1. Spectrophotometers and Concentration Assays Chapter 7

2. Learning Outcomes  Describe how a spectrophotometer operates, compare and contrast ultraviolet and visible (white light) spectrophotometers, and give examples of their uses  Determine which type of spectrophotometer is needed for a particular application and the wavelength to be used  Explain the relationship between absorbance and transmittance in spectrophotometry and interpret the meaning of absorbance measurements  Define the term pH and explain the relationship between the concentration of H + and OH - ions in acids and bases  Describe the proper way to use pH paper and pH meters and which should be used in a specified situation  Justify the need for buffers, describe how buffers are prepared, and calculate the amount of buffering agent needed when making a particular buffer  Explain how protein indicate solutions are used with and without a spectrophotometer  Describe how VIS and UV/VIS spectrophotometers are used to measure protein or DNA concentration.  Use a best-fit standard curve to determine the concentration of an unknown protein sample and explain the usefulness of a protein absorption spectrum when trying to isolate a specific protein

3. 7.1 Using the Spectrophotometer to Detect Molecules Molecules are too tiny to be seen. When an indicator solution changes color, that means a molecule of interest is present.

4. UV SPECTROPHOTOMETER

5. A VIS spectrophotometer uses white light, composed of the visible spectrum, to detect colorful molecules.

6. Absorbance, Transmittance, and Reflection. A spectrophotometer measures how light interacts with atoms or molecules in a sample.

7. Parts of a Spectrophotometer Lamp Prism Sample holder Display How a Spectrophotometer Works White light hits grating or prism Light is split into colors of the rainbow Wavelength knob directs different colors toward sample

8. VIS SPECTROPHOTOMETER

9. How a UV Spectrophotometer Works. Similar to a VIS spectrophotometer, the UV spec shines ultraviolet light or visible light on a sample, and a detector measures the amount of light that passes through, or is absorbed by, the sample. Colors of Light in the Visible Spectrum. Humans can see light with wavelengths of about 350 to 700 nm.

10. ABSORPTION SPECTRA OF CHLOROPHYLL a & b

11. How Concentration Affects Absorbance. If a sample has twice as many molecules as another, it can absorb twice as much light. This is true at any wavelength. It is important to know a sample’s wavelength of maximum light absorbance, so that the difference in absorbance due to concentration is obvious. λ max

12. Ultraviolet light – the high-energy light with wavelengths of about 100 to 350 nm; used to detect colorless molecules Spectrophotometer – an instrument that measures the amount of light that passes through (is transmitted through) a sample Nanometer – 10 -9 meters; the standard unit used for measuring light Visible light spectrum – the range of wavelengths of light that humans can see, from approximately 350 to 700 nm; also called white light Transmittance – the passing of light through a sample Absorbance – the amount of light absorbed by a sample (the amount of light that does not pass through or reflect off a sample) Tungsten lamp – a lamp, used for VIS spectrophotometers, that produces white light (350 – 700 nm) Vocabulary

13. Deuterium lamp – a special lamp used for UV spectrophotometers that produces light in the ultraviolet (UV light) part of the spectrum (100 – 350 nm) % transmittance – the manner in which a spectrophotometer reports the amount of light that passes through a sample Absorbance units – (abbreviated “au”) a unit of light absorbance determined by the decrease in the amount of light in a light beam Absorbance spectrum – a graph of a sample’s absorbance at different wavelengths Lambda max – the wavelength that gives the highest absorbance value for a sample Vocabulary

14. 7.1 Review Questions 1.What is measured in a spectrophotometer? 2.What is the difference between a UV spectrophotometer and a VIS spectrophotometer? 3.What happens to the absorbance of a sample as the concentration of a sample increases or decreases? 4.What color of light has a wavelength of 530 nm? If a molecule absorbs light at 530 nm, what color could it be? What color do we know that it is not?

15. CONCENTRATION OF H + & OH - IONS

16. 7.2 Introduction to pH pH is the measure of the level of hydrogen ions in a solution. The pH value determines whether a solution is an acid, a base, or is neutral. Measuring the pH of a Solution pH paper pH meter Calibrating and Using a pH Meter 1.Turn on the pH meter. Rinse off the electrode with distilled water. If the meter has a temperature setting knob, set it to room temperature. 2.Place the electrode in the pH 7 buffer standard. While swirling the solution, adjust the calibration knob until the display reads “7.” 3.Rinse the electrode with distilled water, being very careful of the very delicate tip. Place the electrode into the solution to be tested. Swirl the solution until the pH display stops changing. Read the pH value. 4.If the solution is a strong acid or base, use a pH 4 buffer (for the strong acid) or a pH 10 buffer (for the strong base) to slope the meter after it has been calibrated to pH 7.

17. This pH paper measures the concentration of the H + ions in a solution.

18. A pH meter is used to adjust the pH of solutions or to watch the pH of a solution change over time. The pH electrode is sitting in a 50-mL tube of storage solution.

19. Vocabulary Acid – a solution that has a pH less than 7 Base – a solution that has a pH greater than 7 Neutral – uncharged Hydrogen – a hydrogen atom which has lost an electron (H + ) Pepsin – an enzyme, found in gastric juice, that works to break down food (protein) in the stomach pH paper – a piece of paper that has one or more chemical indicators on it and that changes colors depending on the amount of H + ions in a solution pH meter – an instrument that uses an electrode to detect the pH of a solution Buffer standards – the solutions, each of specific pH, used to calibrate a pH meter

20. 7.2 Review Questions 1.If a sample has a pH of 7.8, is it considered an acid, a base or neutral? 2.What does pH paper measure? 3.Before a pH meter can be used, it needs to be calibrated. To measure the pH of most solutions, the pH meter is calibrated to what pH? 4.If the pH of a hot tub is too high, say pH 8, then what should be added to bring it to a neutral pH?

21. 7.3 Buffers Buffer solution resists changes in pH.

22. In a buffer where HA + H 2 O H 3 O + + A -, most of the water molecules exist as H 2 O. When HA ionizes, some H 2 O receive an extra H + and become H 3 0 + while A - ions are released. When a small amount of acid, such as carbonic acid (H 2 CO 3 ), is added to the buffer, the acid ionizes to release H +, which would decrease the pH if they were not bound by the A - in the buffer.

23. Vocabulary pI – the pH at which a compound has an overall neutral charge and will not move in an electric field; also called the isoelectric point pKa – the pH at which 50% of a buffering molecule in aqueous solution is ionized to a weak acid and its conjugate base; the point at which there are an equal number of neutral and ionized units.

24. 7.3 Review Questions 1.Why must DNA and proteins be stored in buffered solutions? 2.In what kind of buffer should a DNA sample that was isolated from human cheek cells be stored? 3.The formula weight of TRIS is 121.14 g/mole. How is 100 mL of 0.02 M TRIS, pH 8.0, prepared?

25. 7.4 Using the Spectrophotometer to Measure Protein Concentration Proteins are too small to be seen, they must be visualized in a way other than direct observation. A spectrophotometer can assess changes in light absorbance due to protein molecule concentration and give numerical values to proteins in solution.

26. Determining the Wavelength of Maximum Light Absorbance. The lambda max for this sample is 440 nm. Once lambda max is determined for a molecule, the spec is set to that wavelength, and all readings for the molecule are made at that wavelength. The lambda max value is a characteristic of a protein.

27. Vocabulary Standard curve – a graph or curve generated from a series of samples of known concentration

28. 7.4 Review Questions 1.What is lambda max, and why is it important? 2.What is the lambda max for colorless proteins? 3.How is Bradford reagent used to detect a specific protein in solution? 4.Which graph is used to determine the concentration of unknown protein samples?

29. Questions and Comments?