UV-visible Spectroscopy

By Maria, Pricilla & Samira

The relevant theory behind UV-visible spectroscopy…  A UV-Visible Spectrophotometer works because many substances absorb different light of different wavelengths. This information is used to identify the substance.

Different wavelengths of light promote electrons from a substance to move from low energy levels to higher energy levels. When this happens they absorb light but to our human eye, we see the complementary colours. Different substances have different energy levels and require different wavelengths of light from the electromagnetic spectrum to promote electron movement. The spectrum assists in indentifying a substance.

When a substance absorbs visible light, the observed colour is the complementary colour.

For example: Chlorophyll appears green because it absorbs all visible light besides green.

Wavelength (nm)

Colour Absorbed Colour Observed

380-420 Violet Green-Yellow

420-440 Violet-Blue Yellow

440-470 Blue Orange

470-500 Blue-Green Red

500-520 Green Purple

520-550 Yellow-Green Violet

550-580 Yellow Violet-Blue

580-620 Orange Blue

620-680 Red Blue-Green

680-780 Purple Green

Complementary Colours

Interpreting the UV-visible

spectraThe absorbance of the visible or ultraviolet light is presented in a calibration graph, this is measured by many standard solutions. This absorbance of light measurement of an unknown solution indicates us to determine it’s concentration by reading off the graph.

Examples of the output from the UV-visible spectroscopy 

• Absorbance spectra.• Measures the absorbance of the substance at a

range of wavelengths. As shown in diagram below 445.0 is highest and that when its used to measure the absorbance. It gives you qualitative data to compare this spectra and other kinds of spectra.

This is used for qualitative analysis by comparing this spectra to other known spectra. It is used in quantatitive analysis to find the frequency of maximum absorbance.

Calibration curves Readouts of absorbance's for the standards are used to

produce a calibration curve. It gives you quantitative data.  

Realibility of Data

• This technique is easy and quick to use.

• Generally, this technique is reliable. However through research, it seems that the wavelength seems to have some deviation and can’t be 100% accurate. Therefore affecting the final result.

Uses of UV-Visible Spectroscopy

• Determining the amount of solutions of transition metal ions & organic compounds.

• Identifying an unknown compound.• Assisting in finding out the concentration of a

diluted solution. Applications of UV-Vis…  

• Clinical Analysis, measuring the concentrations of specific substances in body fluids such as urine or blood.

• Determining the amount of coloured dye in plastics• Identifying the presence of metal ions.• Determining the levels of nutrients, additives and

contaminants in water and foods.• Quantitative analysis of DNA and proteins in the field

of molecular biology. 

Safety Precautions• When working with organic solvents you need to be careful when mixing

chemicals together as exposure to organic solvents may effect your:• Respiratory area •        Vapours may irritate the lining of the respiratory area and may affect the

nose, throat and lungs. Reactions similar to asthma have been reported when using some organic solvents. So when using solvents face masks have to be worn at times so you don’t inhale the chemicals.

• Skin•        Skin contact with solvents often cause dryness, cracking, blisters, redness

usually leading to a allergic like reaction. Lab coats and gloves help prevent chemicals from contacting the skin.

• Eyes•        Safety glasses have to be worn as direct contact with vapour or liquid may

cause irritation.• Some solvents that are highly toxic is very dangerous to the human body when

exposed to it. It also affects your Liver, Cardiovascular system, Kidneys and Nervous system. So you need to take into consideration the appropriate clothing or procedures when experimenting with solvents.  

• First aid and safety procedures• If accidental contact with skin or eyes, it should be immediately flushed with

water.• When dealing with spills, you must clear the area and clear the spill by adsorbent

material. Make sure protective equipment is used.   

Exam Question Example…

We would use this wavelength as this has maximum difference.

Answer?

• Statement I and II were correct. 

• Statement III was incorrect because energy is absorbed during the transition of electrons from lower energy levels to higher energy levels. 

• Statement IV was also incorrect because the spectrum shows two absorbance peaks, at wavelengths of 250 nm and 400 nm. Both of these peaks can be used if one has had an interference.

THE END

Hope we didn’t bore you too much