicators Indicators are chemicals that change color when the right conditions occur. There are indicators that depend on the pH level, that is, the H+ concentration, and there are also indicators that react with a chemical specific to that ind...

Instrumental Analysis  Indicators Indicators are chemicals that change color when the right conditions occur. There are indicators that depend on the pH level, that is, the H+ concentration, and there are also indicators that react with a chemical specific to that indicator and change color.  UV (Ultraviolet) Spectroscopy Measures absorbance value (A) of solutions. A is depended on intensity of colors proportional to the concentration of colored chemical.  Monochromator Seperates wavelengths and direct the wanted wavelength to the target. UV Visible Spectrometer and Working Principle  Methodology 1. Prepare several known concentration of a coloured chemical and measure absorbance values of all these solutions. 2. Prepare a calibration curve 3. Measure the absorbance value of a solution including the same type of chemical but at Unknown concentration. 4. Using the calibration curve that we prepared at the 2nd step, estimate the concentration of the solution.  Example Calibration Curve Calibration Curve Chemical Absorbanc 1.2 Concentration (mM) e 1 f(x) = 2.19 x + 0.00900000000000012 Absorbance (A) 0,1 0,23 R² = 0.998937765558611 0.8 0,2 0,45 0.6 0,3 0,65 0.4 0,4 0,9 0.2 0,5 1,1 0 0 0.1 0.2 0.3 0.4 0.5 0.6 Chemical Concentration (mM) Unknown 0,8 concentration 0.8 − 0.009 0.8=2.19 𝑥 +0.009=⇒ 𝑥= =0.36 2.19 The concentration of the sample is 0.36mM Blood Test Question We have an indicator for measuring of glucose concentration in the blood. Firstly, we prepared samples having the known glucose concentratons and we measured their absorbances at 500nm.  Blood Glucose Concentration Glucose Concentration Absorbanc (mg/L) e Calibration Curve 1 0,12 1.2 2 0,25 f(x) =10.109555061179 x + 0.060050055617 3 0,48 Absorbance R² = 0.975841802221934 4,5 0,55 0.8 6 0,7 0.6 8 1 0.4 10 1,1 0.2 0 0 2 4 6 8 10 12 Glucose Concentration (mg/L) Blood from a patieent 0,9  x = (0,9-0,0601)/0,1096 = 7,66 mg/L  Fluorescence Spectroscopy Measures fluoresence (emission) of solutions that was excited at a specific wavelength. The emission is depended on excitance wavelength and the concentration of fluorescent chemical. It is used to estimate the concentration of fluorescent chemicals Antioxidant capacity measurements are usually performed by using fluorescence spectroscopy. Fluorescence is defferent than phosphorescence https://www.youtube.com/watch?v=OzPCzFu472Y Fluorescence Spectrometer and Working Principle Notes on Fluorescence Spectroscopy Calibration Curve for Fluorescence Spectrometer We prepare several samples with known concentrations We already know max excitation and emmision wavelengths so we make the measurements at these wavelengths  Elemental Analysis Flame photometer Atomic absorption ICP