“Blinded By the Light”Determining the Concentration of Thiamine (B1) in Vitamin Supplements using Kinetic Fluorescence

Tim Kellenberger, Tony Juritsch, Clare Fried, Lauren EltringhamTA: Jing Dong, Penn State Chemistry 111-Majors

IntroWhat is Thiamine? Vitamin B1, or thiamine, is a vitamin of the B complex that helps fuel the body by converting blood sugar into energy. This vitamin is required by our bodies to properly use carbohydrates. Thiamine serves a variety of purposes, including keeping the nervous system, cardiovascular system, and muscular systems of the body healthy. Vitamin B is also is useful in boosting the immune system, diabetic pain, heart disease, alcoholism, vision problems, motion sickness, and numerous other conditions (1). Thiamine shots can also be administered to treat thiamine deficiency syndromes in patients who are in critical states, associated with alcohol withdrawal, or patients in comas (1). Thiamine may also be effective for preventing kidney disease in patients with type 2 diabetes and preventing cataracts. However, this vitamin is not effective for the prevention of cervical cancer, AIDS, heart disease, alcoholism, and other related conditions (1).

Good sources of thiamine are yeast, cereal, grains, beans, nuts, and meat. Thiamine is able to react with several foods, such as coffee, tea, and seafood. Some of the chemicals in coffee and tea (called tannins) react with thiamine, converting it to a form that is difficult for one’s body to take in. Raw freshwater fish and shellfish contain certain chemicals that destroy vitamin B1 (1). Fluorescence: Florescence occurs at a molecular level when a compound absorbs light in the form of photons at a certain wavelength and then emits them at a less energetic wavelength. The concentration of the fluorescence in compounds can be determined using a device called a fluorimeter. A fluorimeter illuminates the solution, which holds the particular compound, then measures the amount of light released at wavelengths around the range of the emission wavelength. By measuring the amount of light released, the concentration of the sample can be determined. Thiamine itself doesn’t fluoresce, although the concentration can be found through the process of converting thiamine to thiochrome. This is completed using Hg 2+ and as the thiamine is oxidized throughout this process, the ability of the solution to fluoresce will increase. The fluorimeter is used to determine the kinetic fluorescence levels.  Kinetic Fluorescence: Chemical kinetics is the study of rates of chemical processes. Studying how quickly or slowly the reaction takes place will help measure the increase in fluorescence for the certain concentrations of vitamin B1. The rate of fluorescence determined from this process will provide the rate of the reaction and will also allow a rate law expression to be determined from the information.

ProcedureThe first step was to build the fluorimeter. This was done by soldering various parts to a circuit board. This had to be done very carefully, so that the areas of the circuit board were not connected. If two areas are connected this can short circuit the fluorimeter.. After the fluorimeter was built, it was calibrated to achieve the correct results. This was done by preparing a blank cuvette with one mL of acidified water, one mL of HgCl2 solution, and one mL of sodium phosphate buffer. The cuvette then needed to be placed in the fluorimeter and the black knob on the fluorimeter needed to be turned until it read approximately 0.000. Then, the fluorimeter was used to test known concentrations of thiamine of 50ppm, 100ppm, 150ppm, 200ppm, 300ppm, and an unknown. A reading was taken from the fluorimeter was taken every thirty seconds for four minutes. The data was then placed into a graph of fluorescence versus time, which allowed for the determination of the concentration of the unknown solution.

ResultsReading# Time (s) Fluorescence (mV)

Concentration 50ppm 100ppm 150ppm 200ppm 300ppm Unknown 11 30 0.102 0.159 0.198 0.248 0.262 0.1522 60 0.175 0.274 0.331 0.378 0.41 0.2493 90 0.241 0.365 0.435 0.482 0.536 0.3424 120 0.295 0.444 0.525 0.565 0.626 0.4275 150 0.346 0.509 0.597 0.635 0.726 0.4956 180 0.392 0.569 0.651 0.689 0.758 0.5567 210 0.433 0.614 0.7 0.754 0.804 0.618 240 0.466 0.654 0.739 0.771 0.843 0.652

Fluorescence vs. Time for the Thiamine Oxidation Reaction

0 50 100 150 200 250 3000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9 Fluorescence vs. Time for the Thiamine Oxidation Reaction

50 ppmLinear (50 ppm)100 ppmLinear (100 ppm)150 ppmLinear (150 ppm)200 ppmLinear (200 ppm)300 ppmLinear (300 ppm)

Time (seconds)

Flu

ores

cen

ce (

mil

livo

lts)

0 50 100 150 200 250 300 3500

0.0005

0.001

0.0015

0.002

0.0025

0.003

Calibration Line for the Fluorimeter

Concentration (ppm)

Slo

pe o

f F

luor

esce

nce

vs. T

ime

Lin

e

Concentration (ppm) Equation Slope

50 y=0.0017x+.0736 0.0017

100 y=.0023x+.1355 0.0023

150 y=.0025x+.1817 0.0025

200 y=.0025x+2314 0.0025

300 y=.0027x+.2562 0.0027

Calibration Lines and Slopes

0 50 100 150 200 250 3000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

f(x) = 0.00185912698412698 x + 0.175142857142857

Fluorescence vs. Time for the Unknown

Time (seconds)

Flu

ores

cenc

e (m

illi

volt

s)

y = 3E-06x + 0.0018R² = 0.748

Time Fluorescence

30 0.152

60 0.249

90 0.342

120 0.427

150 0.495

180 0.556

210 0.61

240 0.652

Table 1- Fluorescence vs Time of Knowns

Time vs. Fluorescence of Unknown