1. Electrophoresis:
Movement of a solid phase with respect to a liquid (buffer)
Sample is applied to the medium and under the effect of electric field, group of particles (similar in charge, size & shape) migrate
Used to measure:
Quantity of protein in plasma etc.
Separate enzymes into isoenzymes
Identifies antibodies

2. Factors affecting electrophoresis:
Magnitude of charge (distance per unit field strength)
Ionic strength of buffer
Temperature
Time
Type of support media (cellulose acetate, starch gel, sucrose etc..)

3. Cellulose acetate electrophoresis:
A strip is saturated with the buffer solution and placed in membrane holder (bridge)
Sample is placed on the strip
250 v is applied and 4 – 6 mA is obtained
After 15 – 20 minutes, supply is removed
The membrane is placed in the holder of densitometer
Low voltage is output is amplified and recorded

4. Contd..

5. Colorimeters:
Simplest form of measurement using human eye (400 – 700 nm)
Comparison of color of unknown with standard
Series of standards should be developed
Photoelectric methods have largely replaced and it is precise (photometric / spectro – photometric method)

6. Cuvettes:

7. Contd.. Single beam Double beam configurations
Sample is normally a liquid
Simple in construction & operation
Range of filters is required to cover different wavelength regions
Types:
Single beam
Double beam configurations

8. Multi channel colorimeter (photometer):
Increases number of chemical analysis
Increases the capacity of laboratory
Increases the measuring capacity
Introducing a batch of samples into a single light path
Simultaneous measurement
25 channels : 24 – 3 key eight matrix
25th channel : reference beam

9. Schematic diagram:

10. Spectrometers: Isolates monochromatic radiation
Source light is passed as parallel beam in to prism and diffracted & dispersed at different angles
Light reaching spectrometer is generally much smaller (small spectral bandwidth)
Sensitive ammeter is used to measure
To overcome:
Accurate potentiometric bridge is used
Amplified electronically and displayed in digital form

11. Commercial instruments:
Double beam
Digital reading / recording : automatic sampling
Deuterium source: 190 to 700 nm nm
4 wavelength scanning speeds and seven chart speeds
6 V tungsten lamp is source
Heated : ionization may occur inside the anode , energy decreases
Ultra violet with deuterium lamp
Visible range with tungsten lamp

12. Commercial instruments:
Slit : 0.05 to 2.0 mm
Slit width is approximately 1/10th of bandwidth of the sample

13. Microprocessor based spectrophotometers:
Control functions: wavelength scanning, automatic light source selection, control of slit width, density sensitivity etc..
Signal processing functions : signal baseline correction, calculation of concentration, absorbance etc..
Communication functions: keyboard entry, data presentation, with external systems etc..

14. Block diagram:

15. Contd.. Signal is amplified (preamplifier),
converted into digital form (ADC),
differentiated into sample signal S, reference signal R and zero signal (Z),
stored in memory
Transmittance, T = (S-Z) / (R-Z)
Absorbance = - log T

16. Auto analyzer:
Sequentially measures blood chemistry, displays on a graphic readout
Sampler
Proportioning pump (heart of auto analyzer) & manifold
Dialyzer
Heating bath (37℃)
Colorimeter
recorder

17. Auto analyzer:

18. Proportioning pump: Peristaltic pump
Separates from cleaning fluid and other samples
Sterilization is needed
Frequent calibration
Mechanical & electrical maintenance