15. Chromatography
Terms
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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?
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1. Flat
2. Column
3. Packed
4. Capillary - what are 2 different kinds of flat chromatography?
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Paper
Thin-layer - What are 2 different types of column?
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-Open
-HPLC - Name 4 different mechanisms of seperation on stationary phase:
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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?
- Retention
- So how is general peak resolution, R, calculated?
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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?
- Internal
- What is the difference between normal and reversed-phase chromatography?
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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?
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1. Thinlayer
2. Liquid
3. Gas
4. Mass spectroscopy - What are the 2 applicationso of TLC?
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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?
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1. Tricyclic antidepressants
2. Biogenic amines
3. Hemoglobin A1C
4. Toxicology