Alsharifi

 High-performance liquid chromatography (HPLC), 

formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.

HPLC has been used for manufacturing (e.g., during the production process of pharmaceutical and biological products), legal (e.g., detecting performance enhancement drugs in urine), research (e.g., separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and medical (e.g., detecting vitamin D levels in blood serum) purposes.^[1]

Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made of solid particles (e.g., silica, polymers, etc.), 2–50 μm in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents (e.g., water, acetonitrile and/or methanol) and is referred to as a "mobile phase". Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination.

HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller adsorbent particles (2–50 μm in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.

The schematic of an HPLC instrument typically includes a degasser, sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in an HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature at which the separation is performed.

Operation

The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are a function of specific physical interactions with the adsorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time measured under particular conditions is an identifying characteristic of a given analyte.

Many different types of columns are available, filled with adsorbents varying in particle size, and in the nature of their surface ("surface chemistry"). The use of smaller particle size packing materials requires the use of higher operational pressure ("backpressure") and typically improves chromatographic resolution (the degree of peak separation between consecutive analytes emerging from the column). Sorbent particles may be hydrophobic or polar in nature.

Common mobile phases used include any miscible combination of water with various organic solvents (the most common are acetonitrile and methanol). Some HPLC techniques use water-free mobile phases (see normal-phase chromatography below). The aqueous component of the mobile phase may contain acids (such as formic, phosphoric or trifluoroacetic acid) or salts to assist in the separation of the sample components. The composition of the mobile phase may be kept constant ("isocratic elution mode") or varied ("gradient elution mode") during the chromatographic analysis. Isocratic elution is typically effective in the separation of sample components that are very different in their affinity for the stationary phase. In gradient elution the composition of the mobile phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times with high eluting strength producing fast elution (=short retention times). A typical gradient profile in reversed phase chromatography might start at 5% acetonitrile (in water or aqueous buffer) and progress linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition may be part of any gradient profile. For example, the mobile phase composition may be kept constant at 5% acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.

The chosen composition of the mobile phase (also called eluent) depends on the intensity of interactions between various sample components ("analytes") and stationary phase (e.g., hydrophobic interactions in reversed-phase HPLC). Depending on their affinity for the stationary and mobile phases analytes partition between the two during the separation process taking place in the column. This partitioning process is similar to that which occurs during a liquid–liquid extraction but is continuous, not step-wise. In this example, using a water/acetonitrile gradient, more hydrophobic components will elute (come off the column) late, once the mobile phase gets more concentrated in acetonitrile (i.e., in a mobile phase of higher eluting strength).

The choice of mobile phase components, additives (such as salts or acids) and gradient conditions depends on the nature of the column and sample components. Often a series of trial runs is performed with the sample in order to find the HPLC method which gives adequate separation.

The device consists of 8 main components:

1. Mobile phase reservoir: It is a flask, or just a commercial container, provided that it is clean, clean, emptied of air and gases; So as not to cause an error in the analysis. It should also be cleaned of impurities when preparing the moving medium; To prevent device failure and error in analyzes.

2. Solvent delivery system: It is a pump to ensure the free flow of the moving medium in a continuous, accurate and constant pulse. There are two types used in HPLC: a screw-driven syringe type syringe pump, which although excellent for easy flow rate control, is not suitable for solvent change. The Reciprocating piston double reciprocating pump consists of a cylindrical chamber that is filled and emptied by the forward and backward movement of the pistons. Advantages of this technology include its small internal volume ranging from 35 to 40 microliters, with a large external pressure of 10,000 pounds per square inch (psi). . An important use of the device is gradient elution with constant flow rates where it is not significantly affected by any of the Column back-pressure and no solvent viscosity.

 

3. Sample introduction system: It can be automatic or manual and uses valves. When opened, a loop sample can be filled with a volume of 10 to 50 μl. When the valves are closed, the sample goes into the stream of the high pressure moving medium, where it is sent to the column where it is analyzed. The sample must be in a liquid state and dissolved in a solution if it is in a solid state, where the solvent is in harmony with the moving and stationary medium, and the sample is injected with an amount ranging from 1 to 100 μl.

4. Column: is the heart of the device, where the separation process takes place. It is usually made of stainless steel and anti-corrosion and is divided into two types: 1- Analytical columns: They are the basic type and are found in all devices and their length ranges from 5 to 25 cm and an inner diameter of 3 mm to 5 mm is filled with a fixed medium material which is 5 particles Μm .. and in the 1980s, the separation speed improved due to a reduction in diameter and increased length. 2- precolumns, divided into two types. The first, the scavenger column, is located between the sample injection area and the moving media vessel and improves the quality of the moving medium, and the second, the guard column is located between the analytical column and the sample injection area and removes impurities from the solvent. The column filler used in this device is also divided into two types: coated bundles (pellicular) which are nonporous spherical bead-shaped polymers 30 to 40 mm in diameter coated with a thin, porous layer of silica, alumina, or ion-exchange resin (Ion-exchange resin). ), Which is now used to separate proteins and large biomolecules, and the other type of filler is porous filler, and usually contains small particles with a diameter ranging between 3 to 10 mm and consists of silica, alumina, or ion-exchange resin and silica is the most common material used in filling the column and sometimes surrounding With a thin organic layer that binds to the inner surface of the column (chemically or physically).

5. Detector: its function is to monitor the dissolved materials to be extracted when they come out of the column; It emits electrical signals that are proportional to the level of a specific characteristic of the material in the moving medium or of the extract. There are many types:

U.V. absorbance detectors

Fluoresce detectors

Electrochemical detectors

Conductivity detectors

Refractive index detectors

Mass spectrometer

6. Bonding tubes: They are made of an inert material that does not interact with the moving medium material and solvents, and they are usually made of stainless steel or inert plastic.

7. A computer or recorder device: used as a data gathering device; Where it is connected to the detector to pick up the electronic signals coming from it, then analyze it and output it in the form of graphs called a chromatogram.

8. The litter tray.

Applications

Manufacturing

HPLC has many applications in both laboratory and clinical science. It is a common technique used in pharmaceutical development, as it is a dependable way to obtain and ensure product purity.^[20] While HPLC can produce extremely high quality (pure) products, it is not always the primary method used in the production of bulk drug materials.^[21] According to the European pharmacopoeia, HPLC is used in only 15.5% of syntheses.^[22] However, it plays a role in 44% of syntheses in the United States pharmacopoeia.^[23] This could possibly be due to differences in monetary and time constraints, as HPLC on a large scale can be an expensive technique. An increase in specificity, precision, and accuracy that occurs with HPLC unfortunately corresponds to an increase in cost.

Legal

This technique is also used for detection of illicit drugs in urine. The most common method of drug detection is an immunoassay.^[24] This method is much more convenient. However, convenience comes at the cost of specificity and coverage of a wide range of drugs. As HPLC is a method of determining (and possibly increasing) purity, using HPLC alone in evaluating concentrations of drugs is somewhat insufficient. With this, HPLC in this context is often performed in conjunction with mass spectrometry.^[25] Using liquid chromatography instead of gas chromatography in conjunction with MS circumvents the necessity for derivitizing with acetylating or alkylation agents, which can be a burdensome extra step.^[26] This technique has been used to detect a variety of agents like doping agents, drug metabolites, glucuronide conjugates, amphetamines, opioids, cocaine, BZDs, ketamine, LSD, cannabis, and pesticides.^[27][28] Performing HPLC in conjunction with Mass spectrometry reduces the absolute need for standardizing HPLC experimental runs.

Research

Similar assays can be performed for research purposes, detecting concentrations of potential clinical candidates like anti-fungal and asthma drugs.^[29] This technique is obviously useful in observing multiple species in collected samples, as well, but requires the use of standard solutions when information about species identity is sought out. It is used as a method to confirm results of synthesis reactions, as purity is essential in this type of research. However, mass spectrometry is still the more reliable way to identify species.

Medical

Medical use of HPLC can include drug analysis, but falls more closely under the category of nutrient analysis. While urine is the most common medium for analyzing drug concentrations, blood serum is the sample collected for most medical analyses with HPLC.^[30] Other methods of detection of molecules that are useful for clinical studies have been tested against HPLC, namely immunoassays. In one example of this, competitive protein binding assays (CPBA) and HPLC were compared for sensitivity in detection of vitamin D. Useful for diagnosing vitamin D deficiencies in children, it was found that sensitivity and specificity of this CPBA reached only 40% and 60%, respectively, of the capacity of HPLC.^[31] While an expensive tool, the accuracy of HPLC is nearly unparalleled.

References

1. * Gerber, Frederic (May 2004). "Practical aspects of fast reversed-phase high-performance liquid chromatography using 3 μm particle packed columns and monolithic columns in pharmaceutical development and production working under current good manufacturing practice". Journal of Chromatography. 1036 (2): 127
2. ^
3. * Kolmonen, Marjo; Leinonen, Antti; Pelander, Anna; Ojanperä, Ilkka (2007-02-28). "A general screening method for doping agents in human urine by solid phase extraction and liquid chromatography/time-of-flight mass spectrometry". Analytica Chimica Acta. 585 (1): 94–102.Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam (2015-01-01)

*Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam (2015-01-01)

* Nobilis, Milan; Pour, Milan; Senel, Petr; Pavlík, Jan; Kunes, Jirí; Voprsalová, Marie; Kolárová, Lenka; Holcapek, Michal (2007-06-15). "Metabolic profiling of a potential antifungal drug, 3-(4-bromophenyl)-5-acetoxymethyl-2,5-dihydrofuran-2-one, in mouse urine using high-performance liquid chromatography with UV photodiode-array and mass spectrometric detection". Journal of Chromatography B. 853 (1–2): 10–19. 

*Martin, A J P; Synge, R L M (1941)

*Gerber, F.; Krummen, M.; Potgeter, H.; Roth, A.; Siffrin, C.; Spoendlin, C. (2004). "Practical aspects of fast reversed-phase high-performance liquid chromatography using 3μm particle packed columns and monolithic columns in pharmaceutical development and production working under current good manufacturing practice". Journal of Chromatography A. 1036 (2): 

*

1.  Iler, R.K. (1979) The Chemistry of Silica. John Wiley & Sons. New York.
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