Does anyone know the easiest way to determine the concentration of an unknown using a baush and lomb spectrophotometer 20. Ive seen one used and know it has to do with beers law and the amount of light that is absorbed in a given spectrum but could some give me a stepwise procuedure in say determining the amount of phosphate in a given unknown sample?
2006-07-21 04:38:11 · 3 answers · asked by chemhead102 2 in Science & Mathematics ➔ Chemistry
First of all you need to specify the procedure.Phospate alone won't give you anything... You need a proper assay e.g. one where a complex of phopshate with another compound is formed and gives a coloured soluble compound.
You should measure at the wavelenght that absorption is maximal.
Be careful because in some assays the colour is a function of time in those cases you have to standarize your time as well.
You'll need to prepare a series of solutions with known phosphate concentrations to use as standards.
Measure the absorbance you get for those standards and do the curve Absorbance vs concetration.
It should be a line since A=edC.
Do the linear regression to get the equation of your line (y=slope*x+intercept)
In order to have a good reference curve you should have at least 9 points and make sure that you always stay within the linear range of the method (for colourimetric assays don't go above 1.0)
A good curve should have R^2>0.8 (the closer to 1 the better, blow 0.8 is trash)
Do the same with your sample and measure its absorbance.
Use the equation to determine the concetration.
Highest accuracy is for values of absorbance between 0.2 and 0.8. If tou have more than 1.0 diltute so that you are within the 0.2-0.8 range, calculate the conc and correct with the factor that you dilute (e.g if you add equal volume of water to your sample you need to multiply by 2)
2006-07-21 05:27:00 · answer #1 · answered by bellerophon 6 · 0⤊ 0⤋
Make up some standard solutions with known concentrations. Make them different by powers of 10: 0.001 M, 0.01M, 0.1 M, 1.0M, and add a few others in between these. Observe and record the absorption of each standard solution. Put in an equal amount of the unknown solution and record the data. Graph all points.
2006-07-21 11:44:42 · answer #2 · answered by physandchemteach 7 · 0⤊ 0⤋
The usual method for colourimetric phosphate determination is to convert the phosphate to a blue complex using sodium vanadomolybdate reagent.
Here is a link which gives you the full text of the method :
http://www.reachoutmichigan.org/funexperiments/quick/csustan/phosphat.htm
2006-07-21 19:05:32 · answer #3 · answered by Bruce H 3 · 0⤊ 0⤋