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

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..)

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

Contd..

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)

Cuvettes:

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

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

Schematic diagram:

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

Commercial instruments: Double beam Digital reading / recording : automatic sampling Deuterium source: 190 to 700 nm 0.5 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

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

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..

Block diagram:

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

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

Auto analyzer:

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