BASIC INSTRUMENTAL TECHNIQUES OF ANALYTICAL CHEMISTRY

Thomas J. Bruno

The following section provides a very brief description of the major instrumental methods of chemical analysis. Please note that these paragraphs are general descriptions and are not meant to convey a comprehensive knowledge on these topics. The reader is referred to one of many excellent texts on instrumental methods of chemical analysis for additional details.

Suggested Reading

1. Skoog, D. A., Holler, F. J., and Crouch, S. R., Principles of Instrumental Analysis, Sixth Edition, Thomson Brooks/Cole Publishing, Belmont, CA, 2007.
2. Robinson, J. W., Skelly Frame, E. M., and Frame II, G. M., Undergraduate Instrumental Analysis, Sixth Edition, CRC Press, Boca Raton, FL, 2004.
3. Pungor, E., A Practical Guide to Instrumental Analysis, CRC Press, Boca Raton, FL, 1994.

• Separation Methods

• Gas Chromatography (GC): A separation method in which the sample or solute is vaporized (usually in a solvent, but sometimes neat or free of solvent) and passed through a medium under the influence of a carrier gas. The medium is called the stationary phase, in contrast to the carrier gas, which is mobile. The most common modern stationary phases are based on open tubular or capillary columns, in which the separation medium coats the inside periphery of a tube (typically tenths of millimeters in inside diameter) that is between 25 and 60 meters long. Older media are packed columns, consisting of packed beds, which are still used for gas analysis. In these applications, a solid sorbent is very common, and this is called gas-solid chromatography. Some open tubular columns are available with solid sorbents as well. Interactions of the solute with the separation medium affect the separation of the components of the mixture. A wide variety of detectors is available for general or specific applications. One of the most useful combinations is gas chromatography coupled with mass spectrometry (GC-MS). Solutes amenable to analysis by GC are usually of moderate volatility and relative molecular mass, usually not exceeding a relative molecular mass of 400. The most common stationary phases are cross-linked polymers based on dimethyl polysiloxane, the backbone of which can be derivatized with ligands to provide specific interactions. It is also possible to incorporate stereogenic (chiral) stationary phases as well.
• Liquid Chromatography (LC, HPLC): A separation method in which a sample or solute (usually in a solvent, but sometimes neat or free of solvent) is passed through a medium under the influence of a carrier liquid. The medium is called the stationary phase, in contrast to the carrier, which is a mobile phase. Unlike gas chromatography, where the carrier gas plays little role other than mass transfer, the mobile phase in liquid chromatography is a controllable variable whose polarity and other properties are varied, in addition to the interactions with the stationary phase, to affect separation. The stationary phase in liquid chromatography is usually a micrometer-size particle packed bed that requires a high-pressure solvent system to cause mass flow. Liquid chromatographic systems have therefore been called high-pressure liquid chromatography (HPLC), although the acronym is usually taken to mean high-performance liquid chromatography. Many variations of the method have been developed for specific analysis. For example, gel permeation chromatography is an adaptation used for the separation of polymers. Affinity chromatography is similar in concept to gel permeation chromatography but uses the specific interaction between an antibody and an antigen. The use of stereogenic (or chiral) stationary phases is also an important development, especially in the analysis of pharmaceuticals.
• Thin-Layer Chromatography (TLC): A separation method in which a stationary phase (typically a polar adsorbent such as alumina or silica gel) coated on a sheet of plastic, aluminum, or glass is used with a mobile phase usually consisting of a solvent or mixture of solvents in a beaker. The solute is applied as a blotted spot just above the end of the adsorbent-coated plate, and then the end is immersed into the solvent (but not so far as to immerse the solute spot). Commercial plates are robust, plastic sheets that can be cut to the size desired. In earlier applications, filter paper has been used in TLC, giving rise to the term “paper chromatography,” which is rarely used today. Solvent is then drawn up through the adsorbent coating by capillary action. Separation results from a combination of interactions with the adsorbent and solvent. Commonly, the separated components are rendered visible by spraying stains or reactants on the plate after separation has been completed. It is also common to view the “developed” plate under an ultraviolet lamp, to visualize spots that may be fluorescent.
• Supercritical Fluid Chromatography and Extraction: A separation technique similar to other extraction and chromatographic methods, but in which the mobile phase is actually a fluid in its supercritical fluid state. A supercritical fluid is a fluid that is held above its critical temperature and pressure, and for which no application of additional pressure can result in the development of a liquid phase. Supercritical fluids are unique in that while they are chosen to have liquid-like densities, the mass transfer properties are very much similar to those of liquids. Supercritical fluid chromatography remains a niche method that is applicable to pharmaceuticals and other high relative molecular mass solutes. Supercritical extraction, on the other hand, is more widely used as a sample preparation method, especially in pharmaceutical analyses, polymers, and environmental analyses.
• Electrophoresis: A family of separation methods based on the motion induced in particles by an applied, uniform electric field. The most common application of electrophoresis is gel electrophoresis, in which a sample (typically proteins, amino acids, nucleic acids, etc.) is applied to a channel that is formed in a cross-linked polymer, usually polyacrylamide or agarose (the gel). The speed at which the individual species move through the gel under the influence of the field is determined largely by the size of the species, as expressed by the mass-to-charge ratio. After separation, the individual species usually appear as discrete bands that may be better visualized by staining with ethidium bromide, silver, or Coomassie Brilliant Blue dye. Other related and more specific techniques include isoelectric focusing, pulsed-field gel electrophoresis, immunoelectrophoresis, and isotachophoresis.
• Purge-and-Trap Sampling: Purge-and-trap sampling is a family of methods that are used to capture the headspace above a condensed phase for subsequent analysis, most often for complex mixtures, environmental samples, etc. The headspace is the vapor space that develops above any condensed (solid or liquid) phase. Thermodynamics assures us that the concentration of a particular analyte found in the headspace will be different than that found in the condensed phase, but often the relationship is predictable. The value in the method comes from the simplicity; sample preparation is usually far simpler than the cleanup that is typically required for many complex mixtures. Purge-and-trap methods fall into two general categories: static and dynamic. The dynamic method typically uses a sweep gas to continuously purge vapor analytes into a cold (cryogenic) trap or an adsorbent. Modern dynamic purge-and-trap methods include porous layer open tubular (PLOT) column cryo-adsorption, which uses a combined adsorbent and cryo-trapping approach on a high-efficiency platform. Static methods typically employ a syringe to pressurize the headspace above a condensed phase, followed by uptake of the pressurized headspace into a trap. A modern static method (that usually is done without pressurization) is solid-phase microextraction (SPME). This method utilizes a fiber coated with a stationary phase (similar to stationary phases used for gas and liquid chromatography) at the end of a wire mounted in a syringe needle.

• Spectroscopic Methods

• Mass Spectrometry (MS): An analytical technique in which charged particles or radical ions are produced from a sample by either electron impact (bombardment with a stream of electrons) or chemical ionization (interaction with a small charged ion). Analysis on the basis of mass-to-charge ratio is performed on fragments of the molecule that develop after the initial ionization. The method is very useful for mass determination and structure determination on the basis of the induced fragmentation pattern. The charged fragments can be separated or analyzed by a magnetic sector, a quadrupole, an ion trap, by time of flight, or by cyclotroning. When coupled to separation techniques such as gas or liquid chromatography, a nearly universal qualitative detection capability is provided. Structure determination can be performed by comparison to well-known fragmentation patterns or characteristics. When a mass spectrometer is capable of high mass resolution, a nearly unequivocal identification of a compound formula is possible. While direct interfaces and gas chromatographs are the most common sample introduction techniques, many others are available for specific applications. Matrix-assisted laser desorption and ionization (MALDI) is often used in time-of-flight instruments and is especially useful for the analysis of biopolymers. Inductively coupled plasma ionization is capable of producing mass spectra at high sensitivity for many metals and some nonmetals. When coupled to liquid chromatography instrumentation, thermospray and electrospray methods have been used with HPLC for analytes that are not amenable to gas chromatography separation.
• Ultraviolet Spectrophotometry (UV, UV-Vis): A spectroscopic technique that focuses on electronic transitions within the visible and ultraviolet regions of the electromagnetic spectrum for excitation and detection. The practical ultraviolet region extends from 190 nm to 400 nm in wavelength. The UV region can be divided into subranges: near-UV: 300 nm to 400 nm, mid-UV: 300 nm to 200 nm, far-UV: 200 nm to 122 nm, and vacuum-UV: 200 nm to 100 nm. Other divisions are possible, but these are less important for analytical chemistry. The visible region, so called because of the response of human vision, extends from about 390 nm to 750 nm in wavelength. Although it is possible to use ultraviolet visible spectrophotometry for structure determination, it is most often used as a quantitative tool. The wavelength-structure correlations are not as detailed, nor the spectra as sharp, as with other spectroscopic methods such as infrared spectrophotometry. The utility of UV-Vis absorptions for many organic compounds has led to this instrument being adapted as a detector for liquid chromatography. Related to ultraviolet spectrophotometry are fluorescence spectroscopic methods. In these methods, the energy emitted is at a different wavelength (usually longer, of lower energy), and this is typically detected perpendicularly to the incident excitation beam. This makes fluorescence spectroscopy more sensitive than UV-Vis spectroscopy. This type of instrument is also incorporated as a detector for liquid chromatography.
• Infrared Spectrophotometry (IR, FTIR): A spectroscopic technique that focuses on molecular vibrations (with a concurrent change in dipole moment) within the infrared (IR) region of the electromagnetic spectrum for excitation and detection. This region is further divided into three separate but overlapping ranges. The near-infrared (high-energy IR, approximately 14 000 cm⁻¹ to 4000 cm⁻¹, 0.8 μm to 2.5 μm wavelength) is used to study overtone or harmonic vibrations. The mid-infrared (mid-range energy IR, approximately 4000 cm⁻¹ to 400 ⁻¹, 2.5 μm to 25 μm wavelength) is used to study the fundamental vibrations and associated rotational-vibrational combinations. The far-infrared (low-energy IR, approximately 400 cm⁻¹ to 10 cm⁻¹, 25 μm to 1000 μm wavelength) is close to the microwave region and is used to study rotational transitions. Most modern instruments use the Fourier transform (FT) technique to record the spectrum over all wavelengths, rather than by scanning through the wavelengths. The absorbances of the IR radiation are associated with specific chemical moieties, and a study of the spectra can be used to aid in structure determination. One often uses structure correlation charts to aid in the assignment of absorbance bands. An analysis of the intensity of the absorptions can also be used for quantitative analysis.
• Nuclear Magnetic Resonance Spectrometry (NMR): A spectroscopic method that takes advantage of magnetic nuclei (nuclei with an odd number of protons and/or of neutrons, having an intrinsic magnetic moment and angular momentum), placed in a magnetic field will absorb pulses of electromagnetic radiation and then radiate this energy back out. For these nuclei, the energy and signal intensity are proportional to the applied magnetic field. The power of NMR results from the ability to probe the molecular environment around a particular nucleus, thus making it long a tool of the organic chemist. This is done by measurement of the chemical shift (or frequency) of an absorption, and by the analysis of splitting patterns, which are caused by the influence of adjacent nuclei. New high-field, high-sensitivity instruments have given this technique more applications in analytical chemistry. The most commonly studied nuclei are ¹H (proton, the most NMR-sensitive isotope after the radioactive ³H) and ¹³C. With high-field instruments, additional nuclei are accessible: ²H, ¹⁰B, ¹¹B, ¹⁴N, ¹⁵N, ¹⁷O, ¹⁹F, ²³Na, ²⁹Si, ³¹P, ³⁵Cl, ¹¹³Cd, ¹²⁹Xe, and ¹⁹⁵Pt.
• Raman Spectroscopy: A vibrational spectroscopic method that arises from the inelastic scattering of monochromatic radiation by molecules that undergo a change in polarizability during the vibration. This is in contrast to infrared spectrophotometry, in which a change in the dipole moment occurs during the vibration. When radiation (typically light from a laser in the visible, near-infrared, or near-ultraviolet range) is scattered, a small fraction of the scattered radiation is observed to have a different frequency (the Raman effect). The variations of Raman spectroscopy are used to locate functional groups or chemical bonds in molecules. There are several variations in the approach to Raman spectroscopy. In resonance Raman spectroscopy, the excitation wavelength is matched to an electronic transition of the molecule, enhancing the vibrational modes. In coherent anti-Stokes Raman spectroscopy (CARS), two laser beams are used to generate a coherent anti-Stokes frequency beam. In surface-enhanced Raman spectroscopy (SERS), surface plasmons (a quantum of plasma oscillation) on a silver or gold colloid on a surface (such as a mirror) are excited by the laser, resulting in an increase in the electric fields surrounding the metal.
• Atomic Absorption and Emission Spectroscopy (AA, AES): Two related spectroscopic methods applied primarily to the analysis of inorganic compounds. Atomic absorption procedures use the absorption of optical radiation (light) by free atoms in the gaseous state. The light can be produced by a hollow cathode lamp, an electrodeless discharge lamp, or a deuterium lamp. The light is absorbed by the analyte during an electronic transition, the wavelength of which corresponds to only one element in the analyte, and the width of an absorption line is only of the order of a few picometers. This method can be used for the quantitative determination (on the basis of a calibration curve) of approximately 70 different elements in solution or directly in solid samples. Atomic emission spectroscopy (AES) uses the light emitted by a vaporized sample in a flame, plasma, arc, spark, or laser, at a particular wavelength, to determine the atomic spectrum (for determination of the elemental composition) of an element in a sample. The wavelength of the atomic spectral line gives the identity of the element while the intensity of the emitted light is proportional to the number of atoms of the element. No single source, as described above, is optimal for a given sample, and it is the choice of source that distinguishes the various techniques.

• Miscellaneous Methods

• Colorimetry, Spot Tests, Presumptive Tests: Rapid, simple tests based on color change are frequently used as the basis of preliminary conclusions or approximate concentration measurement. Colorimetric methods use simple comparative instruments to determine the concentration of colored compounds in solution. These devices, called colorimeters, can be manual or automatic, and use filtered light in the visible region (between 400 nm and 700 nm). In both cases, the operation depends upon having multiple solutions, including a blank, for comparison with a solution of unknown concentration. Manual colorimeters function by measuring the variable light path through the unknown solution as compared to a known solution until a match is achieved visually. The product of concentration and path length matches when the concentrations are the same, so an unknown concentration may be obtained by a proportion. Automated devices function similarly but with photocells. Colorimetric tests are usually done following four basic protocols: (1) an unknown can be treated/reacted with a reagent to form a new compound which is colored, (2) a chelate complex is formed having a different color than the starting compound, (3) a colored compound is oxidized or “bleached” by another compound, resulting in less color, or (4) an intermediate is formed that can be oxidized or reduced later to a colored compound. Related to colorimetric methods are spot tests, often called presumptive tests in forensic science (because they are used to establish probable cause for ordering and performing more sophisticated tests), which are the observation of color changes upon the addition of one or more reagents to an unknown. Spot tests are inherently simple and are done with a drop or two of reagent(s), on a filter paper or other suitable medium, and generate minimal waste. Usually instrumentation is not used, one merely notes a color change, although simple devices such as handheld color space analyzers (used in the paint industry) can be helpful. Other approaches include the recording of a digital photograph of the spot and the determination of the L*, a*, and b* axes in LAB color space. An example of spot testing for presumptive purposes includes the addition of cobalt thiocyanate (CoC₂N₂S₂) to cocaine, producing a blue color (the Scott test). These tests are presumptive rather than confirmatory because of the potential of false positives. Thus, the Scott test will also produce a blue coloration in the presence of lidocaine and diphenylhydramine.
• Refractometry: Refractometry is the measurement of the degree to which the path of electromagnetic radiation, specifically light, is bent upon traversing from one medium to another. Indirectly, a refractometer measures the speed of light because the refractive index, the measurand provided by a refractometer, is defined as the speed of light in vacuo divided by the speed of light in a particular medium (which can be a gas, liquid, or solid). Refractive index typically varies between 1.3 and 1.7 for most compounds, and being a ratio, it is dimensionless.  The refractive index of vacuum is by definition unity. Refraction is the result of differing densities of media; light passes through dense media slower than it passes through less dense media. Refractive index is dependent on temperature and the wavelength of light used for the measurement, and when both are known for a media, it can be used as an indication of composition or concentration. Thus, the refractive index is typically reported measured with white light from the sodium D-line (589 nm), and often at a temperature that is near ambient (20 ^oC).  The measurand is thus indicated as n_D^20. As an analytical tool, refractometry is used in four primary ways. First, it is used to help identify compounds by comparison with known values. Second, it is used to assess purity by comparison with the refractive index of a pure material. Third, it is used as a measure of solute concentration of a solution, by reference to a calibration curve. Finally, it is used as the basis of universal detection in HPLC. Refractometers are commercially available as small handheld instruments, both manual and digital (that can be carried in a pocket), Abbe refractometers (the typical lab bench device usually equipped with a thermostat), and in-line refractometers such as those used for HPLC detectors.
• Thermal Analysis (TA) Methods: A family of analytical techniques in which various properties of a sample are examined as a function of changing temperature at a particular rate of change. For chemical analysis, the most common thermal analysis techniques include differential thermal analysis (DTA) and thermogravimetric analysis (TGA). Many other thermal analysis methods are available, perhaps the most common of which is differential thermal analysis, used for determination of phase transitions. In chemical analysis, TGA determines a mass change as a function of temperature. This is used to determine decomposition or degradation temperatures, moisture content of materials (although Karl Fisher coulombic titrimetry is also used for this), the level of inorganic and organic components in materials, decomposition points of explosives, and solvent residues in materials. DTA, on the other hand, monitors temperature change rather than mass change. In this respect, it is useful as a complementary technique to probe the energetics of decomposition, moisture loss, etc. Other thermal analysis methods, such as differential scanning calorimetry (DSC), are not primarily analytical tools but rather thermophysical property measurement tools.
• Electrochemical Methods: A family of techniques that analyze the effect and role of electricity in either creating or serving as an outcome of a chemical change. These techniques include electrolysis, electrogravimetry, cyclic voltammetry, linear sweep voltammetry, electrochemical titrations, and the newer area of nanoelectrochemistry. Electrolysis leads to the separation and isolation of metals originally in a molten or a solution (ionic) mixture on an electrode using a direct electric current (DC) and a voltage called the decomposition potential. Electrogravimetry includes electroplating, electrophoretic deposition, and underpotential deposition. These methods are closely related to coulometry, which quantitatively measures the amount of matter transformed during electrolysis by using Faraday’s laws. Cyclic voltammetry is a potentiodynamic electrochemical measurement where the electrode potential is ramped linearly versus time. Once the set potential is reached, the working electrode’s potential is ramped toward the opposite direction to return to the initial potential thus creating a cycle which can be repeated at will. Linear sweep voltammetry involves the measurement of the current at a working electrode while the potential between the working electrode and a reference electrode is plotted linearly against time. Electrometric titration refers to any technique that uses an electrometer, or an instrument that determines, or even detects, the magnitude of a potential difference or charge by the different electrostatic forces between charged species. Nano-electrochemistry is a recent branch of electrochemistry that studies the electrochemical properties of nanometer-size materials. It uses nano-electrodes whose size is in the order of 1 to100 nm and are made of metals or semiconducting materials. It has created a significant impact in the development of many sensors and the study of reactions that involve extremely low concentrations.
• X-Ray Methods: A family of techniques that utilize radiation in the x-ray region, with wavelengths between 0.01 nm to 10 nm (corresponding to frequencies in the 3 × 10¹⁶ Hz to 3 × 10¹⁹ Hz) to analyze for the presence of elements in a sample. In x-ray fluorescence spectroscopy (XRS), short wavelength x-rays are used to excite secondary or fluorescent x-rays from a sample. The wavelength of x-rays used to excite the sample must be shorter than the expected fluorescence wavelength. The sample is typically presented as a powdered solid on a glass plate. Spectra are generated by changing the incident angle between the source and the sample with a device called a goniometer. The fluorescent signals obtained are very precise and specific and can rival wet chemical methods for the identification of elements. Auger electron spectroscopy (AES) is a surface analysis method that measures the electrons emitted from a surface by electron bombardment of the surface. This method is considered to be in the same family as x-ray methods because during electron bombardment, the surface can lose energy either by electron emission (the Auger effect, via Auger electron emission) or x-ray emission. These methods are in contrast to x-ray diffraction methods, in which a crystal structure measurement is desired.
• Flow Injection Analysis (FIA): An analytical protocol that seeks to replace the manual “test tube and beaker” aspects of wet chemical analysis by injecting an analyte in a flowing stream of a carrier reactant. As the analyte flows with the reactant stream, it diffuses into the reactant and product forms. Ultimately, the product zone, under the influence of the moving reactant, is passed into a detector section. The detection devices can consist of the same wide variety as is used in HPLC. The major advantage of flow injection analysis is the automation and decreased uncertainty associated with sampling and reagent addition. Strict control of reagent concentration, flow rate, and analyte volume is possible. Modern applications of FIA include the sequential addition of analyte and reactant in a stream so that the two are “stacked” in an inert carrier. They then mix by the parabolic flow profile of a laminar flowing stream in a tube. This arrangement can be miniaturized within a sampling valve, forming the so-called lab-in-a-valve approach.