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Flashcards in Exam 3 Deck (75):
1

Separation

Dividing a group into smaller groups that share the same type of similar trait

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2 Common Separation Techniques

A. Chromatography
B. Electrophoresis

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Chromatography
(Define and give three examples)

An interaction between two phases; the mobile phase and the stationary phase.
Ex. Gas Chromatography (GC), Liquid Chromatography, Supercritical Fluid Chromatography

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Supercritical Fluid

Something that moves like a gas, but dissolves like a liquid

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Elution

Describes the separation of analyses on a packed column

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Dilution occurs in a column due to.....

The addition of solvents to push the analyte thru

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Eluent

Portion of the sample in the mobile phase

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Mobile Phase vs. Stationary Phase

The phases that interact in chromatography
- mobile: phase that moves
- stationary: phase that does not move

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Partitions

Formed as analytes move down the column

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Column Chromatography:
The rate that a zone migrates down a column is dependent on.....

How long the analyte spends in the stationary phase.

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Chromatogram

Readout that shows the detector response over time
- x-axis = time
- y-axis can vary

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Formula:
Distribution Constants

K = cs/cm

(cs=ns/vs)
(cm=nm/vm)

- v=volume, s=stationary phase, m=mobile phase

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Parts of the HPLC inlets (3)

- injection system
- pump
- solvent

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Calculating Resolution:

Rs = 2( (tr)B - (tr)A) ) / (WA + WB)

- trA = retention time of A
- trB = retention time of B
- WA = width of peak A
- WB = width of peak B

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Calculating Plate Count:

N = L/H

N = plate count (# of plates)
L = column length
H = plate height

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Calculating Plate Height:

H = sigma^2 / L

H = plate height
sigma = distance from peak, one distribution out
L = column length

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Diffusion

process in which species migrate from a more concentration part of a medium to a more dilute region
*no direction

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The _____ the analytes are on the column, the _____ the longitudinal diffusion.

longer; greater

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Gas Chromatography:
- mobile phase
- stationary phase

- mobile phase: inert gas
- stationary phase: varies (solid, gel)

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Gas Chromatography:
Information about Inlets

- sample injection is complex due to gas rapidly flowing into the column
- injection of either liquid or gas sample
- sample must be volatilized, temperature must be high enough

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Gas Chromatography:
Important parts of the Inlet

- septum: keeps gases separate from atmosphere
- syringe: used to puncture septum and inject sample
- vaporization chamber: vaporizes sample, pushes the injection mix into mobile phase

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Gas Chromatography:
Information about the Column

*piece of instrument that causes separation to occur
- 2 types of columns: Open Tubular & Packed

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Gas Chromatography:
Open Tubular Columns

WCOT and SCOT

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Gas Chromatography:
WCOT

"Wall Coated Open Tubular" column
- capillary tube is coated with stationary phase

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Gas Chromatography:
SCOT

"Support Coated Open Tubular" column
- stuff inside the tube is coated with stationary phase

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Gas Chromatography:
What is more efficient WCOT or SCOT?

WCOT is more efficient than SCOT.

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Gas Chromatography:
Most widely used column
Advantage and Disadvantage

FSWC (fused silica wall coated, open tubular column)
- + thinner, flexible
- - can not add a large amount of sample

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Gas Chromatography:
Packed Columns

tube packed with fine material coated with thin layer of stationary phase

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Generic setup of GC

inlet --> column --> detector

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Examples of common stationary phases

- PEG
- dimethylpolysiloxane
- wax

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2 Methods of Temperature Ramp

A. Isothermal - keep same temperature throughout analysis
B. Gradient - increase temperature at some rate

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Gas Chromatography:
As temperature _______, analytes will spend _____ time in mobile phase, _______ing the total time on the column

Increases; more; decreasing

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Advantage and Disadvantage of Isothermal Temperature Ramp

+ easier to develop method
- longer runtimes and worse separations

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Advantage and Disadvantage of Gradient Temperature Ramp

+ give better resolution and shorter runtime
- tricky to correct

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Gas Chromatography:
Ideal Detector Characteristics

- sensitivity
- good stability/reproducibility
- linear response to solutes over many magnitudes
- short response time
- high reliability + ease of use
- non-destructive

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Gas Chromatography:
Common Detectors

A. FID
B. ECD
C. Thermionic Detectors
D. Conductivity Detectors
E. Photoionization
F. Atomic Emission
G. Flame Photometric
H. Mass Spec.

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Gas Chromatography:
FID

Flame Ionization Detector
- detector in GC
- very common
- burns whatever comes out of column
- mass sensitive
- does not discriminate between hydrocarbons

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Gas Chromatography:
ECD

Electron Capture Detector
- detector in GC
- useful in detection of halogenated compounds

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Gas Chromatography:
Thermionic Detectors

- detector in GC
- specific for phosphorus and nitrogen
- hot gas flows, forms plasma, current is generated and detected

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Gas Chromatography:
Conductivity Detector

- detector in GC
- gas dissolved in liquid to produce conductive solution
- detected in conductivity cell

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Gas Chromatography:
Photoionization

- detector in GC
- uses light to ionize

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Gas Chromatography:
Atomic Emission

- detector in GC
- converts sample to plasma and observes emissions

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Gas Chromatography:
Flame Photometric

- detector in GC
- used for pollution studies

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Liquid Chromatography:
Common Types

- HPLC
- UPLC
- Column Chromatography
- TLC
- Paper Chromatography

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HPLC

High Pressure Liquid Chromatography

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UPLC

Ultra high Pressure Liquid Chromatography

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TLC

Thin Layer Chromatography (Liquid)

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Liquid Chromatography:
Mobile Phase:
Stationary Phase:

Mobile Phase: liquid
Stationary Phase: varies

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Liquid Chromatography:
Information about Columns

- columns generally packed with small particles
- smaller the particles, the better the efficiency of the column

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Isocratic

same solvent at all times during run

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Gradient

mixture of 2 solvents with different polarities; mixture changes during run

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Liquid Chromatography:
Parts of the Inlet

- injection system
- pump
- solvent

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Liquid Chromatography:
Function of Injection System

- allows ability to inject sample; would not be able to inject sample into just the inlet due to high pressure area

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Liquid Chromatography:
Function of Pumps

forces solvent thru the inlet; irregular flow can cause issues that alter data

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Liquid Chromatography:
Function of Column

where separation occurs; polarity based separations

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Liquid Chromatography:
Separations are based on

POLARITY

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Polarity of long CH chains.....

long CH chains are NON-POLAR

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The larger the chain......

the more non-polar the molecule

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Liquid Chromatography:
Common Columns in HPLC

A. Normal Phase
B. Reversed Phase

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Liquid Chromatography:
Normal Phase

Column in HPLC
*polar stationary phase

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Liquid Chromatography:
Reversed Phase

Column in HPLC
*non-polar stationary phase

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Liquid Chromatography:
Rules for Columns in HPLC

***like dissolves like***
molecules with corresponding polarity to stationary phase will remain on column.
If column is polar, polar molecules will remain on column.

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Column Types

A. Guard Column - short, filled with packing material; purpose is to catch anything that will damage analytical column
B. Analytical Column - where separation occurs

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What can affect column performance?

TEMPERATURE; temperature should be kept constant throughout analysis

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Liquid Chromatography:
Selection of Detector is based on

SAMPLE CONCENTRATION

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Liquid Chromatography:
Types of Detectors

A. Bulk Detectors - measures some property of mobile phase
B. Solute-Property Detectors - measures some property of solutes (analytes)

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Liquid Chromatography:
Advantage and Disadvantage of IR and Diode Array Detectors

+ gives structural info about analyte
- 3D graphs can be resource problematic

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Liquid Chromatography:
Advatgage and Disadvantage of Electrochemical Detectors

+ cheap to install, high sensitivity, quick and easy
- pH dependent which impacts redox potentials

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Electrophoresis

Is NOT Chromatography

70

Is electrophoresis Chromatography?

NO

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Electrophoresis is NOT Chromatography

U right

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Types of electrophoresis

1. Capillary electrophoresis
2. Slab gel electrophoresis
3. Combinations

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Slab gel electrophoresis

Uses thin flat layer/slab of porous gel containing aqueous buffer solution in its pores; analytes form bands as electricity is applied

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What field is slab gel electrophoresis used in?

BIOLOGY; separation of DNA/proteins

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Why does pH matter in electrophoresis?

Due to supply of ions; lower pH gives larger supply of H+ ions