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Spectrophotometry
• Determines concentration of a
substance in solution
– Measures light absorbed by solution at a
specific wavelength
Spectrophotometry
• One of the simplest and most widely
used methods to determine the amount
of protein or nucleic acid present in a
given solution
Spectrophotometry
• Proteins do not absorb in visible
wavelength region unless they have a
prosthetic group (e.g., Fe2+), or an
unnatural amino acid
Spectrophotometry
• The amino acids tryptophan, tyrosine &
cytosine absorb light in the UV
wavelength
• Aromatic rings in the bases of nucleic
acids also absorb light in the UV range
Spectrophotometry
• Visible region: low energy electronic transition
due to:
a. Compounds containing transition metals
b. Large aromatic structures & conjugated
double bond systems (vitamin A, retinal,
heme)
• UV region (200-400 nm):
a. Small conjugated ring systems (Phe, Tyr,
Trp)
Spectrophotometry
Io I
A = 0.012
l
lo g Io = cl
I
A = abc = log(100/%T)
Where A = absorbance
a = absorptivity of the compound under standard
conditions
b = light path of the solution
c = concentration of the compound
%T = percent transmittance
Beer-Lambert’s Law
• Absorbance
A = K x C = Log10Io
I
Where: Io = amount of light absorbed by the solution
expressed as absorbance or optical density
K = constant
C = concentration of the substance
Transmittance
• Defined as the ratio of the intensity of
light emerging from the solution (I) to
that of incident light entering (Io)
T=I
Io Io I
Transmittance
• Inversely related to the concentration of
the solution and is expressed in %
% T = 1 x 100
Io
Transmittance
• 100% transmittance means no light is
absorbed by the solution so that
incident light is 100% transmitted
Absorbance & Transmittance
• Absorbance concentration
A = Log I0 = lc
I
Sample Problem
• Solution:
1. A = lc = (6 x 103)x (0.1) x (1 x 10-3)
= 6 x 10-1
= 0.6 (O.D.)
Cu = Cs x A(u) x D
A(s)
Where: Cs = concentration of standard
Cu = concentration of unknown
A(s) = absorbance of standard
A(u) = absorbance of unknown
D = dilution factor
Calibration Curve
Glucose Standard Calibration Curve
Glucose Absorba
Std. Concn. nce 1.2
60 mg% 0.2 1
Absorbance
0.8
120 mg% 0.4 0.6
0.4
0.2 Linear ( )
U 0.5
0
60 120 180 200
180 mg% 0.6
Mg% glucose
Colorimetric determination of
reducing sugars
• Dinitrosalicylate
• Potassium ferric hexacyanid (Prussian
blue)
• Nelson-Somogyi (molybdenum blue)
DNS method
• Developed by Sumner & Sisler (1944)
and modified by Miller (1959)
• Based on reduction of sugars by DNS
under alkaline conditions to yield 3-
amino-5-nitrosalicylate (brown color)
DNS method
• Measured at 540 nm
• Quantity of reducing sugar is
extrapolated from a calibration curve
prepared with D-glucose
• Amylase-catalyzed reactions are
typically buffered at pH5 using acetate
or citrate
DNS method
• Amylase-catalyzed reactions are
typically buffered at pH 5 using acetate
or citrate
• Citrate may interfere with DNS color
development
Principle
• Carbohydrates are essentially
aldehydes or ketones that contain
multiple hydroxyl (-OH) groups
• Monosaccharides can be aldoses
(glucose) or ketoses (fructose
Principle
• Both aldoses & ketoses occur in equilibrium
between the open-chain forms and cyclic
forms (chain lengths of C4)
• These are generated by bond formation
between one of the (-OH) groups of the sugar
chain with the C of the aldehyde or keto
group to form a hemiacetal bond.
Principle
Principle
Principle
Principle
oxidation
Aldehyde group carboxyl group
reduction