It contains a brief knowledge on Introduction, Principle, Laws, Flow representation, Instrumentation, Applications and Mass spectrometer - Principle - Instrumentation

1. Colorimeter , Spectrophotometer
and Mass Spectrometer.
By: Mr. Prachand Man Singh Rajbhandari.
BSc Medical Biochemistry (Nobel College, Pokhara University, Nepal)
MSc Medical Biochemistry (JN Medical College, KLE University, Belgaum)

2. Outlines
• Introduction
• Principle
• Laws
• Flow representation
• Instrumentation
• Applications
• Mass spectrometer
- Principle
- Instrumentation

3. INTRODUCTION
COLORIMETER
• Colorimeter is instrument which is used in the
measurement of the luminious intensity of
light.
• Invented by Louis Jules Duboscq in 1870.

4. PRINCIPLE
COLORIMETER
• Involves the quantitative estimation of colors.
• The difference in color intensity results in the
difference in the absorption of light.
• The intensity of color is directly proportional
to the concentration of the compound being
measured.

5. CONTD.
• The amount of light absorbed or transmitted by
a colored solution is in accordance with two
laws:
• Beer’s law
• Lambert’s law

6. LAWS
Beer’s law :
• AαC
Lambert’s law :
• AαL

7. Io I

8. Derivation of the Formula
• Combining the two laws
AαCxL
OR A=KxCxL
• 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
2/2/2015 12:20 PM

9. 2/2/2015 12:20 PM
AT
AS
KxCTxL
KxCSxL
=
AT
AS
CT
CS
=
CT =
AT
AS
X CS
AS=KxCSxLAT=KxCTxL

10. 2/2/2015 12:20 PM
CT =
AT
AS
X CS
Concentration
of TEST
solution
Absorbance of TEST
Absorbance of STANDARD
Concn of STANDARDX
=
Concentration
of TEST
/100ml
Absorbance of TEST
Absorbance of STANDARD
Concn of Std X 100X
=
Xml

11. 2/2/2015 12:20 PM
Concentration
of TEST
/100ml
Absorbance of TEST
Absorbance of STANDARD
X
=
Xml
Concn of Std X 100
Concentration
of TEST
/100ml
O.D of ‘T’- O.D of ‘B’
O.D of ‘S’- O.D of ‘B’
X
=
Volume of ‘T’
Amount of ‘S’ X 100
Concentration
of TEST /100ml
T - B
S - B
X
=
Volume of ‘T’
Amount of ‘S’ X 100

12. Flow representation of colorimeter

13. Parts of the colorimeter
• Light source
• Slit
• Condensing lenses
• Filter
• Detector (photocell)
• Output :
INSTRUMENTATION

14. Complementary filters for coloured solutions.
The selected filters has the color to the complementary to that of
the color of unknown solution

15. • Cuvette are rectangular cell , square cell or
circular one
• Made up of optical glass for visible wavelength.
• Common one is square,rectangular
to avoid refraction artefacts.
• dimension of cuvette is 1cm.
Cuvette(sample holder)

16. cuvettes

17. • For estimation of biochemical samples , like plasma, serum,
cerebrospinal fluid (csf ) , urine.
• Ex. Determination of blood glucose, blood urea, serum
creatinine, serum proteins, serum cholesterol, serum inorganic
phosphate, urine creatinine & glucose in CSF, etc.
• They are used by the food industry and by manufacturers of
paints and textiles.
APPLICATION OF COLORIMETRIC
ASSAY

18. SPECTROPHOTOMETER

19. Introduction
• compounds absorb light radiation of a specific wavelength.
• the amount of light radiation absorbed by a sample is
measured.
• The light absorption is directly related to the concentration of
the compound in the sample.
• As Concentration increases, light Absorption increases,
linearly, As Concentration increases, light Transmission
decreases, exponentially.
19

20. Introduction
• Spectrophotometer:
a) Single-beam.
b) Double-beam
[4]
20

21. Beer-Lambert law
• Light Absorbance: (A) = log (I0 / I)= ƐLC
• Light Transmission (T) = I/I0 = 10-ƐCL
• I0: Light Intensity entering a sample
• I: Light Intensity exiting a sample
• C: The concentration of analyte in sample
• L: The length of the light path in glass sample cuvette
• Ɛ: a constant for a particular solution and wave length
21
[5]

22. Flow representation of
spectrophotometer
22

23. Parts of spectrophotometer
• Light source:.
23
INSTRUMENTATION

24. Parts of spectrophotometer
• Monochromator : Accepts polychromatic input light from
a lamp and outputs monochromatic light.
24

25. Parts of spectrophotometer
• 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.
25
The more lines
the smaller
the wavelength
resolution.

26. Parts of spectrophotometer
• Focusing devices: Combinations of lenses, slits,
and mirrors.
• relay and focus light through the instrument.
26

27. • 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.
27

28. Parts of spectrophotometer
• 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 .
28

29. Applications
1. Concentration measurement
– Prepare samples
– Make series of standard solutions of known concentrations
29

30. Applications
− Measure the absorption of the unknown, and from the
standard plot, read the related concentration
30

31. Applications
2. Chemical kinetics
• Kinetics of reaction can also be studied using
UV spectroscopy. The UV radiation is passed through
the reaction cell and the absorbance changes can be
observed.
31

32. MASS SPECTROMETER
(Principle and Instrumentation)

33. PRINCIPLES
Technique involves
• - Creating gas phase ions from the analyte atoms or
molecules
• - Separating the ions according to their mass-to-charge
ratio (m/z)
• - Measuring the abundance of the ions

34. PRINCIPLES
Technique can be used for
• - Qualitative and quantitative analysis
• - Providing information about the mass of atoms and
molecules
• - Molecular structure determination (organic & inorganic)
• - Identification and characterization of materials

35. PRINCIPLES
• - Separates gas phase ionized atoms, molecules, and fragments
of molecules
• - Separation is based on the difference in mass-to-charge ratio
(m/z)
• m = unified atomic mass units (u)
• 1 dalton (Da) = 1 u = 1.665402 x 10-27 kg
• z = charge on the ion (may be positive or negative)

36. PRINCIPLES
• - Analyte molecule can undergo electron ionization
• M + e- → M●+ + 2e-
• - M●+ is the ionized analyte molecule called molecular
ion
• - Radical cation is formed by the loss of one electron
• - Permits easy determination of molecular weight of
analyte

37. General Structure of Mass
Spectrometer

39. References
• Text book of biochemistry,
DM Vasudevan and U. Satyanaarayana
• Principle and techniques of biochemistry and
molecular biology, wilson and walker.
• Hand book of Biomedical Instrumentation. R S
Khandpur
• Clinical chemistry. Bishop.
• Wikipaedia.

40. THANK YOU..