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Introduction for Biochemistry Lab -Spectrophotometry

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Submitted By emra
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Every chemical compound absorbs, transmit or reflect light over a certain range of wavelength. In this experiment one of the main purpose is to introduce students to the quantitative and qualitative study of spectrophotometry. Spectrometry is the measurement of light that is absorbed or transmitted by the sample solution (Vo, n.d). It is one of the most commonly used methods in various fields to study the quantitative analysis of light that passes through a solution. Meanwhile, the use of spectrophotometer to determine the extent of absorption of wavelengths of visible of light is known as colorimetry.
The intensity of light that passes through the sample as well as the concentration of the substance is measured by a spectrophotometer. The instrument consists of some basic components: Radiation light source (tungsten for visible region and deuterium lamp for ultraviolet region), wavelength selector (to isolate a desired wavelength form the source), a cuvette or container to hold the sample solution and a detector (photometer) which delivers the signal in a display device. The voltage output is determined by the difference in the amount of light absorbed by the sample. The fraction of light that passes through the solution is called transmittance, T and the proportion of light absorbed is the absorbance, Abs.

Thus, the value of transmittance can be calculated by using the following formula:
Transmittance, I = It/ I0 where, It = intensity of the incident radiation entering the medium, I0 = intensity of the transmitted radiation leaving the medium

Meanwhile transmittance, T, and absorbance, Abs, is related by the formula of absorbance:

Absorbance (Abs) = -log (T) = -log (It/I0)

The data is written in transmittance, % transmittance or absorbance. Absorbance has no unit as it is a ratio of light. However, sometimes, it is also expressed absorbance units.
On the other hand, to determining the unknown concentration of the absorbing solution, the beer lambert law is used as it is the linear relationship between transmittance and absorbance. This law states that absorbance is proportional to the amount of the concentration of analyte, the path length and the molar coefficient. Therefore,
Absorbance, A = εcl
ε= molar extinction coefficient for the absorbing material at wavelength in units of 1/(mol x cm) c= concentration of the absorbing solution (molar) l = light path in the absorbing material (l=1 cm for our purposes)

In the UV/Visible region, compounds absorb light in wavelength of (200nm-700nm). The functional which is responsible to light absorption is known as chromophore. The result obtained is plotted against wavelength which shows the absorption bands corresponding to structural groups within the molecules. The absorption spectra are caused by the transition of electrons in its excited and ground states. Increasing solvent polarity from the effect of increased solvation of lone pair and decreased conjugation results in shorter wavelength which gives rise to the blue shift or hypochromic shift (Clark, 2007). Meanwhile, the inverse, lower polarity solvent and increased conjugation gives rise to red shift that result in increased wavelength. These shifts in wavelengths and intensities are the results of the molecules structure and the interaction between solute and solvents molecules (Robinson, Frame & Frame II).

In colorimetry, calibration curve where absorbance (A) of such solutions can be plotted against the concentration (c) of the test substance producing the color. Some important highlights in this method include that it is a destructive technique in which once the sample is reacted, it cannot be recovered, the chromophore reflects the complementary color that it absorbs and the calibration curves may vary with the different batches of reagents and standards used. Colorimetry is usually used to determine the concentrations of chemicals substance which can give colours either directly or indirectly or after the addition of other chemicals. In addition, with its efficiency in producing large amount of absorbance with relatively small amount of material, it is highly used in biochemistry assay.


Clark, J., (2007). UV-Vis Absorption Spectroscopy: Theorical principals. Retrieved at on 9th May 2014.

Robinson, J, W., Frame, E.M.s., and G.M., (2004). UV spectra and the structure of organic molecules. Undergraduate Instrumental Analysis, 6th Ed, p350.

Vo, K., (n,d). Spectrophotometry. Retrieved at on 9th May 2014.

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