Preparing Phosphate Buffer Solution & Determining Bromophenol Blue Extinction Coefficient | Lab Reports Biochemistry

Carlyn Annunziata

Bio 340 lab

Report: Creating a Buffered solution and Extinction Coefficient of Bromophenol Blue

Introduction

Buffers are aqueous solutions that resist change in hydrogen ion concentration when an acid or a base

is introduced to the system. Buffered solutions are comprised of a weak acid and its conjugate base,

which can be created one of several ways; using weak acids or weak bases and their respective salts,

two salts that provide a conjugate acid-base pair, adding a strong acid or strong base to a weak acid or

weak base(Pietri & Land, 2020). The pKa of a buffered solution is its pH value when the buffering species

concentrations are equivalent. Additionally, pKa represents where the buffer’s maximum capacity is

achieved(Urbansky & Schock, 2000). Biological buffers, such as phosphate buffers, maintain a pH of

approximately 7.4, which closely simulates the internal pH of the human body in the absence of

pathological states (= 7.35-7.45). Phosphoric acid is a polyprotic acid which is capable of donating three

protons, and therefore has three pKa values. Phosphate buffers therefore are effective biological buffers

which may be prepared to maintain a pH near any of the three pKa values(Soderberg, 2021).

In the following two-part experimental report, a buffered solution is prepared using Sodium phosphate

monobasic (NaH2PO4), which acts as the weak acid species and contains a pKa within the range 6.8-

7.20, and Sodium phosphate dibasic (Na2HPO4), which acts as the conjugate base species and contains a

pH ranging from 8 to 11. The mole ratio of the acid and base components of the buffer, the number of

moles for both the acid and base component, and the molar concentrations necessary to prepare the

phosphate buffered solution were determined using the Henderson Hassel Balch equation. From the

number of moles of each buffer component and their respective molar concentrations, the acid and

base volumes required to prepare the buffer solution were calculated using equations 4 and 5.

In the second part of this experiment, the acid base indicator Bromothymol blue and deionized water

were pipetted into 12 different wells in a microtiter plate, with each well containing a constant volume

of the prepared buffer solution from part one. For each subsequent well, the concentration of

Bromothymol blue was decreased. The absorbance, or measure of the ability of a substance to absorb

light, of the Bromothymol blue in the buffered solution was analyzed using a spectrophotometric plate

reader. Bromothymol blue turns yellow when added to an acidic solution, blue in basic solution and

green in neutral solutions (NCBI,2021). For buffered solutions containing Bromothymol blue which are

yellow, the spectrophotometric plate reader wavelength is set to 405nm and for solutions which are

blue the wavelength is set to 570nm(BioTek, 2021). The beer-lambert law was used to determine the

extinction coefficient of Bromothymol blue at the wavelength specified by the color of the solution. The

Beer-Lambert Law is expressed as 𝐴 = 𝜀𝑙𝑐, where 𝐴 represents the absorbance of the Bromothymol

blue, and 𝜀 represents the extinction coefficient of the Bromothymol blue, where both values were

measured at 570nm wavelength and pH of 6.9 for the buffered solution. The extinction coefficient is a

measure of how strongly a chemical species or substance absorbs light at a given wavelength(AAT

Bioquest, 2019). According to the beer-lambert law, absorbance is proportional to the path length, 𝑙,

through the sample and the concentration of the absorbing species, 𝑐 (D.Ball,2006).

Preparing Phosphate Buffer Solution & Determining Bromophenol Blue Extinction Coefficient, Lab Reports of Biochemistry