Victoire Ndong & ELina Shrestha Biochemistry 311 Tuesday lab 10/5/07 Instructor: Laurie Lentz-Marino

LAB REPORT #2: pH AND BUFFERS Many natural reactions that take place in our organism require an environment of optimum pH. Reactions depending on where they happen require more acidic or basic pH. For that reason, there are solutions called buffer which are important in keeping the pH required for those reactions. A buffer is a solution that can keep a narrow range of pH even when strong acids or bases are added. An example of a buffer is blood which is very good at counter acting different substances pH. In our experiment, we try to see the difference between non pH solution and pH solutions. For that, we made buffers like Acetic acid/Acetate and pyrophosphate and we looked for changes that occurred when strong acids or strong bases were added. The change in the pH will determine whether it was really a buffer. We also titrated Glycine in order to get its pKas from the titration curve.

RESULTS & CALCULATIONS: Table1: Measured pH and calculated pH

Measured Substance H2O
H2O + HCl

pH 6.1 2.7 11.2

Calculated pH 7 2.6 11.3

H2O

Victoire Ndong & ELina Shrestha Biochemistry 311 Tuesday lab 10/5/07 Instructor: Laurie Lentz-Marino +NaOH HCl NaOH 1.8 12.4 1.3 12.7

Table 2: Titration of glycine with HCl pH and volume

pH 7 5.6 4.6 4 1.6 Volume (ml) 0 1.3 4.5 6.3 8.7

Table 3: Titration of Glycine with NaOH pH and volume

pH 6.5 8.5 8.8 9.1 10.1 10.6 Volume (ml) 0 0.3 0.4 0.8 0.9 1

Victoire Ndong & ELina Shrestha Biochemistry 311 Tuesday lab 10/5/07 Instructor: Laurie Lentz-Marino 10.9 2

Table 4: Data sheet for the dilution read on the spectrophotometer

Absorbance Tube Letter mmoles of HOAc Initial amount A B C D E F G H I 3.2 1.6 0.8 0.4 0.2 0.1 0.05 0.025 0.0125 Final amount (x) 1.6 0.8 0.4 0.2 0.1 0.05 0.025 0.0125 0.00625 0.0076 0.163 0.3666 0.4231 0.4985 0.3221 0.0798 0.0208 0.095 3.56 3.83 4.14 4.44 4.74 5.04 5.34 5.64 5.94 530nm pH calculated

Graph1: Plot of the pH versus the volume of HCl during the titration of Glycine

Victoire Ndong & ELina Shrestha Biochemistry 311 Tuesday lab 10/5/07 Instructor: Laurie Lentz-Marino

pH versus volume of HCl

10 9 8 7 6 5 4 3 2 1 0 0 2 4 Volume of HCl in ml 6 8 pH of the solution

Series1

Graph 2: Plot of the whole titration of Glycine

Titration of Glycine
12 10 pH value 8 6 4 2 0 -10 -8 -6 -4 -2 0 2 Volume of NaOH and HCl Series1

Graph3: Absorbance versus pH of Casein

Victoire Ndong & ELina Shrestha Biochemistry 311 Tuesday lab 10/5/07 Instructor: Laurie Lentz-Marino

Absorbance versus pH
0.6 Absorbance 530 nm 0.5 0.4 0.3 0.2 0.1 0 0 2 4 pH 6 8 Series2 Series1

Calculation pH of water is 7. When 0.05 N of HCl is added it increases the volume to 21ml and the new concentration is (.05x0.001)/(.021)=0.00238 M So the new pH is log(0.00238)= 2.6 pH+pOH=14 The new concentration of OH when NaOH is added is (.05x0.001)/(.021)=0.0023 so pOH is log(0.00238)=2.6 and pH=14 2.6=11.3pH of HCl=-log(0.05x0.001)= 1.3 pOH of NaOH is -log(0.05x0.001)= 1.3 so the pH= 14 1.3= 12.7 pH = pKa +log([base]/[acid]) pKa is 4.74 ratio= 10^.06=1.148. [Base] + [acid]=0.15 so [acid]= 0.15/2.148=0.069 and [base]= 0.15-0.069=0.080 M. We only want 50ml so we will need 6.9/2=3.5ml of acetic acid and 8.0/2= 4ml of acetate

Victoire Ndong & ELina Shrestha Biochemistry 311 Tuesday lab 10/5/07 Instructor: Laurie Lentz-Marino

Buffer+ 1ml of HCl changes ratio to 0.07995/0.06905=1.22 and the new pH is 4.74 +log (0.063) = 4.8 and when NaOH is added we have the ratio 0.08005/0.06805=1.17 and the new pH is 4.81 For the pyrophosphate we chose the closest pKa which is 8.44 so the log of the ratio is 8.58.44= 0.06 and the ratio is 10^ (0.06) =1.148. We have molarity is 0.01 so [acid] =0.047 and [base] = 0.053. This time we will start with 10ml of NaOH and we will add 4.7 ml of the HCl. When HCl is added the ratio changes: 0.05295 for the base and 0.04705 for the acid. The new pH = 8.44 + log(0.05295/0.0465)= 8.49 When NaOH is added the ratio shifts the other way the new ratio is 0.05305 and 0.04605 so pH= 8.44 +log(0.0535/0.04595)=8.51 For the diluted buffer: the concentration changed and the new pH=pka3 + log ([0.00053]/[0.00047])=6.81 when HCl was added the calculated pH is 6.74 + log (0.00058/0.00465) = Calculation of the pH in each tube: pH= pKa+log([base]/[acid]) the pKa of acetic acid is 4.74 so only the ratios change: the concentration of acid is half for every dilution so we plug it in the equation pH= 4.76 + log (0.1/x) with X= the concentration of the acid. We will have first x=3.2 next is 1.6, then 0.8 and 0.4 and 0.2, then 0.1, 0.05, 0.025, 0.0125, 0.00625. The pI of glycine is pKa1+pKa2/2 it is: 6.6

Victoire Ndong & ELina Shrestha Biochemistry 311 Tuesday lab 10/5/07 Instructor: Laurie Lentz-Marino

DISCUSSION: A buffer is a solution that maintains a narrow range of pH even when strong acid are added. In our experiment, we can see how a substance that is not a buffer like water can undergo very big changes when small amounts of strong acid or strong bases are added. The pH when only iml of HCl was added went from 7 to 2.6 and when iml of NaOH was added it went to 11.3. When a buffer is present, it keeps the pH in a more narrow range. The buffer we made of acetic acid didnt undergo a lot of changes even when we added HCl and NaOH. With 1ml of HCl the pH change was almost nothing (<0.1) same for the NaOH. The pyrophosphate buffer also had the same results. A buffer however can change if diluted. When we diluted our pyrophosphate we noticed that even with a small addition of HCl the pH went very low and it went high when NaOH was added. That means that we didnt have buffer anymore. The dilution changed our solution into a non-buffer substance. In our titration of glycine, we noticed that as we added the HCl, there was not a lot of change. We kept titrating for a very long time without a lot of change but at some point the pH sudently changed drastically. The same happened for the titration with NaOH. The period where there is no change is the buffering range. The solution is able to absorb lots of acid or base without changing a lot. However, big changes can happen as soon as we are out of that buffering range. In our experiment we might have an error caused by a too fast HCl titration. It may have given us an inaccurate titration curve causing the pI to be also wrong. The normal pKa1 of Glycine is

Victoire Ndong & ELina Shrestha Biochemistry 311 Tuesday lab 10/5/07 Instructor: Laurie Lentz-Marino

around 2.1 and in our curve we got 4.1. The other possible source of error is the pH meter which was not very accurate during our titration. The pI is the point at which the net charge of the solution is ) so the ion aggregate and they are able to be detected by the spectrophotometer. The more the aggregation, the highest the absorbance is. The pI of casein corresponds to pH associated with the highest absorbance; in our case the pI is ~4.74.