Week 8 - Chromatography Flashcards
Standard Plate Count
Serial dilution of broth culture to estimate bacterial density
Disciplines that use serial dilutions
Microbio - urine (UTI)
Transfusion science
Clinical chem
CFU calculation
Original [bacteria]
CFU/mL = # colonies / amount pipetted onto plate
Original [bacteria] = CFU/mL / dilution factor
TFTC
Too Few To Count
< 30 CFU
Colony count range
30-300 CFU
Protein Mix Colour explanation
Mixture of Hb and Vit B12 (separate components)
Hb - brown
Vit B12 - pink
Why did the column have to be dry? (Size Exclusion Chromatography)
So buffer solution wouldn’t separate the components
Low interference
Diffusion decreased when column is dry
Why was buffer added after protein mix was added to the column?
Keep mixture in fluid phase
Carry through the column
What molecule size runs through the column faster?
Large molecules run through faster
Small molecules are caught in pores in the column
Gas Chromatography
Separates mixture of volatile liquids
Mobile - gas
Stationary - tube/column
Volatile separate first
High Performance Liquid Chromatography
Separate and purify mixture
Speed depends on affinity to silicone phase.
Strong retention = slower movement
3 Types of Chromatography and their Properties
Ion Exchange - charge
Size Exclusion - porous beads in column
Affinity - non-covalent interaction between analyte and molecules
Chromatogram
Response vs Time
Want narrow peaks (not broad)
Conditions for Resolution
Compound must be:
Retained (retention)
Retained differently (selectivity)
Narrow peaks, no overlap (efficiency)
Dead/Void time
Adjustment in retention time to account for unretained analyte to pass thru entire stationary phase
