chromatography: gc and hplc dr. sc. agr. m. samer | chromatography | 3 cairo university...

Dr. sc. agr. M. Samer | Chromatography | 1Cairo University

Chromatography: GC and HPLC

In the framework of the undergraduate course “Instrumentation and Control Systems”

Mohamed Samer, Dr. sc. agr. Associate ProfessorBioresource EngineeringDepartment of Agricultural Engineering Faculty of Agriculture, Cairo UniversityE-Mails: [email protected]; [email protected]: http://scholar.cu.edu.eg/samer/biocv

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http://scholar.cu.edu.eg/samer/biocv

http://scholar.cu.edu.eg/samer/biocv


Introduction


Chromatography is a laboratory technique for the separation ofa mixture.

The mixture is dissolved in a fluid called the mobile phase,which carries it through a structure holding another materialcalled the stationary phase.

The various constituents of the mixture travel at differentspeeds, causing them to separate.

The separation is based on differential partitioning between themobile and stationary phases.

Subtle differences in a compound's partition coefficient result indifferential retention on the stationary phase and thus affectthe separation.


Source: Anthony Crasto


Chromatography may be preparative or analytical.

The purpose of preparative chromatography is to separate thecomponents of a mixture for later use, and is thus a form ofpurification.

Analytical chromatography is done normally with smalleramounts of material and is for establishing the presence ormeasuring the relative proportions of analytes in a mixture.

Both are not mutually exclusive.


Gas Chromatography (GC)


Gas chromatography is a technique used for the separation ofthe macromolecules based on their volatility and their ability tointeract with the stationary phase.

The mobile phase is an inert gas while the stationary phase canbe either solid or liquid.

The column has liquid with a high boiling point such as silicongrease and placed in an oven between 50°C to 300°C.

The sample injector point of the oven is maintained at hightemperature so that the temperature of the gas entering thecolumn need not be increased.

(Grob and Barry, 2004; Leonard et al., 1999)


The retention time of the molecules in the column depends ontheir ability to interact with the stationary phase.

If the molecules interact with the stationary phase thenretarded and eluted last while the molecules which do notinteract with the stationary phase would elute first.

There are two types of the column used in gas chromatography:Packed Conventional Column & Open Tubular/Capillary Column.



The Packed Conventional Column is made of glass or stainlesssteel and is 1-15 meter long with pore range of 1.6-9.5 mm.

The Open Tubular/Capillary Columns (OTC) are a bit differentthan the packed conventional column. The length of the OTC is15–30 meters while thickness is 1 mm and pore size 0.25 mm.

They are of two types- wall coated open tubular column andsupport coated open tubular column.

In case of wall coated open tubular column, the stationaryphase is coated on the wall while in the later, the inert matrixbonded to the wall holds the stationary phase.



Examples of stationary phase: (1) Polyethylene glycol (veryviscous); (2) BP1, BP2, BP10 etc.; (3) CP wax; and (4) BD wax.

Depending on the boiling point of the liquid, the oventemperature needs to be maintained.

In general, the temperature of the column is maintained atconstant and is known as isothermal elution.

If the sample is complex mixture and separation is difficult, thenthe temperature can be varied so as to get betterresolution/separation. This is called temperature programming.



Expanded-bed chromatography

In this type of chromatography,elution is performed in a normalpacked-bed, but during theadsorption-wash step, the flow isreversed and the column bedexpanded. This allows forparticulate contaminates to passfreely through the column andprevent column clogging.

Magdeldin and Moser (2012)


Source: Anthony Crasto


Kulsing et al. (2015)


Illustration showing different molecules that can be purified using affinity chromatography(Magdeldin and Moser, 2012)


The sample used in gas chromatography needs to volatile.However, if the sample is non-volatile, then they can bevolatized by reacting it with some reactants.

The sample molecule with -OH, or -NH2 groups needs to bederived to a volatile compound. They are very polar and thusthey would be retained in a column for a longer time.

The molecules which are eluted from the column are detectedby highly accurate detectors.

The detectors used are: Thermal Conductive Detectors, FlameIonization Detectors, Nitrogen-Phosphorous Detectors, etc.



In thermal conductive detectors, the temperature of thedetector (filament) is lower than the column.

Thus, the molecule eluted would transfer the heat to thefilament causing the increase in the resistivity.

The limitation of this type of detector is that it is non-specificand less sensitive.

The retention time depends on : (1) Temperature, (2) Flow rateof the carrier gas, (3) Length and diameter of the column, (4)Nature and interaction between mobile and stationary phase,and (5) Volatility of the compound.



Diagram of a gas chromatograph Quora (2018)


Diagram of a gas chromatographOffnfopt (2017)


Structure diagram of gas chromatographWang et al. (2014)


A typical modern gas chromatograph

Source: McShane Firm (2018)


Source: HP AgilentGas Chromatograph


Gas Chromatography is a technique of separation of gases andvolatile liquids. Liquids can also be analyzed using GC, providedthey are made gaseous by derivatization.

For a sample to be analyzed using GC, the sample should be: (1)Thermostable (not degraded while heated); and (2) Volatile.

In GC, the mobile phase used is a gas: e.g. hydrogen, helium,nitrogen.

When GC is attached with a Mass Spectrometry Detector, it isknown as GC-MS which can be used to determine any unknownimpurities or identify unknown samples including theirstructural elucidation and molecular weight determination.


Gas Chromatography Mass Spectrometer Schematic

Source: Chemistry LibreTexts


Schematic diagram of GC-MS

Kumar et al. (2015)


Principles involved in stationary phase:

1. If a solid is used as the stationary phase, then the principleof separation is Adsorption. It is known as Gas-SolidChromatography (GSC).

2. If a liquid (over an inert solid support) is used as thestationary phase, then the principle of separation is Partitionand the method is called Gas-Liquid Chromatography (GLC).

GSC is not widely used because of semi or permanent retentionof active or polar molecules due to adsorption, resulting insevere tailing. Therefore, in general, Gas Chromatography refersto GLC only.


Applications of GC:

1. Qualitative analysis by identifying the compounds using theretention times.

2. Quantitative analysis by comparing the peak area of asample with that of a standard.

3. Separation of volatile oils from mixtures.

4. Food analysis and forensic laboratories.

5. Determination of levels of pollutants in air.

6. Identification and quantification of impurities inpharmaceutical preparations.

7. Determination of residual solvents in pharmaceuticals.


Typical affinity chromatography purificationMagdeldin and Moser (2012)


This diagram of a typical chromatogram shows key concepts such as Rt, and peak area. Different Rt’s will tell us what we have. The different area sizes under the peak mean how much of that

substance at that Rt there is. More area means more substance. Less area, means less substance.

Source: McShane

Firm (2018)


High-Performance Liquid Chromatography (HPLC)


High-Performance Liquid Chromatography (HPLC), also knownas High-Pressure Liquid Chromatography, is a technique used toseparate, identify, and quantify each component in a mixture.

It relies on pumps to pass a pressurized liquid solventcontaining the sample mixture through a column filled with asolid adsorbent material.

Each component in the sample interacts slightly differently withthe adsorbent material, causing different flow rates for thedifferent components and leading to the separation of thecomponents as they flow out of the column.


HPLC is a mass transfer process involving adsorption.

HPLC relies on pumps to pass a pressurized liquid and a samplemixture through a column filled with adsorbent, leading to theseparation of the sample components.

HPLC relies on pumps to pass a pressurized liquid and a samplemixture through a column filled with adsorbent, leading to theseparation of the sample components.

The components of the sample mixture are separated fromeach other due to their different degrees of interaction with theadsorbent particles.


The pressurized liquid is a mixture of solvents (e.g. water,acetonitrile and/or methanol) and is known as "mobile phase".

Its composition and temperature play a major role in theseparation process by influencing the interactions taking placebetween sample components and adsorbent.

These interactions are physical in nature, such as hydrophobic(dispersive), dipole–dipole and ionic, most often a combination.


An HPLC. From left to right: A pumping device generating a gradient of two different solvents- a steel-enforced column and a detector for measuring the absorbance.

Source: Kjaergaard


Source: Agilent

A modern self-contained HPLC


Schematic representation of an HPLC unit: (1) Solvent reservoirs, (2) Solvent degasser, (3)Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6)Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injectionloop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e., IR, UV), (11)Data acquisition, (12) Waste or fraction collector.

YassineMrabet (2009)


Preparative HPLC System YassineMrabet (2012)


Catalogs


Videos


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chromatography: gc and hplc dr. sc. agr. m. samer | chromatography | 3 cairo university...

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