# Spectrophotometric Analysis of Dyes

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Spectrophotometric Analysis of 2 dyes

Alice, Ko

Introduction:

The goal of this experiment is accurately determine the concentration of two types of dye, which are Red #40 and Yellow #5 in an unknown solution by using spectrophotometric analysis. To determine ε, extinction coefficient, of each dye, first we collect absorbance of different concentration of each dye prepared by 2 different methods at 2 different wavelengths: two syringe method and buret & volumetric flask method at 400nm and 520nm. Prior to perform each method, we made the concentration of each red and yellow dye approximately 5.0*10-5, by diluting each dye to 50mL. First for syringe method, we measured amount of both dye and water added by 2 syringes. We prepared each sample in cuvette with total volume of 3mL by increasing amount added of each dye from 0.5mL to 2.5mL. Secondly, for buret & volumetric flask method, we first add certain amount of dye measured by buret, and then dilute it until 5mL line of 5mL volumetric flask. We prepared each sample in cuvette by increasing amount added of each dye from 0.5mL to 4.5mL. By analyzing slope of the trendline of collected data (Abs. vs concentration), according to the Beer’s law, A= ε*l*c, we know that slope of each graph is equal to ε*l. After measure absorbance of red and yellow dyes by two methods, we measured absorbance of orange sports drink which is a mixture of red #40 and Yellow #5 dyes at 2 different wavelengths. Once we determine ε of red and yellow dye at 2 different wavelengths, we could determine concentration of each red and yellow dye in unknown sports drink.

Data and Result (Sample calculation attached in the back)

1. Syringe method

Table 1. Concentration and corresponding absorbance of Red and Yellow dye at the

two different wavelengths for syringe method.

 Syringe Methode Sample # Concentration (M) Abs. Red ( λ400) Abs. Red ( λ520) Abs. Yellow (λ400) Abs. Yellow (λ520) 1 8.3533E-06 0.003 0.250 0.170 -0.075 2 1.6707E-05 0.107 0.404 0.350 0.001 3 0.00002506 0.173 0.566 0.441 0.011 4 3.3413E-05 0.228 0.702 0.619 0.013 5 4.1767E-05 0.278 0.800 0.655 0.014 6 0.00005012 0.313 0.895 0.796 0.021

[pic 1]

Figure 1. (Syringe method) The Beer Law’s Plot showing the relationship between Absorbance vs concentration. The line of plot makes a curve as the concentration increases, which means the saturated points of the dye’s absorbance.

[pic 2]

Figure 2..(Syringe Method) Calibration curve generated from Beer Law’s plot. Only sample 2-4 points are chosen to make calibration curve to avoid saturated sample.

Table 2. (Syringe method) Calculated result of slope derived from calibration curve of yellow and red dyes

 Abs. Red ( λ400) Abs. Red ( λ520) Abs. Yellow (λ400) Abs. Yellow (λ520) Slope 7242.618 17837.19 16101.36 718.2761 Std. Dev. 658.4198 1556.265 5207.502 478.8508 Relative Std. Dev (%) 9.090909 8.724832215 32.34200743 66.66666667 T-value 4.302653 4.302653 4.302653 4.302653 95% Confident Interval 2000 4000 10000 10000 Calculated Value of Slope (M-1 ) 7000 ± 2000 18000 ± 4000 20000 ± 10000 700 ± 1000

Table 3. (Syringe method) Calculated extinction coefficient ε of yellow and red at 400nm and 520nm

 Wavelength (nm) Extinction Coefficient ε (M-1 ,cm-1 ) Red Yellow 400 7000 ± 2000 20000 ± 10000 520 18000 ± 4000 700 ± 1000

-Buret & Volumetric Flask Method

Table 4. Concentration and corresponding absorbance of Red and Yellow dye at the two different wavelengths for buret &volumetric flask method.

 Buret & Volumetric Flask Method Sample # Concentration (M) Abs. Red ( λ400) Abs. Red ( λ520) Abs. Yellow (λ400) Abs. Yellow (λ520) 7 0.00000500 0.029 0.135 0.139 -0.002 8 0.00000999 0.061 0.248 0.206 0.006 9 0.00001499 0.087 0.365 0.281 0.012 10 0.00001998 0.141 0.452 0.433 0.014 11 0.00002498 0.147 0.545 0.478 0.017 12 0.00002997 0.181 0.633 0.517 0.033 13 0.00003497 0.247 0.705 0.554 0.037 14 0.00003996 0.333 0.779 0.576 0.053

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