Apat 2013 gc workshop 2

AnalySys Sciences
2
Instrumental in your success
http://analysciences.com

An Analytical chemist …
… tries to answer only two questions.
Given a sample …
What is it? Qualitative analysis
How much is it? Quantitative analysis

The evolution of analysis
1900‟s Manual titration 1 mg 10-3 0.001 gm
1920‟s TLC 1 µg 10-6 0.000001
1960‟s GC 1 ng 10-9 0.000000001
1980‟s HPLC 1 pg 10-12 0.0000000001
1990‟s GC-MS 1 fg 10-15 0.000000000000001
2008 LCMS 1 ag 10-18 0.000000000000000001
2013 FTMS 1 zg 10-21 0.000000000000000000001

Analytical Chemistry – The road ahead
Increased use of hyphenated techniques, like LC-MS, GC-FTIR
& LC-NMR.
Lower limits of detection.
“Walk-away” automation.
Intuitive software and data handling.
Increasing use single-point control systems via the Internet.

The Analytical Pharmacist in the 21st century
Full-time analytical chemist.
Part-time software engineer and EDP
specialist.
AND…a knowledge of software
platforms, data handling techniques
and preferably, basic electronics.

Chromatography … An introduction
7http://analysciences.com
From: The Universal Etymological Dictionary, 1731

Chromatography … since Biblical times.
So Moses brought Israel from the Red Sea, and they went
out in the wilderness of Shur …and found no water.
And when they came to Marah, they could not drink of the
waters of Marah, for they were bitter;
And the people murmured against Moses, saying, What
shall we drink?
And he cried unto the Lord and the Lord shewed him
a tree, which when he had cast into the waters, the
waters were made sweet.
Exodus, Chapter 15 §22–25 (King JamesVersion).
Source: Article by Leslie Ettre.
Ion exchange
chromatography?

110 years of modern chromatography
March 21, 1903
At the Warsaw Society of Natural Scientists,
Russian botanist, Mikhail Semenovich Tswett
presented the first official lecture on
chromatographic separation.
9
Tswett, MS (1906) Physico-chemical studies on chlorophyll adsorptions. Berichte der Deutschen botanischen Gesellschaft, 24, 316-23
Tswett, MS (1906) Adsorption analysis and chromatographic method. Application to the chemistry of chlorophyll. Berichte der Deutschen
botanischen Gesellschaft, 24, 385 http://www.life.uiuc.edu/govindjee/Part2/34_Krasnovsky.pdf
http://web.lemoyne.edu/~giunta/tswett.html

When a chlorophyll solution in petrol ether is filtered through the column
of an adsorbent …then the pigments will be separated from the top down
in individual colored zones…the pigments which are adsorbed stronger will
displace those which are retained more weakly.
http://analysciences.com 10

"Like light rays in the spectrum, the different components of a
pigment mixture, obeying a law, are separated on the calcium
carbonate column and can thus be qualitatively and quantitatively
determined.
I call such a preparation a chromatogram and the corresponding
method the chromatographic method."

Gas chromatography – the pioneers.
Erika Cremer, Univ of
Innsbruck, Austria,
1944, developed the
theory and use of gas
chromatography.
She was assisted by
her PhD student, Fritz
Prior.

Chromatography is …
“…a method in which the components of a
mixture are separated on an adsorbent
column in a flowing system". M.Tswett
A separation involving a mobile
phase, a stationary phase, and the
sample. The sample undergoes a
series of interactions between these
two phases, resulting in separation of
its components. Sample components
elute in increasing order of
interaction.

What interaction?
Adsorption
…analyte in mobile phase
adsorbed onto stationary phase.
Equilibration between the mobile
and stationary phase results in
separation.

Partition
…thin film of a liquid
stationary phase formed on a
solid support.
Solute molecules partition
between the mobile phase and
stationary phase.

Ion-exchange
Ion-ex resin is used to
covalently attach anions or
cations onto it. Solute ions of
the opposite charge are
attracted to the resin.
Example: Purification of hard
water.

Affinity
specific interaction between a
solute molecule and a
molecule that is immobilized
on a stationary phase. eg.
purification of
immunoglobulins.

Size Exclusion
a porous gel separates
molecules by size.
Example: Purification of
enzymes or proteins.

Mobile
phase
Gas
Gas-solid
(Adsorption)
Gas-Liquid
(Partition)
Liquid
TLC /Planar
chromatography
Column chrom
HPLC
Supercritical
fluid
SFC
Chromatography –
Modes

Mobile phase
Stationary phase
Eluate collection
Sample introduction
Detection
Chromatography
– the system
20
Stationary phase is
packed into a column,
or …
In the form of a thin
layer coated on a glass
or aluminium plate or
… In the form of a
thick sheet of paper.

A typical chromatogram
21
Y axis = Detector
response (usually in
millivolts)
X axis = retention time
(or volume)
A symmetrical peak is
known as a Gaussian
peak.

Some boring equations

Retention Volume / Time
Volume of mobile phase required to elute a particular
analyte from the stationary phase.
Time taken by an analyte to elute from the stationary
phase.
VR = tR x Fc
tR = Retention time
Fc = Flow rate

Retention Time
Dead Time/volume
Retention time / retention volume taken by an
unretained solute to elute from the system.
Represents the combined volume of tubings,
detector flow cell, injector loop, column volume.
Relative (corrected) retention time
0R Rt t t  

Partition Co-efficient
(Distribution / Adsorption co-efficient)
M
sC
K
C

CS = concentration of the analyte in the
stationary phase.
CM = concentration in the mobile phase
Analytes in a sample mixture will separate
in a chromatographic system only if their K
values are significantly different.

Partition Ratio (Capacity Factor)
Measure of the time spent by a
solute in the mobile phase, with
respect to the stationary phase.
For baseline separation, K’ > 2

Relative retention (Selectivity / separation factor)
For baseline separation, a > 1.5
2
1
k
k
a




Selectivity
Depends on
•Nature of the two phases
•Column temperature

Resolution
For baseline separation, Rs >2
2 1
1 2
2
R R
s
t t
R
w w


 
 
 

Peak Width (4s)

Tailing factor (Asymmetry/ Skew factor)
BC
As
CA


Tailing factor - 2

System Suitability Parameters USP
Plate count > 2000 plates/meter
Tailing factor < 2
Resolution > 2
Partition ratio > 2
Relative retention > 1.5
Precision / repeatability RSD </= 1% for n >/= 5

Chromatography Theories
or… why a chromatography column will not do
what it’s told..

Plate theory Martin and Synge (1941)
Nobel in Chemistry, 1952 for “their
invention of partition chromatography”.
Chromatography column assumed to be
similar to a distillation column.
Separation occurs across a series of
theoretical plates.
Higher number of theoretical plates
improves column performance.

Plate theory explained
A distillation column is used for fractional distillation of liquid
mixtures. Higher surface area inside the column improves
distillation efficiency. This is done by putting in a series of
glass plates, with each plate containing glass beads or similar
packing material.
A chromatographic column is similar to a distillation column.
The packing inside the column is considered similar to the
packing inside a distillation column. There are no real plates
inside, hence „theoretical plates‟.
Hence, height equivalent to a theoretical plate (HETP). Higher
number of plates, higher separation efficiency.

Rate theory Dr JJ van Deemter (1956)
Plate theory does not explain band spreading and peak
broadening. Does not take into account packing material
properties, mobile phase flow rate and column geometry.
Rate theory takes into account various factors that cause
chromatographic peak broadening and reduction of
separation efficiency.
37

van Deemter Equation
linear velocity ( flow rate)
C
H A B


  

38
van Deemter took into account several
factors that can affect HETP and column
performance. He formulated a mathematical
equation that defined the relationship
between various chromatographic factors and
HETP.
This equation made it possible to numerically
calculate column performance, design better
chromatography stationary phases and
improve separation efficiency.

A term – Multipath effect or Eddy diffusion
Analyte molecules take different paths
through the packing, leading to band
broadening
To reduce eddy diffusion, reduce
stationary phase particle size.
However, backpressure will increase.
In GC, backpressure is not a major
issue.

B term
Longitudinal diffusion / wall effect
Distortion of the mobile phase front, due
to varying velocity across the column,
especially at the column wall
To reduce wall effect, increase flow rate

C term – mass transfer resistance
Analytes remain trapped in stagnant pockets
in the packing. To improve mass transfer,
decrease mobile phase flow rate.

Van Deemter plot
What does it mean?
In practical terms, it means that for a given
stationary phase and for a given
chromatography column or plate, there is
one optimal mobile phase flow rate.
Increasing or decreasing flow rate might
have an adverse effect on performance.
For example: For an HPLC column with
4.6mm internal diameter and 150mm length,
packed with 5u, spherical particles, the
optimal flow rate is 1ml/min.

HETP Height Equivalent to a theoretical plate
2
2
4
16
2
5.54
R
R
L
H
t
L
H
t
s
s
 
   
 
   

Plate Count
2
2
16
4
25
5
R
R
t
t
s
s
 
   
 
   
2
5.54
2
R
L
N
H
t
s

 
   

Plate count – what it means.
The plate count gives you an idea of the efficiency and separating power of a column.
Higher plate count for a given column implies better performance
(but does not guarantee it !)
Plate count is affected by:
Nature of sample
Flow rate
Detector flow cell volume
Dead volume
Temperature
Detector settings / Data system settings.
Injector reproducibility, etc…
Be wary when comparing plate counts!!

A typical chromatogram
46
Y axis = Detector
response (usually
in millivolts)
X axis = retention
time (or volume)

Quantitation in Chromatography
Area (height) under the peak is
proportional to the injected
amount.
Proportionality constant is the
response factor.

How is peak area determined?
Integration
Data system sub-divides
peak into small rectangles,
calculates area of each,
and adds them up.

Quantitation – External
standards
Inject known concentrations of the
analyte using reference standards.
Analyse the test sample under the
same conditions.
Plot a calibration curve of analyte
concentration v/s peak area (or
height).

Internal Standards
Chemically similar to the analyte.
Added to the sample and external standards.
Same amount added to both.
Accounts for variations in injection volume
and other system variables.
Provides better precision.

Gas Chromatography
51

Gas Chromatography
Mobile phase is a gas
Used for volatile, heat stable samples only. eg.
Petroleum products, volatile oils, perfumeries.
… Or analytes that can be converted to
volatile derivatives, eg. amino acid silyl
derivatives, fatty acid methyl esters.

Why GC?
Minimal sample prep.
Fast analysis time. High separation
efficiency.
Easier to automate. Easier to upgrade
to hyphenated methods like GC-MS.
Lower capital costs and running costs.
Given a choice between HPLC and GC,
choose GC!
Restricted to analytes that are volatile
and thermo-stable … or to analytes that
can be derivatised.

Carrier
gas
Filters/traps
Injector
Detector
Column
oven
Column
Data
system
GC Schematics

GC – Mobile phases / Carrier gases.

GC – Mobile phases
Helium is commonly used as a carrier gas. Nitrogen is
also used.
Hydrogen is becoming a popular alternative to helium.
Gases are stored in high-pressure cylinders.
Gas flow is controlled by regulators.
Sometimes nitrogen and helium generators are used
instead of cylinders.

Hydrogen as carrier gas.
H2 has low viscosity and high diffusivity.
Hence, faster analysis times.
Much cheaper than helium. Lower cost-
per-analysis.
Helium is extracted from natural gas.
Process is very expensive. Not eco-friendly.
Acute shortage of Helium.
H2 can be cheaply produced using H2
generators.

Gas manifolds
Gas manifolds are used to purify and
dehumidify the gases before they enter
the GC.
Dust filters, moisture traps, silica gel
pellets and molecular sieves are used.
58

Sample
Introduction

Injector ports
Samples are injected through sealed,
heated injection ports.
Injection volumes are very small, usually
less than 5 μl.
Injectors should accurately deliver the
vaporised sample on to the head of the
GC column.

Packed column injector
Injector septum provides a leak-tight
seal.
Injector liner protects the inlet seal
from dirt and contaminants.
Inlet seal protects the GC column.
Injector body is heated by a
programmable heater system.

Used with capillary columns.
Injects small sample volumes.
(<1μl)
Splits the injection volume into
smaller volumes, by adjusting the
split ratio.

PTV injector
Programmable temperature vaporising
injector.
Used for large sample volumes and
thermo-labile compounds
Instantly vaporises sample, upto 3000C
Highly reproducible and accurate.

Injector septa
Septa ensure a leak-tight seal
at the injection port.
Available in various materials
– teflon, rubber and silicone.

Sampling Valves
Used for continuous, reproducible injection
of gaseous samples.
Can be configured in several ways:
•Multiple column switching
•Detector switching
•Automated air sampling

Injector liners
Glass liners are used inside the
injector body.
Protect the injector from sample
debris.

GC – sample injection syringes
Septum piercing needle.
Available in various volumes,
from 1ul to 100ul.
Can be automated.

Autosamplers
Two types:
Carousel
XYZ samplers
Can automate many tasks:
Simple injection
Sample prep/derivatisation/filtration/
dilution/heating/cooling /weighing.

Autosamplers – pros & cons
Low cost-per-analysis.
Reagent & solvent consumption is
reduced.
High reproducibility.
Reliable results.
24/7/365 operation.
Chemist is free of repetitive
manual tasks.
High capital costs.

GC – Stationary phases & columns

Packed columns
Made of SS, glass or copper tubing, filled
with porous packing material, which may
be coated with a viscous liquid phase.
Packed columns contain a finely divided,
inert, solid support material (usually
based on diatomaceous earth ) coated
with liquid stationary phase. Most packed
columns are 1.5 - 10m in length and
have an internal diameter of 2 - 4mm.

Packed columns – phases.
The packing usually consists of an inert
porous material such as Celite (a
diatomaceous earth), or calcined Celite (in
the form of powdered fire brick) or a
synthetically polymeric resin.
Glass beads and molecular sieves are also
used.

Packed columns - Kieselguhr
Packings are treated with
dimethylchlorosilane to
remove active silanols.
Washed with HCl to
remove trace metals.
Diatomaceous earth or kieselguhr is
soft, sedimentary rock that contains
fossilised remains of diatoms (hard-
shelled algae). It consists of 80-90%
silica, and small amounts of alumina
and iron oxide. It crumbles easily
into a fine, white powder.
Celite is a brand name, owned by
World Minerals Inc, a division of
Imerys Filtration.
Chromosorb W = Untreated celite
Chromosorb P = Calcined celite
Chromosorb S = Celite calcined with sodium
carbonate.

Packed columns – Molecular sieves
Molecular sieves are synthetic
zeolites (complex alumino-silicates
of sodium, potassium or calcium)
of various pore sizes, usually 4 Å
or so.
Used for separation of fixed gases
like CO, CO2, CH4, Ar, H2, O2.

Packed columns – Polymeric packings
Macroporous, spherical,
ultrapure resins.
Used for difficult separations in
gas chromatography.
Eg. Separation of H2S and H2O.
Separation of gas mixtures.
HayeSep is a popular brand.

Capillary columns Made from fused silica.
Have an internal diameter of a few
tenths of a millimeter, usually
0.32mm and 0.53 mm.
Length between 3m to 30m.
Capillary columns are more efficient
than packed columns.
Much higher plate counts >30,000
plates per meter.

Capillary columns - 2
Liquid stationary phase is
coated or chemically bonded to
the inner wall of the capillary.
Most common phases:
Polysiloxanes
Polyethylene glycols.

Separation mechanisms in GC
Partition: Analyte partitions
between the carrier gas and a
viscous stationary phase.
Adsorption: Analyte
adsorbs/desorbs between the
carrier gas and a solid stationary
phase.

GC - Detection systems

Thermal Conductivity Detector
Detector cell contains a heated filament with an
applied current. As carrier gas containing solutes
passes through the cell, a change in the filament
current occurs. The current change is compared
against the current in a reference cell. The difference
is measured and a signal is generated. (Wheatstone
bridge principle).
Selectivity: All compounds except for the carrier gas
Sensitivity: 5-20 ng Linear range: 105-106
Temperature: 150-250°C

Flame Ionisation Detector
Analytes are burned in a hydrogen-air flame. Carbon
containing compounds produce ions that are attracted
to the collector. The number of ions hitting the
collector is measured and a signal is generated.
Selectivity: Compounds with C-H bonds.
Sensitivity: 0.1-10 ng. Linear range: 105-107
Gases: Combustion - hydrogen and air; Makeup -
helium or nitrogen. Temperature: 250-450°C.

Electron Capture
Detector
Electrons are supplied from a 63Ni foil
lining the detector cell. A current is
generated in the cell. Electronegative
compounds capture electrons, causing a
reduction in current. The amount of
current loss is indirectly measured and a
signal is generated.
Selectivity: Halogens, nitrates and
conjugated carbonyls.
Sensitivity: 0.1-10 pg
Temperature: 300-400°C

Pulsed discharge ionisation detector (PDID)
Pulsed DC discharge creates a plasma
by ionising helium gas inside the
detector body.
Charged helium plasma in turn
ionises analytes eluting from the GC
column.
This results in a current that is
proportional to the amount of the
analyte.

PDID - Advantages
Universal, non-destructive detector.
Very sensitive, can detect analytes in the
femtogram level (10-15).
Good alternative to electron-capture
detector for pesticides and halogenated
compounds, since it is non-radioactive.
More sensitive than FID, and can be
used in settings where a flame is not
safe (like petroleum and gas analyses.)

Flame Photometric Detector.
Uses a photomultiplier tube to
detect spectral lines of analytes,
as they are burned in a flame.
(like in a flame photometer).
Especially useful for sulfur and
phosphorus compounds.

Photoionisation detector
UV lamp ionises analytes from the GC
column eluent.
Useful for volatile organic compounds
like polyaromatic hydrocarbons and
inorganic species that are ionised in
UV light.
Used for environmental pollutants.

Inside the GC
GC columns are mounted in
an oven.
Oven temperature can be
programmed.
Better separations are
achieved with temperature
programming.

Temperature programming – why.
In GC, analytes are separated according to boiling point and polarity.
Molecules with low boiling point will elute early from the GC column. Compounds
with high boiling point will elute later.
Analytes interact with the GC column. If the column is non-polar, analytes with
high polarity will travel faster through the column while more non-polar
compounds will be retained.

Isothermal GC
Isothermal GC is not a good choice for samples
containing analytes with varying boiling points.
For example, petroleum products, silylated amino
acids, methylated fatty acids.
In an isothermal GC analysis, the column
temperature is constant. Fast eluting compounds
may then appear as overlapping peaks and late
eluting compounds will have long retention time
and broad peak shape.
http://ull.chemistry.uakron.edu/chemsep/slide.php?Chapt
er=/chemsep/GC/&Last=100&Slide=56

Temperature programming
By varying column temperature over
time, analytes with different boiling
points can be separated.
Analysis time can be optimised.
http://ull.chemistry.uakron.edu/chemsep/slide.php?Chapt
er=/chemsep/GC/&Last=100&Slide=56

Temperature ramping

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