This exercise will help to develop and test multiple skills, including accuracy of pipetting, handling sample dilutions, transporting and manipulating data, as well as to illustrate useable dynamic range of a spectrometer. Your task is to record a series of concentrations of tryptophan from the lowest detectable limit to the highest. Results will illustrate how absorption changes proportionally with concentration, as well as distortions that occur at the limits of dynamic range.
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From 5 mM or 10 mM stock solution of Trp prepare two additional dilutions: 0.5 mM and 0.025 mM. You will need approximately 2 ml of each.
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Prepare spectrophotometer for measurement.
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For this exercise it is best to use magnetic stirring bar in a standard 3 ml, 1 cm cuvette. Stirring will take care of mixing solutions throughout and make measurements more reliable.
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Place 2.0 ml of MQ water in the cuvette and start stirring; record blank then record sample – since there were no change in the sample composition, there should be no change in the spectrum and you should see flat line with some noise around zero. This noise limits the lower end of measurable spectra.
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Throughout measurement take notes of each step and indicate points where spectra were recorded.
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Beginning with the most dilute stock, start adding aliquots into cuvette recording a spectrum after each addition. Allow at least 10-15 sec for sample to mix thoroughly. Start with 10 mL on first addition and double this volume on each successive addition. Record spectra even if you see no obvious changes. When you reach upper limit of pipette capacity, move to a larger capacity pipette.
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While making additions, keep track of volume added and total volume in the cuvette. Continue to add increasing volumes of stock solution until volume reaches approximately 3 mL. At his point cuvette will be nearly full and you will not be able to add any more.
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Using pipette carefully withdraw 1.0 ml of solution from the cuvette (what pipette do you use?) and dispose it off. Take this volume in to account in your volume tracking record. Measure the spectrum – while concentration will not change with sample withdrawal, it will help to have this measurement when dilutions are calculated.
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Switch to the next more concentrated stock and, re-starting with 10 mL volume, continue to add it to the cuvette exactly the same way as you have just done.
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Once you reach volume close to 3 ml, again withdraw 1.0 ml and continue with the most concentrated stock. As you continue to add concentrated stock, notice that growth of absorption intensity will begin to slow down and spectra will become noisy. This is the upper limit of measurable range.
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Once you complete the measurement convert measured spectra into a matrix form as described here. Confirm that matrix was created in the Matrices folder and save matrix to a file.
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After washing and storing the cuvettee on a rack, proceed to data handling section to port data into data analysis software, to make graph, and find results of titration.
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