Short Report Haemotology Rmit

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Use of spectrophotometric techniques to determine the optical density or absorbance for the sample with different dilution factors

Haematology 1
Genia Burchall
26th July, 2012
10th August, 2012
ABSTRACT: This experiment aimed at determining the optical density in terms of absorbance of a solution using the different dilution factors to obtain a standard curve where interpretation of Beer’s Law is important to get the results. In order to frame this aim, the method of dilution factor was used so that absorbance, using the spectrophotometric techniques, could be measured at different concentrations of the solution. Using the micropipette, three different dilutions were prepared in the labeled tubes with coloured liquid and water. Then the results were interpreted in form of graph and table. Standard curve was drawn that was expected to obey the Beer’s Law. This law states that the concentration and absorbance are directly proportional to each other, which means with the increase in concentration the absorbance also increases and vice-versa. The equation used for Beer’s law is A = ᵋ* l * c, where A is absorbance, l is the path length and c is the concentration. Finally, the conclusion was made based upon the results. At this instant, the results met did the aim as the straight line was obtained on the graph plotted for absorbance and the dilution values. INTRODUCTION: The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of a solution. The general Beer-Lambert law is usually written as: A = ᵋ * l * c

where A is the measured absorbance, ᵋ is a wavelength-dependent molar absorptivity coefficient, b is the path length, and c is the analyte concentration. This law is usually helpful when determining an unknown concentration of an analyte. It can be determined by measuring the amount of light that a sample absorbs and applying Beer's law. If the absorptivity coefficient is not known, the unknown concentration can be determined using a standard curve of absorbance versus concentration derived from standards. The linearity of the Beer-Lambert law is normally limited or affected by chemical and instrumental factors. Causes of nonlinearity include: * Deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity * Scattering of light due to particulates in the sample

* Fluorescence or phosphorescence of the sample
* changes in refractive index at high analyte concentration * shifts in chemical equilibria as a function of concentration * non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band * stray light [1]

The application of Beer’s Law is Spectrophotometry. Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.[2] These types of measurements are performed by a spectrophotometer. A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm - 2500nm using different controls and calibrations. Principles of a spectrophotometer are as follows:

* The light source is imaged upon the sample
* A fraction of the light is transmitted or reflected from the sample * The light from the sample is imaged upon the entrance slit of the monochromator * The monochromator separates the wavelengths of light and focuses each of them onto the photodetector sequentially. [2] Many older spectrophotometers must be calibrated by a...
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