Module 6 Flashcards
What are limitations with simple separations
- When the D of analyte is similar to another species it becomes difficult to separate it from the matrix
- Additional analytes require additional extractions to isolate from matrix
W. To separate, the D must be significantly greater than all other species in solution
How to improve LLE separation
By first extracting the analyte into the extracting phase and then extracting again into a fresh portion of the initial phase
What is counter current extraction
Extracting the analyte into extracting phase then extracting again into fresh portion of the initial phase
How are chromatographic separations achieved
By continuously passing a mobile phase over a phase that remains fixed or stationary
What is the mobile phase
The extracting phase that moves through the system
What is the stationary phase
The extracting phase that remains in a fixed position
Explain the 4 steps to chromatography
- Sample injected into mobile phase
- As sample moves with mobile phase, the samples components Partition themselves between the two phases
- The components whose distribution ratio favours the stationary phase requires a longer time to pass through the system
- Given sufficient time and sufficient stationary and mobile phase, solutes with similar distribution ratios can be separated
3 ways to classify analytical separations
- Physical state of mobile and stationary phases
- Method of contact between mobile and stationary phase
- Chemical or physical mechanism responsible for separation
What can the mobile and stationary phases be
Mobile: gas, liquid, supercritical fluid
Stationary: solid or liquid film coated on a solid surface
What is the physical state of mobile and stationary phases
Chromatographic techniques are names by listening the name of mobile phase then the same of stationary phase
- gas-liquid chromatography
Or just the mobile phase
- gas chromatography
What is the method of contact between mobile and stationary phases
In column, and planar chromatography
Column: stationary phase is placed in narrow column which the mobile phase moves under influence of gravity
- the stationary phase is either a solid or thin liquid film coating on a solid particulate packing material on the columns wall
Planar: the SP coats a flat glass, metal of plastic plate which is placed in developing chamber
- a reservoir containing the MP is placed in contact with SP and the MP moves up by capillary action
What is chemical or physical mechanism responsible for separation
The mechanism by which solutes separate provided and 3rd means for characterizing a separation
What is adsorption chromatography
- solutes separate based on their ability to adsorb on the surface of the solid particles
- the more strongly a solute is adsorbed, the slower it travels through the column
What is partition chromatography
- a thin liquid film coating a solid surface serves as the stationary phase
- separation is based on a difference in the equilibrium partitioning of solutes between the liquid SP and the MP
What is ion exchange chromatography
- stationary phase consists of solid support with covalently attached an ionic or cationic functional groups
- ionic solutes of opposite charge are attracted to the SP by electrostatic forced
What is molecular exclusion chromatography
- porous gels are used as SP and separations are based on the size of solutes
- large solutes are unable to penetrate into the porous SP and quickly pass through column
-smaller solutes enter into porous SP and increase the time spent in the column
What is electrophoretic chromatography
- there is no stationary phase
- charged solutes migrate under the influence of an applied potential field
- differences in ion mobility of ions account for their separation
What is the plot of a chromatogram
Detector signal vs time
What is retention time (tr)
Time it takes for a solute to move from point of injection to detector
Aka. Retention volume
What is retention volume (Vr)
Volume of mobile phase needed to move a solute from its point of injection to the detector
What is baseline width (w) and how is it determined
- width of solutes chromatographic band measured at the baseline (time, or volume)
- determined by intersection with the baseline of tangent lines drawn through inflection points in either side of the chromatographic peak
What is void time (tm) and what does it imply
- time required for an unrestrained solute to move from point of injection to the detector
- implies that solute does not interact with stationary phase
Aka void volume
What is void volume
Volume of mobile phase needed to move an unrestrained solute from point of injection to the detector
What is chromatographic resolution (R)
A quantitive measure of the degrees of separation between two peaks
What is the goal of chromatography
To separate a sample into a series of chromatographic peaks, each representing a single component of all the analytes in the sample
What are the 2 ways to improve R
- Increase deltatr
- Decreasing w1 w2 or both
How to increase deltatr
- Enhance interaction of solutes with the column
- Increase the columns selectivity for one of the solutes
How to decrease wa or what
- capacity factor
Increase the length of the column - column sensitivity
Changing stationary phase in gas ch, leads to increase in R
Liquid chrom. Changing composition of MP can lead to increase in R - column efficiency
Increase k increases r
Ensure solute spends more time on the SP
What is capacity factor (K’) and equation
A measure of how strongly a solute is retained to the stationary phase
-=time spent in SP/time spent in MP
=cs x vs/ cm x vm
= tr-tm/tm
What happens in a solute partitions 3x more in SP than MP
Then there will be 3x more moles in SP as in the MP at any given point
What is column selectivity and formula (alpha)
The relative selectivity if a chromatographic column for a pair of analytes is expressed by the selectivity factor, alpha
= K’a/K’b
What is column efficiency
- quantitatively measure the extent of band broadening
What is the process of band broadening
- the increase in a solutes baseline width as it moves from the point of injection to the detector
- at the beginning of chromatographic separation, the solute occupies a narrow band of finite width
- as the solute travels down column, the width of the band increases
What are theoretical plates
Zones where partitioning of the solutes between the mobile and stationary phase occurs
Equation for number of theoretical plates (N)
N=L/H
What does smaller plate height imply
More plates in column, narrower chromatographic peaks, better resolution
What is peak capacity
A quantitative term that provides an estimate of the number of solutes that might be feasible to separate
What is non ideal solute behaviour(2)
- Fronting
- tail at the beginning of a peak caused by injection of too much sample on column - Tailing
- a tail at end of a peak arising when some sites on the Sp retain the solutes more strongly than other sites
-caused by active sites on the sp which means the sp has deteriorated
What is column overloading and what does it cause
Injection of too much sample into a column
Causes fronting
What happens in gas chromatography
- sample gas or liquid is injected into a stream of inert gaseous Mp
- sample is carried through a column where sample components are separated based on their ability to distribute between the MP and SO
What is the mobile phase in gas chromatography
- a must be chemically inert to stationary phase
-He, Ar or N2 is used - packed w columns that can have flow rates between 125 -150mL/min
-with capillary columns, can have flow rates between 1-10mL/min
What are chromatographic columns
- a column that provides the location for physically retaining the SP
What can the type of colume influence in gC (4)
- amount of sample that can be handled
- efficiency of the separation
- number of analytes that can be separated
- amount of time required for separation
What are the two types of columns used in gas chromatography
- Packed
- Capillary
What are packed columns
- constructed from glass, stainless steel, cu or al
- column filled with particulate solid support
- most common is diatomaceous earth
- solid support is the porous and provides sample contact between MP and SP
Plates=3000-10000
What are capillary columns and what are the two types
- columns constructed from silica coated with a protective polymer
1. Wall coated open tubular columns (WCOT)
Q. Support coated open tubular columns (SCOT)
WCOT characteristics
Open tubular columns in which is SP is coated in the capillary’s inner walls
SCOT characteristics
Open tubular columns which a thin film layer of a solid support is coated with a liquid SP attached to the capillary’s inner wall
What column provides the best separation efficiency and 3 reasons why they’re better
Capillary columns because they contain more theoretical plates
1. Shorter analysis time
2. Increased sensitivity
3. Lower sample capacity
What is selectivity in gas chromatography influenced by
The choice of stationary phase
What is elation order in gas chromatography determined by
Solutes boiling points and to a lesser extent, by the solutes interaction with the SP
How are two solutes with similar boiling points separated
Only if the stationary phase selectively interacts with one of the solutes
Main criteria for selecting a stationary phase in gas chromatography (4)
- Chemically inert
- Thermally stable
- Low volatility
- Similar polarity to the solutes being separated
How to increase polarity of stationary phase
Replacing some of the -CH3 groups with other substituents
What do autosamplers do
Introduce sample doirectly into a heated injector port or directly onto the head of a GC-column
Samples injected rapidly through a septum into the evaporation zone of the heated injector port
What is the injection port temperature heated to to promote?
250C to promote flash vaporization
3 ways to inject sam-les onto an open tubular column
- Split injection
- Split less injection
- On column injection
What is a split injection and how much of a sample goes onto the column
- injecting samples onto a capillary column in which only a small portion of the sample enters the column
- 0.2-2% of sample goes onto column
What is a splitless injection and how much of the sample goes onto column
- injecting a sample onto a capillary column that allows a higher percentage of sample to enter the column
- 80% of sample
What is on column injection and how much sample goes onto column
- direct injection of thermally unstable samples into a capillary column
- 100% of sample
5 main detectors for GG
- Thermal conductivity detector (TCD)
- Flame ionization detector (FID)
- Electron capturing detector (ECD)
- Nitrogen phosphorous detector (N/PD)
- Mass spectrometer (MS)
What is the thermal conductivity detector (TCD)
- measures the ability of a substance to transport heat from a hot region to a cold one
- heat transfer takes place at a slower rate for materials (gases) with low thermal conductivity
How does thermal conductivity detector work
- carrier gas containing solute flows over hot tungsten- rhenium filament
- when solute flows past the filament, the conductivity of the gas stream decreases and so the voltage across the filament decreases
- the detector measures the change in voltage
What element has the 2nd highest thermal conductivity and what happens if an analyte is mixed with it
- He
- an analyte mixed with He lowers the conductivity of the gas stream
4 characteristics of thermal conductivity detectors
- Universal in nature
- Large linear dynamic range
- Non destructive
R. Poor detection limits compared to other detectors
What is the flame ionization detector (FID)
- combustion of an organic compound in H2/air flame results in a flame rich in electrons and ions
What happens in the absence of a solute if a potential of 300V is applied with a FID
A small current of 10-14 A develops
3 characteristics of flame ionization detectors
- Detection limits 1000x smaller than thermal conductivity detectors
- Large linear dynamic range
- Destructive
What is the electron capture detector
- a selective detector, only responds to compounds with electronegative atoms
- consists of a beta-emitter such as radioactive 63Ni
How does the electron capture detector (ECD) work
- the emitted electron ionizes the mobile phase giving rise to a small electric current
- when a solute with a high affinity passes by, they can capture electrons and decrease the electric current which produces a signal
Electron capture detector (ECD) characteristics
- selective for electronegative compounds
- detection limits small for those compounds
- poor linear dynamic range (~2 orders of magnitude)
- radioactive
What is the nitrogen phosphorous detector (NPD)
Modified flame ionization detector but especially sensitive to compounds containing N and P
- response factor is 10^4 to 10^6 times greater for N/P than its response factor for C
- selective
What is electrophoresis used for
- separating technique in which analytes are separated based on their ability to move through a conductive medium under the influence of an applied electric current
How do ions move in electrophoresis
- cations migrate through a conductive medium toward the electric fields negatively charged cathode
- higher charged ions of smaller size migrate faster
What is the conductive medium in electrophoresis
An aqueous buffer
What are the two types of mobility’s that result when an EF is applied in electrophoresis
- Electrophoretic mobility
- Electroosmotic mobility
What is electrophoretic mobility
A measure of a solutes ability to move through a conductive medium in response to an applied EF
- the velocity at which a solute moves in response to an applied electric field
- called Vep
What is electroosmotic mobility
- occurs when the conductive medium moves through a tube in response to an applied EF
- buffered solutions move toward the cathode sweeping most analytes toward the cathode
- measure of velocity Veof
How to analytes pass through detectors in GC, HPLC, and electrophoresis
- in GC AND HPLC, each analyte passes through detector at same rate so peak area is proportional to quantity of analyte
- in electrophoresis, analytes with different mobilities pass through the detector at different rates.
tre mobility= shorter migration time = less time analyte spends in detector
Equation for normalized peak area equation for electrophoresis
- peak area/ migration time
Why are smaller capillaries more desireable in electrophoresis
They generate less heat
What are the two types of injection techniques
- Hydrodynamic
-use pressure to force a small portion of the sample into the capillary tube - Electrokinetic
- done by placing both the capillary and the anode into the sample vial and briefly applying an electric field
What are the 3 forms of capillary electrophoresis
- Micellar electrokinetic capillary chromatography (MEKC)
- Capillary cell electrophoresis
- Capillary electrochromatography
Describe micellar electrokinetic capillary chromatography
- a form of capillary electrophoresis which neutral analytes are separated based on their ability to partition into a charged micelle
- a surfactant is added to buffer solution, at high concentrations, it creates a micelles
- agglomeration of molecules containing ionic heads and hydrophobic tails form into a structure with a hydrophobic interior and hydrophilic exterior
What is capillary cell electrophoresis
A form of capillary electrophoresis in which the capillary column contains a gel enabling separations based on size
Describe capillary electrochromatography
- a form of capillary electrophoresis which a stationary phase is included within the capillary column
-SP consists of 1.5-3um silica particles coated with a bonded non polar liquid film - partitioning between Sp and buffered solutions occurs resulting in HPLC like separation
Pros of capillary electrophoresis compared to GC and HPLC
- similar levels of sensitivity and comparable degree of specificity
- smaller injection volumes (1nL CE, 1uL GC and 20uL HPLC
- fast analysis times and smaller instrument costs
- higher detection limits
Come of capillary electrophoresis compared to GC, HPLC
- poor run to run repeatability as drifts in migration times can occur
