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CHROMATOGRAPHY

Forensic
Chemistry

Identification of Marijuana
Microscopic Examination
Look for Bear Claw cystolythic hair on top
surface of leaf
Duquenois-Levine Color test (Screening)
2% vanillin, 1% acetaldehyde in Ethanol
1. Hydrochloric acid: purple color
2. Chloroform heaver than water forms lower
layer: Will pull purple color into lower layer

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Marijuana
View the sample at varying magnifications
(approximately 10 40x) using a stereomicroscope.
Cystolithic hairs are unicellular, bear claw shaped
hairs with a cystolith of calcium carbonate, CaCO3,
at the base.

Duquenois-Levine Color Test

On the left is the purple HCl step indicating the
presence of THC. Followed by the chloroform step
which also indicates THC. The chloroform layer is
on the bottom

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Chromatography
Mobile Phase

Sample
in

Sample
out

Solid Phase a solid or heavy liquid coated onto a

solid or support system

Chromatography
Definition:

Concentration of component A in stationary phase

Concentration of component A in mobile phase
Different affinity of these 2 components to
stationary phase causes the separation.
Distribution Coefficient (Equilibrium Distribution)

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Kinds of Chromatography
1. Thin-layer
2. Liquid Chromatography
3. Gas Chromatography

Chromatography is Used to:

1. Analyze Examine a mixture, its components, and
their relations to one another
2. Identify Determine the identity of a mixture or
components based on known components
3. Purify Separate components in order to isolate
one of interest for further study
4. Quantify Determine the amount of the a mixture
and/or the components present in the sample

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Chromatography is Used by:

Pharmaceutical Company Determine amount of each
chemical found in new product
Hospital Detect blood or alcohol levels in a patients
blood stream

Law Enforcement Compare a sample found at a

crime scene to samples from suspects
Environmental Agency Determine the level of
pollutants in the water supply
Manufacturing Plant Purify a chemical needed to
make a product

Thin-Layer Chromatography
Separates dried liquid
samples with a liquid
solvent (mobile phase) and
stationary phase of silica gel
or alumina (stationary
phase) deposited on a sheet
of glass or aluminum

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Thin-Layer Chromatography
The analytes are separated
by the effect of capillary
action. The solvent or eluent
is drawn up the TLC plate.
There is now a competition
and material with a greater
attraction to the solvent will
move further up the plate.

Thin-Layer Chromatography
Silica gel is
covered in
hydroxy groups
or can be
further
functionalized

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Thin-Layer Chromatography
Alumina (Al2O3) is found in a wide variety of
particle sizes.

Aluminas surface is
functionalized with a
combination of oxygens and
hydroxyl groups

Thin-Layer Chromatography
With its polar hydroxy groups Silica Gel is
better suited for separating polar compounds,
while alumina is more frequently used for
nonpolar compounds

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Thin-Layer Chromatography
Think about the yellow as the solvent with a
mixture of 3 components (white, blue and red)

Analyze
Identify

Separate

Purify
Mixture

Components

Quantify

Thin-Layer Chromatography
Compound is placed on a stationary phase
Mobile phase passes travels up the stationary
phase via capillary action.
Mobile phase solubilizes components
The mobile phase carries the individual
components through the stationary phase
depending upon the relative attraction of the
components to both the mobile and stationary
phase

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Thin-Layer Chromatography
Selection of an appropriate mobile and
stationary phase is critical to ensure separation

Analyze
Identify

Separate

Purify
Mixture

Quantify

Components

Thin-Layer Chromatography
Stationary Phase

Separation

Mobile Phase

Components

Affinity to Stationary
Phase

Affinity to Mobile
Phase
Insoluble in Mobile Phase

Blue

----------------

White

Red

Yellow

Same as mobile phase

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Thin-Layer Chromatography
Chromatography of a Sharpie

Thin-Layer Chromatography
Chromatography of a Sharpie

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Thin-Layer Chromatography
Chromatography of a Sharpie in 50% Isopropanol

Thin-Layer Chromatography
Chromatography of a Sharpie in 50% Isopropanol

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Thin-Layer Chromatography
Chromatography of a Sharpie in 100%
Isopropanol

Solvent Effect
By varying the solvent or solvent ratio, elution
can be controlled (% Isopropanol)

0%

20%

50%

70%

100%

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Chromatography of Ink
Idealized separation of ink samples using TLC

Calculating Rf Values
A polar solvent will carry a
polar compound further
while a nonpolar solvent will
carry a nonpolar compound
further.
An Rf value can be calculated
for the distance the
compound travels.

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Calculating Rf Values

Calculating Rf Values
An Rf of value of 0.0 to 1.0 is possible.
An Rf value of 0.0 indicates an analyte has bound
to the stationary phase (not mobile)
An Rf value of 1.0 indicates virtually no interaction
with the stationary phase
Values between 0.2 and 0.8 provides the best
separation of compounds

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Liquid Column
Chromatography
The ideas
developed in TLC
are the same in
column
chromatography

Liquid Column Chromatography

The TLC plate is replaced by a
column packed with the
stationary phase
These columns can vary
dramatically in size and length
This technique is suited for
sample purification and isolation

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Liquid Column Chromatography

The silica and alumina used in TLC can be replaced
by a wide range of stationary phases.

Analyte
Stationary
Phase

Liquid Column Chromatography

Three possible interactions must be considered in
all of these chromatography applications
1. Solute Solvent
2. Solute Stationary phase
3. Solvent Stationary phase

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Liquid Column Chromatography

Particularly strong Solute Stationary phase
interactions may produce poor separation with
little or no movement of the solute.
By increasing Solute Solvent interactions the
mobility of the solute can be improved.
It must be remembered that both the solvent and
the stationary phase are competing for the solute
via intermolecular interactions.

Liquid Column Chromatography

Differential adsorption of the solute will result in
the progressive separation of a mixture of
compounds.

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Liquid Column Chromatography

Compounds which interact weakly with the solid
phase will elute first from the column.

Liquid Column Chromatography

Compounds which interact strongly with the solid
phase will have increased retention times and will
elute last.

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Liquid Column Chromatography

If movement of the compounds are too rapid or
too slow little or no separation will be noted.

High Performance Liquid Chromatography

This entire liquid chromatography system can be scaled up and

automated.

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High Performance Liquid Chromatography

Bench-top HPLC

Column
HPLC typically uses a packed
column
Stationary phases are
particles which are usually
about 1 to 20 m in average
diameter (often irregular in
shape)
HPLC is typically used for
molecular weights less than
2,000

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Column Stationary Phase

High surface area and functionalized with a wide variety of groups

High Performance Liquid Chromatography

Mobile Phase Liquid
Stationary Phase Solid, small diameter particles
with a wide range of functionalities available
Pressure A high pressure is used to force the
sample and solute through the system
Advantages High resolution and faster
resolution

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High Performance Liquid Chromatography

Normal Phase The stationary phase is a polar
with a nonpolar solvent (mobile phase)
Reverse Phase The stationary phase is nonpolar
with a polar solvent (mobile phase)

HPLC Normal Phase

NP-HPLC is best suited for separation of non-polar
analytes
Stationary Phase Typically uses a polar
stationary phase
Mobile Phase Uses a non-polar solvent and
works best for separating non-polar compounds
RP-HPLC is more commonly used

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HPLC Reverse Phase

RP-HPLC is best suited for separation of polar
analytes
Stationary Phase Typically a non-polar media
such as silica or styrene covalently modified with
alkyl chains (3 to 18 carbons)
Mobile Phase Includes water, alcohols, CH3CN

TLC vs. NP-HPLC vs. RP-HPLC

Normal Phase (SiO2) TLC

Normal Phase (SiO2)

c
Time

0
b

Reverse Phase (C18)

c
c

x
0

Time

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HPLC Retention Times

Some factors which effect retention times
1. Retention times will be reduced by the use of
higher pressures. Often 4,000+ psi
2. Functionalization of the stationary phase.
Does it contain polar or non-polar groups.
3. The particle size of the stationary phase. Smaller
particles increase retention times
4. Solvent composition

Types of Stationary Phases

Alltech Chromatography Sourcebook, 2004-04 catalog

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Types of
Stationary
Phases

Alltech Chromatography
Sourcebook, 2004-04
catalog

Stationary Phase Size

Consider particle 1 and 2 start at the same point, particle 2 must travel
further through smaller particles

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Solvent Polarity
Hexane
Petroleum Ether
Carbon tetrachloride
Toluene
Diethyl ether
Dichloromethane
Tetrahydrofuran
Acetone
Ethanol
Acetonitrile
Dimethyl sulfoxide
Water

0.0
0.0
1.6
2.4
2.8
3.1
4.0
5.1
5.2
5.8
7.2
10.2

Solvent Problems
Excessively polar solvents such as water, alcohols
and acetonitrile can move all analytes along and
reduce resolution or peak separation
Water and alcohols can react with the analyte.
While an interaction between the solvent and
analyte is desirable, reaction is not.
Once a fraction is collected, separation of very
polar solvents such as water from the analyte can
be difficult.

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Functionalization of Stationary Phase

Residual Si-OH groups can produce tailing in the chromatogram to

minimize this effect these may be capped with chlorotrimethylsilane

Elution Problem

Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998

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Solvent Composition

Gradient Elution - By vary solvent composition separation of

mixtures can be enhanced

Detectors

A number of detection methods exist once separation has been achieved

by HPLC with UV-Visible spectroscopy the most common

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UV-Visible

This is a nondestructive test relatively inexpensive technique. If a

compound does not absorb at a particular frequency, a series of
detectors can be placed in line operating at different frequencies.

Absorption Spectra

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General Factors to Increase

Resolution for HPLC
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Increase column length

Decrease flow-rate
Pack column uniformly
Use smaller stationary phase
Decrease sample size
Select proper stationary phase
Select proper mobile phase
Use a Solvent gradient
Decrease pressure
Use a solvent gradient elution

Gas Chromatography

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Gas Chromatography

GC Advantages
1. Requires very small sample size ml

2. Very good separation/resolution

3. Time (analysis is short 1 to 100 minutes)
4. Greater sensitivity to low concentrations
5. Relatively simple equipment

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GC Column

GC Oven

The column is hung in the oven while temperatures are maintained

within 0.1 C up to 450 C

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Gas Chromatography
The analyte must be volatile
Mobile Phase For the most part the mobile
phase does not interact with the analyte and we
have a limited group of gasses to choose from,
Helium, Argon, Nitrogen, Hydrogen. Helium and
Nitrogen are the most common.
Stationary Phase Typically coated on the wall of
the capillary tube

The Stationary Phase

A thin film of a variety of possible stationary phases coats the interior of

the capillary. These are typically 0.25mm inside diameter and 15
meters, 30 meters or longer.

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The Stationary Phase

The Fused Silica columns are fragile and are coated with a polyimide
coating to provide flexibility and to prevent breakage. They are
suspended in the oven.

Stationary
Phase
Like Dissolves
Like
Nonpolar columns
for nonpolar
solutes
Polar columns for
polar solutes

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Retention Time
The time required for the
sample to travel from the
injection port through the
column to the detector.
A number of factors can
effect Retention Time

3
Inject
1

10

15

20

Column: u Bondapak C18

Solvent: M eOH
Sample: Water-Soluble Vita

Gas Chromatography Retention Time

Volatility is the primary factor in compound
separation.
The least volatile will be retained the longest
In general polar compounds will have the
lowest volatility
Interaction between the compound and the
stationary phase is a secondary factor in retention

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GC Tube Diameter

Resolution can be improved by decreasing the tube diameter

GC Tube Length

Resolution can be improved by increasing column length

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GC Stationary Phase

Retention times can be adjusted by changing the polarity of the column

GC Stationary Phase

Resolution can be improved by increasing the stationary phase thickness,

but this can decrease the stability of the stationary phase

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GC Temperature Gradient

Temperature gradients can be used to increase resolution and reduce

retention time

GC Temperature Gradient

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Temperature Gradient
240
200

Temp (deg C)

While a temperature
gradient can be useful
an excessively cool oven
could lead to
condensation and
temperature which are
too high can lead to
degradation of the
analyte

160
120
80
40
0
0

10

20

30

40

50

60

Time (min)

Gas Chromatography

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GC Split Injector
The sample is
injected through the
septum where it is
heated to ensure that
the sample is
vaporized instantly

Thermal Conductivity Detector

A nondestructive detector,
it is a filament which is
cooled by the carrier gas.
Helium passes across the
filament as the analyte
passes over the filament it
heats in a way proportional
to its concentration.

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GC of Gasoline
1. Isobutane
2. n-Butane
3. Isopentane
4. n-Pentane
5. 2,3-Dimethylbutane
6. 2-Methylpentane
7. 3-Methylpentane
8. n-Hexane
9. 2,4-Dimethylpentane
10.Benzene
11.2-Methylhexane
12.3-Methylhexane
13.2,2,4-Trimethylpentane
14.n-Heptane
15.2,5-Dimethylhexane
16.2,4-Dimethylhexane
17.2,3,4-Trimethylpentane
18.Toluene
19.2,3-Dimethylhexane
20.Ethylbenzene
21.m-Xylene
22.p-Xylene
23.o-Xylene

Identification of an Unknown
Response
Mixture of known compounds

Octane

Decane

1.6 min = RT
Hexane

GC Retention Time on Carbowax-20 (min)

Response

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)

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General Factors to Increase Resolution

1.

Increase column length

2.
Decrease column diameter
3.
Decrease flow-rate
4.
Vary thickness of stationary phase
5.
Vary particle size of stationary phase
6.
Decrease sample size
7.
Select proper functionalization of stationary phase
8.
Select proper mobile phase
9.
Decrease pressure
10. Use temperature gradient
11. Reduce oven temperature (must not to allow the oven to
become so cool as to permit the sample condenses in the column).

Standards
To verify GC operation standards are used

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1. Phenyl-2-propanone
2. Amphetamine
3. Methamphetamine
4. N,N-Dimethylamphetamine
5. Norpseudoephedrine
6. Pseudoephedrine
7. 3,4-Methylenedioxyamphetamine
8. 3,4-Methylenedioxymethamphetamine
9. Benzocaine
10.Methylenedioxyethyl amphetamine
11.Mescaline
12.Caffeine
13.Phencyclidine
14.Procaine
15.Cocaine
16.Diazepam
17.Acetylcodeine
18.Flunitrazepam
19.Heroin
20.Quinine
21.LSD
22.Strychnine

Abused Drugs and

Relative Retention
Times

Club Drugs
1. Methamphetamine (0.18mg/mL)
2. 4-Methoxyamphetamine (0.18mg/mL)
3. 2,3-MDA (0.18mg/mL)
4. 3,4-MDA (0.18mg/mL)
5. 2,3-MDMA (0.18mg/mL)
6. 3,4-MDMA (0.18mg/mL)
7. 4-Bromo-2,5dimethoxyphenethylamine (0.18mg/mL)
8. Ketamine (0.18mg/mL)
9. Phencyclidine (0.18mg/mL)
10. Cocaine (0.18mg/mL)
11. Flunitazepam (0.18mg/mL)

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