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/molDensity 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

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

(a)Calculate the weight of dibasic sodium phosphate dodecahydrate (Na2HPO4 ∙ 12H2O) powder required to prepare 1 liter of 1M stock of Na2HPO4 solution.

(b)Calculate the weight of dibasic sodium phosphate dihydrate (Na2HPO4 ∙ 2H2O) 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 solutionB. 333 mL of monobasic solution and 667

mL of dibasic solutionC. 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 glucoseA.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 - 4

1. 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 mLV(glucose) = (50 g) x (1 mL/1.54 g) = 32.5 mL

Total Volume = V(H2O) + V(glucose) = 82.5 mLBUT: 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 = log10 I0 / I

Transmission

T = I / I0

%T = 100 T

Beer Lamert’s Law

Relationship between A(OD) and %T

Transmittance, T = P / P0%

Transmittance, %T = 100 T

Absorbance, A = log10 P0 / PA = log10 1 / T A = log10

100 / %TA = 2 - log10 %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

Absorbance Fluorescence

Ene

rgy

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

reflection

Emission

scattering

Exitation

reflection

Emission

scattering

Exitation

Detector

monochromator

EmissionExcitation

Dichroic Mirror

Microscope and Plate Reader

reflection

Emission

scattering

Exitation

reflection

Emission

scattering

Exitation

DetectorFilter

Microscope and Plate Reader

reflection

Emission

scattering

Exitation

reflection

Emission

scattering

Exitation

Emission

Excitation

Dichroic MirrorEmission

Excitation

Dichroic Mirror

Emission

Excitation

Dichroic Mirror

Detector

Filter

http://www.chroma.com/products/catalog/11000_Series/11000v3

Filter and Dichroic Mirror

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