‫بسم هللا الرحمن الرحيم‬

Principles of analytical
toxicology
Prof. Dr/ USAMA
M. EL-BARRANY
DEPARTMENT OF FORENSIC MEDICINE
AND clinical TOXICOLOGY
Faculty of medicine
Cairo University
 Introduction

Analytical toxicology is concerned

with the detection and identification of
drugs and toxins, and their
metabolites in biological fluids and
related specimens.
 Applications of analytical
toxicology
Clinical toxicology
Forensic toxicology
Occupational and environmental
toxicology
Therapeutic drug monitoring (TDM)
Drug abuse screening
Analytical toxicology sequence
Physical Examination of the Sample

Sample Preparation

Screening Tests

Confirmatory Tests

Interpretation of the Results & Writing the Report

 Types of samples
Biological Samples:
- Antemortem
- Postmortem samples
Scene residues e.g. pills, powders,
bottles etc…..
Types of samples
Types of samples
Types of samples
 Dealing with samples
Physical Examination of samples:
Color, smell, pH. microscopic examination of
stomach contents, all may yield valuable
diagnostic information.
 Sample preparation
Homogenization, centrifugation, protein
precipitation, hydrolysis
Extraction (liquid–liquid extraction, solid phase
extraction, supercritical fluid extraction)
Preparation for Headspace analysis
Preparation for Chromatographic analysis
 Separatory Funnel Extraction Procedure

Liquid-Liquid Extraction (LLE)

1. Support the 2. Pour in liquid to 3. Add extraction 4. Add ground glass

separatory funnel in a be extracted solvent Stopper (well greased)
ring on a ringstand.
Make sure stopcock is
closed
 Separatory Funnel Extraction Procedure

Liquid-Liquid Extraction (LLE)

Shake the separatory funnel vigorously.
Now, shake the funnel vigorously for a few seconds. Release
the pressure, then again shake vigorously. About 30 sec
total vigorous shaking is usually sufficient to allow solutes to
come to equilibrium between the two solvents.

Vent frequently to prevent pressure buildup,

which can cause the stopcock and perhaps
hazardous chemicals from blowing out. Take
special care when washing acidic solutions with
bicarbonate or carbonate since this produces a
large volume of CO2 gas
 Liquid-Liquid Extraction (LLE)
Separate the layers.

Let the funnel rest
undisturbed until the layers
are clearly separated
While waiting, remove the Carefully open the stopcock and
stopper and place a beaker allow the lower layer to drain
or flask under the sep into the flask. Drain just to the
funnel. point that the upper liquid
barely reaches the stopcock
Solid phase extraction
 Solid phase extraction
Principle:
The samples is applied to SPE tube, , most
of the contaminants are held the packing
material then, and the analytes of interest
are collected while passing through the
adsorbent.
Advantages:
Simple rapid, and clean extract.
Steps in SPE

17
Analytical toxicology procedures
Screening (qualitative) Tests:
– Color tests, TLC, Toxi-lab system,
immunoassay (semiquantitative).
Confirmation (quantitative) Tests:
– immunoassay (semiquantitative)
– Spectrophotometer
– GC, HPLC
– GC-MS, GC-MS-MS
– HPLC-MS, HPLC-MS-MS
 Color tests
Principle:
Reagent + sample extract → characteristic color
Advantages:
Simple
Generally performed on urine samples, stomach
contents or other samples after their extraction
Can be performed in clear glass test-tubes or
more preferably in a large, white ceramic plate.
 Disadvantages
1. Compounds containing similar functional
groups will also react.
2. The colors produced usually vary in
intensity or hue with concentration.
3. When performing color tests, it is always
important to analyze concurrently with
the test sample:
 A blank reagent, i.e., an appropriate sample known not
to contain the compound(s) of interest.
 A known positive sample at an appropriate
concentration.
 Disadvantages
4. Need confirmation.
5. Some compounds are not detected by
the screening procedures:
- Inorganic ions e.g. lead, arsenic, iron.
- Organic chemicals e.g. CO, ethylene glycol, phenols,
drugs :cannabis, digoxin ,clonidine, salbutamol,
pesticides e.g. organophosphates, carbamates,
organochlorines.
 Color Tests
Marquis reagent
Mecke reagent
Simon reagent
Dragendorff reagent
Iodoplatinate reagent
Liebermann reagent
Ninhydrin reagent
Forrest reagent
Trinder reagent
Zwicker reagent
Ferric chloride reagent
Millon’s reagent
Marquis reagent
 Marquis test
Reagent:
Marquis test is a useful broad–spectrum
test used mostly for opium alkaloids and
amphetamines.
Carefully mix 100 mL of concentrated
sulfuric acid with 1 mL of 40% (v/v)
formaldehyde solution (stable for several
weeks if protected from light).
 Marquis test
Method:
Marquis test is conducted by placing a drop of reagent
liquid onto a small sample of the material being tested
(using a large, white ceramic plate).
Morphine, codeine, heroin, opium – Purple/violet.
Amphetamine, methamphetamine - Orange/brown.
MDMA, MDA, MDEA - Purple/black.
2C-B, DOM, DOB - Yellow/green.
 Marquis test
Marquis Test:
– Turns purple in the
presence of Heroin,
morphine, codeine,
opium

– Turns orange-brown in
presence of
Amphetamines
Mecke reagent testing kit
(Red label)
 Mecke test
Reagent:
Mecke Reagent is a solution of selenious
acid in sulfuric acid. Mecke Reagent
produces clearly different color results for
DXM and MDMA, which is why it is now a
preferred reagent for MDMA testing. It can
also be used to test for the presence of
2C-T-x variants and certain opiates.
 Mecke test
Method:
Mecke test is conducted by placing a drop of reagent
liquid onto a small sample of the material being tested
(using a large, white ceramic plate).
Codeine/morphine/hydrocodone - Very dark bluish green
Oxycodone – Olive.
MDMA, MDA, MDEA - Green turning to blue.
DXM - Yellowish-brown.
2C-T-x - Yellow through red to purple.
 Handling instructions
Mecke Reagent is a mixture of acids, and is
strong enough to burn skin and clothing. Keep
out of eyes and mouth. Wear latex gloves when
handling the bottle and cap.  If you get some on
you then wash quickly with soap and water.
Wash testing surfaces with soap and water as
well. Dispose of any unwanted reagent down the
sink with running water.
Simon reagent testing kit (Yellow
& green labels)
 Simon test
Reagent:
Simon reagent is sodium nitroprusside in a
basic buffer. It works by detecting
'secondary amines', such as the 'meth' in
METHamphetamine and in
mythylenediaoxy-METHamphetamine
(MDMA). We use it after testing a
substance with Marquis or Mecke
Reagent.
 Simon test
Important notes: This test should be used only
after you have first received a positive result with
Mecke or Marquis reagent. It should never be used
alone.
The presence of MDMA/MDEA/Methamphetamine
will be indicated by a change to a bright cobalt blue.
If no color change is observed, then the substance
contains either MDA or amphetamine.
 Simon test
Important notes: In some kits, solution A may
be a dull sea-green color, or it may be clear. This
is normal. What you're looking for is a change in
color. In all cases, the presence of
MDMA/MDEA/Methamphetamine will be
indicated by a change to a bright cobalt blue. If
no color change is observed when solution B is
added, then the substance contains either MDA
or amphetamine.
 Other color tests
Dragendorff reagent:
An orange, red–orange or brown–orange
precipitate suggests the presence of an
alkaloidal base.
This reagent is commonly used as a spraying
agent to detect alkaloids on thin–layer
chromatographic plates.
 Other color tests
Iodoplatinate reagent: (for basic extracts)
A general test for alkaloids and nitrogenous
heterocyclic compounds.
A violet, blue–violet, brown–violet or grey–violet
precipitate suggests the presence of an
alkaloidal base
 Other color tests
Ferric chloride:
Red, orange, green, blue, violet or brown colors indicate
the presence of a phenolic compound, fatty acid or a
phenylpyrazoline. High quantities of phenothiazines can
also cause this test to be positive.
Salicylates give a violet color. Aspirin (acetylsalicylic
acid) does not give a positive result unless first
hydrolyzed with concentrated sodium hydroxide to give
salicylates.
 Other color tests
Forrest reagent:
A variety of colors, from pink, red, orange, violet
to blue, indicate the presence of phenothiazines.
FPN reagent:
A variety of colors, from pink, red, orange, violet
to blue, indicate the presence of phenothiazines.
 Other color tests
Liebermann reagent:
A wide range of colors is given by compounds
that contain –OH, O–alkyl or –O–CH2O– groups
attached to a benzene ring or to a ring in a
polycyclic structure that contains a benzene ring.
Paracetamol gives a violet color, cocaine gives a
yellow color, ephedrine gives an orange color,
and codeine gives a black color with Liebermann
reagent.
 Other color tests
Mandelin reagent: (for basic extracts)
It shows different colors for Ecstasy-like substances (MDMA,
MDEA and MDA), speed, Ketamine and para-methoxy-
amphetamine (PMA).
Tricyclic antidepressants give fluorescent spots if viewed
under ultraviolet light (366 nm) after spraying with this
reagent.
Ibuprofen: grey/tan, Indomethacin: grey, Ketoprofen: yellow
Mefenamic acid: blue/green, Paracetamol: white/grey
Propranolol: green, Strychnine: blue → orange
 Other Color Tests
Mercurous nitrate reagent: (for acidic
extracts)
Mercurous nitrate reagent gives white spots with
a grey centre on a darker background with
barbiturates and related compounds such as
glutethimide.
 Other color tests
Ninhydrin reagent:
A violet color that appears rapidly indicates the
presence of an aliphatic primary amine or an
amino acid group.
The presence of an aromatic ring inhibits the
response, and the inhibition increases the
nearer the amino group is to the ring, as for
amphetamine (pink–orange).
Gentamicin gives a violet color after heating for
4 min.
 Other color tests
Sulfuric acid: (for basic extracts)
Sulfuric acid 500 ml/l alone gives red, purple or blue
spots with many phenothiazines and their metabolites.
Trinder reagent:
A purple color indicates the presence of salicylates.
Zwicker reagent (alkaline cobalt test):
This is a general test for barbiturate–like compounds.
The presence of a violet–blue color indicates
barbiturates
 Thin layer chromatography
(TLC)
Principle:
- Thin Layer chromatography (TLC) involves the movement
by capillary action of a liquid phase (usually an organic
solvent) through a thin, uniform layer of stationary phase
(usually silica gel, SiO2) held on a rigid or semi-rigid
support, normally a glass, aluminum or plastic sheet or
plate.
- Compounds are separated by partition between the
mobile and stationary phases.
- Now, Toxi-lab System is available in commercial kits.
 How TLC works
Sample solution is dissolved by the solvent
The solution sample will travel at different
distances based on solubility, polarization,
size
Silica gel
– Polar substances do not move far
– Non polar substances move farther up the plate
HO OH OH

R Si Si Si R
O O O O
R R R
 Thin layer chromatography

Spread a thin layer of absorbent (Silica

gel) on an unreactive hard surface
– Glass, plastic, thick aluminium
Heat in oven at 110°C for 30 minutes to
activate and dry the plate
 TLC Procedure
Place a small amount of
solvent in a beaker

In pencil, draw a
straight line across the
plate about 1 cm from
the end of the plate

Place a drop of sample

solution on the line
 TLC procedure

Place in solvent-
sealed container
as shown in the
figure.
 Solvents
The solvent can be a mixture of compounds
Choose a solvent depending on the polarity of the
compound
Petroleum ether
Least Polar
Cyclohexane
Toluene
Chloroform
Acctone
Ethanol
More polar
Methanol
 Recommended TLC
visualization reagents
Ninhydrin reagent: 1° or 2° amines (sympathomimetics)
Mercurous nitrate reagent (acidic extract)
Mercuric Sulfate: barbiturates, glutethimide, phenytoin
(white ppt)
Sulfuric acid (500 ml/l) (basic extract)
Mandelin reagent (basic extract)
Acidified iodoplatinate reagent (basic extract): 3° amines
Dragendorff reagent: methaqualone
UV absorption at 254 nm: benzodiazepines, barbiturates,
methaqualone
Fluorescence at 366 nm : Benzodiazepines, quinine,
quinidine
 Results
Multiple spots from one sample can be
achieved.
 Interpretation
Calculation of the Rf value

D istan ce th at th e S p o t T rav eled

 R f V alu e
S o lv en t F ro n t D istan ce
 Rf value
Rf (The retention factor) value is the ratio
of the distance from the center of the spot
for a given mixture component to
the distance travelled by the mobile phase.
Calculation the Rf value
 Rf Value
What affects the Rf value?
– Temperature
– Solvent
– Thickness and amount of spot
– Other compounds
 Thin layer chromatography
Advantages:
TLC is relatively inexpensive, simple & sensitive
test to perform, and can be a powerful
qualitative technique.
Disadvantages:
Slow, not quantitative, poorly specific.
TLC, Toxi-lab system
 Immunoassay tests
Principle:
Use antibodies to detect the presence of drugs. In using
immunoassays in drug testing, the targeted drug acts
as the antigen.
Disadvantages:
Relatively nonspecific i.e. molecules of similar
structure cross react e.g. can’t distinguish
between the drug or its metabolite or the
family of tricyclic antidepressants
 Immunoassay tests

Radioimmunoassay
Enzyme Linked Immunosorbent Assay
(ELISA)
Enzyme Multiplied Immunoassay
Technique (EMIT)
 Radioimmunoassay
Principle: Uses an immune reaction [Antigen –
Antibody reaction] to estimate a ligand

*Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab

– Unbound Ag* and Ag is washed out

– Radioactivity of bound residue measured
– Ligand conc is inversely related to radioactivity

Ag : ligand to be measured ; Ag* radiolabelled[

]ligand
Advantages & Disadvantages of RIA
Advantages
– Highly specific: Immune reactions are specific
– High sensitivity : Immune reactions are sensitive
Disadvantages
– Radiation hazards: Uses radio-labelled reagents
– Requires specially trained persons
– Labs require special license to handle radioactive
material
– Requires special arrangements for:
Requisition, storage of radioactive material
Radioactive waste disposal.
 ELISA
Principle:
– Uses an immune reaction like RIA
– Differs from RIA in detection method
– Detection based on
Enzyme catalysed reaction OR
Fluorescent probe
NOT radioactivity [great advantage!]
ELIZA
 Advantages of ELISA
Sensitive: nanogram levels or lower
Reproducible
Minimal reagents
Qualitative & Quantitative
– Qualitative  E.g HIV testing
– quantitative assays  E.g Ther. Drug Monitoring
Greater scope : Wells can be coated with
Antigens OR Antibodies
Suitable for automation high speed
NO radiation hazards
 Principle of Enzyme Multiplied
Immunoassay Technique (EMIT)
- After the addition of substrate, absorbance
measurements are taken at time intervals to
determine the speed of the enzyme reaction.  
- The more free drug in the sample, the faster the
enzyme reaction because only the unbound
enzyme-drug complexes are capable of binding
the substrate. The method can be used for
whole blood, serum, or urine.

65
EMIT Technique
 Advantages Of EMIT
 Homogeneous means there is no need of
washing excess of labelled enzymes.
 Long shelf-life
 Assays are well established
 More specialty assays are available
 It is a competitive assay
 Reagent separation is not required
 Is faster
 Smoller molecules are measured

67
 Disadvantages Of EMIT
 Many interferences
 Less sensitive compared to Elisa in
hormone determination
 False negatives are common
 Tolmetin metabolites
 Aspirin metabolites

68
Fluorescence polarization
immunoassay
Automated TLC
Toxi-lab system
 Immunoassay Tests
Principle:
Use antibodies to detect the presence of drugs .
In using immunoassays in drug testing, the
targeted drug acts as the antigen.
Radioimmunoassay
Enzyme Linked Immunosorbent Assay
(ELISA)
Enzyme Multiplied Immunoassay
Technique (EMIT)
Enzyme-multiplied immunoassay
technique (EMIT)
Fluorescence polarization
immunoassay
 Recommendations developed by the National Academy of Clinical Biochemists
(NACB) involving clinical biochemists, medical toxicologists, forensic
toxicologists, and emergency medicine physicians

Serum assays, Quantitative   Urine assays, Qualitative

Acetaminophen   Amphetamines
Salicylates Cocaine
Ethanol, cyclosporine & tacrolimus (in blood) Cannabis
Theophylline   Opiates
Digoxin Tramadol
Aminoglycosides (Amikacin, gentamycin, tobramycin)
Barbiturates
Vancomycin
  Benzodiazepines
Phenobarbital, phenytoin, Valproic acid, Carbamazepine
  Tricyclic antidepressants
Lithium, methotrexate, lidocaine Propoxyphene

Tricyclic antidepressants   Phencyclidine

Co-oximetry (carboxyhemoglobin, methemoglobin)
Iron (plus transferrin or unfilled iron-binding capacity)
Detection Limits Drug/Class

300 ng/mL Opiates

300 ng/mL Propoxyphene
300 ng/mL Methadone

200 ng/mL Tramadol

50 ng/mL THCA Cannabinoids

25 ng/mL Phencyclidine
Detection Limits Drug/Class

1000 ng/mL Amphetamines

300 ng/mL Cocaine

200 ng/mL Barbiturates

200 ng/mL Benzodiazepines

Expected duration for a positive urine
drug screening

• 2 -4 days • Amphetamine

•2 -4 days •Methamphetamine

•2 -4 days •Barbiturates (short acting)

•up to 30 days •Barbiturates (long acting)

•up to 30 days •Benzodiazepines

•1 - 3 days •Cocaine
Expected duration for a positive urine
drug screening
• 1 - 3 days • Heroine/morphine

• up to 70 days •Marijuana (chronic use)

• 1 - 3 days •Marijuana (occasional use)

• 2 - 4 days •Methadone

• up to 30 days •PCP (chronic use)

• 2 - 7 days •PCP (occasional use)

 Immunoassay screening tests
False positive results
Substance False positive
Alcohol Isopropyl alcohol
Pseudo-ephedrine, phenylpropanolamine
Amphetamine and phenylephrine, dexedrine (d-amphetamine),
methamphetamine Vick's inhaler (contains l-methamphetamine), ranitidine,
amantadine.
Barbiturates Ibuprofen
Benzodiazepines Oxaprozin (nonsteroidal anti-inflammatory drug).
Cannabinoids Ibuprofen, fenoprofen,
Dextromethorphan (cough suppressant), diphenhydramine,
Opiates
ofloxacin.
Methadone Clomipramine, diphenhydramine, verapamil.

Phencyclidine Dextromethorphan, diphenhydramine, Ibuprofen, imipramine

 False negative results

– If tests taken are too late

– If adulteration, dilution or substitution is
successful and undetected
If in routine screening:
– MDMA (ecstasy),LSD, psilocybin are not detected by
all screening.
– Opiate screening focus on heroin, morphine &
codeine use, but miss propoxyphene, meperidine,
methadone, pentazocine and oxycodone.
 False negative results
One class includes commonly available
household substances like:
Water
Bleaching agents
Detergents
Baking soda
Vinegar
Eye drops
 Trying to beat the test
The use of diuretics to remove drugs
 False negative results

Definitions according to SAMHSA

U.S. Substance of Abuse and Mental Health Services
Administration
Urine is considered to be diluted if:
• Creatinine < 1.8 mmol/L (20 mg/dL), but > 0.4 mmol/L (4.52 mg/dL)
In Europe < 2 mmol/L (22.6 mg/dL), but > 0.4 mmol/L (4.52 mg/dL)
• Specific gravity < 1.003 kg/L, but > 1.001 kg/L

Urine is considered to be adulterated if:

• The nitrite concentration is > 500 mg/L
• The pH value is < 3 or > 11
• Exogenous substances that lead to adulteration are detectable.
 Cut-off values, sensitivity and specificity of
chromatographic methods
Cut-off values
- As with immunoassays, the cut-off value means the limit for a decision on whether a
result is to be interpreted as positive or negative

Sensitivity
- As a rule, the sensitivity of the confirmation method should be greater than that of the
screening test.
- GC/MS also has greater sensitivity than the screening immunoassays. The high
sensitivity afforded by the combination of the retention time and the mass spectrum
makes false-negative results extremely unlikely.

Specificity
- As a rule, the specificity of the confirmation analysis should be better than that of the
screening test.
- The most common confirmatory method is GC/MS. The high specificity afforded by the
combination of the retention time and the mass spectrum makes false-positive results
extremely unlikely.
- Some states require GC/MS confirmation for workplace drug screening and it may be
legally required for all drug screening.
The breathalyzer
 The breathalyzer
Once the alveolar breath is trapped it is
allowed to undergo a chemical reaction:

 2K2Cr2O7 + 3C2H5OH + 8H2SO4  2Cr2(SO4)3 + 2K2SO4 + 3CH3COOH + 11H2O

Potassium Ethyl Sulfuric Chromium Potassium Acetic Dihydrogen

dichromate alcohol acid sulfate sulfate acid oxide

The Breathalyzer indirectly determines the quantity of alcohol

consumed by measuring the absorption of light by potassium
dichromate before and after its reaction with alcohol using the
principle of spectrophotometry
 Spectroscopy

Spectral Distribution of Radiant Energy

 
Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)
 Spectroscopy

g-rays X-rays UV IR Microwave Radio

nuclear core electronic molecular molecular Nuclear Magnetic
excitation electron excitation vibration rotation Resonance (NMR)
excitation

Visible
 Conventional
spectrophotometer

Schematic of a conventional single-beam

spectrophotometer
Absorbance and path length:
Beer-Lambert law

A   log T   logI / I 0   logI 0 / I     b  c

Transmittance and path length:
Beer-Lambert law

Concentration

T  I / I 0  e  ConstConcentration
UV/VIS-Spectrophotometer
UV spectrophotometer application
Protein
Amino acids (aromatic)
Pantothenic acid
Glucose determination
Enzyme activity (hexokinase)
Visible spectrophotometer application
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (Choline
esterase enzyme, glucose oxidase)
 Absorption spectra of
hemoglobin derivatives
Radiometer Co-oximeter
 Infrared (IR) Spectroscopy

Infrared Spectrometer determines the

wavelength and absorbance of a sample
in the infrared region of the
electromagnetic spectrum.
 IR Spectroscopic Process
There are two types of bond vibration:
• Stretch – Vibration or oscillation along the line of the bond
H H
C
C
H
H

symmetric asymmetric

• Bend – Vibration or oscillation not along the line of the bond

H H H H
C C C C
H H H
H
scissor rock twist wag
in plane out of plane
 IR Spectroscopic Process
As a covalent bond oscillates – due to the oscillation of the dipole of the
molecule – a varying electromagnetic field is produced

The greater the dipole moment change through the vibration, the more
intense the electromagnetic (EM) field that is generated
 IR Spectroscopic process
When a wave of infrared light encounters this oscillating EM field
generated by the oscillating dipole of the same frequency, the two waves
couple, and IR light is absorbed

The coupled wave now vibrates with twice the amplitude

IR beam from spectrometer

“coupled” wave

EM oscillating wave
from bond vibration
Infrared Spectrum of Cocaine
 Sample
Atomic Absorption Compartment
Spectrophotometer

Light Source Detector

Atomic Absorption Spectrophotometer

ATOMIZER

FLAME GRAPHITE TUBE

ATOMIZERS ATOMIZERS
Atomic Absorption Spectrophotometer
In most of the cases, air-acetylene flame or nitrous oxide-acetylene flame is used.
Plateau Graphite Tube
Atomic Absorption Spectrophotometer

 The cathode lamps are

stored in a compartment
inside the AAS.
 The specific lamp needed
for a given metal analysis is
rotated into position for a
specific experiment.
Atomic Absorption Spectrophotometer
Liquid sample is aspirated to become aerosols of fine particles (nebulization)
Flame vaporizes the aerosols (atomization)
Elevated temperatures in a flame or furnace changes the chemistry of atoms
so that they can absorb light.
Each metal atoms absorbs light at a characteristic frequency.
Beer’s law is used to calculate concentration

Metal Zn Fe Cu Ca Na
λ (nm) 214 248 325 423 589
Gas chromatography
 Instruments for GC

Carrier Gas-Supply:
- Carrier gases, which must be chemically inert, include
helium, nitrogen, and hydrogen.
- Associated with the gas supply are pressure regulators,
gauges, and flow meters.
- In addition, the carrier gas system often contains a
molecular sieve to remove water or other impurities.
 Sample Injection System
The most common method of sample injection involves the
use of microsyringe to inject a liquid or gaseous sample.
The sample port is ordinarily about 50oC above the boiling
point of the least volatile component of the sample.
 GC Columns
There are two main types of supports used in GC:

Packed columns
large sample capacity
preparative work

Capillary (open-tubular) columns

higher efficiency
smaller sample size
analytical applications
Common Stationary Phases for GC
 Column Ovens
Temperature Control

Isothermal Gradient

240

200

Temp (deg C)
160

120

80

40

0
0 10 20 30 40 50 60
Time (min)
 Common GC Detectors

Flame ionization detector

Mass spectrometer detector

Thermal-conductivity detector
Intensity of detector signal
Chromatogram

tR
Peak tR : Retention time
t0 t0 : Non-retention time
h
A A : Peak area
h : Peak height

Time

120
 Common GC Applications
Alcohols in blood
Aromatics (benzene, toluene, ethylbenzene,
xylene)
Flavors and Fragrances
Permanent gases (H2, N2, O2, Ar, CO2, CO, CH4)
Hydrocarbons
Pesticides, Herbicides, PCBs, and Dioxins
Solvents
GC-MS
Headspace GC-MS
High Performance Liquid Chromatography
(HPLC)
 HPLC Pump

HPLC pumps which have

pistons that pull the mobile
phase in and push it out
into the head of the
column.
HPLC Autosampler and Injector
 Mobile Phase in HPLC
Ideal Characters:
Purity of the solvents
Detector compatibility
Solubility of the sample
Low viscosity
Chemical inertness
Reasonable price
 Mobile Phase in HPLC
Isocratic Mode: of HPLC means that:
the composition of mobile phase remains
constant throughout the run.
Isocratic mode is most commonly used for
assay, dissolution, and routine analysis where
you want to separate 2 or 3 compounds in a
single run. 
 Mobile Phase in HPLC

Gradient mode: of HPLC means that:

composition of mobile phase varies and is not
constant throughout the run.
Gradient mode is useful when we want to
analyse mixture of analytes having differences in
the polarity so that they can not be separated in
a single mobile phase and can be separated by
varying polarities of solvents.
 HPLC Degasser
It is recommended to degas the chosen solvents for
several minutes before use.
Regulator Vacuum chamber
Helium Polymeric film tube
cylinder

To pump
To pump
To draft

Drain valve

Eluent container Eluent container

Helium purge method Gas-liquid separation membrane method

131
 Stationary Phase in HPLC
Normal Phase:
This is where the stationary phase is strongly
polar (silica gel) and the mobile phase is largely
non-polar such as hexane.
Reverse Phase:
The stationary phase is non-polar (C18, C8) and
the mobile phase is polar liquids such as
methanol, acetonitrile, or water.
 HPLC Columns
The reason why C-18 is more
hydrophobic than the other
reverse phases is because the
length of the carbon chains are
longer (i.e. C18 is longer than
C8, and C8 is longer than C4).
C8 may be used instead of C18
when shorter retention times are
required.
C4 provides even less retention
towards non-polar compounds
than both C18 and C8.
 Common HPLC Detectors

Diode array detector

Fluorescence detector Mass spectrometer detector

 Fields in which HPLC is used
Food products
– Vitamins,substances
Biogenic food additives, sugars, organic acids, amino acids, etc.
Environmental samples
– Sugars, lipids, nucleic
– Inorganic ionsacids,
acids, amino
– Hazardous
proteins, peptides, steroids,
organic substances, etc.
amines,industrial
Organic etc. products
Medical products
– Synthetic polymers, additives, surfactants, etc.
– Drugs, antibiotics, etc.

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HPLC-MS
Ultra High Performance Liquid
Chromatography (UPLC)
Contrasting HPLC and UPLC
The pump pressure, which is 40Mpa (400
atmospheres) in HPLC, can go up to 100MPa in UPLC.
UPLC also allows for much smaller particle size,
normally less than 2um whereas it is normally 5um in
HPLC.
UPLC uses less of valuable solvents like acetonitrile
which lowers cost.
The reduction of solvent use is more environmentally
friendly.
UPLC gives faster results with better resolution
Increased productivity can increase your revenue
(income) in an industrial setting.
UPLC-MS
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