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Tuesday, September 4, 2012

Introduction to Spectrophotometry

I am often queried as to how things work and how to explain big concepts in a broader way. Sometimes, humorously, I am asked to explain things that are in no way within my capabilities, such as the nature of the soul or concepts of time travel. Generally, those complicated, though interesting, topics are best handled by someone who thinks on those things more often than I do.


I recently appeared on WCIA's ciLiving TV program (ci stands for Central Illinois) to talk about "Science you can do with school supplies". I covered a simple chromatography experiment with paper towel strips, colored markers and water. I demonstrated the capacity of graphite in your pencil "lead" to conduct electricity and followed that with how someone won a Nobel Prize by playing with graphite and tape to make a super thin substance called graphene and finally how to make an eraser/superball/silly putty out of school glue and a few other ingredients.

The piece can be viewed here. The protocols are listed just below the video so you can recreate the experiments yourself



Recently someone asked me about spectrophotometry, and since I've used this measurement technique many, many times in lab, I figured I'd pull together some resources for those of you curious about it.

Spectrophotometry is a pretty long word, but breaking it down to its roots is helpful.

Spectr- comes from Latin and means "to watch, see, or observe".

Photo- comes from Greek and means "light". 

Meter- comes from Greek and means "to measure". 

Let's play around with the word roots a bit before we get to the entire word, spectrophotometry.

Spectrometry has been widely defined across science, but for what I'll describe refers to optical spectrometry as a technique for measuring the distribution of light across the optical spectrum, from the UV spectral region to the visible and infrared. (Do you want your own DIY spectrometer? That link goes to a cool project at kickstarter.)

And, just for fun, here is a photo, whose attribution I do not have, but will share with you regardless. Someone created a demonstration of the visible light spectra in an array of nailpolish in bottles:



I think the only way to make that image more compeling would be if the bottles became wider as the wavelengths increased (as they did with the final red bottle)

Continuing on, then,

Photometry is the science of measuring light. 

So putting those roots together, spectrophotometry "is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance.

Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications." (ChemWiki)

spectrophotometer is used to measure the amount of light that a sample absorbs. (note, that link goes to a fancy handheld one)

"The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry and molecular biology. They are widely used in many industries including semiconductors, laser and optical manufacturing, printing and forensic examination, as well in laboratories for the study of chemical substances. Ultimately, a spectrophotometer is able to determine, depending on the control or calibration, what substances are present in a target and exactly how much through calculations of observed wavelengths." (wikipedia)

As a cell biologist, I have used spectrophotometry to analyze the following:

  • How much protein a group of cells has produce or how much a culture of cells has grown (this works because more cells or proteins will make the sample more opaque, obscuring light)
  • To calculate how much DNA and RNA I have purified from a sample of cells and PCR
  • The activity of an enzyme on a given substance.
  • The toxicity of substances on cells.
  • The rate of proliferation of cells and their viability.

Generally, I'd be running experiments with multiple variables and in tiny volumes, so I'd most often use


a multiplate reader, which performs spectrophotometry on samples in a plate with multiple wells, and works on the same principles that are spelled out in the videos below.

This link, How does a spectrophotometer work?  , takes you to a very well done video that I could not embed on this site for some reason. I highly suggest clicking through and taking a look.

And for the science and some math behind how spectrophotometry works, here is the Khan Academy explanation. It should really get you up to speed on how this all works. 

Khan Academy, Spectrophotometry Introduction:

Finally, here is an online science simulation on spectrophotometry which gives you practice in the steps and the essential parts and concepts of a spectrophotometer. Very well done, even if it moves a lot slower than the actual procedure. I don't think anything replaces actual hands on work, but this is a good place to begin.

Please contact me if you have a science topic in particular that you'd like me to explain and find resources that I can write up in a blog post for the future. Soon, I'll have a video on eponymously named structures and concepts in neurobiology. 

Tue, September 4, 2012 | link 

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