SPECTROSCOPY . Spectral Distribution of Radiant Energy Wave Number (cycles/cm). SPECTROSCOPY. V = Wave Number (cm-1) l = Wave Length C = Velocity of Radiation (constant) = 3 x 10 10 cm/sec. u = Frequency of Radiation (cycles/sec) The energy of photon:
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Spectral Distribution of Radiant Energy
Wave Number (cycles/cm)
V = Wave Number (cm-1)
l = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
u = Frequency of Radiation (cycles/sec)
The energy of photon:
h (Planck's constant) = 6.62 x 10-27 (Ergsec)
C = u
Prism - spray out the spectrum and choose the certain wavelength
(l) that you want by slit.
1. Spectrophotometer - an instrument which can measure the optical density of a sample at any wavelength.
2. Fluorometer - measures the intensity of fluorescent light emitted by a sample exposed to UV light under specific conditions.
As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases.
R = I0 - original light intensity
I- transmitted light intensity
% Transmittance = 100 x
Absorbance (A) or optical density (OD) = Log
= Log = 2 - Log%T
Log is proportional to C (concentration of solution) and is also proportional to L (length of light path through the solution).
A CL = KCL by definition and it is called the Beer Lambert Law.
A = KCL
K = Specific Extinction Coefficient ---- 1 g of solute per liter of solution
A = ECL
E = Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution
E differs from K (Specific extinction Coefficient) by a factor of molecular weight.
A = ECL
A = No unit (numerical number only)
L = Cm
C = Moles/Liter
A = KCL
A = No unitC = Gram/Liter L = Cm
NEVER extrapolate beyond point known where becomes non-linear.
Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line
Quartz (crystalline silica)
1. Hydrogen Gas Lamp
2. Mercury Lamp
1. Tungsten Lamp
1. Carborundum (SIC)
Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultra-violet region.
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Amino Acids (aromatic)
Enzyme Activity (Hexokinase)
Thiamin (365 nm, 435 nm)
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)
1. A solution of purified DNA isolated from Escherichia coli gives an absorbance of 0.793 at 260 Mm in a 1 Cm cell at pH 4.5. If E1%1Cm is 197, calculate the concentration of the solution in milligrams per milliliter.
2. Calculate the Molar Extinction Coefficient E at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the following data which were obtained in 1 Cm cell.
SolutionC x 105 MIoI
A 2.23 93.1 27.4
B 1.90 94.2 32.8
3 x 103 Liter/Cm x Mole
If the absorbance reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the concentration of compound x in sample?
4. The concentration of compound Y was 2 x 10-4 moles/liter and the absorption of the solution at 300 nm using 1 Cm quartz cell was 0.4. What is the molar extinction coefficient of compound Y?
5. Calculate the molar extinction coefficient E at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from the following data which were obtained in 1 Cm cell.
SolutionC x 105 MI0I
A 2.0 100 30
Question 6.A = 0.01E = 10000L / mole x cmL = 1cmA = ECL0.01= 10000L/mole X Cm X C (Concentration) x 1CmC = mole / LiterC = X mole / Liter = X mole (236 g/mole) / Liter (1000 Cm3) x PPM (10-6 g/Cm3)= X mole (236 g / mole) / Liter x 1 Liter / 1000 Cm3 x ( PPM) 10-6g / Cm3)=x PPMPPM = 1ug / Cm31ug = 10-6 g