Experiment 6 (Chromatographic Separations) Flashcards
(12 cards)
Chromatography
a method of separation base on the principle of phase distribution
Stationary phase (chromatography)
-solid particles or liquid coating on solid particles
-before elution
Elution
-the process of separating a substance that is adsorbed (bound) to another material by washing it with a solvent
-mobile phase passes through stationary phase, causing individual components of mobile phase to separate due to interactions with stationary phase
Mobile phase
-carrier
-liquid or gas carrying the sample mixture
-moves through stationary phase and interacts, causing different components to move at different rates through column
Different types of chromatography
-Gas chromatography (mobile phase is gas)
-Liquid chromatography (mobile phase is a liquid)
—Paper chromatography (stationary phase is a piece of paper)
—Thin layer chromatography (stationary phase is spread in thin layer)
-Column chromatography: stationary phase is packed in a column
—Ion-exchange chromatography: stationary phase is ion-exchange resins
Column Chromatography
-a method that involves a column where the solutes from the given solution migrate through the entire system followed by solute detection as it emerges from the column
-Glass wool prevents stationary phase from leaving column/clogging stopcock
-mobile phase is added first, then stationary phase
-never let liquid drop below the resin
-sample is added last with two components with varying degrees of affinity for stationary phase (allows visualization of different components)
Cation-exchange chromatography
-separation technique that utilizes a negatively charged resin to bind and separate positively charged molecules
-Contains dowex50 cation exchange resin
–Polystyrene beads with sulfonic acid groups
–H+ is in solution with the mobile phase
–Metal cation will have stronger affinity for resin than H+
-Binding preference for Dowex50:
—M3+ > M2+ > M+ > H+
-Allows us to separate things based on their charge and magnitudes of charges
-Anions have lowest affinity for the resin, will travel through column fastest
-Proceeded by component with highest affinity, then eleunt
-Measuring absorbance of each aliquot allows us to see a gradual increase then decrease in concentration of each component
Chromatogram
-a display of the elution pattern of the components of a mixture
-Shows how components in a mixture are separated and the quantity of each component (which is proportional to the area under the curve)
-Distinct peaks for each component mean good separation
-Overlap is considered poor separation
-Absorbance vs well numbers
Spectrophotometer
-measures how much light is transmitted and absorbed
-White light shines through sample and blank and reaches prism
–CCD Array detector measures how much of each wavelength reached this point (I0)
–Absorbance A = log(I0 / I) = log(I0) - log(I)
—Proportional to Aλα c (concentration of the light absorbing solute)
—Aλα b (path-length of sample cell)
Beer-lambert law
-A= εbc
-ε is extinction coefficient (1 / M cm) (molar absorptivity)
-c is the concentration of absorbing substance in solution (M)
-A is absorbance (L / mol cm)
-b is path length (usually 1.0 cm)
-Only good for a specific molecule at a specific wavelength
-should be wiped with kimwipe, capped, and pushed firmly into sample holder
Calibration curve
-a graph used to determine the concentration of an unknown substance by comparing its absorbance to the absorbance of known standards
-Standard solutions are prepared and then converted into complexes and measuring their absorbances
-Absorbances are plotted vs molarity (concentration) of standard
-c = A / slope
-Concentration can be used to calculate number of moles eluted (moles = MV)
Slope of calibration curve
-slope = A / c
-Concentration can be used to calculate number of moles eluted (moles = MV)
