Spectrofotometry Flashcards
Spectrofotometry
photo = light
The priciple of the spectrofotometry
is a method to measure how much a coloured solution absorbs light in a certain wavelength.
is in accordance with the Beer-Lambert law.
Beer-Lambert law
used to measure the amount of absorbed or transmitted light by the coloured soultion.
Formula Beer-Lambert law
A = absorbance, represents ..
the amount of light absorbed - have no units
Epsilon, represents
molar absorption coefficient = is a constant for a given compound at a given wavelength (1/cmM)
Concentration (C) (Beer-Lambert law)
concentration of the absorbing molecules (in M).
d (Beer-Lambert law)
distance of light path through the solution (in cm).
The absorbance (A) of a solution is defined as:
incident light = einfallende Licht
Calculation the plot factor
Calculation the concentration using a single standard protein
- Regarding the linear relationship between absorbance and concentration, it is possible to relate the concentration of the unknown compound using a single standard protein
result in g/dL
Determination of total serum proteins:
The principle of the Gornall method (Biruet method)
**Principle:** Cupric ions (Cu2+) react with the peptide bonds of proteins in an alkaline medium and disassembled the protein.
The result:
a violet-colored complex, with an absorption maximum at 540nm.
Hyperproteinemia
high serum protein level > 8 g/dl
monoclonal gammopathie: develop =>
multiple myeloma, Waldenstrom macroglobulinemia etc
Hypoproteinemia
low serum protein levels < 6 g/dl
liver diseases: hepatic cirrhosis, chronic hepatitis, liver tumors
kidney diseases: glomerulonephritis, nephrotic syndrome
Reference range of total serum protein
6-8 g/dl
Calculate the concentration of serum proteins (in g/dl) and compare with the reference range knowing that, for this determination, the following results were obtained:
= 0,47/ 0,53 * 75 mg/ml = 66,51 mg/dl
mg/ml in g/dl
66,51 mg/dl * 0,1 = 6,651 g/dl
