Unit 5 Flashcards
Elution chromatography
Column is filled with stationary phase .
Sample is added at the inlet and moved over the stationary phase by a mobile phase (ie solvent).
Analyte partitions between the mobile and stationary phase, moving down the column because of the time spent in the mobile phase.
KA = [A]stat/[A]mob
Describe solid-phase micro-extraction
Support coated with a hydrophobic extraction phase (ex.polymer)
Amount of analyte extracted to its concentration in a sample.
Simple, suitable for on-site sampling.
Can pre-concentrate analyte
Larger partition coefficient means
More partitions into the stationary phase
Define components of the partition coefficient
KA = [A]stationary/[A]mobile
Give examples and properties of solid adsorption media
Silica (SiO2): Polar, slightly acidic
Alumina (Al2O3): Polar, neutral or slightly basic
Steps of thin-layer chromatography
Spot a mixture onto plate of silica/alumina (stationary phase)
The bottom big the plate is immersed in solvent, which then travels up the plate by capillary action (mobile phase)
The mixture separated into spots or bands
Visualizing colourless compounds (uv-shadowing, charring, chemical staining
Elution Adsorption chromatography (preparatory technique for chemical synthesis): steps
Column packed with silica gel or alumina
Load sample mixture as narrow band at top of column.
Elute with organic solvent as mobile phase bands for each component separate out
Collect volume fractions
What can separation science do?
Separate and isolate components of the mixture. The amount of each analyte can then be determined.
What can chromatographic techniques mitigate?
Poor intrinsic selectivity of other analytical techniques.
Benefits/downsides of tlc
Quick, simple, low cost, but only qualitative
Block diagram: HPLC instrumentation
HPLC pump (solvents;mobile phase in), injector (sample in), HPLC Column, Detector (waste out), electronics/computer
HPLC pump function
Forces mobile phase through column at high pressure
Injector
Introduces a reproducible volume of sample onto the column
Column
Effects the separation of analytes
Detector function (HPLC)
Produces a measurable signal when analytes elute from the column
Function: inlet and inline solvent filter
Removes particulate matter and air bubbles (ie. Degas) in mobile phase
Pre-column filter
Remove particulate matter introduced with sample
Guard column
Protects analytical column. Removes particulate matter and chemical components that would irreversibly bind to the analytical column
What would happen without HPLC filters?
Top segment of stationary phase in the analytical column would be rapidly deteriorated
Back pressure regulator
Prevents bubbles from forming in the detector cell as the mobile phase exits the column
Draw out an HPLC diagram
See slide 5 under high performance liquid chromatography
Benefits and trade-offs of decreased particle size
Column more densely packed, separation efficiency increase
Tradeoff: very high back pressures. Gravity insufficient to move mobile phase through column. Pumps required
Slide 6 under high performance liquid chromatography
Analytical column
Used for trace analysis and detection
Diameters between 1-10mm and lengths on order of 10-10^2 um
Preparative and semi-preparative columns
Used for purification of synthesized compounds (laboratory scale)
Column diameters and particle sizes are typically about an order of magnitude larger than analytical columns
Bonded stationary phase: column is packed with:
Silica particles. Functional particles are bonded to silica particles to modify their polarity. Slide 10 high performance chromatography
What type of stationary and mobile phase is in normal phase HPLC? Also provide examples.
Polar stationary phase: silica, amino, cyano
Non-polar mobile phase: hexanes, diethyl ether, Ethyl acetate, dichloromethane, etc.
What type of stationary and mobile phase are in reverse HPLC? Also provide examples
Non-polar stationary phase: C18, C8, phenyl, etc.
Polar mobile phase: water, methanol, acetonitrile, propanol, etc.
Predict the order of elution of the following compounds on a reverse phase column for LC.
Benzene, 1-hydroxybenzene, 1,3-dihydroxybenzene
A. Benzene (first), 1-hydroxybenzene, 1,3-dihydroxybenzene (last)
B. Benzene, 1,3-dihydroxybenzene,1hydroxybenzene
C. 1,3-dihydroxybenzene, 1-hydroxybenzene, benzene
D. 1,3-hydrobenzene, benzene, 1-hydroxybenzene
E. The 3 compounds co-elute
C. 1,3-dihydroxybenzene, 1-hydroxybenzene, benzene
Which of the following methods will extract an analyte from a 50 mL aqueous solution with the highest efficiency?
A. One 50 mL portion of organic solvent
B. One 150 mL portion of organic solvent
C. Two 75 mL portions of organic solvent
D. Three 50 mL portions of organic solvent
D. Three 50 mL portions of organic solvent
Examples of Commercial Systems
- Solvent reservoirs
- Pumps and valves
- Autosampler and injector
- Column in thermostated oven
- UV-vis detector
Sparging
Helium is bubbled through the solvent reservoirs to sweep out dissolved air. Helium is insoluble in most solvents. Slide 16 under high performance liquid chromatography.
Solvent proportioning valve
Mixes different solvents together in defined and controllable proportions.
Draw and Label: Single wavelength detector (HPLC UV-Vis Detector)
Slide 17 under High Performance Liquid Chromatography
Draw and label: Diode Array (HPLC UV-Vis Detector)
Slide 18 under High Performance Liquid Chromatography
Design (shape) of the flow cell and what its good for
Z-shaped. offers long path length (better sensitivity) without diluting the sample or significantly affecting flow.
Advantages of Diode Array
- Speed
- The diode array permits acquisition of full spectra as analytes elute from the column (or acquisition of several wavelengths in parallel)
Commonly monitored wavelengths and the groups they are associated with:
- 190-210 nm
- 210-220 nm
- 240-260 nm
- 190-210 nm: Isolated double bonds
- 210-220 nm: Carbonyl groups, dienes
- 240-260 nm: Aromatic groups
UV-visible detectors are sensitive to
Concentration and non-destructive
Fluroescence detector
Very sensitive, but often requires derivatization of non-fluorescent analytes
Refractive index detector
Universal detector, but not very sensitive
Evaporative light scattering detector
Mass sensitive. Useful with almost all non-volatile analytes.
Charged aerosol detector
Mass sensitive. Useful with almost all analytes
Electrochemical detector
Voltammetric measurements on eluent. Sensitive to species that can be oxidized or reduced.
Mass spectrometer
HPLC-MS is a hyphenated technique that combines HPLC with mass spectrometry. The method is both sensitive and powerful, providing information about molecular structure and identity while also permitting quantitative analysis.
HPLC Chromatogram
Analytes can be differentiated and identified on the basis of their retention time.
The size of the peaks indicates something about the quantity of the corresponding analyte.
Draw and label: HPLC Chromatogram
Slide 25 under High Performance Liquid Chromatography
Dead Time (tm)
Time required for the mobile phase to travel the length of the column.
Retention time (tr)
Time required for the analyte to elute from the column
Adjusted retention time, with formula
Retention time corrected for dead time.
t’R = tR - tM
Retention factor (k):
A measure of the relative amount of time an analyte spends in the stationary phase.
k = (tR - tM)/tM
k = KVs/VM
k = time in stationary phase/time in mobile phase
Resolution
The degree to which two peaks are separated in a chromatogram. See slide 27.
Theoretical plate
Conceptual representation of a separation step. More theoretical plates, better separation.
See slide 28
Tailing
Ubiquitous (found everywhere) in liquid chromatography. Often results when there is more than 1 retention mechanism for the analyte. For ex, acidic silanol groups (w/o C18 modification) can support ion exchange and hydrogen bonding interactions (in addition to partitioning with C18).
Other common causes include a sample dilution solvent that is a stronger eluent than the mobile phase, contamination of the stationary phase, and sample overload. Slide 29
Fronting
Opposite of failing; relatively rare in liquid chromatography. Usually caused by bad column packing (fix by replacing the column)
Rate theory
Band broadening and its impact on separation efficiency
The longer an analyte remains on column, the…
Broader the peak shape becomes. Total area remains constant
Higher number of plates (N); higher plate height means
There are narrower distribution of carbon numbers from each trap (or plate). Higher number of plates means the narrower “peak” obtained from that trap
Plate height formula
N = L/H
Abbreviated Form of the Van Deemter Equation, define each term
H = A + B/v + Cv where A,B,C are constants A = "Eddy" Diffusion (Anastomosis) B = Molecular Diffusion v = linear flow rate C = Rate of Mass-Transfer (Non-Equilibrium Effect)
Draw a Van Deemter Grraph
Slide 36 under high performance liquid chromatography
As particle size decrease and monodispersity increases, what happens to various paths through the column?
The various paths through the column become more uniform in distance
As particle size decreases, what happens to diffusion lengths?
Decrease.
Diffusion length: the distance an analyte must travel to reach the stationary phase.
Isocratic elution + examples
The same mobile phase composition is used throughout the separation. Example: Reverse phase HPLC, Solvent A = water, Solvent B = acetonitrile. Slide 40
Gradient Elution
The mobile phase composition is gradually changed during the separation, used to maintain good resolution all around
In reverse phase HPLC, the mobile phase is gradually made…
Less polar
Draw linear gradient, segmented gradient, and step gradient
Slide 40 under higher performance liquid chromatography
How to identify: Low organic content in RP-HPLC
Good early peak resolution, Poor late peak resolution (lag)
How to identify: High organic content in RP-HPLC
Poor early peak resolution (mass transfer is inadequate), good late peak resolution
Draw and label: Gas Chromatography column
Slide 3 under GC
GC: does the gaseous mobile phase act as a solvent
No
GC: what drives analytes into the gas phase
Temperature
Analytes of GC can remain in…. condense in….. dissolve in…
Remain in vapour phase, condense on stationary phase, dissolve in stationary phase
Predict the order of elution of the following compounds on a GC column with a moderately polar stationary phase:
A. Hexane (bp 68C), 1-butanol (bp 117C), octane (bp 125C)
B. Hexane, octane, 1-butanol
C. Octane, hexane, 1-butanol
D. 1-butanol, octane, hexane
E. The butanol and octane will co-elute
C. Octane, hexane, 1-butanol
Compounds with lower b.p. move through column more quickly; have shorter retention times.
More polar = more likely to remain on stationary phase
Describe Gas Chromatographs
Gaseous Mobile phase, liquid/solid stationary phase, partitioning generally independent of mobile phase gas, temperature is critical
Draw and label: Gas chromatograph block diagram
See slide 5 GC
Draw and label: GC Capillary Column
Slide 7 GC
FSCOT/WCOT
Fused silica/Wall-coated open tubular column:
High efficiency, low capacity, most common type
For narrow capillary columns, what is required for sample injection?
Splitter system
Split/Splitless injection
Inject liquid sample into a heated port with a syringe.
Rapid vaporization of sample.
Some of sample bled off to waste, some enters column, the amount of which is controlled by valve
What does flame ionization detector do?
- Air/hydrogen flame.
- Hydrocarbons burned to produce ions (most prominent =formylium) and electrons
- ions and electrons collected by electrode assembly help at high potential
- current measured is proportional to amount of material
Flame ionization detector is insensitive to
Most inorganic compounds, eg. O2, N2, SO2, NH3, CO2
Flame ionization detector has no response to
Fully oxidised carbons, ex. carbonyl, carboxyl
Detection limit and dynamic range of flame ionization detector
Detection limit as low as 10^-12 g C/s;
Dynamic range of ~10^7
Draw the effect of flow rate on Van Deemter Plot
Slide 10 GC
Temperature programming is the GC equivalent to
Gradient elution in LC
Difference between isothermal and temperature ramp plot
Slide 11 GC
Comparison: Liquid Chromatography and Gas Chromatography
Liquid Chromatography:
- Applicable to any soluble compound (eg ions, small molecules, polymers, biomolecules)
- More versatile optimization than GC (can vary mobile phase and stationary phase)
- purified compounds can be collected afterwards
- Often less preparation than GC
Gas Chromatography:
- Requires volatile and thermally stable compounds
- Faster than HPLC (minutes vs. tens of minutes)
- More sensitive, better resolution than HPLC
- More “universal” detectors, less expensive
