Quantitative Analysis of Blue Dye in Apple Juice Using Visible Spectroscopy

Quantitative Short Analysis of Blue Dye in Apple Juice Using Visible Spectroscopy

                                                                                                                                                        Jihyun Yoo

Objective

Explain the amount of light absorbed by a solution of dyes in relationship to the concentration, path length, and molar absorptivity. Find the relationship between transmission and solution concentration for blue dye #1 at 630nm. Find the concentration of blue dye in a sample. 

The amount of light absorbed by the dye. Beer's Law is used to determine the amount of dye in the sample by comparing the absorbance data of the sample with those of prepared solutions of blue dye of known concentration called standard solutions. 

A is the absorbance, c is the concentration of solutions, b is the length at which light passes through and e is a constant called molar absorptivity. The value of e depends on the wavelength used for analysis and the nature of species in solution. 

Materials

- 2 cuvettes

- spectrophotometer 

- deionized water (DI)

- stock dye

- pipettes: one for each solution

- wipes

- parafilm

- test tube racks

- cuvette covers

- two test tubes

- graduated cylinder

Procedure

1. Prepare a cuvette with only distilled water 

2. Load it into the spectrophotometer to calibrate

3. Preparation of samples: for sample 1, measure 10 mL of stock dye and then 0 mL of water into the test tube

4. Transfer to cuvette and cover

5. Cover the remaining sample with parafilm

6. For sample 2, measure 8 mL of stock dye and 2 mL of water

7. Repeat 4 and 5

Calibrate → Experiment → Calibrate → Spectrophotometer 1 → Put in cuvette (DI water) → Finish calibration → Experiment → Load sample → Start collection

Data Table

Solutions	Dillution (mL stock dye/H20)	Absorbance (630nm)	Molar Concentration	Measured  Transmittance %	Transmittance Decimal
1	10mL/0mL	1.193 abs	9.15nm	6%	0.06
2	8mL/2mL	1.008 abs	7.32nm	10%	0.10
3	6mL/4mL	0.768 abs	5.49nm	17%	0.17
4	4mL/6mL	0.506 abs	3.66nm	31%	0.31
5	3mL/7mL	0.451 abs	2.75nm	35%	0.35
6	2mL/8mL	0.259 abs	1.83nm	55%	0.55
7	1mL/9mL	0.190 abs	0.915nm	65%	0.65
8	0mL/10mL	0 abs	0nm	100%	1

Calculation Works

Molar Concentration 

(ML of stock dye x molar mass)/(ML of solution)

Solution 1: 10ml x 91.5 mol/1 = 91.5/10 = 9.15nm

Soulution 2: 8ml x 9.15mol/1 = 73.2/10= 7.32nm

Soulution 3: 6ml x 9.15mol/1= 54.9/10= 5.49nm

Solution 4: 4ml x 9.15mol/1= 36.6/10= 3.66nm

Soulution 5: 3ml x 9.15mol/1= 27.45/10= 2.75nm

Solution 6: 2ml x 9.15mol/1= 18.3/19= 1.83nm

Soulution 7: 1ml x 9.15mol/1= 9.15/10= 0.915nm

Solution 8: 0ml x 9.15mol/1= 0

Transmittance Decimal

(inv log (-abs) = T)

Solution 1: 2nd log (-1.193)= 0.06

Soulution 2: 2nd log (-1.008)= 0.10

Soulution 3: 2nd log (-0.768)= 0.17

Soulution 4: 2nd log (-0.506)= 0.31

Soulution 5: 2nd log (-0.451)= 0.35

Solution 6: 2nd log (-0.259)= 0.55

Soulution 7: 2nd log (-0.190)= 0.65

Solution 8: 2nd log (-0)= 1

Transmittance %

(Transmittance Decimal  x 100)=Transmittance %

Solution 1: 0.06 (100) = 6%

Solution 2: 0.10 (100) = 10%

Solution 3: 0.17 (100) = 17%

Solution 4: 0.31 (100) = 31%

Solution 5: 0.35 (100) = 35%

Solution 6: 0.55 (100) = 55%

Solution 7: 0.65 (100) = 65%

Solution 8: 1 (100) = 100%

Concentration vs Log transmittance

Log (transmittance decimal) 

	Concentration	-Log Transmittance
Solution 1	9.15uM	-log(0.06) = 1.22
Solution 2	7.32uM	-log(0.10) = 1
Solution 3	5.49uM	-log(0.17) = 0.77
Solution 4	3.66uM	- log(0.31) = 0.51
Solution 5	2.75uM	-log(0.35) = 0.46
Solution 6	1.83uM	-log(0.55) = 0.26
Solution 7	0.915uM	-log(0.65) = 0.19
Solution 8	0uM	-log(1) = 0

Graph

By testing the concentration of different solutions with different volumes of stock dye and water in a spectrophotometer, we were able to gather results that reflected that absorbance decreases when the stock dye and water solution has a lower concentration.

I took the inverse absorbance log, which yielded a transmittance decimal number.

Our -log vs. transmittance graph shows a linear relationship between these two factors.

This means that when the concentration of the stock dye solution increases, the transmittance is also increasing.

Transmittance increases when concentration increases because it is easier for light to be transmitted through a more concentrated solution because there are more molecules for light to pass through.