Effective Date: 02July2013 Review Date: 24February2014

Author: E. Lawrence

SOP #1254
Page 1 of 5


Background
Chromatography is a technique that separates mixtures to identify the individual components. There are
many variations to this technique, but usually a mixture is placed on a filter and a solvent is allowed to
pass through the filter. Different types of molecules interact with the filter differently depending on what
the molecules look like. As the solvent passes through the filter, some of the molecules may dissolve and
travel with the solvent through the filter. The remainder of the molecules will get stuck on the filter.
Based on the mixture and the abilities of its molecules to dissolve in different solvents and attach to the
filter, the components in the mixture will start to separate. This makes it easy to determine what was
present in the original mixture.

Vocabulary
Mixture: A substance that is made when two substances are blended or mixed together.
Solvent: The liquid a substance is dissolved in to form a solution
Dissolve: A solid is incorporated into a liquid to form a solution.
Solution: A liquid mixture.
Molecule: A group of atoms bonded together. A molecule represents the smallest unit of an element or compound.
Elute: To remove by dissolving.

Shark Science Group Develops GrapeX to combat MS Symptoms
Multiple Sclerosis (MS) is a disease that affects the brain and spinal cord. Patients with MS will
experience tingling, numbness, bladder problems, difficulty walking, dizziness, fatigue, and muscle
spasms. GrapeX is a drug designed to help people with MS. GrapeX, designed by the Shark Science Group,
combats the mild side effects of MS and makes living with the disease manageable. With the future
looking bright, the Shark Science Group quickly acquired GrapeX approval by the FDA and is being
manufactured as we speak. However, with short time lines and high demand for the drug, Shark Science
Group has to make sure they maintain quality.

Maintaining Quality - Putting the Patient First
As a member of the Microbiology Quality Control team you need to keep the Shark Science Group on their
toes. GrapeX was designed to

contain two molecules, Red #40 and Blue #1 in a water solution. You can
see from the picture that the molecules are a little different.

Figure 1. GrapeX Molecules
Effective Date: 02July2013 Review Date: 24February2014
Author: E. Lawrence

SOP #1254
Page 2 of 5


You will need to test each batch of drug that is being released to market for environmental
contamination. You will be testing each batch to ensure that it only contains the two molecules that are
supposed to be in it. Batches have the potential to contain endotoxin, a harmful chemical given off, due to
a bacterial contamination. If a batch of GrapeX is contaminated, it can cause substantial side effects
including fever, vomiting, diarrhea, and memory loss. This batch should not be packaged and sent to
market. The Shark Science Group needs you!

You will complete the technique of reverse phase liquid chromatography to separate the GrapeX mixture.
You will use the Sep-pak C-18 columns that will bind molecules that do not dissolve in water. Both the
Red #40 and the Blue #1 molecules will dissolve in water, but when they are passed through a Sep-pak
C18 filter they prefer to stick to this filter instead of staying dissolved in the water. When you pass two
solutions of varying percentages of isopropanol alcohol across the filter the molecules will become more
attracted to the isopropanol alcohol and will elute from the column.

These columns are widely used for sample preparation in the laboratory and are used to remove
substances from water solutions. The Waters Corporation that makes the columns states that the Sep-
pak C-18 columns are used to separate drugs and their metabolites in serum, plasma or urine, desalting
of peptides, trace organics in environmental water samples, and organic acids in beverages.
Effective Date: 02July2013 Review Date: 24February2014
Author: E. Lawrence

SOP #1254
Page 3 of 5


1.0 Purpose
The purpose of this SOP is to specify the procedures for running a liquid chromatography experiment to determine
the purity of a batch of GrapeX.
2.0 Scope
This SOP describes the methods used to run one chromatography column.
3.0 Responsibility
All scientists in the QC-Microbiology group will be responsible for following the correct protocol for testing drug
GrapeX for purity.
4.0 References
Liquid Chromatography Test Record
5.0 Safety
Safety glasses/goggles, lab coats, gloves must be worn at all times and all materials must be handled with care.
6.0 Materials and equipment
6.1 Sep-Pak C18 cartridge (aka Cartridge)
6.2 Sample to test
6.3 Distilled Water
6.4 70%, 35% and 8% Isopropyl Alcohol
6.5 6-well collection plate
6.6 Marker
6.7 Waste beaker
6.8 Syringe, 10mL

7.0 Procedure
NOTE: Before beginning you must fill out your Liquid Chromatography Test Record and fill in your results as you
complete the experiment
7.1 Preparing Cartridge
7.1.1 Using the 10mL syringe, collect 5mL of 70% isopropyl alcohol
7.1.2 Firmly attach the Sep-Pak C18 cartridge to the end of the syringe
7.1.3 Slowly and carefully, pass the 70% isopropyl alcohol through the cartridge, colleting the waste
material in the beaker labeled Waste
7.1.4 Remove the cartridge
7.1.5 Using the 10mL syringe, collect 5 mL of distilled water
7.1.6 Firmly attach the cartridge to the end of the syringe
7.1.7 Slowly and carefully, pass the water through the cartridge, colleting the waste material in the
beaker labeled Waste
7.1.8 Repeat steps 7.1.4 through 7.1.7
7.1.9 Remove the cartridge It is now ready for your test sample

Effective Date: 02July2013 Review Date: 24February2014
Author: E. Lawrence

SOP #1254
Page 4 of 5



7.2 Prepare Test Sample
7.2.1 With the marker, label the six wells in your 6-well collection plate with the numbers 1 through 6.
Each well will collect the following items:
7.2.1.1 Well #1: Testing sample
7.2.1.2 Well #2: 8%
7.2.1.3 Well #3: 8% Repeat
7.2.1.4 Well #4: 35%
7.2.1.5 Well #5: 70%
7.2.2 Using the 10mL syringe, collect 5mL of your test sample
7.2.3 Firmly attach the cartridge to the end of the syringe
7.2.4 Slowly and carefully, pass the 5mL of sample through the cartridge, collecting the material in
Well #1 (NOTE: the band of color should be concentrated near the inlet of the cartridge)
7.2.5 Remove cartridge

7.3 Well #2: 8%
7.3.1 Using the 10mL syringe, collect 5mL of 8% isopropyl alcohol.
7.3.2 Firmly attach the cartridge to the end of the syringe
7.3.3 Slowly and carefully, pass the 5mL of 8% isopropyl alcohol through the cartridge, collecting the
liquid material in Well #2.
7.3.4 Remove cartridge

7.4 Well #3: 8% Repeat
7.4.1 Using the 10mL syringe, collect 5mL of 8% isopropyl alcohol.
7.4.2 Firmly attach the cartridge to the end of the syringe
7.4.3 Slowly and carefully, pass the 5mL of 8% isopropyl alcohol through the cartridge, collecting the
liquid material in Well #3
7.4.4 Remove cartridge

7.5 Well #4: 35%
7.5.1 Using the 10mL syringe, collect 5mL of 35% isopropyl alcohol.
7.5.2 Firmly attach the cartridge to the end of the syringe
7.5.3 Slowly and carefully, pass the 5mL of 35% isopropyl alcohol through the cartridge, collecting the
liquid material in Well #4
7.5.4 Remove cartridge





Effective Date: 02July2013 Review Date: 24February2014
Author: E. Lawrence

SOP #1254
Page 5 of 5


7.6 Well #5: 70%
7.6.1 Using the 10mL syringe, collect 5mL of 70% isopropyl alcohol.
7.6.2 Attach the cartridge to the end of the syringe
7.6.3 Slowly and carefully, pass the 5mL of 70% isopropyl alcohol through the cartridge, collecting the
liquid material in Well #5
7.6.4 Remove cartridge
8.0 Results
8.1 Well #1: Sample This well should be light pink/clear. The color should be captured in the column.
8.2 Well #2: 8% This well should contain red dye
8.3 Well #3: 8% Repeat This well should contain both red and blue dye and it will be purple
8.4 Well #4: 35% This well should contain blue dye
8.5 Well #5: 70% This well should contain the clear/light blue-flavoring oils