Spectrophotometer Principle

Hello World, I’m Saiful Islam and I’m a paramedic and today I’m talking to you how to use a spectrophotometer.

The Spectrophotometer is a ubiquitously used instrument in biologically, clinically, chemically, environmentally research. A spectrophotometer is a device used to measure light at a specific wavelength. It consists of two parts: a spectrometer and a photometer. The spectrometer provides light at a specific wavelength. The photometer measures how intense the light is. By calculating the amount of light that a solution is able to absorb and applying Beer’s Law, the spectrophotometer can determine the concentration of a colored solution.

parts of spectrophotometer
parts of spectrophotometer

Parts of the Spectrophotometer:

  • The sample compartment the lid opens and closes to picture sample inside
  • The wavelength control knob to change the wavelength this is called the zero control on the left hand side
  • The transmittance absorbance control on the right hand side
  • Right on top is the mode select button and when you push the mode select button you’ll notice that the LED will change positions.

How to sample ready to Use

The readout to begin using the spectrophotometer and your samples is ready to use.

First

You should prepare a blank solution and the blank solution has everything in it except for the molecule or compound. You wish to measure then you should also have a tube which contains a sample which you want to measure when taking readings it’s best to go ahead and use the transmittance as the way to set up the machine.

 And also to take your readings the transmittance scale is linear. It’s easy to set the zero and the 100% .

When you’re ready to calibrate the machine be sure to push the mode button several times until you have the transmittance value highlighted. Before you begin in order to start taking a reading you have to first set the Machine up. Do first thing you must do is using the wavelength control

knob and adjust the wavelength until the wavelength that you want to measure at is reached and

let’s go ahead and try taking a measurement at 420 nano-meters.

Second

Thing you need to do is using the zero control. Turn the zero control until you get zero point zero showing up on the display.

Three

Thing you should do is now open up the sample compartment and put the blank solution in we should always wipe off the bottom of your cuvettes. Before you insert them in the sheen to make sure that nothing interferes with the reading. Also notice that there’s a little line shown in the front of the cuvette line.

That up with the dash on the front of the sample compartment and then insert it all the way inside and then close the sample compartment door.

This time we’re going to use the absorbance transmittance control and turn it appropriately one way or the other until you get 100 on the dial. Once you’ve set the Machine at a 100%. Then you can go ahead and take your cuvette out and then put your sample in again making sure that you line up the mark on the cuvette with the mark on the sample compartment.

Result Analysis of Spectrophotometer

Here you can see that the transmittance value is 93.0. You can also send an absorbance reading by simply pushing the mode button to go down to where it says absorbance. when the absorbance LED is lit you can see that at the same wavelength. The absorbance is 0.032 you want to do that the Machine has to be set up specifically in order to use that option and this one is not. Do not write this concentration down and you need to know the correct factor in order to set the concentration. So ignore those just use the transmittance and absorbance on the machine.

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After test occur in Spectrophotometer

When you remove your sample the machine should return back to zero transmittance. You can go ahead and continue to put new samples into the sample compartment and continue to take readings at the same wavelength that you just set up the Machine. If you have ten test tubes you can now put all ten of them in one at a time and note the readings.

But once you decide that you want to record a wavelength at a different wavelength. We want to switch from 420 to 480 nano-meters. Now we have to go through the entire calibration method one more time.

Then we’ll have to begin by first changing the wavelength to the desired 480. We’ll have to zero the Machine using the zero-control knob on the left. Then we’ll have to put the blank cuvette back in and then we’ll have to use the knob on the right.

Conclusion

The absorbance transmittance control in order to set the machine back at 100% transmittance and then after that’s complete. Then you can go ahead and put your sample in to read the absorbance or transmittance at the new wavelength.

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