Chromatography
Main article: chromatography
In paper chromatography, the movement of each substance in the mixture
depends on two factors: solubility of the substance in the solvent and adsorption
of the substance on the filter paper. The substance moves with the solvent
easily if the substance is very soluble in the solvent, and some solids can
attract other substances and hold them on their surface. This is called
adsorption, and such solids are called adsorbents. The substance does not move
with the solvent easily if the substance in the mixture is strongly absorbed by
the filter paper. Since neither substance has the same adsorption and
solubility, each travels a different distance along the filter paper—and the
two separate.Substances separated by chromatography need not be colored. Colorless substances can be made visible by spraying the paper with a locating agent that reacts with the colorless substances to produce a color. Labs use chromatography to identify the substances in a mixture. Hospital labs, for example, use the technique to determine if a patient has diabetes by identifying sugar in urine. Chromatography also identifies dyes used in food.
Ion chromatography
Ion-exchange chromatography (or ion chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids. It is often used in protein purification, water analysis, and quality control.PRINCIPAL-
Ion-exchange chromatography retains analyte molecules
on the column based on coulombic
(ionic) interactions. The stationary phase surface displays ionic functional
groups (R-X) that interact with analyte ions of opposite charge. This type of
chromatography is further subdivided into cation exchange chromatography and anion-exchange
chromatography. The ionic compound consisting of
the cationic species M+ and the anionic species B- can be retained by the
stationary phase.
Cation exchange chromatography
retains positively charged cations because the stationary phase displays a negatively charged
functional group:
Anion exchange chromatography
retains anions using positively charged functional group:
Note that the ion strength of either
C+ or A- in the mobile phase can be adjusted to shift the equilibrium position,
thus retention time.
The ion chromatogram shows a typical
chromatogram obtained with an anion exchange column.
TYPICAL TECHNIQUE
A sample is introduced, either
manually or with an autosampler, into a sample loop of known volume. A buffered aqueous
solution known as the mobile phase carries the sample from the loop onto a
column that contains some form of stationary phase material. This is typically
a resin or gel matrix consisting of agarose or cellulose
beads with covalently bonded charged functional groups. The target analytes
(anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged
species that displaces the analyte ions from the stationary phase. For example,
in cation exchange chromatography, the positively charged analyte can be
displaced by adding positively charged sodium ions. The analytes of interest
must then be detected by some means, typically by conductivity or UV/Visible light absorbance.
Control an IC system usually
requires a chromatography
data system (CDS). In addition to IC systems,
some of these CDSs can also control gas chromatography (GC) and HPLC
SEPARATING PROTIEN
Proteins have numerous functional
groups that can have both positive and negative charges. Ion exchange
chromatography separates proteins by their net charge, which depends on the
composition of the mobile phase. By adjusting the pH or the ionic concentration
of the mobile phase, various protein molecules can be separated. For example,
if a protein has a net positive charge at pH 7, it binds to a column of
negatively charged beads—whereas a negatively charged protein does not. By
changing the pH so that the net charge on the protein is negative, it too is
eluted.
Elution by increasing ionic strength
of the mobile phase is more subtle. It works because ions from the mobile phase
interact with the immobilized ions on the stationary phase, thus
"shielding" the stationary phase from the protein, and letting the
protein elute.
Separation can be achieved based on
the natural isoelectric point of the protein. Alternatively a peptide tag can be
genetically added to the protein to give the protein an isoelectric point away
from most natural proteins (e.g., 6 arginines for binding to a cation-exchange
resin or 6 glutamates for binding to an anion-exchange resin such as DEAE-Sepharose).
Elution from ion-exchange columns
can be sensitive to changes of a single charge- chromatofocusing.
Ion-exchange chromatography is also useful in the isolation of specific
multimeric protein assemblies, allowing purification of specific complexes
according to both the number and the position of charged peptide tags
Uses
Clinical utility
Used in measurement of HbA1c, porphyrin & water purification.Industrial Applications
Allows for quantitative testing of electrolyte and proprietary additives of electroplating baths.[4] It is an advancement of qualitative hull cell testing or less accurate UV testing. Ions, catalysts, brighteners and accelerators can be measured.[4]High-performance liquid chromatography
High-performance liquid
chromatography (HPLC; formerly referred to
as high-pressure liquid chromatography), is a technique in analytical chemistry used to separate, identify, and quantify each component in
a mixture. It relies on pumps to pass a pressurized liquid solvent
containing the sample mixture through a column filled with a solid adsorbent material.
Each component in the sample interacts slightly differently with the adsorbent
material, causing different flow rates for the different components and leading
to the separation of the components as they flow out the column.
HPLC has been used for medical (e.g.
detecting vitamin D levels in blood serum), legal (e.g. detecting performance
enhancement drugs in urine), research (e.g. separating the components of a
complex biological sample, or of similar synthetic chemicals from each other),
and manufacturing (e.g. during the production process of pharmaceutical and
biological products) purposes.[1]
Chromatography can be described as a mass transfer
process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a
sample mixture through a column filled with a sorbent, leading to the
separation of the sample components. The active component of the column, the
sorbent, is typically a granular material made of solid particles (e.g. silica,
polymers, etc.), 2–50 micrometers in size. The components of the sample mixture
are separated from each other due to their different degrees of interaction
with the sorbent particles. The pressurized liquid is typically a mixture of
solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a
"mobile phase". Its composition and temperature
play a major role in the separation process by influencing the interactions
taking place between sample components and sorbent. These interactions are
physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic,
most often a combination.
HPLC is distinguished from
traditional ("low pressure") liquid chromatography because operational pressures are significantly higher
(50–350 bar), while ordinary liquid chromatography typically relies on the
force of gravity to pass the mobile phase through the column. Due to the small
sample amount separated in analytical HPLC, typical column dimensions are
2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made
with smaller sorbent particles (2–50 micrometer in average particle size). This
gives HPLC superior resolving power (the ability to distinguish between
compounds) when separating mixtures, which makes it a popular chromatographic
technique.
The schematic of an HPLC instrument
typically includes a sampler, pumps, and a detector. The sampler brings the
sample mixture into the mobile phase stream which carries it into the column.
The pumps deliver the desired flow and composition of the mobile phase through
the column. The detector generates a signal proportional to the amount of
sample component emerging from the column, hence allowing for quantitative
analysis of the sample components. A digital microprocessor
and user software control the HPLC instrument and provide data analysis. Some
models of mechanical pumps in a HPLC instrument can mix multiple solvents
together in ratios changing in time, generating a composition gradient in the
mobile phase. Various detectors are in common use, such as UV/Vis, photodiode
array (PDA) or based on mass spectrometry.
Most HPLC instruments also have a column oven that allows for adjusting the
temperature the separation is performed at.
Operation
The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are function of specific physical interactions with the sorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time measured under particular conditions is considered an identifying characteristic of a given analyte.Many different types of columns are available, filled with sorbents varying in particle size, and in the nature of their surface ("surface chemistry"). The use of smaller particle size packing materials requires the use of higher operational pressure ("backpressure") and typically improves chromatographic resolution (i.e. the degree of separation between consecutive analytes emerging from the column). In terms of surface chemistry, sorbent particles may be hydrophobic or polar in nature.
Common mobile phases used include any miscible combination of water with various organic solvents (the most common are acetonitrile and methanol). Some HPLC techniques use water-free mobile phases (see Normal-phase chromatography below). The aqueous component of the mobile phase may contain acids (such as formic, phosphoric or trifluoroacetic acid) or salts to assist in the separation of the sample components. The composition of the mobile phase may be kept constant ("isocratic elution mode") or varied ("gradient elution mode") during the chromatographic analysis. Isocratic elution is typically effective in the separation of sample components that are not very different in their affinity for the stationary phase. In gradient elution the composition of the mobile phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times with high eluting strength producing fast elution (=short retention times). A typical gradient profile in reversed phase chromatography might start at 5% acetonitrile (in water or aqueous buffer) and progress linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition may be part of any gradient profile. For example, the mobile phase composition may be kept constant at 5% acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.
The chosen composition of the mobile
phase (also called eluent) depends on the intensity of interactions between
various sample components ("analytes") and stationary phase (e.g.
hydrophobic interactions in reversed-phase HPLC). Depending on their affinity
for the stationary and mobile phases analytes partition between the two during
the separation process taking place in the column. This partitioning process is
similar to that which occurs during a liquid–liquid
extraction but is continuous, not step-wise.
In this example, using a water/acetonitrile gradient, more hydrophobic
components will elute (come off the column) late, once the mobile phase gets more
concentrated in acetonitrile (i.e. in a mobile phase of higher eluting
strength).
The choice of mobile phase
components, additives (such as salts or acids) and gradient conditions depends
on the nature of the column and sample components. Often a series of trial runs
is performed with the sample in order to find the HPLC method which gives
adequate separation.
IN FIGURE –
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 injection loop, (8) Pre-column (guard column), (9) Analytical
column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or
fraction collector.
Parameters
Theoretical
HPLC separations have theoretical parameters and equations to describe the separation of components into signal peaks when detected by instrumentation such as by a UV detector or a mass spectrometer. The parameters are largely derived from two sets of chromatagraphic theory: plate theory (as part of Partition chromatography), and the rate theory of chromatography / Van Deemter equation. Of course, they can be put in practice through analysis of HPLC chromatograms, although rate theory is considered the more accurate theory.They are analogous to the calculation of retention factor for a paper chromatography separation, but describes how well HPLC separates a mixture into two or more components that are detected as peaks (bands) on a chromatogram. The HPLC parameters are the: efficiency factor(N), the retention factor (kappa prime), and the separation factor (alpha). Together the factors are variables in a resolution equation, which describes how well two components' peaks separated or overlapped each other. These parameters are mostly only used for describing HPLC reversed phase and HPLC normal phase separations, since those separations tend to be more subtle than other HPLC modes (e.g. ion exchange and size exclusion).
- Void volume is the amount of space in a column that is occupied by solvent. It is the space within the column that is outside of the column's internal packing material. Void volume is measured on a chromatogram as the first component peak detected, which is usually the solvent that was present in the sample mixture; ideally the sample solvent flows through the column without interacting with the column, but is still detectable as distinct from the HPLC solvent. The void volume is used as a correction factor.
- Efficiency factor (N) practically measures how sharp component peaks on the chromatogram are, as ratio of the component peak's area ("retention time") relative to the width of the peaks at their widest point (at the baseline). Peaks that are tall, sharp, and relatively narrow indicate that separation method efficiently removed a component from a mixture; high efficiency. Efficiency is very dependent upon the HPLC column and the HPLC method used. Efficiency factor is synonymous with plate number, and the 'number of theoretical plates'.
- Retention factor (kappa prime) measures how long a component of the mixture stuck to the column, measured by the area under the curve of its peak in a chromatogram (since HPLC chromatograms are a function of time). Each chromatogram peak will have its own retention factor (e.g. kappa1 for the retention factor of the first peak). This factor may be corrected for by the void volume of the column.
- Separation factor (alpha) is a relative comparison on how well two neighboring components of the mixture were separated (i.e. two neighboring bands on a chromatogram). This factor is defined in terms of a ratio of the retention factors of a pair of neighboring chromatogram peaks, and may also be corrected for by the void volume of the column. The greater the separation factor value is over 1.0, the better the separation, until about 2.0 beyond which an HPLC method is probably not needed for separation.
Internal diameter
The internal diameter (ID) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. It also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low-ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.
- Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large loading capacity.
- Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-Vis absorbance detector.
- Narrow-bore columns (1–2 mm) are used for applications when more sensitivity is desired either with special UV-vis detectors, fluorescence detection or with other detection methods like liquid chromatography-mass spectrometry
- Capillary columns (under 0.3 mm) are used almost exclusively with alternative detection means such as mass spectrometry. They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ.
Particle size
Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5 µm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.[6][7][8]This means that changing to particles that are half as big, keeping the size of the column the same, will double the performance, but increase the required pressure by a factor of four. Larger particles are used in preparative HPLC (column diameters 5 cm up to >30 cm) and for non-HPLC applications such as solid-phase extraction.
Pore size
Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.Pump pressure
Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure may reach as high as 60 MPa (6000 lbf/in2), or about 600 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography" systems or UHPLCs can work at up to 120 MPa (17,405 lbf/in2), or about 1200 atmospheres.[9] The term "UPLC" is a trademark of the Waters Corporation, but is sometimes used to refer to the more general technique of UHPLC.Detectors
HPLC most commonly uses a UV-Vis absorbance detector, however a wide range of other chromatography detectors can be used. One commonly utilized detector includes refractive index detectors, which provide readings by measuring the changes in the refractive index of the effluent as it moves through the flow cell. In certain cases it is possible to use multiple detectors, for example LCMS normally combines UV-Vis with a Mass spectrometer.GAS LIQUID CHROMATOGRAPHY –
Gas-Liquid Chromatography
CHROMATOGRAPHY is a method of separating and identifying the components of a mixture. There are multiple types of chromatography: paper, thin-layer chromatography, column, gas-liquid, high-pressure liquid chromatography.
All types of chromatography depend upon the equilibrium set up when the components of a mixture distribute themselves between the stationary phase and the mobile phase. Components with a higher affinity for the stationary phase move more slowly than those with a lower affinity.
☛ IN GAS-LIQUID CHROMATOGRAPHY:
The stationary phase is a non-volatile (high boiling point) liquid coated on the surface of finely divided particles of an inert solid, packed inside of a long, thin column.
The mobile phase is an inert (unreactive) carrier gas (for example, nitrogen gas) which carries the mixture through the column- acting like the solvent in thin layer chromatography, which carries the mixture up the thin layer plate (the stationary phase).
1. The sample to be analysed is injected into a stream of the inert carrier gas.
2. Each component in a mixture applied to the column sets up an equilibrium between the mobile and stationary phases. The more volatile the compound (lower boiling point), the more time it spends in the mobile phase, so the faster is gets carried through the column.
3. When the compounds leave the column they are detected, and a GAS CHROMATOGRAM is produced. This displays how long it takes each compound in the mixture to travel through the column: THE RETENTION TIME (units=minutes).
4. The RETENTION TIME is the time it takes a compound to pass through the column (from injection into the detector). Under given conditions, the retention time for a substance is CONSTANT, so helps to identify a compound in a mixture.
Factors which affect RETENTION TIME:
- How hot the thermostatically controlled oven is - The RATE OF FLOW of the GAS (mobile phase) - The COLUMN LENGTH and PACKING of the COLUMN (stationary phase)
As each component emerges from the column, a peak is recorded on the CHROMATOGRAM. The area beneath each peak is proportional to the amount of that component in the mixture applied to the glc column. If the peaks are sharp, peak height can be used as a measure of the amount of each substance.
Size-exclusion chromatography (SEC)-
Size-exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight.[1] It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
Applications
The main application of gel-filtration chromatography is the fractionation of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic-soluble polymers. Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges.Advantages
The advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate,[2] and that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to be separated. The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. With size exclusion chromatography, there are short and well-defined separation times and narrow bands, which lead to good sensitivity. There is also no sample loss because solutes do not interact with the stationary phase. Disadvantages are, for example, that only a limited number of bands can be accommodated because the time scale of the chromatogram is short, and, in general, there has to be a 10% difference in molecular mass to have a good resolution[2]Theory and method
SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping smaller molecules in the pores of the adsorbent materials adsorption ("stationary phases"). The larger molecules simply pass by the pores because those molecules are too large to enter the pores. Larger molecules therefore flow through the column more quickly than smaller molecules, that is, the smaller the molecule, the longer the retention time.One requirement for SEC is that the analyte does not interact with the surface of the stationary phases, with differences in elution time between analytes ideally being based solely on the solute volume the analytes can enter, rather than chemical or electrostatic interactions with the stationary phases. Thus, a small molecule that can penetrate every region of the stationary phase pore system can enter a total volume equal to the sum of the entire pore volume and the interparticle volume. This small molecule will elute late (after the molecule has penetrated all of the pore- and interparticle volume -- approximately 80% of the column volume). At the other extreme, a very large molecule that cannot penetrate any the smaller pores can enter only the interparticle volume (~35% of the column volume) and will elute earlier when this volume of mobile phase has passed through the column. The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near-simultaneously, particles of the same size should elute together.
However, as there are various measures of the size of a macromolecule (for instance, the radius of gyration and the hydrodynamic radius), a fundamental problem in the theory of SEC has been the choice of a proper molecular size parameter by which molecules of different kinds are separated. Experimentally, Benoit and co-workers found an excellent correlation between elution volume and a dynamically based molecular size, the hydrodynamic volume, for several different chain architecture and chemical compositions.[10] The observed correlation based on the hydrodynamic volume became accepted as the basis of universal SEC calibration.
Still, the use of the hydrodynamic volume, a size based on dynamical properties, in the interpretation of SEC data is not fully understood.[11] This is because SEC is typically run under low flow rate conditions where hydrodynamic factor should have little effect on the separation. In fact, both theory and computer simulations assume a thermodynamic separation principle: the separation process is determined by the equilibrium distribution (partitioning) of solute macromolecules between two phases --- a dilute bulk solution phase located at the interstitial space and confined solution phases within the pores of column packing material. Based on this theory, it has been shown that the relevant size parameter to the partitioning of polymers in pores is the mean span dimension (mean maximal projection onto a line).[12] Although this issue has not been fully resolved, it is likely that the mean span dimension and the hydrodynamic volume are strongly correlated.
Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit defines the molecular weight at the upper end of the column 'working' range and is where molecules are too large to be trapped in the stationary phase. The lower end of the range is defined by the permeation limit, which defines the molecular weight of a molecules that is small to penetrate all pores of the stationary phase. All molecules below this molecular mass are so small that they elute as a single band[2]
This is usually achieved with an apparatus called a column, which consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the faster the elution.
The filtered solution that is collected at the end is known as the eluate. The void volume includes any particles too large to enter the medium, and the solvent volume is known as the column volume.
Factors affecting filtration
A cartoon illustrating the theory behind size exclusion
chromatography
In real-life situations, particles in solution do not have a fixed size,
resulting in the probability that a particle that would otherwise be hampered
by a pore passing right by it. Also, the stationary-phase particles are not
ideally defined; both particles and pores may vary in size. Elution curves,
therefore, resemble Gaussian distributions. The
stationary phase may also interact in undesirable ways with a particle and
influence retention times, though great care is taken by column manufacturers
to use stationary phases that are inert and minimize this issue.Like other forms of chromatography, increasing the column length will enhance the resolution, and increasing the column diameter increases the capacity of the column. Proper column packing is important to maximize resolution: An overpacked column can collapse the pores in the beads, resulting in a loss of resolution. An underpacked column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores. Unlike affinity chromatography techniques, a solvent head at the top of the column can drastically diminish resolution as the sample diffuses prior to loading, broadening the downstream elution.
Analysis
In simple manual columns, the eluent is collected in constant volumes, known as fractions. The more similar the particles are in size the more likely they will be in the same fraction and not detected separately. More advanced columns overcome this problem by constantly monitoring the eluent.
Standardization of a size exclusion column.
The collected fractions are often examined by spectroscopic
techniques to determine the concentration of the particles eluted.
Common spectroscopy detection techniques are refractive
index (RI) and ultraviolet (UV). When eluting spectroscopically
similar species (such as during biological purification), other techniques may
be necessary to identify the contents of each fraction. It is also possible to
analyse the eluent flow continuously with RI, LALLS, Multi-Angle Laser Light
Scattering MALS, UV, and/or viscosity measurements.
SEC Chromatogram of a biological sample.
The elution volume (Ve) decreases roughly linear with the logarithm
of the molecular hydrodynamic volume. Columns
are often calibrated using 4-5 standard samples (e.g., folded proteins of known
molecular weight), and a sample containing a very large molecule such as
thyroglobulin to determine the void volume.
(Blue dextran is not recommended for Vo determination because it is
heterogeneous and may give variable results) The elution volumes of the
standards are divided by the elution volume of the thyroglobulin (Ve/Vo) and
plotted against the log of the standards' molecular weights.Applications
Biochemical applications
In general, SEC is considered a low resolution chromatography as it does not discern similar species very well, and is therefore often reserved for the final "polishing" step of a purification. The technique can determine the quaternary structure of purified proteins that have slow exchange times, since it can be carried out under native solution conditions, preserving macromolecular interactions. SEC can also assay protein tertiary structure, as it measures the hydrodynamic volume (not molecular weight), allowing folded and unfolded versions of the same protein to be distinguished. For example, the apparent hydrodynamic radius of a typical protein domain might be 14 Å and 36 Å for the folded and unfolded forms, respectively. SEC allows the separation of these two forms, as the folded form will elute much later due to its smaller size.Polymer synthesis
SEC can be used as a measure of both the size and the polydispersity of a synthesised polymer, that is, the ability to be able to find the distribution of the sizes of polymer molecules. If standards of a known size are run previously, then a calibration curve can be created to determine the sizes of polymer molecules of interest in the solvent chosen for analysis (often THF). In alternative fashion, techniques such as light scattering and/or viscometry can be used online with SEC to yield absolute molecular weights that do not rely on calibration with standards of known molecular weight. Due to the difference in size of two polymers with identical molecular weights, the absolute determination methods are, in general, more desirable. A typical SEC system can quickly (in about half an hour) give polymer chemists information on the size and polydispersity of the sample. The preparative SEC can be used for polymer fractionation on an analytical scale.Drawback
In SEC, mass is not measured so much as the hydrodynamic volume of the polymer molecules, that is, how much space a particular polymer molecule takes up when it is in solution. However, the approximate molecular weight can be calculated from SEC data because the exact relationship between molecular weight and hydrodynamic volume for polystyrene can be found. For this, polystyrene is used as a standard. But the relationship between hydrodynamic volume and molecular weight is not the same for all polymers, so only an approximate measurement can be obtained.[13] Another drawback is the possibility of interaction between the stationary phase and the analyte. Any interaction leads to a later elution time and thus mimics a smaller analyte size.Affinity chromatography
From Wikipedia, the free encyclopedia
Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction
such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.Uses
Affinity chromatography can be used to:- Purify and concentrate a substance from a mixture into a buffering solution
- Reduce the amount of a substance in a mixture
- Discern what biological compounds bind to a particular substance
- Purify and concentrate an enzyme solution.
Principle
The stationary phase is typically a gel matrix, often of agarose; a linear sugar molecule derived from algae. Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate, growth medium or blood serum. The molecule of interest will have a well known and defined property, and can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in the mobile phase will not become trapped as they do not possess this property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution. Possibly the most common use of affinity chromatography is for the purification of recombinant proteins.Batch and column setup
Column chromatography
Batch chromatography
Binding to the solid phase may be achieved by column chromatography whereby
the solid medium is packed onto a column, the initial mixture run through the
column to allow setting, a wash buffer run through the column and the elution
buffer subsequently applied to the column and collected. These steps are
usually done at ambient pressure. Alternatively, binding may be achieved using
a batch treatment, for example, by adding the initial mixture to the solid
phase in a vessel, mixing, separating the solid phase, removing the liquid
phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and
removing the eluate.Sometimes a hybrid method is employed such that the binding is done by the batch method, but the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column.
A third method, expanded bed adsorption, which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensure that the solid phase does not exit the column with the liquid phase.
Affinity columns can be eluted by changing salt concentrations, pH, pI, charge and ionic strength directly or through a gradient to resolve the particles of interest.
Specific uses
Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts, and purification from blood.Immunoaffinity
Another use for the procedure is the affinity purification of antibodies from blood serum. If serum is known to contain antibodies against a specific antigen (for example if the serum comes from an organism immunized against the antigen concerned) then it can be used for the affinity purification of that antigen. This is also known as Immunoaffinity Chromatography. For example if an organism is immunised against a GST-fusion protein it will produce antibodies against the fusion-protein, and possibly antibodies against the GST tag as well. The protein can then be covalently coupled to a solid support such as agarose and used as an affinity ligand in purifications of antibody from immune serum.For thoroughness the GST protein and the GST-fusion protein can each be coupled separately. The serum is initially allowed to bind to the GST affinity matrix. This will remove antibodies against the GST part of the fusion protein. The serum is then separated from the solid support and allowed to bind to the GST-fusion protein matrix. This allows any antibodies that recognize the antigen to be captured on the solid support. Elution of the antibodies of interest is most often achieved using a low pH buffer such as glycine pH 2.8. The eluate is collected into a neutral tris or phosphate buffer, to neutralize the low pH elution buffer and halt any degradation of the antibody's activity. This is a nice example as affinity purification is used to purify the initial GST-fusion protein, to remove the undesirable anti-GST antibodies from the serum and to purify the target antibody.
A simplified strategy is often employed to purify antibodies generated against peptide antigens. When the peptide antigens are produced synthetically, a terminal cysteine residue is added at either the N- or C-terminus of the peptide. This cysteine residue contains a sulfhydryl functional group which allows the peptide to be easily conjugated to a carrier protein (e.g.Keyhole Limpet Hemocyanin (KLH)). The same cysteine-containing peptide is also immobilized onto an agarose resin through the cysteine residue and is then used to purify the antibody.
Most monoclonal antibodies have been purified using affinity chromatography based on immunoglobulin-specific Protein A or Protein G, derived from bacteria.[1]
Immobilized metal ion affinity chromatography
Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent bond of amino acids, particularly histidine, to metals. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt, nickel, copper for the purification of histidine containing proteins or peptides, iron, zinc or gallium for the purification of phosphorylated proteins or peptides. Many naturally occurring proteins do not have an affinity for metal ions, therefore recombinant DNA technology can be used to introduce such a protein tag into the relevant gene. Methods used to elute the protein of interest include changing the pH, or adding a competitive molecule, such as imidazole.
A chromatography column containing nickel-agarose beads used
for purification of proteins with histidine tags
See also: Polyhistidine-tag
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