Date: July 29, 2014
High performance liquid chromatography (HPLC) is aslo known as liquid chromatography. HPLC uses a
liquid moblie phase.There are three basic types of liquid chromatographic columns:
1.liquid-liquid chromatographic columns,
2. liquid-solid chromatographic columns and
3. ion-exchange chromatographic columns
Liquid-liquid chromatographic columns have the liquid stationary phase bonded or
absorbed to the surface of the column, or packed material. liquid-liquid chromatographic columns are not as popular because they have limited stability and they are inconvenient. Partitioning occurs between the two different liquids of the mobile and stationary phases. In liquid-solid chromatographic columns the stationary phase is a solid and the analyte absorbs onto the stationary phase which separates the components which separates the components exchange chromatographic columns the stationary phase is an ion-exchange resin and
partitioning occurs with ion exchanges that occur between the analyte and stationary phase.
Usually HPLC has a guard column ahead of the analytical column to protect and extend the life of the analytical column. The guard column removes particulate matter, contaminants, and molecules that bind irreversibly to the column. The guard column has a stationary phase similar to the analytical column.
Column Dimensions & Packing material
The most common HPLC columns are made from stainless steel, but they can be also made out of thick glass, polymers such as polyetherethelketone, a combination of stainless steel and glass, or a combination of stainless steel and polymers. Typical HPLC analytical columns are between 3 and 25 cm long and have a diameter of 1 to 5 mm. The columns are usually straight unlike GC columns. Particles that pack the columns have a typical diameter between 3 to 5 µm. Liquid chromatographic columns will increase in efficiency when the diameter of the packed particles inside the column decreases.
«HPLC columns are usually packed with pellicular, or porous particles. Pellicular
particles are made from polymer, or glass beads. Pellicular particles are surrounded by a thin uniform layer of silica, polystyrene-divinyl-benzene synthetic resin, alumina, or other type of ion-exchange resin. The diameter of the pellicular beads is between 30 and 40 µm. Porous particles are more commonly used and have diameters between 3 to 10 µm. Porous particles are made up silica, polystyrene-divinyl-benzene synthetic resin, alumina, or other type of ion-exchange resin. Silica is the most common type of porous particle packing material.
«Partition chromatography uses liquid bonded phase columns, where the liquid stationary phase is chemically bonded to the packing material. The packing material is usually hydrolyzed silica which reacts with the bond-phase coating. Common bond phase coatings are siloxanes. The relative structure of the siloxane is shown in Figure 1.
|R group attached to siloxane||Chromatography method application|
|Cyano||Normal and reverse phase|
|Amino||Normal and reverse phase|
|Dimethylamine||Weak anion exchanger|
|Quaternary Amine||Strong anion exchanger|
|Sulfonic Acid||Strong anion exchanger|
|Carboxylic Acid||Weak cation exchanger|
|Raheja Constructions , Building no.11||Malad-Mumbai|
The above table shows the R groups that can be attached to the siloxane and what chromatographic method it is commonly applied to
Reverse and Normal Phase HPLC Columns
A polar stationary phase and a non-polar mobile phase are used for normal phase HPLC. In normal phase, the most common R groups attached to the siloxane are: diol, amino, cyano, inorganic oxides, and dimethylamino. Normal phase is also a form of liquid-solid chromatography. The most non-polar compounds will elute first when doing normal phase HPLC. Reverse phase HPLC uses a polar mobile phase and a non-polar stationary phase. Reverse phase HPLC is the most common liquid chromatography method used. The R groups usually attached to the siloxane for reverse phase HPLC are: C8, C18,or any hydrocarbon. Reverse phase can also use water as the mobile phase, which is advantageous because water is cheap, nontoxic, and invisible in the UV region. The most polar compounds will elute first when performing reverse phase HPLC. Check the animation on the principle of reversed-phase chromatography to understand its principle.
Ion Exchange Chromatographic Columns
Ion exchange columns are used to separate ions and molecules that can be easily ionized. Separation of the ions depends on the ion’s affinity for the stationary phase, which creates an ion exchange system. The electrostatic interactions between the analytes, moble phase, and the stationary phase, contribute to the separation of ions in the sample. Only positively or negatively charged complexes can interact with their respective cation or anion exchangers. Common packing materials for ion exchange columns are amines, sulfonic acid, diatomaceous earth, styrene-divinylbenzene, and cross-linked polystyrene resins. Some of the first ion
exchangers used were inorganic and made from aluminosilicates (zeolites). Although aluminosilicates are not widely used as ion exchange resins used.R group attached to siloxane Chromatography method application Alkyl Reverse phaseFluoroalkyl Reverse phaseCyano Normal and reverse phase Amide Reverse phase Amino Normal and reverse phase Dimethylamine Weak anion exchanger
Quaternary Amine Strong anion exchanger Sulfonic Acid Strong anion exchanger
Carboxylic Acid Weak cation exchanger Diol Reverse phase Phenyl Reverse phase
Carbamate Reverse Phase Figure 1: Basic structure of a siloxane. The R groups can be varied depending on the type of column and analyte being analyzed.
Size Exclusion Chromatographic Columns
Size Exclusion Chromatographic columns separates molecules based upon their size, not molecular weight. Acommon packing material for these columns is molecular sieves. Zeolites are a common molecular sieve that is used. The molecular sieves have pores that small molecules can go into, but large molecules cannot. This allows the larger molecules to pass through the column faster than the smaller ones. Other packing materials for size exclusion chromatographic columns are polysaccharides and other polymers, and silica. The pore size for size exclusion separations varies between 4 and 200 nm.
Chiral columns are used to separate enantiomers. Separation of chiral molecules is based upon stereochemistry. These columns have a stationary phase that selectively interacts with one enantiomer over the other. These types of columns are very useful for separating racemic mixtures.
Peak or band broadening causes the column to be less efficient. The ideal situation would to have sharp peaks that are resolved. The longer a substance stays in the column it will cause the peaks to widen. Lengthening the column is a way to improve the separation of different species in the column. A column usually needs to remain at a constant temperature to remain efficient. Plate height and number of theoretical plates determines the efficiency of the column. Improving the efficiency would be to increase the number of plates and decrease the plate height.
The number of plates can be determined from the equation:
where L is the length of the column and H is the height of
each plate. N can also be determined from the equation:
N=16(tR/W)2 or N=5.54(tR/W1/2)2
where tR is the retention time, W is the width of the peak
and W1/2 is half the width of the peak.
Height equivalent to a theoretical plate (HETP) is
determined from the equation:
or HETP can also be determined by the equation:
where H equals HETP, A is the term for eddy diffusion, B is the term for longitudinal diffusion, C is the coefficient for mass-transfer between the stationary and mobile phases, and u is the linear velocity. The equation for HETP is often
used to describe the efficiency of the column. An efficient column would have a minimum HETP value.
Gas chromatographic columns have plate heights that are at least one order of magnitude greater than liquid chromatographic column plates. However GC columns are longer, which causes them to be more efficient. LC columns have a maximum length of 25 cm
whereas GC columns can be 100 meters long
|Stationary Phase||Method(s) Used|
|Metal Chelates||GC, LC|
|Amino Acid Derivatives||GC, LC|
|Amino||Normal and reverse phase|
In its earliest form, liquid chromatography was used to separate the pigments of chlorophyll by a Russian botanist. Decades later, other chemists used the procedure for the study of carotins. Liquid chromatography was then used for
the isolation of small molecules and organic compounds like amino acids, and most recently has been used in peptide and DNA research. Monolith columns have been instrumental in advancing the field of biomolecular research. In recent trade shows and international meetings for HPLC, interest in column monoliths and biomolecular applications has grown steadily, and this correlation is no coincidence. Monoliths have been shown to possess great potential in the “omics” fields- genomics, proteomics, metabolomics, and pharmacogenomics, among others.
The reductionist approach to understanding the chemical pathways of the body and reactions to different stimuli, like drugs, are essential to new waves of healthcare like personalized medicine. Pharmacogenomics studies how responses to pharmaceutical products differ in efficacy and toxicity based on variations in the patient’s genome; it is a correlation of drug response to gene expression in a patient. Jeremy Nicholson of the Imperial College, London, used a postgenomic viewpoint to understand adverse drug reactions and the molecular basis of human disesase. His group studied gut microbial metabolic profiles and were able to see distinct differences in reactions to drug toxicity and metabolism even among various geographical distributions of the same race. Affinity monolith chromatography provides another approach to drug response measurements. David Hage at the University of Nebraska binds ligands to monolithic supports and measures the equilibrium phenomena of binding interactions between drugs and serum proteins.A monolith-based approach at the University of Bologna, Italy, is currently in use for high-speed screening of drug candidates in the treatment of Alzheimer’s. In 2003, Regnier and Liu of Purdue University described a multi-dimensional LC procedure for identifying single nucleotide polymorphisms (SNPs) in proteins. SNPs are alterations in the genetic codethat can sometimes cause changes in protein conformation, as is the case with sickle cell anemia. Monoliths are particularly useful in these kinds of separations because of their superior mass transport capabilities, low backpressures coupled with faster flow rates, and relative ease of modification of the support surface.
Bioseparations on a production scale are enhanced by monolith column technologies as well. The fast separations and high resolving power of monoliths for large molecules means that real-time analysis on production fermentors is possible.
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