Concepts in Instrumentation

Spectrophotometry

Method of determining the concentration of substance in solution by measuring the amount of light absorbed by that solution after appropriate treatment.

Wavelength

It is the distance between two peaks

Beer-Lambert Law (Beer’s Law)

It states that the concentration of a substance is DIRECTLY PROPORTIONAL to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light

Incandescent tungsten / Tungsten-Iodide lamp

Most commonly used light source for work in the visible and near-infrared regions

Deuterium discharge lamp / Mercury arc lamp

Most commonly used light source for UV work

Diffraction gratings

Most commonly used monochromators

Square

Type of sample cell that has an advantage over round cuvettes in that there is less error from the lens effect, orientation in the spectrophotometer, and refraction

Glass

Type of sample cell used for applications in the visible range

Quartz

Type of sample cell used for applications requiring UV radiation

Barrier (Layer cell or photo cell)

It is the least expensive photodetector

Photomultiplier tube

Photodetector used in instruments designed to be extremely sensitive to very low light levels and light flashes of very short duration

Reflectometry

It measures the quantity of light reflected by a liquid sample that has been dispensed onto a grainy or fibrous solid support

• Urine dipstick analysis

• Dry slide chemical analysis

Reflectometry is applied in:

Measurement of concentration is done by detecting the absorption of electromagnetic by atoms rather than molecules.

What is the principle of AAS?

Monochromator

It is used to isolate the desired wavelength

• also protects photodetector from excessive light

Measurement of unexcited trace metals

AAS is used for:

Flame

In AAS, it serves as the sample cell instead of a cuvette

Measurement of light emitted by excited atoms

What is the principle of Flame Photometry?

Measurement of the concentration of solutions that contain fluorescing molecules

What is the principle of Fluorometry

Xenon lamp

It is the most common light source in Fluorometry

Drugs

Fluorometry is used to measure small particles such as:

• Detection of bacterial growth and bacterial culture

• Antibiotic sensitivity

• Coagulation studies

• Protein concentration in CSF and urine

Applications of Turbidimetry:

Ab-Ag complexes formed in enzyme immunoassays

Nephelometers measure particles which are too large for spectrophotometry. These particles include:

Involves measurement of the current or voltage generated by activity of specific ions

What is the principle of Electrochemistry

Measurement of potential (voltage) between two electrodes in a solution to measure analyte concentration

What is the principle of Potentiometry?

Measurement of the current flow produced by an oxidation-reduction reaction

What is the principle of Amperometry?

Amperometry

Which technique measures pO2 (Clarke Electrode)?

Electrochemical titration in which the titrant is electrochemically generated

What is the principle of Coulometry?

Potential is applied to an electrochemical cell and the resulting current is measured

What is the principle of Voltammetry?

Anoding stripping voltammetry

An electrochemical technique that is used for determining lead and iron

Electroendosmosis

It is the movement of buffer ions and solvent relative to the fixed support

Densitometry

Most common and reliable way for quantitation of separated protein fractions

Separation of complex mixtures on the basis of different physical attractions between the individual compounds and the stationary phase of the system

What is the principle of Chromatography?

High-Performance Liquid Chromatography (HPLC)

Type of chromatography which uses pressure for faster separations

Gas Chromatography (GC)

Type of chromatography which separates mixtures of compounds that are volatile or can be made volatile

Column

It holds the stationary phase in chromatography

Mass Spectrometry

It is the gold standard for drug testing when coupled with GC

MALDI-TOF MS

A two-step procedure used for the analysis of biomolecules such as peptides and proteins