UV/VIS Spectroscopy

Ultraviolet-Visible (UV-VIS) Spectroscopy is an analytical technique that can quantitate the amount of analyte based on the amount of light the analyte absorbs.  If the analyte in solution is colored, analysis in the visible spectrum is typically used.  This is because the physics involved with color.  Visible light is called visible light because when different wavelengths of light are separated (by a prism for example), the light can visualized as a color.  Light travels as photons and photons move in waves. The wavelengths of light that can be visualized by humans are tabulated below.

Color of Light Wavelength Complimentary Color
Violet 390-450 nm Yellow
Blue 450-495 nm Orange
Green 495-570 nm Red
Yellow 570-590 nm Violet
Orange 590-620 nm Blue
Red 620-750 nm Green

 

Complementary colors are also important when discussing the perceived color of objects. If the object reflects all colors of light but absorbs yellow, the eye will perceive the object as violet which is the complementary color yellow. The color of an object is determined by what wavelengths of light the object absorbs and reflections.

Now back to UV-VIS spectroscopy. If a solution of red liquid is placed in a cuvette and a visible light source shines on that red liquid, the detector on the other side the cuvette will show that the red solution reflects all color of light, but it absorbs green light, the complementary color to red.  Therefore, the absorbance peak of a red solution generated by the spectrophotometer will be in the green wavelength range.

To humans, light in the UV range is not perceived as color.  Other animals can perceive UV light as color, but humans cannot.  To us humans, solutions that absorb UV light appear to be clear. However, many different molecules can absorb light in the UV range.  For example, DNA is known to absorb light at 260nm and most proteins can absorb light at 280nm. UV/VIS spectroscopy is the most common type of detector for HPLC.  The spectrum and retention time can provide useful information regarding identity and purity of compounds.

 

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