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

A spectrophotometer is calibrated by using known standards, such as neutral density filters or certified reference materials, to verify the accuracy and precision of the instrument. The calibration process involves adjusting the instrument's settings to match the expected absorbance values of the standards at specific wavelengths. Regular calibration helps ensure the spectrophotometer provides accurate and reliable measurements over time.

Answer 5

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 6

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 7

Using standard solutions with different concentrations.

Answer 8

To get accurate readings