To larn how to do a spectrophotometer accurately and construe the informations recorded to build a graph and obtain a standard curve utilizing excel.

To larn how mathematical computations of the optical density readings and of terra incognitas to acquire the standard curve of a concentration value from the curve.

Method:

Refer to Proc 2048 Biochemical Engineering Lab Manual

Exercise 1- The Spectrophotometer

Absorbance readings of Methyl Orange and Bormophenol Blue were recorded for a scope of wavelengths from 400 to 700nm intervals, zeroing the setup with a distilled H2O space after each alteration in wavelength.

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Concentration

Methyl Orange

Bormophenol Blue

400

0.463

0.031

450

0.761

0.037

500

0.541

0.089

550

0.043

0.218

600

0.001

1.005

650

0.001

1.562

700

0

0.183

Table 1: The Spectrophotometer

Graph 1:

Graph 2:

Exercise 2: Determination of Glucose Concentration

Each of the criterion glucose solutions and the unknown solutions were tested in spectrophotometer utilizing a wavelength of 580nm and optical density readings were taken for each.

Concentration g/L

Optical density

0

0

0.5

0.077

1

0.177

1.5

0.253

2

0.371

A

0.084

Bacillus

0.125

C

0.21

Calciferol

0.142

Tocopherol

0.269

Table 2: Determination of Glucose Concentration

Graph 3:

Exercise 3- Determination of Yeast concentration

Similarly to the glucose experiment optical density readings of different criterion and unknown barm concentrations were recorded at a wavelength of 600nm. Unknown solutions U and V were besides diluted to a 1:2 ratio with distilled H2O as the concentrations are excessively high and autumn outside the acceptable optical density scope.

Concentration g/L

Optical density

0

0

0.2

0.115

0.4

0.231

0.6

0.337

0.8

0.446

Tungsten

0.056

Ten

0.169

Yttrium

0.017

Omega

0.411

Uracil

0.518

Volt

0.725

Table 3: Determination of Yeast concentration

Graph 4:

Calculations:

Unknown glucose computations

Y = 0.1836x – 0.008, Solve for x gives

Substituting in optical density values for each unknown glucose solution gives:

Unknown yeast computations

Y = 0.557x + 0.003 Solve for x gives

Substituting in optical density values for each unknown glucose solution gives:

U and V need to be multiplied by 2 after computation as they were diluted in a 1:2 dilution

Discussion:

In exercising 1 different wavelengths was used and the optical density in the graph increasing so lessenings and so somewhat increasing. The coloring material contributes to where the soap optical density occurs and the concentration affects the strength of the extremums. For instant the parts blue and the parts xanthous visible radiation is a green coloring material. This is the light that we see, and hence the wavelengths of visible radiation to go through through the optical density with the lower limit in footings of all other moving ridges of light optical density higher.

Spectrum analysis of pure sugar solution would be impossible for any optical density that can go on is the solution to be crystalline and any suspended atoms. This means that solutions need glucose to the reaction with 1 milliliters of 3.5 acid Dinitrosalicylic ( Domain Name System ) to organize amino 3, 5 – Nitrosalicylic acid, a compound colour absorbs light strongly in all parts of 580nm. This enables us to utilize spectral analysis to find the focal point. This applies merely if the strength of the colour of the merchandise is straight relative to the concentration of the reactants. In this instance, glucose concentration is straight relative to the sum of amino – 3, 5 – Nitrosalicylic acerb manufacturers such as the Stoichiometry of the reaction is 1:01, and most of this focal point is non to make a balance It is of import that the space or zero concentration used for this experiment is non merely distilled H2O but 1ml of DNS and distilled H2O made up to the same volume as the other samples, as the unreacted DNS in our glucose solutions is lending to the coloring material of the solution every bit good as the 3-Amino,5-Nitrosalicylic Acid.

In exercisings 3 barm is basic in footings of chemical science is based on the natural philosophies. By increasing the concentration, the optical density will increases in barms solutions nevertheless they are non coloured but they are block and spread, so some the visible radiation will non travel through them. And this because we are covering with suspended particulate affair, and non resolved ions. Blot out the light commensurate with the focal point so that we can happen the concentrations of unknown values of optical density. It is of import to agitate good before taking the sample optical density reading such as barm, atoms and settle to the underside, that average if we are non shaken them, so they will give us a lower optical density reading.

Questions

Exercise2

The cuvettes have different surfaces for two grounds. The frosted ridged sides are so no light flights out the sides of the cuvette giving a false reading. The other ground they have 2 different sides is so that you do n’t manage the transparent sides straight as oils or soil from your fingers could increase the optical density and give inaccurate consequences

Atoms in solution ( merely like in the yeast experiment ) affect the soaking up reading by barricading or debaring light off from the sensor therefore the atoms in a coloured would increase the optical density and give inaccurate consequences, unless the concentration and size of the atoms is changeless with all trials conducted so it would non impact the deliberate consequences.

A standard curve in spectrophotometric analysis is a additive trendline that fits through your experimental information. It is calculated by mensurating optical density readings at a scope of different concentrations and plotting them against each other. A additive arrested development done my excel or other agencies is calculated for the points and an equation in footings of optical density ( Y ) and concentration ( X ) is formed and you can utilize this equation to cipher unknown concentrations from optical density readings.

Exercise 3

The cuvettes have different surfaces for two grounds. The frosted ridged sides are so no light flights out the sides of the cuvette giving a false reading. The other ground they have 2 different sides is so that you do n’t manage the transparent sides straight as oils or soil from your fingers could increase the optical density and give inaccurate consequences

First dilute the dye to an equal concentration with distilled H2O.Find the maximal optical density of the diluted dye by proving optical density ‘s at a scope of different wavelengths guaranting you zero with distilled between each wavelength. Take some of the dye and thin it with distilled H2O to about 6 – 8 different concentrations i.e. 1:100 i? 1:10, depending on what optical density readings you get set the dilutions to suit in a scope of 0 i? 0.8 as that is where the Beer-Lambert Law applies. Using the soap soaking up wavelength you would so fix a standard curve for the dye by mensurating optical density of each of the diluted concentrations. Plot the optical density V concentration and utilize a additive arrested development to organize an equation. Take a sample of the waste H2O and filtrate off any suspended atoms to increase the truth of the optical density reading. Finally mensurate the optical density of the sample thining consequently if optical density is non in the needed scope. Using this optical density value in your standard curve equation calculate the concentration and multiply by your dilution factor if the sample was diluted.

Assuming you already have a standard curve and equation for the nitrate composite for a scope of 0 i? 1.5 mg/L. All you need to make is thin your sample from your suspected 55 mg/L to fall in the concentration scope of 0 i? 1.5 mg/L. So a 1:50 or a 1:100 dilution would be adequate to give you 1.1 mg/L or 0.55 mg/L severally. Then the sample is ready for spectrophotometer analysis.

Decision:

All of the barm and sugar were obtaining accurate trial consequences with the value of R2 ( 0.99 ) for each of the tendency lines and graphs. 99 % truth significance that lines tantrums for most the points. The procedure was a good cognition to larn and it is utile to understand the Beer-Lambert jurisprudence and his applications.

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