Organismos e instituciones que prestan apoyo a nuevos emprendedores

(A quantitative analysis of Salicylates in Blood by the Beer-Lambert equation¹⁵)

A Summary of this Week’s Lab During this lab period, you will conduct a quantitative analysis for salicylates (aspirin) in blood. Again, water will be used as a surrogate for blood, and aspirin will be determined as sodium salicylate. You will prepare a calibration curve as a plot of % transmittance (% T) on the vertical axis and concentration (C) in mg/dL on the horizontal axis, using the Excel program. You will the % T for an unknown and use the calibration curve to find the concentration of the unknown. Salicylic acid is a white solid, which reacts with dilute iron(III) nitrate (also called ferric nitrate) to produce a violet color. The intensity of the violet color is a function of the concentration of the salicylate. Therefore, the % T of the solution is directly proportional to the violet color or concentration of the salicylate. Because violet is in the visible region of the

electromagnetic spectrum, we will be using the visible portion of the UV-Vis instrument. The first step is to determine the wavelength of maximum absorption. For the violet color, this wavelength is known to be 540 nm, which will be used throughout for this

experiment. In the following paragraphs, we will conclude our study of the interpretation of ultraviolet spectra by studying the rules formulated by R. B. Woodward in the 1940’s.

The Interpretation of UV Spectra

Historically, UV predates IR, NMR, and mass spectrometry as instrumental techniques. In general, these latter instruments give more information than UV. Hence, UV is not studied to the extent it once was by chemists. However, there are some interesting structure-absorption properties that are worthy of study by sophomore students. These are known collectively as Woodward’s rules. They are empirically derived rules. That is they are based on the observartions of Professor Woodward.

These rules will be developed, starting with the absorption observed for the simple diene system present in 1,3-butadiene.

Types of groups and the effect of substituents on the UVmax.

Two groups that are commonly encountered are called chromophores—groups that absorb in the UV region of the electromagnetic spectrum and auxochromes—groups that do not themselves absorb but do alter either λmax or ε for a chromophore with which it is bonded in a molecule. As noted earlier, a shift in λmax to a longer wavelength is called a bathochromic shift, and a shift to a shorter wavelength is called a hypsochromic shift.

Dienes and Polyenes

A conjugated diene might exist in one of two major conformations, an cis conformation or an s-trans conformation. Recall that an s-cis conformation is required for the Diels-Alder reaction. A

15 This experiment was modified from: Meloan, C.E, James, R.E, and Saferstein, R., Lab Manual for Criminalistics: An Introduction to Forensic Science, 7th ed.

compound that is free to rotate is considered to adopt the s-trans or more stable conformation for these calculations. In general, we start with a given structure and assign a base value for λmax. Then, adjustments are made to the base value for each substituent we add to the conjugated system. The base value for 1,3-butadiene in the s-trans conformation is 217 nm. For each alkyl group bonded to 1,3-butadiene, we add 5 nm to λmax. Figure 1 shows butadiene and six derivatives with the predicted and actual values of λmax.

Figure 1 Acyclic 1,3-Butadienes

In the examples of Figure 1, all of the compounds can freely rotate to attain the s-trans conformation shown. Some compounds are locked into an s-cis conformation as shown in Figure 2. For

six-membered conjugated cyclohexadienes the base value is 253 nm, plus 5 nm for each alkyl substituent.

Figure 2 Conjugated Cycohexadienes

The presence of an additional double bond in conjugation with an existing diene causes a bathochromic shift of 30 nm for either conformation.

s-trans

Actual λmax in black, predicted value in red for conjugated dienes in s-trans conformations.

cyclic s-cis In most instances, a ring is needed to lock a diene into an s-cis conformation.

Figure 3 The Bathochromic Shift of Adding a New Conjugated Double Bond

Exocyclic vs Endocyclic Double Bonds

Figure 4 shows two kinds of double bonds. In one double bond, both C atoms of the double bond are part of the ring; it is called an endocyclic double bond. In the other double bond only one C atom is part of the ring; it is called an exocyclic double bond.

Figure 4 Endocyclic and Exocyclic Double Bonds

The bathochromic shifts caused by various auxochromes are shown in Table 1 below.

Table 1 Bathochromic Shift of Various Groups

Group added to Base Correction in nm

Conjugated double bond 30 nm

Alkyl group 5 nm

Exocyclic double bond 5 nm

Conjugated Aldehydes and Ketones base = 217 nm

extra db = 30 nm predicted 247 nm actual = 258 nm

base = 253 nm extra db = 30 nm two alkyl = 10 nm predicted = 293 nm An extra conjugated db adds 30 nm (bathochromic shift).

A B

endocyclic double bond

exocyclic double bond

double bond exocyclic to ring A endocylcic to ring B

The same kind of empirical rules apply to conjugated aldehydes and ketones. The base value for an α,β-unsaturated aldehyde is 210 nm and for an α,β-unsaturated ketone is 215 nm. The general structures for a,b-unsaturated aldehydes and ketones are shown in Figure 5.

Figure 5 Conjugated Aldehydes and Ketones

Table 2 shows the bathochromic shifts caused by the addition of alkyl groups at the α or β carbon atoms, an exocyclic double bond at the β carbon and an additional conjugated π bond.

Table 2 Bathochromic Effects in α,β-Unsaturated Carbonyl Compounds

Group Correction (add)

α-alkyl substituent 10 nm β-alkyl substituent 12 nm Exocyclic double bond, β carbon 5 nm

Extended conjugation 30 nm

The strong UV absorptions in α,β-unsaturated aldehydes and ketones are due to π to π* transitions that start at 210 and 215 nm, respectively. For the n to π* or weak transitions that are also present, the absorption occurs between 280 and 300 nm.

Lab Preparations:

Stockroom Prepartions:

Stock solution of sodium salicylate: Add 5 drops of chloroform, a preservative, to a 1-L volumetric flask. Then, add 1.16 g of sodium salicylate. Dissolve the solid by the addition of water. Continue adding water to the 1-L mark on the volumetric flask.

Ferric Nitrate: Dissolve 1.0 g ferric nitrate in 99 mL of water to make a 1% solution of ferric nitrate. (Total volume = 100 mL of 1% ferric nitrate)

Nitric Acid I: Make 100 mL of 0.07 M nitric acid.

Dilute Ferric Nitrate: Mix 5 mL of 1% ferric nitrate with 4 mL of 0.07 M HNO3 (nitric acid I) and label the container “dilute ferric nitrate.”

Nitric Acid II: Make 100 mL of 0.039 M nitric acid.

C C C

Student Preparations:

1. Transfer 5.00 mL of stock solution to a 150 erlenmeyer flask and add 95.00 mL of water. This is your standard solution. Its concentration is 5.0 mg/dL.

Procedure for Spectronic 20 instrument in the visible range.

1. Turn on the instrument and allow it to warm up for about 20 min.

2. Set the wavelength to 540 nm.

3. Depress the “mode” control key and set the mode to “transmittance.”

4. Fill six cuvettes with varying amounts of your standard solution and adjust the volumes of each to 2.0 mL as shown in Table 1 below.

5. The zero transmittance will be determined with the sample holder empty. With the sample compartment empty and the lid closed, set the transmittance to zero by adjusting the on-off knob.

6. Determine 100 % transmittance (% T) for the blank. Place the cuvette all the way into the sample holder. Make sure the marking on the cuvette is aligned with the mark on the instrument. Adjust the on-off knob until the % T is 100%.

7. Replace the blank in turn with each of the numbered samples and determine the %T for each.

Carefully record the % T as you go into your lab notebook.

8. Prepare your unknown for analysis by mixing 1.0 mL of the unknown with 1.0 mL of the dilute ferric nitrate solution.

9. Determine the % T for your unknown and record it in your notebook.