Spectrophotometry Flashcards
Spectrophotometry
Physical chemical investigation based on the properties of interaction between light and materials
Why is phosphate buffer good for biological reactions
Because of it’s high stability, buffering capacity and non-toxicity to most cells
pH 7.4 (which is required for cells) can easily be achieved with this buffer
Chromatography
Separation of mixture into individual components
The movement of molecules is determined by a molecule’s affinity for either phase
Stationary phase
A solid support that traps or retains molecules
Mobile phase
A buffer that flows on a stationary phase
Fick’s first law
J = - D (dy/dx)
Differential of concentration over concentration of space
What is another word for the
mobile phase?
Eluent
What can you call a mixture of buffer and solute or molecules exiting the stationary phase?
Eluate
What is an analyte?
A single molecule of a mixture
The average time it takes for a particular analyte to pass through the system is the ___ ___
Retention time
Which size molecules move faster in a sepharose gel column? Why?
Large
Smaller molecules enter the gel pores so they enter thre stationary phase and take a longer path down, entering and exiting through the gel beads.
Larger molecules are too large to enter the pores so they stay in the mobile phase and move down the column with the buffer.
Void volume
The volume of buffer that passes through the gel filtration column first and doesn’t contain any protein
Internal volume
The liquid within the pores of
the beads
What are the light ranges used in spectrophotometry?
UV: 185-400nm
Visible: 400-700nm
IR: 700-15000nm
?The rationale of spectrophotometry is to …
Measure interaction parameters (light intensity) to extrapolate chemical
information
