Lab 1 Biochem

Lab 1: Determining the pKa of PNP PNP or paranitrophenol is an organic compound that undergoes changes when the pH is manipulated of the solution. PNP is also an acid/ base indicator. The compounded starts off as internal conjugated ring with the hydroxyl group and the nitro group single bonded to the aromatic ring. This occurs at the pH of 5. At this point the aromatic compound is colorless and is in the ultraviolet range. However, as the pH begins to increase the hydroxyl group deprotonates, leaving the oxygen with a negative 1 charge. The molecule compensates for the negative charge by conforming to a more stable resonance structure. The structure now has the oxygen and the nitrogen for the nitro group double bonded to the ring. The negative charge lies with one the oxygen atom that is a part of the nitro group. This lead to a highly conjugated system. If a system is conjugated the compound shows the presence by giving off a color. In PNP the color change is most visible at the pH of 10. The Henderson-Hasselbalch equation is useful in determining the pH of weak acid. The equation is as follows: pH= pKa+ log ([A-]/[HA]). The pKa is the negative log of the acid disassociation constant. This equation is extremely useful figuring out the pH of a buffer solution. A buffer solution contains the mixture a weak acid and its conjugate base or a weak base and its conjugate acid. A buffer solution is useful because there resistant to pH change. The pH changes very little when a small amount of an acid or a base is added. The blood in living organisms serves as buffer solution. In humans the blood pH is 7.4. The equation is also helpful in finding the pH when acid-base reaction are at equilibrium. Once the the pH at equilibrium is known, it can be used to calculate the isoelectric point in different proteins. Light is absorbed due to the interaction of light with electronic and vibrational nodes of molecules. The energy of light is E=hv. The h is Planck’s constant and v is the frequency of td light. The wavelength is represented as the λ=c/nv. The c is the speed of light 3.0*10^8, and n is the refractive index. This is when Beer’s law is useful in understanding how the light absorption is proportional to the number of molecules in the path. T=10-εcl -logT=log(1/T)=εcl=A, T is transmittance, ε is absorption coefficient or absorptivity, c is concentration, and A is the molar absorbance factor.
Results:
Part 1

The λmax=x when in the first derivative. y = -0.0002x2 + 0.1626x - 30.972 y’=(2)-0.0002x+0.1626+C 0=-0.0004x+0.1626
-0.1626=-0.0004x
-0.1626/(-0.0004)=x
X=406.5=λmax
Part 2: pH Absorbance
Blank
0
5
0
6
0.033
7
0.23
7.5
0.293
8
0.438
8.5
0.429
9
0.426
10
0.476

y = -0.0035x3 + 0.0637x2 - 0.2406x + 0.0014 y = -0.0035x3 + 0.0637x2 - 0.2406x + 0.0014 y’= (3)-0.0035x2 + (2)0.0637x - 0.2406 +C y’=-0.0105x2+0.1274x-0.02406 y”=(2) -0.0105x+0.1274+C y”=-0.021x+0.1274 0=-0.021x+0.1274
-0.1274=-0.021x
-0.1274/(-0.021)=x
X=6.06=pKa