High Performance Liquid Chromatography (HPLC)

The high-performance liquid chromatography ( HPLC ) or high performance liquid chromatography ( HPLC ) is a type of column chromatography used frequently in biochemistry and analytical chemistry . Also it is sometimes called liquid chromatography high pressure or high performance liquid chromatography resolution ( high pressure liquid chromatography ) ( HPLC), although this terminology is considered old and is not in use. HPLC is a technique used to separate the components of a mixture based on different types of chemical interactions between the analyzed substances and the chromatographic column. [one]

Summary

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  • 1 Principle
    • 1 High Performance Liquid Chromatography (HPLC)
  • 2 Types of chromatography
    • 1 Normal phase chromatography
    • 2 Reverse phase chromatography (or reverse)
    • 3 Molecular Exclusion Chromatography
    • 4 Ion exchange chromatography
    • 5 Chromatography based on bioaffinity
    • 6 High performance liquid chromatography under denaturing conditions (DHPLC)
  • 3 Parameters
    • 1 Internal diameter
    • 2 Measurement of particles
    • 3 Pore ​​size
    • 4 System pressure
  • 4 HPLC apparatus manufacturers
  • 5 Manufacturers of HPLC columns and accessories
  • 6 See also
  • 7 References
  • 8 External links

Beginning

High Performance Liquid Chromatography (HPLC)

HLPC 580 from GIBNIK.

In isocratic HPLC the compound passes through the chromatographic column through the stationary phase (usually a cylinder with small rounded particles with certain chemical characteristics on its surface) by pumping liquid ( mobile phase ) at high pressurethrough the column. The sample to be analyzed is introduced in small quantities and its components are differentially delayed depending on the chemical or physical interactions with the stationary phase as they advance through the column. The degree of retention of the components of the sample depends on the nature of the compound, the composition of the stationary phase and the mobile phase. The time it takes for a compound to elute from the column is called the retention time.and it is considered a characteristic identifying property of a compound in a certain mobile and stationary phase. The use of pressure in this type of chromatography increases the linear speed of the compounds within the column and thus reduces their diffusion within the column, improving the resolution of the chromatography. The most widely used solvents are water , methanol and acetonitrile . The water may contain buffers, salts, or compounds such as trifluoroacetic acid , which aid in the separation of the compounds.

An improvement introduced in the described HPLC technique was the variation in the composition of the mobile phase during the analysis, known as gradient elution . A normal gradient on reverse phase chromatography can start at 5% acetonitrileand progress linearly up to 50% in 25 minutes. The gradient used varies depending on the hydrophobicity of the compound. The gradient separates the components of the sample as a function of the affinity of the compound for the mobile phase used with respect to the affinity for the stationary phase. In the example, using a water / acetonitrile gradient, the more hydrophilic compounds will elute at a higher concentration of water, while the more hydrophobic compounds will elute at high concentrations of acetonitrile. Often a series of pretests is required to optimize the gradient so as to allow good separation of the compounds.

Types of chromatography

Normal phase chromatography

HPLC manufacturers.

Normal phase chromatography or normal phase HPLC (NP-HPLC) was the first type of HPLC system used in the field of chemistry, and is characterized by separating compounds on the basis of their polarity. This technique uses a polar stationary phase and an apolar mobile phase, and is used when the compound of interest is quite polar. The polar compound associates and is retained by the stationary phase. The adsorption force increases as the polarity of the compound increases and the interaction between the polar compound and the polar stationary phase (compared to the mobile phase) increases the retention time.

The strength of interaction depends not only on the functional groups of the compound of interest, but also on steric factors such that structural isomers can often be distinguished from each other. The use of more polar solvents in the mobile phase decreases the retention time of the compounds, while the more hydrophobic solvents tend to increase the retention time.

NP-HPLC fell into disuse in the 1970s with the development of reversed-phase or reversed-phase HPLC due to the lack of reproducibility of retention times since protic solvents changed the hydration state of silica or alumina of chromatography. [2]

Reverse phase chromatography (or reverse)

Reverse phase HPLC (RP-HPLC) consists of a stationary apolar phase and a mobile phase of moderate polarity. One of the most common stationary phases of this type of chromatography is RMe 2 SiCl treated silica , where R is an alkyl chain such as C 18 H 37 (octadecyl) or C 8 H 17 (octyl). The retention time is longer for molecules of a nonpolar nature, while molecules of a polar nature elute more quickly.

The retention time increases with the addition of polar solvent to the mobile phase and decreases with the introduction of more hydrophobic solvents. Reverse phase chromatography is so widely used that it is often called HPLC without any further specification. Reverse phase chromatography is based on the principle of hydrophobic interactions resulting from the repulsive forces between a relatively polar solvent, a relatively apolar compound, and an apolar stationary phase. The driving force in binding the compound to the stationary phase is the decrease in the area of ​​the apolar segment of the analyte exposed to the solvent. This hydrophobic effect is dominated by the increase in entropy , and the consequent decrease in free energy., associated with the minimization of the polar compound-solvent interface. The hydrophobic effect decreases with the addition of apolar solvent to the mobile phase. This modifies the partition coefficient so that the compound moves through the column and elutes.

The characteristics of the compound of interest play a very important role in retention. In general, a compound with a long alkyl chain is associated with a longer retention time because it increases the hydrophobicity of the molecule. Still, very large molecules may see reduced interaction between the compound’s surface and the stationary phase. The retention time increases with the hydrophobic surface area, which is usually inversely proportional to the size of the compound. Branched compounds tend to elute faster than their linear isomers since the total surface area is reduced.

Apart from the hydrophobicity of the immobile phase, other modifications of the mobile phase can affect the retention of the compound; for example, the addition of inorganic salts causes a linear increase in surface tension, and since the entropy of the compound-solvent interface is precisely controlled by surface tension, the addition of salts tends to increase retention time.

Another important variable is pH since it can change the hydrophobicity of the compound. For this reason, most methods use a buffer such as sodium phosphate > [3] to control the pH value. These buffers control the pH, but also neutralize the charge or any residue of silica from the stationary phase that has been exposed and act as counter ions that neutralize the charge of the compound. The effect of buffers on chromatography can vary, but generally they improve chromatographic separation.

Reverse phase columns spoil less easily than normal silica columns. Even so, many reversed phase columns are made of modified silica with alkyl chains and should never be used with bases in aqueous medium since these could damage the underlying silica skeleton. The columns can be used on acids in aqueous media but should not be exposed to the acid too long because it can corrode the metal parts of the HPLC apparatus.

Molecular exclusion chromatography

The size exclusion chromatography , also known as gel filtration chromatography, separates particles from the sample according to their size. Generally it is a low resolution chromatography so it is usually used in the final steps of the purification process. It is also very useful for determining the tertiary and quaternary structures of purified proteins.

Molecular filtration chromatography is a method of column chromatography whereby molecules are separated in solution according to their molecular weight, or more precisely, according to their Stokes radius .

In this chromatography, the stationary phase consists of long crosslinked polymers that form a porous three-dimensional network. For practical purposes, the columns are packed with small spheroidal particles formed by these crosslinked polymers. Consequently, these particles are porous, and the size of the pores is such that some molecules (too large) will not be able to enter those pores, while others (small enough) will be able to pass freely. The pores are connected forming a mesh or network, which determines a series of paths to be traveled by the molecules that access the interior of it.

Ion exchange chromatography

Main article: Ion exchange chromatography .

In ion exchange chromatography, retention is based on the electrostatic attraction between ions in solution and charges immobilized to the stationary phase. Ions of the same charge are excluded while those of the opposite charge are retained by the column. Some types of ion exchangers are: i) Polystyrene resins, ii) cellulose and dextran ion exchangers (gels) and iii) Porous silica or controlled pore size glass. In general, ion exchangers favor the union of high charge and small radius ions. An increase in the concentration of the counterion (with respect to the functional groups of the resin) reduces the retention time. An increase in pH reduces retention time on cation exchange chromatographs while a decrease in pH reduces retention time on anion exchange chromatography. This type of chromatography is widely used in the following applications: water purification, concentration of trace components,Ligand-exchange chromatography , Ion-exchange chromatography of proteins , High-pH anion-exchange chromatography of carbohydrates and oligosaccharides , etc.

Bioaffinity based chromatography

This type of chromatography is based on the ability of biologically active substances to form stable, specific and reversible complexes. The formation of these complexes involves the participation of molecular forces such as interactions of Van der Waals , electrostatic interactions , dipole-dipole interactions , hydrophobic interactions and hydrogen bonds between the particles of the sample and the stationary phase.

High performance liquid chromatography under denaturing conditions (DHPLC)

It is a method that is used for the screening of mutations (already almost totally obsolete due to the boom in sequencing), which allows detecting the presence of variations in DNA, although they are not specifically determined which ones. In this case, the chromatographic technique is used for the detection of DNA heteroduplex, instead of using a gel to run the nucleic acid molecules.

The procedure consists of denaturing (increasing the temperature normally) a sample that contains both the problem DNA and a control DNA, which is only the same problem DNA but in its wild or normal version (without mutations). Then it proceeds to renaturate (lowering the temperature), so that those DNA strands that rejoin their respective complements (wild “a” chain with wild “b” chain; or “a” mutant chain “b “mutant) will not show difference with the original state; however, if for example a hybrid wild “a” chain with a mutant “b” chain, that region (more or less broad) in which there is a mutation will not complement, forming a loop or hairpin, that is, a region in which the nitrogenous bases are not complementary and do not establish the characteristic bonds by hydrogen bridges in the DNA double helix. These structures are called heteroduplexes (hybrid DNA duplexes). These heteroduplexes migrate differently than homoduplexes on the reverse phase chromatography column (just as they do on an agarose or acrylamide gel). The separation is carried out under variable denaturing conditions, the DNA molecules being detected by measuring the absorbance at 260 nm. This causes a peak of elution at a characteristic time. As denaturing conditions increase, heteroduplexes migrate ahead of homoduplexes, with peaks corresponding to heteroduplex appearing earlier in the resulting graph, the chromatogram. In this way,

Prior to the initial denaturation process, a PCR is usually carried out to amplify the template DNA into fragments of about 150-450 bp.

With this system, base substitutions, insertions or deletions can be easily detected, with a low cost and quickly (approximately 16 minutes). [4]

Parameters

Internal diameter

The inside diameter of an HPLC column is a critical aspect that determines the amount of sample that can be loaded onto the column and also influences its sensitivity. Columns with a larger internal diameter (> 10mm) are normally used in compound purification for later use. In contrast, columns with a smaller internal diameter (4-5 mm) are used in the quantitative analysis of the samples, and are characterized by the increased sensitivity and the minimization of the consumption of solvents that they entail. These columns are usually called analytical range columns and are normally associated with a UV-VIS detector . Besides, there are other types of columns, such as capillary type, with a diameter of less than 0.3 mm, used mainly in mass spectrometry.. [5]

Particle measurement

Most traditional HPLCs are performed with a stationary phase attached to the outside of spherical silica particles. These particles can have different measures, being the ones of 5 µm in diameter the most used. Smaller particles offer a larger surface area and better separation, but the pressure required to obtain an optimal linear velocity increases inversely proportional to the cube of the particle diameter. This means that decreasing the particle size by half would increase the resolution of the column, but at the same time increase the necessary pressure by a factor of eight.

Pore ​​size

Many stationary phases are porous to provide a greater surface area. Small pores provide a larger surface area while larger pores provide better kinetics, especially for larger size compounds; for example, a protein that is slightly smaller than the pore size can get in, but it will hardly come out easily.

System pressure

Pump pressure varies by model and manufacturer, but their performance is measured in their ability to generate a consistent and reproducible flow. The pressure can achieve values ​​of up to 40 MPa (or about 400 atmospheres). The most modern HPLC devices incorporate improvements to be able to work at higher pressures and, therefore, to be able to use smaller particles in the columns (<2 micrometers). These new devices, called ultra performance liquid chromatography (UPLC) can work with values ​​of up to 100 MPa of pressure (about 1000 atmospheres). (Note that UPLC is a registered trademark of the Waters Corporation, although it is sometimes used generally to designate this type of device.)

 

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