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Preparation of Buffers - 1

Preparation of Buffers - 1. Calculate the volume of sulfuric acid (H 2 SO 4 ) necessary to prepare 600 milliliter 0.5M H 2 SO 4 from concentrated H 2 SO 4 stock (assume 100%). MW H 2 SO 4 : 98.1 g/mol Density H 2 SO 4 : 1.84 g/cm 3. Calculation:

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Preparation of Buffers - 1

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  1. 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

  2. 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).

  3. 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

  4. 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.

  5. 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)

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

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

  8. Beer Lamert’s Law

  9. 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 

  10. Light scattering

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

  12. Prism Diffraction grating

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

  14. Single Beam - Spectrophotometer

  15. Dual Beam – Single Detector

  16. Diode Array - Spectrophotometer

  17. NanoDrop

  18. Bradford Assay

  19. ELISA Enzyme-Linked Immunosorbent Assay

  20. Endpoint vs Kinetic

  21. Coupled Assays

  22. Molecular Orbital

  23. Factors that influence on Fluorescence pH Solid state or Solution state Solvent

  24. Energy Absorbance Fluorescence Vibrational and rotational relaxation

  25. The excitation and emission spectra of a fluorophore and the correlation between the excitation amplitude and the emission intensity. General diagram of the excitation and emission spectra for a fluorophore (left). The intensity of the emitted light (Em1 and Em2) is directly proportional to the energy required to excite a fluorophore at any excitation wavelength (Ex1 and Ex2, respectively; right).

  26. The Stokes shift of the excitation and emission spectra of a fluorophore. Fluorophores with greater Stokes shifts (left) show clear distinction between excitation and emission light in a sample, while fluorophores with smaller Stokes shifts (right) exhibit greater background signal because of the smaller difference between excitation and emission wavelengths.

  27. scattering Exitation reflection Emission

  28. Spectrofluorometer Detector monochromator Emission Excitation

  29. Microscope and Plate Reader Detector Filter Emission Excitation Dichroic Mirror

  30. Microscope and Plate Reader Detector Filter Emission Dichroic Mirror Excitation

  31. Filter and Dichroic Mirror http://www.chroma.com/products/catalog/11000_Series/11000v3

  32. http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.htmlhttp://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html

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