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: 0.5M H 2 SO 4 x 98.1 g/mol = g/liter g/liter x 0.6 L = g H 2 SO g H 2 SO 4 / 1.84 g/mL = mL H 2 SO 4 ALWAYS ADD ACID TO WATER!!! Take mL water, add 16 mL concentrated sulfuric acid, then add water to 600 mL

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

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

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 A.Mix 50 g glucose with 50 mL of water B.Mix 50 g glucose with 100 mL of water C.Mix 50 g glucose enough water to dissolve it completely, then add water to 100mL total volume.

Preparation of Buffers 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(H 2 O) = (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(H 2 O) + 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)

absorbance A (also called optical density) is defined as Absorbance A = log 10 I 0 / I

Transmission T = I / I 0 %T = 100 T

Beer Lamert’s Law

Relationship between A(OD) and %T Transmittance, T = P / P 0 % Transmittance, %T = 100 T Absorbance, A = log 10 P 0 / P A = log 10 1 / T A = log / %T A = 2 - log 10 %T

Light scattering

reflection scattering For Solution: Scattering  1/ 4

Prism Diffraction grating

Spectrophotometer types -Single beam -Dual beam -Diode array

Single Beam - Spectrophotometer

Dual Beam – Single Detector

Diode Array - Spectrophotometer

NanoDrop

Bradford Assay

Enzyme-Linked Immunosorbent Assay ELISA

Endpoint vs Kinetic

Coupled Assays

Molecular Orbital

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

Vibrational and rotational relaxation AbsorbanceFluorescence Energy

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).

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.

reflection Emission scattering Exitation

Emission Excitation Spectrofluorometer Detector monochromator

Emission Excitation Dichroic Mirror Microscope and Plate Reader Detector Filter

Microscope and Plate Reader Emission Excitation Dichroic Mirror Detector Filter

Filter and Dichroic Mirror