Biochem Lab Quiz 4

Gel Filtration Chromatography

Size based

Affinity Chromatography

Based on specific interaction between two molecules (ex. enzyme-substrate)

Spectrophotometer Components

Light source, Monochromator, Sample Cell, and Detector

Lamda Max

Wavelength at which a peak of absorbance occurs (absorbance measurements should be preformed at this value for greatest sensitivity)

Beer Lambert Law

I = I₀(10^-ECB);  B = the path length, E = Extinction coefficient, C = Concentration of solution through which light passes

I_o

Intensity of the Incident; Found by placing a cuvette containing a suitable blank in the light beam

I

Intensity of transmitted light

Transmittance

I/I_o

Extinction Coefficient Equation

E = A/BC

Cuvettes in lab have a path length of ___ cm

1 cm

Instrumental Deviation from Beer Law

Slit width determines the width of the wavelength range

Sample-Related Deviations from Beer Law

Molecule can exist in equilibrium between weak acid and conjugate base form; If absorbance of one form is significantly different from the other, small changes in solution pH will cause large changes in absorbance 

Standards

Absorbance of a series of solutions of a known concentration (should always be prepared in duplicate)

Absorbance Plot

y-axis Absorbance at Wavelength; x-axis Amount of protein

Data Beyond Standard Curve

Prepare a dilution of the sample that will fall on the curve

Acid Form of P-Nitrophenol

Clear, Unionized, Colorless

Basic Form of P-Nitrophnol 

Yellow