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(Note: The following is not intended to represent an exhaustive treatise on chromatographic methods, nor does it take into account numerous variations in procedures which may be necessary, depending upon the nature of analytes, particular reagents or instruments used. Though the fundamental principles on which the instruments work remain the same, the handling and operation of instruments vary from one manufacturer to the other. It is therefore recommended that, for more detailed instructions, the analyst follow the operation and maintenance manuals provided by the instrument manufacturers and directions provided by the supplier of the reagents for the particular additive under analysis.)


Chromatography is defined as an analytical technique whereby a mixture of chemicals may be separated by virtue of their differential affinities for two immiscible phases. One of these, the stationary phase, consists of a fixed bed of small particles with a large surface area, while the other, the mobile phase or "eluant", is a fluid that moves constantly through, or over the surface of, the fixed phase. Chromatographic systems achieve their ability to separate mixtures of chemicals by selectively retaining some compounds in the stationary phase for varying times, while permitting others to move more freely. Therefore, the chromatogram may be evaluated qualitatively by determining the Rf, or retention factor, for each of the eluted substances. The Rf is a measure of that fraction of its total elution time that any compound spends in the mobile phase. Since this fraction is directly related to the fraction of the total amount of the solute that is in the mobile phase, the Rf can be expressed as


where Vm and Vs are the volumes of the mobile and stationary phase, respectively, and Cm and Cs are the concentrations of the solute in either phase at any time. This can be simplified to


where K = Cs/Cm and is an equilibrium constant that indicates this differential affinity of the solute for the two phases. Alternatively, a new constant k', the capacity factor, may be introduced, giving another form of the expression:


where k' = KVs/Vm. The capacity factor, k', which is normally constant for small samples, is a parameter that expresses the ability of a particular chromatographic system to interact with a solute. The larger the k' value, the more the sample is retained.

Both the retention factor and the capacity factor may be used for qualitative identification of a solute or for developing strategies for improving separation. In terms of parameters easily obtainable from the chromatogram, the Rf is defined as the ratio of the distance travelled by the solute band to the distance travelled by the mobile solvent in a particular time. The capacity factor, k', can be evaluated by the expression


where tr, the retention time, is the elapsed time from the start of the chromatogram to the elution maximum of the solute, and to is the retention time of a solute that is not retained by the chromatographic system.

Chromatography Principles

Retention of the solutes by the stationary phase may be achieved by one or a combination of mechanisms. Certain substances, such as alumina or silica gel, interact with the solutes primarily by adsorption, either physical adsorption, in which the binding forces are weak and easily reversible, or chemisorption, where strong bonding to the surface can occur. Another important mechanism of retention is partition, which occurs when the solute dissolves in the stationary phase, usually a liquid coated as a thin layer on the surface of an inert material or chemically bonded to it.

If the liquid phase is a polar substance (e.g. polyethylene glycol) and the mobile phase is nonpolar, the process is termed normal-phase chromatography. When the stationary phase is nonpolar (e.g. octadecylsilane) and the mobile phase is polar, the process is reversed-phase chromatography. For the separation of mixtures of ionic species, insoluble polymers called ion exchangers are used as the stationary phase. Solute ions in the mobile phase selectively displace an electrically equivalent amount of less strongly bound ions of the ion exchanger in order to maintain the electro neutrality of both phases.

The chromatographic separation of mixtures of large molecules such as proteins may be accomplished by a mechanism called size exclusion chromatography or gel permeation chromatography. The stationary phases used are highly cross-linked polymers that have incorporated a sufficient amount of solvent to form a gel. The separation is based on the physical size of the solutes; those that are too large to fit within the interstices of the gel are eluted rapidly, while the smaller molecules permeate into the pores of the gel and are eluted later.

Separation of certain molecules is accomplished by a mechanism called affinity chromatography in which specific binding between an antibody (stationary phase) and antigen (analyte) occur. In any chromatographic separation, more than one of the above mechanisms may be occurring simultaneously.

Chromatography Techniques

Chromatographic separations may also be characterized according to the type of instrumentation or apparatus used. The types of chromatography that may be used are column, paper, thin-layer, gas and high-performance liquid chromatography. Each will be discussed below.

Column Chromatography


The equipment needed for column chromatography is not elaborate, consisting only of cylindrical glass or Teflon tube that has a restricted outflow orifice. The dimensions of the tube are not critical and may vary from 10 to 40 mm in inner diameter and from 100 to 600 mm in length. For a given separation, greater efficiency may be obtained with a long narrow column, but the resultant flow rate will be lower. A fritted-glass disk may be seated in the end of the tube to act as a support for the packing material. The column is fitted at the end with a stopcock or other flow-restriction device in order to control the rate of delivery of the eluant.


The stationary phase is introduced into the column either as a dry powder or as slurry in the mobile phase. Since a homogeneous bed free of void spaces is necessary to achieve maximum separation efficiency, the packing material is introduced in small portions and allowed to settle before further additions are made. Settling may be accomplished by allowing the mobile phase to flow through the bed, by tapping or vibrating the column if a dry powder is used, or by compressing each added portion using a tamping rod. The rod can be a solid glass, plastic, or metal cylinder whose diameter is slightly smaller than the column, or it can be a thinner rod onto the end of which has been attached a disk of suitable diameter. Ion-exchange resins and exclusion polymers are never packed as dry powders since after introduction of the mobile phase they will swell and create sufficient pressure to shatter the column. When the packing has been completed, the sample is introduced onto the top of the column. If the sample is soluble, it is dissolved in a minimum amount of the mobile phase, pipetted onto the column and allowed to percolate into the top of the bed. If it is not soluble or if the volume of solution is too large, it may be mixed with a small amount of the column packing. This material is then transferred to the chromatographic tube to form the top of the bed.

The chromatogram is then developed by adding the mobile phase to the column in small portions and allowing it to percolate through the packed bed either by gravity or under the influence of pressure or vacuum. Development of the chromatogram takes place by selective retardation of the components of the mixture as a result of their interaction with the stationary phase. In column chromatography, the stationary phase may act by adsorption, partition, ion exchange, exclusion of the solutes, or a combination of these effects.

When the development is complete, the components of the sample mixture may be detected and isolated by either of two procedures. The entire column may be extruded carefully from the tube, and if the compounds are coloured or fluorescent under ultraviolet light, the appropriate segments may be cut from the column using a razor blade. If the components are colourless, they may be visualized by painting or spraying a thin longitudinal section of the surface of the chromatogram with colour-developing reagents. The chemical may then be separated from the stationary phase by extraction with a strong solvent such as methanol and subsequently quantitated by suitable methods.

In the second procedure, the mobile phase may be allowed to flow through the column until the components of the mixture successively appear in the effluent. This eluate may be collected in fractions and the mobile phase evaporated if desired. The chemicals present in each fraction may then be determined by suitable analytical techniques.

Paper Chromatography

In this type of chromatography, the stationary phase ordinarily consists of a sheet of paper of suitable texture and thickness. The paper used is made from highly purified cellulose, which has a great affinity for water and other polar solvents since it has many hydroxyl functional groups. The tightly bound water acts as the stationary phase, and therefore the mechanism that predominates is liquid-liquid or partition chromatography. Adsorption of solutes to the cellulose surface may also occur, but this is of lesser importance. Papers especially impregnated to permit ion-exchange or reverse-phase chromatography are also available.


The essential equipment for paper chromatography consists of the following:

Procedure for Descending Paper Chromatography

Separation of substances by descending chromatography is accomplished by allowing the mobile phase to flow downward on the chromatographic sheet.

The substance or substances to be analyzed are dissolved in a suitable solvent. Convenient volumes of the resulting solution, normally containing 1 to 20 μg of the compound, are placed in 6 to 10 mm spots along the pencil line not less than 3 cm apart. If the total volume to be applied would produce spots of a diameter greater than 6 to 10 mm, it is applied in separate portions to the same spot, each portion being allowed to dry before the next is added.

The spotted chromatographic sheet is suspended in the chamber by use of the antisiphoning rod and an additional heavy glass rod that holds the upper end of the sheet in the solvent trough. The bottom of the chamber is covered with a mixture containing both phases of the prescribed solvent system. It is important to ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber without touching the rack or the chamber walls. The chamber is sealed to allow equilibration (saturation) of the chamber atmosphere and the paper with solvent vapour. Any excess pressure is released as necessary. For large chambers equilibration overnight may be necessary.

A volume of the mobile phase in excess of the volume required for complete development of the chromatogram is saturated with the immobile phase. After equilibration of the chamber, the prepared mobile solvent is introduced into the trough through the inlet. The inlet is closed, and the mobile phase is allowed to travel down the paper the desired distance. Precautions must be taken against allowing the solvent to run down the sheet when opening the chamber and removing the chromatogram. The location of the solvent front is quickly marked, and the sheets are dried.

The chromatogram is observed and measured directly or after suitable development to reveal the location of the spots of the isolated components of the mixture.

Procedure for Ascending Paper Chromatography

In ascending chromatography, the lower edge of the sheet (or strip) is dipped into the mobile phase to permit the mobile phase to rise on the chromatographic sheet by capillary action.

The test materials are applied to the chromatographic sheet as directed under Procedure for Descending Paper Chromatography. Enough of both phases of the solvent mixture to cover the bottom of the chamber are added. Empty solvent troughs are placed on the bottom of the chamber, and the chromatographic sheet is suspended so that the end near which the spots have been added hangs free inside the empty trough.

The chamber is sealed, and equilibration is allowed to proceed as described under Procedure for Descending Paper Chromatography. Then the solvent is added through the inlet to the trough in excess of the quantity of solvent required for complete moistening of the chromatographic sheet. The chamber is resealed. When the solvent front has reached the desired height, the chamber is opened and the sheet is removed, the location of the solvent front is quickly marked, and the sheet is dried.

Small cylinders may be used without troughs so that only the mobile phase is placed on the bottom. The chromatographic sheet is suspended during equilibration with the lower end just above the solvent, and chromatography is started by lowering the sheet so that it touches the solvent.

Detection of Chromatographic Bands

After the chromatogram has been fully developed, the bands corresponding to the various solutes may be detected by means similar to those described in Column Chromatography. If the compounds are coloured or fluorescent under ultraviolet light, they may be visualized directly. Colourless compounds may be detected by spraying the paper with colour-developing reagents. The bands corresponding to the individual components can be cut from the paper, and the chemical substances eluted from the cellulose by the use of a strong solvent such as methanol.

Identification of Solutes

Since the chromatographic mobilities of the solutes may change from run to run due to varying experimental conditions, presumptive identification of a substance should be based on comparison with a reference standard. The Rf values of the unknown substance and the standard on the same chromatogram must be identical. Alternatively, the ratio between the distances travelled by a given compound and a reference substance, the Rr value, must be 1.0. Identification may also be made by mixing a small amount of the reference substance with the unknown and chromatographing. The resulting chromatograph should contain only one spot. Definitive identification of solutes may be achieved by eluting them from the paper and subjecting them to UV, IR, NMR, or mass spectrometry.

Thin-layer Chromatography

In thin-layer chromatography (TLC), the stationary phase is a uniform layer of a finely divided powder that has been coated on the surface of a glass or plastic sheet and that is held in place by a binder. The capacity of the system is dependent on the thickness of the layer, which may range from 0.1 to 2.0 mm. The thinner layers are used primarily for analytical separations, while the thicker layers, because of their greater sample-handling ability, are useful for preparative work.

Substances that are used as coatings in TLC include silica gel, alumina, cellulose and reversed phase packings. Separations occur due to adsorption of the solutes from the mobile phase onto the surface of the thin layer. However, adsorption of water from the air or solvent components from the mobile phase can give rise to partition or liquid-liquid chromatography. Specially coated plates are available that permit ion-exchange or reverse-phase separations.


Acceptable apparatus and materials for thin-layer chromatography consist of the following:


Clean the plates scrupulously, as by immersion in a chromic acid cleansing mixture, and rinse them with copious quantities of water until the water runs off the plates without leaving any visible water or oily spots, and then dry.

Arrange the plate or plates on the aligning tray, and secure them so that they will not slip during the application of the adsorbent. Mix an appropriate quantity of adsorbent and liquid, usually water, which when shaken for 30 sec gives a smooth slurry that will spread evenly with the aid of a spreader. Transfer the slurry to the spreader, and apply the coating at once before the binder begins to harden. Move the spreader smoothly over the plates from one end of the tray to the other. Remove the spreader, and wipe away excess slurry. Allow the plates to set for 10 min, and then place them in the storage rack and dry at 105° for 30 min or as directed in the monograph. Store the finished plates in a desiccator.

Equilibrate the atmosphere in the developing chamber as described under the Procedure for Descending Paper Chromatography in the section on Paper Chromatography.

Apply the Sample Solution and the Standard Solution at points about 1.5 cm apart and about 2 cm from the lower edge of the plate (the lower edge is the first part over which the spreader moves in the application of the adsorbent layer), and allow to dry. A template will aid in determining the spot points and the 10 to 15 cm distance through which the solvent front should move.

Arrange the plate on the supporting rack (sample spots on the bottom), and introduce the rack into the developing chamber. The solvent in the chamber must be deep enough to reach the lower edge of the adsorbent, but must not touch the spot points. Seal the cover in place, and maintain the system until the solvent ascends to a point 10 to 15 cm above the initial spots, this usually requiring from 15 min to 1 h. Remove the plates, and dry them in air. Measure and record the distance of each spot from the point of origin. If so directed, spray the spots with the reagent specified, observe, and compare the sample with the standard chromatogram.

Detection and Identification

Detection and identification of solute bands is done by methods essentially the same as those described in Paper Chromatography and Column Chromatography. However, in TLC an additional method called fluorescence quenching is also used. In this procedure, inorganic phosphorus is mixed with the adsorbent before it is coated on the plate. When the developed chromatogram is irradiated with ultraviolet light, the surface of the plate fluoresces with a characteristic colour except in those places where ultraviolet-adsorbing solutes are situated. These quench the fluorescence and are detectable as dark spots.

Quantitative Analysis

Two methods are available if quantitation of the solute is necessary. In the first, the bands are detected and their position marked. Those areas of adsorbent containing the compounds of interest are scraped from the surface of the plate into a centrifuge tube. The chemicals are extracted from the adsorbent with the aid of a suitable strong solvent, the suspension is centrifuged, and the supernatant layer is subjected to appropriate methods of quantitative analysis.

The second method involves the use of a scanning densitometer. This is a spectrophotometric device that directs a beam of monochromatic radiation across the surface of the plate. After interaction with the solutes in the adsorbent layer, the radiation is detected as transmitted or reflected light and a recording of light intensity versus distance travelled is produced. The concentration of a particular species is proportional to the area under its peak and can be determined accurately by comparison with standards.

Gas Chromatography

This type of chromatography differs from the others in that the mobile phase is a gas and therefore the solutes must be vaporized in order to allow their movement through the chromatographic column. Gas chromatography is further divided into:

In gas-solid chromatography, the stationary phase is an active adsorbent, such as alumina, silica, carbon or a polyaromatic porous resin, packed into a column. The passage of solute through the column will be retarded by adsorption or exclusion mechanisms. In gas-liquid chromatography, the stationary phase is a high boiling point liquid which is finely coated on an inert solid support, such as diatomaceous earth or a porous polymer which is packed into a column (packed column) or is coated as a thin film on the inside of a column (capillary column).

When a volatile compound is introduced into the carrier gas and carried into the column, it is partitioned between the gas and stationary phases by a dynamic countercurrent distribution process. The compound is carried down the column by the carrier gas, retained to a greater or lesser extent by sorption and desorption in the stationary phase. The elution of the compound is characterized by the partition ratio, k, a dimensionless quantity also called the capacity factor. It is equivalent to the ratio of the time required for the compound to flow through the column (the retention time) to the retention time of an unretained compound. The value of the capacity factor depends on the chemical nature of the compound; the nature, amount, and surface area of the liquid phase; and the column temperature. Under a specified set of experimental conditions, a characteristic capacity factor exists for every compound. Separation by gas chromatography occurs only if the compounds concerned have different capacity factors


(Note: Most gas chromatographic methods presented in this manual are based on capillary columns as they provide better separation and have replaced the traditional packed columns. As a result, manufacturers of gas chromatographs (GC) have discontinued the manufacture of packed column GCs. However, certain methods using packed columns are listed under individual monographs either due to non-availability of a suitable capillary column or an equivalent method using capillary column has not been developed as yet. In the absence of availability of a packed column GC, analyst may choose an equivalent capillary column, develop and validate proper method for use. The JECFA Secretariat highly appreciates receiving updates on such developments to include in future publications).

The essential components of a basic gas chromatograph are a carrier gas supply, an injection port, column oven, column, detector, and a suitable data-recording device. The injection port, column and detector are carefully temperature controlled.

   Carrier gas supply: Typical carrier gas is helium or nitrogen, depending on the column and detector in use. The gas is supplied from a high-pressure cylinder, suitable pressure or flow controllers are used to regulate the pressure or flow of the carrier gas. Carrier gas shall be highly pure (minimum of 99.999%, water < 1ppm, oxygen <0.1 ppm) and free from any particulate matter. Additionally, gas purifiers such as activated charcoal to remove hydrocarbons, molecular sieve to remove traces of water and oxygen trap may be used to further purify the carrier gas. On line filters (2 Ám) may also be used to remove any particulate matter.

    Sample injection device: Sample injectors depend on the type of column connected to the injector. They can be classified into packed column injectors (for use with packed columns) and capillary injectors (for use with capillary columns). Sample injection devices range from simple syringes to fully programmable automatic injectors. The amount of sample that can be injected into a capillary column without overloading is small compared with the amount that can be injected into a packed column, and may be less than the smallest amount that can be manipulated satisfactorily by syringe. The injected sample is required to be split into two fractions prior to reaching the column.

Capillary injectors have the capability to split sample into two fractions, a small one that enters the column and a large one that goes to waste (split injector). Injector can be operated either in split mode or splitless mode depending on the quantity of the sample injected. Temperature programmable injectors are also available where the sample is injected into an injector at low temperature (about 50°) and the injector temperature is quickly raised (250o/sec) to the required temperature. This helps in preventing thermal degradation of solutes in the injector.

Compounds to be chromatographed, either in solution or as gases, are injected into the gas stream at the injection port. Depending on the configuration of the instrument, the test mixture may be injected directly into the column or be vaporized in the injection port and mixed into the flowing carrier gas before entering the column. Purge and trap injectors are equipped with a sparging device by which volatile compounds in solution are carried into a low-temperature trap. When sparging is complete, trapped compounds are thermally desorbed into the carrier gas by rapid heating of the temperature-programmable trap.

Headspace injectors are equipped with a thermostatically controlled sample-heating chamber. Solid or liquid samples in tightly closed containers are heated in the chamber for a fixed period of time, allowing the volatile components in the sample to reach equilibrium between the nongaseous phase and the gaseous or headspace phase. After this equilibrium has been established, the injector automatically introduces a fixed amount of the headspace in the sample container into the gas chromatograph.

    Column oven: Chromatographic columns are housed in an oven and its temperature is carefully controlled. Column oven may be operated either in isothermal or temperature programming modes. Compounds in the test mixture are separated by virtue of differences in their capacity factors, which in turn depend on their vapor pressure and degree of interaction with the stationary phase. The capacity factor, which governs resolution and retention times of components of the test mixture, is also temperature dependent. The use of temperature programmable column ovens takes advantage of this dependence to achieve efficient separation of compounds differing widely in vapor pressure. As resolved compounds emerge from the column, they pass through a detector, which responds to the amount of each compound present. The type of detector to be used depends on the nature of the compounds to be analyzed.

    Columns: Two types of columns, packed or capillary are available for use in gas chromatography. Packed columns consist of tubes made up of glass, stainless steel or aluminium which are packed with the stationary phase. Columns of various dimensions may be used, but they usually range from 0.6 to 1.8 m in length and from 2 to 4 mm id.

Capillary columns with 0.25 mm inner diameter and lengths of 30 m or more, have replaced traditional packed columns due to their high efficiency. They are usually made of fused silica or aluminum clad. Fused silica columns are externally coated with polyamide to prevent breakage when they are coiled. Capillary columns are classified into three categories depending on their id. 0.15 - 0.25 id (narrow bore), 0.30-0.53 id (wide bore), .0.53 id (megabore). Wide bore and megabore columns withstand relatively high sample loading as compared to narrow bore columns. The liquid or stationary phase coated is 0.1 to 1.0 mm thick, although nonpolar stationary phases may be up to 5 mm thick. Three types of capillary columns are available:

WCOT columns are the most popular. Because of the absence of a solid support, capillary columns are much more inert than packed columns. Retention time and the peak efficiency depend on the carrier gas flow rate; retention time is also directly proportional to column length, while resolution is proportional to the square root of the column length.

Solid support materials must be as inert as possible in order to prevent interaction of the solutes with active surfaces, resulting in degradation, rearrangement, or loss of peak symmetry (tailing). The most commonly used supports are derived from silicates, usually diatomaceous earth. Before use they are acid-washed, calcined, and treated with a silanizing reagent to render surface hydroxyls inactive. They are available in various particle sizes from 30- to 120-mesh, with the 80- to 100-mesh and 100- to 120-mesh fractions most often used. Porous polymeric materials, which may be coated if desired or used as supplied, are available for the separation of low-molecular-weight compounds.

Liquid phases for partition chromatography may be chosen from a large variety of compounds, ranging from the very polar polyethylene glycols to the nonpolar methyl silicone gums. The choice of a liquid phase for a particular separation is mainly empirical, but usually polar phases are used for the analysis of mixtures of polar compounds. Chemically bonded and cross-lined phases can be used as they withstand higher temperatures (little 'bleeding' at about 300°) and can also be rinsed to restore column performance. They are useful for the analysis of high-boiling-point compounds. Capillary columns with stationary phases with varying polarities, lengths and id are commercially available.

For packed columns, the carrier gas flow rate is usually expressed in milliliters per minute at atmospheric pressure and room temperature. It is measured at the detector outlet with a soap film flow meter while the column is at operating temperature. Unless otherwise specified in the individual monographs, flow rates for packed columns are 60 to 75 ml/min for 4-mm id columns and ~30 ml/min for 2-mm id columns.

Before use, a packed column should be conditioned in the chromatograph to reduce the level of extraneous detector signals produced by the bleeding of volatile substances from the support and the liquid phase. This can be accomplished by increasing the column temperature gradually above its expected operating temperature, while maintaining a low flow of carrier gas through it and leave it overnight at the maximum temperature. During this process, the column should be disconnected from the detector. A suitable test for support inertness should be done.

Capillary columns can be protected by connecting them using a 'retention gap' (an empty or low polar capillary column of about 1.5 m in length depending on the polarity of the analytical column) through a quick seal connector. This will retain some unwanted matrix components and protect the column from contamination. For capillary columns, linear flow velocity is often used instead of flow rate. This is conveniently determined from the length of the column and the retention time of a dilute methane sample, provided a flame-ionization detector is in use. Typical linear velocities are 20 to 60 cm/s for helium. At high operating temperatures there is sufficient vapor pressure to result in a gradual loss of liquid phase, a process called "bleeding."

Capillary columns must be tested to ensure that they comply with the manufacturers' specifications before they are used. These tests consist of the following injections: a dilute methane sample to determine the linear flow velocity; a mixture of alkanes (e.g., C14, C15, and C16) to determine resolution; and a polarity test mixture to check for active sites on the column. The latter mixture may include a methyl ester, an unsaturated compound, a phenol, an aromatic amine, a diol, a free carboxylic acid, and a polycyclic aromatic compound, depending on the samples to be analyzed.

    Detectors: GC detectors can be classified into two groups, universal and selective detectors based on their general response or its response to specific elements or ions. Flame-ionization detector (FID) is the most commonly used detector in GC. The other detectors include electron-capture detector (ECD), thermal conductivity detector (TCD), nitrogen-phosphorus or thermionic specific detector (NPD or TSD), flame photometric detector (FPD) and mass spectrometric detectors (MSD). For quantitative analyses, detectors must have a wide linear dynamic range: the response must be directly proportional to the amount of compound present in the detector over a wide range of concentrations.

(Note: Refer to the section on gas chromatography- mass spectrometry for details on mass specific detectors).

    Data Collection Devices: Modern data stations receive the detector output, calculate peak areas, and print chromatograms, complete with run parameters and peak data. Chromatographic data may be stored and reprocessed, with integration and other calculation variables being changed as required. Data stations are used also to program the chromatograph, controlling most operational variables and providing for long periods of unattended operation. Data can also be collected using integrators whose capabilities range from those providing a printout of chromatogram and peak areas/ heights and data stored for possible reprocessing. Simple recorders are available for manual measurement.

Qualitative Analysis

Since it is impracticable in gas chromatography to measure the Rf, presumptive identification of a solute should be done by comparing its position on the chromatogram with that of a reference standard. The position of a solute is characterized by its retention time, the time from injection to the peak maximum; its retention volume, the product of retention time and carrier gas flow rate; or its retention distance, the distance from injection to the peak maximum. Since conditions may vary between determinations, it is more appropriate to identify a substance by its relative retention,


Where t2 is the retention time, volume, or distance of the desired chemical, t1 is that of the reference compound, preferably determined on the same chromatogram, and t0 is the retention of an inert compound that is not retained by the column.

A method of definitive identification is to trap and condense the effluent for each peak and subject the condensate to analysis by IR, NMR, mass spectrometry, or other suitable methods.

A measure of the efficiency of a column is the number, N, of theoretical plates it contains for a given compound:

A measure of the efficiency of the separation of two adjacent peaks is given by the dimensionless constant R, the resolution factor, which can be calculated by the equation:

Quantitative Analysis

In a gas chromatography, the parameter that is proportional to the concentration of any solute is the area under its peak or height. The following techniques can be used for the quantitation of solutes.

Area normalization

This method is based on the assumption that a peak is obtained on the chromatogram for each component of the mixture. The areas of all the peaks, each corrected by multiplying by its response factor, are added together to obtain the total area. Then the percentage of any component is equal to its corrected area divided by the total area and multiplied by 100. This method is reliable only if all components of the sample give a peak and if the various response factors are known.

External standard

A series of standards containing known amounts of the analyte are chromatographed under identical conditions. From the data obtained, a standard or working curve can be constructed by plotting area versus amount of standard that passes through the origin. If the compound to be analyzed is adsorbed within the system, the calibration curve will intersect the abscissa at a nonzero value. This may result in error, particularly for compounds at low concentrations determined by a procedure based on a single reference point. At high concentrations, the liquid phase may be overloaded, leading to loss of peak height and symmetry. A major source of error is irreproducibility in the amount of sample injected, notably when manual injections are made with a syringe. Auto samplers provide better precision.

Internal standard

In order to correct for errors that might occur when injection volumes vary or the chromatographic conditions change slightly from run to run, the internal standard method may be used. In this method, another standard, which is chemically similar to the unknown component and which elutes separately from all other peaks, is added in a constant amount to all standard and test solutions of the analyte. After chromatographing, a standard curve is constructed by plotting the area ratios of the standard solutions (area of analyte standard per area of internal standard) versus the weight or concentration ratios of each standard. The unknown is then chromatographed, its area ratio is determined, and the corresponding weight ratio is found by interpolation using the calibration curve. Since the amount of internal standard is constant and known, the concentration of the unknown component can be calculated.

Headspace-Gas Chromatography

Headspace-gas chromatography (headspace-GC) is an analytical technique in which the analyte in its gas phase and its GC analysis have been combined. The method can be applied to the analysis of compounds with a low boiling point, which therefore vapourize easily at low temperatures.

There are two different forms of headspace-GC: static and dynamic headspace-GC. In static headspace-GC the gaseous sample is taken from a sealed headspace vial into a loop and the analytes are transferred to the GC with the help of carrier gas. In dynamic headspace-GC the gaseous sample is forced out of the headspace vial with the help of an external source, usually the same gas as that used as the carrier gas. Headspace injectors are equipped with a thermostatically controlled sample-heating chamber. Solid or liquid samples in tightly closed headspace vials incubated in the chamber for a fixed period of time, allowing the volatile components in the sample to reach equilibrium between the nongaseous phase and the gaseous or headspace phase. After incubation at a specified temperature a gas sample from the head-space vial is sampled.

Purge and Trap Analysis

Purge and trap injectors are equipped with a sparging device by which volatile compounds in solution are carried into a low-temperature trap. When sparging is complete, trapped compounds are thermally desorbed into the carrier gas by rapid heating of the temperature-programmable trap.

Analysis of Samples - General Method Instructions

(Note: The instructions detailed below provide guidance on performing gas chromatographic analysis. However, analyst should consult individual monographs for sample preparation, appropriate GC column, detector and other conditions)

The following need careful consideration during the analysis:

    Analyte characteristics: The selection of a proper gas chromatographic system depends on the analyte characteristics such as its molecular weight, polarity, boiling point, thermal stability and other properties (refractive index, functional groups, presence of nitrogen, phosphorus, halogens etc. which help in choosing a selective detector).

    Column and detector selection: The selection of the right chromatographic column depends on the polarity of analytes and other matrix components. The general rule is that polar compounds can be separated on polar columns. Capillary columns with stationary phases in different polarities are commercially available. Kovat's index, McReynolds constants and CP Index guide selection of an appropriate column. Choice of selective detectors is based on the analyte characteristics (analytes containing halogens- ECD, nitrogen and phosphorus - NPD/FPD, sulphur-FPD). FID is the most commonly used. The analyst may use either the columns or equivalent as specified in the monograph. Use the detector as specified in the monograph. Use of a different detector requires establishing the limit of detection and limit of determination to ensure that the sensitivity of the method meets the specification requirements.

    Optimizing separation: The analyst may follow the GC operating conditions (injector, detector, column temperature - isothermal/temperature programming) detailed in the monographs. Temperature programming is suitable with most capillary columns to achieve optimum separation of analytes and other sample components. However, changes in column length, slight changes in polarity and flow rates demand slight adjustments to the temperatures. It is recommended that the analyst optimize the GC conditions to achieve better separation and peak symmetry by injecting analyte mixture.

    Sample preparation: Sample preparation is considered as one of the critical steps in GC analysis and generally involves extraction, cleanup and concentration steps. Sample cleanup involves either single or a combination of steps which include: liquid-liquid extraction, column cleanup, GPC cleanup, SPE cleanup etc. It is recommended to follow the sample preparation method specified in the monograph.

    Derivatization: Derivatization is carried out improve volatility, thermal stability, changes in separation properties or paves way for selective detection. General derivatization methods include:

Most analytes are volatile at the specified GC temperatures and do not require derivatization. It is recommended to follow the derivatization method specified in the monograph.

    Qualitative analysis: Comparison of retention times of standard and the analyte is most commonly used for the identification of analytes. Wrong identification is possible in complex matrices where matrix components interfere. Techniques such as use of another column or mass spectral confirmation may be used in such cases. For such cases, specific instructions are detailed in the additive specifications. In the absence of any instructions, follow comparison of retention times for identification.

    Quantitative analysis: The external standard method is most commonly used. Instructions are detailed in the additive specifications if alternate methods (normalization technique or internal standard method) are used. Follow the external standard method in the absence of any instructions.

    Method validation: It is recommended to validate each test method to ensure accurate and reliable results. Method performance characteristics such as resolution, peak asymmetry, precision, accuracy, limit of detection, limit of determination, linearity and percent recovery provide data on method selectivity, sensitivity and applicability.

    Reporting Results: Results need to be reported to three significant figures, unless otherwise specified.

Gas Chromatography - Mass Spectrometry (GC-MS)

Gas chromatography-mass spectrometry is a hyphenated technique widely used for the confirmation and quantitation of analytes. In this technique, effluents from a GC column are passed into a mass selective detector or mass spectrometer and subjected to analysis. The gas chromatography part is detailed in the above section.

Mass Spectrometry

Mass spectrometry is perhaps the most widely used analytical tool to provide information on molecular weight, identification and confirmation of compounds through their mass spectra as well as quantitation of compounds. In the mass spectrometry, the analyte molecule is bombarded with a stream of electrons that lead to the loss of an electron from the analyte molecule forming a charged molecular ion (M+). The collision between electrons and analyte molecules usually imparts enough energy to excite the molecules to the higher energy states. Relaxation then often occurs by fragmentation of part of the molecular ion to produce ions of lower masses. The positive ions produced on electron impact are attracted through the slit of a mass spectrometer where they are sorted according to their mass-to-charge ratios and displayed in the form of a mass spectrum.

The plot is in the form of a bar graph that relates the relative intensity (abundance) of mass peaks to their mass-to-charge ratio. The peak having highest intensity is termed as a base peak, and is arbitrarily assigned a value of 100. The heights of the remaining peaks are then computed as a percentage of the base-peak height. Mass spectrometers have the capability to identify base peak and normalize the remaining peaks to that peak. Figure 1 shows a typical mass spectrum of ethyl benzene.


Figure 1


The essential components of a basic mass spectrometer are a sample inlet system, an ion source, a mass analyzer, a detector, a vacuum system, and a data processing device. Figure 2 shows the block diagram of a typical quadrupole GCMS.


Figure 2

    Sample inlet system: The sample inlet system permits introduction of a representative sample into the ion source with minimal loss of vacuum. These include batch inlets, direct probe inlets, chromatographic inlets and capillary electrophoretic inlets. In GCMS, chromatographic inlets are used where the tip of the capillary column is precisely inserted into the inlet.

    Ion Source: The purpose of an ion source is to ionize the molecule to produce gaseous analyte ions. Electron impact ionization (EI), chemical ionization (CI) and field ionization (FI) are used to ionize the analytes.

EI is the most common ionization technique used, in which the sample is brought to a temperature high enough to produce molecular vapour, which is then ionized by bombarding with a beam of energetic electrons. Despite certain disadvantages (excessive fragmentation leading to disappearance of molecular ion peak at times and the need to volatilize the sample), this technique produces a reproducible mass spectra of a compound and is the basis on which many mass spectral libraries are built.

In CI, also termed as soft ionization, a gaseous sample is ionized by collision with ions produced by electron bombardment of a reagent gas such as methane or ammonia. Collision between the sample molecule (M) and highly reactive reaction products aroused from the reagent gas usually involves proton or hydride transfer leading to formation of either (M+1)+ or (M-1)+ ions. Relative to EI spectrum, CI spectrum is simple and provides molecular weight information.

    Mass Analyzer: The mass analyzer separates the mass fragments produced by the ionization sources. The capability of a mass spectrometer to differentiate between masses is usually stated in terms of its resolution (R) which is defined as R = m/m, where m is the mass difference between two adjacent peaks that are just resolved and m is the nominal mass of the first peak (mean of the two peaks is some times used). The mass spectrometer with a resolution of 4000 would resolve peaks with m/z values of 400.0 and 400.1 or 40.00 and 40.01. Several low and high resolution mass analyzers are available which include single stage quadrupole, triple stage quadrupole, ion trap, magnetic sector and time-of-flight.

Single stage quadruple and ion trap are most commonly used. In a quadrupole mass spectrometer, the mass analyzer consists of four parallel cylindrical rods that serve as electrodes, one pair being attached to positive side of a variable dc source and the other pair to the negative terminal. Variable radio-frequency ac potentials are applied to each pair of rods. Meanwhile, ac and dc voltages on the rods are increased simultaneously while maintaining their ratio constant. Fragment ions are accelerated into the space between the rods by a potential of 5 to 10 V. All the ions except those having the m/z value strike the rods and are converted into neutral molecules. Thus, the resonance ions will pass through the quadrupole and reach the transducer. Quadrupole resolves ions that differ in mass by one unit. Quadrupole mass analyzers are termed as mass filters.

A ion trap analyzer consists of a central doughnut-shaped ring electrode and a pair of end cap electrodes. A variable radio-frequency is applied to the ring electrode while the end cap electrodes are grounded. Fragment ions circulate in a stable orbit within the cavity surrounded by the ring. As the radio-frequency energy is increased, the orbits of heavier ions become stabilized, while those with lighter ions become destabilized, causing them to collide with the walls of the ring electrode. When radio-frequency is scanned, the destabilized ions leave the cavity of the ring electrode via openings in the lower end cap and pass into a transducer.

    Detectors: Detectors used in the mass spectrometer include electron multiplier detectors, Faraday cup collector and photomultiplier detectors. Electron multiplier detectors are most commonly used.

    Vacuum system: The ion source, mass analyzer and the detector must be kept under high vacuum (10-4 to 10-8 torr) because charged particles interact with components of the atmosphere and are annihilated as a consequence. Several vacuum pumps are used in achieve the required vacuum. Pumps include rotary pumps, oil diffusion pumps and turbo molecular pumps. Differential pumping is recommended using rotary/oil diffusion pump at the first stage and turbo molecular pump at the second stage to crate a proper vacuum environment.

    Data processing device: The data processing devices have the capability to control the instrument as well as process a large quantity of data and provide mass spectrum of compounds.

    Identification of compounds: Independent identification of compounds is achieved by comparing the sample mass spectrum with several mass spectra available in the mass spectral library. Several EI mass spectral libraries are available. Most modern mass spectrometers have the facility of making custom mass libraries for use. Modern mass spectrometers have the facility to shift from EI to the CI mode which will provide molecular weight information. Tandem mass spectrometry (MS/MS) provides additional confirmation in identification of compounds. In MS/MS, a mass ion (usually the ion with high abundance) is selected and is subjected to further ionization followed by analyzing its mass spectrum. The ion selected for further ionization is termed as parent ion and the ions produced are termed as daughter ions. This process requires a triple quadrupole or ion trap-quadrupole system. In triple quadrupole, the first and third ones work as analyzers, where as the middle one works as an ionizer. The daughter ions so produced are very specific to the parent ions, providing a highly reliable confirmation of the presence of the analyte.

    Quantitation: Quantitation is usually carried out using a single ion or a group of ions following selective ion monitoring. The area under the curve for standard as well as sample is used in the quantitation.

High Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is a separation technique based on a solid stationary phase and a liquid mobile phase. Separations are achieved by partition, adsorption, exclusion, or ion-exchange processes, depending on the type of stationary phase used. HPLC has distinct advantages over gas chromatography for the analysis of nonvolatile organic compounds. Compounds to be analyzed are dissolved in a liquid, and most separations take place at room temperature.

As in gas chromatography, the elution time of a compound can be described by the capacity factor, k, which depends on the chemical nature of the composition and flow rate of the mobile phase, and the composition and surface area of the stationary phase. Column length is an important determinant of resolution. Only compounds having different capacity factors can be separated by HPLC.

HPLC is divided into two types namely normal phase chromatography and reversed phase chromatography. In the normal phase chromatography, non-polar to polar stationary phases and non-polar to polar mobile phases separate compounds by their increasing polarity. In the reversed-phase chromatography, the stationary phase has been modified to be a non-polar substance (silica gel has been bonded with a long-chain non polar substance e.g. octadecylsilyl), and polar solvents are used as mobile phase. The order of elution is from polar to non polar compounds.

Reversed-phase chromatography has become a highly powerful technique because its selectivity over a wide range of solutes that can be adjusted by varying the polarity of the mobile phase. In most cases, mobile phases consist of water:methanol or water: acetonitrile. Adjustment of the pH of a mobile phase prevents ionization of weak acids and bases (ion suppression). Addition of an ionic reagent (e.g. heptane-sulfonate) to form a less polar ion pair with a charged solute (ion pairing) aids the retention of polar compounds (e.g. food colours).


The basic components of a liquid chromatograph are a solvent delivery system, a sample injection device, a chromatographic column, a detector, and a suitable data-recording device.

    Solvent delivery systems: consists of one or more pumps capable of delivering a pulse-less flow of mobile phase at pressures up to 6000 psig. Flow rates up to 10 ml/min with increments of 0.01 ml/min are typical. In the isocratic mode, where a mobile phase of constant composition is used throughout the run, a single pump and solvent reservoir are required. The mobile phase needs to be prepared externally by mixing the liquids in the required proportion and degassing it by sparging with helium.

For the separation of mixtures where the k' values vary over a wide range, gradient-elution analysis may be used. In this method, the composition of the mobile phase is constantly changed during the chromatographic run. Modern gradient HPLCs (binary, ternary and quaternary systems) have the advantage of internally mixing the liquids, in a mixing chamber, in the required proportions, and have the facility for continuous degassing using a vacuum degasser.

    Injectors: After dissolution in a mobile phase or another suitable solution, compounds to be chromatographed are injected into the mobile phase, either manually by syringe or loop injectors, or automatically by auto samplers. The latter consist of a carousel or rack to hold sample vials with tops that have a pierce able septum and an injection device to transfer sample from the vials to a loop from which it is loaded into the chromatograph. Auto samplers are programmed to control sample volume, the number of injections and loop rinse cycles, the interval between injections, and other operating variables. Some valve systems incorporate a calibrated sample loop that is filled with test solution for transfer to the column in the mobile phase. In other systems, a test solution is transferred to a cavity by syringe and then switched into the mobile phase.

    Columns used for analytical separations usually have internal diameters of 2 to 4.6 mm and lengths from 5.0 - 30 cm and are usually made of stainless steel. Glass cartridge columns are also used with a cartridge column holder. Larger diameter columns are used for preparative chromatography.

Stationary phases for modern reversed-phase liquid chromatography typically consist of an organic phase chemically bound to silica or other materials. Particles are usually 3, 5, or 10 Ám in diameter. Column polarity depends on the polarity of the bound functional groups, which range from relatively nonpolar octadecyl silane to very polar nitrile groups. The percent carbon load (amount of bonded phase material loaded on to the silica support, measured as weight percentage of bulk silica) is important in determining the polarity of reversed phase columns. Increasing carbon load and chain length increases retentivity. End capping of columns (a chemical process that reduces the number of free silanol groups attached to the base silica support material) minimizes competing mechanisms.

Columns used for normal phase chromatography are polar in nature (silica). Use of guard columns in front of analytical column protects it and extends its life. Guard columns retain the non-polar substances and any particulate matter that may be in the sample. Guard columns in different sizes (0.5 - 5.0 cm in length) are available for both normal and reversed-phase columns.

Columns are housed in column housing with a thermostatic system to control the temperature. Columns may be heated to give more efficient separations, but only rarely are they used at temperatures above 60° because of potential stationary phase degradation or mobile phase volatility (Certain resin based columns e.g. carbohydrate columns are heated to 90° to achieve efficient separation of sugars). Unless otherwise specified, columns are used at an ambient temperature.

Ion-exchange chromatography is used to separate water-soluble, ionizable compounds of molecular weights that are less than 2000. The stationary phases are usually synthetic organic resins. Cation-exchange resins contain negatively charged active sites and are used to separate basic substances such as amines, while anion-exchange resins have positively charged active sites for separation of compounds with negatively charged groups such as phosphate, sulfonate, or carboxylate groups. Water-soluble ionic or ionizable compounds are attracted to the resins, and differences in affinity bring about the chromatographic separation. The pH of the mobile phase, temperature, ion type, ionic concentration, and organic modifiers, all affect the equilibrium. These variables can be adjusted to obtain the desired degree of separation.

In size-exclusion chromatography, columns are packed with a porous stationary phase. Molecules of the compounds being chromatographed are filtered according to size. Those too large to enter the pores pass unretained through the column (total exclusion). Smaller molecules enter the pores and are increasingly retained as molecular size decreases. These columns are typically used to remove high molecular weight matrices or to characterize the molecular weight distribution of a polymer or separation of a mixture of proteins with varying molecular weights.

    Detectors: The types of detectors most frequently used in HPLC are spectrophotometric, fluorometric, refractometric, potentiometric, voltammetric, or polarographic, electrochemical and mass detectors. The detectors consist of a flow-through cell (8-16 Ál) to which the column outlet is connected.

In spectrometric detectors, a beam of ultraviolet radiation passes through the flow cell and into the detector. As compounds elute from the column, they pass through the cell and absorb the radiation, resulting in measurable energy level changes. Fixed, variable, and photodiode array (PDA) detectors are widely available. Fixed wavelength detectors operate at a single wavelength, typically 254 nm, emitted by a low-pressure mercury lamp. Variable wavelength detectors contain a continuous source, such as a deuterium or high-pressure xenon lamp, and a monochromator or an interference filter to generate monochromatic radiation at a wavelength selected by the operator. Modern variable wavelength detectors can be programmed to change wavelength while an analysis is in progress.

Multi-wavelength detectors measure absorbance at two or more wavelengths simultaneously. In diode array multi-wavelength detectors, continuous radiation is passed through the sample cell, and then resolved into its constituent wavelengths, which are individually detected by the photodiode array. These detectors acquire absorbance data over the entire UV-visible range, thus providing the analyst with chromatograms at multiple, selectable wavelengths and spectra of the eluting peaks. Since the absorption spectrum is continuously collected (from start to the end of the peak) PDA has the additional capability to overlay normalized absorption spectra at different points of the peak and assesses peak purity. Signals also can be extracted at different wavelengths depending on the absorption maximum of each analyte, which provides improved sensitivity. Diode array detectors usually have lower signal-to-noise ratios than fixed or variable wavelength detectors, and thus are less suitable for analysis of compounds present at low concentrations.

Differential refractometer detectors measure the difference between the refractive index of the mobile phase alone and that of the mobile phase containing chromatographed compounds as it emerges from the column. Refractive index detectors are used to detect non-UV absorbing compounds, but they are less sensitive than UV detectors. They are sensitive to small changes in solvent composition, flow rate, and temperature, so that a reference column may be required to obtain a satisfactory baseline.

Fluorometric detectors are sensitive to compounds that are inherently fluorescent or that can be converted to fluorescent derivatives either by chemical transformation of the compound or by coupling with fluorescent reagents at specific functional groups. If derivatization is required, it can be done before chromatographic separation or, alternatively, the reagent can be introduced into the mobile phase just before it enters the detector.

Potentiometric, voltammetric, or polarographic electrochemical detectors are useful for the quantitation of species that can be oxidized or reduced at a working electrode. These detectors are selective, sensitive, and reliable, but require conducting mobile phases free of dissolved oxygen and reducible metal ions. A pulse-less pump must be used, and care must be taken to ensure that the pH, ionic strength, and temperature of the mobile phase remain constant. Working electrodes are prone to contamination by reaction products with consequent variable responses.

Electrochemical detectors with carbon-paste electrodes may be used advantageously to measure nanogram quantities of easily oxidized compounds, notably phenols and catechols.

Mass spectrometric detectors provide additional advantage of independent identification of compounds However, the mobile phase needs to be removed and compounds need to be ionized. This is achieved by connecting the column outlet to an inlet using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). For further details, refer the section on mass spectrometry.

    Data Collection Devices: Modern data stations receive and store detector output and print out chromatograms complete with peak heights, peak areas, sample identification, and method variables. They are also used to program the liquid chromatograph, controlling most variables and providing for long periods of unattended operation. Data also may be collected on simple recorders for manual measurement or on stand-alone integrators, which range in complexity from those providing only a printout of peak areas to those providing a printout of peak areas and calculated peak heights plus storing the data for possible use in subsequent reprocessing.

System Suitability

System suitability tests are an integral part of both gas and liquid chromatographic methods. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such.

Figure 3 shows the chromatographic separation of two substances. The resolution, R, is a function of column efficiency, N, and is specified to ensure that closely eluting compounds are resolved from each other, to establish the general resolving power of the system, and to ensure that internal standards are resolved from the analyte. Column efficiency may be specified also as a system suitability requirement, especially if there is only one peak of interest in the chromatogram; however, it is a less reliable means to ensure resolution than direct measurement. Column efficiency is a measure of peak sharpness, which is important for the detection of trace components. Replicate injections of a standard preparation used in the assay or other standard solution are compared to ascertain whether requirements for precision are met. Data from five replicate injections of the analyte are used to calculate the relative standard deviation if the requirement is 2.0% or less; data from six replicate injections are used if the relative standard deviation requirement is more than 2.0%.


Figure 3: Chromatographic Separation of Two Substances.

Figure 4 shows an asymmetrical chromatographic peak with tailing. The tailing factor, T, a measure of peak symmetry, is unity for perfectly symmetrical peaks, and its value increases as tailing becomes more pronounced. In some cases, values less than unity may be observed. As peak asymmetry increases, integration, and hence precision, becomes less reliable. The calculation is expressed by the equation


Where W 0.05 is the width of the peak at 0.05 height and f is the width of theCOMBINED COMPENDIUM OF FOOD ADDITIVE SPECIFICATIONS first half peak (see details in Figure 4).

Figure 4: Asymmetrical Chromatographic Peak

These tests are performed by collecting data from replicate injections of standard or other suitable solutions. Adjustments of operating conditions to meet system suitability requirements may be necessary. It is common to determine system suitability parameters from the analyte peak.

To ascertain the effectiveness of the final operating system, it should be subjected to a suitability test before use and during testing whenever there is a significant change in equipment or in a critical reagent or when a malfunction is suspected.



Absorption Spectrophotometry

Is the measurement of the selective absorption by molecules or ions, of electromagnetic radiation having a definite and narrow wavelength range, approximating monochromatic light.

Absorption spectrophotometry encompasses the following wavelength and wave number regions: ultraviolet (185 nm to 380 nm), visible (380 nm to 780 nm), near-infrared (0.78 - 2.5 urn or 12,800 - 4,000 cm -1) and mid-infrared (2.5 - 50 μm or 4000 - 200 cm-1).


Has been commonly accepted as the measurement of "filtered" light in the visible region; however, it is more prudent to restrict its use to those applications where human perception of colour is involved, i.e., the visible region.

Atomic Absorption Spectroscopy

Is the measurement of the radiation absorbed by the unexcited atoms in their gaseous phase, that have been aspirated into a flame or, in the absence of a flame, directly into the path of the radiation.

Flame Emission Spectroscopy (Flame Photometry)

Is the measurement of the intensity of radiation emitted from electronically excited atoms or molecular species. The excitation is brought about by aspirating a solution of the sample into a hot flame.

Fluorescence Spectrophotometry

Or "fluorometry", is the measurement of light emitted from a chemical substance while it is being exposed to electromagnetic radiation. The maximum intensity of the emitted fluorescence is usually at a wavelength longer (i.e., of lower energy) than the exciting radiation.

Turbidimetry and Nephelometry

Are two light-scattering techniques that involve the measurement of light scattered due to its passage through a transparent medium containing a suspended particulate phase. As a result of this scattering, an attenuation or decrease in intensity is suffered by the beam along its axis of travel. Turbidimetry involves the measurement of the degree of attenuation of the light beam by particles suspended in a medium, the measurement being made in the axis of the transmitted beam. Nephelometry involves the measurement of the light scattered by the suspended particles, the measurement being made at right angles to the incident beam.


Radiant power, P

Is the energy of radiation per sec that reaches certain areas of a detector. Incident radiant power is usually given the symbol Po. Alternate terminology is radiation intensity with symbols I and Io.

Absorbance, A = Log10 (Po/P)

Is the logarithm to the base 10 of the quotient of the incident radiant power upon a specimen divided by the radiant power transmitted by the specimen. Former terms were optical density "OD", absorbancy, and extinction.

Specific absorbance, A1%1 cm = A/bc x 10

Is the quotient of the absorbance, A, divided by the product of the adsorption path length, b, in cm, and the concentration, c, of the specimen, expressed in g per 100 ml. In general the specific absorbance of a substance is a constant and is independent of the intensity of the incident radiation, path length and concentration. Previously designated by the symbol


Transmittance, T = (P/Po)

Is the quotient of the radiant power transmitted by a specimen divided by the incident power upon the specimen. Transmittance is often expressed as a percentage and is related to the absorbance by the equation Log10T = A, or A = 2 - Log10%T. Other terms are transmission and transmittancy.

Absorptivity, a = A/bc

Is the quotient of the absorbance, A, divided by the product of the absorption path length, b, in cm, and the concentration, c, of the specimen, expressed in g per 1,000 ml. In general, the absorptivity of a substance is a constant and is independent of the intensity of the incident radiation, path length, and concentration.

Molar absorptivity.

Is the quotient of the absorbance, A, divided by the product of the absorption path length, in cm, and the specimen concentration, expressed in moles per liter. Former terms were molar absorbancy index, molar extinction coefficient and molar absorption coefficient.

Absorption spectrum

Is a graphic representation of the absorbance of a specimen or any of its functions, e.g., transmittance, as the ordinate and the wavelength of the incident radiation as the abscissa.

Fluorescence intensity, I

Is a descriptive term for the fluorescence activity of a substance and is commonly expressed in units related to the detector response. An alternate term is fluorescence power, with the symbol F.

Fluorescence excitation spectrum

Is a graphic representation of the incident (activating) radiation intensity as the ordinate and its wavelength as the abscissa.

Fluorescence emission spectrum

Is a graphic representation of the radiation intensity emitted by an activated species for a specific excitation wavelength as the ordinate and its wavelength as the abscissa.


Is the light-scattering effect of the suspended particles in a turbid medium.


Is a measure of the attenuation in the incident beam power per unit length of a turbid medium. The former term is turbidity coefficient.

Theory and Formulas for Calculations

When electromagnetic radiation travels through a medium containing atoms, molecules, or ions of a chemical substance, radiation at certain frequencies may be partially or totally removed in a process called "absorption". As a result of this absorption, these species are activated from their lowest energy state (ground state) to higher energy states (excited states). For absorption to occur, the energy of the exciting radiation must match the quantized energy difference between the ground state and one of the excited states of the specimen. In atomic absorption, excitation occurs only through electronic transition. Visible and ultraviolet radiation can excite only the outermost or bonding electrons to a higher energy level. Inner-shell electrons are excited only by X-ray radiation (less than 1 nm).

In the case of polyatomic molecules, vibrational and rotational transitions can occur in addition to electronic excitation, and as a result the molecular spectrum consists of closely spaced absorption bands instead of the sharp lines generally observed in the atomic absorption spectrum. Pure vibrational transitions can be achieved by infrared radiation in the range of 1 to 15 μm, while changes in rotational levels are detectable in the region from 10 to 100 μm.

The decrease in the radiant power of a monochromatic beam of light has been found to be proportional to both the distance the radiation travelled through the absorbing medium and the concentration of the absorbing species encountered in that medium. This decrease in energy can be described quantitatively by the Beer-Lambert law:


Therefore, if the absorptivity and the cell thickness are kept constant during a specific determination, a plot of the absorbance as the ordinate versus concentration as the abscissa should yield a linear relationship. The practical application of the Beer-Lambert law, however, necessitates the use of a reference standard solution of known concentration in order to compare its absorbance with that of the sample solution of unknown concentration. If absorption measurements are conducted in two matching cells having the same path lengths, the absorptivity, a, and the cell thickness, b, will be the same. Therefore the following general formula can be used for the calculation of the unknown concentration of the sample solution,



Cu = the concentration of the sample solution

Cs = the known concentration of the standard solution

Au = the absorbance of the sample solution

and As = the absorbance of the standard solution.

The Beer-Lambert law is usually satisfactory, provided a thorough understanding of its limitations is taken into consideration. Some of these are of such a fundamental nature that they constitute a real limitation of the law. They are due to the fact that the law does not take into consideration the effects of temperature, wavelength, or solute-solvent and solute-solute interactions, e.g., association, dissociation, chemical reaction, etc. Due to these limitations, the law usually applies only to dilute solutions, where these interactions are insignificant. Another limitation to the Beer-Lambert law is due to the inability of most instruments to provide monochromatic radiation.

Fluorescence can be observed in a number of gaseous, liquid, or solid substances. However, it is only applied analytically to a relatively small number of organic compounds. Fluorescence occurs when a molecule absorbs sufficient radiation at a certain wavelength to promote it to an excited singlet state with higher levels of energy. The gained energy is released as radiation or "fluorescence" of wavelengths longer than the incident radiation. In most cases, in order for fluorescence to occur the electronic transition involved is a pipi* system. To a lesser extent, pi pi and pi sigma* transitions occur. There is a delay between the absorption and emission of radiation of about 10-9 sec. This short delay period distinguishes fluorescence from phosphorescence, which has a delay period of about 10-3 sec and is due to release of weaker radiations from an excited triplet state and not a singlet state as is true of fluorescence. The effect of concentration on the fluorescence intensity can be described by a slightly modified version of the Beer-Lambert law. A linear relationship exists between the fluorescence intensity, I, of the solution and the concentration of the emitting species:


where K is a constant dependent upon the quantum efficiency of the fluorescence process and instrumental parameters. At constant Po, a simple relationship as in the Beer-Lambert law can be obtained: I = Kc. Thus a plot of the fluorescence intensity of a solution as the ordinate versus concentration of the emitting species as the abscissa should be linear at low concentrations (see Figure 5).


Figure 5

When light passes through a transparent medium containing a suspended particulate phase, scattering occurs in all directions, and as a result the beam loses power along its axis of travel. For dilute suspensions and under fixed conditions (particles, shape, size, refractive index, wavelength of radiation), the loss in radiation intensity can be related to the number of particles (or concentration, c) by an equation similar to the Beer-Lambert law.


where tau = kc/2.303. Therefore, in turbidimetric analysis a plot is constructed with standard solutions with Log10(Po/P) as the ordinate and c as the abscissa (Po is determined by using the solvent as reference). In nephelometric analysis, the radiation intensity scattered at right angles to the incident beam is plotted as the ordinate versus concentration as the abscissa.


The fundamental principles of optics and electronics that are used in manufacturing spectrophotometers are common to all regions of the spectrum from the vacuum ultraviolet to the far-infrared. However, due to important differences in detail, spectrophotometers are commercially available for use in the visible; in the visible and ultraviolet; in the visible, ultraviolet, and near-infrared; and in the infrared regions of the spectrum. In selecting the type of spectrophotometer to be employed, several factors have to be considered, including the nature of the specimen to be analyzed, the degree of accuracy required, sensitivity, and selectivity.

The essential parts of all spectrophotometers include a stable source of radiant energy; a device that permits the selection of a defined wavelength region such as a prism or grating monochromator: a slit for limiting the suitable bandwidth; a sample compartment; a radiation detector; and an indicator that may be a meter, a recorder, a digital counter, a printer, or a computer.

Radiation sources commonly employed are hydrogen or deuterium lamps for the ultraviolet region, tungsten lamps for the visible, and a Nernst glower, a globar, or an incandescent wire for the infrared. Quartz or fused-silica cells or cuvettes can be used in the ultraviolet, visible, or near-infrared regions. For infrared spectrophotometry, cells or plates made of sodium chloride are usually used. The radiation detector of ultraviolet and visible radiation is usually a photomultiplier tube with associated amplifiers.

UV/Visible Spectrophotometers:

Two types of spectrophotometers are available: a single-beam spectrophotometer, which adapts well to quantitative analysis that involves single-wavelength measurements, and a double-beam spectrophotometer, which is particularly useful for qualitative analysis and where continuous monitoring of absorbance is required. Some spectrophotometers are manually operated, while others are equipped for automatic and continuous recordings. Spectrophotometers employing the latest technology can be interfaced to a digital computer through an analog-digital converter for the direct determination of different spectra of analytes as well as for the storage of reference spectra. Fourier transformed infrared spectrophotometry is different from the regular dispersion type in that it employs an interferometric technique, whereby polychromatic radiations pass through the specimen to a detector on an intensity and frequency basis. Interfacing with a digital computer is required in order to process such complicated spectral data.

Atomic Absorption Spectrophotometers (AAS):

Instruments for atomic absorption measurements have the same basic components as other spectrophotometers except for the radiation source and the sample container. The most common radiation source is the hollow-cathode lamp, the cathode of which is usually made of the element to be analyzed. The sample is aspirated as a fine mist into a flame that is produced by an optimized mixture of air and acetylene or other suitable gases. The flame vaporizes the solvent molecules and brings the solutes into a gaseous phase. Monochromatic light emitted from the hollow-cathode lamp is selectively absorbed by the analytes. Photomultiplier tubes are used as detectors, with the electronics designed to accept the modulated radiation source output, thereby negating the continuous signal from the flame. Therefore only changes in the signal from the hollow-cathode lamp are monitored by the detector. These changes are proportional to the number of atoms in the analyte.

Both single-and double-beam atomic absorption spectrophotometers are available. One of the major limitations of Flame AAS is its low sensitivity for certain elements. For determination of elements in low concentrations, electro-thermal atomization technique (graphite furnace atomization) is useful. In the electro-thermal atomization technique, the laminar flow burner is replaced by a graphite furnace. A known volume of analyte solution is placed at the centre of a graphite tube placed in the furnace, the solution is dried and ashed using a controlled heating from electrodes attached to the furnace. Solutes in the dry matter are atomized to a vapour state in a short period to absorb light from the hallow-cathode lamp. Certain elements can be selectively brought to gaseous atomic state (e.g. mercury vapour) without using the flame. Such techniques use vapour generation accessory, in which mercury ions in the solution are reduced to metallic mercury and the mercury vapour is flushed with an inert gas such as nitrogen into an optical cell.

Inductively Coupled Plasma - Atomic Emission Spectrophotometers (ICP-AES):

Instruments for inductively coupled plasma - atomic emission measurements consist of a sample solution aspiration system, a high temperature (~ 6000O) plasma source (a torch in which argon gas is subjected to collisions using a radio frequency source to produce high temperature plasma). The plasma vaporizes the solvent molecule, brings solutes to an atomic state and excites them to higher energy levels. The emitted light from the atoms is measured using a specific emission wavelength. The torch can be operated either in the axial or radial mode depending on the type and nature of elements to be quantitated and type of sample matrix.

Modern ICP instruments use a charge coupled detector (CCD) for the fast measurement of the intensity of emitted light consisting of different wavelengths. Two types of ICP systems are currently available: (a) The sequential system scans the emission spectrum from lower to higher wave lengths during a sample run, making the determination of elements sequentially and; (b) the simultaneous system that collects emission data from all wave lengths simultaneously. ICP instruments have the added advantage of having high linearity of standard curves as compared to AAS and more sensitivity than flame AAS for several elements.


The apparatus for fluorescence intensity measurement is either a fluorometer, which employs filters to restrict the bandwidth of both the excitation and emission beams, or a spectrofluorometer, where prism or grating monochromators are used to limit the excitation beam, the emission beam, or both.

Since a spectrofluorimeter requires a more intense radiation source than a spectrophotometer, either a mercury lamp with its strong discrete lines or a xenon lamp with its energy continuum from the ultraviolet to the infrared is used. Cells for fluorometric measurement are constructed of silica, and the cell compartment is designed to allow a minimum of scattered light to reach the photomultiplier. To minimize scattering interferences, the detector is placed at right angles to the incident excitation beam.

For turbidimetric measurements, a conventional photometer with a tungsten source is usually employed. However, it is preferable to make the measurements in the blue region of a mercury arc. For nephelometric measurements, standard fluorometers are commonly used.

Infrared Spectrometers:

Infrared spectroscopy deals with vibrational and rotational frequencies in the molecules. Infrared measurements may be carried out in the region of near-infrared (0.78 - 2.5 μm or 12,800 - 4,000 cm -1) or mid-infrared ( 2.5 - 50 μm or 4000 - 200 cm -1 ). The most commonly used region, however, is 2.5 - 15 μm or 4000 - 670 cm-1.

Two types of instruments are available: (a) dispersive grating spectrophotometers mainly used for qualitative analysis and (b) multiplex instruments, employing Fourier transform that are used for both qualitative and quantitative measurements. The dispersive grating spectrophotometers are replaced by Fourier transform instruments due to their speed, reliability and convenience. Most commercial Fourier transform infrared spectrometers (FTIR) are based on the Michelson interferometer.

The essential components of FTIR include a radiation source, interferometer, sample holder and a detector. Several infrared sources such as Nernst glower, globar source, incandescent wire source, high pressure mercury arc source (used in classical dispersion instruments), and tunable diode laser (helium-neon) sources may be used in FTIR. Michelson interferometer consists of a moving mirror, a fixed mirror and a beam splitter. Radiation from an infrared source is collimated by a mirror and the resultant beam is divided at the beam splitter, half the beam passes to a fixed mirror and the other half is reflected to the moving mirror. After reflection, the two beams recombine at the beam splitter and, for any particular wavelength, constructively or destructively interfere, depending on the difference in optical paths between the two arms of the interferometer. With a constant mirror velocity, the intensity of the emerging radiation at any particular wavelength modulates in a regular sinusoidal manner. In case of a broad band source the emerging beam is a complex mixture of modulation frequencies that after passing through the sample compartment, is focused into the detector. Detectors such as thermal detector (deuterated triglycine sulphate, DTGS) and quantum detector (mercury cadmium telluride, MCT) are used. Detector signal is sampled at precise intervals during the mirror scan. Both the sampling rate and the mirror velocity are controlled by the reference signal incident upon a detector, which is produced by modulation of beam from the helium-neon laser. The resulting signal from the detector is known as a interferogram which contains all the information to reconstruct the spectrum via the mathematical process known as Fourier transformation.

Nuclear Magnetic Resonance Spectrometers (NMR):

Nuclear magnetic resonance spectrometers consist of a continuously spinning super conducting magnet capable of producing a magnetic field (~ 11 tesla) cooled with liquid nitrogen in a double jacketed closed system. Sample solutions using liquid inlets or solids directly using solid inlets are introduced into the core of the magnetic field. The magnetic field produced by the spinning nucleus of elements such as 1H or 13C depend on their environment (functional moieties) in which they exist. Resonance takes place when the magnetic field of the nucleus matches with that of external magnetic field, producing a signal.

The NMR spectrum of a molecule consists of signals produced by a specific nucleus existing in different functional groups (environments) which help in its identification. A study of NMR spectrums helps in the structural elucidation of an unknown molecule. The intensity of the signal produced by a nucleus at a particular resonating frequency is dependent on the number of such resonating atoms, which makes it possible to quantify the number of atoms and provides a way for the quantitative determination. Instruments ranging from 60 MHz to 1000 MHz are currently available for NMR measurements.


Instruction manuals supplied by manufacturers should always be consulted for such matters as care, calibration, handling techniques, and operating procedures. Calibration of both the wavelength and the photometric scales should be conducted at fixed intervals. For wavelength calibration in the ultraviolet and visible regions, a quartz-mercury arc and a holmium oxide glass filter are the most common standards employed. For the near-infrared and infrared regions, a polystyrene film may be used. The photometric scale can be checked by a number of standard inorganic glass filters or by standard solutions of known transmittance.

In absorption spectrophotometry, comparisons of the sample and reference standard are best made at or within ± 1 nm of the wavelength at which maximum absorbance occurs. If matched cells are unavailable, both cells are filled with the selected solvent and any difference in absorbance should be corrected instrumentally or mathematically. The solvent should be transparent in the spectral range of interest. Water, lower alcohols, chloroform, aliphatic hydrocarbons, and many other organic solvents can be used as solvents for ultraviolet and visible measurements. For best results, the concentration of the sample solution should produce an absorbance in the range of about 0.2 to 0.7. For the infrared region, however, few solvents are suitable for sample preparation.

The solvent used in infrared spectrophotometry must not affect the material, usually sodium chloride, of which the cell is made. No solvent in appreciable thickness is completely transparent throughout the infrared spectrum. Infrared spectral grade solvents such as carbon tetrachloride R is practically transparent (up to 1 mm in thickness) from 4,000 to 1,700 cm-1 (2.5 to 6 μm). Chloroform R, dichloromethane R, and dibromomethane R are other useful solvents. Carbon disulfide IR (up to 1 mm in thickness) is suitable as a solvent to 250 cm-1 (40 μm), except in the 2,400-2,000 cm-1 (4.2-5.0 μm) and the 1,800-1,300 cm-1 (5.5-7.5 μm) regions, where it has strong absorption. Its weak absorption in the 875-845 cm-1 (11,4-11,8 μm) region should also be noted. Other solvents have relatively narrow regions of transparency (carbon disulfide, chloroform, and carbon tetrachloride are the most frequently used). (Note: These solvents are hazardous and appropriate precautions should be taken).

In some cases, the sample can be dispersed in mineral oil to form a mull, which is transferred to the salt plates. In most cases, however, the sample is dispersed in dried potassium bromide and the mixture is compressed into a tablet or pellet. The proportion of substance to the halide should be about 1 to 200. The amount taken should be such that the weight of substance per area of the disc is about 5-15 μg per mm2 , varying with the molecular weight and to some degree with the type of apparatus used. However, the concentration of the substance should be such that the strongest peak attributable to the substance reaches to between 5% and 25% transmittance. Although the infrared region extends from 2 to 40 μm, for purposes of ascertaining compliance with a reference spectrum, the range from 2.5 to 15 urn (3,800 to 650 cm-1) is usually satisfactory.

For atomic absorption measurements, the solvent should not seriously interfere with the absorption or emission processes or with the production of neutral atoms. Also, both the analyte solution and the standard solution should be as much alike as possible, especially with respect to concentration, viscosity, and surface tension.

In fluorescence spectrophotometry, test solutions are usually very dilute (10-3 to 10-7M) in order to minimize the "inner filter" effect caused by significant absorption of incident radiation by the sample near the cell surface. Other undesirable effects of highly concentrated solutions in fluorometry are the "self-quenching" and "self-absorption" phenomena that cause significant deviation from linearity. Test solutions used in fluorometry should also be free from any dust and solid particles, as they cause interference in the measurement. In some cases, before any measurement it is advisable to remove dissolved oxygen from the test solutions, due to its quenching effect. Temperature control is usually needed for extremely sensitive determinations, and baseline correction may be critical.

In turbidimetric and nephelometric measurements, it is important to minimize the settling of the suspended particles. This is generally achieved through the addition of protective colloids.

When visual colour and turbidity comparisons are made, matched colour-comparison tubes that are of the same internal diameter must be used. The solutions to be compared should be at the same temperature (preferably room temperature). For colour comparisons, the tubes are usually held vertically and illuminated from below. Viewing is done from above along the axis of the tube, against a white background. If the colours to be compared are too dark to be viewed downward through the depth of the solutions, they may be viewed horizontally across the diameter of the tubes, with the aid of a light source directed from the back of the tubes. If two layers are present, the designated layer must be viewed horizontally across the diameter of the tube.

For visual turbidity comparisons, the tubes should be viewed horizontally across the diameter of the tubes, with the aid of a light source directed at a right angle against the sides of the tubes.

When conducting limit tests involving the comparison of colour or turbidities, suitable detection instruments may be used in place of the unaided eye.


Ultraviolet and visible spectra provide only limited information about the chemical structure of a substance. However, because of the sensitivity of these techniques and the high degree of precision and accuracy in their measurements, they are employed extensively in assays and other quantitative determinations.

Near-infrared and infrared spectra, on the other hand, are unique for a given chemical compound, except for optical isomers, which have identical spectra in solution. Polymorphism and other factors, such as variations in crystal size and orientation, the grinding procedure, and the possible formation of hydrates may, however, be responsible for a difference in the infrared spectrum of a given compound in the solid state. The infrared spectrum is usually not greatly affected by the presence of small quantities of impurities (up to several percent) in the tested substance. For identification purposes the spectrum may be compared with that of a reference substance, concomitantly prepared or with a standard reference spectrum. Specificity makes the infrared spectrum one of the most valuable tools for structure elucidation and positive identification of complex organic molecules. Correlation charts and reference spectra of thousands of chemicals are readily available. The sensitivity of infrared analysis, however, is poor (about 1/100 to 1/1,000 of ultraviolet), and therefore it has only a very limited application in quantitative analysis.

Atomic absorption is the technique of choice for the quantitative determination of most of the common elements, even those in complex matrices. Although interferences may occur in the determination of some elements due to chemical interaction between different atoms in the flame (e.g., cation-anion interference), they can usually be circumvented by preliminary treatment (e.g., addition of a complexing agent) or by the optimization of the instrumentation parameters (e.g., increasing the temperature of the flame to decrease anion-cation attraction). High background signals can be corrected using deuterium background correctors or use of Zeeman furnace techniques. Use of chemical modifiers also helps in reducing background in the furnace analysis.

Fluorescence spectrophotometry has the most inherent sensitivity of all the absorption and light-scattering techniques. Concentrations as low as 10-7M can be quantitatively determined with high precision and accuracy. Fluorescence, however, is not as widespread as the other techniques because of the limited number of organic compounds in which fluorescence can be induced.

Light-scattering techniques, including turbidimetry and nephelometry, are very useful in the determination of weight-average molecular weights in dispersed colloidal systems. Several common ions can be determined using these techniques after their precipitation with suitable reagents. Generally, turbidimetry is adequate for the analysis of heavy suspensions where excessive scattering occurs. Nephelometry, on the other hand, is more suitable for the analysis of cloudy liquids where the attenuation of the radiant power is minimal.

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