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15. Chromatography


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ok; what in the world is HPLC?
High pressure liquid chromatography - thank you!
What are the branches of chromatography based on mobile phase?
Liquid vs. Gas
What 4 types of apparatus' are used?
1. Flat
2. Column
3. Packed
4. Capillary
what are 2 different kinds of flat chromatography?
What are 2 different types of column?
Name 4 different mechanisms of seperation on stationary phase:
1. Adsorption
2. Electrostatic
3. Partition
4. Steric exclusion
How does adsorption seperation work?
Analytes have different attractions for the stationary or mobile phase.
How does electrostatic separation work?
Based on +/- charges
What is partition seperation?
Seperation of solutes based on how they distribute between 2 LIQUID phases.
What is steric exclusion seperation based on?
Molecular size
What is the general GOAL of chromatography?
To seperate analytes by producing fractions that each contain a higher concentration of one analyte than the other.
What is the physiochemical basis of chromatography?
The distribution equilibrium; Kd
What is Resolution
mathematical representation of how well a method seperates 2 analytes
What represents resolution on an isotherm plot?
Distance between peaks divdied by average width of peak bases
What is "Theoretical plates"?
A description of the method's efficiency.
What is "Retention"?
The amount of compound that is retained by the column; represented by k
What does the selectivity factor describe?
A method's ability to separate two solutes.
What does the capacity factor describe?
So how is general peak resolution, R, calculated?
By multiplying
-Column efficiency
-Selectivity factor
-Capacity factor
How are analytes QUANTITATED in chromatography?
By analyzing the peak height and area
Which is better, peak height or peak area?
peak area - it is more theoretically correct.
Why would you use peak height instead of area then?
Because height is easier to measure - areas have sloping bases and broad peaks.
What is needed for peak area measurement?
High resolution
Which is better; external or internal standardization?
What is the difference between normal and reversed-phase chromatography?
Normal: stationary phase is polar, mobile is non
Reversed: stationary is NONPOLAR to attract nonpolar analytes.
What are the 4 type of chromatoraphy used in clinics?
1. Thinlayer
2. Liquid
3. Gas
4. Mass spectroscopy
What are the 2 applicationso of TLC?
1. to measure the L:S ratio (lecithin-sphingomelin) to determine fetal lung maturity
2. In toxicology/drug screening
What are the applications of open liquic chromatography?
Glycated hemoglobin
What are the 4 applications of closed liquid chromatography?
1. Tricyclic antidepressants
2. Biogenic amines
3. Hemoglobin A1C
4. Toxicology

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