Brief description of instruments used in "Biochemistry Practical Lab"

1. DEMONSTRATION OF DIFFERENT INSTRUMENTS
USED IN BIOCHEMISTRY LABORATORY
Amit Jha
Lecturer
UCMS, Bhairahawa

2. Pipettes
Blow out Non blow out Volumetric
Measuring pipette Transfer pipette
Ostwald’s
Micropipette
1. Fixed
2. Variable
Pipettes are suction devices that are used to either
suck liquids into or expel liquids out of pipettes

3. Serological (Blow out) Pipette
• Calibrated all the way down to tip.
• Last drop of liquid needs to be blown out of the
tip to deliver full volume of pipette

6. Mohr (Non blow out) pipette
• Calibration marks don’t extend upto tip
but at a point above the tip.

7. Volumetric pipette
• Transfers a specific
volume of a given liquid.
• Shaped like a rolling pin,
with two thinner ends & a
thicker bulge in the middle.
• Have single calibration
mark.

8. Ostwald Follin pipette
• Similar to volumetric pipette, but bulb closer to
delivery tip.
• Used for accurate measurement of viscous fluids
[blood, serum]

9. Water bath [Constant temp. bath]
• Used to provide specific temp. to carry out different
chemical rxns.
Serum enzymes estimation 37◦C
Enzymatic methods 37◦C
Serological tests 56◦C
Saponification 60◦C-70◦C
• Components
• Nickel plated tank
• Strip heater: provide heat [below tank]
• Control knob: regulate temp.

10. Incubator
• Uses:
– Provide constant temp. during:
• Enzyme estimation
• Glucose, urea, creatinine, etc estimation
• Components:
– Doubled walled cabinet of mild steel
– Heating element
– Ventilation system [passes expanded inner air]
– Control knob [regulate temp.]

12. Electronic balance
• Principle:
– When a mass is kept on pan, an electromagnetic
force is generated by magnet & coil.
– This electromagnetic force is converted into an
electric signal & displayed on a digital display.

14. Centrifuge
• Centrifugation: process of using centrifugal force to
separate lighter particle suspended in a solution.
• Principle:
– When a particle suspended in a liquid is subjected to
centrifugal force (rotating the medium at high speed),
particles gets sedimented at bottom of tube.
– F = ω2.r
F: Centrifugal force
ω: Angular velocity
r: Radius

15. • Relative centrifugal force
– Centrifugal force expressed in terms of ‘g’
RCF = (1.19 x 10-5) . (rpm)2 . r
– RCF in terms of no. of ‘g’ is determined using a
nomogram.
– Eg: r = 7cm & rpm = 20,000
• RCF = 32000 x g

16. • Application of centrifuge
1. Separation of plasma or serum from blood.
2. Concentrate particles in urine sample, helpful for
microscopical examination
3. Ultracentrifugation
• To separate lipoproteins in serum sample
• To separate cell organelles

17. Colorimeter
• Principle:
– Beer's law: states that concentration of a subst. is
directly proportional to amount of light absorbed
• A ∞ C
– Lambert’s law: states that absorbnce is directly
proportional to the thickness of Pathlight.
• A ∞ T
– Beer-Lambert Law
• A ∞ C T
• A = E . C . T
A: Absorbance
C: Concentration
T: Thickness of Pathlight
E: Molar extinction coefficient

18. Parts

19. • Standard solution:
– As: Absorbance
– Cs: concentration
As = E . Cs . T ----------- [i]
• Unknown test solution
– At: Absorbance of test solution
– Ct: Concentration of a subst. in test solution [unknown]
At = E . Ct . T ------------- [ii]
As = E . Cs . T
At = E . Ct . T
• Ct = [At/As] . Cs
• Concentration of analyte in test solution is given by: ?????

20. • Application of Colorimeter
– Used for routine biochemical analysis of different
analytes in serum sample
• [Glucose, Total Protein, Albumin, Uric Acid, Total
Calcium, Phosphorus, Total Cholesterol, Triglyceride,
Urea, Creatinine, Bilirubin, etc]

21. Flame emission photometer
• Fast, simple & sensitive analytical method used to
determine inorganic metal ions [Na, K, Li, Ca etc] in
solution.
• Using hot flame metal is energized to higher energy state,
as isn’t stable, when gets back to normal low energy state
produces specific radiation.

22. Flame emission photometer
• Under const. & controlled condition, light intensity of
characteristics wavelength produced is directly
proportional to no. of atoms (concentration of metal of
interest in solution).
Na Yellow
K Violet
Li Red
Mg Blue

23. Flame emission photometer
• Instrumentation
Nebulizer For spraying of solution
Flame Generates heat for excitation of metal ions
Monochromator select light of single wavelength
Photo detector Quantifies emitted light by converting into electrical
impulse.
• Application
– Estimation of Na, K, Ca, Li, in various body fluids.
– Used in agricultural science to know about fertilizer
requirement of soil.

24. pH meter
• Principle:
– Based on measurement of potential difference
generated between reference (known potential) &
unknown electrode (glass electrode) (sensitive to H+).
– AgCl electrode: most commonly used.
– Thin glass membrane separates two electrodes.
– When electrode dipped in a solution, due to H+
movement, an electric potential is developed.
– Electric potential developed is amplified & converted into
direct pH reading on display unit.

25. • Electrophoresis
– Method used for separation of charged particle
– Principle
• When charged particle is run in an electric field,
charged particle moves towards oppositely charged
electrode.
• Rate of Migration ∞ e/m ratio
» e-: no. of charge
» m: mass of charged particle
– Due to differ in e/m ratio, rate of migration is
different for different charged particle.

26. • Procedure of separation of serum proteins:
– Sample [serum] is apllied to a strip of filter paper or
cellulose acetate.
– Both edges of strip is dipped in alkaline buffer solution.
– Being amphoteric, protein carry –ve charge in alkaline
medium.
– When current passed, proteins will move towards anode.
– Rate of migration of diff. proteins depends on:
• No. of charge on protein
• Mol. wt. of protein molecule.

27. • Procedure of separation of serum proteins:
– Serum proteins are separated into different bands:
» Albumins
» α1 globulins
» α2 globulins
» β globulins
» γ globulins
• Density of each band ∞ its concentration in serum

28. Electrophoresis apparatus 28

30. Multiple Myeloma

32. • Application of electrophoresis
– Used to separate charged particles like proteins, nucleic
acids, lipo-proteins.
– Used for diagnosis of:
» Multiple myeloma
» Nephrotic syndrome
» Cirrhosis of liver etc.

33. Chromatography
• Tech. used for separation of mixture of group of similar
substances by their continuous distribution & redistribution
into two phases. [stationary & mobile phase]
• Mobile phase: mixture of substance dissolved in liquid or
gas.
• Stationary Phase: porous solid matrix through which
mobile phase pass. [Solid / liquid / liquid coated on Solid
Surface]

34. Chromatography
• When mobile phase pass through stationary phase,
solutes gets distributed between two phase.
• High affinity to mobile phase: move faster & separated
from solutes having high affinity to stationary phase
• Distribution depends on physical property [Mass, Shape,
Size, Charge, Solubility, Adsorption property]

35. • Partition coefficient
conc. Of substance in Mobile phase
conc. Of substance in Stationary phase
Kd =
• Kd = 1: subst. gets distributed equally between two
phase
• Kd > 1: subst. have more affinity to mobile phase
• Kd <1 : subst. have more affinity to stationary phase

36. • Relative front
Dist. Covered by solvent from origin
Dist. Covered by solute from origin
Rf =

37. • Applications
– Used for identification & quantitative estimation of:
• Amino acids
• Proteins
• FA etc.