i've got some questions on spectrophotometers... please help...?

Question

thanks! here they are:

i. what is the purpose in doing the blank sample? why not go directly to the standard solutions?

ii. What is the purpose of pressing the "Autozero" button?

iii. why do you have to specify the wavelength in the determination of absorbance?

iv. what is the difference in using different types of light sources?

v. what is the difference in using different types of cuvettes?

Answer 1

1. Because you prepared the solution using the blank solvent. And the blank may itself contain some of the molecules you want to determine by this instrument. So you have to run it first and then the standard, so that the instrument will correct the absorbance and show you the exact absorbance of the solution.

2. The autozero, corrects if there will be any other absoption and reads only the absoption of the standard.

3. Because, it will directly allow the specific wave length ray to pass through it and the corresponding absorbance. If not then you can also specify a range and it will scan the whole region and find you the absorption peak. It is time consuming.

4. Generally the light sources are deuterium and tungsten lamp. Deuterium takes care of the UV and near UV region whereas the Tungsten in the visible region.

5. Cuvettes also affect the absoption process so You may use the quartz or glass cuvettes.

Answer 2

1. The blank Si the same as any of the sample except that it contains no added analyte. It corrects for any analyte in the reagents and should be matched ( as the standards should) to the matrix of the sample. The blank sets your zero.

2. The "auto zero" will reurn the the instrument back to the position set by usually the blank. It can be used to correct for some instrument instability. With good quality equipment this should not be necessary.

3. You will need different light sources to get to different parts of the EM spectrum from far infrared to the ultraviolet. An absorption spectrum will show show marked peaks if it is to useful for analysis.

4. Different cuvettes (cells) will generally give longer or shorter light paths and this is necessary to get respectable absorbance readings. A good range is from ~ 0.3 to 0.8.

Spectrophotometry revolves around the the application of Beer's Law ie absorbance is proportional to concentration and light path length.