colorimetry....spectrophotometry

2. COLORIMETRY
&
SPECTROPHOTOMETRY
Dr.Tasnim Ara Jhilky
MD(Part-1)
Department of Biochemistry
Sir Solimullah Medical College

3. Introduction
• Photometry is the most common analytical
technique used in the biochemical laboratory.
It is designed to measure the intensity of a
beam of light.
• Photometric principles are applied to the
several kinds of analytical techniques:
(a) where absorbed or transmitted light is measured:
•Colorimetry
•Spectrophotometry
•Atomic absorption, and
•Turbidometry
(b) where emitted light is measured:

4. Introduction (cont.)
• The components of most photoelectric
colorimeters are basically the same and the
basic method of operation is also similar for
all the instruments.
• In analytical chemistry, Colorimetry is a
technique “used to determine the
concentration of colored compounds
(analytes) in sample solution” at visible
spectrum of light (400 – 700 nm).

5. Properties of Light
Light is the visible spectrum of
electromagnetic radiation,emitted in the
form of waves of different wave lengths
ranging from380nm to 750nm.

6. Electromagnetic Spectrum

7. Colors & Wavelengths
COLOR WAVELENGTH (λ in nm)
Ultraviolet < 380
Violet 380 – 435
Blue 436 – 480
Greenish-blue 481 – 490
Bluish-green 491 – 500
Green 501 – 560
Yellowish-green 561 – 580
Yellow 581 – 595
Orange 596 – 650
Red 651 – 780
Near Infrared > 780

8. Principle of Photometry
Substance to be measured by photometry must be
colored to begin with or can be made to produce
color derivatives by using certain reagents and
reactions.
Intensity of colour produced is propotional to the
concentration of the colour producing subs.present
in solution.
Colored subs.absorbs light of a particular wave
length and the extent of light absorption depends on
the conc .of color producing subs.in solution.

9. Principle (con.)……
A characteristic wavelength of absorption spectrum
is isolated from light passing it through filter
monochromator
Solution with colored subs. is kept in a cuvet&
allowed the subs. to absorb light.
Degree of light absorption by a solute of unknown
conc. Is propotional to degree of light absorption
by same solute in a solution of known conc.
Subs.of unknown conc. is measured by comparing
with same subs .in another solu. Of known conc.

10. Colorimetry
Principle
.Colored solutions have the property of
absorbing certain wavelength of light
when a monochromatic light is passed
through them.
.The amount of light absorbed or
transmitted by a colored solution is in
accordance with two laws:
Beer’s law
Lambert’s law

11. Beer’s law :
 This law states that,the intensity of
transmitted light decreases exponentially
with the increase in concentration of
colored substance in the solution.
i.e. the amount of light absorbed by a
colored solution is directly proportional to
the conc. Of substance in the solution.

12. Beer’s law
Beer’s law

13. Lambert’s law :
This law states that,the intensity of
transmitted light decreases exponentially
with increase in length of light pathway.
(diameter of the cuvette)
i.e. the amount of light absorbed by a colored
solution is directly propotional to the length
of light path.

14. Transmittance
 Transmittance: It is the ratio of intensity of
transmitted light (It) to the intensity of incident
light (lo) across a solution. It is expressed as %
 Transmittance (T) = It/ 1o
 Transmittance is inversely and logarithmically
proportional to the concentration.i.e Tα log1/C.

15. Absorbance
• Optical density: it is the amount of light
absorbed by the colored substance. OD
may be defined as the logarithmic ratio
of incident light to that of transmitted
light.
• So A = log ( I/T ) ;= log10 ( 100/T); = 2 -
log10T
Absorbace is directly and linearly
propotional to con.

16. Relationship between absorbance
and transmittance
2 - log10T
OD %T
OD=

17. Combined Beer’s- Lambert’s law
Combining the two laws:
A α C x L
A = K x C x L
Let AT=absorbance of the test solution
CT=concentration of the test solution
AS=absorbance of the standard solution
CS=concentration of the standard solution

18. AT
AS
K x CT x L
K x CS x L
=
AT
AS
CT
CS
=
CT =
AT
AS
x CS
AS = K x CS x LAT = K x CT x L

19. CT =
AT
AS
xCS
Concentration
of TEST sol.
Absorbance of TEST
Absorbance of STANDARD
Con. of STANDARDx=
Concentration
of TEST/100ml
Absorbance of TEST
Absorbance of STANDARD
Concn of Std X 100
x=
X ml
=
ODT
ODS
x CS

20. Standard (calibration curve)
• The standard curve is prepared to check
whether the method of assaying a particular
substance follows Beer’s Law, i.e. whether the
absorbance of the substance increases in a
linear way with its concentration.
• The standard curve is constructed by plotting
a vertical axis (y – axis, ordinate) for optical
densities (absorbance) and a horizontal axis
(x – axis, abscissa) the concentration of
standard solution.
• The concentration of the test/unknown can be

21. Standard Curve / Calibration curve

22. Preparation of solution for investigation
• In colorimetric estimation it is necessary to
prepare 3 solutions:
BLANK(B)
STANDARD(S)
TEST(T)
10/3/2016 4:36 PM

23. BLANK
.To compensate any non specific
color
.To set the instrument 100%T
and zero %OD
Water BLANK
Reagent BLANK

24. STANDARD
Solution of known concentration of
the substance
Both O.D and
concentration are
known
So concentration of
unknown can be
calculated

25. TEST
Test solution is made by treating
a specific volume of the test
sample with reagents
As per procedure

26. Complimentary color
• Wavelength between 400nm to 700 nm
form the visible spectrum of light
• Light passed through a solution which
selectivity absorbs radiation at fixed wave
lengths,then the color of the transmitted
light is complementary to that of the
absored light.

27. Colors and complimentary colors of visible
spectrum
Color of the
solution/ solution
color transmitted
Filter used/ color
absorbed
Wavelength (nm)
Yellow Blue 450 – 479
Red Green 505 – 534
Blue yellow 640 – 689
Green Red 620 - 689

28. Colorimeter

29. Components of Colorimetry
1. Light source:
The light source is usually a tungten lamp, for
wavelength in the visible range (320 – 700nm) and a
deutarium or hydrogen lamps for ultraviolet light
(below 350nm).
a) Tungsten lamp  Visible range
b) Deutarium/hydrogen lamp (preferred)  UV Rays
c) Black body radiators (Nerst glower)  Infrared radiations

30. Monochromators/Filters
• This device used for spectral isolation(light of
single wavelength)
• this means of selecting a sufficiently narrow
wave band.
Filter SBW about 50nm (wide band pass
monochromator)
Prism SBW is 5-10 or <5nm (narrow
band pass monochromator)
Diffraction grating SBW is 15-20nm.

31. Monochromators
• Early colorimeters used Absorption filters (i.e. glass
filter, Gelatin filter) that transmitted a wide segment of
spectrum (50nm or more).
• Newer instrument use Interface filters that consist of
thin layer of magnesium fluoride crystals with a
semitransparent coating of silver on each side.
• Monochromator consists of:
– Entrance slit
– Absorption/ interface filter and
– Prisms or diffraction grating for wavelength selection
– Exit slit

32. Sample Holder/ Cuvette
• Cuvettes are rectangular cell , square cell or
circular one.
• Made up of optical glass for visible wavelength
(quartz or fused silica for UV).
• Common one is square, rectangular to avoid
refraction artifacts.
• Optical path (length) of cuvette is always1cm.
• Capacity may be 3ml/2ml/1ml depending upon
the thickness of the wall of the cuvette.
• For accurate and precise reading cuvette must be
transparent, clean, devoid of any scratches and
there should be no bubble adhering to the inner
surface of the filled cuvette.

33. Photosensitive detectors
Detectors are the transducers, which convert
light energy to electrical enagery. A detector
should be possess follwing characteristics:
 Should be sensitive,stable
 Should have linear response,short response.
• Different detectors used are:
 Barrier layer cells (photocells)
– simpliest
 Photoconductive cells
(photodiodes) – newest

34. Read out devices
• The detector response can be measured by any
of the following devices:
a) Galvanometer
b) Ammeter
c) Recorder
d) Digital readout.
The signal may be transmitted to computer or print
out devices.

35. Criteria for satisfactory colorimetric
estimations
Stability of color
Intensity of color The color of the solution should
be intense.
 Clarity of the solution Substance under
investigation should be completely soluble.
 Specificity Color produced should be specific for the
desired constituent.
 Validity of Beer’s law The intensity of color
should be proportional to concentration.

36. Applications Of Colorimeter
• Estimation of biochemical compounds in blood,
plasma, serum, CSF, urine, etc.:
– Glucose
– Urea
– Creatinine
– Uric Acid
– Bilirubin
– Lipids
– Total Proteins
– Enzymes [e.g. ALT, AST, ALP]

37. Spectrophotometry :
Instruments & Applications
37

38. Principle of Spectrophotometer
– Solutes in a solution show characteristic absorption
spectrum in UV or visible or infrared region of
electromagnetic radiation.
– Characteristic absorption spectrum can be
isolated by passing the electromagnetic
radiation through a prism monochromator.
– Degree of absorption of electromagnetic
radiation depends on the condensation of solute
in solution.

39. The Spectrophotometer

40. The Spectrophotometer

41. Introduction
• Spectrophotometer:
a) Single-beam
b) Double-beam
[4]41

42. Instruments
• Light source: provide a sufficient of light which is
suitable for marking a measurement.
• The light source typically yields a high output of
polychromatic light over a wide range of the
spectrum.[4]
42

43. • Monochromator : Accepts polychromatic input light from
a lamp and outputs monochromatic light.
• Monochromator consists of these parts:
I. Entrance slit
II.Collimating lens or mirror
III.Dispersion element
IV.Focusing lens or mirror
V.Exit slit [6]
43
Common monochromators:
 Filter
 Prism
 Diffraction grating
 Interference filter

44. Instruments
• Dispersion devices: A special plate with hundreds
of parallel grooved lines.
• The grooved lines act to separate the white light into
the visible light spectrum.
44
The more lines
the smaller
the wavelength
resolution.[5]

45. Instruments
• Focusing devices: Combinations of lenses, slits,
and mirrors.
• relay and focus light through the instrument.[2]
45

46. Instruments
• Cuvettes: designed to hold samples for spectroscopic
experiments. made of Plastic, glass or optical grade
quartz
• should be as clear as possible, without impurities that
might affect a spectroscopic reading.[2]
46

47. Instruments
• Detectors: Convert radiant energy (photons) into an
electrical signal.
The photocell and phototube are the simplest
photodetectors, producing current proportional to the
intensity of the light striking Them .[1],[2]
47

48. Instruments
• Display devices: The data from a detector are
displayed by a readout device, such as an analog meter,
a light beam reflected on a scale, or a digital display , or
LCD .
• The output can also be transmitted to a computer or
printer. [3]
48

49. Applications
1. Concentration measurement
– Prepare samples
– Make series of standard solutions of known concentrations
[4]
49

50. Applications
− Set spectrophotometer to the λ of maximum light
absorption
− Measure the absorption of the unknown, and from the
standard plot, read the related concentration[4]
50

51. Applications
2. Detection of Impurities
• UV absorption spectroscopy is one of the
best methods for determination of impurities in organic
molecules. [7]
51
Additional peaks can be
observed due to impurities
in the sample and it can be
compared with that of
standard raw material.

52. Applications
3. Structure elucidation of organic compounds.
• From the location of peaks and combination of peaks
UV spectroscopy elucidate structure of organic
molecules:
o the presence or absence of unsaturation,
o the presence of hetero atoms.[7]
52

53. Advantages:
Ensure higher degree of spectral purity.
Minimum stray light into the exit beam
(wave length outside the desired light is
called stray light).
Greater accuracy.
More sensitivity, specificity and precision.

54. Source of errors in
spectrophotometer
Stray light
Low resolution of light source
Lacking linearity
Variation in temp.
Low sample volume

55. Difference between colorimeter and
spectrophotometer
Traits Colorimeter Spectrophotometer
Monochromator Filter Prism
Spectral bandwidth Broad band Narrow band
Spectral purity Less More
Spectral isolation Filter has to be
changed
Desired wave length
can be adjusted
Stray light More Minimum
Accuracy Less More
Sample Larger volume needed Small volume needed
Cost Cheaper More costly
Light source Visible range of light Beyond visible range