Spectrophotometry can be used to determine the concentrations of light-absorbing

Question

Spectrophotometry can be used to determine the concentrations of light-absorbing chemicals, such as chlorophyll-a (Experiment 8) or the red dye used in this project. Briefly describe the information that you need to collect in order to determine the concentration of a sample using spectrophotometry. (Review and consider the steps in Experiment 8.) What wavelength(s) will you need to consider for the dye in Project 3? Your dye stock solution is 0.05 mM. How many dilutions will you need to perform? What concentrations do you hope to use? Provide the detailed procedure for making these concentrations.

Explanation / Answer

Informations required for spectrophotometric estimation-

1. Suitable wavelength of light which could show maximum absorbance.

Although any wavelength can be chosen to measure concentration of the sample , but the

magnitude of absorbance is significant. It is only at a particular wavelength when absorbance

is maximum. Hence , the choice of wavelength is important. This is usually done by measuring absorbance at different wavelengths for a standard solution. A plot showing absorbance vs. concentration for a standard solution of the sample is helpful to interpret the suitable wavelength.

A wavelength showing maximum absorbance is selected.

2. Concentration or dilution of the sample solution.

Spectrphotometric measurements are based on Beer Lambert law,

A = Extinction coefficient . l .c

where A represents absorptivity ( dimensionless number ) , extinction coefficient or molar

absorptivity with unit liter/ mol. cm. , c is concentration in moles / L , l represents length in cms.

It has been observed that higher concentrations of the sample solutions are less accurate in absobance measurements . The accuracy goes on decreasing at higher concentrations. hence proper dilution of the sample solution should be known & is significant for accurate determinations. The law is applicable for only dilute solutions ,the extent of dilution of the unknown solution should be known.

Further , a calibration curve ( showing a plot of absorbance vs. concentrations ) is helpful to

measure / interpret absorbance of any unknown sample.

---------------------------------------------------------------------------------------------------------------------

The second and third question call for project & experimental details. Please post the questions separately as fresh questions. Glad to help.

Related Questions

Navigate

• Browse (All)
• Browse S
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.