Chromatography is an analytical technique that separates components in a mixture. Chromatographic columns are part of the instrumentation that is used in chromatography. Five chromatographic methods that use columns are gas chromatography (GC), liquid chromatography (LC), Ion exchange chromatography (IEC), size exclusion chromatography (SEC), and chiral chromatography. The basic principals of chromatography can be applied to all five methods.
In gas chromatography the mobile phase is a gas. Gas chromatographic columns are usually between 1 and 100 meters long.
Gas liquid chromatography(GLC): The liquid stationary phase is bonded or adsorbed onto the surface of an open tubular (capillary) column, or onto a packed solid support inside the column.
Gas solid chromatography (GSC): The stationary phase is a solid and the analyte absorbs onto the solid. GSC uses a packed or open capillary column. GSC is used for analytes that do not absorb onto a liquid stationary phase. GSC is a popular method for analyzing carbon disulfide, hydrogen sulfide, carbon monoxide, carbon dioxide, nitrogen oxide, volatile hydrocarbons, halo-carbons, solvents, and other gases found in the air. The solid stationary phase is usually made of graphite carbon blocks, alumina, silica, molecular sieves, or porous polymers beads. Molecular sieves are composed of aluminum and silicate ion exchangers. Porous polymer beads are made up of styrene cross-linked by divinylbenzene.
When the stationary phase is uniformly distributed on the interior surface of column it is called an open tubular (capillary) column. Open tubular columns are longer, smaller in diameter, and more efficient than packed columns. Open tubular columns have less flow resistance which allows for them to be longer and have a lot of theoretical plates. Capillary columns are between 3 and 100 meters long and form a helical shape. The most common stationary phases used for open tubular columns are polysiloxanes. Polysiloxanes are silicon atoms which have attached oxygen and R groups. The R groups can vary, which makes polysiloxanes very versatile (see Figure 2). There are three types of open tubular columns: wall-coated (WCOT), support-coated (SCOT), and porous-layer (PLOT). WCOT is the most popular type of open tubular column.
The wall coated open tubular column consists of a capillary tube with its interior surface coated in a tiny layer of stationary phase. The most common type of wall coated open tubular column used is fused-silica, because it is stronger, inert, reliable, easy to use, and flexible. Fused silica capillary tubes are made from purified silica that has a small quantity of metal oxides dispersed throughout the silica. The fused silica column also has a layer of polyimide on the outside of the column, which makes the column flexible and extends the life of the column. Wall-coated open tubular columns can also be made out of plastic, glass, stainless steel, aluminum, or copper.
Figure 1: Diagram of a fused-silica open tubular column. This figure was created with Microsoft Paint.
A support-coated open tubular column has a thin layer (approximately 30 µm) of liquid support matter. This type of open tubular column has a greater amount of stationary phase than the wall coated column, so it can handle a larger quantity of sample.
A porous-layer open tubular (PLOT) column is very similar to a support-coated open tubular column. The only difference between the two types of columns is that a PLOT does not have a liquid stationary phase. PLOT columns are used for gas solid chromatography. PLOT columns have a solid layer of carbon, molecular sieves, cyclodextrins, inorganic oxides, or porous polymers, coating the inner wall of the column. PLOT columns can be up to 100 meters long. The inner diameter of a PLOT column is between 0.25 and 0.53 mm. The stationary phase coating is between 5 and 50 micrometers thick.
A packed column is dense and evenly packed by solid support. The solid support usually has a liquid stationary phase bonded to it. The solid support allows for the liquid stationary phase to be exposed to the maximum amount of the mobile phase. The solid support and stationary phase must be inert at high temperatures and allow for the mobile phase to be evenly distributed as it moves through the column. The packed columns are shorter in length and wider in diameter than the open tubular columns. The diameter of a packed column is usually between 2 and 4 mm. Packed columns are typically 1 to 5 meters long and also form a helical shape.The packing particles typically have a diameter of 100 to 250 micrometers. Micro-packed columns are packed capillary tubes and are packed with the same material as larger packed columns. The most common stationary phase used for packed columns is diatomaceous earth (diatomite). Diatomite is made up of diatom (single-celled algae) skeletons. The skeletons are composed of mostly silica, and small quantities of alumina and metallic oxides. Other popular stationary phases are pure silica (SiO2) and alumina (Al2O3). Alumina is great for separating aromatic hydrocarbons.
The liquid stationary phase must be inert, thermally stable, and not volatile at high temperatures. The right type of stationary phase is necessary for separation of molecules. Remember “like dissolves like”, because the analyte must be somewhat soluble into stationary phase. In other words, the polarity of the analyte must be equivalent or closely resemble the polarity of the stationary phase.
|Column Stationary Phase||Polarity|
|Diatomaceous Earth (SiO2, Al2O3, Fe2O3, CaO, MgO, Na2O, and K2O)||relatively non-polar|
|Squalane (C30H62) (purified with charcoal and alumina)||non-polar|
|Apiexon Greases (purified with charcoal and alumina)||non-polar|
|Dialkyl Phthalates||moderately polar|
|Polyethylene Glycol (Carbowax 20M)||moderately polar|
|Polysiloxanes||non-polar-polar (depending on the R group attached)|
Table 1: List of common stationary phases and their polarities. The polarity of the stationary phase should resemble the polarity of the analyte.
|Stationary Phase Functional Group || |
|Polyester Phases||Highly Polar|
Table 2: List of functional groups attached to the stationary phase and their polarities. These polarities should resemble the polarities of the analyte and can be compared to the analyte functional groups in Table 3.
|Analyte Functional Groups Ranked From Most Polar to Non-polar|
Table 3: This table shows the order of relative polarity of different functional groups. The most polar compounds are at the top of the table and the least polar compounds are at the bottom. The polarity of the analyte should resemble the polarity of the stationary phase in Table 2.
Matching the polarities of the analyte and stationary phase is not an exact science. The two should have similar polarities. The thickness of the stationary phase ranges between 0.1 and 8 µm. The thicker the layer the more volatile the analyte can be.
High performance liquid chromatography (HPLC) is aslo known as liquid chromatography. HPLC uses a liquid moblie phase. The same basic principals from gas chromatography are applied to liquid chromatography. There are three basic types of liquid chromatographic columns: liquid-liquid, liquid-solid, and ion-exchange. 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 of the mixture. In ion-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.
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 HPLC 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 2.
|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 cation exchanger|
|Carboxylic Acid||Weak cation exchanger|
Table 4: This table shows the R groups that can be attached to the siloxane and what chromatographic method it is commonly applied to.
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.
Figure 2: Basic structure of a siloxane. The R groups can be varied depending on the type of column and analyte being analyzed. This figure was created with ChemBioDraw Ultra 12.0.
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 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.
Size Exclusion Chromatographic columns separates molecules based upon their size, not molecular weight. A common 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.
Figure 3: Schematic of a size exclusion column. The larger particles will elute first because they are too big to fit inside the pores. The smallest particles will elute last because they fit very well inside the pores. This figure was created with Microsoft Paint.
Chiral columns are used to separate enantiomers. Separation of chiral molecules is based upon steriochemistry. 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.
Some Stationary Phases Used to Separate Enantiomers
|Stationary Phase||Method(s) Used|
|Metal Chelates||GC, LC|
|Amino Acid Derivatives||GC, LC|
|Cyclodextrin Derivatives||GC, LC|
Table 5: This table shows some stationary phases that are used to separate enantiomers and the corresponding chromatographic methods that they are applied to.
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.
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