How is a spectrophotometer calibrated?

Answer 1

Spectrophotometers

are useful devices that allow you to mainly find out the concentration of molecules within a reaction mixture. This mixture could be an enzyme and its substrate, or much simpler chemicals.

Most spectrophotometers

work using light from the U.V-visible

part of the electromagnetic spectrum (150-700nm). The radiation hits the sample. Some parts of the sample molecules, called chromophores,

absorb some of this radiation. A detector measures how much radiation has been absorbed by the sample.

One can then use the Beer-Lambert

law to calculate the concentration of your chemical sample. The law is given by A=Ecl

A=Absorbance,

E=Molar

extinction coefficient (constant unique to different chromophores)

c=concentration

l=path

length (usually 1cm- the size of the reaction vessel). From the absorbance

value given by our sample, and its molar extinction coefficient, one can then rearrange the equation to find c:
c=A

/E.l

From this, one can work out the rates of chemical reactions etc.

N.B-

As a side-note,

the technique is cheap, easy and reliable. Also, The 'E' above is notated as a Greek letter 'Eta' in other reference sources, just in case you read up more about the Beer-Lambert

Law.

Answer 2

Spectrophotometer need to be calibrated against a blank solution so that measurements after it can use the blank solution's absorbance as a zero reference. For example, if you want to measure the concentration of a variety of protein solutions prepared in 1% ammonium sulphate, you need to blank the spectrophotometer with a 1% ammonium sulphate solution (no protein) so that the measurements only reflect the concentration of protein.

Answer 3

I have been reading numerous threads debating whether to use RO/DI or a calibration fluid. I bought mine from BRS and the insructions that came with the refractometer said to calibrate to zero with RO/DI. But BRS had put a sticker on the case saying no to do that, and included a fluid. There argument is to calibrate it to a range to where you will actually be using it. and ZERO ain't it!

Answer 4

Calibrating a microscope simplifies the process of obtaining quick and accurate measurements. It is important to calibrate the microscope for each object you plan to view with the lens.

Answer 5

It needs to be calibrated against a blank solution so that measurements after it can use the blank solution's absorbance as a zero reference.

Answer 6

Using standard solutions with different concentrations.

Answer 7

To get accurate readings