Download Introduction to Analytical Techniques

Introduction to Analytical Techniques
I. Analytical Techniques
A. Def.- Any procedure that allows one to determine the molecular composition
of an unknown substance.
1. May be qualitative or quantitative
2. Must be accompanied by control substances that allow the experimenter
to assess the proper function of the procedure.
3. Frequently requires the separation of substances from a mixture. (Cell
extracts, tissue extracts, and biological fluids represent a complex mixture
of biological molecules.)
B. Types of techniques
1. Separation based on molecular size
* a.. Agarose electrophoresis
i. frequently used to separate and identify DNA fragments
from a mixture of fragments extracted from a biological
sample
ii. Required for DNA fingerprinting, DNA sequencing,
DNA cloning, gene mapping, etc.
iii. Involves “pulling” components of a mixed sample
through a mesh network of agarose gel. An electrical field
generates the “pull”. The “mesh network” provides an
“obstical course” or series of pores through which the
molecules in the sample must travel. Small molecules move
quickly, large molecules move slowly.
b. Gel filtration chromatography
i. Frequently used to separate and purify large proteins
from biological samples.
ii. Involves separation of molecules within a mixture by
“pulling” them through a gel matrix of pores that has been
packed into a vertical column. The “pull” is provided by
gravity. A column of beads that contain specified pore sizes
provides the matrix of pores.
c. Differential sedimentation- centrifugation
i. Relies on the concept that heavier substances will
sediment at a faster rate when spun at high speeds in a
centrifuge.
ii. Frequently used to prepare cell fractions as a preparative
step prior to one of the other analytical techniques
discussed.
2. Separation based on specific characteristics
a. differential solubility.
* i. Thin layer chromatography
ii. Paper chromatography
iii. Requires a mobile phase and an immobile phase.
iv. Solubility in the mobile phase allows for characteristic
mobility across the immobile phase. The mobile phase is
the solvent, the immobile phase is the solid support (either
a thin layer of silica or paper)
v. Solubility is based on the “Like dissolves like” rule.
Polar substances will dissolve in polar solvents, nonpolar
substances will dissolve in nonpolar solvents.
b. Electrical charge
i. Some forms of electrophoresis (Isoelectric focusing)
- Used to separate proteins based on the nature of
the R groups found in their specific amino acid
sequence.
- Can be used to separate isotypes of the same
protein.
ii. Ion exchange chromatography
- Used to separate proteins or nucleic acids based on
their electrical charge.
- Frequently used as a preparative technique.
c. Antigen/antibody reaction
* i. Enzyme linked immunosorbent assay (ELISA)
- Antibodies are used to “catch” an specific
substance from an unknown sample.
- The presence of the substance is then detected by a
visible enzymatic reaction that is attached to the
specific antibody.
ii. Affinity chromatography
- Antibodies or other specific “sticky” chemical
groups are used to “catch” a substance from an
unknown sample.
- The “catching” substance is attached to beads that
are placed into a vertical column.
- Sample is poured over the column allowing the
specific “target” molecule to be “caught”
- The “caught” molecules are then eluted by
disrupting the “sticky” reaction that allowed them to
become “caught”
3. Analysis of light wave absorption or scatter
* i. Visible spectrophotometry
- Qualitative- what wavelengths of light are
absorbed by the sample?
- Quantitative- How much light of a specific
wavelength is absorbed by a sample containing an
unknown quantity of a known substance?
ii. Ultraviolet spectrophotometry
- Same as above but with ultraviolet wavelengths of
light
- Most common mechanism for quantitating DNA
and RNA in research and clinical settings.
iii. Infrared spectroscopy
- Same as above but using infrared wavelengths of
light
- Commonly used to determine the identity of
substances in an unknown mixture.
C. Web addresses for additional information
1. Chromatography
http://www.chem.ubc.ca/courseware/154/tutorials/exp3A/columnchrom/
2. Thin layer chromatography.
http://www.chem.ucla.edu/~bacher/General/30BL/tips/TLC1.html
3. Gel electrophoresis.
http://www.life.uiuc.edu/molbio/geldigest/electro.html
4. ELISA http://www.chemicon.com/resource/ANT101/a2C.asp
5. Spectroscopy http://chemistry.about.com/library/weekly/aa021302a.htm