Information

What is the difference between HPLC and FPLC and why is FPLC preferable for protein purification?


I've used HPLC (high performance liquid chromatography) before (once, so I'm barely even qualified to know what it stands for) so I was surprised when my labmate told me she would be using an alternate technique to isolate a protein.

What exactly are differences in HPLC and FPLC (fast protein liquid chromatography) instruments, and why would FPLC be a better technique than HPLC to use with proteins? What situations make HPLC preferable?


The only difference between FPLC and HPLC is the amount of pressure the pumps apply to the column. FPLC columns have a maximum pressure of about of 3-4 MPa, whereas HPLC columns can withstand or require much higher pressures. As a general rule, HPLC columns won't work with old FPLC equipment; FPLC columns can go on HPLCs as long as the pressure can be regulated.

Manufacturers have been marketing separate equipment to handle these different classes of columns, but the trend seems to be heading towards machines that can handle both types of columns without issue. A GE rep told me a few years ago that they've improved the pumps on the AKTAs to the point that "they're technically HPLCs now." The term "FPLC" is probably on its way out.


Fast Protein Liquid Chromatography

3.2.3.7 Fast protein liquid chromatography

Fast protein liquid chromatography (FPLC, formerly named “fast performance liquid chromatography”) is a form of medium pressure chromatography originally developed for purifying proteins with high resolution and reproducibility. Its distinguishing feature is that the stationary phase is composed of small-diameter beads (generally cross-linked agarose) that are packed in glass or plastic columns and have high loading capacity. Resins for FPLC are available in a wide range of particle sizes and ligand surfaces, which are selected on the basis of their application.

The FPLC system allows the use of a wide range of aqueous buffers (the mobile phase) and different stationary phases to perform the main chromatography modes (ion exchange, gel filtration, affinity, chromatofocusing, hydrophobic interaction, reverse phase). However, anion exchange and gel filtration chromatography are the modes most commonly used.

In general, the mobile phase is an aqueous buffer solution, whose flow rate through the stationary phase is controlled by a pump (normally kept constant), while the composition of the buffer may vary by mixing two or more solutions contained in external reservoirs. In most common FPLC strategies, eg, ion exchange, the resin is selected in a way that the given protein can be bound to the resin through charge interaction in a buffer A (running buffer), and subsequently, it can be dissociated and taken back to the solution in a buffer B (elution buffer). In contrast to high-performance liquid chromatography (HPLC), the buffer pressure used is low, typically 5 bar, but the flow rate is high (eg, 1–5 mL·min −1 ). FPLC chromatography can be scaled up, allowing the analysis of samples containing from milligrams of proteins in 5 mL-columns to preparative production of kilograms of purified proteins using columns of several liters of volume.


Which One is Right For Me?

There are many different factors one should consider before purchasing an FPLC system like an ӒKTA. The first is how heavily your lab relies on protein purification services. For labs that seldom require producing their protein and have been running columns manually, a lower-end system like the ӒKTA start will likely suit their needs with its automation of many different types of chromatography. For labs that do purify proteins regularly and want to automate more advanced chromatography techniques like size exclusion, the ӒKTA go would be a good system to start with. If more advanced purification protocols with complex readouts are a common occurrence in your lab, a highly customizable system like the ӒKTA pure will satisfy all of those conditions while providing opportunities for future upgrades. For labs that purify proteins around the clock and need to scale up their workflow while increasing throughput, a high-end system like the ӒKTA Avant would be most ideal. While many intricate details of all ӒKTA systems have been omitted from this article for the sake of brevity, the general descriptions of the capabilities these systems offer should be sufficient to get prospective buyers looking in the right direction.


General Considerations

This section provides general considerations for hydrophobic interaction chromatography including factors such as the ligand, matrix, salt concentration, pH, and temperature.

Ligand
A protein's adsorption behavior is determined by the type of immobilized ligand. In general, straight chain alkyl ligands demonstrate hydrophobic character while aryl ligands show a mixed mode behavior where both aromatic and hydrophobic interactions are possible (Hofstee and Otillio, 1978). The choice of ligand type is empirically determined.

Degree of substitution
The protein binding capacity increases with an increased degree of substitution of the immobilized ligand. With a high level of ligand substitution, the binding capacity remains constant however, the affinity of the interaction increases (Jennissen and Heilmeyer, 1975). Proteins bound under these conditions are difficult to elute due to multi-point attachment (Jennissen, 1978).

Matrix
The most widely used supports are hydrophilic carbohydrates: cross-linked agarose and synthetic copolymer materials. The selectivity between different supports will not be identical though the ligands may be the same. Modify adsorption and elution conditions to achieve similar results when moving from one media to another.

Salt concentration
The addition of structured salts to the equilibration buffer and sample promotes ligand-protein interactions in HIC (Porath et al.,1973). As the salt concentration increases, the amount of bound protein increases as does the risk of protein precipitation at the higher ionic strength.

The figure below represents the Hofmeister series on the effect of some anions and cations on protein precipitation. Though sodium, potassium or ammonium sulphates produce relatively higher precipitation effects, these salts effectively promote ligand-protein interactions in HIC. Most bound proteins are eluted by washing with water or dilute buffer at near neutral pH.

Effect of anions and cations on protein precipitation.

pH
HIC mobile phases are typically in the neutral pH range from 5&ndash7 and buffered with sodium or potassium phosphate. In general, the strength of the interaction between proteins and the media decreases with increasing pH as a result of increased charge of the protein due to the titration of acidic groups. This effect can vary from protein to protein. Thus, pH can impact the level of protein binding and the selectivity of the media. However, changes in pH do not have a significant effect over moderate ranges. Though it is useful to determine the optimal pH, pH gradients are not generally used as an elution method.

Temperature
The affinity of hydrophobic interactions increases with temperature. Temperature also impacts protein structure, solubility, and the interaction with the HIC matrix. Because temperature effects can be difficult to predict, it is generally not used to modulate separation using HIC. Not surprisingly, experiments conducted at room temperature may not be reproduced in a cold room.


Types of HPLC

The two most common variants are normal-phase and reversed-phase HPLC.


Normal-Phase HPLC
The column is filled with tiny silica particles, and a non-polar solvent, for example, hexane. A typical column has an internal diameter of 4.6 mm or smaller and a length of 150 to 250 mm. Non-polar compounds in the mixture will pass more quickly through the column, as polar compounds will stick longer to the polar silica than non-polar compounds will.

Reversed-Phase HPLC
The column size is the same. The column is filled with silica particles which are modified to make them non-polar. This is done by attaching long hydrocarbon chains (8–18 C atoms) to its surface. A polar solvent is used, for example, a mixture of water and an alcohol such as methanol. Polar compounds in the mixture will pass more quickly through the column because a strong attraction occurs between the polar solvent and the polar molecules in the mixture.

Non-polar molecules are slowed down on their way through the column. They form varying degrees of attraction with the hydrocarbon groups principally through van der Waals dispersion forces and hydrophobic interactions. They are also less soluble in the aqueous mobile phase components facilitating their interactions with the hydrocarbon groups.


The role of HPLC in analysing AAV gene therapy vectors

Gene therapy products exploit the biology of viruses, transferring the genetic sequences from a therapeutic into targeted cells. 1 Viral vectors are tools commonly used in genetics as they enable the movement of genetic material inside cells in a process called transduction the infected cells are described as transduced. 1 Once a genetic sequence is transduced, the therapeutic is expressed by the cell from the gene. Within this framework, there are two key strategies: ex vivo and in vivo. Ex vivo involves the removal and transduction of defective cells from the patient, before being reintroduced, whereas in vivo sees this process happening without cell removal. 1

The first approved gene therapy clinical research involved the ex vivo methodology. It took place in the USA in 1990, at the National Institute of Health. 2 The treatment involved a four-year-old girl who suffered from a genetic disease, adenosine deaminase (ADA) deficiency, which left her with a severe immune system deficiency. 2 White blood cells were taken from her and corrected with normal genes for making adenosine deaminase. They were then reintroduced, effectively repairing the patient&rsquos defective cells.

In vivo involves the introduction of genetic material directly into a patient&rsquos cells. To make this therapy possible, a highly specific vector is needed to avoid delivery to undesired cells and tissues, as this risks an immune response. Since adeno associated virus (AAV) can be targeted to specific tissue types it has become a focus for this type of gene therapy.

It&rsquos not simple, however, and the development of AAV gene therapy faces a number of challenges. HPLC can play a part in alleviating these challenges through characterisation.

AAV as a vector for gene therapy

Jesse Gelsinger was the first person to die in a clinical trial of gene therapy. 3 The investigation into his death suggested that previous infection with adenovirus had caused a supercharged immune reaction towards the adenovirus gene therapy vector. With this discovery, future research turned towards gene-delivery vehicles which patients would be unlikely to have an immune reaction towards. AAV was identified as the answer and quickly became the focus for development. 3

Many different variants of the virus exist that have different tissue specificity within the body for example, some will penetrate cardiac tissue more efficiently compared to others that may penetrate the brain. 3 The difference in tissue selectivity is conferred by variations in the proteins that form the capsid coat of the virus. Worldwide, this vector is being used in more than 200 ongoing clinical studies to treat a wide variety of diseases and disorders and this year the FDA approved the first AAV gene therapy (Zolgensma) for a lethal disorder of spinal muscular atrophy. 3,4

AAV is very promising and prevalent in the most commonly used platforms for gene delivery in preclinical and clinical studies. 4,5 However, the potential of AAV gene therapy is limited by several factors. It&rsquos small, meaning the size of therapeutic gene it can deliver is limited. Also, despite the initial thoughts, the virus was minimally immunogenic&mdashanti-AAV neutralising antibodies have been found in the general population and the AAV capsid proteins themselves can be immunogenic in some cases. 4 Producing a large supply can prove difficult which is exacerbated by the requirement for a large dose of highly purified vector for treatment.

There are plenty of challenges facing AAV gene therapy. In addition, the past failures in gene therapies highlight the requirement for stringent characterisation of these products before progressing to clinical trials.

Characterising AAV vectors

In the virus genome, it is the cap and rep genes that control capsid production and gene replication, respectively. These genes are removed from gene therapy vectors so the virus cannot replicate when its in human cells. 5 Although this is necessary for safety, it also presents a challenge producing the therapeutic. Since the virus can usually only replicate in the presence of another virus, &ldquohelper&rdquo genes are also needed during production. There are several methods employed to do this but each can lead to considerable heterogeneity within preparations. 5 Due to the co-expression of these genes and the viral assembly process, rAAV (recombinant AAV) can be produced that lack any genetic material. There is also the risk of the encapsulation of non-desirable genetic material, such as viral genes, which is risky if present in the final therapeutic. 5

The testing requirements for assessing the safety and purity of these gene therapy products are still being established but some of the main parameters are listed in Table 1. Clearly, with the increase in AAV vectors entering clinical trials there is a need to develop robust QC assays to distinguish between different viral variants and ensure consistent production of these therapies. The discussion below focuses on the role of HPLC in the analysis of AAV samples for gene therapy.

Table 1 Testing requirements for AAV-Based products (adapted from Pharmacology of Recombinant Adeno-associated Virus Production, Budloo et al, Methods & Clinical Development, 2018)

There are many impurities introduced into the AAV sample during the purification process that need to be carefully monitored using HPLC. 5 For example, Iodixanol is often used for density gradient purification of AAV. 4 Iodixanol is an iodinated density gradient originally used as an X-ray contrast compound for clinical use. Unlike other compounds used to generate gradients for fractionalisation, iodixanol solutions are non-ionic and inert, so fractions of AAV can be used directly in electrophoretic analysis and viral infectivity assays. 4 However, iodixanol is one of many residual reagents that are important quality attributes to monitor in the final AAV vector sample. While there is already a USP monograph for the quantification of iodixanol 6 (and other residual impurities) the role of HPLC in other areas of AAV analysis is still evolving.

Vector capsid purity and identity

The AAV capsid is composed of 3 proteins, VP1, VP2, and VP3. 5 Traditionally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been used to analyse the purity of these proteins within a viral sample. 7 This method involves denaturing the proteins in the presence of SDS. The SDS binds to the proteins and results in a negative charge that is proportional to the chain length. The proteins can then be separated based on charge in the polyacrylamide gel, where an electrical current is applied. Although this is a traditional technique, quantitation is often an unreliable and resource-intensive process. 7 Since reverse phase liquid chromatography (RPLC) is also a denaturing method it is well suited to replace this method for the analysis of protein purity for viral vectors. Good method development can produce chromatography that is much more reproducible and allows robust quantification. 7 In addition, RPLC is generally cheaper and directly amenable to on-line mass spectrometry (MS) for the identification of capsid proteins and impurities.

Alongside achieving capsid purity, it is important to characterise the capsid identity to ensure the correct vector has been produced. Traditionally, SDS-PAGE would be combined with western blotting, where an antibody that binds to an epitope in each of the viral proteins is used to identify each band in the SDS-PAGE. 7 Again, this is time-consuming and RPLC can provide much more reliable quantitative techniques. The use of peptide mapping can also be used to pick up on variations in the protein sequence that would not be recognised by an antibody-based assay.

Empty/full capsids analysis

During the production of viral vectors, there will be a population of viral particles produced that has failed to package the vector DNA (Figure 1). These empty capsids will represent varying proportions of the crude harvest for rAAV preparations. 5 Incomplete encapsulation can also occur, leading to capsids containing truncated genomes or incorrect DNA (i.e. from plasmids, cells, or helper viruses). 5 Given the history of problems with immune system responses to gene therapy vectors, it is important to reduce the sources of unnecessary, potentially antigenic material, that could elicit an unwanted immune response. 2 Although the impact of these species is not fully understood, they are undesirable and, thus, a carefully regulated quality attribute. 5

One challenge for analysing empty/full capsid vectors is that the DNA is protected by the capsid shell. There is little change in shape/size between empty, full ,and partially full capsid particles. Therefore, the ratio of empty to full capsids is an important attribute to monitor in viral vector preparations. Regulatory requirements in this area are still being established and there is interest in developing traditional HPLC methods for this purpose. Current methods to evaluate the capsids are transmission electron microscopy (TEM) and analytical ultracentrifugation (AUC). 7 Cryogenic TEM involves a sample vitrified by rapid freezing to preserve the structure of a biological specimen. When imaged, there is a clear morphological distinction between packed and empty particles. 7 Since the empty capsids have a different density and/or mass to the correctly packaged particles they can be separated under centrifugal force. AUC is a tool to distinguish and quantify different AAV species either by mass (sedimentation velocity) or density (sedimentation equilibrium). Innovations in AUC mean it is now possible to monitor multiple wavelengths of absorbance to properly quantify both genomic DNA and viral capsid content in a single experiment. The main advantage of this technique is that it is reproducible, can quantify viral particles in the formulation buffer (no sample preparation), works independently of the AAV variant (or the size of the transgene), and requires no standard comparison. 7

Figure 1: example of different capsid assemblies produced during AAV vector manufacturing

HPLC has a role in the characterisation of capsid particles. Conformational changes occur to the capsid proteins during encapsulation of the DNA that causes a change in the overall surface charge on the capsid particle. 5 Efforts are made to utilise this physiochemical difference between empty/full capsids to develop analytical HPLC methods ion exchange, though it is difficult to establish one optimal method for all rAAV variants because their capsid physiochemical properties differ. This has led to some success since AAV is relatively small for a virus (20-25 nm) making it amenable to both traditional and monolithic columns. Monolithic columns are sometimes preferred when dealing with very large viruses and biomolecules as there is less risk of clogging, higher flow rates, and fewer shear forces. 8 However, with a careful choice of column, there is no reason traditional ion-exchange chromatography cannot be used to analyse AAV particles on an analytical scale. Furthermore, different column dimensions and chemistry is useful during method development to try and maximise resolution for different variants. Viral particles are sensitive to their environment, for example, capsid uncoating is common at high temperatures therefore, it may be important to cross-validate ion exchange results with TEM or AUC. Assuming accurate results are attained, ion exchange HPLC could provide a cheap, easy, and high-throughput method of monitoring capsid assembles.

Aggregation analysis

Due to the immunogenicity of aggregates, characterisation is a critical quality attribute for gene therapy vectors, as it is for any biological therapeutic. Aggregates can vary greatly from nm (subvisible) to mm (visible) in diameter and their formation during production, storage, and shipment can be caused by numerous factors. 7 One method alone can&rsquot cover the large size range needed, but we can combine different techniques to evaluate the level of aggregates in a sample. This ranges from basic techniques such as a visual inspection to methods such as dynamic light scattering (DLS), AUC, size exclusion chromatography (SEC), TEM, and field-flow fractionation with multi-angle static light scattering (FFF-MALS). 7

SEC is particularly attractive for aggregate analysis because it is inexpensive to quantify aggregates in a high-throughput manner. 7 SEC is unusual compared to other chromatography techniques due to absence of a retentive mechanism. However, secondary analyte/stationary phase interactions can occur as a result of surface silanol species or hydrophobic Interactions. 9 Secondary interactions between an analyte and the column are troublesome for SEC analysis as they can give rise to poor peak shape, resolution and recovery. When dealing with large biomolecules, choosing the right column chemistry and mobile phase conditions is particularly important during method development. Secondary interactions, temperature and the SEC column itself could impact the amount of aggregate observed. 9 However, using complementary techniques such as AUC should provide confidence in the results generated from SEC analysis, and the additional biophysical information it generates supports the production and process development.

Conclusions

This article has outlined some of the issues facing AAV as a gene therapy vector and some of the different safety and quality attributes that need to be considered during product development. AAV testing poses challenges since the parameters for what makes a cell and gene therapy product safe for patients are still emerging. As experts in chromatography, Crawford Scientific can provide support with all aspects of HPLC method development. We have a wide range of high-quality analytical columns suitable for a broad range of biomolecule applications. If you do need any advice or run into any problems with one of the HPLC methods discussed above, our in-house technical team are always on hand to provide expert advice to our customers.

Get in touch with our technical team ( [email protected] ) to find out more!


Protein Purification Strategies

Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction. Protein purification is a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. A variety of protein purification strategies exist to address desired scale, throughput and downstream applications. The optimal approach often must be determined empirically.

Protein Purification

The best protein purification protocol depends not only on the protein being purified but also on many other factors such as the cell used to express the recombinant protein (e.g., prokaryotic versus eukaryotic cells). Escherichia coli remains the first choice of many researchers for producing recombinant proteins due to ease of use, rapid cell growth and low cost of culturing. Proteins expressed in E. coli can be purified in relatively high quantities, but these proteins, especially eukaryotic proteins, may not exhibit proper protein activity or folding. Cultured mammalian cells might offer a better option for producing properly folded and functional mammalian proteins with appropriate post-translational modifications (Geisse et al. 1996). However, the low expression levels of recombinant proteins in cultured mammalian cells presents a challenge for their purification. As a result, attaining satisfactory yield and purity depends on highly selective and efficient capture of these proteins from the crude cell lysates.

To simplify purification, affinity purification tags can be fused to a recombinant protein of interest (Nilsson et al. 1997). Common fusion tags are polypeptides, small proteins or enzymes added to the N- or C-terminus of a recombinant protein. The biochemical features of different tags influence the stability, solubility and expression of proteins to which they are attached (Stevens et al. 2001). Using expression vectors that include a fusion tag facilitates recombinant protein purification.

Isolation of Protein Complexes

A major objective in proteomics is the elucidation of protein function and organization of the complex networks that are responsible for key cellular processes. Analysis of protein:protein interactions can provide valuable insight into the cell signaling cascades involved in these processes, and analysis of protein:nucleic acid interactions often reveals important information about biological processes such as mRNA regulation, chromosomal remodeling and transcription. For example, transcription factors play an important role in regulating transcription by binding to specific recognition sites on the chromosome, often at a gene’s promoter, and interacting with other proteins in the nucleus. This regulation is required for cell viability, differentiation and growth (Mankan et al. 2009 Gosh et al. 1998).

Analysis of protein:protein interactions often requires straightforward methods for immobilizing proteins on solid surfaces in proper orientations without disrupting protein structure or function. This immobilization must not interfere with the binding capacity and can be achieved through the use of affinity tags. Immobilization of proteins on chips is a popular approach to analyze protein:DNA and protein:protein interactions and identify components of protein complexes (Hall et al. 2004 Hall et al. 2007 Hudson and Snyder, 2006). Functional protein microarrays normally contain full-length functional proteins or protein domains bound to a solid surface. Fluorescently labeled DNA is used to probe the array and identify proteins that bind to the specific probe. Protein microarrays provide a method for high-throughput identification of protein:DNA interactions. Immobilized proteins also can be used in protein pull-down assays to isolate protein binding partners in vivo (mammalian cells) or in vitro. Other downstream applications such as mass spectrometry do not require protein immobilization to identify protein partners and individual components of protein complexes.


What is the difference between HPLC and FPLC and why is FPLC preferable for protein purification? - Biology

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC

High performance liquid chromatography is a powerful tool in analysis. This page looks at how it is carried out and shows how it uses the same principles as in thin layer chromatography and column chromatography.

Note: It is important to read the introductory page about thin layer chromatography before you continue with this one - particularly the part about how thin layer chromatography works. High performance liquid chromatography works on the same basic principle. HPLC is essentially an adaptation of column chromatography - so it might be a good idea to have a (very quick) look at that as well.

Use the BACK button on your browser to return quickly to this page.

High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.

It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture.

The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive.

The column and the solvent

Confusingly, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase.

Normal phase HPLC

This is essentially just the same as you will already have read about in thin layer chromatography or column chromatography. Although it is described as "normal", it isn't the most commonly used form of HPLC.

The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm.

Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column.

Reversed phase HPLC

In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol.

In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent.

Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column.

That means that now it is the polar molecules that will travel through the column more quickly.

Reversed phase HPLC is the most commonly used form of HPLC.

Note: I have been a bit careful about how I have described the attractions of the non-polar molecules to the surface of the stationary phase. In particular, I have avoided the use of the word "adsorpion". Adsorption is when a molecule sticks to the surface of a solid. Especially if you had small molecules in your mixture, some could get in between the long C18 chains to give what is essentially a solution.

You could therefore say that non-polar molecules were more soluble in the hydrocarbon on the surface of the silica than they are in the polar solvent - and so spend more time in this alternative "solvent". Where a solute divides itself between two different solvents because it is more soluble in one than the other, we call it partition.

So is this adsorption or partition? You could argue it both ways! Be prepared to find it described as either.

Looking at the whole process

A flow scheme for HPLC

Injection of the sample

Injection of the sample is entirely automated, and you wouldn't be expected to know how this is done at this introductory level. Because of the pressures involved, it is not the same as in gas chromatography (if you have already studied that).

The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.

Different compounds have different retention times. For a particular compound, the retention time will vary depending on:

the pressure used (because that affects the flow rate of the solvent)

the nature of the stationary phase (not only what material it is made of, but also particle size)

the exact composition of the solvent

the temperature of the column

That means that conditions have to be carefully controlled if you are using retention times as a way of identifying compounds.

There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption.

Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, you can get a direct reading of how much of the light is absorbed.

The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time.

You might wonder why the solvents used don't absorb UV light. They do! But different compounds absorb most strongly in different parts of the UV spectrum.

Methanol, for example, absorbs at wavelengths below 205 nm, and water below 190 nm. If you were using a methanol-water mixture as the solvent, you would therefore have to use a wavelength greater than 205 nm to avoid false readings from the solvent.

Note: If you are interested, there is a whole section about UV-visible spectroscopy on the site. This explores the question of the absorption of UV and visible light by organic compounds in some detail.

Interpreting the output from the detector

The output will be recorded as a series of peaks - each one representing a compound in the mixture passing through the detector and absorbing UV light. As long as you were careful to control the conditions on the column, you could use the retention times to help to identify the compounds present - provided, of course, that you (or somebody else) had already measured them for pure samples of the various compounds under those identical conditions.

But you can also use the peaks as a way of measuring the quantities of the compounds present. Let's suppose that you are interested in a particular compound, X.

If you injected a solution containing a known amount of pure X into the machine, not only could you record its retention time, but you could also relate the amount of X to the peak that was formed.

The area under the peak is proportional to the amount of X which has passed the detector, and this area can be calculated automatically by the computer linked to the display. The area it would measure is shown in green in the (very simplified) diagram.

If the solution of X was less concentrated, the area under the peak would be less - although the retention time will still be the same. For example:

This means that it is possible to calibrate the machine so that it can be used to find how much of a substance is present - even in very small quantities.

Be careful, though! If you had two different substances in the mixture (X and Y) could you say anything about their relative amounts? Not if you were using UV absorption as your detection method.

In the diagram, the area under the peak for Y is less than that for X. That may be because there is less Y than X, but it could equally well be because Y absorbs UV light at the wavelength you are using less than X does. There might be large quantities of Y present, but if it only absorbed weakly, it would only give a small peak.

Note: You will find a useful industry training video which talks through the whole process by following this link.

Linking to other sites is always a little bit hazardous because sites change. If you find that this link doesn't work, please contact me via the address on the About this site page.

Coupling HPLC to a mass spectrometer

This is where it gets really clever! When the detector is showing a peak, some of what is passing through the detector at that time can be diverted to a mass spectrometer. There it will give a fragmentation pattern which can be compared against a computer database of known patterns. That means that the identity of a huge range of compounds can be found without having to know their retention times.

Note: If you have forgotten about mass spectrometry, explore the mass spectrometry menu - particularly how a mass spectrometer works, and the formation of fragmentation patterns.

Questions to test your understanding

If this is the first set of questions you have done, please read the introductory page before you start. You will need to use the BACK BUTTON on your browser to come back here afterwards.


Contents

Chromatography, pronounced / ˌ k r oʊ m ə ˈ t ɒ ɡ r ə f i / , is derived from Greek χρῶμα chroma, which means "color", and γράφειν graphein, which means "to write". The combination of these two terms was directly inherited from the invention of the technique first used to separate pigments. [3]

Chromatography was first devised in Russia by the Italian-born scientist Mikhail Tsvet in 1900. [4] He developed the technique, he coined chromatography, in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. Since these components separate in bands of different colors (green, orange, and yellow, respectively) they directly inspired the name of the technique. New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes. [5]

Chromatography technique developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s, for which they won the 1952 Nobel Prize in Chemistry. [6] They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several chromatographic methods: paper chromatography, gas chromatography, and what would become known as high-performance liquid chromatography. Since then, the technology has advanced rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in many different ways, resulting in the different varieties of chromatography described below. Advances are continually improving the technical performance of chromatography, allowing the separation of increasingly similar molecules.

  • Analyte – the substance to be separated during chromatography. It is also normally what is needed from the mixture.
  • Analytical chromatography – the use of chromatography to determine the existence and possibly also the concentration of analyte(s) in a sample.
  • Bonded phase – a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
  • Chromatogram – the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

Chromatography is based on the concept of partition coefficient. Any solute partitions between two immiscible solvents. When we make one solvent immobile (by adsorption on a solid support matrix) and another mobile it results in most common applications of chromatography. If the matrix support, or stationary phase, is polar (e.g. paper, silica etc.) it is forward phase chromatography, and if it is non-polar (C-18) it is reverse phase.

Column chromatography Edit

Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample. [8] [9] In 1978, W. Clark Still introduced a modified version of column chromatography called flash column chromatography (flash). [10] [11] The technique is very similar to the traditional column chromatography, except that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.

In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of broken cells.

Phosphocellulose chromatography utilizes the binding affinity of many DNA-binding proteins for phosphocellulose. The stronger a protein's interaction with DNA, the higher the salt concentration needed to elute that protein. [12]

Planar chromatography Edit

Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin-layer chromatography). Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.

Paper chromatography Edit

Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip of chromatography paper. The paper is placed in a container with a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample mixture, which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel further if they are less polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.

Thin-layer chromatography (TLC) Edit

Thin-layer chromatography (TLC) is a widely employed laboratory technique used to separate different biochemicals on the basis of their relative attractions to the stationary and mobile phases. It is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. TLC is very versatile multiple samples can be separated simultaneously on the same layer, making it very useful for screening applications such as testing drug levels and water purity. [13] Possibility of cross-contamination is low since each separation is performed on a new layer. Compared to paper, it has the advantage of faster runs, better separations, better quantitative analysis, and the choice between different adsorbents. For even better resolution and faster separation that utilizes less solvent, high-performance TLC can be used. An older popular use had been to differentiate chromosomes by observing distance in gel (separation of was a separate step).

The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) competes effectively for binding sites, and thus displaces all molecules with lesser affinities. [14] There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired for maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than "peaks". Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.

Gas chromatography Edit

Gas chromatography (GC), also sometimes known as gas-liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatographic separation is always carried out in a column, which is typically "packed" or "capillary". Packed columns are the routine work horses of gas chromatography, being cheaper and easier to use and often giving adequate performance. Capillary columns generally give far superior resolution and although more expensive are becoming widely used, especially for complex mixtures. Further, capillary columns can be split into three classes: porous layer open tubular (PLOT), wall-coated open tubular (WCOT) and support-coated open tubular (SCOT) columns. PLOT columns are unique in a way that the stationary phase is adsorbed to the column walls, while WCOT columns have a stationary phase that is chemically bonded to the walls. SCOT columns are in a way the combination of the two types mentioned in a way that they have support particles adhered to column walls, but those particles have liquid phase chemically bonded onto them. [15] Both types of column are made from non-adsorbent and chemically inert materials. Stainless steel and glass are the usual materials for packed columns and quartz or fused silica for capillary columns.

Gas chromatography is based on a partition equilibrium of analyte between a solid or viscous liquid stationary phase (often a liquid silicone-based material) and a mobile gas (most often helium). The stationary phase is adhered to the inside of a small-diameter (commonly 0.53 – 0.18mm inside diameter) glass or fused-silica tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat denatures them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring and remediation, and industrial chemical fields. It is also used extensively in chemistry research.

Liquid chromatography Edit

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. It can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high-performance liquid chromatography (HPLC).

In HPLC the sample is forced by a liquid at high pressure (the mobile phase) through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is termed reversed phase liquid chromatography (RPLC).

Specific techniques under this broad heading are listed below.

Affinity chromatography [16] is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labeled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.

Affinity chromatography often utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Traditional affinity columns are used as a preparative step to flush out unwanted biomolecules.

However, HPLC techniques exist that do utilize affinity chromatography properties. Immobilized Metal Affinity Chromatography (IMAC) [17] [18] is useful to separate aforementioned molecules based on the relative affinity for the metal (i.e. Dionex IMAC). Often these columns can be loaded with different metals to create a column with a targeted affinity. [19]

Supercritical fluid chromatography Edit

Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.

Ion exchange chromatography Edit

Ion exchange chromatography (usually referred to as ion chromatography) uses an ion exchange mechanism to separate analytes based on their respective charges. It is usually performed in columns but can also be useful in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion-exchange resin that carries charged functional groups that interact with oppositely charged groups of the compound to retain. There are two types of ion exchange chromatography: Cation-Exchange and Anion-Exchange. In the Cation-Exchange Chromatography the stationary phase has negative charge and the exchangeable ion is a cation, whereas, in the Anion-Exchange Chromatography the stationary phase has positive charge and the exchangeable ion is an anion. [20] Ion exchange chromatography is commonly used to purify proteins using FPLC.

Size-exclusion chromatography Edit

Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention such species are the first to be eluted. It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.

Expanded bed adsorption chromatographic separation Edit

An expanded bed chromatographic adsorption (EBA) column for a biochemical separation process comprises a pressure equalization liquid distributor having a self-cleaning function below a porous blocking sieve plate at the bottom of the expanded bed, an upper part nozzle assembly having a backflush cleaning function at the top of the expanded bed, a better distribution of the feedstock liquor added into the expanded bed ensuring that the fluid passed through the expanded bed layer displays a state of piston flow. The expanded bed layer displays a state of piston flow. The expanded bed chromatographic separation column has advantages of increasing the separation efficiency of the expanded bed.

Expanded-bed adsorption (EBA) chromatography is a convenient and effective technique for the capture of proteins directly from unclarified crude sample. In EBA chromatography, the settled bed is first expanded by upward flow of equilibration buffer. The crude feed, a mixture of soluble proteins, contaminants, cells, and cell debris, is then passed upward through the expanded bed. Target proteins are captured on the adsorbent, while particulates and contaminants pass through. A change to elution buffer while maintaining upward flow results in desorption of the target protein in expanded-bed mode. Alternatively, if the flow is reversed, the adsorbed particles will quickly settle and the proteins can be desorbed by an elution buffer. The mode used for elution (expanded-bed versus settled-bed) depends on the characteristics of the feed. After elution, the adsorbent is cleaned with a predefined cleaning-in-place (CIP) solution, with cleaning followed by either column regeneration (for further use) or storage.

Reversed-phase chromatography Edit

Reversed-phase chromatography (RPC) is any liquid chromatography procedure in which the mobile phase is significantly more polar than the stationary phase. It is so named because in normal-phase liquid chromatography, the mobile phase is significantly less polar than the stationary phase. Hydrophobic molecules in the mobile phase tend to adsorb to the relatively hydrophobic stationary phase. Hydrophilic molecules in the mobile phase will tend to elute first. Separating columns typically comprise a C8 or C18 carbon-chain bonded to a silica particle substrate.

Hydrophobic interaction chromatography Edit

Hydrophobic interactions between proteins and the chromatographic matrix can be exploited to purify proteins. In hydrophobic interaction chromatography the matrix material is lightly substituted with hydrophobic groups. These groups can range from methyl, ethyl, propyl, octyl, or phenyl groups. [21] At high salt concentrations, non-polar sidechains on the surface on proteins "interact" with the hydrophobic groups that is, both types of groups are excluded by the polar solvent (hydrophobic effects are augmented by increased ionic strength). Thus, the sample is applied to the column in a buffer which is highly polar. The eluant is typically an aqueous buffer with decreasing salt concentrations, increasing concentrations of detergent (which disrupts hydrophobic interactions), or changes in pH.

In general, Hydrophobic Interaction Chromatography (HIC) is advantageous if the sample is sensitive to pH change or harsh solvents typically used in other types of chromatography but not high salt concentrations. Commonly, it is the amount of salt in the buffer which is varied. In 2012, Müller and Franzreb described the effects of temperature on HIC using Bovine Serum Albumin (BSA) with four different types of hydrophobic resin. The study altered temperature as to effect the binding affinity of BSA onto the matrix. It was concluded that cycling temperature from 50 to 10 degrees would not be adequate to effectively wash all BSA from the matrix but could be very effective if the column would only be used a few times. [22] Using temperature to effect change allows labs to cut costs on buying salt and saves money.

If high salt concentrations along with temperature fluctuations want to be avoided you can use a more hydrophobic to compete with your sample to elute it. [source] This so-called salt independent method of HIC showed a direct isolation of Human Immunoglobulin G (IgG) from serum with satisfactory yield and used Beta-cyclodextrin as a competitor to displace IgG from the matrix. [23] This largely opens up the possibility of using HIC with samples which are salt sensitive as we know high salt concentrations precipitate proteins.

Hydrodynamic chromatography Edit

Hydrodynamic chromatography (HDC) is derived from the observed phenomenon that large droplets move faster than small ones. [24] In a column, this happens because the center of mass of larger droplets is prevented from being as close to the sides of the column as smaller droplets because of their larger overall size. [25] Larger droplets will elute first from the middle of the column while smaller droplets stick to the sides of the column and elute last. This form of chromatography is useful for separating analytes by molar mass, size, shape, and structure when used in conjunction with light scattering detectors, viscometers, and refractometers. [26] The two main types of HDC are open tube and packed column. Open tube offers rapid separation times for small particles, whereas packed column HDC can increase resolution and is better suited for particles with an average molecular mass larger than 10 5 > daltons. [27] HDC differs from other types of chromatography because the separation only takes place in the interstitial volume, which is the volume surrounding and in between particles in a packed column. [28]

HDC shares the same order of elution as Size Exclusion Chromatography (SEC) but the two processes still vary in many ways. [27] In a study comparing the two types of separation, Isenberg, Brewer, Côté, and Striegel use both methods for polysaccharide characterization and conclude that HDC coupled with multiangle light scattering (MALS) achieves more accurate molar mass distribution when compared to off-line MALS than SEC in significantly less time. [29] This is largely due to SEC being a more destructive technique because of the pores in the column degrading the analyte during separation, which tends to impact the mass distribution. [29] However, the main disadvantage of HDC is low resolution of analyte peaks, which makes SEC a more viable option when used with chemicals that are not easily degradable and where rapid elution is not important. [30]

HDC plays an especially important role in the field of microfluidics. The first successful apparatus for HDC-on-a-chip system was proposed by Chmela, et al. in 2002. [31] Their design was able to achieve separations using an 80 mm long channel on the timescale of 3 minutes for particles with diameters ranging from 26 to 110 nm, but the authors expressed a need to improve the retention and dispersion parameters. [31] In a 2010 publication by Jellema, Markesteijn, Westerweel, and Verpoorte, implementing HDC with a recirculating bidirectional flow resulted in high resolution, size based separation with only a 3 mm long channel. [32] Having such a short channel and high resolution was viewed as especially impressive considering that previous studies used channels that were 80 mm in length. [31] For a biological application, in 2007, Huh, et al. proposed a microfluidic sorting device based on HDC and gravity, which was useful for preventing potentially dangerous particles with diameter larger than 6 microns from entering the bloodstream when injecting contrast agents in ultrasounds. [33] This study also made advances for environmental sustainability in microfluidics due to the lack of outside electronics driving the flow, which came as an advantage of using a gravity based device.

Two-dimensional chromatography Edit

In some cases, the selectivity provided by the use of one column can be insufficient to provide resolution of analytes in complex samples. Two-dimensional chromatography aims to increase the resolution of these peaks by using a second column with different physico-chemical (chemical classification) properties. [34] [35] Since the mechanism of retention on this new solid support is different from the first dimensional separation, it can be possible to separate compounds by two-dimensional chromatography that are indistinguishable by one-dimensional chromatography. Furthermore, the separation on the second dimension occurs faster than the first dimension. [34] An example of a two-dimensional TLC separation is where the sample is spotted at one corner of a square plate, developed, air-dried, then rotated by 90° and usually redeveloped in a second solvent system. Two-dimensional chromatography can be applied to GC or LC separations. [34] [35] This separation method can also be used in a heart-cutting approach, [36] where specific regions of interest on the first dimension are selected for separation by the second dimension, or in a comprehensive approach, [34] [35] where all the analytes from the first dimension undergo the second dimension separation.

Simulated moving-bed chromatography Edit

The simulated moving bed (SMB) technique is a variant of high performance liquid chromatography it is used to separate particles and/or chemical compounds that would be difficult or impossible to resolve otherwise. This increased separation is brought about by a valve-and-column arrangement that is used to lengthen the stationary phase indefinitely. In the moving bed technique of preparative chromatography the feed entry and the analyte recovery are simultaneous and continuous, but because of practical difficulties with a continuously moving bed, simulated moving bed technique was proposed. In the simulated moving bed technique instead of moving the bed, the sample inlet and the analyte exit positions are moved continuously, giving the impression of a moving bed. True moving bed chromatography (TMBC) is only a theoretical concept. Its simulation, SMBC is achieved by the use of a multiplicity of columns in series and a complex valve arrangement, which provides for sample and solvent feed, and also analyte and waste takeoff at appropriate locations of any column, whereby it allows switching at regular intervals the sample entry in one direction, the solvent entry in the opposite direction, whilst changing the analyte and waste takeoff positions appropriately as well.

Pyrolysis gas chromatography Edit

Pyrolysis–gas chromatography–mass spectrometry is a method of chemical analysis in which the sample is heated to decomposition to produce smaller molecules that are separated by gas chromatography and detected using mass spectrometry.

Pyrolysis is the thermal decomposition of materials in an inert atmosphere or a vacuum. The sample is put into direct contact with a platinum wire, or placed in a quartz sample tube, and rapidly heated to 600–1000 °C. Depending on the application even higher temperatures are used. Three different heating techniques are used in actual pyrolyzers: Isothermal furnace, inductive heating (Curie Point filament), and resistive heating using platinum filaments. Large molecules cleave at their weakest points and produce smaller, more volatile fragments. These fragments can be separated by gas chromatography. Pyrolysis GC chromatograms are typically complex because a wide range of different decomposition products is formed. The data can either be used as fingerprint to prove material identity or the GC/MS data is used to identify individual fragments to obtain structural information. To increase the volatility of polar fragments, various methylating reagents can be added to a sample before pyrolysis.

Besides the usage of dedicated pyrolyzers, pyrolysis GC of solid and liquid samples can be performed directly inside Programmable Temperature Vaporizer (PTV) injectors that provide quick heating (up to 30 °C/s) and high maximum temperatures of 600–650 °C. This is sufficient for some pyrolysis applications. The main advantage is that no dedicated instrument has to be purchased and pyrolysis can be performed as part of routine GC analysis. In this case quartz GC inlet liners have to be used. Quantitative data can be acquired, and good results of derivatization inside the PTV injector are published as well.

Fast protein liquid chromatography Edit

Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid (the "mobile phase") and a porous solid (the stationary phase). In FPLC the mobile phase is an aqueous solution, or "buffer". The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.

Countercurrent chromatography Edit

Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids and the liquid stationary phase is held stagnant by a strong centrifugal force.

Hydrodynamic countercurrent chromatography (CCC) Edit

The operating principle of CCC instrument requires a column consisting of an open tube coiled around a bobbin. The bobbin is rotated in a double-axis gyratory motion (a cardioid), which causes a variable gravity (G) field to act on the column during each rotation. This motion causes the column to see one partitioning step per revolution and components of the sample separate in the column due to their partitioning coefficient between the two immiscible liquid phases used. There are many types of CCC available today. These include HSCCC (High Speed CCC) and HPCCC (High Performance CCC). HPCCC is the latest and best-performing version of the instrumentation available currently.

Hydrostatic countercurrent chromatography or centrifugal partition chromatography (CPC) Edit

In the CPC instrument, the column consists of a series of cells interconnected by ducts attached to a rotor. This rotor rotates on its central axis creating the centrifugal field necessary to hold the stationary phase in place. The separation process in CPC is governed solely by the partitioning of solutes between the stationary and mobile phases, which mechanism can be easily described using the partition coefficients (KD) of solutes. CPC instruments are commercially available for laboratory, pilot, and industrial-scale separations with different sizes of columns ranging from some 10 milliliters to 10 liters volume.

Periodic counter-current chromatography Edit

In contrast to Counter current chromatography (see above), periodic counter-current chromatography (PCC) uses a solid stationary phase and only a liquid mobile phase. It thus is much more similar to conventional affinity chromatography than to counter current chromatography. PCC uses multiple columns, which during the loading phase are connected in line. This mode allows for overloading the first column in this series without losing product, which already breaks through the column before the resin is fully saturated. The breakthrough product is captured on the subsequent column(s). In a next step the columns are disconnected from one another. The first column is washed and eluted, while the other column(s) are still being loaded. Once the (initially) first column is re-equilibrated, it is re-introduced to the loading stream, but as last column. The process then continues in a cyclic fashion.

Chiral chromatography Edit

Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers, these have no chemical or physical differences apart from being three-dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes. Chiral chromatography HPLC columns (with a chiral stationary phase) in both normal and reversed phase are commercially available.


How Does High Performance Liquid Chromatography Work?

The components of a basic high-performance liquid chromatography [HPLC] system are shown in the simple diagram in Figure E.

A reservoir holds the solvent [called the mobile phase, because it moves]. A high-pressure pump [solvent delivery system or solvent manager] is used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute. An injector [sample manager or autosampler] is able to introduce [inject] the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column contains the chromatographic packing material needed to effect the separation. This packing material is called the stationary phase because it is held in place by the column hardware. A detector is needed to see the separated compound bands as they elute from the HPLC column [most compounds have no color, so we cannot see them with our eyes]. The mobile phase exits the detector and can be sent to waste, or collected, as desired. When the mobile phase contains a separated compound band, HPLC provides the ability to collect this fraction of the eluate containing that purified compound for further study. This is called preparative chromatography [discussed in the section on HPLC Scale].

Note that high-pressure tubing and fittings are used to interconnect the pump, injector, column, and detector components to form the conduit for the mobile phase, sample, and separated compound bands.

Figure E: High-Performance Liquid Chromatography [HPLC] System

The detector is wired to the computer data station, the HPLC system component that records the electrical signal needed to generate the chromatogram on its display and to identify and quantitate the concentration of the sample constituents (see Figure F). Since sample compound characteristics can be very different, several types of detectors have been developed. For example, if a compound can absorb ultraviolet light, a UV-absorbance detector is used. If the compound fluoresces, a fluorescence detector is used. If the compound does not have either of these characteristics, a more universal type of detector is used, such as an evaporative-light-scattering detector [ELSD]. The most powerful approach is the use multiple detectors in series. For example, a UV and/or ELSD detector may be used in combination with a mass spectrometer [MS] to analyze the results of the chromatographic separation. This provides, from a single injection, more comprehensive information about an analyte. The practice of coupling a mass spectrometer to an HPLC system is called LC/MS.

Figure F: A Typical HPLC [Waters Alliance] System

HPLC Operation
A simple way to understand how we achieve the separation of the compounds contained in a sample is to view the diagram in Figure G.

Mobile phase enters the column from the left, passes through the particle bed, and exits at the right. Flow direction is represented by green arrows. First, consider the top image it represents the column at time zero [the moment of injection], when the sample enters the column and begins to form a band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of the column as a single black band. [In reality, this sample could be anything that can be dissolved in a solvent typically the compounds would be colorless and the column wall opaque, so we would need a detector to see the separated compounds as they elute.]

After a few minutes [lower image], during which mobile phase flows continuously and steadily past the packing material particles, we can see that the individual dyes have moved in separate bands at different speeds. This is because there is a competition between the mobile phase and the stationary phase for attracting each of the dyes or analytes. Notice that the yellow dye band moves the fastest and is about to exit the column. The yellow dye likes [is attracted to] the mobile phase more than the other dyes. Therefore, it moves at a faster speed, closer to that of the mobile phase. The blue dye band likes the packing material more than the mobile phase. Its stronger attraction to the particles causes it to move significantly slower. In other words, it is the most retained compound in this sample mixture. The red dye band has an intermediate attraction for the mobile phase and therefore moves at an intermediate speed through the column. Since each dye band moves at different speed, we are able to separate it chromatographically.

Figure G: Understanding How a Chromatographic Column Works – Bands

What Is a Detector?
As the separated dye bands leave the column, they pass immediately into the detector. The detector contains a flow cell that sees [detects] each separated compound band against a background of mobile phase [see Figure H]. [In reality, solutions of many compounds at typical HPLC analytical concentrations are colorless.] An appropriate detector has the ability to sense the presence of a compound and send its corresponding electrical signal to a computer data station. A choice is made among many different types of detectors, depending upon the characteristics and concentrations of the compounds that need to be separated and analyzed, as discussed earlier.

What Is a Chromatogram?
A chromatogram is a representation of the separation that has chemically [chromatographically] occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis. Each peak represents the detector response for a different compound. The chromatogram is plotted by the computer data station [see Figure H].

Figure H: How Peaks Are Created

In Figure H, the yellow band has completely passed through the detector flow cell the electrical signal generated has been sent to the computer data station. The resulting chromatogram has begun to appear on screen. Note that the chromatogram begins when the sample was first injected and starts as a straight line set near the bottom of the screen. This is called the baseline it represents pure mobile phase passing through the flow cell over time. As the yellow analyte band passes through the flow cell, a stronger signal is sent to the computer. The line curves, first upward, and then downward, in proportion to the concentration of the yellow dye in the sample band. This creates a peak in the chromatogram. After the yellow band passes completely out of the detector cell, the signal level returns to the baseline the flow cell now has, once again, only pure mobile phase in it. Since the yellow band moves fastest, eluting first from the column, it is the first peak drawn.

A little while later, the red band reaches the flow cell. The signal rises up from the baseline as the red band first enters the cell, and the peak representing the red band begins to be drawn. In this diagram, the red band has not fully passed through the flow cell. The diagram shows what the red band and red peak would look like if we stopped the process at this moment. Since most of the red band has passed through the cell, most of the peak has been drawn, as shown by the solid line. If we could restart, the red band would completely pass through the flow cell and the red peak would be completed [dotted line]. The blue band, the most strongly retained, travels at the slowest rate and elutes after the red band. The dotted line shows you how the completed chromatogram would appear if we had let the run continue to its conclusion. It is interesting to note that the width of the blue peak will be the broadest because the width of the blue analyte band, while narrowest on the column, becomes the widest as it elutes from the column. This is because it moves more slowly through the chromatographic packing material bed and requires more time [and mobile phase volume] to be eluted completely. Since mobile phase is continuously flowing at a fixed rate, this means that the blue band widens and is more dilute. Since the detector responds in proportion to the concentration of the band, the blue peak is lower in height, but larger in width.


Conclusions and future directions

Currently, mRNA vaccines are experiencing a burst in basic and clinical research. The past 2 years alone have witnessed the publication of dozens of preclinical and clinical reports showing the efficacy of these platforms. Whereas the majority of early work in mRNA vaccines focused on cancer applications, a number of recent reports have demonstrated the potency and versatility of mRNA to protect against a wide variety of infectious pathogens, including influenza virus, Ebola virus, Zika virus, Streptococcus spp. and T. gondii (Tables 1,2).

While preclinical studies have generated great optimism about the prospects and advantages of mRNA-based vaccines, two recent clinical reports have led to more tempered expectations 22,91 . In both trials, immunogenicity was more modest in humans than was expected based on animal models, a phenomenon also observed with DNA-based vaccines 171 , and the side effects were not trivial. We caution that these trials represent only two variations of mRNA vaccine platforms, and there may be substantial differences when the expression and immunostimulatory profiles of the vaccine are changed. Further research is needed to determine how different animal species respond to mRNA vaccine components and inflammatory signals and which pathways of immune signalling are most effective in humans.

Recent advances in understanding and reducing the innate immune sensing of mRNA have aided efforts not only in active vaccination but also in several applications of passive immunization or passive immunotherapy for infectious diseases and cancer (Box 4). Direct comparisons between mRNA expression platforms should clarify which systems are most appropriate for both passive and active immunization. Given the large number of promising mRNA platforms, further head-to-head comparisons would be of utmost value to the vaccine field because this would allow investigators to focus resources on those best suited for each application.

The fast pace of progress in mRNA vaccines would not have been possible without major recent advances in the areas of innate immune sensing of RNA and in vivo delivery methods. Extensive basic research into RNA and lipid and polymer biochemistry has made it possible to translate mRNA vaccines into clinical trials and has led to an astonishing level of investment in mRNA vaccine companies (Table 4). Moderna Therapeutics, founded in 2010, has raised almost US$2 billion in capital with a plan to commercialize mRNA-based vaccines and therapies 172,173 . The US Biomedical Advanced Research and Development Authority (BARDA) has committed support for Moderna's clinical evaluation of a promising nucleoside-modified mRNA vaccine for Zika virus (NCT03014089). In Germany, CureVac AG has an expanding portfolio of therapeutic targets 174 , including both cancer and infectious diseases, and BioNTech is developing an innovative approach to personalized cancer medicine using mRNA vaccines 121 (Box 2). The translation of basic research into clinical testing is also made more expedient by the commercialization of custom GMP products by companies such as New England Biolabs and Aldevron 175 . Finally, the recent launch of the Coalition for Epidemic Preparedness Innovations (CEPI) provides great optimism for future responses to emerging viral epidemics. This multinational public and private partnership aims to raise $1 billion to develop platform-based vaccines, such as mRNA, to rapidly contain emerging outbreaks before they spread out of control.

The future of mRNA vaccines is therefore extremely bright, and the clinical data and resources provided by these companies and other institutions are likely to substantially build on and invigorate basic research into mRNA-based therapeutics.

Box 4: mRNA-based passive immunotherapy

Recombinant monoclonal antibodies are rapidly transforming the pharmaceutical market and have become one of the most successful therapeutic classes to treat autoimmune disorders, infectious diseases, osteoporosis, hypercholesterolemia and cancer 188,189,190,191,192 . However, the high cost of protein production and the need for frequent systemic administration pose a major limitation to widespread accessibility. Antibody-gene transfer technologies could potentially overcome these difficulties, as they administer nucleotide sequences encoding monoclonal antibodies to patients, enabling in vivo production of properly folded and modified protein therapeutics 193 . Multiple gene therapy vectors have been investigated (for example, viral vectors and plasmid DNA) that bear limitations such as pre-existing host immunity, acquired anti-vector immunity, high innate immunogenicity, difficulties with in vivo regulation of antibody production and toxic effects 193,194 . mRNA therapeutics combine safety with exquisite dose control and the potential for multiple administrations with no pre-existing or anti-vector immunity. Two early reports demonstrated that dendritic cells (DCs) electroporated with mRNAs encoding antibodies against immuno-inhibitory proteins secreted functional antibodies and improved immune responses in mice 195,196 . Three recent publications have described the use of injectable mRNA for in vivo production of therapeutic antibodies: Pardi and colleagues demonstrated that a single intravenous injection into mice with lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNAs encoding the heavy and light chains of the anti-HIV-1 neutralizing antibody VRC01 rapidly produced high levels of functional antibody in the serum and protected humanized mice from HIV-1 infection 197 Stadler and co-workers demonstrated that intravenous administration of low doses of TransIT (Mirus Bio LLC)-complexed, nucleoside-modified mRNAs encoding various anticancer bispecific antibodies resulted in the elimination of large tumours in mouse models 198 and Thran and colleagues 199 utilized an unmodified mRNA–LNP delivery system 12 to express three monoclonal antibodies at levels that protected from lethal challenges with rabies virus, botulinum toxin and a B cell lymphoma cell line. No toxic effects were observed in any of these studies. These observations suggest that mRNA offers a safe, simple and efficient alternative to therapeutic monoclonal antibody protein delivery, with potential application to any therapeutic protein.


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