1. SPECTROPHOTOMETRY IN BIOTECHNOLOGY

2. LIGHT IS A TYPE OF ELECTROMAGNETIC RADIATION Imagine electromagnetic radiation like waves on a pond – But instead of water, electromagnetic radiation is energy moving through space – Distance from one crest to the next is the wavelength

3. WAVELENGTH AND COLOR Different wavelengths of light correspond to different colors All colors blended together is called white light The absence of all light is black Light of slightly shorter wavelengths is ultraviolet – Eyes do not perceive UV light

5. WAVELENGTH OF VISIBLE LIGHT AND COLOR WAVELENGTHCOLOR PERCEIVED 380-430Violet 430-475Blue 475-495Greenish Blue 495-505Bluish Green 505-555Green 555-575Yellowish Green 575-600Yellow 600-650Orange 650-780Red

6. INTERACTION OF LIGHT WITH MATERIALS IN SOLUTION When light shines on a solution, it may pass through – be transmitted OR Some or all of the light energy may be absorbed

7. THE ABSORPTION OF LIGHT AND COLOR OF SOLUTIONS WAVELENGTH OF LIGHT ABSORBED COLOR OF LIGHT ABSORBED COLOR OF SOLUTION 380-430VioletYellow 430-475BlueOrange 475-495Greenish BlueRed-Orange 495-505Bluish GreenOrange-Red 505-555GreenRed 555-575Yellowish GreenViolet-Red 575-600YellowViolet 600-650OrangeBlue 650-780RedGreen

8. BIOLOGICAL SOLUTIONS Usually appear clear to our eyes – have no color DNA, RNA, most proteins do not absorb any visible light But they do absorb UV light, so UV spectrophotometers are useful to biologists – Example, can use a detector that measures absorbance at 280 nm, or 254 nm to detect proteins

9. SPECTROPHOTOMETERS Are instruments that measure the interaction of light with materials in solution

10. THE BLANK Spectrophotometers compare the light transmitted through a sample to the light transmitted through a blank. The blank is treated just like the sample The blank contains everything except the analyte (the material of interest) – Contains solvent – Contains whatever reagents are added to the sample

11. WHEN OPERATING A SPEC Blank is inserted into the spectrophotometer Instrument is set to 100% transmittance or zero absorbance

12. PROPER SELECTION, USE, AND CARE OF CUVETTES 1. Cuvettes are made from plastic, glass, or quartz. a. Use quartz cuvettes for UV work. b. Glass, plastic or quartz are acceptable visible work. c. There are inexpensive plastic cuvettes that may be suitable for some UV work.

13. Cuvettes are expensive and fragile (except for “disposable” plastic ones). Use them properly and carefully a. Do not scratch cuvettes; do not store them in wire racks or clean with brushes or abrasives b. Do not allow samples to sit in a cuvette for a long period of time c. Wash cuvettes immediately after use

14. Disposable cuvettes are often recommended for colorimetric protein assays, since dyes used for proteins tend to stain cuvettes and are difficult to remove. Matched cuvettes are manufactured to absorb light identically so that one of the pair can be used for the sample and the other for the blank. Do not touch the base of a cuvette or the sides through which light is directed. Make sure the cuvette is properly aligned in the spectrophotometer. Be certain to only use clean cuvettes.

15. Handling a Cuvette

17. EXAMPLES Some examples of qualitative spectrophotometry – The absorbance spectra of various common solvents. Note that some solvents absorb light at the same wavelengths as DNA, RNA, and proteins – Hemoglobin bound to oxygen versus carbon monoxide – Native versus denatured bovine serum albumin (a protein commonly used in the lab)

19. OVERVIEW OF QUANTITIVE SPECTROPHOTOMETRY Measure the absorbance of standards containing known concentrations of the analyte Plot a standard curve with absorbance on the X axis and analyte concentration on the Y axis Measure the absorbance of the unknown(s) Determine the concentration of material of interest in the unknowns based on the standard curve

21. LINEAR RANGE If there is too much or too little analyte, spectrophotometer cannot read the absorbance accurately

22. COLORIMETRIC ASSAYS Quantitative assays of materials that do not intrinsically absorb visible light Combine the sample with reagents that make the analyte colored The amount of color is proportional to the amount of analyte present

23. BRADFORD PROTEIN ASSAY A quantitative colorimetric assay Used to determine the concentration, or amount, of protein in a sample

24. Prepare standards with known protein concentrations Add Bradford Reagent to the samples and to standards – Read absorbances – Create a standard curve Determine the concentration of protein in the samples based on the standard curve Running a Protein Assay

25. MORE ABOUT THE CALIBRATION LINE ON A STANDARD CURVE Three things determine the absorbance of a sample: – The concentration of analyte in the sample – The path length through the cuvette – The intrinsic ability of the analyte to absorb light at the wavelength of interest

26. BEER-LAMBERT LAW A =  B C Where: A = absorbance at a particular wavelength  = E = absorptivity constant – intrinsic ability of analyte to absorb light at a particular wavelength B = path length through cuvette C = concentration of analyte

27. UV METHODS These UV methods for estimating concentration and purity of DNA, RNA, and proteins are very commonly used, are very quick, and easy to perform However, they values obtained are not very accurate – they are rough estimates

28. CALIBRATION OF A SPECTROPHOTOMETER Brings the readings of the spectrophotometer into accordance with nationally accepted values Part of routine quality control/maintenance

29. CALIBRATION Two parts: 1. Wavelength accuracy, the agreement between the wavelength selected by the operator and the actual wavelength of light that shines on sample 2. Photometric accuracy, or absorbance scale accuracy, the extent to which a measured absorbance or transmittance value agrees with an accepted reference value

30. Wavelength accuracy is determined using certified standard reference materials (SRMs) available from NIST or traceable to NIST – An absorbance spectrum for the reference material is prepared – The absorbance peaks for reference standards are known, so the wavelengths of the peaks generated by the instrument can be checked

31. Manufacturers specify the wavelength accuracy of a given instrument – For example, a high performance instrument may be specified to have a wavelength accuracy with a tolerance of + 0.5 nm – A less expensive instrument may be specified to have a wavelength accuracy of + 3 nm

32. PHOTOMETRIC ACCURACY Assures that: – If the absorbance of a given sample is measured in two spectrophotometers at the same wavelength and under identical conditions then the readings will be the same and the readings will correspond to nationally accepted values

33. Photometric accuracy is difficult to achieve due to different instrument designs and optics Usually photometric accuracy is not critical if the same instrument is used consistently and if its readings are linear and reproducible Photometric accuracy is required where values from different labs and instruments are compared Required if rely on published absorptivity constants Likely required in a GMP-compliant facility