Review on Lab Instrumentation, Clinical Chemistry 1

Spectrophotometer

An instrument used for measuring the transmission or reflection of light by comparing various wavelengths of the light. It also makes use of the Beer-Lambert’s principle which is “Light transmitted is inversely proportional to the concentration, while the absorbance is directly proportional to it.”

Some of the precautions in this test:

1.    Never brush cuvettes because it will lead to scratches and give inaccurate results

2.    Use rather a clean soft cloth to wipe the cells before reading it

3.    Ideal amount of the sample is ¾ full so that the light will strike the given sample and not the empty space.

4.    Let the machine be warmed up first for about 10-15 min before use, so that fluctuation is avoided.

There are four essential parts of a spectrophotometer

            1. Light source

            2.  Monochromator or filter

            3.  Sample cell with holder

            4.  Detector

Turbidimetry

-      The determination of the quantity of insoluble suspended matter in a liquid by measuring the loss of intensity of light in the direction of propagation of the incident beam, with reference to a standard solution

Gravimetry

-      Gravimetric methods of analysis are used where weights of reactants and products of chemical reactions are reproducible, stable and reflect the presence of constituents which are important in the establishment of identity. 

-      Two important methods deal with the trapping and weighing of products in the solid and gaseous phases.

Osmometry

-      Measurement of the amount of osmotic pressure that a particular solution  exerts

-      Can be vapor pressure osmometry, freezing point depression osmometry or membrane osmometry

Emission Flame Photometry

A type of flame photometry in which molecules in a flame are volatilised to generate free atoms that are excited to higher energy levels. When these atoms return to the ground state, they produce a characteristic emission spectrum. These typically use a low-temperature, air-gas, laminar flame burner which is inherently subject to drift and therefore lithium (Li), which is not a normal serum constituent, is excited as a known added constituent to "standardize" the results despite the drifting of the flame output. This approach does not permit addition of Ca as a third determined constituent as the emission of the Ca at serum levels in the low-temperature flame is below measurable intensity; furthermore the Li would interfere optically with the Ca determination.           

Atomic Absorption Spectrophotometry

             Atomic Absorption Spectrophotometry is the measurement of the concentration of an element in a given sample. This technique can be done in three ways: Flame type, Electrothermal type, and Color Vapor type. In the flame type, gas and combustible substance are used to ignite the mixture of gases.

            The second type which is electrothermal makes use of electric current and slit width. On the other hand is the cold vapor method wherein a reducing agent is added and vaporized.

Volumetry

            Volumetry is t he measurement of volumes of liquids, solids, and gas.

-the quantitative analysis of an unknown chemical solution by determining the amount of reagent of known concentration necessary to effect a reaction in a known volume of the solution

Chromatography

            It is a broad range of physical methods used to separate and or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed.

1. Thin Layer Chromatography

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the R_f of a compound is compared with the R_f of a known compound (preferably both run on the same TLC plate).

A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action.

B.   Gas chromatography 

    specifically gas-liquid chromatography - involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

C.   ‘High Performance Chromatography 

       High-performance liquid chromatography   (HPLC) is a form of liquid chromatography  to  separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting a plug of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase.

Electrophoresis

Electrophoresis is a separations technique that is based on the mobility of ions in an electric field. Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode. For safety reasons one electrode is usually at ground and the other is biased positively or negatively. Ions have different migration rates depending on their total charge, size, and shape, and can therefore be separated.

Instrumentation

An electrode apparatus consists of a high-voltage supply, electrodes, buffer, and a support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or a capillary tube. Open capillary tubes are used for many types of samples and the other supports are usually used for biological samples such as protein mixtures or DNA fragments. After a separation is completed the support is stained to visualize the separated components. 

Resolution can be greatly improved using isoelectric focusing. In this technique the support gel maintains a pH gradient. As a protein migrates down the gel, it reaches a pH that is equal to its isoelectric point. At this pH the protein is neutral and no longer migrates, i.e., it is focused into a sharp band on the gel.