Instruments & Application
- 1. UV-visible spectroscopy How They Work
- 2. What is Spectroscopy? • The study of molecular structure and dynamics through the absorption, emission and scattering of light.
- 3. Spectroscopy Spectral Distribution of Radiant Energy Wave Number (cycles/cm) X-Ray UV Visible IR Microwave 200nm 400nm 800nm WAVELENGTH(nm)
- 4. Spectrophotometer (Spec) An instrument that measures the amount of light that passes through (is transmitted through) a sample.
- 5. Transmission and Color The human eye sees the complementary color to that which is absorbed
- 7. Molecules are whatever color of light that they do not absorb. Green molecules appear green because they absorb most wavelengths of visible light, except the green wavelengths.
- 8. Ultraviolet (UV) Spectrophotometers. Uses ultraviolet light of wave lengths from 200 nm to 350 nm. Visible (VIS) Light Spectrum Spectrophotometers. Uses visible light (white light) of wave lengths from 350 nm to 700 nm.
- 9. Conventional Spectrophotometer Schematic of a conventional single-beam spectrophotometer
- 10. Conventional Spectrophotometer Optical system of a double-beam spectrophotometer
- 11. Cells UV Spectrophotometer Quartz (crystalline silica) Visible Spectrophotometer Glass
- 12. Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume Cell Types I
- 13. Cell Types II Micro cell (a) for very small volumes and flow-through cell (b) for automated applications
- 14. Light Sources UV Spectrophotometer Hydrogen Gas Lamp Visible Spectrophotometer Tungsten Lamp
- 15. The concentration of an unknown sample can be determined by comparing the absorbance data to standards of known concentration. The data generated with the set of known standards is called a standard curve.
- 16. Transmittance and Path Length: Beer’s Law Concentration
- 17. The Beer-Bouguer- Lambert Law cbIIIITA /log/loglog 00
- 18. R- Transmittance R = I0 - original light intensity I- transmitted light intensity % Transmittance = 100 x Absorbance (A) or optical density (OD) = Log Log is proportional to C (concentration of solution) also proportional to L (length of light path through the solution). I I0 I I0 1 T I I0
- 19. STEPS IN DEVELOPING A SPECTROPHOTOMETRIC ANALYTICAL METHOD 1. Run the sample for spectrum 2. Obtain a monochromatic wavelength for the maximum absorption wavelength. 3. Calculate the concentration of your sample using Beer Lambert Equation: A = KCL Wavelength (nm) Absorbance 0.0 2.0 200 250 300 350 400 450
- 20. Slope of Standard Curve = A C 1 2 3 4 5 1.0 0.5 Concentration (mg/ml) Absorbance at 280 nm There is some A vs. C where graph is linear. NEVER extrapolate beyond point known where becomes non-linear.
- 21. SPECTROMETRIC ANALYSIS USING STANDARD CURVE 1 2 3 4 0.4 0.8 1.2 Absorbance at 540 nm Conc entration (g/l) glucose Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line
- 22. • Every instrument has a useful range for a particular analyte. • Often, you must determine that range experimentally. • This is done by making a dilution series of the known solution. • These dilutions are used to make a working curve.
- 23. What concentration do you think the unknown sample is?
- 24. In this graph, values above A=1.0 are not linear. If we use readings above A=1.0, graph isn’t accurate.
- 25. Spectrophotometry 1. Turn instrument on 2. Select correct wavelength 3. Choose and clean cuvette 4. Open light, insert Blank (maximum light = no absorption = 100% T) 5. Measure absorption of Standards, Controls and Patient samples to 3rd decimal place
- 26. Standards • Precisely prepared = known concentration • Usually pure solution of single compound • Plot absorbance vs concentration: standard curve
- 28. How to Ensure Accuracy? • Repeat tests many times and take average • Run another sample that was tested before along with patient samples and make sure its result is close to what it should be
- 29. Control Samples • Similar in composition to patient sample • Usually pooled from many donors • Tested at least 30 times to calculate the average (target value) and allowable range of variation