# Structural Biochemistry/Proteins/Purification/High Pressure Liquid chromatography
## Introduction

High Pressure Liquid Chromatography (also known as High Performance Liquid Chromatography, or simply HPLC) is an enhanced form of column chromatography that is commonly used in biochemistry to separate and purify compounded samples. Instead of the solvent dripping through the column as a result of gravity as is the case in other methods of chromatography, the solvent is pushed through with high pressures.
The column materials of HPLC are much more neatly and greatly divided, and so there are more interaction opportunities and greater resolving (separating) power. Since the columns are made of materials of better quality, constant pressure must be applied to the column to obtain acceptable flow rates. Therefore, the final result is high resolution and very fast separation.

## History

HPLC was developed and improved with new column technologies in the mid-1970's, replacing the other primeval column chromatographic techniques which failed when it came to quantifying and purifying similar compounds. Pressure liquid chromatography proved to be much less time consuming than the old methods. Compared with classical column chromatography, in which the columns are powered by gravity and a separation can take hours or even up to days, HPLC was able to produce results as fast as five to thirty minutes.
HPLC was used frequently for the compound purification by the 1980's. Computers and other improved technology added to the convenience of HPLC. Improvements in the types of columns and consequently, reproducibility of HPLC, led to developments of micro-columns, affinity columns, and fast HPLC.
The past decade has seen a vast advancement in the development of micro-columns, now commonly used for HPLC, and other specialized columns. The diameter of the typical HPLC column is about 3-5 mm. But the usual diameter of micro-columns, or capillary columns, ranges from 3 µm to 200 µm, so it is considerably smaller. Fast HPLC utilizes a column that is shorter than the typical column, and so they are packed with smaller particles.
These days, one has the option of considering several types of columns for the Purification of mixtures, as well as a variety of detectors to work with the HPLC in order to get the best possible analysis of the compound.

## Theory

A small volume of the sample is put into the High-Pressure Liquid Chromatography where a mobile phase will move it through the stationary phase. The mobile phase is usually a gas or a liquid and the stationary phase is immobile and immiscible. The stationary phase will slow down the flow of the sample because of it physical or chemical properties (size, net charge, or other differences depending on the type HPLC) where it will be filtered or purified. Because of the difference in how the stationary phase affects the impurities from the desired compound, the different components of the sample will come out at different times. The time that a component comes out of the column is called the retention time. The retention time should be unique to the component in the particular sample, so that no two components being analyzed elute at the same time and obscure each other. If solvent composition cannot be tweaked to effectively separate components in HPLC analysis, then a different type of chromatography might be better suited. HPLC, unlike other column chromatography techniques, uses pressure via pumps to push components through the more finely packed columns to speed up analysis and enable analysis of component and column combinations that take longer to elute on their own.

## Mobile Phase

The mobile phase is a solvent or mixture of solvents that carries the sample through the stationary phase. As it moves through the stationary phase, molecular interactions between the sample's components and the column material determine the retention time of the different components. The components that have stronger interactions with the mobile phase than the column will "prefer" the mobile phase and elute quicker with a shorter retention time while components that have stronger interactions with the stationary phase than the solvent will "prefer" the column and elute slower with a longer retention time. This is how HPLC separates, filters, and aids in purification of the compound. There are different techniques in regards to mobile phases that are tweaked to optimize retention time, separation, and peak clarity. These are isocratic, gradient, and polytyptic.

### Isocratic

Isocratic elution involves a constant mobile phase composition. For example, a mobile phase of 50% acetonitrile and 50% water for a reversed phase HPLC (RP-HPLC) run that remains unchanged through the entire analysis. A solvent system is chosen and it will be used for the entire duration of the HPLC run. The sample is injected as the mobile phase flows through, enters the HPLC at a constant flow rate, and passes through the chosen column. This method is generally used when the sample being analyzed is simple enough that all the components of the sample come out at different times with sufficient clarity, and do not have impractically long retention times.

### Gradient

Most samples are not so easy to work with. In these cases, a gradient elution method is set up. The mobile phase mixture will shift as the run proceeds, and the concentrations of the solvents are modified so that the run begins with the "weaker" solvent, and the "stronger" of the solvents will be the most concentrated at the end. One such example is a reversed phase HPLC run that begins with more mobile phase A, which is composed of a 95% water and 5% acetonitrile mixture, and will gradually increase mobile phase B, which is a 100% acetonitrile mixture, until at the end of the run the majority of mobile phase flowing through the column is mobile phase B. Usually for reversed phase HPLC, the mobile phase will begin with the more polar solvent combination and increase the concentration of the less polar solvent combination as the run proceeds. This is so that the less polar molecules (relative to the mobile phase and stationary phase being used) will eventually elute due to a higher concentration of a less polar solvent and the necessary run time for the analysis can be shortened. An isocratic mobile phase can have a polarity too close to the stationary phase, resulting in components eluting out together immediately and their peaks overlapping, or a polarity too different from the nonpolar stationary phase, resulting in nonpolar components taking too long to elute. This is why a gradient mobile phase is often used in analysis, where concentration of less-polar to more-polar solvents can be modified to obtain optimal peak separation.

### Polytyptic

The polytptic elution, also known as mixed-mode chromatography, involves the use of a special column that can switch modes of analysis depending on the solvent. The same column can perform size exclusion, ion exchange, or affinity chromatography depending on the type of solvent that flows through it.

## Retention Time

Retention times depend on the interaction of the component of the sample, the mobile phase, and the stationary phase to each other. Therefore, a well-designed HPLC run relies on choosing the correct type of column for the analysis desired and the right combination of mobile phases for the analyte and the column.

## Column Efficiency

Column efficiency describes how well the stationary phase filters or purifies, basically how packed it is and how well things move along it. There are a couple of ways to measure column efficiency but they all use the same formula:
N=number of theoretical plates a=constant that depends on the height of a graph t$_r$=retention time W=width of a peak

## Applications of HPLC
### Normal phase chromotography

Normal phase chromatography, or NP-HPLC is the first kind of HPLC developed. In this method a polar stationary phase and a non-polar mobile phase is used in order to separate analytes based on their polarity. Since the polar phase is stationary, polar analytes will bind to that phase. Their adsorption strength and elution time depend on the strength of the analyte polarity and the analyte’s steric factors. Since the elution time depends on steric clashes, it is then possible to differentiate and separate structural isomers since each isomer has a different steric clash. One can increase the elution time by adding a non-polar solvent to the non-polar mobile phase. One can also able to decrease the retention time of the analytes by adding polar substances to the non-polar mobile phase and even occupy the stationary phase surface preventing the polar analytes from binding to the polar surface. In the past, this method is unfavorable due to the fact that water or protic organic solvents changed the hydration state of the media in the system. However, this problem was solved with another version of NP-HPLC called hydrophilic interaction liquid chromatography, which uses a variety of phases that had better retention times.

### Reversed phase chromotography

Reverse phase chromatography, as the name suggests, is the opposite of normal phase chromatography, where it now has a non-polar stationary phase and a polar mobile phase. Consequently, the non-polar analytes will bind to the non-polar phase, and its elution time will also depend on how non-polar it is. One can still also increase the elution time by adding a polar solvent to the mobile phase or decrease the elution time by adding a non-polar solvent to the same phase. However, unlike NP-HPLC, the method depends on hydrophobic interactions.
Some factors can influence hydrophobic interactions. One of those factors is surface area. An analyte with a larger hydrophobic surface area would consequently have a longer retention time since there would be more bonds interacting between the analyte and the non-polar surface. However, too large of an analyte surface won’t be able to enter the pores of the non-polar phase and have no interactions with the phase. This strengthening in bonds is also due to the force of water for “cavity-reduction” around the analyte, and the energy released in this process depends on the surface tension of the eluent, which in this case is water.
Another factor that can affect the hydrophobic interactions is the pH. An ideal environment is one that is uncharged. As a result, chemists use buffering agents, such as sodium phosphate, to regulate the pH and neutralize the charge on exposed media, which usually is composed of silica, on the stationary phase and the charge on the analyte.
Reverse phase columns are stronger than normal silica columns, but still have some weaknesses. Aqueous bases shouldn’t be used with columns consisting of alkyl derivatized silica particles since the base will destroy the underlying silica particle. Also, if an aqueous acid is used, it should be exposed too long to the column in order to prevent corrosion.

### Gel filtration

Gel-filtration chromatography separates proteins based on differing in size. The process involves a gel in a buffer solution that is packed into a column. This gel has many porous carbohydrate polymer bead-like particles. The size of the pores is selected so that it can only allow proteins with a certain size to diffuse through them. The movement of the molecules that are small enough to enter through the pores of the beads is then slowed down because it is forced to enter the stationary phase of the column. The larger molecules on the other hand, end up moving through the column faster because they cannot enter the internal volume of the beads.
The most important advantage of gel-filtration chromatography is its ability to separate the proteins in its original, non-denatured condition, giving you a sample that is in a suitable form for possible further analysis. Another advantage as well is the high resolution that is obtained by applying pressure into the column to get adequate flow. Improved resolution is achieved with slower flow rates. An optimum flow rate for protein fractionation of approximately 5mL/cm2/h is recommended for most gels.
Reference: Aguilar, Marie-Isabel. HPLC of Peptides and Proteins Methods and Protocols. volume 251. Humana Press.

### Ion exchange

Ion-exchange chromatography separates proteins based on their charge. It is efficient enough to be able to resolve proteins that differ only by one single charged group. It depends on the formation of ionic bonds between the charged groups on the proteins and an ion-exchange gel carrying the opposite charge in a column. Proteins that do not have an electrical charge and are neutral are removed by washing. Those proteins that can form ionic bonds, though, are recovered by elution with a buffer of either higher ionic strength or changing pH. An increase in oppositely charged ions (those of the protein being analyzed and those of the gel medium) increases the retention time, which is based on the attraction between the protein ions and charged ions of the gel medium.
There are two types of ion-exchangers. One is the anion exchanger, which has positively charged groups that are stationary in a gel-medium and will interact and bind to negatively charged ions in the protein. The other is the cation exchanger, which has negatively charged groups that are stationary in a gel-medium as well but interact and bind to positively charged ions in the protein.
The pH of the solution can also alter how the ionization process between the protein ions and the ions in the gel-medium. When the pH is equal to the isoelectric point of the protein (the point where the net charge is zero). However, when the pH is less than the isolectric point, the net electric charge on the protein will be positive and it will bind to the cation exchangers. Finally, if the pH is greater than the isoelectric point, the net charge on the protein will be negative and it will bind to the anion exchangers. Therefore, by controlling the pH of the solution we can control how the protein gets separated since it is these exchangers that separate the protein
Reference: Aguilar, Marie-Isabel. HPLC of Peptides and Proteins Methods and Protocols. volume 251. Humana Press.

### Affinity chromatography

Affinity chromatography is the method of the separation of biochemical mixtures, based on a highly specific biologic interaction. It is used to purify a molecule from a mixture and concentrate it into a buffering solution, and also to recognize what biological compounds bind to another molecule, like drugs. It was discovered in 1968 by Pedro Cuatrecasas and Meir Wilcheck.
The process involves the trapping of the target protein (or molecule) that one wants separated from the mixture onto a solid or a medium. A column is filled with beads that contain covalent glucose residues, which are chosen to correspond with the target protein. The proteins will travel down through the beads as they are poured into the column, and when the target protein is recognized, it will get trapped to the column by covalent bonds due to its affinity for glucose. The rest of proteins will run down the column and become successfully separated. The portion of buffer will be added to the column to wash out the unbounded protein. Lastly, a concentrated solution of glucose is added to separate the target protein from the column-attached glucose residues, resulting with the protein being completely purified out of the mixture.
Adsorption Chromatography
Adsorption, meaning the accumulation of solutes of the surface of a solid or liquid, chromatography is useful in separating a mixture of solutes based on their different polarities. It is based on the notion that polar solute will form a tighter bond with the polar stationary phase than a less polar solute will. An insoluble, polar material like silica gel (a derivative of silica gel, Si(OH)¬4) is filled into a glass column, making it the stationary phase. The sample containing the mixture is the mobile phase, which can be a liquid or gas, is poured onto the glass column, where each solute with a different polarity will bind differently to the solute. The polar solutes will bind tightly to the stationary phase, the less polar ones will bind more loosely, and the neutral ones will pass right through the column. The solute can be eluted with solvents of progressively higher polarity, where the solutes will be eluted with increasing polarity. So, neutral solutes will pass right through the column, the less polar ones will be eluted first, and very polar solutes will be eluted last.
Reference: Principles of Biochemistry 4th Edition.Nelson, David L.; Cox,Michael M.W.H Freeman and Company. New York

## Additional References

* Practical HPLC Method Development 2nd Edition. Snyder, Lloyd R.; Kirkland, Joseph Jack; Glajch, Joseph L. New York.
* Handbook Of Pharmaceutical Analysis By HPLC. M. W. Dong. Elsevier.