Spectroscopic methods

Spectrophotometry

Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry deals with visible light, near-ultraviolet, and near-infrared, but does not cover time-resolved spectroscopic techniques.

Spectrophotometry uses photometers that can measure a light beam’s intensity as a function of its color (wavelength) known as spectrophotometers. Important features of spectrophotometers are spectral bandwidth, (the range of colors it can transmit through the test sample), and the percentage of sample-transmission, and the logarithmic range of sample-absorption and sometimes a percentage of reflectance measurement.

A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm – 2500nm using different controls and calibrations.

UV-visible spectrophotometry

Visible region 400–700nm spectrophotometry is used extensively in colorimetry science. It is a known fact that it operates best at the range of 0.2-0.8 O.D. Ink manufacturers, printing companies, textiles vendors, and many more, need the data provided through colorimetry. They take readings in the region of every 5–20 nanometers along the visible region, and produce a spectral reflectance curve or a data stream for alternative presentations. These curves can be used to test a new batch of colorant to check if it makes a match to specifications, e.g., ISO printing standards.

Traditional visible region spectrophotometers cannot detect if a colorant or the base material has fluorescence. This can make it difficult to manage color issues if for example one or more of the printing inks is fluorescent. Where a colorant contains fluorescence, a bi-spectral fluorescent spectrophotometer is used. There are two major setups for visual spectrum spectrophotometers, d/8 (spherical) and 0/45. The names are due to the geometry of the light source, observer and interior of the measurement chamber. Scientists use this instrument to measure the amount of compounds in a sample. If the compound is more concentrated more light will be absorbed by the sample; within small ranges, the Beer-Lambert law holds and the absorbance between samples vary with concentration linearly. In the case of printing measurements two alternative settings are commonly used- without/with uv filter to control better the effect of uv brighteners within the paper stock.

Samples are usually prepared in cuvettes; depending on the region of interest, they may be constructed of glass, plastic (visible spectrum region of interest), or quartz (Far UV spectrum region of interest).

Applications

Atomic absorption spectroscopy

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. Atomic absorption spectroscopy was first used as an analytical technique, and the underlying principles were established by Robert Wilhelm Bunsen and Gustav Robert Kirchhoff. Atomic absorption spectrometry has many uses in different areas of chemistry such as:

Clinical analysis: Analyzing metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain tissue, liver, muscle tissue, semen

Pharmaceuticals: In some pharmaceutical manufacturing processes, minute quantities of a catalyst that remain in the final drug product

Water analysis: Analyzing water for its metal content.

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 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.

FTIR

Fourier transform infrared spectroscopy (FTIR) is a technique which is used to obtain an infrared spectrum of absorption, emission, photoconductivity or Raman scattering of a solid, liquid or gas. An FTIR spectrometer simultaneously collects spectral data in 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. FTIR has made dispersive infrared spectrometers all but obsolete, opening up new applications of infrared spectroscopy.FTIR is a method of measuring an infrared absorption spectrum.

Beam splitter

The beam-splitter can not be made of common types of glass, as they are opaque to infrared radiation of wavelengths longer than about 2.5 μm. A thin film, usually of a plastic material, is used instead.

Applications

FTIR can be used in all applications where the multiplex and throughput advantages have opened up new areas of application. These include:

  • GC-IR (gas chromatography-infrared spectrometry). A gas chromatograph can be used to separate the components of a mixture. The fractions containing single components are directed into an FTIR spectrometer, to provide the infrared spectrum of the sample. This technique is complementary to GC-MS (gas chromatography-mass spectrometry). The GC-IR method is particularly useful for identifying isomers, which by their nature have identical masses. FTIR has also been applied to the analysis of liquid chromatography
  • TG-IR (thermogravimetry-infrared spectrometry) IR spectra of the gases evolved during thermal decomposition are obtained as a function of temperature.
  • Micro-samples. Tiny samples, such as in forensic analysis, can be examined with the aid of an infrared microscope in the sample chamber. An image of the surface can be obtained by scanning. Another example is the use of FTIR to characterize artistic materials in old-master paintings.
  • Emission spectra. Instead of recording the spectrum of light transmitted through the sample, FTIR spectrometer can be used to acquire spectrum of light emitted by the sample. Such emission could be induced by various processes and the most common ones are luminescence and Raman scattering. Little modification is required to an absorption FTIR spectrometer to record emission spectra and therefore many commercial FTIR spectrometers combine both absorption and emission/Raman modes.
  • Photocurrent spectra. This mode uses a standard, absorption FTIR spectrometer. The studied sample is placed instead of the FTIR detector, and its photocurrent, induced by the spectrometer’s broadband source, is used to record the interferrogram, which is then converted into the photoconductivity spectrum of the sample.

NMR

Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a magnetic field absorb and re-emit electromagnetic radiation. This energy is at a specific resonance frequency which depends on the strength of the magnetic field and the magnetic properties of the isotope of the atoms. NMR allows the observation of specific quantum mechanical magnetic properties of the atomic nucleus. Many scientific techniques exploit NMR phenomena to study molecular physics, crystals, and non-crystalline materials through NMR spectroscopy. NMR is also routinely used in advanced medical imaging techniques, such as in magnetic resonance imaging (MRI).

All isotopes that contain an odd number of protons have an intrinsic magnetic moment and angular momentum, in other words a nonzero spin, while all nuclotides with even numbers of both have a total spin of zero.

A key feature of NMR is that the resonance frequency of a particular substance is directly proportional to the strength of the applied magnetic field. It is this feature that is exploited in imaging techniques; if a sample is placed in a non-uniform magnetic field then the resonance frequencies of the sample’s nuclei depend on where in the field they are located. Since the resolution of the imaging technique depends on the magnitude of magnetic field gradient, many efforts are made to develop increased field strength, often using superconductors. The effectiveness of NMR can also be improved using hyperpolarization, and using two-dimensional, three-dimensional and higher-dimensional multi-frequency techniques.

The principle of NMR usually involves two sequential steps:

  • The alignment (polarization) of the magnetic nuclear spins in an applied, constant magnetic field H0.
  • The perturbation of this alignment of the nuclear spins by employing an electro-magnetic, usually radio frequency (RF) pulse. The required perturbing frequency is dependent upon the static magnetic field (H0) and the nuclei of observation.

The two fields are usually chosen to be perpendicular to each other as this maximizes the NMR signal strength. The resulting response by the total magnetization (M) of the nuclear spins is the phenomenon that is exploited in NMR spectroscopy and magnetic resonance imaging. Both use intense applied magnetic fields (H0) in order to achieve dispersion and very high stability to deliver spectral resolution, the details of which are described by chemical shifts, the Zeeman effect, and Knight shifts (in metals).

It is a powerful technique that can provide detailed information on the topology, dynamics and three-dimensional structure of molecules in solution and the solid state. Thus, structural and dynamic information is obtainable (with or without “magic angle” spinning (MAS)) from NMR studies of quadrupolar nuclei (that is, those nuclei with spin S > 1⁄2) even in the presence of magnetic “dipole-dipole” interaction broadening (or simply, dipolar broadening) which is always much smaller than the quadrupolar interaction strength because it is a magnetic vs. an electric interaction effect.

Additional structural and chemical information may be obtained by performing double-quantum NMR experiments for quadrupolar nuclei such as 2H. Also, nuclear magnetic resonance is one of the techniques that has been used to design quantum automata, and also build elementary quantum computers.

X-ray Crystallography

X-ray crystallography is a tool used for identifying the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.

Since many materials can form crystals—such as salts, metals, minerals, semiconductors, as well as various inorganic, organic and biological molecules—X-ray crystallography has been fundamental in the development of many scientific fields. In its first decades of use, this method determined the size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences among various materials, especially minerals and alloys. The method also revealed the structure and function of many biological molecules, including vitamins, drugs, proteins and nucleic acids such as DNA. X-ray crystallography is still the chief method for characterizing the atomic structure of new materials and in discerning materials that appear similar by other experiments. X-ray crystal structures can also account for unusual electronic or elastic properties of a material, shed light on chemical interactions and processes, or serve as the basis for designing pharmaceuticals against diseases.

In a single-crystal X-ray diffraction measurement, a crystal is mounted on a goniometer. The goniometer is used to position the crystal at selected orientations. The crystal is bombarded with a finely focused monochromatic beam of X-rays, producing a diffraction pattern of regularly spaced spots known as reflections. The two-dimensional images taken at different rotations are converted into a three-dimensional model of the density of electrons within the crystal using the mathematical method of Fourier transforms, combined with chemical data known for the sample. Poor resolution (fuzziness) or even errors may result if the crystals are too small, or not uniform enough in their internal makeup.

X-ray crystallography is related to several other methods for determining atomic structures. Similar diffraction patterns can be produced by scattering electrons or neutrons, which are likewise interpreted as a Fourier transform. If single crystals of sufficient size cannot be obtained, various other X-ray methods can be applied to obtain less detailed information; such methods include fiber diffraction, powder diffraction and small-angle X-ray scattering (SAXS). If the material under investigation is only available in the form of nanocrystalline powders or suffers from poor crystallinity, the methods of electron crystallography can be applied for determining the atomic structure.

The oldest and most precise method of X-ray crystallography is single-crystal X-ray diffraction, in which a beam of X-rays strikes a single crystal, producing scattered beams. When they land on a piece of film or other detector, these beams make a diffraction pattern of spots; the strengths and angles of these beams are recorded as the crystal is gradually rotated. Each spot is called a reflection, since it corresponds to the reflection of the X-rays from one set of evenly spaced planes within the crystal. For single crystals of sufficient purity and regularity, X-ray diffraction data can determine the mean chemical bond lengths and angles to within a few thousandths of an angstrom and to within a few tenths of a degree, respectively. The atoms in a crystal are not static, but oscillate about their mean positions, usually by less than a few tenths of an angstrom. X-ray crystallography allows measuring the size of these oscillations.

Procedure

The technique of single-crystal X-ray crystallography has three basic steps. The crystal should be sufficiently large (typically larger than 0.1mm in all dimensions), pure in composition and regular in structure, with no significant internal imperfections such as cracks or twinning.

In the second step, the crystal is placed in an intense beam of X-rays, usually of a single wavelength (monochromatic X-rays), producing the regular pattern of reflections. As the crystal is gradually rotated, previous reflections disappear and new ones appear; the intensity of every spot is recorded at every orientation of the crystal.

In the third step, these data are combined computationally with complementary chemical information to produce and refine a model of the arrangement of atoms within the crystal. The final, refined model of the atomic arrangement—now called a crystal structure—is usually stored in a public database.

MALDI-TOF

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become an important tool in the analysis of biomolecules. The matrix-assisted laser desorption/ionization technique, or MALDI, which is based on an ultraviolet absorbing matrix, was developed in 1987. The method offers a rapid and accurate means for genotyping DNA samples and is a promising emerging technique.

Mass spectrometry (MS) has been widely used in forensic science in the identification of compounds, particularly illicit drugs. MS is a technique that allows the detection of compounds by separating ions by their unique mass (mass-to-charge ratios) using a mass spectrometer. The method relies on the fact that every compound has a unique fragmentation pattern (mass spectrum). The sample is ionized; the sample ions are separated based on their differing masses and relative abundance.

A typical mass spectrometer is comprised of these components:

  • ion source
  • mass analyzer
  • detector

U.S. Department of Energy Human Genome Program

Biomolecules and synthetic polymers have low volatility and are thermally unstable, which has limited the use of MS as a means of characterization. These problems have been minimized through the development of MALDI-TOF MS, which allows for the mass determination of biomolecules by ionization and vaporization without degradation.

A laser beam is used to ionize the sample bound in a matrix that is used to protect the DNA from being destroyed during the process. One common matrix is 3-hydroxypicolinic acid, which when mixed with the sample and dried under vacuum conditions, leaves a recrystallized matrix. The DNA is homogeneously spread throughout the recrystallized matrix. The laser is pulsed onto the crystals which absorb the laser energy and are partially vaporized, carrying intact DNA into the vapor phase. The matrix crystals transfer part of their charge to the DNA, allowing for ionization, while protecting the DNA from degradation.

At the time of each laser pulse, a voltage is applied to accelerate the sample towards a time-of-flight mass analyzer. The ions enter a vacuum where they are accelerated by a strong electric field in a flight tube towards the detector. Separated in the flight tube, the smaller DNA ions arrive at the detector in a shorter amount of time than the larger ions. An analyzer measures the time-of-flight (TOF) taken for particular ions to hit the detector. Separated ion fractions arriving at the end of the flight tube are detected by a recorder that produces a signal upon the impact of each ion group. The digitized data generated from successive laser shots is summed yielding a TOF mass spectrum. The flight time of an ion is related to its mass-to-charge ratio.

The method has the ability to accurately determine molecular weight information without the use of allelic ladders as size standards. MALDI was originally designed for ionization of large polypeptides and proteins; the techniques have been modified to include the analysis of oligonucleotide single nucleotide polymorphism (SNP) DNA and other macromolecules. It has been shown to be capable of separating STR amplicons at the rate of several thousand samples per day. MALDI-TOF MS is amenable to automation and rapid analysis. It is not only a viable technique for STR fragment analysis, but also SNP analysis.

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