Gas & Affinity

chromatography
Ahsham Ali (23125) Sherdil Abdullah (22564)
Abdul Wahab (22554) Musab Adeem (22758)
Raja Hasnain (22781) Furqan Naveed (22780)
Husnain Riaz (23480)
M. Hashir (23129)
Salman Saleem (23471)
Malik Shaheer (23474)
 Introduction of Gas chromatography

 In this technique we can separate out components which are volatile, heavy molecular
weight compound, fatty acids and polymers.
 In GC Mobile phase is Gas and stationary phase is liquid/solid.
 In GC carrier gas such as Nitrogen, Helium, Argon, Neon is used. Carrier gas is a
solvent which means it will not allow any modification in a sample.
 GC has further two types which are Gas solid and Gas liquid chromatography.
 In gas solid chromatography, solid is simply packed in a column.
 In gas liquid chromatography, support is used. First we add solid support and then
liquid support is applied.
 Basic principle
 If we describe in one word, the principle of GC is partition.
 Component of mixture is vaporized and flow through the column.
 Component of mixture which have high affinity with Mobile phase elute earlier which
means that they will come out of column earlier.
 And component of mixture which have high affinity with stationary phase, they come
out of column late.
Gas chromatography
 Instrumentation of GC

 Carrier gas:
 Helium, N2, H, Argon are used as carrier gases.
 Helium is preferred for thermal conductivity detectors because of its high thermal
conductivity relative to that of most organic vapors.
 N2 is preferable when a large consumption of carrier gas is employed.
 Carrier gas from the tank passes through a toggle valve, a flow meter, (1-1000 ml/min),
capillary restrictors, and a pressure gauge (1-4 atm).
 Flow rate is adjusted by means of a needle valve mounted on the base of the flow meter
and controlled by capillary restrictors.
 GC instrumentation
Sample injection system: Liquid samples are injected by a micro syringe with a needle inserted
through a self-scaling, silicon-rubber septum into a heated metal block by a resistance heater.
 Gaseous samples are injected by a gas-tight syringe or through a by-pass loop and valves.
 Typical sample volumes range from 0.1 to 0.2 ml.
Separating column: The heart of the gas chromatography is the column which is made of metals
bent in U shape or coiled into an open spiral or a flat pancake shape.
Support
Detector: Detectors sense the arrival of the separated components and provide a signal.
These are either concentration-dependent or mass dependent.
The detector should be close to the column exit and the correct temperature to prevent
decomposition.
Recorder: The recorder should be generally 10 mv (full scale) fitted with a fast response pen (1 sec
or less). The recorder should be connected with a series of good quality resistances connected across
the input to attenuate the large signals.
 Working-procedure of GC

 Gas chromatography works by separating compounds in a mixture by passing a

gaseous or liquid sample through a column with a stationary phase and a mobile phase.
 The sample is injected into the column with a syringe and vaporized by heating.
 The mobile phase is an inert gas that carries the sample molecules through the column.
 The stationary phase is a liquid or solid that retains some molecules longer than others.
 The temperature of the column and the gas can be controlled to affect the separation.
 The separated molecules are detected by a computerized detector at the end of the
column.
 Comparison of GC with other
chromatography's
 Other types of chromatography can have certain characteristics while we can also compare
limitations of GC with these chromatography such as:
 Not suitable for detecting semi-volatile compounds
 Only indicates if volatile organic compounds are presents.
 High concentration so methane are required for higher performance.
 Frequent calibration are required.
 Units of parts per million range
 Environmental distraction, especially water vapor.
 Strong electrical fields Rapid variation in temperature at the detector and naturally
occurring compounds may affect instrumental signal.
 For these cases we can use liquid, HPLC, Column chromatography.
 Applications of GC

 GC analysis is used to calculate the content of a chemical product, for example in assuring the
quality of products in the chemical industry; or measuring toxic substances in soil, air or water.
 Gas chromatography is used in the analysis of:
 (a) air-borne pollutants
(b) performance-enhancing drugs in athlete’s urine samples
(c) oil spills
(d) essential oils in perfume preparation
 GC is very accurate if used properly and can measure Pico moles of a substance in a 1 ml liquid
sample, or parts-per-billion concentrations in gaseous samples.
 Gas Chromatography is used extensively in forensic science. Disciplines as diverse as solid drug
dose (pre-consumption form) identification and quantification, arson investigation, paint chip
analysis, and toxicology cases, employ GC to identify and quantify various biological specimens
and crime-scene evidence.
 Advancements in GC

 Now the GC setup allows the analytical column to be kept under vacuum conditions,
resulting in a faster GC run time, higher sample throughput, lower detection limits,
reduced analyte degradation, and greater robustness compared to conventional GC
analysis.
 Nowadays, GC×GC can be considered as a mature technique adopted as a routine food
control technique. The advantages mostly rely on the possibility of performing detailed
and sensitive targeted and untargeted sample profiling.
 The analytical strategy of GC and non target screening (NTS) is gaining popularity. GC
separations can be coupled to mass spectrometry with gentle and soft ionization
techniques, including chemical ionization (CI), atmospheric chemical ionization
(APCI), and dielectric barring direct ionization (DBD).
 Affinity chromatography
 Introduction: Affinity chromatography is a method of separating a biomolecule from a
mixture, based on a highly specific macromolecular binding interaction between the
biomolecule and another substance.
 The stationary phase consists of a support medium, on which the substrate (ligand) is
bound covalently, in such a way that the reactive groups that are essential for binding of
the target molecule are exposed.
Principle:
 As the crude mixture of the substances is passed through the chromatography column,
substances with binding site for the immobilized substrate bind to the stationary phase,
while all other substances is eluted in the void volume of the column.
 Once the other substances are eluted, the bound target molecules can be eluted by methods
such as including a competing ligand in the mobile phase or changing the pH, ionic
strength or polarity conditions.
 Ligands and matrix materials for affinity
chromatography
Ligand: It refers to the molecule that binds reversibly to a specific target molecule.
 The ligand can be selected only after the nature of the macromolecule to be isolated is known.
 When a hormone receptor protein is to be purified by affinity chromatography, the hormone itself
is an ideal candidate for the ligand.
 For antibody isolation, an antigen or hapten may be used as ligand.
 If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or effector may be used as a
the immobilized ligand.
Matrix: Matrix should be chemically and physically inert.
 It must be insoluble in solvents and buffers employed in the process
 It must be chemically and mechanically stable.
 It must be easily coupled to a ligand or spacer arm onto which the ligand can be attached.
 It must exhibit good flow properties and have a relatively large surface area for attachment.
 The most useful matrix materials are agarose and polyacrylamide.
Instrumentation of Affinity chromatography
 Working and Column preparation in affinity
chromatography
Working:
 The ligand is first covalently coupled to a matrix, such as agarose beads.
 The matrix is poured into a column.
 An impure mixture containing biomolecule of interest is loaded on the affinity column
 Biomolecules sieve through matrix of affinity beads and interact with affinity ligand.
Molecules that do not bind to ligand elute from the column
 Wash off contaminant molecules that bind to ligand loosely
 Column preparation: The column is loaded with solid support such as sepharose,
agarose, cellulose etc.
 Ligand is selected according to the desired isolate.
 Spacer arm is attached between the ligand and solid support.
 Applications of affinity chromatography
 Affinity chromatography is one of the most useful methods for the separation and
purification of specific products.
 It is essentially a sample purification technique, used primarily for biological molecules
such as proteins.
 Its major application includes:
 Separation of mixture of compounds.
 Removal of impurities or in purification process.
 In enzyme assays
 Detection of substrates
 Investigation of binding sites of enzymes
 In in vitro antigen-antibody reactions
 Detection of Single Nucleotide polymorphisms and mutations in nucleic acids.
Comparison of affinity chromatography with
other chromatographic techniques
 As compared to other chromatographic techniques, affinity chromatography High specificity
 Target molecules can be obtained in a highly pure state
 Single step purification
 The matrix can be reused rapidly.
 The matrix is a solid, can be easily washed and dried.
 Give purified product with high yield.
 Affinity chromatography can also be used to remove specific contaminants, such as
proteases.
Thank you!
6th semester, section-A