Instrumental Chemical Analysis

Today chemical analysis has progressed from the initial days of volumetric and gravimetric determinations to chemical instrumentation. Instrumental analysis is more precise, reliable, and easier to carry out for both chemical analysis of constituents and, particularly, residue analysis. Initially colorimetric methods became the most dominant tools.

By taking advantage of the Beer-Lambert law of absorption, and comparing with a standard solution of known strength, the concentration of a chemical constituent can be determined at the absorption maxima in the visible region. Subsequently spectrophotometry, wherein either UV or visible spectra can be used, has become the major instrumental method for determinations.

In recent years, chromatography has become the most powerful tool for the determination of chemical compounds in plant products or extracts.

Gas Chromatography (GC)

For volatile constituents, gas-liquid chromatography, or more often simply called gas chromatography, has become very valuable for separating and analyzing chemical constituents that can be transformed into a volatile gaseous phase through controlled heating. The specific compound is then separated from other constituents while passing through a column. The mobile phase, which carries the volatile component through the column, is a carrier gas, usually an inert gas such as nitrogen or helium. The column is tubing made from glass, a polymer, or metal, which is coated with a stationary phase. The stationary phase is a microscopic layer of a suitable liquid or polymer on a nonreactive solid support. The volatile analyte of interest is then detected, using various types of detectors, depending on the class of compounds of interest.

Thermal Conductivity Detector (TCD) This is based on the change in thermal conductivity to the reference flow of a carrier gas as a result of the volatile compound. When the volatile compound emerges from the column, the thermal conductivity in the chamber, where one of the arms of a Wheatstone bridge is positioned, is reduced. This results in a detectable signal due to an upset in the electrical balance of the Wheatstone bridge.

Flame Ionization Detector (FID) The principle of an FID is based on the detection of ions formed during the combustion of organic compounds in a hydrogen flame. The ions generated are proportional to the concentration of organic compounds in the gas stream. Hydrocarbons generally have molar response factors corresponding to the carbon atoms in their molecules, whereas oxygenated and other heteroatoms tend to have a lower response factor. FID cannot detect inorganic molecules. Because it oxidizes organic molecules, it is not useful in preparatory work.

Electron Capture Detector (ECD) This is a device for detecting electronabsorbing components of high electronegativity, for example, halogenated compounds. It has a р-particle (electron) emitter, in conjugation with a make-up gas such as nitrogen flowing through the detection chamber. A typical electron emitter consists of a metal foil holding 10 pCi (370 MBq) of the radionuclide 63Ni. The electrons from the electron emitter collide with the make-up gas molecules and move towards the positively charged anode, resulting in the production of a current. As the volatile compound is carried into the detector, electron-absorbing molecules of the volatile compound under analysis capture electrons, resulting in a proportionate reduction in the current.

These detectors are very sensitive to halogenated compounds such as chlorinated pesticides.

Nitrogen-Phosphorus Detector (NPD) In this type of detector, thermal energy ionizes a volatile compound. Nitrogen and phosphorus can be selectively detected with a high degree of sensitivity, and therefore an NPD is useful for analyzing phosphorus-containing pesticides.

A concentration of hydrogen gas below the minimum required for ignition is employed. A rubidium or cesium bead ignites the hydrogen and forms a cold plasma. When excited by an alkali metal, ejection of electrons results, which are detected as a current flow between an anode and a cathode in the chamber. A nitrogen or phosphorus volatile leaving the chromatography column causes an increase in current, which can be detected.

Flame Photometric Detector (FPD) Phosphorus-containing pesticides can also be determined using FPD. This allows sensitive and selective measurement of volatile sulfur and excited hydrogen phosphorus oxide species in a reducing flame. A photomultiplier tube measures the chemiluminescent emissions from these species. By using an appropriate filter, the FPD can determine either sulfur (394 nm) or phosphorus (526 nm).

High-Performance Liquid Chromatography (HPLC) Originally known as high-pressure liquid chromatography, HPLC is a technique to detect, quantify, and even identify nonvolatile components. Here the sample is dissolved in a suitable solvent and passed through a column packed with a stationary adsorbent material. Unlike in conventional column chromatography, where passage of the dissolved material through the adsorbent material occurs through the use of gravity, HPLC relies on pumps to pass a pressurized liquid solvent containing the sample mixture through long, thin columns filled with adsorbents. Each component of the sample moves at a different speed due to differences in the intensities of adsorption on the stationary phase. This results in separation of the components as they emerge from the column.

The active component of the stationary phase in the column is usually a granular material such as silica or a polymer, of 2-50 pm in size. The separated molecules leaving the column are detected by a suitable detector.

Typically, the columns were long and thin, of 4.5 mm diameter and 250 mm length. However, more recently, columns of 2.5 mm diameter and 50 mm length have been used. To increase the efficiency, sub-2 pm diameter particles, compared with the conventional 5 pm, are being used as adsorbents. Since very high pressures are used to pass the solution through the column, this technique is often referred to as ultra high- pressure liquid chromatography (UHPLC).

UV or UV/Visual Detector UV detectors, which are frequently used, use a deuterium discharge lamp with the wavelength ranging between 190 and 380 nm. When longer wavelengths are required, an additional tungsten lamp (range 390-700 nm) is used. Combination detectors are currently available (photodiode array).

Almost all the chemical constituents that are analyzed may have absorptions in both ranges. It should be noted that not all components have similar spectra. The concentration may not be proportional to the peak size, as compounds with greater molar extinction coefficients can produce bigger peaks, even if present at a low dose.

Refractive Index Detector This can be considered as a universal detector for HPLC. The principle involved is the change in refractive index of the effluent when the compound under investigation passes the detector along with solvent. Naturally it is advantageous to have a large difference between the refractive index of the compounds and the mobile phase solvent.

Fluorescence Detector This is the most sensitive of all HPLC detectors. About 15% of all compounds fluoresce. Conjugated n-electrons in aromatic compounds produce the highest fluorescence activity. Fluorescence sensitivity is usually 10-1000 times greater than for UV detectors, even for strong UV-absorbing compounds. Moreover, fluorescence detectors are selective and specific. When compounds have specific functional groups that are excited by shorter wavelengths but emit at higher wavelengths, they are credited with having fluorescence. Aflatoxins, which can be excited in this manner and produce fluorescence emissions, are detected using fluorescence detectors as they are required to be measured at the parts per billion level.

Mass Spectrometer (MS) This produces an ion signal as a function of the mass-to-charge ratio. In order to do this, mass spectrometry works by ionizing chemical compounds to generate charged molecular fragments. The spectra are useful for determining the elemental or isotopic character of the sample and for elucidating the chemical structure of the molecule.

The ionization is achieved by bombarding a solid, liquid, or gaseous sample with electrons. The ions that emerge are separated according to their mass-to-charge ratio. The ions are detected by an electron multiplier. The atoms or molecules in the sample are then identified by correlating known masses with the identified masses or through a character fragmentation pattern.

Chromatography Combined with Mass Spectrometry

A development is combining GC or HPLC with MS, providing both mass resolving and mass determining capability. MS can not only detect, but also indicate the properties of the molecule, so much so it can even be useful in identification. Thus a complex mixture of volatile or nonvolatile compounds can be separated effectively by GC or HPLC, respectively, and the structure of individual components can be arrived at by comparing with the corresponding data for standard reference compounds. Thus in GC-MS, a stream of separated compounds is fed into the ion source, which is a metallic filament to which a voltage is applied. The filament emits electrons, which ionize the compounds that are being analyzed. The ions formed are further fragmented, yielding expected patterns of intact ions and fragments, which are passed on to the MS analyzer, resulting in the identification of the compounds.

However, in LC-MS ions are generated either by the loss or by the gain of charge from a neutral species. Here the ionization is effected by electron spray ionization.

Both GC and LC can be connected to a system with two MS instruments working in tandem to form GC-MS-MS or LC-MS-MS. Here the compounds are ionized in the first MS. The resulting chosen ion can be further fragmented by the second MS, resulting in daughter ions, which can be measured to quantify the original compound, even when present at very low levels of parts per billion or even parts per trillion.

Atomic Absorption Spectroscopy (AAS)

This is very useful for determining the concentration of an element, as a spectroscopic analytical procedure using the absorption of element-specific optical radiation (light) by free atoms in the gaseous state is utilized.

Atomic absorption is so sensitive that it can measure concentrations with an accuracy of parts per billion. The technique makes use of the wavelengths of light specifically absorbed by the element in question. This corresponds to the energies needed to promote electrons from one energy level to a higher level.

AAS is particularly useful for determining the level of heavy metals in plant products or their extracts. Besides food, it is also used in clinical, pharmaceutical, and environmental analysis.

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