1 Chromatography Definition: Chromatography is a technique for separating a sample into various fractions. The heart of any chromatography is the stationary phase (liquid or solid). The stationary phase is attached to a support, a solid inert material. The sample (gas, liquid or solid which may or may not dissolved in solvents) is carried across the stationary phase by a mobile phase (gas or liquid). The sample components undergo a series of exchanges (partitions) between the two phases due to the differences in their chemical and physical properties. These differences govern the rate of movement (called migration) of the individual components. Purpose of Chromatography: Analytical - determine chemical composition of a sample Preparative - purify and collect one or more components of a sample Classification of Chromatographic Methods Chromatography is classified according to three ways: 1. According to the physical state of the mobile phase: Liquid chromatography : This subdivided according to the stationary phase into liquid-liquid or liquid-solid chromatography. Gas chromatography : This subdivided according to the stationary phase into Gas-liquid or Gas-solid chromatography. 2. According to the method of contact between the mobile phase and stationary phase: Column chromatography : the stationary phase is placed in a column through which the mobile phase moves under the influence of gravity or pressure. The stationary phase is either a solid or a thin, liquid film coating on a solid particulate packing material or the column’s walls. Planar chromatography : the stationary phase coats a flat glass, metal, or plastic plate and is placed in a reservoir containing the mobile phase which moves by capillary action carrying with it the sample components 3. According to the chemical or physical mechanism responsible for separating the sample’s constituents. ( attractive forces) Adsorption chromatography : for polar non-ionic compounds Ion Exchange chromatography : for ionic compounds - Anion: analyte is anion; bonded phase has positive charge - Cation: analyte is cation; bonded phase has negative charge Partition chromatography : based on the relative solubility of analyte in mobile and stationary phases - Normal: analyte is non-polar organic; stationary phase MORE polar than the mobile phase - Reverse: analyte is polar organic; stationary phase LESS polar than the mobile phase Size Exclusion chromatography : stationary phase is a porous matrix.
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1
Chromatography Definition:
Chromatography is a technique for separating a sample into various fractions. The heart of any chromatography is the stationary phase
(liquid or solid). The stationary phase is attached to a support, a solid inert material. The sample (gas, liquid or solid which may or may
not dissolved in solvents) is carried across the stationary phase by a mobile phase (gas or liquid). The sample components undergo a
series of exchanges (partitions) between the two phases due to the differences in their chemical and physical properties. These
differences govern the rate of movement (called migration) of the individual components.
Purpose of Chromatography:
Analytical - determine chemical composition of a sample
Preparative - purify and collect one or more components of a sample
Classification of Chromatographic Methods
Chromatography is classified according to three ways:
1. According to the physical state of the mobile phase:
Liquid chromatography: This subdivided according to the stationary phase into liquid-liquid or liquid-solid chromatography.
Gas chromatography: This subdivided according to the stationary phase into Gas-liquid or Gas-solid chromatography.
2. According to the method of contact between the mobile phase and stationary phase:
Column chromatography: the stationary phase is placed in a column through which the mobile phase moves under the influence of
gravity or pressure. The stationary phase is either a solid or a thin, liquid film coating on a solid particulate packing material or
the column’s walls.
Planar chromatography: the stationary phase coats a flat glass, metal, or plastic plate and is placed in a reservoir containing the
mobile phase which moves by capillary action carrying with it the sample components
3. According to the chemical or physical mechanism responsible for separating the sample’s constituents.( attractive forces)
Adsorption chromatography: for polar non-ionic compounds
Ion Exchange chromatography: for ionic compounds
- Anion: analyte is anion; bonded phase has positive charge
- Cation: analyte is cation; bonded phase has negative charge
Partition chromatography: based on the relative solubility of analyte in mobile and stationary phases
- Normal: analyte is non-polar organic; stationary phase MORE polar than the mobile phase
- Reverse: analyte is polar organic; stationary phase LESS polar than the mobile phase
Size Exclusion chromatography: stationary phase is a porous matrix.
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Planner chromatography
1- Paper chromatography (PC)
Principle
In paper chromatography, the mobile phase (solvent) carries the components of the sample on the stationary phase (filter paper)
separating them according to the differences in the migration rate (depends on the molecular weight , polarity and adsorption ability)
Components
For one-dimensional paper chromatography, either ascending or descending development can be carried out in simple units. Descending
development is more often used because it is faster and more suitable for long paper sheets.
- The stationary phase (filter paper) - The mobile phase (solvent may be in a reservoir)
Procedures
1. Make the initial line on the paper.
2. Apply the solvent alone on the initial line.
3. Wait till the solvent migration is stopped, then make the final line.
4. Spot the sample, and then apply the solvent either in ascending or descending or concentric manner.
5. In case of colored sample: Calculate the rate of flow (Rf) directly then compare it with stander in order to know the unknown
sample (qualitatively).
6. In case of the colorless sample: use UV-lamb to detect the spot position then determine the (Rf).
Rf depends on the temp., solvent, type of paper
Rf = distance of sample migration / distance of solvent migration
Applications
1. Separation of amino acids
2. Separation of the plant pigments
Advantages
1. Simple
2. Cheap
Disadvantages
1. Time consuming.
2. Need high quantity of sample.
3. With weak solvent power.
4. limited use
5. Difficulty detection of spots
6. Difficulty isolation of separated substances.
3
2- Thin layer chromatography (TLC)
Principle (As in paper chromatography)
Components
1- glass or plastic plate: as a support to the stationary phase
2- stationary phase (silica gel, alumina or agar)
3- mobile phase solvent system
Procedures
(a) Preparing the plate
1- Prepare a glass plate. for example (20X20)Cm
2- Dissolve suitable amount of the silica gel in water path.
3- Spread it on glass plate homogeneously. Then wait till solidification.
(b) Running the sample 1- Make the initial and final line as in PC
2- Spot the sample on the initial line and then apply the solvent either in ascending or descending or concentric manner
3- For example: add the plate on a tank containing the solvent (ascending) where the solvent move by the capillary action carrying
with it the components of the sample.
4- The plate is removed from the tank and dried. (Additional separation can be achieved in two dimensional TLC)
5- According to the sample type ( if colored, if colorless, if florescent …) , identification (qualitative) occurs either by Rf , UV,
spraying colored reagent or autoradiography.
6- Compare it with stander in order to know the unknown sample components.
7- We can separate the sample by cutting the silica layer by spatula, then dissolve it with the same solvent then filter for further
purification.
Applications
1- Environmental application from water analysis (especially pesticides) to plant residues
2- Pharmaceutical applications from stability and impurity studies to drug monitoring in biological fluid
3- Biomedical compounds (organic acids, lipids, carbohydrates and steroids)
4- Food analysis from carcinogens, drug residues , preservatives and flavors
Disadvantages
1- Time consuming
2- Limited to non-volatile compounds
3- Less accurate and less sensitive
Advantages
1- Need small quantity of sample.
2- With greater solvent power.
3- easy detection of spots
4- easy isolation of separated substances
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Column chromatography
1- Gel filtration chromatography (Size-exclusion chromatography)
Principle
This technique separate proteins according to their size and shape, as they pass through a stationary phase (cross-linked polymer
=sephadex) by the help of mobile phase (without binding). Larger proteins or molecules, which can not penetrate the sephadex pores,
move around the sephadex in space between them faster than the smaller molecules which may penetrate the sephadex pores taking long
time to elute from the column.
Components
1. column: as a support to the stationary phase
2. stationary phase (pours matrix in the form of spherical particles, stable, inert e.g. sephadex or agarose)
3. mobile phase (buffer system)
Procedures
1. (loading step): spherical particles of the sephadex are packed into the column
2. (Sampling step): sample is applied to the column
3. Buffer (mobile phase) and sample move through the column. The sample components diffuse in and out
of the pores of the matrix (sephadex) according to their size.
4. Larger proteins or molecules move faster than the smaller molecules and leave the column first
5. Separation completed as the entire buffer volume is passed.
Applications
1. Separation of neutral proteins and larger molecules including polymers and biomolecules according to size.
2. The determination of formula weights.
Disadvantages
1. limited applications
2. low purification
Advantages
1. provides a rapid means for separating larger molecules
2. Use only one buffer (coast effective)
3. Do not need elution step because there are no bonds formed.
Lecture (2)
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Relation between pH, Pi & ion exchange
pH: is aquantitative description of the acidity of
an aqueous solution.
pH= -Log[H]+ =Log 1/[H]
+
Isoelectric point (Pi): is the point at which the
protein charge reach zero.
If pH > Pi use anion exchange
If pH < Pi use cation exchange
pH = pK + Log conjugated base / conjugated acid
2- Ion-exchange chromatography
Principle
Ion exchange chromatography separates molecules (proteins) according to their differences between the overall charges. The proteins to
be separated must have a charge opposite to that of stationary phase in order to bind.
Ion exchange has two types according to the stationary phase charge:
1. Cation-exchanger: in which the stationary phase is charged negatively in order to binds with positive molecules (cations)
2. Anion-exchanger: in which the stationary phase is charged positively in order to binds with negative molecules (anions)
Cation-exchange chromatography Cation-exchange chromatography can be classified as: either strong or weak. A strong cation exchanger contains strong acid which
stable along pH1-14. Whereas, weak cation exchanger contains weak acid which loss its charge as the pH decrease below 4-5 The sample must be charged positive in order to bind with the negative matrix (strong or weak acid). H
+
Anion-exchange chromatography Anion-exchange chromatography can be classified as: either strong or weak. A strong anion exchanger contains strong base which
stable along pH1-14. Whereas, weak anion exchanger contains weak base which loss its charge as the pH increase over 9 The sample must be charged negative in order to bind with the positive matrix (strong or weak base).OH
-
Components
1. The column containing the stationary phase (anion or cation exchanger) on suitable matrix
2. washing and eluting buffer
3. pump to withdrew the buffer
4. Detector
Procedures
Before carry out the process, you must answer two important questions:
a) What is the sample charge? If +Ve, use cation exchanger. if –Ve, use anion exchanger
b) What is the suspected strength of the charge? If weak +Ve, use weak cation exchanger, if strong +Ve, use strong cation
exchanger, if weak –Ve, use weak anion exchanger, if strong –Ve use strong anion exchanger.
basicفإذاا كاتإم مع إح اضماضإه ايمينيإة , ؟ طبعا من خالل تركيبه(قوية او ضعيفة)او موجبة( قوية اوضعيفة)كيف تستطيع معرفة ان هذا البروتين يحمل شحنة سالبة : ملحوظة
الشحنةومن خالل عدد ايضماض تستطيع توقع قوة او ضعف . سيكون موجب acidicسيكون سالب وااا كاتم
e.g. the sample is weak negative proteins. So we will use anion exchanger contain weak base.
1. (loading step): the column is packed with the matrix that charged with weak positive charge by adding weak base e.gDEAE-
cellulose (stationary phase)
2. (Sampling step): apply the sample in the column: the negatively charged proteins bind to positively charged matrix whereas;
the positively charged proteins flow down to the exterior. Some negative charged contaminants can bind to matrix.
3. (Washing step): apply washing buffer (Tris-HCL) to remove the contaminants remaining the target proteins.
4. (Elution step): now, we need to separate the target proteins from the matrix, so we apply an eluting buffer that has the same
charge of protein in order to substitutes it (ion exchange). Separation can be done also by ion exclusion and ion pairing.
5. (Gradient step): make gradient elution with different buffer till you obtain 100% correct proteins. i.e. repeat washing and
eluting steps with different buffer
6. (detection step): after separation carry out detection by electrophoresis
Applications
(Separation of Ions and ionized sp) القاعدة هنا
1. Separation and detection of ions and ionized species.
2. separation and purification of components from mixture
3. identification of ionic impurities
Disadvantages
1. Analytes can be misidentified
2. Analytes are performed sequentially
3. Analysis consume eluent
Advantages
1. Selective to charge
2. Separation and detection of ions and ionized species
Note
Lecture (5)
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3- Affinity chromatography
Principle
Affinity chromatography depends on separation of proteins or molecules by their binding specificities. So the proteins of interest bind
specifically to a ligand (which attached to the matrix) such as antigen-antibody interaction.
Components
1. stationary phase: affinity matrix (ligand attached to matrix)
2. suitable column
3. mobile phase: buffer
Procedures
1. (Loading step): the column is packed with suitable matrix to which ligand (or antigen) has been coupled.
2. (Sampling step): sample (may be serum) is applied to the column. The protein or antibody of interest will bind tightly to ligand
(because they are specific to each others) whereas; other contaminants similar to target proteins bind loosely.
3. (Washing step): the unwanted proteins or antibodies do not bind to ligands so; it is easy to be washed off. Also the
contaminants that bind loosely washed off by another buffer.
4. (Elutiion step): proteins or antibodies that bind tightly to ligands are eluted by eluting buffer.
5. (Detection step): after separation carry out detection to ensure the correct proteins.
Applications
(Separation of samples that can carry out KEY & LOCK mechanism with stationary phase) القاعدة هنا
1. Separation of biomacromolecules from other biological compounds.
o Separation of proteins
o Separation of mRNA (poly A) from RNAs using oligo dT cellulose (poly T)
2. it can be used to determine dissociation constants of ligands and molecules
Advantages
1. it is more specific method for the target analyte
2. simple and practical
3. fast
Lecture (4)
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4- Gas chromatography (GC)
Principle
In gas chromatography (GC) the sample, which may be a gas or liquid, is injected into a stream of an inert gaseous mobile phase (often
called the carrier gas). The sample is carried by mobile phase through a packed or capillary column where the sample’s components
separate based on their ability to distribute themselves between the mobile and stationary phases.
Components Gas chromatography has two types according to the stationary phase:
a) Gas-liquid chromatography b) gas-solid chromatography
The main components are:
1. Gas supply: it is usually helium, hydrogen or nitrogen. This serves as mobile phase that moves the sample towards column.
2. Injector: it is a hollow heated glass cylinder where the sample is introduced and vaporized into the mobile phase stream.
3. The column: it is the heart of GC. It is coated with either solid or liquid stationary phase.
4. Oven: it contain the column where the temp. can be controlled giving constant heat along the run.
5. Detector: it detects the separating components passing from the column. Detectors have many types e.g. flame ionization