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Chapter 29: Problem 1

Write a protocol for using a spectrophotometer. After reading this chapter, prepare a detailed stepwise protocol explaining how to use one of the spectrophotometers in your department. Ask another student or a tutor to evaluate your protocol and provide you with feedback.

Short Answer

Expert verified
A detailed guide to use a spectrophotometer involves setting up, calibration, sample preparation, taking measurements, cleaning & maintenance and troubleshooting & safety measures.

Step by step solution

01

Setting Up

Start by making sure that the spectrophotometer is plugged in correctly. Power the device on and wait for it to boot up (this can take a few seconds to a few minutes). Ensure that the sample holder or cuvettes are clean and free of dust or scratches.
02

Calibration

Before you start to take measurements, the spectrophotometer needs to be calibrated properly. This usually involves the use of a blank solution for measuring. Place the blank solution in the sample holder and adjust the machine so it's measuring 100% transmission or 0 absorbance.
03

Sample Preparation

Prepare your sample by diluting it as necessary. It is important that the solution is homogenous and free of bubbles. Fill a cuvette with the solution, taking note to clean the outside of the cuvette to remove any fingerprints or smudges.
04

Taking Measurements

Place the prepared sample in the sample holder and take note of the absorbance or transmission reading. It's also recommended to run the same sample multiple times and find an average, as this can help combat minor inconsistency issues.
05

Cleaning and Maintenance

Once you have finished, clean the machine following the manufacturer's instructions and store it in a clean and safe place. Ensure the sample holder or cuvettes are cleaned and dried properly before storage.
06

Troubleshooting and Safety

If something goes wrong, do not panic. Most spectrophotometer issues can be addressed by checking the manual or consulting with a supervisor or knowledgeable colleague. Always observe safety procedures and use personal protective equipment where necessary.

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Most popular questions from this chapter

Use the Beer-Lambert relationship in quantitative spectrophotometric analysis. Calculate the following (express your answer to 3 significant figures): (a) The concentration \(\left(\mu \mathrm{g} \mathrm{mL}^{-1}\right)\) of pentachlorophenol in a test solution giving an absorbance at \(300 \mathrm{~nm}\left(A_{300}\right)\) of \(0.57\) in a cuvette of path length \(5 \mathrm{~mm}\), based on an absorptivity of \(20 \mathrm{~L} \mathrm{~g}^{-1} \mathrm{~cm}^{-1}\). (b) The amount (ng) of pentachlorophenol in a \(50 \mu \mathrm{L}\) sub-sample from a test solution where \(A_{300}=0.31\) in a cuvette of path length \(1 \mathrm{~cm}\), based on an absorptivity of \(20 \mathrm{~L} \mathrm{~g}^{-1} \mathrm{~cm}^{-1}\).

Determine the molar absorptivity of a substance in aqueous solution. A solution of \(p\)-nitrophenol containing \(8.8 \mu \mathrm{g} \mathrm{mL}^{-1}\) gave an absorbance of \(0.535\) at \(404 \mathrm{~nm}\) in a cuvette of path length \(1 \mathrm{~cm}\). What is the molar absorptivity of a \(p\)-nitrophenol at \(404 \mathrm{~nm}\), expressed to 3 significant figures? (Note: \(M_{r}\) of \(p\)-nitrophenol is \(291.27\).)
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