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An Overview Of Spectrometery Biology Essay

A spectrometer (spectrophotometer, spectrograph or spectroscope) is an instrument used to measure properties of light over a specific portion of the electromagnetic spectrum, typically used in spectroscopic analysis to identify materials. The variable measured is most often the light's intensity but could also, for instance, be the polarization state. The independent variable is usually the wavelength of the light or a unit directly proportional to the photon energy, such as wave number or electron volts, which has a reciprocal relationship to wavelength. A spectrometer is used in spectroscopy for producing spectral lines and measuring their wavelengths and intensities. Spectrometer is a term that is applied to instruments that operate over a very wide range of wavelengths, from gamma rays and X-rays into the far infrared. If the region of interest is restricted to near the visible spectrum, the study is called spectrophotometry.

In general, any particular instrument will operate over a small portion of this total range because of the different techniques used to measure different portions of the spectrum. Below optical frequencies (that is, at microwave and radio frequencies), the spectrum analyzer is a closely related electronic device.

   The earliest picture of a spectrometer I have found is this cut from Hauksbee and Whiston's Course of Lectures, ca. 1705. 

   The liquid-filled glass prism was fixed in position, and the light admitted through the cross-shaped aperture on the left arm. The eye looks through a sighting tube on the end of the right-hand arm. Provision is made for slow-motion drive screws at I and K. 

Spectrometers for Telescopes

   The spectrometer on the left was built by John A. Brashear of Allegany, Pennsylvania, for the Smith Observatory at Hobart College. The date is 1888. The device at the bottom was used to attach the spectrometer to the telescope: the ring clamped around the telescope tube and the tubes above and below the collimator arm on the right-hand side slipped into the two rods on the connector. The spectrometer can also be used off the telescope.

   Brashear (1840-1910) was a self-taught astronometer and instrument maker who produced, among other projects, the 72 inch mirror for the Dominion Observatory in Victoria, British Columbia.

Spectroscopes

Spectroscopes are often used in astronomy and some branches of chemistry. Early spectroscopes were simply prisms with graduations marking wavelengths of light. Modern spectroscopes generally use a diffraction grating, a movable slit, and some kind of photo detector, all automated and controlled by a computer. The spectroscope was invented by Joseph von Fraunhofer.

When a material is heated to incandescence it emits light that is characteristic of the atomic makeup of the material. Particular light frequencies give rise to sharply defined bands on the scale which can be thought of as fingerprints. For example, the element sodium has a very characteristic double yellow band known as the Sodium D-lines at 588.9950 and 589.5924 nanometres, the color of which will be familiar to anyone who has seen a low pressure sodium vapour lamp.

In the original spectroscope design in the early 19th century, light entered a slit and a collimating lens transformed the light into a thin beam of parallel rays. The light then passed through a prism (in hand-held spectroscopes, usually an Amici prism) that refracted the beam into a spectrum because different wavelengths were refracted different amounts due to dispersion. This image was then viewed through a tube with a scale that was transposed upon the spectral image, enabling its direct measurement.

With the development of photographic film, the more accurate spectrograph was created. It was based on the same principle as the spectroscope, but it had a camera in place of the viewing tube. In recent years the electronic circuits built around the photomultiplier tube have replaced the camera, allowing real-time spectrographic analysis with far greater accuracy. Arrays of photo sensors are also used in place of film in spectrographic systems. Such spectral analysis, or spectroscopy, has become an important scientific tool for analyzing the composition of unknown material and for studying astronomical phenomena and testing astronomical theories.

In modern spectrographs, the spectrum is generally given in the form of photon number (in the UV, visible, and near-IR spectral ranges) or Watts (in the mid- to far-IR) and is displayed with an abscissa given in terms of wavelength, wave number, or eV.

A comparison of the three abscissa types typically used for visible spectrometers.

A comparison of the three abscissa types typically used for infrared spectrometers.

Photospectrometer

A spectrophotometer consists of two instruments, namely a spectrometerfor producing light of any selected color (wavelength), and a photometerfor measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes.

If development of color is linked to the concentration of a substance in solution then that concentration can be measured by determining the extent of absorption of light at the appropriate wavelength. For example hemoglobin appears red because the hemoglobin absorbs blue and green light rays much more effectively than red. The degree of absorbance of blue or green light is proportional to the concentration of hemoglobin.

When monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is,

where I sub 0 is the intensity of transmitted light using the pure solvent, I is the intensity of the transmitted light when the colored compound is added, c is concentration of the colored compound, l is the distance the light passes through the solution, and k is a constant. If the light path l is a constant, as is the case with a spectrophotometer, Beer's law may be written,

where k is a new constant and T is the transmittance of the solution. There is a logarithmic relationship between transmittance and the concentration of the colored compound. Thus,

The O.D. is directly proportional to the concentration of the colored compound. Most spectrophotometers have a scale that reads both in O.D. (absorbance) units, which is a logarithmic scale, and in % transmittance, which is an arithmetic scale. As suggested by the above relationships, the absorbance scale is the most useful for colorimetric assays.

Spectrographs

A spectrograph is an instrument that separates an incoming wave into a frequency spectrum. There are several kinds of machines referred to as spectrographs, depending on the precise nature of the waves. The first spectrographs used photographic paper as the detector. The star spectral classification and discovery of the main sequence, Hubble's law and the Hubble sequence were all made with spectrographs that used photographic paper. The plant pigment phytochrome was discovered using a spectrograph that used living plants as the detector. More recent spectrographs use electronic detectors, such as CCDs which can be used for both visible and UV light. The exact choice of detector depends on the wavelengths of light to be recorded.

An echelle spectrograph uses two diffraction gratings, rotated 90 degrees with respect to each other and placed close to one another. Therefore an entrance point and not a slit is used and a 2d CCD-chip records the spectrum. Usually one would guess to retrieve a spectrum on the diagonal, but when both gratings have a wide spacing and one is blazed so that only the first order is visible and the other is blazed that a lot of higher orders are visible, one gets a very fine spectrum nicely folded onto a small common CCD-chip. The small chip also means that the collimating optics need not to be optimized for coma or astigmatism, but the spherical aberration can be set to zero.

A spectrograph is sometimes called polychromator, as an analogy to monochromator.

Types Of Spectrometeres

A. Two-arm Spectrometers

 

   The basic spectrometer has a light source Silluminating a slit that acts as an object for lens C. This produces a parallel beam of light illuminating the prism P. After refraction by the prism, the light is focussed by lens Oon cross-hairs R. The eyepiece lens Eis then used to examine the various images of the slit in the various colors present in the source. The cut is from Wm. S. Franklin and Barry MacNutt, Light and Sound(Bethlehem, PA, 1909)

B. Three-Arm Spectrometers

 

A present-day user of a spectrometer employs a diffraction grating; if the grating spacing of the grating is known with precision the wavelengths may be obtained directly from the angle at which the particular lines appear.

   For most of the nineteenth century the typical spectroscope used a prism to separate the spectral lines. Calibration was obtained by projecting the lines of a known spectral source onto the same plane as the unknown lines

 C. One Arm Spectrometers (Direct-Vision Spectrometers)

This direct-vision spectrometer at St. Mary's College in Notre Dame, Indiana, is by John Browning of London. 

   The cut below, from pg 134 of Franklin and MacNutt, shows the arrangement of two crown glass and one flint glass equilateral prisms. This provides zero refraction of the middle part of the prism, but does give dispersion to produce the spectrum. The images of slit Sare located at RV, the plane on which the eyepiece Eis focussed. There may also a graduated reticule at this point. 

   The side-arm on the spectroscope at the left is probably designed to project an image of a reticule on plane RV.

D. Multiple-Prism Spectrometers

  The two-prism spectrometer by John Browning of London produces twice the dispersion as one using a single prism. The 1888 Queen Catalogue of Instruments

The two-prism spectrometer by John Browning of London produces

E. Wavelength Spectrometers

 

  

The Mass Spectrometry

In order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields. The three essential functions of a mass spectrometer, and the associated components, are:

1.   A small sample is ionized, usually to cations by loss of an electron.   The Ion Source

2.   The ions are sorted and separated according to their mass and charge.   The Mass Analyzer

3.   The separated ions are then measured, and the results displayed on a chart.   The Detector

Because ions are very reactive and short-lived, their formation and manipulation must be conducted in a vacuum. Atmospheric pressure is around 760 torr (mm of mercury). The pressure under which ions may be handled is roughly 10-5 to 10-8 torr (less than a billionth of an atmosphere). Each of the three tasks listed above may be accomplished in different ways. In one common procedure, ionization is effected by a high energy beam of electrons, and ion separation is achieved by accelerating and focusing the ions in a beam, which is then bent by an external magnetic field. The ions are then detected electronically and the resulting information is stored and analyzed in a computer. A mass spectrometer operating in this fashion is outlined in the following diagram. The heart of the spectrometer is the ion source. Here molecules of the sample (black dots) are bombarded by electrons (light blue lines) issuing from a heated filament. This is called an EI (electron-impact) source. Gases and volatile liquid samples are allowed to leak into the ion source from a reservoir (as shown). Non-volatile solids and liquids may be introduced directly. Cations formed by the electron bombardment (red dots) are pushed away by a charged repeller plate (anions are attracted to it), and accelerated toward other electrodes, having slits through which the ions pass as a beam. Some of these ions fragment into smaller cations and neutral fragments. A perpendicular magnetic field deflects the ion beam in an arc whose radius is inversely proportional to the mass of each ion. Lighter ions are deflected more than heavier ions. By varying the strength of the magnetic field, ions of different mass can be focused progressively on a detector fixed at the end of a curved tube (also under a high vacuum).

When a high energy electron collides with a molecule it often ionizes it by knocking away one of the molecular electrons (either bonding or non-bonding). This leaves behind a molecular ion (colored red in the following diagram). Residual energy from the collision may cause the molecular ion to fragment into neutral pieces (colored green) and smaller fragment ions (colored pink and orange). The molecular ion is a radical cations, but the fragment ions may either be radical cations (pink) or carbonations (orange), depending on the nature of the neutral fragment. An animated display of this ionization process will appear if you click on the ion source of the mass spectrometer diagram.

  Isotopes

Since a mass spectrometer separates and detects ions of slightly different masses, it easily distinguishes different isotopes of a given element. This is manifested most dramatically for compounds containing bromine and chlorine, as illustrated by the following examples. Since molecules of bromine have only two atoms, the spectrum on the left will come as a surprise if a single atomic mass of 80 amu is assumed for Br. The five peaks in this spectrum demonstrate clearly that natural bromine consists of a nearly 50:50 mixture of isotopes having atomic masses of 79 and 81 amu respectively. Thus, the bromine molecule may be composed of two 79Br atoms (mass 158 amu), two 81Br atoms (mass 162 amu) or the more probable combination of 79Br-81Br (mass 160 amu). Fragmentation of Br2 to a bromine cations then gives rise to equal sized ion peaks at 79 and 81 amu.

Bromine

vinyl chloride

methylene chloride

The centre and right hand spectra show that chlorine is also composed of two isotopes, the more abundant having a mass of 35 amu, and the minor isotope a mass 37 amu. The precise isotopic composition of chlorine and bromine is:

        Chlorine:   75.77% 35Cl and 24.23% 37Cl

        Bromine:   50.50% 79Br and 49.50% 81Br

The presence of chlorine or bromine in a molecule or ion is easily detected by noticing the intensity ratios of ions differing by 2 amu. In the case of methylene chloride, the molecular ion consists of three peaks at m/z=84, 86 & 88 amu, and their diminishing intensities may be calculated from the natural abundances given above. Loss of a chlorine atom gives two isotopic fragment ions at m/z=49 & 51amu, clearly incorporating a single chlorine atom. Fluorine and iodine, by contrast, are monoisotopic, having masses of 19 amu and 127 amu respectively. It should be noted that the presence of halogen atoms in a molecule or fragment ion does not change the odd-even mass rules given above.

d by the three mechanisms shown above. Plausible assignments may be seen by clicking on the spectrum, and it should be noted that all are even-electron ions. The m/z = 42 ion might be any or all of the following: C3H6, C2H2O or C2H4N. A precise assignment could be made from a high-resolution m/z value (next section).

Odd-electron fragment ions are often formed by characteristic rearrangements in which stable neutral fragments are lost. Mechanisms for some of these rearrangements have been identified by following the course of isotopically labeled molecular ions

  High Resolution Mass Spectrometry

In assigning mass values to atoms and molecules, we have assumed integral values for isotopic masses. However, accurate measurements show that this is not strictly true. Because the strong nuclear forces that bind the components of an atomic nucleus together vary, the actual mass of a given isotope deviates from its nominal integer by a small but characteristic amount (remember E = mc2). Thus, relative to 12C at 12.0000, the isotopic mass of 16O is 15.9949 amu (not 16) and 14N is 14.0031 amu (not 14).

Formula

C6H12

C5H8O

C4H8N2

Mass

84.0939

84.0575

84.0688

By designing mass spectrometers that can determine m/z values accurately to four decimal places, it is possible to distinguish different formulas having the same nominal mass. The table on the right illustrates this important feature, and a double-focusing high-resolution mass spectrometer easily distinguishes ions having these compositions. Mass spectrometry therefore not only provides a specific molecular mass value, but it may also establish the molecular formula of an unknown compound.

Tables of precise mass values for any molecule or ion are available in libraries; however, the mass calculator provided below serves the same purpose. Since a given nominal mass may


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