Overview of Chromatography

S.E. Van Bramer, March 9, 1996

  1. Gas chromatography (GC) is a well developed technique that is very widely used. In the early 1980's the fused silica capillary column was developed. This lead to a significant increase in the separating power of gas chromatography, but it also places extreme demands upon the injection and detection systems.

    1. Injection systems. Keep in mind that the sample must be vaporized prior to analysis by GC, this is a limiting factor for many inlets.
      1. The most common method is to introduce a small volume of sample with a syringe. This injection may be done by hand (like you will do in the lab) or by an automatic sampler. Other sampling systems discussed have special applications.
      2. Headspace sampling involves taking a sample of the gas above a liquid sample (the headspace) and injecting it into the chromatograph.
      3. Purge and trap is a variation on headspace analysis. A gas is bubbled through the sample and the analyte is trapped on a special kind of filter (or in a cold trap), this concentrates the analyte, the trap is then heated to desorb the analyte off of the trap and into the column.

    2. Derivative formation. This is a technique for extending GC analysis to compounds that are not very volatile. This is accomplished by chemically adding functional groups that make the analyte volatile. This technique is very useful, but improvements in liquid chromatography now make it possible to separate these non-volatile compounds in the condensed phase without derivitization.

    3. Column selection. Keep in mind that the capillary columns are capable of much more efficient separation (so more complex mixtures are separated or resolved. Alternatively, a simple mixture can be separated faster). Packed columns have extremely high surface area, which is an advantage for large amounts of analyte (like when separating gases). Capillary columns, however, have much greater resolution so they are more widely used for analysis.
      1. The most important consideration is the stationary phase (this is the liquid that is coated onto the inside of a capillary column or on the packing material of a packed column). The stationary phase is selected to separate the compounds of interest. One way to look at this is using polarity. A polar column will retain polar molecules longer (since like dissolves like) so it is better for separating polar compounds. Likewise a non-polar column is used for non-polar analytes. Other stationary phases are designed to interact with different types of functional groups. A very large selection of stationary phases are available from different vendors. One of the easiest ways to select a column is by looking for a similar application in the chromatography catalogs.

    4. Detectors, the differences and advantages of each is very important in selecting an analysis technique.
      1. Thermal Conductivity is simply how much heat is conducted by the effluent (the mobile phase after it exits the column). When the analyte exits the column and passes through the detector it changes the amount of heat conducted by the effluent. This detector is sensitive to almost any compound (this may be an advantage or a disadvantage). It the detector is not very sensitive.

      2. Flame Ionization Detector (FID). The FID is essentially a small H2 flame that the column effluent flows into. When an organic compound (or anything that burns) enters the detector the combustion produces ions that are easily detected. This detector is very sensitive and responds to most compounds (anything that burns). It does not respond to Air and Water so background levels of these compounds to interfere with analysis.

      3. Thermionic Emission Detector. This is a slight variation on the FID. The flame is adjusted so that the combustion temperature is very low and most organic compounds do not undergo combustion to form ions. A special bead (rubidium silicate) is inserted into the flame to catalyze the combustion of compounds with nitrogen or phosphorous. This detector is only sensitive to nitrogen and phosphorous containing compounds.

      4. Electron Capture Detector (EC). This is probably the strangest and most difficult detector to understand, but it is very useful for environmental analysis. In this detector electrons are created by a radioactive source, then they travel across the detector and are detected. The effluent flows through the detector. When a compound that absorbs electrons enters the detector, the electron current decreases. Because of the way it works this detector is extremely sensitive to certain compound classes (particularly chlorinated hydrocarbons).

      5. Fourier Transform Infrared (FT-IR) and Mass Spectrometry (MS) detectors. These detectors also provide structural information about the analyte. This is very useful for identification of unknowns, but the techniques are not usually as sensitive as other detectors.

  1. High Pressure Liquid Chromatography(1) (HPLC). This technique is very widely used. It is useful for the analysis of compounds that are not volatile enough for GC analysis. The mobile phase is a liquid and separation is dependent upon the solubility of an analyte in the mobile phase. Typical types of liquid chromatography include: Normal phase (use organic solvent for the mobile phase), Reverse phase (use an aqueous mobile phase), and Ion chromatography (separation of ions).

    1. Column efficiency is highly dependent upon the packing size (5-10m diameter is common). The smaller the packing the higher the pressure required for a certain flow rate (typical pressures are several thousand PSI).

    2. Gradient Elution. This is a very important for liquid chromatography. In Gas Chromatography the temperature is often increased during a run to elute compounds with increasing boiling points. The equivalent in HPLC is to change the solvent strength. This is often done using a mixture of water and an organic solvent, by changing the mixture different compounds will be soluble in the mobile phase and then pass through the column.
    3. Derivatization. This is simply reacting the analyte to make it easier to detect. This often involves placing a fluorescent tag on the analyte so that it is easily detected.

    4. Instrumentation used for HPLC.
      1. Pump. HPLC is usually done at relatively high pressure (several thousand PSI). This requires high pressure pumps. Several different designs are used, each has advantages and disadvantages.
        1. Piston pumps are the most common. The have the advantage of being continuous (see syringe pumps) and capable of relatively high pressure. The disadvantage is that they produce pressure pulses each time the piston moves forward. This causes pressure and flow fluctuations which cause band broadening (decrease resolution). These pressure fluctuations are minimized in two ways. One is to use a dual-head pump, this is a two piston pump arranged so that one piston is always pumping. The other method is to use a pressure dampener to absorb the shock of the pulse.
        2. Syringe pumps are sometimes used. This is simply a motorized syringe. These pumps are capable of very high pressure and produce no pressure pulse. The disadvantage is that they are not continuous. After the syringe is empty, you have to turn it off and reload.

      1. Sample Introduction. Because HPLC operates under high pressure sample introduction is more difficult. The most common method is to use a sample loop. Typical injection volumes are 10 to 20 L for packed column HPLC.

      2. Columns
        1. A typical column is made from a piece of 1/4" stainless steel tubing, is 6 to 12 inches long and packed with 5 to 20m particles for the stationary phase.
        2. Narrow bore and capillary columns are sometimes used, they have and advantage of greater separation but less sample can be injected This makes the injection technique difficult and it provides less material for detection. Since HPLC detectors are not extremely sensitive this is a serious disadvantage.
        3. Guard Columns are used to protect the analytical (regular) column. This is simply a very short column, anything that would stick to the analytical column strongly enough to ruin it will be trapped on the guard column (which is small and therefor much less expensive).

      3. Detectors. Important considerations are: sensitivity (how much analyte is required for detection), dead volume (smaller is better) and selectivity (what does it detect and not detect). Common detectors are
        1. UV-VIS. This is a UV-VIS spectrometer. The eluent flows into a very small cuvette that is part of a spectrometer. This cuvette is small so the path length is short (reduces sensitivity). This detector may be designed for a fixed wavelength, variable wavelength, or simultaneous detection at many wavelengths (using a diode array). The tradeoff between these different detectors is price. This detector responds to most compounds (anything that absorbs light at the set wavelength).
        2. Fluorescent detectors. Detects fluorescence signal. Usually more sensitive than UV-VIS and more selective (only observe fluorescent compounds).
        3. Electrochemical. This detector can be extremely sensitive and selective. However, it is also more difficult to use than UV-VIS or Fluorescent detectors.
        4. Refractive index. This detects changes in the refractive index of the mobile phase as things elute. It is a very universal detector, but it is not very sensitive and it is difficult to align. (It is roughly equivalent to the thermal conductivity detector for GC)
        5. Conductivity. This measures the resistance of the mobile phase. It is very useful for detecting ions (in ion chromatography). It is extremely sensitive for some ionic compounds.
        6. HPLC/MS. This is an area of current development. The mass spectrometer provides structural information about the analyte as the peaks elute. The instrumentation is rather complex and expensive (> $100,000).

1. Also called: High Performance Liquid Chromatography, High Price Liquid Chromatography, Hewlett Packard Liquid Chromatography or High Probability of Leaky Connectors.