1. SPECTROPHOTOMETRY

2. Spectrophotometry Determines concentration of a substance in solution
Measures light absorbed by solution at a specific wavelength

3. Spectrophotometry
One of the simplest and most widely used methods to determine the amount of protein or nucleic acid present in a given solution

4. Spectrophotometry
Proteins do not absorb in visible wavelength region unless they have a prosthetic group (e.g., Fe2+), or an unnatural amino acid

5. 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

6. 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 ( nm):
a. Small conjugated ring systems (Phe, Tyr, Trp)

7. Spectrophotometry Detector Cuvette Io I A = 0.012  l  Lamp
Monochromator
Detector
Cuvette

8. Spectrophotometers Light source (Lamp) Optical filters or prism
Tube or cuvette
Photocell or photomultiplier tube

9. Light source (Lamp) Visible region = tungsten or tungsten-halogen
UV light = deuterium or hydrogen lamp

10. 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

11. Tubes or cuvettes Visible range = glass cuvette
UV range = quartz cuvette

12. Photocell
To detect transmitted light

13. Spectrophotometry Beer-Lambert’s Law lo
g Io = cl I

Where: 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)

14. 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

15. Visible region wavelength
Color
Wavelength (nm)
Ultraviolet
400 and under
Violet
Blue
Green
Yellow
Orange
Red
Infrared
750 & over

16. 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

17. 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

18. 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

19. Transmittance
Inversely related to the concentration of the solution and is expressed in %
% T = 1 x 100 Io

20. Transmittance
100% transmittance means no light is absorbed by the solution so that incident light is 100% transmitted

21. Absorbance & Transmittance
Absorbance  concentration
Transmittance 1/  to concentration and absorbance

22. 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

23. Sample Problem Solution: 1. A = lc = (6 x 103)x (0.1) x (1 x 10-3)
= 0.6 (O.D.)
O.D. between 0.1 and 2 are most reliable

24. Spectrophotometry Clinical applications:
1. Aromatic amino acids have characteristic strong absorbance of light at a wavelength of 280 nm ex. Tryptophan & tyrosine

26. 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

27. Calibration Curve Glucose Std. Concn. Absorbance 60 mg% 0.2 120 mg%
0.4 U
0.5
180 mg%
0.6

28. Colorimetric determination of reducing sugars
Dinitrosalicylate
Potassium ferric hexacyanid (Prussian blue)
Nelson-Somogyi (molybdenum blue)

29. 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)

30. 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

31. DNS method
Amylase-catalyzed reactions are typically buffered at pH 5 using acetate or citrate
Citrate may interfere with DNS color development

32. Principle
Carbohydrates are essentially aldehydes or ketones that contain multiple hydroxyl (-OH) groups
Monosaccharides can be aldoses (glucose) or ketoses (fructose

33. 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.

34. Principle

35. Principle

36. Principle

37. Principle
When salivary amylase is added to starch, a hydrolysis reaction is initiated in which water breaks bonds, releasing maltose

38. 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

39. 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

40. Principle Aldehyde group carboxyl group
3,5 Dinitrosalicylic amino, 5 nitrosalicylic
oxidation
reduction

41. Standard Absorbance Curve
Done by reacting know concentration of glucose with DNS then determining absorbance at 540 nm
Plot absorbance vs. glucose concentration

42. 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