Principles of Scientific Instrumentation Design of Experiments and Analysis of Results

1. Principles of Scientific InstrumentationDesign of Experiments and Analysis of Results 80-801 Bldg 102 Room 3 12:00-14:00 Thurs Lecturers: Dr. Alexander Varvak Dr. Chaim Wachtel Dr. Refael Minnes Dr. Itay Lazar

2. Structure of the course • Weekly lectures • Requirements: Course is geared for MS/PhD students who conduct experimental research in Biological Sciences • Course materials can be found at http://lifefaculty.biu.ac.il/FacultyInstruments/ • Grade is based on the class presentation (date to be determined)

3. Preparation of Buffers - 1 Calculate the volume of sulfuric acid (H2SO4) necessary to prepare 600 milliliter 0.5M H2SO4 from concentrated H2SO4 stock (assume 100%). MW H2SO4: 98.1 g/mol Density H2SO4: 1.84 g/cm3 Calculation: 0.5M H2SO4 x 98.1 g/mol = 49.05 g/liter 49.05 g/liter x 0.6 L = 29.43 g H2SO4 29.43 g H2SO4 / 1.84 g/mL = 15.99 mL H2SO4 ALWAYS ADD ACID TO WATER!!! Take 550-580 mL water, add 16 mL concentrated sulfuric acid, then add water to 600 mL

4. Preparation of Buffers - 2 Preparation of monocomponent buffer stocks. Given: MW of Na2HPO4 ∙ 12H2O = 358.14 g/mol MW of Na2HPO4 ∙ 2H2O = 177.99 g/mol • Calculate the weight of dibasic sodium phosphate dodecahydrate (Na2HPO4 ∙ 12H2O) powder required to prepare 1 liter of 1M stock of Na2HPO4 solution. • Calculate the weight of dibasic sodium phosphate dihydrate (Na2HPO4 ∙ 2H2O) required to prepare the same solution as in (a).

5. Preparation of buffers - 3 Monobasic potassium phosphate has pKa of 7 at room temperature. To prepare 1 liter of 0.5M potassium phosphate buffer at pH 7.5 by mixing stocks of 0.5M monobasic potassium phosphate (pH 4.5) and 0.5M dibasic potassium phosphate (pH 9.5), you will need approximately (choose the best answer): A. 500 mL of each solution B. 333 mL of monobasic solution and 667 mL of dibasic solution C. 667 mL of monobasic solution and 333 mL of dibasic solution

6. Preparation of Buffers - 4 Preparation of glucose solution. • Density of water = 1 g/mL • Solubility of glucose: 91g in 100 mL of water • Density of glucose 1.54 g/mL In order to prepare 100 mL of 50% (weight / vol) solution of glucose • Mix 50 g glucose with 50 mL of water • Mix 50 g glucose with 100 mL of water • Mix 50 g glucose enough water to dissolve it completely, then add water to 100mL total volume.

7. Preparation of Buffers - 4 • What is the volume of 100 g of 50% weight/weight solution of glucose in water at room temp (25C)? In principle, it would be calculated as follows: V(H2O) = (50 g) x (1 mL/1 g) = 50 mL V(glucose) = (50 g) x (1 mL/1.54 g) = 32.5 mL Total Volume = V(H2O) + V(glucose) = 82.5 mL BUT: At room temp, 50 g of glucose will not dissolve in 50 mL of water (solubility exceeded, 45 g will dissolve only)

8. Scientific Method

9. History of Instrument Use

10. Light electromagnetic radiation

11. Visible Light Spectrum

12. In 1900, Max Planck developed a new theory of black-body radiation that explained the observed spectrum A photon has an energy, E, proportional to its frequency, f, by E = h = hc/ where h is Planck's constant, λ is the wavelength and c is the speed of light E ~ ~ 1/

14. ~

15. Electron orbital transitions

16. Characteristic UV-vis absorption spectrum

17. Absorbance absorbanceA (also called optical density) is defined as A = log10I0/ I

18. Transmission T = I / I0%T = 100 T

19. Beer Lamert’s Law

21. Relationship between A(OD) and %T Transmittance, T = P / P0% Transmittance, %T = 100 T Absorbance, A = log10P0/ P A = log10 1 / T A = log10 100 / %T A = 2 - log10 %T 

23. Polarization

24. Reflection

25. Light scattering

26. reflection scattering For Solution: Scattering ~ 1/4

28. Prism Diffraction grating

29. Spectrophotometer types -Single beam-Dual beam-Diode array

30. Single Beam - Spectrophotometer

31. Dual Beam - Spectrophotometer

32. Dual Beam – Single Detector

33. Diode Array - Spectrophotometer

36. NanoDrop