Podcast
Questions and Answers
In column chromatography, what role does the matrix play?
In column chromatography, what role does the matrix play?
- It provides mechanical stability and chemical stability within the column.
- It directly interacts with the solute components for separation.
- It is the finely divided material through which the liquid percolates.
- It is the material supporting the stationary phase. (correct)
Why is chemical inertness an important property of the matrix material in column chromatography?
Why is chemical inertness an important property of the matrix material in column chromatography?
- To prevent the support from reacting with the solutes, ensuring separation is based on intended interactions. (correct)
- To facilitate a higher flow rate through the column.
- To allow the matrix material to be available in different particle sizes.
- To increase mechanical stability of the matrix.
What structural feature does cellulose have that makes it highly hydrophilic?
What structural feature does cellulose have that makes it highly hydrophilic?
- It consists of -1, 4-linked D-glucose units.
- It's cross-linked with epichlorohydrin.
- Its inherent polysaccharide structure. (correct)
- It has a fibrous form.
Which characteristic distinguishes dextran from cellulose as a matrix in column chromatography?
Which characteristic distinguishes dextran from cellulose as a matrix in column chromatography?
Why should agarose matrix materials be suspended in water or a suitable buffer solution?
Why should agarose matrix materials be suspended in water or a suitable buffer solution?
What is the primary cross-linking agent used in polyacrylamide matrices for column chromatography?
What is the primary cross-linking agent used in polyacrylamide matrices for column chromatography?
What property makes polystyrene matrices particularly suitable for ion exchange and exclusion chromatography?
What property makes polystyrene matrices particularly suitable for ion exchange and exclusion chromatography?
Why are excess silanol groups removed from silica matrices intended for column chromatography?
Why are excess silanol groups removed from silica matrices intended for column chromatography?
What is the purpose of allowing the gel to swell in a solvent before packing a column?
What is the purpose of allowing the gel to swell in a solvent before packing a column?
Why is it important to de-aerate the slurry under vacuum if it is prepared cold?
Why is it important to de-aerate the slurry under vacuum if it is prepared cold?
What procedural step helps prevent uneven packing when pouring the slurry into the column?
What procedural step helps prevent uneven packing when pouring the slurry into the column?
After settling the gel in the column, what is the purpose of running the eluent through it for an extended period before sample application?
After settling the gel in the column, what is the purpose of running the eluent through it for an extended period before sample application?
What is the primary reason for applying the sample to the prepared column carefully?
What is the primary reason for applying the sample to the prepared column carefully?
In the context of sample application in column chromatography, what is the purpose of using a high-density solution?
In the context of sample application in column chromatography, what is the purpose of using a high-density solution?
What is considered the most satisfactory method for sample application among the methods that leverage dense solutions?
What is considered the most satisfactory method for sample application among the methods that leverage dense solutions?
How does increasing the length/width ratio of the column typically affect column efficiency?
How does increasing the length/width ratio of the column typically affect column efficiency?
What is the general relationship between the particle size of the column packing material and the resulting separation in column chromatography?
What is the general relationship between the particle size of the column packing material and the resulting separation in column chromatography?
Why is it important to select a solvent with low viscosity in column chromatography?
Why is it important to select a solvent with low viscosity in column chromatography?
How does increasing the temperature generally affect the speed of elution in column chromatography, and what temperature range is typically used?
How does increasing the temperature generally affect the speed of elution in column chromatography, and what temperature range is typically used?
What is an effect of a very slow flow rate in column chromatography?
What is an effect of a very slow flow rate in column chromatography?
What is the term for the removal of the sample from the solid matrix using a suitable solvent?
What is the term for the removal of the sample from the solid matrix using a suitable solvent?
Which term defines the total volume of material, both solid and liquid, within the column?
Which term defines the total volume of material, both solid and liquid, within the column?
What is the void volume in the context of column chromatography?
What is the void volume in the context of column chromatography?
What is the 'elution volume' in chromatography?
What is the 'elution volume' in chromatography?
What is the relationship between elution volume, retention time, and the flow rate of the mobile phase?
What is the relationship between elution volume, retention time, and the flow rate of the mobile phase?
What defines isocratic elution?
What defines isocratic elution?
In stepwise or batch elution, what is the key procedural difference compared to isocratic elution?
In stepwise or batch elution, what is the key procedural difference compared to isocratic elution?
What is the main purpose of gradient elution?
What is the main purpose of gradient elution?
Which variable is plotted against detector signal in a chromatogram generated from column chromatography?
Which variable is plotted against detector signal in a chromatogram generated from column chromatography?
What information can be derived from the area under the peaks in a chromatogram?
What information can be derived from the area under the peaks in a chromatogram?
What phenomenon primarily drives the separation of substances in adsorption column chromatography?
What phenomenon primarily drives the separation of substances in adsorption column chromatography?
What determines the position of a component as it migrates through an adsorption column?
What determines the position of a component as it migrates through an adsorption column?
In adsorption chromatography, where does the most strongly adsorbed component end up?
In adsorption chromatography, where does the most strongly adsorbed component end up?
What dictates the degree of separation in adsorption chromatography?
What dictates the degree of separation in adsorption chromatography?
What is represented by 'K' in the distribution coefficient formula for chromatography?
What is represented by 'K' in the distribution coefficient formula for chromatography?
What is one common application of Fuller's earth in adsorption chromatography?
What is one common application of Fuller's earth in adsorption chromatography?
What is a significant limitation of using powdered charcoal as an adsorbent in chromatography?
What is a significant limitation of using powdered charcoal as an adsorbent in chromatography?
What property of polystyrene beads makes them function by adsorption?
What property of polystyrene beads makes them function by adsorption?
What is a critical consideration when working with hydroxyapatite in chromatography?
What is a critical consideration when working with hydroxyapatite in chromatography?
For what type of molecules does silica gel have a particularly strong adsorptive property?
For what type of molecules does silica gel have a particularly strong adsorptive property?
What treatment determines the type of alumina (acidic, basic, or neutral) used in column chromatography?
What treatment determines the type of alumina (acidic, basic, or neutral) used in column chromatography?
Flashcards
Column Chromatography
Column Chromatography
A separation process involving uniform percolation of liquid through a column packed with finely divided material.
Chromatography Matrix
Chromatography Matrix
The material supporting the stationary phase in column chromatography, needs high stability and inertness.
Cellulose Matrix
Cellulose Matrix
A matrix made from -1, 4-linked D-glucose units, cross-linked with epichlorohydrin, hydrophilic nature.
Dextran Matrix
Dextran Matrix
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Agarose Matrix
Agarose Matrix
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Polyacrylamide Matrix
Polyacrylamide Matrix
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Polystyrene Matrix
Polystyrene Matrix
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Silica Matrix
Silica Matrix
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Column Packing
Column Packing
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Sample Application
Sample Application
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Column Dimensions
Column Dimensions
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Packing Particle Size
Packing Particle Size
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Solvent Quality
Solvent Quality
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Column Temperature
Column Temperature
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Flow Rate
Flow Rate
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Column Packing Quality
Column Packing Quality
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Elution
Elution
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Bed Volume
Bed Volume
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Void Volume
Void Volume
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Elution Volume
Elution Volume
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Retention Time
Retention Time
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Isocratic Elution
Isocratic Elution
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Stepwise Elution
Stepwise Elution
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Gradient Elution
Gradient Elution
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Chromatogram
Chromatogram
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Column Chromatography
Column Chromatography
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Adsorption Chromatography
Adsorption Chromatography
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Adsorption Chromatography Principle
Adsorption Chromatography Principle
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Distribution Coefficient (K)
Distribution Coefficient (K)
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Common Adsorbents
Common Adsorbents
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Fuller's Earth
Fuller's Earth
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Powdered Charcoal
Powdered Charcoal
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Polystyrene Beads
Polystyrene Beads
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Hydroxyapatite
Hydroxyapatite
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Silica or Silica Gel
Silica or Silica Gel
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Column Preparation
Column Preparation
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Separation Procedure
Separation Procedure
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Liquid Chromatography
Liquid Chromatography
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Affinity Chromatography
Affinity Chromatography
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Affinity Chromatography Principle
Affinity Chromatography Principle
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Study Notes
- Column chromatography separates substances through liquid percolation in a packed column.
- Separation occurs via direct interaction of solute components with the stationary phase or solute adsorption.
Matrices in Column Chromatography
- The matrix supports the stationary phase.
- Matrix material should have high mechanical and chemical stability, allow good flow rate, have variable particle sizes and be chemically inert.
- Commonly used matrices: cellulose, dextran, agarose and polyacrylamide and Polystyrene and Silica.
- Cellulose is a hydrophilic polysaccharide of -1,4-linked D-glucose units.
- For matrix purposes cellulose is cross-linked with epichlorohydrin, controlling pore size.
- Dextran is a hydrophilic polysaccharide of -1,6-linked D-glucose units.
- For matrix purposes Dextran is cross-linked with epichlorohydrin.
- Dextran is less stable to acid hydrolysis but stable up to pH 12, and marketed as Sephadex and Sephacryl.
- Agarose is a polysaccharide of 3,6-anhydro-1-galactose and D-galactose units.
- Agarose is cross-linked with 2,3-dibromopropanol, forming gels stable in pH 3-14, and has good flow.
- Agarose materials must be suspended in water or buffer to avoid irreversible changes; include Sepharose and Bio-gel A.
- Polyacrylamide is a polymer of acrylamide monomer, cross-linked with N,N-methyl-bisacrylamide.
- Polyacrylamide is stable in pH 2-11, e.g., Bio-gel P.
- Polystyrene is a polymer of styrene, cross-linked with divinylbenzene.
- Polystyrene matrices have high stability over all pH ranges, used in ion exchange and exclusion chromatography.
- Silica is a polymeric material from orthosilicic acid, hydrophilic due to silanol groups.
- For matrix use, excess silanol groups are removed with trichloromethyl silane and are stable in pH 3-8.
Column Packing
- A column (2.5 x 100 cm) is held vertically with a glass wool plug at the end.
- Before packing, the gel swells in a solvent; heat-accelerated swelling requires cooling before packing.
- Cold-prepared slurry de-aerated under vacuum.
- Allow gel settling; remove half the supernatant and mix with solvent into a slurry/suspension.
- Constant stirring is needed while carefully pouring the slurry into the column using a glass rod.
- Add all slurry at once to ensure even packing.
- After the gel settles, run eluent/buffer for an extended time before sample application to achieve fine packing.
Sample application
- The application of sample to a prepared column should be done carefully.
- Mobile phase can be removed by suction or drained to the column bed.
- The sample should then be carefully applied with a pipette for it to slowly run into the column.
- A small volume of mobile phase should be slowly released into the column a few times for it to increase 2-5cm in the column.
- The column is then connected to a large reservoir with adjusted flow and steady height for the mobile phase.
- Application of a high-density sample (e.g., 1% sucrose) from 2 cm above, will allow it to sink to the bottom immediately.
- A capillary tube of syringe can be used to pass the sample to the column surface
- Capillary tubes of syringes are the preferred method of sample application
Separation Efficiency Factors
- Separation efficiency of column chromatography can be affected by drastic factors; dimension of column, particle size of column packing, quality of solvents, temperature of the column, flow rate, packing of the column.
- Column efficiency is improved by increasing the length/width ratio.
- Length to width ratio of 10:1 to 100:1 has been found most satisfactory for preparative separations.
- Long columns are used for closely related compounds.
- Large column diameters are used to accommodate large amounts of samples.
- Decreasing the particle size of the adsorbent improves separation.
- Particle sizes of 100 to 200 mesh range is recommended for both adsorption and partition.
- Solvents with low viscosity are generally used in high efficiency separations.
- The flow rate of solvent is inversely proportional to viscosity, so solvents with low viscosity should be selected.
- Adsorption decreases at higher temperatures, increasing the speed of elution.
- Column chromatography is done at room or low temperatures (5-10°C).
- Medium flow rates are preferred; slow flow rates cause zone spreading; high flow rates lead to non-equilibrium and tailing.
- Column packing is very important, with poorly packed columns leading to zone spreading or tailing.
Elution Techniques
- Elution is removing the sample from the solid matrix using a suitable solvent.
- Total volume of material (solid and liquid) in the column is the bed volume.
- The volume of the mobile phase is known as void volume.
- The amount of liquid to produce a peak of a particular solute in the effluent is the elution/effluent volume.
- Retention time is the time taken for each material to emerge from the column.
- Elution volume and retention time are related to the flow rate of the mobile phase through the column.
- Columns can be eluted by simple isocratic elution, stepwise elution, or gradient elution methods.
- An isocratic elution uses a single liquid mobile phase.
- In an isocratic elution, a single solvent flows continually through the column.
- Isocratic elution is mainly used in gel permeation chromatography.
- Stepwise/batch elution is employed for preparative purposes.
- Stepwise elution is when the column is eluted with one solvent until a predetermined volume is used.
- A second solvent is added to elute separation in the column via stepwise elution.
- Stepwise elution is used in ion exchange and affinity chromatography.
- Gradient elution is done to increase the resolving power of the mobile phase and a continuous change to its pH, ionic strength or polarity.
- Gradient elution is a method where a solution is used to elute the column with a gradually increased concentration of one of the components.
- Gradient elution is used in adsorption, affinity and ion exchange columns.
Chromatogram
- Placing a detector at the column's end in column chromatography creates a symmetrical peak signal when plotted as a function of time/volume.
- A plot like this is a chromatogram.
- Peak positions show sample components and peak areas quantify their amounts.
- Column chromatography involves ion exchange, molecular sieve, and adsorption or partition phenomenon.
- In adsorption column chromatography the substances are preferentially adsorbed by the adsorbent (stationary phase) packed in the column
- In partition column chromatography, the components of a mixture distribute then get separated.
Adsorption Chromatography
- Developed by D.T. Day (1900) and M.S. Tswett (1906) in plant pigment investigations.
- The procedure was not commonly used by chemists until 1930.
- The rate of adsorption varies with the adsorbent used on different materials.
- Separation relies on the principle of selective adsorption.
- The mixture to separate is dissolved in a solvent and passed through a tube with an adsorbent.
- Components with greater adsorbing power adsorb in the upper column.
- The next component is adsorbed in the lower portion of the column, which has lesser adsorbing power than the first component.
- The process is continued as materials are separated and adsorbed in the various parts of the column.
- Passing the original or a suitable solvent slowly through the column can improve the separation.
- As the various bands become defined, the banded adsorbent is termed a chromatogram.
- A zone is the occupied portion of a column by a particular substance.
- Adsorption chromatography separates mixture components by adsorption efficiency onto the solid stationary phase.
- The most strongly adsorbed component forms the topmost band while the least strongly adsorbed material forms the lowermost band on the adsorbent column.
- The separation depends on the surface area because adsorption is a surface phenomenon.
- Distribution coefficient (K) is obtained by the formula: Amount of solute per unit of mobile phase/Amount of solute per unit of stationary phase.
- The distribution coefficient mainly depends upon temperature and substance concentration.
Types of Adsorbents
- Commercial Adsorbents; Fuller's earth, powdered charcoal, polystyrene beads, hydroxyapatite, silica or silica gel, and alumina or aluminium oxide gel
Fuller's Earth
- Mixture of minerals from clay deposits like Kaolinite, Montmorillonite, and Halloysite.
- Decolorizes food oils and removes wine pigments.
- Fuller's Earth's low cost, it is sold as Florisil.
Powdered Charcoal
- A very powerful adsorbent with properties that are highly dependent on the origin processes.
- Bone, blood, and coconut can be a resource material.
- Reproducibility is a problem with charcoal.
- Applying finely divided charcoal and preparing a densely packed column is troublesome.
Polystyrene Beads
- Modified Polystyrene polymers or polystyrene-divinyl benzene copolymers create tiny beads without charged groups.
- Controlled cross-linking creates beads that have known porosity and swelling characteristics.
- They function by adsorption as they lack ionized groups.
- Used commercially under the trade name “XAD resins" for alkaloids, steroids, etc.
Hydroxyapatite
- A basic calcium phosphate with the formula 3Ca3(PO4)2·Ca(OH)2.
- Hydroxyapatite preparation is tedious, the quality varies from batch to batch.
- The gel must not be allowed to dry out an freeze.
- Drying or freezing alters hydration and gel structure, useful in protein separation.
Silica or Silica Gel
- Very popular, binding a wide range of substances with moderate strength.
- Preparing Silica gel is done by precipitating hydrated silica from sodium silicate by adding acid.
- Then it is exhaustively washed dried and sized precisely.
- Handle with caution as it is not as stable as alumina to changes from mechanic damage.
- Silica can be dried at relatively modest temperatures.
- Silica comes in the form of crushed and powdered glass, which has very strong adsorptive properties for some proteins.
Alumina or aluminum oxide gel
- Alumina is a very powerful adsorbent that is widely used.
- It is prepared as a precipitate of aluminium hydroxide by addition of sodium hydroxide to some soluble aluminium salts.
- The gelatinous precipitate is then washed, dried and sized.
- Can be made acidic, basic or neutral.
- Anhydrous state (Al2O3) is achieved by heating it strongly
- Alumina is mechanically strong.
- It has the advantage of not crumbling to still finer particles when manipulated for packing into columns.
Preparation of Column
- Dry Packing Method is used when pouring in powdered substance into the tube in small lots and the tube is then tapped.
- Wet Method is the second method and it closes the bottom of the tube and is then filled with the solvent.
- In wet method powdered material is added in small quantities and settles against the solvent's viscosity.
- Interlocking of air bubbles must be avoided in both methods.
Separation Procedure
- The components can be separated as distinct bands along the adsorbent column.
- Extrusion method stops the running solvent when separation of bands has occurred, this is how elution is carried out.
- The column is drained, extracted and kept horizontally on a table with knife cuts separating the bands.
- The scrapings are then extracted by a suitable technique using solvent.
- Liquid chromatography involves continuous elution for subsequent component washing.
Applications of Adsorption Chromatography
- Adsorption chromatography is used for the separation of the following:
- Polycyclic aromatic compounds, phenols, amines, etc.
- Urinary 17-ketosteroids and their glucuronides.
- Plasma cortisol.
- Aliphatic hydrocarbons from aromatic hydrocarbons.
- Geometrical isomers.
- Technical products in a highly purified state.
Affinity Chromatography
- Useful for the isolation of proteins, polysaccharides, nucleic acids and other classes of naturally occurring compounds
- Affinity chromatography exploits functional specificities of biological systems
- Affinity chromatography can achieve separations that are otherwise difficult using other techniques rapidly.
- A complex mixture of substances can be purified simply using an affinity column.
- Separations use specific affinity of a substance and a specifically binding molecule ligand.
- Covalently coupling a binding molecule (macromolecule or small molecule) to an insoluble matrix synthesizes the column.
- The column can specifically adsorb the substance to be isolated.
- Elution of the desired substance is done by changing the conditions to avoid binding.
- The ligand can be directly attached to the matrix.
Matrix Properties
- Contains groups suited for ligand binding.
- Exhibits good flow properties.
- Stable during macromolecule binding and elution.
- Low interaction with other macromolecules.
- Matrix materials: cross-linked dextrans, agarose, polyacrylamide gel, and polystyrene.
- Cross-linked dextrans are used for Commercial Sephacryl S.
- Agarose is used for Commercial Sepharose or Bio-Gel A.
- Polyacrylamide gel is for Commercial Bio-Gel P.
- Polystyrene is used for Commercial Bio-Beads.
Experimental Procedure
- The column packing has a ligand-treated matrix.
- The buffer must contain cofactors needed for ligand-macromolecule interaction.
- The sample is applied and fractionated.
- The macromolecule binds and gets immobilized.
- Non-specifically bound contaminants are removed with buffer.
- The purified compound is recovered from the either specifically or non-specifically.
- Non-specific elution is achieved by a change in either pH or ionic strength.
- Acetic acid or ammonium hydroxide can be used in elution to change the state of ionization of groups.
- Collected fractions are readjusted to have reduced chances in protein denaturation.
- An ionic strength change causes macromolecule elution, with 1M NaCl used towards that purpose.
- Protein is purified from low molecular species through exclusion chromatography.
Applications of Affinity Chromatography
- Used to purify a wide range of enzymes and other proteins, including receptor proteins and immunoglobulins
- Lectin-sepharose isolates glycoproteins, separates lymphocytes, and fractionates T-cells.
- 5'-AMP- and 2',5'-ADP-sepharose purifies NAD+, NADP+-, and ATP-dependent dehydrogenases and kinases.
- Protein A-agarose is used to purify human immunoglobulin G (IgG) from serum or cell extracts.
- Cibacron-blue agarose can bind with affinity nucleotides as its structurally similar.
- Cibacron-blue agarose is used for kinases, dehydrogenases, albumin, DNA polymerase and coagulation factors isolation.
- Poly(A)-agarose binds nuclear mRNA and fractionates ribonucleoproteins and viral mRNA.
- Lysine agarose binds ribosomal RNA and the protein, plasminogen.
- Heparin-agarose is used to purify bone collagenase, hepatitis B surface antigen, uterine estradiol receptor, murine myeloma DNA polymerase, ribosomes, adrenal tyrosine hydroxylase and several androgen receptors.
Gas Liquid Chromatography (GLC)
- Also known as Gas Chromatography (GC)
- Gas Liquid Chromatography (GLC) is a type of partition chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or nitrogen.
- A gas chromatograph uses a thin capillary fiber known as the column, through which different chemicals which pass at different rates depending on various chemical and physical properties.
- As chemicals exit the column, they are detected and identified electronically.
- The Function to separate and concentrate different components in order to maximize detection signal.
- A stationary phase of a high-boiling liquid material is supported on an inert granular solid.
- The column is maintained in an oven at a temperature to separate compounds in an evaporating state.
- The basis for separation depends on the partition coefficients of volatilized compounds between the liquid and gas phases.
- Compounds are carried through the column by the carrier; a recorder then marks the peak as each separable compound passes.
Instrumental Components of GLC
- Carrier gas: nitrogen, helium, argon, and carbon dioxide.
- Sample injection port
- Columns: Packed and capillary (open tubular).
- Stationary chemicals: high-boiling organic compounds
- Stationairy phase examples: polyethylene glycols, methyl phenyl silicon gum and esters of adipic, succinic and phthalic acids.
Advantages of GLC
- The great length of the column in Gas Liquid Chromatography allows for easier lower molecular weight compound separation.
- Sensitivity enables a concentration of 10-12 gram to be detectable for many substances.
- Separation is frequently achieved in less than a minute which dramatically increases the speed.
- Using a non-destructive detector, GLC can be used for preparative purposes.
Uses of GLC
- GLC is widely used for both qualitative and quantitative analyses.
- GLC provides a high speed of resolution, reproducibility and high sensitivity.
- GLC separates partition compounds between liquid/gas phases.
- Thousands of volatile organic compounds can be separated.
- Non-volatile substances can be can also be separated if converted into volatile ones via oxidation, acylation, and alkylation.
- Biological samples of Alcohols, esters, fatty acids and amines can get separated by GLC
- High sensitive reading concentrations of individual elements of Carbon and hydrogen.
- Identification of functional groups
- For example, to identify alkoxy groups, use iodination to form an alkyl iodide
- The position of the double bond can then be determined by cleaving the bond by oxidation.
High Performance Liquid Chromatography (HPLC)
- High-Performance Liquid Chromatography is a chromatographic technique used to separate, identify, and quantify components in a liquid sample.
- HPLC uses high pressure to pass liquid through a stationary phase-packed column.
- HPLC Provides high precision and sensitivity in separating complex mixtures.
- Mobile phase is a liquid solvent in a solvent reservoir.
- The mobile phase is moved through the system by a pump at high pressure.
- As separation occurs, the compounds pack into a stationary phase (silica/polymer beads) to form a column.
- As the components leave this column, they are detected by a detector (e.g., UV, fluorescence, and conductivity).
- Data processors process the signal and generates a chromatogram.
- During operation the sample is injected into the mobile phases.
- The mobile phase moves the sample through the column with the stationary phases.
- The components of the sample interact differently, causing them to separate.
- As the separated components leave the column, they are detected and analyzed by the detector.
HPLC Types
- Normal Phase HPLC has Polar stationary phase and Non-polar mobile phase.
- Normal Phase HPLC is mainly used for polar compounds.
- Reversed Phase HPLC has Non-polar stationary phase with a polar mobile phase.
- Reversed Phase HPLC is more commonly used for non-polar compounds.
Applications of HPLC
- Pharmaceutical: High analytical for active ingredients, impurities, and drug formation.
- Environmental: Helpful for detection in pollutants across water, soil, and air samples.
- Food and Beverage Industry: Monitors quality and analyzes food additives, preservatives, and contaminants.
- Forensic Science: Important to analyse toxins, drugs in biological samples.
- Biotechnology: Used in separation for nucleic acids, protein and peptides.
Advantages of HPLC
- High resolution and sensitivity.
- Capable of separating complex mixtures.
- Quick and reliable.
- Wide range of applications in different fileds.
Limitations of HPLC
- Expensive for both equipment and maintenance.
- Requires skilled operation and maintenance.
- Limited volatile components analysis.
HPLC Chromatogram Example
- The description should be an example chromatogram.
- X-axis: Retention time.
- Y-axis: Detector response (peak height or area).
- An explanation of each peak represents a different compound eluting from the column at different times.
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