SPECTROPHOTOMETRY
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
Lamp
Monochromator
Detector
Cuvette
Spectrophotometers
•
•
•
•
Light source (Lamp)
Optical filters or prism
Tube or cuvette
Photocell or photomultiplier tube
Light source (Lamp)
• Visible region = tungsten or tungstenhalogen
• UV light = deuterium or hydrogen lamp
Optical filters/prisms
• To limit light to a certain wavelength
• Monochromator can isolate a specific
wavelength of white light and allow it to
pass through the solution being
analyzed
Tubes or cuvettes
• Visible range = glass cuvette
• UV range = quartz cuvette
Photocell
• To detect transmitted light
Spectrophotometry
• Beer-Lambert’s Law
lo
Where:
g Io = cl
I
Io = intensity of incident light
I = intensity of transmitted light
 = molar extinction coefficient
c = concentration of the absorbing species (mol/L)
l = path length of the light-absorbing sample (cm)
Beer-Lambert’s Law
• The fraction of the incident light
absorbed by a solution at a given
wavelength is related to
a. thickness of the absorbing layer
(path length) and
b. concentration of the absorbing
species
Visible region wavelength
Color
Ultraviolet
Violet
Blue
Green
Yellow
Orange
Red
Infrared
Wavelength (nm)
400 and under
400 - 450
450 - 500
500 - 570
570 - 590
590 - 620
620 - 650
750 & over
Beer-Lambert’s Law
• Concentration  amount of light absorbed
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
• Transmittance 1/  to concentration and
absorbance
Sample Problem
• Cytosine has a molar extinction
coefficient of 6 x 103 mol-1 cm-1 at 270
nm at pH 7. Calculate absorbance of
1 x 10-3 M cytosine solution in 1mm cell at
270 nm
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.)
O.D. between 0.1 and 2 are most reliable
Spectrophotometry
• Clinical applications:
1. Aromatic amino acids have
characteristic strong absorbance of light
at a wavelength of 280 nm ex.
Tryptophan & tyrosine
Calculation
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
Std. Concn.
Absorba
nce
60 mg%
0.2
120 mg%
U
0.4
0.5
Absorbance
Glucose Standard Calibration Curve
1.2
1
0.8
0.6
0.4
0.2
Linear ( )
0
180 mg%
0.6
60
120
180
Mg% glucose
200
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 3amino-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
• When salivary amylase is added to
starch, a hydrolysis reaction is initiated
in which water breaks bonds, releasing
maltose
Principle
• DNS tests for the presence of free
carbonyl groups (C=O), the so-called
reducing sugars
• Involves oxidation of the aldehyde
functional groups in glucose and the
ketone functional groups in fructose
Principle
• Simultaneously, 3,5 DNS is reduced to
3-amino, 5 nitrosalicylic acid under
alkaline conditions
• As hydrolysis proceeds, more reducing
sugar will be available to react with the
3,5 DNS
Principle
oxidation
Aldehyde group
carboxyl group
reduction
3,5 Dinitrosalicylic
3-amino, 5 nitrosalicylic
Standard Absorbance Curve
• Done by reacting know concentration of
glucose with DNS then determining
absorbance at 540 nm
• Plot absorbance vs. glucose
concentration
Absorbance
• Absorbance corresponds to
0.1 ml of test = x mg of glucose
10 ml contains = x (10 mg of glucose)
0.1
= % of reducing sugars