DEFINITION OF CHROMATOGRAPHY:

Technique used for the separation of the components of a mixture through a

column, in which these are distributed between two phases, one stationary and
another mobile.

CLASSIFICATION OF CHROMATOGRAPHY

 On paper.
 In thin layer
Chromatograph  In Column
y  Of gases.
 Of liquids.

 Gas-solid
Liquid chromatography chromatography
 Gas-liquid
chromatography

 Gel permeation
(GPC).
Gas chromatography
 Reverse phase.
 Ionic exchange

APPLICATIONS OF GAS CHROMATOGRAPHY.

 Pharmaceutical  Food Chemistry.

 Petrochemical  Analysis of polymers.
 Environmental analysis.  Sports medicine.
 Forensic medicine.
DIAGRAM OF A GAS CHROMATOGRAPH.

The basic instrument configuration consists of a mobile phase supply, an injection

port, an oven / separation column containing the stationary phase, a detector and a
data recording and management system.

TRAWL GAS

Also called = Carrier or Mobile phase.

Its function is to move the sample to through the whole system.

Characteristics:

 High purity.
 Low weight
 molecular.
 Inherte.

Example

 Nitrogen
 Helio
 Argon
 Hydrogen

INJECTORS

It is the point of introduction of the sample to the system, where it is vaporized by

heating, being dragged by the mobile phase towards the column.

COLUMNS

The column is the heart of the system, where the separation of the components of
a mixture by the liquid phase takes place.

Packed 1/4 -1/8 inch.

Semi-pillars.0.53mm in diameter and Capillaries 0.32-0.1mm diameter.

MIGRATION OF ANALYTES.

A slow migration through the column inevitably causes a lengthening of its band:
The retention time is the time that a solute takes to travel the column

EFFICIENCY OF THE COLUMN.

Efficiency: Elution capacity with a minimum dispersion of the analyte.

SELECTION OF COLUMNS

There are four main column parameters that must be taken into account: stationary
phase, diameter, length and thickness of the film.

SEASONAL PHASE:

The polarity exerts a very remarkable influence on the retention of the compound
and, consequently, on the separation. Use a stationary phase with a polarity similar
to that of the solutes.

DIAMETER:

The smaller the column diameter, the greater the number of theoretical plates. It
gives better resolution, The capacity of the column increases parallel to the column
diameter. The actual column capacity depends on the stationary phase, the solute
and the thickness of the film.

LENGTH:

The column efficiency (N) is proportional to the length of the column. Longer
columns are used when the peak separation is small and high column efficiency is
needed.

FILM THICKNESS:

Under isothermal temperature conditions, the solute retention is directly

proportional to the thickness of the film. In the case of temperature program
conditions, the change is 1 / 3-1 / 2 of the isothermal value. The thick film columns
serve to obtain a higher retention in highly volatile solutes.

INTRODUCTION OF SAMPLES.

Samples problems in liquid state, the problem mixture is introduced to the column
by means of a syringe and through a rubber stopper (septa) located in the injection
port, which is maintained at a temperature between 150 and 250 ° C. which causes
the volatile sample solutes to vaporize.

The systems used to introduce the sample to the CG, in the first instance can be
manually or automated and depending on the physical state of the sample:

a) solid in solution, c) steam and

b) liquid state, d) gas.

The means for introducing the sample can be a micro syringe (cases a and b);
syringe or sampling valve (cases c and d).

The gas separation is carried out mainly through the use of adsorbents solids
(CGS).

DETECTORS

It senses the output of the compounds eluted from the column according to some
physical or chemical property of the analyte generating an electrical signal.

Represented by a SIGNAL graph (Area or Height) vs TIME.

CLASSIFICATION OF DETECTORS.

UNIVERSAL: Generate a signal for any substance eluted.

SELECTIVES: Detect substances with some Physical-chemical property.

SPECIFIC: Detect substances that have a certain element or functional group in

their structures
 DCT TCD DIC FID
Detector by Detector by
Conductivity Ionization of
Thermal Flame

EM MS
DCE ECD
Mass
Detector by
Spectrometer
Capture of
Detector
Eletrones

Thermal Conductivity Detector. It measures the thermal conductivity of the

carrier gas, caused by the presence of eluded substances. It responds to
everything except the carrier gas and is considered the universal detector.

Flame Ionization Detector. Based on the measurement of the variations of the

ionization current in an oxygen-hydrogen flame due to the presence of eluted
substances. It responds to almost all organic compounds and is considered the
universal detector for organic compounds.

Electron Capture Detector. Based on the electronegativity of the eluted

substances, and their ability to form negative ions by electron capture. It responds
to a limited range of compounds, mainly to holocarbons and is used for the
analysis of pesticides and herbicides at the trace level.

Photoionization detector. Based on the selective ionization of certain compounds

that elute from the column by a lamp that produces radiation in the UV spectrum
(typically at 10.0 eV), the ions produced are collected and the resulting current flow
is measured and amplified. Frequently it is used for the analysis of aromatic
compounds (BTEX, Organic Gasoline Range and unsaturated, aromatic and
polyaromatic hydrocarbons).

Nitrogen-Phosphorus detector. Also called Alkaline Flame Ionization detector.

Responds to compounds with nitrogen or phosphorus and is used in
pharmaceutical and environmental analyzes.

Flame Photometry detector. Based on the measurement of the intensity of the

molecular emission of the fluorescence of hetero-atoms in the organic molecules. It
responds to compounds with sulfur or phosphorus and is used in environmental
and bio-scientific analyzes.

Mass spectrometry detector. It is a detector that in addition to quantitative

information provides qualitative information, based on the identification of the
compound through its characteristic spectrum of mass fragmentation. It responds
to everything except the carrier gas and is used in research, pharmaceutical and
environmental analyzes.

CHROMATOGRAPHY OF GASES-MASSES.

It is an analytical technique that combines the properties of:

 Separation of compounds by CG.

 The identification of said compounds by Mass Spectrometry.

MASS SPECTRUM

It is a graphic representation of the mass of an ion vs. its abundance.

Ionization methods

Electron impact and chemical ionization are the two methods most widely used in
GC/MS work
Electron impact (EI)

most common

The sample for analysis is introduced into the ion source, either through a solids
inlet or through a gas chromatography column. It is essential that the sample
enters the ion source in the gaseous state and the ability to heat the source and
solids probe are important for successful sample analysis. A beam of electrons
produced by a heated filament of either Tungsten or Rhenium collides with the
sample gas molecules, removes an electron and produces a positively charged ion
corresponding to the relative molecular mass of the sample being analysed.

Electron impact is an energetic ionisation technique and also produces fragment

ions which are smaller parts of the original molecule. The ions are accelerated out
of the source and pass through a series of slits to produce a focussed beam. The
beam of ions pass through a flight tube which is located between the poles of an
electromagnet, and are seperated according to their mass/charge ratio by scanning
the magnetic field. The separated ions are detected by electron or photomultipliers
to produce a plot of intensity versus mass/charge ratio respresentative of the
sample being analysed .ie the mass spectrum.

Chemical ionization (CI) a modification of EI wich results in “softer”

ionization-less fragmentation

Chemical ionization mass spectrometry (CIMS) is a technique for forming ions of

the compound of interest (analyte, A) by ion/molecule reactions from reactant ions
of a reagent gas that is generally present in a much greater abundance than the
analyte. The reactant ions are generally by electron ionization (EI) of the reagent
gas. The ions produced by EI often react with the large excess of the reagent gas
to form the actual reagent ions that react with the analyte. CIMS is performed with
both positively and negatively charged reactant ions. The most common
ion/molecule reactions in CIMS are proton transfer (which forms AH + or
[A − H]− ions), hydride transfer (which forms [A − H]+ ions), charge or electron
transfer (which forms A+•or A−•ions), and adduct formation or attachment (which
forms [A + R]+ or [A + R]− ions). Fragment ions from decompositions of these AH+,
A±•, [A − H]±, and [A + R]± ions are frequently observed. The extent of
fragmentation can be controlled by the choice of reagent gas used and can be
predicted to some extent from ionic thermochemical data. Collisionally stabilized
electron capture at high pressures (to form A−• ions) is often classified as chemical
ionization (CI).

The most common use of CIMS in analytical mass spectrometry is to obtain

simplified mass spectra of compounds, often one species spectra, which can be
used for quantitative analysis of mixtures. CIMS can be performed with any type of
mass spectrometer (quadrupole, magnetic, time-of-flight (TOF), Fourier transform
ion cyclotron resonance (FTICR), ion trap) and CIMS capabilities are routinely
available on many commercial instruments. With ion trap or FTICR mass
spectrometers, it is possible to select specific reactant ions. It is generally
considered that CI and EI sensitivities are approximately the same in the positive
ion mode and that electron capture CI at high pressures for compounds with high
electronegativities gives a much greater sensitivity than other EI or CI techniques.
CIMS has the same general limitations as electron ionization mass spectrometry
(EIMS) on the volatility and thermal stability of the compound being analyzed.
However, direct insertion of the sample into the source of the mass spectrometer
allows the analysis of relatively involatile and thermally unstable compounds.

Quadrupole

 Consists of four bars

 Operates in pairs (X or Y) each pair has a voltage
 Only ions with m / z can successfully pass through the filter (z-axis)
 Bars with high current eliminates ions with low m / z ratio
 Bars with low current eliminate ions with excessive m / z ratio
Advantages electronic ionization

 Simple construction.
 High efficiency in ionization.
 Highly characterized fragmentation models "fingerprints".
 Spectrum libraries EI.
 It presents few side reactions due to shocks.

Disadvantages electronic ionization.

 The sample must be in the gaseous state.

 It can cause excessive fragmentation.
 Some compounds do not generate molecular ion.
 Poor difference between isomers.

PHOTOTUBOMULTIPLIER DETECTOR.

 Phosphor plate.

Conversion of e to photons.

 Photomultiplier

Sealing in glass

Eliminates pollution

10 years of life

You can perform the detection of a only ion

VACUUM GENERATION SYSTEMS.

The vacuum allows to ensure that the ions form a beam stable enough to reach the
detector.

The vacuum must be so low that it prevents the collision of the ions with molecules
of the sample, gas or other ions.
ION MONITORING

Spectra of complete massas collected and archived at regular intervals of time.

• TIC: Total Ionn Chromatogram

• SIM: Single Ion Monitoring

TIC: Universal

SIM: Selective, Greater sensitivity

QUALITATIVE ANALYSIS.

TIC: It is the total representation of ions or fragments detected according to the

ratio m / z as a function of time.

SIM: Select a fraction of a species of interest represented by the number of ions

detected for a particular mass as a function of time.

QUALITATIVE ANALYSIS

TIC: The peaks corresponding to all substances eluted appear

SIM (m / z = 149): Chromatogram constructed from the same data using

fragements with mass = 149 (phthalates: plasticizer)

CALIBRATION OF THE MASSES.

The internal calibration of the masses is carried out using a reference compound
HEPTACOSA (PFTBA Perfluoro Terbutil Amina).

EXTERNAL TUNE

It has the function of verifying that the ionization and fragmentation in the source of
ions is carried out correctly.
 DFTPP: Deca Fluoro Triphenyl Phosphine
Semi-volatile compounds.

TUNE
BFB: Bromine Fluoro Benzene
Volatile compounds.

SURROGATED.

They are compounds chemically similar to the analytes of interest, but are not
expected to be found in the sample.

Types of Calibration by Chromatography

It is the process by which the height and / or area of a particular peak is related to
the concentration or amount of a compound.

Calibration Methods

 Area %
 Normalization%
 External Standard
 Internal Standard

CALIBRATION

Calibration methods

Calibrated calculations

 % Area

If Area% and Height% are not adequate, the calibrated calculations use data from
standard analyses to create individual peak calibrations.
The simplest calibration is the Response Factor, which is calculated by dividing the
known amount of a component by the size of the peak it produces.

Graphically, it is the slope of a plot of component amount versus peak size. as

shown in Figure 25

Response Factors can be determined by analyzing a single standard mixture

containing all of the components to be calibrated. However, the Response Factor
approach makes two important assumptions:

• The amount/size line passes through the origin.

• The amount/size line is straight.

For a trustworthy calibration, both assumptions must be demonstrated

experimentally. If the line is really straight and really does pass through the origin,
then the response factor is valid.

In Figure 25, Response Factors can be used for peaks A and B, but not for peak C.
The two forms of calibration correction are shown in Equation 2 and Equation 3.

For Peaks A and B:

CR of peak = MR of peak x Response Factor of peak (2)

For Peak C:

CR of peak = <Response Curve amount> of MR of peak(3)Peak C can only be

corrected by using the entire calibration curve. This is laborious by hand, but is
easily done using a data system.

 Normalization

The normalization percent is similar to Area% and Height%, but uses Corrected
Responses instead of Measured Responses, as shown in Equation 3.

Amount of peak n = [CR of peak n / Sum of all CRs in the run] x 100 (3)

Advantages

• This calculation corrects for component sensitivity differences, which yields more
accurate results for early peaks.

• Moderate sample size variation does not affect results.

Disadvantages

• The method must be calibrated.

• All peaks must be detected. Any peaks not detected or not flushed from the
column will reduce the sum of CRs. This causes overestimation of all measured
peaks.

• All peaks must be identified and calibrated to achieve the highest accuracy.
Unknown (and therefore uncalibrated) peaks reduce the absolute accuracy of the
calculation.

Common uses

• Provides very accurate results if there are no high-boilers to worry about.

 External standard
The great advantage of external standard is that only the peaks of interest need to
be calibrated. The calculation is very simple; see Equation 4.

Amount of peak n = CR of peak n (4)

Advantages

• Only the peaks of interest must be calibrated.

• Only the peaks of interest must be eluted and measured.

• Each calibrated peak is computed independently.

Disadvantages

• Peaks of interest must be calibrated.

• The calculation assumes that instrumental drift is negligible. Known check

samples must be run regularly to confirm this.

• Constant sample size is essential, since this is an absolute (rather than relative)
calculation. This is very difficult to achieve using manual injection. In practice, a
gas or liquid sampling valve or an automatic liquid sampler is a necessity.

Common uses

Gas analyses using a sampling valve. As instrument stability improves, and with
the help of automatic injection devices to ensure constant sample size, ESTD is
taking over many analyses that formerly required ISTD.

 Internal standard

Internal standard provides independent calculation of each calibrated peak. It also

corrects for variation in sample size, instrument drift, and other factors.

ISTD is considered the most accurate chromatographic calculation, although ESTD

with modern equipment is rapidly improving.

The basic calculation is shown in Equation 5.

Amount of peak n = [CR of peak n / CR of ISTD peak] x Amount of ISTD peak
(5)

The quantity Amount of ISTD peak is a known amount of the internal standard
compound that is added to each sample before analysis.

This is generally considered to be the most accurate of the calculations.

Advantages

• Only the peaks of interest must be calibrated.

• Only the peaks of interest must be eluted and measured.

• Each calibrated peak is computed independently.

• Minor variation in sample injection size does not affect results.

• Minor instrumental drift does not affect results.

Disadvantages

• Peaks of interest must be calibrated.

• A known amount of an internal standard substance must be added to every

sample.

Common uses

Liquid sample analysis where high accuracy is required.

Types of Calibration

-A level

 One data per peak

 A single straight line from the origin to the reference point

-Multi-Level

 In the example, 4 data per  Running a series of dilutions

compound  Various types of curves