Electromagnetic radiation travels through space in the form of a wave, with the distance between two crests of a wave termed a wavelength. At one end of the spectrum are gamma rays with short wavelengths and high energy. At the other end of the spectrum are low energy radio waves with long wavelengths.

Energy detected by the human eye as light occupies a small portion of this spectrum, from approximately 380 – 750 nm.

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Substances possess color because of their ability to absorb and transmit certain wavelengths of visible light. Chlorophyll, present in the leaves of plants, absorbs a high percentage of the wavelengths of red and blue light. Green light is not absorbed, and instead is transmitted from the surface of the leaf to our eyes. For this reason plants appear to be green. This ability of molecules to absorb and transmit light energy is the basis for one of the most widely used procedures for determining the concentration of substances in a solution. It is called spectrophotometry, and the instrument used is the spectrophotometer.

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Above the 700 visible light are those of TV, radio, microwave.

Below the 380 visible light are those of the X-rays, Gamma rays, UV rays.

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digital display sample holder mode button wavelength control button

0% Transmittance knob 100% Transmittance knob5

When light of a specific wavelength passes through a solution, some of the light will be absorbed and the rest will be transmitted. A spectrophotometer can measure the absorbance or % transmittance of a solution.

BLANK tube: water + indicator SAMPLE tube: water + indicator + proteincontains all reactants except the Contains all components including the absorbing molecule. It is used to absorbing molecule. Indicator changes calibrate the machine. color with the presence of the protein.

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Set the wavelength to 540nm

With the sample holder empty and closed lid, using the 0%T knob and calibrate to 0%T.

Place blank tube in sample holder and close lid

Set to 100% T with 100%T knob

*Whenever wavelength is changed, you must re-calibrate the machine. 7

http://www.youtube.com/watch?feature=player_embedded&v=MkUGz6kEQMg

Follow these instructions to learn how to use the Spectronic 200, the new machine used to measure transmittance and absorbance, before the lab.

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Making the serial dilution

Tube 1 has 1 ml of a known protein concentration (10.0 mg/ml). Tubes 2-5 have 1 ml of distilled water (fig. a).

1 ml of the protein solution (10 mg/ml) was added to tube 2 and mixed.

1 ml from tube 2 was removed and placed into tube 3 and mixed.

1 ml from tube 3 was removed and placed into tube 4 and mixed.

1 ml from tube 4 was removed and placed into tube 5 and mixed.

1 ml from tube 5 was removed and discarded.

Each tube then received 5 ml of the indicator solution (fig. b).

1 32 4 5

Fig. a

Fig. b

1 2 3 4 5

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The absorbance (A) of each sample tube was measured by placing the tube in the sample holder and reading the absorbance.

Absorbance readings:

A= 0 0.490 0.250 0.120 0.060 0.030

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A new tube is prepared using a sample of protein with unknown concentration. It is prepared exactly as tube 1 was with the standard protein.

Its absorbance is then measured using the spectrophotometer.A= 0.350

To determine the concentration of this sample you must first draw the standard curve using the absorbencies and concentrations of the first 5 tubes you made, and using that graph, find the concentration of the unknown.

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The concentration of an unknown solution can be determined by comparing the absorbance reading of the unknown to your standard curve. Draw a straight line from the recorded A, on the Y-axis, to the curve. Drop your line from the curve to protein concentration on the X-axis.

1 3 5 6.5 8 10

Protein concentration mg/ml

Abs

orba

nce

0.37

In this case the A of your sample measures 0.37 then your protein concentration is approximately 6.5 mg/ml.

Best fit line

End

0.5

0.3

0.2

0.1

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