Analytical Laboratory Facilities
All Keystone biology students are encouraged to make use of the facilities and equipment available to them as part of their coursework and their individual mentored research projects.
Cary 50 UV-VIS Spectrophotometer
A spectrophotometer is an instrument that measures the relative transmittance or absorbance of light passed through a chemical sample. Through a mathematical relationship known as Beer’s Law, the number of molecules dissolved in a liquid sample can be determined by carefully measuring the amount of light absorbed by a chemical in solution. Light is transmitted from the source to sensitive detector and quantified with no sample present. This is the calibration step. Then when a chemical sample is added, it is assumed that any light from a calibrated source that does not reach the detector was absorbed by the molecules in the light path. This particular instrument has a light source that emits light in the ultraviolet through the visible light spectrum, which makes it capable of detecting many organic molecules of chemical, environmental and biological interest.
Cary Eclipse Fluorescence Spectrophotometer
This instrument functions in much the same way as the Cary 50, but with one variation. Some chemical samples have the ability to absorb light energy to become excited, and remain stable for a measurable time in the excited state. After a time the molecules re-emit photons of light that are not of the same wavelength but of lesser energy than the light that excited the molecule. Some of the missing energy is dissipated as heat or through molecular vibration. The energy differences from the excitation photons of light and the emission photons of light give us clues regarding molecular structure and chemical reaction mechanisms. This instrument will enable us to study chemical reactions of interest in the environment and biology.
High Pressure Liquid Chromatograph
The HPLC is used to separate and quantify molecules in solution in a complex mixture. Molecules in a mixture are injected into a flowing solvent stream where they are carried to a separation column. The column captures the molecules by adhesion to the inner surfaces. An elution solvent, usually an organic solvent, is introduced with the aqueous solvent which causes the molecules to be removed from the column at a particular time. The difference in retention times on the column causes the molecules to be separated by size, hydrophobicity and charge. Once the molecule is eluted off the column, it is detected and quantified using visible or UV light just as a spectrophotometer functions.
Varian Gas Chromatograph
The gas chromatograph is used to identify volatile organic compounds. Molecules are introduced to the GC oven, where the temperature is raised until all molecules of the compound have become a gas. The hot gas is passed through a fused silica glass column which is coated with a resin that interacts chemically with the gas molecules. Some gas molecules will adhere to the resin, and will elute off of the resin if the temperature is raised further. By this process the molecules in a mixture can be separated. Once eluted, the gases reach the Flame Ionization Detector (FID). The molecules in the gas are ionized once combusted in the flame. Charged ionic molecular fragments are drawn to cathode or anode near the flame, and an electrical current is established. Molecules are quantified and identified by the electrical signatures and retention/elution times on the column.
Varian GC/LC Mass Spectrometer
The mass spectrometer is useful for sensitive detection of trace quantities of molecules. Molecules are introduced to the MS detector via the Gas Chromatograph or the Liquid Chromatograph interfaces. The molecular vapors or aerosols are ionized by a high voltage electron beam. The ions may then break apart into ionized fragments. Ionized molecules or fragments are accelerated through a high voltage, and then encounter a uniform magnetic field. The ionized molecules and molecular fragments encounter a central force as they attempt to traverse the magnetic field, which forces them into a curved trajectory. The radius of curvature for each trajectory determines the molecular mass. (r=qB/mv) Molecular ions and fragments are then separated, detected and quantified based on the location of their impact upon a detection grid. More massive fragments will have smaller radii curved trajectories. Through this technique, femtomole (10-15 mole) to picomole (10-12 mole) quantities of a molecule can be detected.
The Inductively Coupled Plasma Optical Emission Spectrometer is useful for detecting trace quantities of metals in a sample. An aqueous sample is introduced to plasma at 7000 Kelvin degrees. Aqueous metal ions are nebulized and introduced to the plasma via argon carrier gas. Metal ions introduced to the plasma undergo a rapid electron excitation. As electrons return to the ground state, light is emitted from the metal. Each metal emits a characteristic spectrum or excitation emission lines. The ICP-OES detects the optical emission spectra, and quantifies the metals in the sample. This instrument can simultaneously detect more than 70 metals in a single sample to sub-part per billion levels.