Name: Mafe Nenia A.

Mejias

Student Number: 2020-04903

Section: A

1. Read the journal article entitled, “Separation techniques: Chromatography” and answer the question
that follows (9 pts):

Given the following mixtures, which type of chromatography would you use to separate the individual
components? Explain your answer.

a. A mixture containing basic (positively charged) proteins

The type of chromatography that we will use to separate basic (positively charged) proteins is the
Ion Exchange Chromatography. Ion exchange Chromatography is used in electrostatic interactions
between charged protein groups, and solid support material (matrix). The matrix should be negatively
charged since the protein is positively charged because to separate the mixture, the matrix should have
an ion load opposite to the protein. The affinity of the protein is achieved with ionic ties. Proteins can be
separated into the column by changing pH, the concentration of ion salts, or the ionic strength of the
buffer solution.

b. A mixture containing enzymes, hormones, or nucleic acids

The type of chromatography that we will use to separate a mixture containing enzymes,
hormones, or nucleic acid is Affinity Chromatography. Affinity Chromatography is used for the
purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins. In this separation
process, we isolate a single compound from a complex mixture. The technique is based on the specific
binding of that one compound to the stationary phase. When the sample is passed through the column,
only one solute is bound. After everything else has washed through, the one adhering solute is eluted by
changing a condition such as pH or ionic strength to weaken its binding.

c. A mixture containing 1500 kDa, 1000 kDa, and 500 kDa proteins

In this mixture we can observe the varying sizes of proteins. The type of chromatography that we
will use to separate this mixture is the Gel-permeation (molecular sieve) chromatography. This
separation process is used to separate macromolecules using their size. Small molecules penetrate the
pores in the stationary phase, but large molecules do not. Because small molecules must pass through an
effectively larger volume, large molecules are eluted first.
2. The level of Fe in an industrial waste stream can be determined by reacting it with the o-
phenanthroline at a pH range of 3.0–3.5 to form the deep orange-red Fe(phen)32+ complex. The
absorbance of which is recorded at a wavelength of 510 nm in a 1.00 cm cell. Given the following data
below, calculate the concentration of Fe in mg/L present in the waste stream sample (5 pts).

ppm Fe Absorbance
0 0
1 0.183
2 0.364
3 0.546
4 0.727
Sample 0.459
Solution:

a. Plot a calibration curve and determine the equation of the line. (Used spreadsheet)

Calibration Curve
0.8
0.7 y = 0.1817x + 0.0006
0.6
Absorbance

0.5
0.4
0.3
0.2
0.1
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Concentration (ppm or mg/L)
b. Find the concentration of the unknown using the equation of the line.

ppm Fe Absorbance
0 0
1 0.183
2 0.364
3 0.546
4 0.727
Sample 0.459
y= 0.1817x + 0.0006

y- 0.0006 = 0.1817x
0.1817 0.1817

x= y- 0.0006
0.1817

x= 0.459- 0.0006
0.1817

x= 2.522839846 mg/L

x= 2.52 mg/L

• The concentration of Fe present in the waste stream sample is 2.52 mg/L.

3. In light of what you have learned in this course, propose a study that you are interested in that will use
any of the different analytical techniques. Write the study objective and identify the method/s or
technique/s of your choice. Explain how the method can be used to meet the objective. Outline your
answers as shown below. (6 pts).

Study objective: Chemical Analysis of Fire Retardant Paint Coatings

Method/s: The process involved in this study is the Gas Chromatography and Mass Spectrometry.

Explanation: The aim of this research is determined and observed the toxicity of fire-retardant paints and
their possible harmful effects on humans after inhalation. The research will be conducted by getting
samples from different commercialized fire-retardant paints. The different compounds in the mixture of
paints will be determined by various temperatures separated by Gas Chromatography and identified the
mass ratio using mass spectrometry. After, determining the compounds the researchers will be able to
identify the toxins and their possible effect on humans.