Medical Technology- Spectrophotometry"

Spectrophotometry.

What is a spectrophotometer? A spectrophotometer is an instrument designed to detect the amount of radiant light energy absorbed by molecules. To do this, the instrument must have five basic components: a light source; a prism or diffraction grating; an aperture or slit; a detector (a photoelectric tube); and a digital meter to display the output of the phototube. The arrangement of these parts is shown below.

When light is reflected from a diffraction grating, it is split into its component colors or wavelengths, which then diverge. Sections of the projected spectrum can be either blocked or allowed to pass through the slit so that only one wavelength will pass to the other sections of the spectrophotometer (The position of the grating is adjustable so that the region of the spectrum projected on the slit can be changed.). Light that passes through the slit travels to the phototube, where it creates an electric current proportional to the number of photons striking the phototube. If a digital meter is attached to the phototube, the electric current output can be measured and recorded. The scale is usually calibrated in two ways: percent transmittance, which runs on a scale from 0 to 100; and absorbance, or optical density units, which runs from 0 to 2.

Before the light-absorbing properties of a solution can be measured, two adjustments on the spectrophotometer are necessary. First, the diffraction grating must be adjusted so that the desired wavelength of light passes through the slit. This is usually the wavelength of light that is most absorbed by the compound under consideration. Secondly, the output of the phototube must be adjusted or calibrated to correct the drift in the electronic circuits and dirt or contaminating the material in the light path between the source and the detector.

Because all solutions of chemical compounds absorb light of specific wavelengths, spectrophotometry can be useful in identifying compounds. Furthermore, because the amount of light absorbed is proportional to the concentration of a compound, spectrophotometry is also useful in determining concentrations.

Beer's Law

Beer's Law explains the relationship between absorbance, at a given wavelength and concentration.
A=ebC

Where:

A=absorbance

e=molar exticntion coefficient

b=length of the light path

C=concentraion of the solute

Note that the relationship between absorbance and concentration is linear. As concentration increase the absorbance also increases. This relationship allows one to convert an absorbance value into a concentration.

Operating Procedure

A. Turn on the spectrophotometer (left-hand knob on the front of the instrument (A)) and allow it to warm up for at least 15 min.

B. Adjust the wavelength to the appropriate value. The knob on the right top (C)of the instrument controls the wavelength, which is indicated at the left of the digital display.

C. With the sample holder empty and the lid closed, adjust the Zero Adjust Knob (A) until the instrument reads 0% on the transmittance scale. Be sure that the display function is set to transmittance, if not push the "Mode" button until the display is set to transmittance.

D. Carefully insert the appropriate blank tube (cuvette) into the sample holder (E) and close the cover. Be sure you are using a cuvette with white markings. The cuvette's outside surface must be dry and clean, including free of fingerprints!! Use a Kimwipe to clean the cuvette before inserting. The white markings should line up with the notch on the sample holder. It is important to line up the markings. The cuvettes will be scratched otherwise.

E. Adjust the 100% Adjust Knob (B), on the right front of the instrument, until the display reads 100% on the transmittance scale.

F. Remove the blank cuvette and immediately insert the sample cuvette as described in step d above. Do not change any instrument setting! Switch the display to read absorbance by pushing the "Mode"(D) button.

G. Record the value indicated on the absorbance scale.

H. Repeat this procedure for additional cuvettes or wavelengths as required. Always adjust the blank transmittance to 100% before inserting and reading a new set of cuvettes.

Type of Photometry

Many compounds of chemical interest are colorless. In the early part of the 20th century, analytical methods were developed using color-forming reactions and measurement of color for quantitation. With the development of instrumentation and based on an understanding of optics, ultraviolet and visible spectrophotometric techniques advanced further. However, with rapid development of atomic absorption spectroscopy, plasma atomic spectroscopy, and GC and HPLC techniques, the interest in spectrophotometry waned in the late 1980s and in 1990s. HPLC methods and HPLC instruments advanced further. As a result, rapid scan techniques and microprocessor control of such instruments led to the development of sophisticated UVndashvis spectrophotometric detectors. With renewed interest in the application of UVndashvis spectrophotometric techniques, a whole new generation of spectrophotometers with microprocessor instrument control, data acquisition, data handling, and data smoothing are currently available.

Photometry is divided into three types which are absorption, emission, and luminescence spectroscopy, all yielding line spectra. Flame photometry is an atomic emission technique which may be regarded as the simplest of atomic spectroscopic methods and is very similar to the flame test which is applied for detection of alkali metals.

Thus, emission spectra are produced by thin gases in which the atoms do not experience many collisions (because of the low density). The emission lines correspond to photons of discrete energies that are emitted when excited atomic states in the gas make transitions back to lower-lying levels.

A continuum spectrum results when the gas pressures are higher. Generally, solids, liquids, or dense gases emit light at all wavelengths when heated.

An absorption spectrum occurs when light passes through a cold, dilute gas and atoms in the gas absorb at characteristic frequencies; since the re-emitted light is unlikely to be emitted in the same direction as the absorbed photon, this gives rise to dark lines (absence of light) in the spectrum.

Flame photometry involves excitation of atoms to emit energy once excited.

A flame photometer instrument is extremely simple where the sample in solution is aspirated through an aspirator or nebulizer into the flame which is usually a propane / air fuel or, even, a purified natural gas/air mixture. The sample matrix evaporates followed by atomization of the sample. Atoms present in the high temperature zone of the flame are excited to higher energy levels by absorbing energy from the flame. As excited atoms return to the ground state they emit radiation in definite wavelength depending on the energy level from which each atom drop. This gives rise to a line spectrum. However, in flame photometry a pre-selected filter (depending on the atom in question) is used and it is the intensity of the emission line that is practically measured and is related to the original concentration of the sample in solution. The detector is usually a phototube or a photomultiplier tube depending on the quality of the instrument.

fig.1 Shematic diagram of a Flame photometer

Atomic absorption methods measure the amount of energy (in the form of photons of light, and thus a change in the wavelength) absorbed by the sample. Specifically, a detector measures the wavelengths of light transmitted by the sample (the "after" wavelengths), and compares them to the wavelengths, which originally passed through the sample (the "before" wavelengths). A signal processor then integrates the changes in wavelength, which appear in the readout as peaks of energy absorption at discrete wavelengths.

fig.2 Diagram of an Atomic Absorption Photometer

<<<<BACK NEXT>>>>