Experiment 1: UV-Vis spectrophotometer practical lab report answer PDF

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Summary

Laboratory Experiment 1 that uses UV Visible spectrophotometer to examine the concentration of protein in samples and diluted tubes....

Description

What is the principle of spectrophotometry? Spectrophotometry is a common and very valuable biochemical technique. It can be used to confirm or establish the identity of a biomolecule, or to determine the concentration of a particular biomolecule in solution. When light passes through a solution, components of that solution will absorb some of the incident light. The light scattering will also occur. In some cases, the absorption of light is followed by the emission of light of a different (longer) wavelength. This is termed fluorescence. Biochemists are primarily concerned with absorption spectrophotometry.

Visible wavelength The visible wavelength = 400 - 800 nm, which is the visible light range. Colored compounds absorb some of the components of visible light. It is the light, which they reflect which gives them their apparent color. For example, a red-colored substance absorbs light at the blue/violet region of the spectrum and reflects red light. Thus, we can see it as a red color. If the amount of absorbing substance in solution is increased, the amount of light absorbed (and reflected) increases, hence the apparent color becomes deeper.

Ultraviolet (UV) wavelength The UV wavelength = 100 - 400 nm, which is the UV region. Colorless substances often absorb light outside the visible range, such as in the ultraviolet (UV) region. Many biological compounds have characteristic absorption spectra. The heterocyclic bases of nucleic acids absorb strongly around 260 nm, while proteins absorb at 280 nm (due to the presence of tyrosine and tryptophan residues) and in the far UV (due to peptide bonds).

What is spectrophotometer? A spectrophotometer is an instrument used in spectrophotometry, that measures light absorbed (or transmitted), providing information, which can be used to calculate the concentration of an absorbing species. By definition: A = log (Io/I)

where:

A = absorbance Io= intensity of incident light I = intensity of transmitted light

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produce intensive light with A range from Ultraviolet to visible

via a slit into spectrography

→ glass fiber • allow compact layout & high light intensities ° beam passes through cuvette containing the sample • specific wavelength is absorbed...