Preparation of Buffers - 1 Calculate the volume of sulfuric acid (H 2 SO 4 ) necessary to prepare...
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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
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