Absorbance Spectroscopy_InstructorPPT_Fall2017 719 Why do you need to calibrate
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Question
Absorbance Spectroscopy_InstructorPPT_Fall2017 719 Why do you need to calibrate the instrument every time the wavelength is changed? What precautions need to be taken when calibrating the instrument? What could be the reason for the difference between the experimentally determined concentration and the true concentration for your unknown? - ary to measure the absorbance at the Whv is it necess wavelength of maximum absorbance? Can you visually estimate the concentration from the relative color intensity? Explain why or why not. What is the wavelength of maximum absorbance from the spectrum graph? Is it the same as what you used for the absorbance measurements of solutions? According to Beer's Law, absorbance is proportional to concentration. Does your calibration curve support this? Type here to searchExplanation / Answer
1) Calibration of the instrument refers to adjusting the accuracy of measurements. Like, you teach the instrument that when you place a cuvette with a known solution (standard) , instrument records it in the memory as a reference line. So everytime you change the wavelength, calibration maintains the accuracy and reliability of your measurements. Precaution like accuarate standard preparation with no impurities is a key thing. Everytime an experiment is performed, right calibration would achieve comparable values across biological replicates. Also, always allow the lamps to warm up atleast for 20 min each time an experiment is performed.
2) Certain degree of differences usually within 5% do exists between experimentally determined and theoretical calculations and are acceptable. These errors could be due to human handling, pipetting accurate volumes, physical parameters like temperature. If the instrument is calibrated well, and the standards give you a decent R2 or slope value, your measuremnts for unknown will remain reliable.
3) Though the spectrophotometer has been calibrated to the best possible, there would be a minor variation when sample measurements are made. This minor variation is next to neglible (called background value) at lambda max. However, the error gets amplified between the background and the sample, if one moves farther away from this wavelength.
4) Visual inspection of colored solutions and thereby concentration estimation is definitely not possible. when a light is incident on a solution with certain amount of absorbing species, only a part of the incident beam is transmitted and with change in concentration of the solute this value changes, obeying Beer-Lambert's law. A spectrophotometer serves as a detector for this mathematical treatment and reflects the amount of light being absorbed, thereby concentration. Whereas visual cues are just the reflections on our retina of any image that we see .
Last two questions are incomplete as no reference has been pointed out.