Science of Chromatography

Flame ionization detector is the most common type of detector used in GC. In flame ionization detector Hydrogen (H2) and Oxygen (O2) are present in the gas form. This will create a flame. The components that are eluting out will burn by the flame and will turn into ions. The formation of ions will ultimately create an electrical current. Electrical current depends on the components present in the sample. The following reaction will occur in order to form the electrical current.

Compounds elute out from the column is detected by this system. Simply detector tells the user that a compound has been eluted out. Detector generates a response which indicates the presence of a compound eluting from a GC column. The respond varies with the compound. Thus on the other hand detector not only detects the compound but also identifies the compound. This emphasizes that needfulness of a detector to GC is of utmost importance. There are two types of detectors. Those are as following, Flame ionization detector (FID) Thermal conductivity detector (TCD)

The oven is a special feature present in Gas Chromatography where absent in other chromatography systems. This basically fulfill two main requirements, first is that column is installed in the oven. Second is adjusting a suitable environment for the chromatogram development.  The column temperature must be uniform throughout the time period until the components are eluted out. Thus to maintain the temperature of the column the oven is an essential system in a GC.

Liquidized stationary phases are used in capillary columns. Capillary column has a unique structure compared to the packed column . Capillary columns are also very tiny tube like structures which are highly coiled. The internal diameter of a capillary column is around 0.1-0.53 mm.  The liquid stationary phase is coated as a thin film on the solid adsorbent which has been inserted into the coiled tube. This column type is longer than the packed column. Thus capillary columns may take more time to separate the test sample. On the other hand capillary columns do not need a large space in the GC system. 

Liquidized stationary phases are used in packed columns. Packed columns are very tiny, erect, tube like structures. The internal diameter of a packed column is around 2-4 mm. The stationary phase is packed inside this tiny tube, thus calling this structure as packed column. The stationary phase can be a single solid adsorbent or liquid coated solid adsorbent. Packed columns are not long as capillary columns . Thus the path length for the separation is slightly lower than the capillary column. Hence we can guess the packed columns separate the test sample much quicker than the capillary column. On the other hand use of packed columns takes more space than the capillary columns.

The stationary phase is the non-movable phase in chromatographic techniques. The mobile phase together with the test sample is moved through the stationary phase. During this passage of the mobile gas and the test sample the components in the test sample is separated. The stationary phase is packed inside a column in most chromatographic techniques. In Gas Chromatography two types of columns are used. Those are namely, Packed column   Capillary column   Depending on the packed material the column differs.

The test sample is usually carried with the mobile phase. Then the test sample is gone through the stationary phase together with the mobile phase. In GC the test sample is inserted into the system via an injection port.  Like in HPLC  the test samples are injected into the chromatographic procedure. Injection is done through a self-sealing rubber system. And the test sample is injected using a gas-tight syringe. The gas tight syringe allows only micro liter amounts to be inserted in into the GC when using liquids as the testing sample. Then this is carried by the mobile gas. Together with the test sample the mobile gas is gone into the column where the stationary phase is present.

Carrier gas supply Every chromatographic technique includes a mobile phase and a stationary phase. Mobile phase is interacted with the stationary phase in order to proceed the separation. Basically the mobile phase is gone through the mobile phase together with the test sample. Which means the test sample is carried by the mobile phase. In gas chromatography the mobile phase is a gas. Thus in GC the mobile phase or the mobile gas is termed as the carrier gas. The carrier gas should perform several characteristics. Carrier gas should be inert, dry and free from impurities. The testing sample is either a gas or a liquid in GC. This test sample is carried onto the stationary phase by the carrier gas or the mobile gas. If carrier gas is a very reactive one, then it will perform a reaction with the test sample. If carrier gas contains moisture then of course the water molecules will react with the test sample. And also the presence of impurities will also interact with the test gas

Gas Chromatography is another type of technology used in separation of gases. This is a complex set of sections joined together to form a single instrument, thus making much easier to separate, identify and quantify the gases of interest. Unlike other chromatographic techniques in GC there is no need of developing chromatograms and other difficult time consuming things! It only matters a scientist or a technician who is capable of handling the instrument as well as the handler should perform good knowledge in chromatography.   Gas chromatography consists with a carrier gas supply, an injection port, a column, an oven and a detector. The detector output will be received by an integrator which is an instrument that will integrate the output signal. And the integrated signal is then recorded from a recorder. 

Gas chromatography or GC is a type of adsorption chromatography. The mobile phase of gas chromatography is a gas. This is the reason why this is termed as “gas chromatography”. The stationary phase can be either a solid adsorbent or a liquid adsorbent. If it is a solid adsorbent then this is called as “gas-solid chromatography” where as if liquid adsorbent then this is called as “gas-liquid chromatography”. In gas-liquid chromatography a non-volatile liquid has to use. Gas chromatography helps in many ways to detect explosive air pollutants, essential oils and very useful forensic studies.  The following topics related to GC will be discussed in detail.  Parts of GC Carrier gas in Gas Chromatography Injection port and sample injection  in Gas Chromatograph y Columns in Gas Chromatography Capillary column in Gas Chromatography Packed column in Gas Chromatography The oven in Gas Chromatography Detectors in Gas Chromatography Flame Ionization detector in Gas Chromat

HPLC instrument has four main systems and three other systems which help to proceed HPLC accurately. The four main systems include mobile phase reservoirs, injector system, column and detector. The other three are the pump, waste and the recorder. The four main systems will be discussing in detailed in next pages. Mobile phase reservoirs in HPLC Injector system in HPLC Column in HPLC Detector in HPLC Pump mainly serves to move the mobile phase through the column. For this movement high pressure is required and it is given by the pump. The detector is connected to the waste and the recorder. Recorder will read the output given from the detector and it will produce peaks to indicate the detected compounds as relevant. Waste is to carry the unwanted outputs given from the detector.

In HPLC the detected compounds are identified. Identification of these compounds is done by comparing the retention time of the compounds with that of standard substances. Like in other methods after the identification the identified compounds are quantified. Quantitative analysis of compounds is done on the basis of the detector output (Chromatogram).

In HPLC the compounds are detected by the section detector. Detection is based on different physical principals. These are UV absorption, fluorescence, radioactivity, IR absorption and electrochemical gradient. Detection by UV absorption is mainly based upon the ultraviolet-visible spectroscopy. Some compounds can absorb or reflect light which is in between the UV spectral region. Such compounds are detected through UV absorption spectroscopy. The compounds absorb light and the molecules of these compounds then undergo electronic transitions. This happens when these molecules contain non bonding electrons. These non bonding electrons can absorb energy in the spectrum of UV or visible light to excite these electrons to higher energy states. During electronic transitions of some compounds they undergo fluorescence. Fluorescence is a type of electromagnetic radiation. Also there are radioactive compounds which can be detected by relevant radioactive detectors. IR absorptio

Detector Detector records the relative concentrations of different components of the test sample with respect to their retention time. Retention time is the time taken by the compound to elute from the column. Retention time is calculated from the time of injection until the compound is eluted. Detection is based on several different physical principals .  Those are UV absorption, fluorescence, radioactivity, IR absorption and electrochemical gradient. Depending on the characteristics of the mixture the physical principals will be different.

HPLC tube The separation of the compound mixture takes place in this column or HPLC tube. The HPLC tube is made up of stainless steel. The stationary phase is packed in the column and it is usually a solid adsorbent. In some instances the solid adsorbent is packed with a thin layer of water. Thus the physical principle or the mode of separation in HPLC is partition. As in other chromatographic techniques in HPLC the mobile phase with the test sample is gone through the stationary phase. And the compounds in the test sample are separated in here. Mobile phase serves only as a carrier of the test sample. According to the solubility differences (in other words polarity differences) the mixed compounds are separated.

Sample injection Like in other techniques, in HPLC the test sample is loaded into the stationary phase. But for this application HPLC has an injector system. The injector system loads the test sample into the stationary phase which is a column. Injector system contains an injector port, the place where the sample is injected.  In modernized equipments the injector port is auto sealed section which gradually seals itself after the sample is injected. This injector port is made from a slightly thick flexible material. Auto sealed injector ports should be changed with time by the user, because with time its auto sealing ability reduces. The sample is usually injected using a micro liter syringe.

Solvent Reservoir Mobile phase reservoir will carry the mobile phase solution and through the pump it will be pumped into the column. This movement is according to the gravitational force. The mobile phase reservoir is a solvent with different polarities such as water, methanol, and acetonitrile.  These solvents should be highly pure. For an example, water should be used as de-ionized water. Hygienic conditions are facilitated with the use of 0.45 µm filter. The solvent is filtered through this filter mesh to remove all particulate matter. This is termed as Millipore filtering. Degas is done to minimize errors which occur when compounds interact with several gases. If degas or the removal of all the gases, is not done correctly then it may spoil the column .

A sophisticated HPLC machine controlled by software   HPLC is unique highly sophisticated chromatographic equipment used to separate compounds that are dissolved in a solution. This is the reason why it is called High performance LIQUID chromatography. Also it is termed as High PRESSURE liquid chromatography is due to its mobile phase. Its mobile phase moves due to gravity. Unlike other methods HPLC includes all three sections in chromatographic equipment which are separation, identification and quantification. HPLC is an instrument or a machine which has all these three sections itself. At present HPLC methods are used that are controlled by software.

High performance liquid chromatography is another method used for the compound separation. It is also termed as high pressure liquid chromatography. Unlike other chromatographic techniques this method is somewhat different due to its mobile phase’s unique character. Unlike other procedures the mobile phase of this HPLC moves due to pressure. In other words we can say mobile phase is moving in correlation with the gravitational force. In HPLC we will be focusing on the following topics. What is HPLC? Parts of HPLC Mobile phase reservoirs in HPLC Injector system in HPLC Column in HPLC Detector in HPLC Different physical principals for detecting compounds in HPLC Identification and quantification in HPLC

Readymade TLC plates TLC technique requires less time and fewer amounts of substances. More distinct individual spots make determinations easy. Strong reagents can use for the purpose of identification. TLC plates can be heated to higher temperatures without any damage.