FST Lab 8 (Measuring the Absorbance of different food solutions by UV-Visible Spectrophotometer)

 LAB-8

OBJECTIVE: 

To measure the absorbance of different food solutions by UV-VISIBLE Spectrophotometer

APPARATUS: 

• UV-VISIBLE Spectrophotometer

• Beaker

• Spatula

• Weighing balance

Reagent:

• Distilled water

THEORY: 

A spectrophotometer is an instrument used for detecting the presence of any light-absorbing particles dissolved in a solution and for measuring the concentration of those particles. A light source inside the spectrophotometer emits a full spectrum of white light toward a compartment where a sample liquid is placed. The samples are prepared in cuvettes that are made using specialized plastics or quartz so that they do not absorb any light and will not affect our measurements. Before the light passes through the sample in the cuvette, an adjustable prism and diffraction grating filter the light so that only a single wavelength of light can be selected and allowed to pass through the sample. All molecules differ in how strongly they absorb each wavelength of light in the visible spectrum because of differences in their molecular structure and composition. This allows us to use a specific wavelength of light to detect the presence of and quantify, one molecular compound in a simple or complex liquid mixture. Spectrophotometers are also calibrated by using a “blank” solution that we prepare to contain all of the components of the solution to be analyzed except for the one compound we are testing for so that the instrument can zero out these background readings and only report values for the compound of interest.

Light passing through a sample solution will partially be absorbed by molecules present in the sample. The amount of light unable to pass through a sample is measured as the absorbance value. Absorbance is directly proportional to the concentration of the molecules and is measured on a logarithmic scale from 0 to infinity. The amount of light that is not absorbed is transmitted or passed through the sample. Compared to the amount of light entering the sample, the amount that exits is measured as a percentage of the light transmitted. Percent transmittance is inversely proportional to the concentration of the molecules in the sample and is measured on a linear scale from 0% to 100%.

TYPES OF SPECTROPHOTOMETER:

There are different types of spectrophotometers which two are the most common:

1. UV-Visible spectrophotometer: this type of spectrophotometer operates in the ultraviolet (UV) and visible light range typically from 190-1100 nm. It is widely used for a variety of applications, including quantitative analysis of substances, and determining the absorption spectra of compounds.

2. Infrared (IR) Spectrophotometer: IR spectrophotometer measures a sample's absorption, transmission, or reflection of infrared light. They are used to study molecular vibrations and identify functional groups in organic compounds.

BEER-LAMBERT LAW: 

Spectrophotometers obey the Beer-Lambert Law. This law states that “The absorbance of light by a sample is directly proportional to the concentration of the absorbing substance and the path length of the light through the sample. 

Mathematically, the Beer-Lambert law is expressed as: 

A= Cl

Where, 

• A is the absorbance of the sample

• (Epsilon) is the molar absorptivity or the extinction coefficient, which is a constant specific to the absorbing substance in the sample

• l is the Path length of the light through the sample. 

The Beer-lambert law provides the basis for the quantitative analysis of substances using spectrophotometry. By measuring the absorbance of light at a specific wavelength, the concentration of a substance in a sample can be determined if the molar absorptivity and path length are known or kept constant. 

PROCEDURE: 

• Rinse the beaker with distilled water as a precautionary step

• Weigh the beaker and add a sample to it

• Reweigh the beaker with the sample. 

• Dilute the sample with distilled water. 

• Fill a cuvette 2/3 full with DI water to serve as the “BLANK” cuvette

• Calibrate the spectrophotometer: 

            (Wipe the outside of the BLANK cuvette with a Kim Wipe or soft tissues

• Place the cuvette into the machine so that the clear portions of the cuvette are oriented left to right. (The light needs to pass through a clear area on the cuvette).

• Press "Zero" and Remove the blank.

• Place a cuvette with a sample into the spectrophotometer

• Take the absorbance at different wavelengths. 

OBSERVATION:

Wavelength	Absorbance
400	1.075
450	1.559
500	2.010
550	1.236
600	0.077
650	0.018
700	0.003

 

DISCUSSION: 

The experiment aimed to measure the absorbance of different food solutions using a UV-visible spectrophotometer. A spectrophotometer is an instrument that detects light-absorbing particles dissolved in a solution and measures their concentration. It enables precise quantification of compounds such as vitamins, pigments, flavors, and additives present in food samples.

By measuring absorbance at specific wavelengths, UV-visible spectrophotometers provide valuable information about the concentration and composition of these substances, aiding in quality control, nutritional assessment, and ensuring food safety and compliance with regulatory standards. Their efficiency in rapidly analyzing multiple samples makes them indispensable in food research, production, and quality assurance processes.

STUDY QUESTIONS: 

Q1) What is the difference between % transmittance and absorbance?

Ans: Transmittance refers to an amount or percentage of incident light transmitted through a sample, whereas Absorbance is how much could not get through and was absorbed. 

Q2) How did you determine which wavelength was absorbed at the highest level? How is this process useful in determining the identity of a molecule?

Ans: By plotting absorbance against wavelength and identifying the peak absorbance. Useful for identifying molecules based on their unique absorption spectra.

Q3)How do you clean a cuvette?

Ans: Never Use sulfochromic acid or strong bases, such as potassium or sodium hydroxide, they can damage the cuvettes. Simply rinse with purified water followed by acetone or ethanol and blow dry with clean, dry, compressed air or nitrogen. Make sure that you do not build up pressure in the cuvette.