Biochem lab 1 - Lab report PDF

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Lab #1: Photometry I. Introduction: The measurement of intensity of visible light as the human eye perceives it is called photometry. A typical human eye can see wavelengths between 380 and 740 nanometers; when light interacts with different objects, light may be transmitted at a different color. When interacting with an object, light can be transmitted, absorbed, or reflected. When it is transmitted through an object, it shows a certain color, depending upon the wavelength of the light. Light can also be absorbed when photons have an appropriate orbital of energy and are able to excite another object by causing its electrons to move to a higher energy orbital, moving to an excited state. The absorption spectra is a graph which compares the amount of light absorption of a specific compound with the wavelength used. Absorbance depends on the wavelength of a compound, altering the amount of light that it can absorb. The extinction coefficient is a constant value that measures how much a compound can absorb light at a given wavelength. The BeerLambert law is an equation which explains how much light that a substance can absorb directly correlates with the concentration, extinction coefficient, and the length that light travels through. With a higher concentration, and a larger length, the amount of light absorbed increases. The standard curve is used in this experiment to compare the concentration of PNP to the absorbance at a wavelength, which is typically a straight diagonal line. The PNP assay shows how pH effects the PNP and its absorption spectra. With a pKa of 7.08, PNP is over 50% protonated when the pH is below 7.08, and vice versa. When the pH is 4, the PNP will be fully protonated, and will no longer be able to absorb light. As PNP also has a maximum absorption level at around 450 nanometers, those that are above 450 nanometers can transmit through PNP and result in a color depending on the wavelength. There are four aims in this experiment are for us to learn. They are: to be able to determine an unknown quantity of PNP using its absorbency, create a standard curve based on the absorbance spectrum and amount of PNP in each tube, to better understand the Beer-Lambert law through the use of an absorbance spectrum, and lastly to determine the molar extinction coefficient using the equation from the Beer-Lambert Law. II. Experimental Procedure: This lab uses Epoch 2, a microplate reader to find the absorbance of several wells. Using a 96-well plate, two rows with 10 columns were filled with buffer B (pH 4), sodium carbonate (0.1M, pH 11), and a solution containing PNP. Column 1 is filled as a control, and columns 2-6 contain a 0.12µmole/mL PNP solution, while columns 7-10 have an unknown concentration of PNP. Using the given table, each well plate is filled with 300 µL. From the data obtained from the Epoch 2 machine for absorbance at 405 nanometers, a standard curve is created to determine the concentration of the unknowns. In order to create the curve, the average absorbances of well A and B were taken and subtracted from the average absorbance of the controls. The µmoles of

PNP is found through multiplication of PNP concentration with mL PNP used in each well. The amount of PNP in columns 7-10 are found by using the standard curve by looking at the absorbances and drawing a line to the y-axis, µmoles of PNP in each tube. The Beer-Lambert Law is used to find the molar extinction coefficient of PNP, E=A/cl. For the second part of the experiment, one control is used, and one column contains PNP to determine the peak absorbance levels for each wavelength. III. Results of the experiment: 1) The absorbance peak is located at a wavelength of 400nm. At a pH of 4, the solution is clear. At a pH of 10, however, the solution changes to a yellow color. 2) Worksheet #1: p-Nitrophenol Assay Wells

Volume of 0.12 µmole/ml PNP (µls)

A1

Volume of Unknown #_X__ (µls)

Volume of

Volume of

Absorbance

Amount

pH 4

0.1M, pH 11,

(at 405 nm)

Buffer B

Na carbonate

of PNP in tube

(µls)

(µls)

(µmoles)

0.0

XXXX

150 µl

150 µl

0.063

0

0.0

XXXX

150 µl

150 µl

0.056

0

A2

25 µl

XXXX

125 µl

150 µl

0.218

0.003

B2

25 µl

XXXX

125 µl

150 µl

0.283

0.003

A3

50 µl

XXXX

100 µl

150 µl

0.306

0.006

B3

50 µl

XXXX

100 µl

150 µl

0.283

0.006

A4

75 µl

XXXX

75 µl

150 µl

0.441

0.009

B4

75 µl

XXXX

75 µl

150 µl

0.462

0.009

(Blank) B1 (Blank)

Row

A5

100 µl

XXXX

50 µl

150 µl

0.569

0.012

B5

100 µl

XXXX

50 µl

150 µl

0.575

0.012

A6

125 µl

XXXX

25 µl

150 µl

0.710

0.015

B6

125 µl

XXXX

25 µl

150 µl

0.770

0.015

A7

XXXX

25 µl

125 µl

150 µl

0.236

0.0060486

B7

XXXX

25 µl

125 µl

150 µl

0.220

0.0058848

A8

XXXX

50 µl

100 µl

150 µl

0.394

0.0089242

B8

XXXX

50 µl

100 µl

150 µl

0.392

0.0090152

A9

XXXX

75 µl

75 µl

150 µl

0.531

0.0114176

B9

XXXX

75 µl

75 µl

150 µl

0.561

0.012091

A10

XXXX

100 µl

50 µl

150 µl

0.687

0.0142568

B10

XXXX

100 µl

50 µl

150 µl

0.701

0.014639

Average Absorbance

PNP Absorbance

2

0.0595

0.003

3

0.191

0.006

4

0.235

0.009

5

0.5125

0.012

6

0.6805

0.015

3)

Standard Curve (PNP) Amount of PNP in tube (micromoles)

0.02 f(x) = 0.02 x + 0

0.01 0.01 0.01 0.01 0.01 0 0 0

0

0.1

0.2

0.3

0.4

0.5

0.6

Absorbance (at 405 nm)

4) Sample (A7) Unknown Average Concentration Calculation: 0.236-0.063=0.173 Au Standard Curve: 0.173*0.0182+0.0029=0.0060486 µmoles Concentration: 0.0060486 µmoles / .25mL= 0.024194 µmoles/mL Well

Absorbance PNP (µmoles)

Concentration (µmoles/mL)

A7

0.236

0.0060486

0.024194

B7

0.220

0.0058848

0.02354

A8

0.394

0.0089242

0.017848

B8

0.392

0.0090152

0.01803

A9

0.531

0.0114176

0.015224

B9

0.561

0.012091

0.016121

A10

0.687

0.0142568

0.14257

0.7

0.8

Wavelength

Absorbance wavelength

340

Absorbance

0.391

510

0.056

0.415

01520

390.054

350

0.447

530

0.054

355

0.482

540

0.054

360

0.52

550

0.053

365

0.563

560

0.054

370

0.618

570

0.053

375

0.671

580

0.053

380

0.717

590

0.053

385

0.76

600

0.053

390

0.795

610

0.053

395

0.814

620

0.053

400

0.817

630

0.053

405

0.798

640

0.052

410

0.755

650

0.052

415

0.692

660

0.052

420

0.62

670

0.052

425

0.541

680

0.052

430

0.458

690

0.051

435

0.375

700

0.051

440

0.301

445

0.235

450

0.184

455

0.142

460

0.111

465

0.091

470

0.076

475

0.067

480

0.062

345

1

4639 Unknown Average Concentration = 0.05049 µmoles/mL *1000mL/L=504.9µmoles/L Extinction Coefficient: Point (0.6805, 0.015) c= 0.015 moles/125 mL=1.2x10^-4 moles/mL or 1.2x10^-7 moles/L E=A/cl, E=0.015/(1.2*10^-7) (1cm)=12500 L/(moles*cm)

Worksheet #2 Graph (Data on following page)

IV. Discussion:

The pH change effected the color of PNP. Increasing the pH from 4 to 10 caused it to change from clear to yellow. This is because PNP has a pKa of 7.08, and at 4, the PNP is fully protonated, but at a higher pH, there is more deprotonation. As there is a delocalized electron present after deprotonation, any light passing through the PNP will excite this electron to a higher energy level, and result in absorption. Next, the standard curve created from the data that was given follows the Beer’s law. As it states that absorbance and the concentration is directly proportional, there should be a straight diagonal line. Though the given data is not fully straight, the R2 value is 0.95, which is showing slight error. This inconsistency may be due to human error such as a slight pipetting error or unclean equipment. Based on the standard curve, the concentration of the PNP unknown solution seems to be about 0.05049 µmoles/mL. As there is a small standard deviation of 0.0031, the data should be relatively consistent. From the given data, the molar extinction coefficient for PNP is 12500 L/mole*cm. Literature states that this number should be 17,500 L/mole*cm. As such, our error is roughly 28%, and can be improved, but the values are relatively similar.

Works Cited: 1) Otto A. Bessey and Ruth H. Love PREPARATION AND MEASUREMENT OF THE PURITY OF THE PHOSPHATASE REAGENT, DISODIUM p-NITROPHENYL PHOSPHATE J. Biol. Chem. 1952 196: 175-. 2) National Center for Biotechnology Information. "PubChem Compound Summary for CID 980, 4-Nitrophenol" PubChem, https://pubchem.ncbi.nlm.nih.gov/compound/4Nitrophenol. 3) Stimson, A. “Photometry: The Answer to How Light Is Perceived.” Photonics Media, 8 Mar. 2009, ww.photonics.com/Articles/Photometry_The_Answer_to_How_Light_Is_Perceived/a2511 9....