Atomic absorption spectroscopy (AAS)
Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state.
In analytical chemistry the technique is used for determining the concentration of a particular element (the analyte) in a sample to be analyzed. AAS can be used to determine over 70 different elements in solution or directly in solid samples used in pharmacology, biophysics and toxicology research.
Atomic absorption spectrometry has many uses in different areas of chemistry such as clinical analysis of metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain tissue, liver, muscle tissue, semen, in some pharmaceutical manufacturing processes, minute quantities of a catalyst that remain in the final drug product, and analyzing water for its metal content.
Principles
The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte content to establish the relation between the measured absorbance and the analyte concentration and relies therefore on the Beer-Lambert Law.
In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given wavelength). This amount of energy, i.e., wavelength, is specific to a particular electron transition in a particular element. In general, each wavelength corresponds to only one element, and the width of an absorption line is only of the order of a few picometers (pm), which gives the technique its elemental selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a detector, and the ratio between the two values (the absorbance) is converted to analyte concentration or mass using the Beer-Lambert Law.
Instrumentation

Atomic absorption spectrometer block diagram
In order to analyze a sample for its atomic constituents, it has to be atomized. The atomizers most commonly used nowadays are flames and electrothermal (graphite tube) atomizers. The atoms should then be irradiated by optical radiation, and the radiation source could be an element-specific line radiation source or a continuum radiation source. The radiation then passes through a monochromator in order to separate the element-specific radiation from any other radiation emitted by the radiation source, which is finally measured by a detector.
Inductively coupled plasma atomic emission spectroscopy (ICP-AES)
Inductively coupled plasma atomic emission spectroscopy (ICP-AES), also referred to as inductively coupled plasma optical emission spectrometry (ICP-OES), is an analytical technique used for the detection of trace metals. It is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. It is a flame technique with a flame temperature in a range from 6000 to 10000 K. The intensity of this emission is indicative of the concentration of the element within the sample.
The ICP-AES is composed of two parts: the ICP and the optical spectrometer. The ICP torch consists of 3 concentric quartz glass tubes The output or “work” coil of the radio frequency (RF) generator surrounds part of this quartz torch. Argon gas is typically used to create the plasma.
When the torch is turned on, an intense electromagnetic field is created within the coil by the high power radio frequency signal flowing in the coil. This RF signal is created by the RF generator which is, effectively, a high power radio transmitter driving the “work coil” the same way a typical radio transmitter drives a transmitting antenna. Typical instruments run at either 27 or 40 MHz. The argon gas flowing through the torch is ignited with a Tesla unit that creates a brief discharge arc through the argon flow to initiate the ionization process. Once the plasma is “ignited”, the Tesla unit is turned off.
The argon gas is ionized in the intense electromagnetic field and flows in a particular rotationally symmetrical pattern towards the magnetic field of the RF coil. A stable, high temperature plasma of about 7000 K is then generated as the result of the inelastic collisions created between the neutral argon atoms and the charged particles
A peristaltic pump delivers an aqueous or organic sample into an analytical nebulizer where it is changed into mist and introduced directly inside the plasma flame. The sample immediately collides with the electrons and charged ions in the plasma and is itself broken down into charged ions. The various molecules break up into their respective atoms which then lose electrons and recombine repeatedly in the plasma, giving off radiation at the characteristic wavelengths of the elements involved.
Examples of the application of ICP-AES include the determination of metals in wine, arsenic in food, and trace elements bound to proteins.
ICP-OES is widely used in minerals processing to provide the data on grades of various streams, for the construction of mass balances.
ICP-AES is often used for analysis of trace elements in soil, and it is for that reason it is often used in forensics to ascertain the origin of soil samples found at crime scenes or on victims etc. Taking one sample from a control and determining the metal composition and taking the sample obtained from evidence and determine that metal composition allows a comparison to be made. While soil evidence may not stand alone in court it certainly strengthens other evidence.
It is also fast becoming the analytical method of choice for the determination of nutrient levels in agricultural soils. This information is then used to calculate the amount of fertiliser required to maximise crop yield and quality.
ICP-AES is used for motor oil analysis. Analyzing used motor oil reveals a great deal about how the engine is operating. Parts that wear in the engine will deposit traces in the oil which can be detected with ICP-AES. ICP-AES analysis can help to determine whether parts are failing. In addition, ICP-AES can determine what amount of certain oil additives remain and therefore indicate how much service life the oil has remaining. Oil analysis is often used by fleet manager or automotive enthusiasts who have an interest in finding out as much about their engine’s operation as possible. ICP-AES is also used during the production of motor oils (and other lubricating oils) for quality control and compliance with production and industry specifications.
Fourier transform infrared spectroscopy (FTIR)
Fourier transform infrared spectroscopy (FTIR)is a technique which is used to obtain an infrared spectrum of absorption or emission of a solid, liquid or gas. An FTIR spectrometer simultaneously collects high spectral resolution data over a wide spectral range. This confers a significant advantage over a dispersive spectrometer which measures intensity over a narrow range of wavelengths at a time.
The term Fourier transform infrared spectroscopy originates from the fact that a Fourier transform (a mathematical process) is required to convert the raw data into the actual spectrum.
IR sources
FTIR spectrometers are mostly used for measurements in the mid and near IR regions. For the mid-IR region, 2−25 µm (5000–400 cm−1), the most common source is a silicon carbide element heated to about 1200 K. The output is similar to a blackbody. Shorter wavelengths of the near-IR, 1−2.5 µm (10000–4000 cm−1), require a higher temperature source, typically a tungsten-halogen lamp. The long wavelength output of these is limited to about 5 µm (2000 cm−1) by the absorption of the quartz envelope. For the far-IR, especially at wavelengths beyond 50 µm (200 cm−1) a mercury discharge lamp gives higher output than a thermal source.
Applications
FTIR can be used in all applications where a dispersive spectrometer was used in the past (see external links). In addition, the improved sensitivity and speed have opened up new areas of application. Spectra can be measured in situations where very little energy reaches the detector and scan rates can exceed 50 spectra a second.

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis)
Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent (near-UV and near-infrared [NIR]) ranges.The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, atoms and molecules undergo electronic transitions. Absorption spectroscopy is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.
Applications
UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.
• Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. The colour of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the colour of a dilute solution of copper sulfate is a very light blue; adding ammonia intensifies the colour and changes the wavelength of maximum absorption (λmax).
• Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water-soluble compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. Tyrosine, for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases.
• While charge transfer complexes also give rise to colours, the colours are often too intense to be used for quantitative measurement.
Microwave digestion
Microwave digestion is a common technique used by elemental scientists to dissolve heavy metals in the presence of organic molecules prior to analysis by inductively coupled plasma, atomic absorption, or atomic emission measurements.
This technique is usually accomplished by exposing a sample to a strong acid in a closed vessel and raising the pressure and temperature through microwave irradiation. This increase in temperature and pressure of the low pH sample medium increases both the speed of thermal decomposition of the sample and the solubility of heavy metals in solution. Once these heavy metals are in solution, it is possible to quantify the sample through elemental techniques.
High-performance liquid chromatography (HPLC)
High-performance liquid chromatography (HPLC); formerly referred to as high-pressure liquid chromatography), is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.
HPLC has been used for manufacturing (e.g. during the production process of pharmaceutical and biological products), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and medical (e.g. detecting vitamin D levels in blood serum) purposes
Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made of solid particles (e.g. silica, polymers, etc.), 2–50 micrometers in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a “mobile phase”. Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination.
Applications
Manufacturing
As briefly mentioned, HPLC has many applications in both laboratory and clinical science. It is a common technique used in pharmaceutical development as it is a dependable way to obtain and ensure product purity.While HPLC can produce extremely high quality (pure) products, it is not always the primary method used in the production of bulk drug materials. According to the European pharmacopoeia, HPLC is used in only 15.5% of syntheses. However, it plays a role in 44% of syntheses in the United States pharmacopoeia. This could possibly be due to differences in monetary and time constraints, as HPLC on a large scale can be an expensive technique. An increase in specificity, precision, and accuracy that occurs with HPLC unfortunately corresponds to an increase in cost.
Legal
This technique is also used for detection of illicit drugs in urine. The most common method of drug detection is an immunoassay. This method is much more convenient. However, convenience comes at the cost of specificity and coverage of a wide range of drugs. As HPLC is a method of determining (and possibly increasing) purity, using HPLC alone in evaluating concentrations of drugs is somewhat insufficient. With this, HPLC in this context is often performed in conjunction with mass spectrometry. Using liquid chromatography instead of gas chromatography in conjunction with MS circumvents the necessity for derivitizing with acetylating or alkylation agents, which can be a burdensome extra step.This technique has been used to detect a variety of agents like doping agents, drug metabolites, glucuronide conjugates, amphetamines, opioids, cocaine, BZDs, ketamine, LSD, cannabis, and pesticides. Performing HPLC in conjunction with Mass spectrometry reduces the absolute need for standardizing HPLC experimental runs.
Research
Similar assays can be performed for research purposes, detecting concentrations of potential clinical candidates like anti-fungal and asthma drugs. This technique is obviously useful in observing multiple species in collected samples, as well, but requires the use of standard solutions when information about species identity is sought out. It is used as a method to confirm results of synthesis reactions, as purity is essential in this type of research. However, mass spectrometry is still the more reliable way to identify species.
Medical
Medical use of HPLC can include drug analysis, but falls more closely under the category of nutrient analysis. While urine is the most common medium for analyzing drug concentrations, blood serum is the sample collected for most medical analyses with HPLC.Other methods of detection of molecules that are useful for clinical studies have been tested against HPLC, namely immunoassays. In one example of this, competitive protein binding assays (CPBA) and HPLC were compared for sensitivity in detection of vitamin D. Useful for diagnosing vitamin D deficiencies in children, it was found that sensitivity and specificity of this CPBA reached only 40% and 60%, respectively, of the capacity of HPLC. While an expensive tool, the accuracy of HPLC is nearly unparalleled.

Gas chromatography (GC)
Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.
In gas chromatography, the mobile phase (or “moving phase”) is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. Helium remains the most commonly used carrier gas in about 90% of instruments although hydrogen is preferred for improved separations.[3] The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column (an homage to the fractionating column used in distillation). The instrument used to perform gas chromatography is called a gas chromatograph (or “aerograph”, “gas separator”).
The gaseous compounds being analyzed interact with the walls of the column, which is coated with a stationary phase. This causes each compound to elute at a different time, known as the retention time of the compound. The comparison of retention times is what gives GC its analytical usefulness.
Applications
In general, substances that vaporize below 300 °C (and therefore are stable up to that temperature) can be measured quantitatively. The samples are also required to be salt-free; they should not contain ions. Very minute amounts of a substance can be measured, but it is often required that the sample must be measured in comparison to a sample containing the pure, suspected substance known as a reference standard.
Various temperature programs can be used to make the readings more meaningful; for example to differentiate between substances that behave similarly during the GC process.
Professionals working with GC analyze the content of a chemical product, for example in assuring the quality of products in the chemical industry; or measuring toxic substances in soil, air or water. GC is very accurate if used properly and can measure picomoles of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in gaseous samples.
In practical courses at colleges, students sometimes get acquainted to the GC by studying the contents of Lavender oil or measuring the ethylene that is secreted by Nicotiana benthamiana plants after artificially injuring their leaves. These GC analyse hydrocarbons (C2-C40+). In a typical experiment, a packed column is used to separate the light gases, which are then detected with a TCD. The hydrocarbons are separated using a capillary column and detected with a FID. A complication with light gas analyses that include H2 is that He, which is the most common and most sensitive inert carrier (sensitivity is proportional to molecular mass) has an almost identical thermal conductivity to hydrogen (it is the difference in thermal conductivity between two separate filaments in a Wheatstone Bridge type arrangement that shows when a component has been eluted). For this reason, dual TCD instruments used with a separate channel for hydrogen that uses nitrogen as a carrier are common. Argon is often used when analysing gas phase chemistry reactions such as F-T synthesis so that a single carrier gas can be used rather than two separate ones. The sensitivity is less, but this is a trade off for simplicity in the gas supply.
Gas Chromatography is used extensively in forensic science. Disciplines as diverse as solid drug dose (pre-consumption form) identification and quantification, arson investigation, paint chip analysis, and toxicology cases, employ GC to identify and quantify various biological specimens and crime-scene evidence.

Ion Chromatography
Ion chromatography (or ion-exchange chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids. It is often used in protein purification, water analysis, and quality control.] The water-soluble and charged molecules such as proteins, amino acids, and peptides bind to moieties which are oppositely charged by forming ionic bonds to the insoluble stationary phase.The equilibrated stationary phase consists of an ionizable functional group where the targeted molecules of a mixture to be separated and quantified can bind while passing through the column. This method applies the idea of the interaction between molecules and the stationary phase which are charged oppositely to each other. Cation exchange chromatography is used when the desired molecules to separate are cations; and anion exchange chromatography is used to separate anions, meaning that the beads in the column contain positively charged functional groups to attract the anions. The bound molecules then can be eluted and collected using an eluant which contains anions and cations by running higher concentration of ions through the column or changing pH of the column. One of the primary advantages for the use of ion chromatography is only one interaction involved during the separation as opposed to other separation techniques; therefore, ion chromatography may have higher matrix tolerance. However, there are also disadvantages involved when performing ion-exchange chromatography, such as constant evolution with the technique which leads to the inconsistency from column to column.
Uses
Clinical utility
A use of ion chromatography can be seen in the argentation ion chromatography. Usually silver and compounds containing acetylenic and ethylenic bonds have very weak interactions. This phenomenon has been widely tested on olefin compounds. The ion complexes the olefins make with silver ions are weak and made based on the overlapping of pi, sigma, and d orbitals and available electrons therefore cause no real changes in the double bond. This behavior was manipulated to separate lipids, mainly fatty acids from mixtures in to fractions with differing number of double bonds using silver ions. The ion resins were impregnated with silver ions, which were then exposed to various acids (silicic acid) to elute fatty acids of different characteristics.
Detection limits as low as 1 μM can be obtained for alkali metal ions. It may be used for measurement of HbA1c, porphyrin and with water purification. Ion Exchange Resins(IER) have been widely used especially in medicines due to its high capacity and the uncomplicated system of the separation process. One of the synthetic uses is to use Ion Exchange Resins for kidney dialysis. This method is used to separate the blood elements by using the cellulose membraned artificial kidney.Another clinical application of ion chromatography is in the rapid anion exchange chromatography technique used to separate creatine kinase (CK) isoenzymes from human serum and tissue sourced in autopsy material (mostly CK rich tissues were used such as cardiac muscle and brain)These isoenzymes include MM, MB, and BB, which all carry out the same function given different amino acid sequences. The functions of these isoenzymes are to convert creatine, using ATP, into phosphocreatine expelling ADP. Mini columns were filled with DEAE-Sephadex A-50 and further eluted with tris- buffer sodium chloride at various concentrations (each concentration was chosen advantageously to manipulate elution). Human tissue extract was inserted in columns for separation. All fractions were analyzed to see total CK activity and it was found that each source of CK isoenzymes had characteristic isoenzymes found within. Firstly, CK- MM was eluted, then CK-MB, followed by CK-BB. Therefore, the isoenzymes found in each sample could be used to identify the source, as they were tissue specific.
Using the information from results, correlation could be made about the diagnosis of patients and the kind of CK isoenzymes found in most abundant activity. From the finding, about 35 out of 71 patients studied suffered from heart attack (myocardial infarction) also contained an abundant amount of the CK-MM and CK-MB isoenzymes. Findings further show that many other diagnosis including renal failure, cerebrovascular disease, and pulmonary disease were only found to have the CK-MM isoenzyme and no other isoenzyme. The results from this study indicate correlations between various diseases and the CK isoenzymes found which confirms previous test results using various techniques. Studies about CK-MB found in heart attack victims have expanded since this study and application of ion chromatography.
Industrial applications
Since 1975 ion chromatography has been widely used in many branches of industry. The main beneficial advantages are reliability, very good accuracy and precision, high selectivity, high speed, high separation efficiency, and low cost of consumables. The most significant development related to ion chromatography are new sample preparation methods; improving the speed and selectivity of analytes separation; lowering of limits of detection and limits of quantification; extending the scope of applications; development of new standard methods; miniaturization and extending the scope of the analysis of a new group of substances. Allows for quantitative testing of electrolyte and proprietary additives of electroplating baths.It is an advancement of qualitative hull cell testing or less accurate UV testing. Ions, catalysts, brighteners and accelerators can be measured.Ion exchange chromatography has gradually become a widely known, universal technique for the detection of both anionic and cationic species. Applications for such purposes have been developed, or are under development, for a variety of fields of interest, and in particular, the pharmaceutical industry. The usage of ion exchange chromatography in pharmaceuticals has increased in recent years, and in 2006, a chapter on ion exchange chromatography was officially added to the United States Pharmacopia-National Formulary (USP-NF). Furthermore, in 2009 release of the USP-NF, the United States Pharmacopia made several analyses of ion chromatography available using two techniques: conductivity detection, as well as pulse amperometric detection. Majority of these applications are primarily used for measuring and analyzing residual limits in pharmaceuticals, including detecting the limits of oxalate, iodide, sulfate, sulfamate, phosphate, as well as various electrolytes including potassium, and sodium. In total, the 2009 edition of the USP-NF officially released twenty eight methods of detection for the analysis of active compounds, or components of active compounds, using either conductivity detection or pulse amperometric detection.
Drug Development
There has been a growing interest in the application of IC in the analysis of pharmaceutical drugs. IC is used in different aspects of product development and quality control testing. For example, IC is used to improve stabilities and solubility properties of pharmaceutical active drugs molecules as well as used to detect systems that have higher tolerance for organic solvents.IC has been used for the determination of analytes as a part of a dissolution test. For instance, calcium dissolution tests have shown that other ions present in the medium can be well resolved among themselves and also from the calcium ion. Therefore, IC has been employed in drugs in the form of tablets and capsules in order to determine the amount of drug dissolve with time. IC is also widely used for detection and quantification of excipients or inactive ingredients used in pharmaceutical formulations. Detection of sugar and sugar alcohol in such formulations through IC has been done due to these polar groups getting resolved in ion column. IC methodology also established in analysis of impurities in drug substances and products. Impurities or any components that are not part of the drug chemical entity are evaluated and they give insights about the maximum and minimum amounts of drug that should be administered in a patient per day.

X-ray Spectroscopy
Characteristic X-ray Spectroscopy
When an electron from the inner shell of an atom is excited by the energy of a photon, it moves to a higher energy level, when it returns to the low energy level the energy which it previously gained by the excitation is emitted as a photon which has a wavelength that is characteristic for the element (there could be several characteristic wavelengths per element). Analysis of the X-ray emission spectrum produces qualitative results about the elemental composition of the specimen. Comparison of the specimen’s spectrum with the spectra of samples of known composition produces quantitative results (after some mathematical corrections for absorption, fluorescence and atomic number). Atoms can be excited by a high-energy beam of charged particles such as electrons (in an electron microscope for example), protons (see PIXE) or a beam of X-rays (see X-ray fluorescence, or XRF). These methods enable elements from the entire periodic table to be analysed, with the exception of H, He and Li. In electron microscopy an electron beam excites X-rays; there are two main techniques for analysis of spectra of characteristic X-ray radiation: energy-dispersive X-ray spectroscopy (EDS) and wavelength dispersive X-ray spectroscopy (WDS).
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