Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431

Biochemistry Laboratory

Fall 2009

 

 
 
© R. Paselk 2007
 
 

Lab Exam Study Guide, F 2009

Chromatography:  What are the basic types of chromatography (partition, absorption, Ion exchange and Gel-filtration) and how does each work (on the molecular level)? What are the basic methods of doing chromatography (Paper, TLC, Column (including HPLC and GLC) and what types of chromatography are commonly utilized by each? Given two molecules of known properties be able to design (in general terms) a chromatographic system to separate them. Given appropriate Gel-fitration column chromatography data, be able to determine the “MW” of a protein.

Electrophoresis: What is the basic principle underlying electrophoresis? Determine the direction of migration of a typical protein at low and high pH when given the pH of its physiological environment. How might you try to “sharpen up” diffuse bands on an electrophoretogram? What problems might this cause? Why don’t particles continue to accelerate during an entire electrophoresis? What equipment is required to do electrophoresis? (power supply, cell, etc.) What are advantages/disadvantages of SDS-PAGE electrophoresis? Given appropriate data, be able to determine the “MW” of a protein. How might this MW differ from that found with Gel-filtration chromatography? What basic difference of electrophoretic conditions is there in determining Nucleic acid vs. Protein MW’s? Why the difference? What is 2-D electrophoresis? What two techniques are generally involved?

Spectrophotometry:  What are some advantages of spectrophotometric methods?   Define:  wavelength, wavenumber, Power, Intensity, quanta. What do we mean when we say the energy of light is independent of the intensity of the light?  What difference does this make? What types of quantum transitions are characteristic of energy absorption or emission in the various regions of the electromagnetic spectrum?  What types of spectroscopy characteristically are used in the various regions of the electromagnetic spectrum (and vice-versa)? What types of interactions give rise to 1) absorption, 2) emission and 3) Fluorescence phenomena?  What type of relaxation phenomena is characteristic of each?  What type of excitation process(es) is(are) characteristic of each?

Absorption Spectroscopy and Colorimetry (or photometry): Explain the relationship between absorbance and path length.  Memorize:  A = acl (or abc); A = cl and know the meaning of each term in these equations; log T = A, 2-log %T = A. Know how to do all calculations, plots etc. encountered in lab work. What are the limitations of Beer's Law (We discussed 3) and their physical or chemical basis. What do we mean by  “Spectra?” What considerations are important in picking a wavelength for determining concentrations by absorbance measurements?

      For critical work with the Spectronic 20, why should you limit acceptable instrument readings to between 0.1 and 1.0 A? Is this true for the Beckman DU? the HP? Why or why not? What should you do if your sample gives readings outside of this range?   (diff. dilutions, change blank.)

Enzyme Kinetics: Be able to construct (given data) and interpret the following plots:

Lab Work: general questions regarding the work we have done in lab are also fair game. For example: How did we desalt Mb? What should you do if you drop a 2 l bottle of acetone, breaking it, and others are in the lab? What should you do if you spill 500 ml of methanol when staff and faculty are not available? What do you need to know about a chemical before using it? How might you find the information needed to make up a chemical label? What info is needed on a waste label?  Be able to calculate concentrations of unknowns given colorimetric data graphically or by calculation using the Beer-Lambert Law. Be able to determine MW from SDS PAGE data, etc.

You may bring a data/information sheet to the exam, however you must not exceed one side of a single sheet of 8.5"x 11" paper (total surface of one-half of one standard sheet of paper) for this sheet! GOOD LUCK!


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