2012 UV/VIS Spectroscopy Assisstant : Marcin PAWLAK Group 1 : Romain LAVERRIERE (romainl0@etu.unige.ch) Tatiana PACHVA (pachova0@etu.unige.ch)
Table of content: 1 Abstract... 3 2 Introduction... 3 3 Methodology... 3 3.1 Determination of λ max... 3 3.2 Determination of the equilibrium constants... 3 4 Results... 4 4.1 Determination of λ max... 4 4.2 Determination of the equilibrium constants... 5 5 Discussion... 7 6 Conclusion... 7 7 References... 7 8 Appendices... 8 8.1 Resultats des absorbance a 241 nm et 293 nm... 8 8.2 Notebook s scan... 9 2
1 Abstract By UV/VIS spectrometry, equilibrium constants of every forms of tyrosine amino acid were determined besides the fraction of the two neutral forms. The macroscopic equilibrium constant obtained are pβ 1 = 10.5025 ± 0.0372 and pβ 2 = 19.8192 ± 0.0802. The microscopic equilibrium constant found are pk 10 = 10.2775 ± 0.0213, pk 01 = 10.1091 ± 0.0610, pk 10 = 9.6189 ± 0.1261 and pk 01 = 9.6433 ± 0.0473. Finally, the fractions obtained x zwitterionic = 0.60 ± 0.02 and x uncharged= 0.40 ± 0.02. 2 Introduction Tyrosine is an amino acid, which is synthetized in-vivo and contains three site of protonation (cf : Figure 1): Figure 1: Tyrosine (neutral form) The aim of this experiment is to determine all the constants that describe the equilibrium of tyrosine in aqueous solution. For this, we will us the UV/VIS spectroscopy based on the fact that tyrosine will change its absorbance depending on its state of protonation (function of the ph). For that reason, we will work principally under neutral and basic conditions. Thus, by measuring the absorbance as a function of the ph, all the equilibrium constants will be determined. 3 Methodology [1] First of all, three sodium chloride 100 mm solutions are prepared: one containing 1 mm of tyrosine, another containing ammonium chloride 50 mm and the last with ammonia 50 mm. 3.1 Determination of λ max 3 solutions of 25 ml are prepared: the first (buffer) at ph 9 made with 10 ml of ammonia solution and 10 ml of ammonium chloride solution, adjusted to the mark with sodium chloride 100 mm. The second at ph 6 containing 1 ml of tyrosine, 20 ml of ammonium chloride and adjusted to the mark with sodium chloride 100 mm. The last one made from 1 ml of tyrosine, 20 ml of ammonia and adjusted with NaH to ph 12. Two cuvettes containing buffer solution are required to take a zero value. Then, one of the cuvette is replaced by the sample to be analysed. The measurement has been done for the solution at ph 6 and the other at ph 12. The results are used to find the two λ max. 3.2 Determination of the equilibrium constants 16 solutions containing 1 ml of tyrosine 1mM completed to 10 ml with ammonium buffer at a ph 7 to 12, by approximate steps of 0.33. The absorbance of each solution is determined by UV/VIS spectroscopy. 3
Then, the absorbance at the maximal wavelengths (241 nm and 293 nm) and the corresponding ph are extracted to igor. By making a curve fitting of the two plots, the equilibrium constants (cf: Figure 2) are determined. K 01 ' NH 3 - K 01 H NH 3 - ß 2 NH 2 - ß 1 neutral form K 10 ' K 10 - H NH 2 Figure 2 : Equilibrium constante scheme 4 Results 4.1 Determination of λ max The measurement of the two solutions (ph 6 and ph 12) gives the following plot: 1.2 1.0 0.8 Absorbance 0.6 0.4 0.2 A(pH 6) A (ph 12) 0.0 220 240 260 280 300 320-0.2 λ [nm] Figure 3 : Absorbance at ph 6 and ph 12 From the plot, we can easily see that the two maximum absorbances are gained at 241 nm and 293 nm. 4
4.2 Determination of the equilibrium constants The results of absorbance against wavelength were plotted and gave the following graph (Figure 4): 1.2 Absorbance 1.0 0.8 0.6 0.4 0.2 0.0 220 230 240 250 260 270 280 290 300 310 320-0.2 λ [nm] Figure 4 : Solutions of tyrosine at ph = [7 ; 12] A (ph 7.02) A (ph 7.34) A (ph 7.66) A (ph 8.01) A (ph 8.33) A (ph 8.65) A (ph 9.01) A (ph 9.34) A (ph 9.66) A (ph 10.04) A (ph 10.34) A (ph 10.64) A (ph 11.04) A (ph 11.34) A (ph 11.70) A (ph 12.02) By considering only the absorbance at the wavelength corresponding to the maximum absorbance, the absorbance against the ph at 241 nm and 293 nm are plotted in igor, and curve fittings are done providing the following graphs with their coefficient values (Figure 5 & 6): Figure 5 : Absorbance vs ph at 241 nm Figure 6 : Absorbance vs ph at 293 nm 5
The relation between absorbance and ph is given by the following equation: α = " [ ] [ ] (1) The absorbance can be written as the following equation: A = A + (A " A )α (2) where A R- is the absorbance of the deprotonated form given in the figure 3 at ph 12, and A RH is the absorbance of the protonated form given in the figure 3 at ph 6. The results of the curve fitting gave the following values (Table 1): A(241) vs ph SD A(293) vs ph SD K 10 1.80E+10 1.13E+09 1.9875E+10 2.39E+09 β2 5.40E+19 1.54E+19 7.7947E+19 3.70E+19 β1 2.91E+10 3.39E+09 3.4521E+10 7.57E+09 Table 1 : Curve fitting With these three equilibrium constants, all the other parameters can be defined by the following relations : β = K " + K " K " = β K " (3) K " = (4) " K " = (5) " The final result of each constant is the mean of the two values obtained at the two considered wavelengths. Besides, since the constants have a high order of magnitude, one would express them in terms of pk = logk. Thus, we obtain the following results: Macroscopic Cste : Microscopic Cste : x σ x pβ 1 10.5025 0.0372 pβ 2 19.8192 0.0802 pk 10 10.2775 0.0213 pk 01 10.1091 0.0610 pk 01' 9.6433 0.0473 pk 10' 9.6189 0.1261 Table 2 : Equilibrium constants Standard deviation for the K 01 is given by the propagation of error for an addition [2] : σ " = σ " σ (5) Standard deviation of K 01 and K 10 is obtained by the propagation of error for a multiplication [2] : σ = K + (6) 6
Then, the propagation of error for a logarithm [2] is: σ " = 0.434 (7) Finally, the standard deviation of the mean is given by the relation [2] : σ = ( ) () (8) The fraction of the neutral forms are obtained by the following equations: x "#$$%&#'(#) = " (9) x "#"#$% = " (10) As before, the result of the fractions is the mean of the two values obtained at the two considered wavelengths. Thus, we obtain this fraction of each neutral form: x σ x x uncharged 0.40 0.02 x zwitterionic 0.60 0.02 Table 3 : Neutral fraction The standard deviation for the fractions is given by the following equation [2] : σ = x + " " (11) 5 Discussion The results for the equilibrium constants show relatively low standard deviation, meaning that the results are quite the same at 241 nm and 293 nm. Thus, we can say that UV/VIS spectrometry is a very valuable analysis method, although the determination of the constants in igor is a little bit imprecise. Besides, we can see from the fraction of the neutral species that tyrosine shows itself more in a zwitterionic form. The reason is that this form allows a resonance in the structure. 6 Conclusion Finally, the results seem to be good and were obtained with a good precision, quickly and with few solutions. From them, we are able to consider the favourite form for the tyrosine, which is the zwitterionic structure. 7 References [1] M. Borkovec, TP_AtomicSpectroscopy_rev2012, Travaux pratiques de chimie analytique / Chimistes et Biochimistes 3ème année, 2012 [2] M. Borkovec, Error Analysis Introduction, Travaux pratiques de chimie analytique / Chimistes et Biochimistes 3ème année, 2012 7
8 Appendices 8.1 Resultats des absorbance a 241 nm et 293 nm A(241) vs ph SD px = log(x) SD(pX) β1 2.91E+10 3.39E+09 10.4637 0.0506 β2 5.40E+19 1.54E+19 19.7320 0.1239 K10 1.80E+10 1.13E+09 10.2557 0.0272 K01 1.11E+10 3.57E+09 10.0440 0.1401 K01' 4.88E+09 2.10E+09 9.6880 0.1870 K10' 2.99E+09 8.75E+08 9.4763 0.1268 x01 0.38 1.31E-01 x10 0.62 8.20E-02 A(293) vs ph SD px = log(x) SD(pX) β1 3.452E+10 7.570E+09 10.5381 0.0952 β2 7.795E+19 3.700E+19 19.8918 0.2060 K10 1.988E+10 2.390E+09 10.2983 0.0522 K01 1.465E+10 7.938E+09 10.1657 0.2352 K01' 3.922E+09 2.826E+09 9.5935 0.3127 K10' 5.322E+09 2.606E+09 9.7261 0.2125 x01 0.42 2.48E-01 x10 0.58 1.44E-01 8.2 Notebook s scan 8