Liquid Chromatography

Dr Adel Mohamed Ahmed

Chromatographic parameters
1- Retention time tR
 The time between sample injection and an analyte peak reaching a
detector at the end of the column

 The time taken for the mobile phase to pass through the column is
called tM
 Chromatographic parameters
2- Resolution Rs
 The most important thing in HPLC is to obtain the optimum resolution in
the minimum time. A resolution value of 1.5 or greater between two
peaks will ensure that the sample components are well (baseline)
separated to a degree at which the area or height of each peak may be
accurately measured.
 Chromatographic parameters
3- Retention/capacity factor (K)
The retention (or capacity) factor (k) is a means of measuring the retention
of an analyte on the chromatographic column
When an analytes retention factor is less than one, elution is so fast that
accurate determination of the retention time is very difficult. High retention
factors (greater than 20) mean that elution takes a very long time. Ideally,
the retention factor for an analyte is between one and five.
 Chromatographic parameters
4- selectivity factor, α
which describes the separation of two species (A and B) on the column;
α = k 'B / k 'A
High α values indicate good separating power and a good separation
between the APEX of each peak. However, the alpha value is NOT directly
indicative of the resolution.
 Chromatographic parameters
4- Efficiency (N)
The efficiency of a chromatographic peak is a measure of the dispersion of
the analyte band as it travels through the HPLC system and column. The
plate number (N) is a measure of the peak dispersion on the HPL Ccolumn,
which reflects the column performance.
Higher values for the Plate Number (N) are expected for subsequent peaks
within a chromatogram. Later eluting peaks that look broad in comparison to
early eluting peaks may have a higher plate count.
Basic LC Terminology and Separation Modes
● Adsorption chromatography
• The stationary phase is an adsorbent (like silica gel or any other
silica-based packing)
• The separation is based on repeated adsorption-desorption
steps.

● Normal-phase chromatography
• The stationary bed is strongly polar in nature (e.g., silica gel),
and the mobile phase is nonpolar (such as n-hexane or
tetrahydrofuran).
• Polar samples are retained on the polar surface of the column
packing longer than less polar materials.

● Reversed-phase chromatography
• The stationary bed is nonpolar (hydrophobic) in nature.
• The mobile phase is a polar liquid, such as mixtures of water
and methanol or acetonitrile.
• The more nonpolar the material is, the longer it will be retained.
Basic LC Terminology and Separation Modes
● Size exclusion chromatography (SEC)
• The column is filled with material having precisely controlled
pore sizes, and the sample is simply sieved or filtered
according to its solvated molecular size.
• Larger molecules are rapidly washed through the column;
smaller molecules penetrate inside the pores of the packing
particles and elute later.
• Also called gel permeation chromatography (GPC)
although the stationary phase is not restricted to a "gel"

● Ion-exchange chromatography (IC)

• The stationary bed has a charged surface of opposite charge
to the sample ions.
• Used almost exclusively with ionic or ionizable samples.
• The stronger the charge on the sample, the stronger it will be
attracted to the ionic surface and thus, the longer it will take
to elute
• The mobile phase is an aqueous buffer, where both pH and
ionic strength are used to control elution time
 Analytical Applications of LC
 The “branches” of the LC family:
Note – this means analyte polarity
Basic Mechanisms in LC Separations
High Performance Liquid Chromatography (HPLC)

● HPLC utilizes a high-pressure liquid mobile phase (ca.

100-300 bar) to separate the components of a mixture

● These analytes are first dissolved in a solvent, and then

forced to flow through a packed small-particle
chromatographic column, where the mixture is resolved
into its components

● HP = high pressure and high performance

● Resolution depends upon the extent of interaction

between the solute components and the stationary
phase
Differences between HPLC and “Classical” LC
 Small ID (2-5 mm), reusable stainless steel columns
 Column packings with very small (3, 5 and 10 m)
particles and the continual development of new
substances to be used as stationary phases
 Relatively high inlet pressures and controlled flow of the
mobile phase
 Precise sample introduction without the need for large
samples
 Special continuous flow detectors capable of handling
small flow rates and detecting very small amounts
 Automated standardized instruments
 Rapid analysis
 High resolution
 From now on, LC refers to HPLC
 Advantages and Disadvantages of LC

Advantages:
• Speed (minutes)
• High resolution
• Sensitivity
• Reproducibility
• Accuracy
• Automation

Disadvantages:
• Cost
• Complexity
• Low sensitivity for some compounds
• Irreversibly adsorbed compounds not detected
• Co-elution difficult to detect
 More on Reversed-phase (RP) LC
 RP is the most widely used mode of HPLC (75%?)
 Separates molecules in solution on basis of their
hydrophobicity
– Non-polar stationary phase
– Polar mobile phase
 In practice: non polar functional group bonded to silica
– Stationary phase
 functional group bonded to silica
 this corresponds to a volume (Van deemter)
 Alkyl groups ( C4, C8, C18)
 retention increases exp. with chain length
 Mobile Phases
– Polar solvent (water) with addition of less polar solvent (acetonitrile
or methanol)
 The Packed Column and the Stationary Phase

 Packed LC columns, usually made of stainless steel and

carefully filled with material, are the heart of the LC
experiment

 The stationary phase fills the column – its properties are

critical to the separation
 Review of Molecular Interactions
 The basis of separations (and most of chemistry)…
Name Energy (kcal/mol) Description

Hold molecules together, orbital

Covalent 100-300 overlap

Ionic 50-200 Electrostatic attraction

Polar
Vary from electrostatic-type
• Hydrogen bonding
3-10 interactions (e.g. hydrogen
• Dipole-dipole bonds) to much weaker
• -stacking

Non-Polar
• Van der Waals 1-5 Weak, induced dipole
(dispersion)
 Retention Mechanisms in LC
● LC is a dynamic partition/adsorption process. Analyte molecules, while
moving through the porous packing bead, tend to interact with the
surface adsorption sites or partition. Depending on the LC mode,
different types of the adsorption “forces” may be involved in the retention
process

● Hydrophobic interactions are the main ones in reversed-phase

separations (partition LC)

● Dipole-dipole (polar) interactions are dominant in normal phase mode.

● Ionic interactions are responsible for the retention in ion-exchange

chromatography.

● Retention in LC is competitive:
● Analyte molecules compete with the eluent molecules for the
adsorption sites. So, the stronger analyte molecules interact with
the surface, and the weaker the eluent interaction, the longer
analyte will be retained on the surface.
 Elution Order in LC
 Remember the elution order!
 Elution order in normal-phase vs. reversed-phase LC:
Physical Properties of Stationary Phase Particles
 HPLC separations are based on the surface interactions,
and depends on the types of the adsorption sites (surface
chemistry). Modern HPLC adsorbents are the small rigid
porous particles with high surface area.

 Key parameters:
• Particle size: 3 to 10 µm
• Particle size distribution: as narrow as possible, usually within 10%
of the mean
• Pore size: 70 to 300 Å
• Surface area: 50 to 250 m2/g
• Bonding phase density (number of adsorption sites per surface
unit): 1 to 5 per 1 nm2
 The Most Popular Particle: Silica
 Different morphology for different applications:

Macroporous spherical silica particle. [K.K.Unger, Electron microphotograph of spherical and irregular silica particles. [W.R.Melander, C.Horvath,
Porous silica, Elsevier, 1979] Reversed-Phase Chromatography, in HPLC Advances and Perspectives, V2, Academic Press, 1980]

 Different chemistry:
H OH Si Si O
Si OH Si OH O Si O O O H
H OH Si Si O
H
Free Silanol Adsorbed Water Geminal Silanol
Dehydrated Bound and
Oxide Siloxane Reactive
Silanols
 Chemical Modifications to Silica
 Silica (or zirconia, or alumina) by itself cannot do the job needed by
modern LC users – it must be functionalized and modified to suit the
analytical problem

Functionalized
Residual groups
silanols

Si Si
O OH OH Si
OH O
Si Si Si O OH
O Si Si
O O O O O
Si Si

Diagram from Crawford Scientific

 Chemical Modifications to Silica
 Groups are usually attached via reaction
of an organosilane (which can be pre-
polymerized in solution)
 Besides attaching groups, it is also
possible to polymerize the silica (or the
attached group)
 Purpose: stability at low pH, more
coverage
– High-carbon load
 Monomeric phases are more
reproducible (easier reactions to control)
– Monomeric phases are also known
as “sterically-protected”
 Endcapping: fully react the silica
surface, remove silanols and their
acidity, more coverage

Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861

 Common LC Stationary Phases
Name Structure Description

Normal phase, for separating polar, non-ionic

Silica Si OH
organics

Reversed-phase, for hydrophobic interaction

Propyl Si C3H7
chromatography (proteins, peptides)

Reversed-phase, like C18 but less retentive,

C8 Si C8H17 used for pharmaceuticals, steroids,
nucleotides
Reversed-phase, retains non-polar solutes
C18 Si C18H37 strongly. When bonded to 300A silica can be
used for large proteins and macromolecules

Reversed-phase and normal-phase, more

Cyano Si CH2CH2CH2CN
polar than C18, unique selectivity

Reversed-phase, normal-phase, and weak

Amino Si CH2CH2CH2NH2 anion exchange. RP used to separate
carbohydrates
 Common LC Stationary Phases

Name Structure Description

Reversed-phase, retains aromatic

Phenyl Si CH2CH2CH2 molecules. Also used for HIC
(proteins)

Both reversed-phase and normal-

phase utility. Used for RP SEC,
Diol Si O OH also used for NP separations as a
OH
more robust alternative to silica
(not ruined by trace water)

Normal-phase, separates aromatic

Nitro Si NO2
and alkene-containing molecules
 Polar Stationary Phase Interactions

Sorbents Interactions

CN
H
Dipole/Dipole
Si C
N

Hydrogen-
H
NH2
Si N
H
O Bonding
H

Si O
Hydrogen-
2OH
O O H

Bonding
H

H
O

Source: Crawford Scientific.

 Ionic Stationary Phase Interactions

Sorbents Interactions

H3+N
PRS Electrostatic
Si SO3-

H3+N
CBA O
Electrostatic
Si O-

-O S

SAX
3
Electrostatic
Si N+(CH3)3

Source: Crawford Scientific.

Non-Polar Stationary Phase Interactions

Sorbents Interactions
Si

C8 van der Waals

Si

PH van der Waals

Si
C2 van der Waals

Source: Crawford Scientific.

A Good Choice of Stationary Phase Depends on
the Analyte

Functionality Analyte Mechanism

N
NHH22
Hydrophobic Non-Polar

N
NHH22
H-Bonding Polar

++
N
NHH33
Ionic Ion-Exchange

Source: Crawford Scientific.

 More Subtle Effects

 Shape selectivity (correlates with stationary phase order), temperature,

coverage (and the role of bonding chemistry):

Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861

 More Subtle Effects
 The effects of
temperature on the
order of the stationary
phase are often
surprising:

Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861

 LC Instrumentation
Pumps, Mixers
Column Detector(s) Computer
and Injectors
 LC Instrumentation
 The Agilent 1100, a typical modern LC system

Solvent reservoirs

Solvent degasser
Pump

Autosampler

Column oven

DAD
Review: The Purpose of Key LC Components

column •separation chemistry

tubing to detector flow cell

•signal transduction
detector •amplification/scaling
•filtering
analog output

•data acquisition
A/D
•digitization
digital output

•digital processing
chromatogram
•data analysis
 The LC Pump(s)
Modern pumps have the following parameters:
Flow rate range: 0.01 to 10 ml/min
Pressure range from 1-5,000 psi
Pressure pulsations : less than1 %
Types of Pumps
Constant pressure pumps
Constant flow pumps

Reciprocating Piston Pump (90% of HPLC’s)

small internal volume
pulsed flow

Syringe type pumps (Displacement Pumps)

limited solvent capacity

Pneumnatic Pumps (pressure)

 Temperature Control in LC
 Thermoelectric heating/cooling
– the ability of a surface to
produce or absorb heat
when current is applied
across the junction of two
dissimilar conductors or
semiconductors
 The effect can be reversed (i.e.
heating turned to cooling) by
reversing the DC current
through the junction
 Also known as the Peltier effect
after its 1834 discoverer, a
French watch maker
 Overview of LC Detectors

 Common HPLC detectors

– Refractive Index
– UV/Vis
 Fixed Wavelength
 Variable Wavelength
 Diode Array
– Fluorescence Detector

 Less common:
– Conductivity
– Mass-spectrometric (LC/MS)
– Evaporative light scattering (ELSD)
 Desirable Features of an LC Detector

1. Low drift and noise level

2. High sensitivity (ability to discriminate between
small differences in analyte concentration)
3. Fast response
4. Wide linear dynamic range
5. Low dead volume
6. Cell design that eliminates remixing of separated
bands
7. Insensitivity to changes in types of solvent, flow
rate, temp
8. Operational simplicity and reliability
9. Non-destructive
 Flow Cell Design

Things to note:
parallel light beam
flow cell volume <1/10 of peak volume
optimization of cell geometry
 Fixed / Variable Wavelength Detectors

Mercury vapor lamps emit very intense light at 253.7

nm. By filtering out all other emitted wavelengths,
manufacturers have been able to utilize this 254 nm
line to provide stable, highly sensitive detectors
capable of measuring subnanogram quantities of
any components which contains aromatic ring. The
254 nm was chosen since the most intense line of
mercury lamp is 254 nm, and most of UV absorbing
compounds have some absorbance at 254 nm.
 Other Detectors
Fluorescence Detector
Evaporative Light Scattering

Electrochemical Detector
 Putting it All Together: LC Method Development
 The importance – without a good method:
– Co-elution can be missed
– Unable to detect/assay key components
 Basic consequences of method changes:
 Choosing an LC Approach
 Goals of a separation:
– Resolution (Rs) > 1.5
– Short separation time (5-30 minutes)
– Good quantitative precision/accuracy
– Acceptable backpressure
– Narrow peaks
– Minimal solvent use
 Overall Strategy

 First select an
appropriate method
 If LC is best, then
determine nature of
the sample
 “Exploratory” RP
runs, i.e. fast simple
gradients with C18
phases, are usually
helpful in assessing
retention and polarity
Gas Chromatography
 GC Very Basic Definitions

 Gas chromatography – chromatography using a gas as

the mobile phase and a solid/liquid as a stationary phase
– In GC, the analytes migrate in the gas phase, so their
boiling point plays a role
– GC is generally applicable to compounds with masses
up to about 500 Da and with ~60 torr vapor pressure
at room temp (polar functional groups are trouble)
 Summary of Chromatographic Relationships
 From Skoog:
 GC Theory

 Mobile-phase flow rates are

higher in GC (pressure drop is
much less for a gas) than in LC
 The effect of mobile-phase flow
rate on the plate height (H) is
dramatic
– Lower plate heights yield
better chromatography
– However, much longer
columns can be used with
GC
 GC Instrumentation
 Basic layout of a GC:

 See pg 703 of Skoog et al. for a similar diagram

 GC Instrumentation

 A typical modern GC – the Agilent 6890N:

Diagram from Agilent promotional literature.

 GC Instrumentation

 Typical carrier gases (all are chemically inert):

helium,
nitrogen and hydrogen. The choice of gas affects the
detector.
 Injectors: most desirable to introduce a small “plug”,
volatilize the sample evenly
– Most samples introduced in solution: microflash injections
“instantly” volatilize the solvent and analytes and sweep them into
the column
 Splitters: effectively dilute the sample, by splitting off a
portion of it (up to 1:500)
 Ovens: Programmable, temperature ranges from 77K
(LN2) up to 250 C.
 Detectors: wide variety, to be discussed shortly…
 Headspace GC

 A very useful method for analyzing Needle

volatiles present in non-volatile solids

and liquids

 Sample is (usually) equilibrated in a Headspace

sealed container at elevated

temperature

 The “headspace” in the container is

sampled and introduced into a GC
Liquid/solid
 Columns for GC

 Two major types of columns

used in GC
– Packed
– Open

 Open columns
work better at
higher mobile
phase velocities
 Columns for GC
 Open tubular columns: most
common, also known as capillary
columns (inner diameters of
<0.25 mm)
– up to 150 m long
– 1000-3000 plates/m
– pressure limits particle size in packed
columns
– No “A” term (Eddy or multipath) in van
Deemter equation A Phenomenex Zebron capillary GC column
www.phenomenex.com
– N up to 600000
 Packed columns: contain packing, like HPLC columns
– typical particle sizes 100-600 um
– 3 m long
– 1000-3000 plates/m
– difficult to overload
– N up to 12000
 Types of Columns for GC
 GLC: Gas-liquid chromatography (partition) – most
common
 GSC: Gas-solid chromatography (adsorption)
 FSWC: fused-silica wall-coated open tubular columns,
very popular in modern applications (a form of WCOT
column)
 WCOT (GLC): wall-coated open tubular – stationary
phase coated on the wall of the tube/capillary
 SCOT (GLC): support-coated open tubular – stationary
phase coated on a support (such as diatomaceous earth)
– More capacity that WCOT
 PLOT (GSC): porous-layer open tubular
 Packed columns
 Mobile Phases for GC
 Common mobile phases:
– Hydrogen (fast elution)
– Helium
– Argon
– Nitrogen
– CO2
 The longitudinal diffusion (B)
term in the van Deemter
equation is important in GC
– Gases diffuse much faster than
liquids (104-105 times faster)
 A trade-off between velocity
and H is generally observed
– This is equivalent to a trade-off
between analysis time and
separation efficiency
 Columns and Stationary Phases for GC
 Modern column design emphasizes inert, thermally stable
support materials
– Capillary columns are made of glass or fused silica
 The stationary phase is designed to provide a k and  that
are useful. Polarities cover a wide range (next slide).
– Stationary phases are usually a uniform liquid coating on the wall
(open tubular) or particles (packed)
– When the polarity of the stationary phase matches that of the
analytes, the low-boilers come off first…
– Bonded/cross-linked phases – designed for more robust life, less
“bleeding” – often these phases are the result of good polymer
chemistry
 Adsorption onto silicates (via free silanol groups) on the
silica column itself: avoided by deactivation reactions,
usually leaving an OCH3 group instead.
 Stationary Phases for GC
 Target: uniform liquid coating of thermally-stable, chemically
inert, non-volatile material on the inside of the column or on
its particles.
 Polysiloxanes R R R

– Polydimethylsiloxane R Si O Si O Si R
 (R = CH3)
R R R
– phenyl polydimethylsiloxane n
Backbone structure of
 (R = C6H5, CH3) polydimethylsiloxane
– trifluoropropyl polydimethylsiloxane (PDMS)
 (R = C3H6CF3, CH3)
– cyanopropyl polydimethylsiloxane
 (R = C3H6CN, CH3) HO OH
O
– polyethylene glycol n
structure of polyethylene
 Chiral glycol (PEG)
– amino acids, cyclodextrins
 Common Stationary Phases for GC
Stationary Maximum
Common Trade
phase Stationary Phase Temperature Common Applications
Name
polarity (C)
polydimethylsiloxane OV-1, SE-30 350 General-purpose nonpolar
phase; hydrocarbons,
steroids, PCBs
5% phenyl OV-3, SE-52 350 Fatty acid methyl esters,
polydimethylsiloxane alkaloids, drugs,
halogenated compounds
50% phenyl OV-17 250 Drugs, steroids, pesticides,
polydimethylsiloxane glycols

50% trifluoropropyl OV-210 200 Chlorinated aromatics,

polydimethylsiloxane nitroaromatics, alkyl-
substituted benzenes
polyethylene glycol Carbowax 20M 250 Free acids, alcohols,
ethers, essential oils,
glycols
50% cyanopropyl OV-275 240 Polyunsaturated fatty acids,
polydimethylsiloxane rosin acids, free acids,
alcohols

 High-temperature columns work to 400C, include Agilent’s

DB-1ht (100% polydimethylsiloxane), DB-5ht (5% phenyl).
 Temperature Effects in GC
 Temperature programming (normally a ramp) can be used
to speed/slow elution. It helps handle compounds with a
wide boiling point range. Newer “low thermal mass”
systems allow for fast ramps.
 Comparison of GC Detectors
Selective or
Detector Sensitivity Common Applications
Universal

Flame ionization (FID) 1 pg Universal Hydrocarbons

“carbon”/sec
Thermal conductivity (TCD) 500 pg/mL Universal Virtually all compounds

Electron capture (ECD) 5 fg/sec Selective Halogens

Mass spectrometry (MSD) 0.25 to 100 pg Universal Ionizable species

Thermionic (NPD) 0.1 pg/s (P) Selective Nitrogen and phosphorus

1 pg/s (N) compounds (e.g. pesticides)

Electrolytic conductivity 0.5 pg/s (Cl) Selective Nitrogen, sulfur and halogen-
(Hall) 2 pg/s (S) containing compounds
4 pg/s (N)
Photoionization 2 pg/s Universal Compounds ionized by UV

Fourier transform IR (FTIR) 0.2 to 40 ng Universal Organics

 See pg. 793 of Skoog et al. 6th Ed.

 GC Detectors: FID
 The flame ionization detector
(FID), the most common and
useful GC detector
 Process: The column effluent
is mixed with hydrogen and air
and is ignited. Organic
compounds are pyrolyzed to
make ions and electrons,
which conduct electricity
through the flame (current is
detected)
 Advantages: sensitive (10-13
g), linear all the way up to 10-4
g), non-selective
 Disadvantages: Destructive,
certain compounds (non-
combustible gases) don’t give
signals in the FID.
 GC Detectors: Thermal Conductivity
 Thermal conductivity
detector (TCD): a non-
selective detector like the
FID
 Also known as the
katherometer
(catherometer) or “hot
wire”
– Works by detecting the
changes in thermal
conductivity (also the
specific heat) of a gas
containing an analyte
– About 1000x < sensitive
than FID
– Non-destructive
 GC Detectors: Electron Capture Detector
 Electron capture: selectively detects halogen-containing compounds
(e.g. pesticides)
– Works by ionizing a sample using a radioactive material (63Ni). This material
ionizes the carrier gas – but this ionization current is quenched by a
halogenated compound
– Detects compounds via electron affinity – e.g. I (most sensitive) > Br > Cl > F
 GC Detectors: Other
 Atomic emission detector: plasma systems (like ICP, but
often using microwaves) – elemental analysis
 Sulfur chemiluminescence detector (SCD): reaction
between sulfur and ozone, follows an FID-like process
 Thermionic detector: like an FID, optimized and
electrically charged to form a low-temp (600-800 C)
plasma on a special bead. Leads to large ion currents for
phosphorous and nitrogen – a selective detector that is
500x as sensitive as FID
 Flame photometric detector: specialized form of UV
emission from flame products
 Photoionization detector: UV irradiation used to ionize
analytes, detected by an ion current.
 And, of course, the mass spectrometer (MS)…
Examples of GC Detection: Petroleum Analysis
 An example of atomic
spectroscopy, using
microwave-induced
plasma (MIP), to
selectively detect lead
(Pb) containing
compounds in gasoline

 See pg 710 of Skoog for

an example of oxygen
(O) and carbon (C)
detection for separating
hydrocarbons…
Examples of ECD Detection: Pesticide Analysis

Data from Agilent, http://www.chem.agilent.com/cag/graphics/445a.jpg

 Interpretation of GC Data
 Common use: develop a method to separate compounds
of interest by spiking, and use retention times to determine
whether a compound is present or not in unknowns
– Watch out for compounds with the same retention time!
– GC can function as a negative test – e.g. “rule out the presence of
ethyl acetate in my sample”….
 Relative retention time:
r  (t R ) A /( t R ) std
 Quantitative – Kovats’ retention index (I) – based on
normal alkanes
– the retention index of these compounds is independent of
temperature and packing
– I = 100z (z is the number of carbons in a compound)
– Relative retention index:
I  100 z 

100 log( t  )  log( t 
R B )
R z 
log( t R ) z 1  log( t R ) z 
Purge and Trap GC for Volatile Organic Compounds
 Invented 30 years ago by T. A. Bellar at the US EPA
 Principle:
– Inert gas is bubbled through an aqueous sample
– Gas carries analytes to headspace above sample, through to a
sorbent trap
– After a collection period, the sorbent trap is heated to desorb the
analytes
– The desorbed analytes are injected into a GC
 Results:
– ppb detection of VOC’s like benzene, decane, halomethanes,
etc… in water samples
 Commercialized by Teledyne Tekmar (e.g. the Velocity
XPT) and used worldwide
 Legally-mandated for water analysis in many areas
See C&E News December 12th, 2005, page 28, for more info on the 30th anniversary of Purge and Trap GC
 Chemical Derivatization for GC Analysis
 GC is only applicable to lower molecular weight
compounds with significant (> ~60 torr) volatility
– Polar functional groups reduce volatility
– For other compounds, another separations approach can be used
(LC, etc…) or derivatization can be explored
 Derivatization: chemical reaction(s) that modify an analyte
so that it is easier to separate or detect
 Advantages:
– Can lower LOD (increase sensitivity)
– Can stabilize heat-sensitive compounds
– Can avoid tailing in GC caused by on-column reactions (carbonyl,
amino, imino)
– Can improve the separation of closely-related molecules
 Disadvantage:
– Requires running a reaction, with all its complexities
 Chemical Derivatization for GC Analysis
 A typical derivitization reaction – silylation of an alcohol:
CH3 CH3

OH + Cl Si CH3 O Si CH3 + HCl

CH3 CH3

 Common derivatives that reduce polarity:

Groups Derivative
Alcohol (–OH) Alkyl ester, alkyl ether, silyl ether
Carboxylic acid (–COOH) Alkyl ester, silyl ester
Amino (-NH2) Acyl derivative, silyl derivative
Imino (=NH) Silyl derivative
Aldehyde (COH) Dimethyl acetal
Thiol (SH) Thioether, silylthioether

 Other derivatives contain halogens for ECD detection

S. Ahuja, “Derivatization for Gas and Liquid Chromatography”, in Ultratrace Analysis of Pharmaceuticals and Other Compounds of Interest, Wiley, 1986.
Applications of Derivatization and GC in Doping

 Example: derivatization of androgens (like testosterone)

for GC-MS analysis. Detection limits can be as low as 0.2
ng/mL
 In one procedure, derivitization with TMS is used in
conjunction with a series of pretreatment and extraction
steps, followed by GC-MS:

OH
Si

O
H

H
H H

O H H
testosterone
O

K. Shimada , K. Mitamura, T. Higashi, J. Chrom. A., 935, 2001, 141–172.

 Hyphenation of GC and MS
 The first useful “hyphenated” method?

 Continuous monitoring of the column effluent by a mass

spectrometer or MSD

 Very easy to interface – capillary GC columns have low

enough flow rates, and modern MS systems have high
enough pumping rates, that GC effluent can be fed directly
into the ionization chamber of the MS (for EI or CI, etc…)
– Larger columns require a “jet separator”

 Most common systems use quadrupole or ion trap mass

analyzers (MSD)