Last Quarter Student
Dr. Lopez
11/16/2011
Paper
Chromatography refers to several different techniques used in separating,
isolating, and purifying compounds from a complex mixture. The process of
chromatography was first invented by a Russian botanist named Mikhail Tsvett in 1906
with his study of plant pigments.1Tsvett used calcium carbonate as the adsorbent and
petrol ether/ethanol mixture as the eluent to isolated chlorophylls and carotenoids using a
liquid-adsorption column chromatography.1 Through his research, Tsvett discovered
various colored bands as the solvent washed through the column because each substance
attached to the calcium carbonate at different degrees of strength.1 Each band represented
a different component of the plant pigment, thus Tsvett knew he successfully separated
each component from the plant.1 The band of colors was known was the chromatogram
for the isolation of each substance in a plate pigment.1 Chromatography comes from the
Greek word of chromatos meaning color and graphein meaning to write. These are strict
components in the use of chromatography and how the process is illustrated.
The process of chromatography involves separating substances from a complex
mixture and identifying them based on their chemical properties. Chromatography is used
widely in various studies, but the process is mainly used in chemical laboratories to
isolate components from a mixture, forensic studies for evidence at a crime scene, and
analytical studies for the environment. There are various areas that use chromatography
to isolate different components but they all essentially have the same process. As much as
every type of chromatography is different, every chromatography includes a mobile
phase and a stationary phase. The mobile phase is a part of the chromatographic system
that carries the solutes through the stationary phase. This component can either be a gas
or a liquid, depending on the type of chromatography used. The second part of the
chromatography system is the stationary phase. The stationary phase could be a solid or
liquid, consisting of particles or the walls of a capillary tube. The stationary phase is the
part of the system in which the mobile phase flows through, where the separation of each
component occurs. Each substance are separated due to differences in partitioning
behavior between the mobile and stationary phase allowing for the use of
chromatography to identify the substance based on chemical properties and elution time.
A chromatographic technique is chosen based on the chemical characteristic of the
mobile phase, stationary phase, and the overall chemical properties of the mixture
because separated.
The main types of chromatography are Gas Chromatography (GC), HighPerformance Liquid Chromatography (HPLC), Thin-Layer Chromatography (TLC), and
Supercritical Fluid Chromatography (SFC). Gas Chromatography is a technique used to
isolate compounds based on their volatilities. This technique is used for drug analysis,
arson investigation, toxicology, analyze organic compounds, etc. High-Performance
Liquid Chromatography is a technique similar to column chromatography but HPLC
allows the mobile phase to be forced through the column under extreme high pressures
for faster elution.2 This allows for a much tighter packing material for the stationary
phase creating a greater interaction between the stationary and mobile phase.2 This
intense interaction between the stationary and mobile phase allows for a better separation
of each component in a mixture.2 This technique is used in the analysis of food samples,
ink and drug analysis, explosive investigation, forensics, etc. Thin-Layer
Chromatography is a technique used to separate components in a mixture using capillary
action. The TLC consists of a glass, metal or plastic plate coated with solid adsorbent and
a small amount of solvent in the chamber near the bottom.3 The mixture being analyzed is
placed near the bottom of the plate, allowing the solvent to rise slowly up the plate,
passing the mixture. Capillary action is achieved by the solvent (the mobile phase)
absorbing through the stationary phase.3 The separation of the mixture is achieved using
this technique because each analyte travels the TLC plate at different rates, leaving
reference bands along the way.3 TLC is used in ink analysis, determination of purity of a
mixture and DNA research. Supercritical Fluid chromatography is a technique used in
separating thermally unstable molecules in a mixture by carrying out the sample through
a separating column using a supercritical fluid.4 The mobile phase is usually carbon
dioxide; therefore the flow path for the analytes must be pressurized.4 SFC is a fairly new
technique that is applied in the pharmaceutical industry for purification. The two
techniques that is further discussed in this report, using applications and examples in
Forensic Science, is the Gas chromatography (GC) and the High-Performance Liquid
Chromatography (HPLC).
Forensic Science is an application of scientific methods to the law.5 This specific
application plays an important role in the judicial system by providing the scientific
components to investigators and courts.5 Forensic Science provides the different
techniques to analyze crime scenes and answer questions that are of interest of the court
system. Chromatography is used to provide specific information in regards to a crime
scene, such as blood, DNA, hair samples, etc. Two main types of chromatography used in
Forensic Science is Gas Chromatography and High- Performance Liquid
Chromatography
Gas Chromatography is a specialized technique used to separate analytes based
solely on their volatilities.6 This chromatography provides specific information of each
individual component that is present in a complex mixture, such as purity and quantity.
Gas Chromatography, like all chromatography techniques, consists of a mobile phase and
a stationary phase. The mobile phase is the inert gas, usually helium or hydrogen, and the
stationary phase is the high boiling liquid or solid that the gas travels through.6 The
sample is introduced to the GC using an injector. An injector is a glass cylinder that is
thermally stable, allowing all the components in the mixture to vaporize.6 A small portion
of the sample is injected into the GC column followed by the carrier gas. The compounds
travel through the stationary phase as a gas, if the compound is not a gas then the
compound is then heated and vaporized to the gaseous form.6 The stationary phase is
composed of a heated column containing a high boiling liquid allowing the inert gas to
travel back and forth along the column wall. This interaction time between the inert gas
and the stationary phase allows for different rates of each component to elute, therefore
each component is separated in time and space based on their interaction behavior.6 As
each compound elutes from the column, the detector records each passing analyte and
sends a message to the designated recorded.6 The recorded will respond to the
information by marking a peak for each component in the mixture.
The use of Gas Chromatography and its techniques were used in a forensics crime
case involving the death of Ryan Stallings. Ryan’s mother, Patricia Stallings, was
convicted and blamed for the death of her infant by poisoning him with ethylene glycol,
the main component in antifreeze.7 When Ryan was rushed to the hospital for his ill-like
symptoms, the hospital reported Ryan having metabolic acidosis (low pH in his blood)
and also reported him have 180mg/L of ethylene glycol in his system.7 Ryan was put in
foster care with having weekly visits from his parents.7 On one unsupervised visit Patricia
gave Ryan a bottle and after Ryan fell extremely ill, dying 3 days later of metabolic
acidosis.7 The hospital reported Ryan having 911mg/L of ethylene glycol in Ryan’s blood
and baby bottle, which was found in the dishwasher.7 As the court went on, the
prosecutor argued how there was no rationally explanation for these findings; rather this
was all deliberate poisoning.7 Patricia Stallings was arrested after investigators found a
half bottle of antifreeze in her basement.7 Patricia’s other son, David, was placed into
foster care where later he experienced the same ill-like symptoms.7 The hospital
diagnosed him with a genetic disorder Methylmalonic acidemia.7 This disease is when
one enzyme in a particular biological pathway is defective, causing a build up of
compounds in the bloodstream.7 This is an extremely rare disease, but this meant Ryan
had a 1 in 4 chance of having it as well.7 This idea was not presented to the trial of
Patricia Stallings.7 A biochemist professor, William Sly, became involved in the case
calling the investigation suspicious.7 Sly and his team examined Ryan’s blood using
GC/MS, and the substance the prosecutors thought to be ethylene glycol was actually
propionic acid.7 Propionic acid is a substance that should have been expected to be in
Ryan’s system if he did in fact have the same genetic disorder as his brother, David.7 For
further testing, the team spiked Ryan’s blood sample with ethylene glycol and discovered
the unknown sample they thought was ethylene glycol and the known sample of ethylene
glycol did not have the same retention time.7 Later, Dr. Rinaldo reexamined Ryan’s blood
and it was discovered that he had methylmalonic acid and proionic acid contents in his
blood.7 Both of these components are found in a person suffering from Methylmalonic
Acidemia.7 Dr. Rinaldo further explained that the commercial lab that first studied
Ryan’s blood using Gas Chromatography found that the unknown sample in his blood
had a retention time within 30 seconds of the standard ethylene glycol, assuming that the
30 seconds was experimental error.7 Another suspicious evidence that was found was the
baby bottle testing positive with ethylene glycol.7 The baby bottle had been washed in the
dishwasher before the testing was done raising a red flag since ethylene glycol is highly
water-soluble.7 The case of Patricia Stalling was reopened and all of her charges were
dropped.7
The second technique further discussed is High-Performance Liquid
Chromatography. High-Performance Liquid Chromatography is a technique used to
separate components in a mixture using high pressures. In regular Column
Chromatography, the solvent is eluted out of the column under gravitation forces whereas
in HPLC, the solvent is forced through the column under high pressures for a much faster
elution. The higher pressure allows for the stationary phase to consist of smaller particles
for the packing material, which then gives a greater interaction between the stationary
phase and mobile phase.2 This greater interaction results in a better separation of each
component in a mixture. HPLC consist of two different types of chromatography based
on the polarity of the mobile and stationary phase, Normal-Phase Chromatography and
Reversed-Phase Chromatography. In Normal-Phase Chromatography the stationary
phase is polar and the mobile phase, the solvent, is non-polar. As the solvent travels
through the stationary phase, the polar components in the mixture will attach longer to
the polar silica column than the non-polar components.2 Thus, the non-polar components
in the mixture will elute from the column more quickly.2 In Revered-Phase
Chromatography the stationary phase is non-polar and the mobile phase is a polar
solvent. In this phase, there is a strong attraction between the polar components in the
mixture and the polar solvent, resulting in the polar components in the mixture moving
through the column at the same rate as the polar solvent.2 Due to Van Der Waals forces,
the non-polar components will form an attraction with the hydrocarbon groups of the
modified non-polar silica.2 This attraction decreases the elution rate for the non-polar
components in the mixture, resulting in a slower elution than the polar components.2 In
HPLC, the Reversed-Phase is more commonly used than the Normal-Phase.
An example of HPLC and its techniques were used in the case and trial of O.J.
Simpson. During the O.J. Simpson investigation, investigators concluded that DNA
results found that blood evidence on a gate at the crime scene belonged to O.J. Simpson
and blood found on socks at the Simpson’s residence was from the murder victim Nicole
Simpson.8 During the investigation, blood samples were taken from both O.J. Simpson
and the murder victim Nicole Simpson in test tubes containing ethylenediamine
tetraacetic acid (EDTA), a blood preservative.8 As the prosecutors claimed that the blood
at the crime scene belong to O.J. Simpson, the defense argued that the police purposely
placed the blood in these designate areas by using the blood samples they collected from
O.J. and Nicole Simpson after the murder.8 Chief Roger Martz worked to develop a
method for the identification of EDTA in a blood sample.8 Martz used HPLC to isolate
the EDTA in the original blood sample of O.J. Simpson and Nicole Simple, the blood
found on the gate of the crime scene and the sock found at the Simpson residence that
contained the blood of Nicole Simpson.8 From his investigations, Martz found that
EDTA was only present in the blood samples from O.J. and Nicole after the murder, but
did not find any evidence of EDTA in the blood on the gate and the sock that had the
blood of the murder victim.8 Martz did testify against O.J. Simpson based on the
information he discovered, but the court was not able to make Martz accusations credible
because Martz couldn’t provide them with all the digital data regarding the HPLC testing
that took place.8 O.J. Simpson was found not guilty of the murder of Nicole Simpson.
The use of chromatography is widely used throughout all studies.
Chromatography is constantly developing into different techniques in order to separate
components from a mixture based on their chemical properties. The techniques have
evolved from using just a simple chromatography of separating two components to using
a gaseous state mobile phase for the isolation of several components in a mixture.
Although the techniques are widely changing, chromatography will always have a
stationary phase and a mobile phase. There are several techniques used in Forensic
Science but the use of chromatography is one of the key processes used within the court
system. The examples involving criminal cases against Patricia Stallings and O.J.
Simpson were only two cases that the process of chromatography took place. For Patricia
Stallings, the use of chromatography opened a new investigation that later over turned her
initial sentence. Forensic Science will always need chromatography techniques to help
investigators in a crime and as new technology develops, more precise processes will
soon be discovered.
References
1
"Chromatography - Used, First, Blood, Body, Plant, Uses, History, The First
Chromatograph, Ion-Exchange Chromatography, Paper Chromatography, Martin and
Synge, Gas Chromatography." Medical Discoveries. Web. 09 Nov. 2011.
<http://www.discoveriesinmedicine.com/Bar-Cod/Chromatography.html>.
2
"High Performance Liquid Chromatography - Hplc." Chemguide: Helping You to
Understand Chemistry - Main Menu. Web. 09 Nov. 2011.
<http://www.chemguide.co.uk/analysis/chromatography/hplc.html>.
3
"Thin Layer Chromatography." CU Boulder Organic Chemistry Undergraduate Courses.
Web. 09 Nov. 2011. <http://orgchem.colorado.edu/hndbksupport/TLC/TLC.html>.
4
"Supercritical Fluid Chromatography (SFC)." The Charles Edward Via, Jr. Department
of Civil and Environmental Engineering. Web. 09 Nov. 2011.
<http://www.cee.vt.edu/ewr/environmental/teach/smprimer/sfc/sfc.html>.
5
"Welcome - What Is ?" Forensic Science. Web. 09 Nov. 2011.
<http://forensics.shsu.edu/welcome/fsis.html>.
6
"Gas Chromatography." CU Boulder Organic Chemistry Undergraduate Courses. Web.
09 Nov. 2011. <http://orgchem.colorado.edu/hndbksupport/GC/GC.html>.
7
¨“How Does the Patricia Stallings Case Shed Light on the Murder of Meredith
Kercher?” View-from-Wilimington. Web. 09 Nov. 2011.
http://viewfromwilmington.blogspot.com/2010/06/how-does-patricia-stallings-case-shedlight.html>.
8
1995, Early February. “USDOJ/OIG FBI Labs Report.” Welcome to
States Department of Justice. Web. 09 Nov. 2011.
http://www.justice.gov/oig/special/9704a/07simpso.htm.
the United