What is gas chromatography?
gaseous analyte is transported through the column by a gaseous mobile phase.
A gaseous mobile phase is also referred to as _________ ____.
carrier gas
Describe the GC process.
1) a volatile liquid or gas sample is injected through a septum into the heated port
2) Sample evaporates and is pulled through the column with a carrier gas
3) the column is heated to provide sufficient vapor pressure to elute analytes
4) analytes separately flow through a heated detection for observation.
The mobile phase in GC is usually a (solid/liquid/gas).
gas
What is usually the stationary phase in GC?
nonvolatile liquid, but sometimes a solid.
What is the usually the analyte in GC?
gas or volatile liquid
Name the three different carrier gasses.
- He
- N2
- H2
What does the choice of carrier gas depend on?
the detector and the desired separation efficiency and speed
What are the advantages of using open tubular column rather than packed columns?
- higher resolution
- shorter analysis time
- greater sensitivity
- lower sample capacity
Name the three types of open tubular columns.
- Wall-coated open tubular column (WCOT)
- Support coated open tubular column (SCOT)
- Porous-layer open tubular column (PLOT)
Describe a wall-coated open tubular column (WCOT)
A 0.1 -5 µm film of liquid stationary phase on the inside wall of the column
Describe a support-coated open tubular column
(SCOT)
solid particles coated with liquid stationary phase and attached to the inner wall.
Describe a porous-layer open tubular column (PLOT)
solid particles are the active stationary phase on inside wall of column
(narrow/wider) columns provide higher resolution than (narrow/wider) columns.
narrower; wider
what are the disadvantages of a narrower column?
require higher operating pressure and have less sample capacity.
What are the two types of columns?
1) Open Tubular Column
2) Packed Column
What is the range of a open tubular columns inner diameter, length, and thickness of stationary phase?
diameter: 0.10 to 0.53 mm
length: 10-100 mm
thickness: 0.1 -5 µm thick
Diameters greater than ____ mm tend to overload the vacuum system of a mass spectrometer.
0.32
what does increasing the thickness of the stationary phase of an open tubular column do at a constant linear velocity.
increases the retention time and sample capacity and increases resolution of early eluting peaks with a retention factor of k ≤ 5
Thick films of staionary phase can _______ tailing.
reduce
thick films of staionary phase can __________ bleed (decomposition and evaporation)
increase
List the three different types of open tubular columns in order of increasing surface area.
WCOT < SCOT < PLOT
List the three different types of open tubular columns in order of greater separation efficiency.
PLOT < SCOT < WCOT
For an Open tubular column, Rs increases with a ________ column
longer
Describe Packed Columns.
Solid fine porous particles coated with non-volatile liquid stationary phase.
How do Packed columns compare to Open tubular columns (OPTC)
- Packed columns are shorter in length and wider in diameter
- Has greater sample capacity
- Give borader peaks
- have less separation efficiency
- longer retention times
Packed columns are usually used for what type of separations
Preparative
What is the range of diameter and length for a packed column?
diameter: 3-6 mm
length: 1-5 m
In a packed column, unifrom particle size (increases/decreases) the multiple path term in the van Deemter equation.
decreases
The Kovats retention index for Linear alkanes are equal to _____ times the number of carbon atoms.
100
For gas chromatography, _____________ temperature will decrease retention times of late-eluting components.
increasing
For gas chromatography, _____________ temperature will increase analyte vapor pressure
For gas chromatography, increasing temperature will _________ peaks for late-eluting components.
sharpen
What are the four signs of column degradation?
- Increased baseline noise at low temperature
- Peak boradening
- Tailing
- Retention time changes
What is column “bleeding”?
decomposition of stationary phase at high temperatures
For gas chromatography, Increasing inlet pressure (increases/decreases) the flow of the mobile phase and (increases/decreases) retention time.
increases; decreases
What does the low end of temperature limit mean for the column?
column can be kept for a long time if used at this temprature
What does the high end of temperature limit mean for the column?
column should only be exposed for a few mintues, short lifetime if used at this temperature. Column bleeding more likely to occur.
For analytes that are unstable at high temperatures, what elution technique should be used?
Pressure Programming
Name the two types of elution techniques.
- Temperature Programming
- Pressure Programming
Kovats retention index, I, for any given solute can be calculated from a chromatogram of a mixture of that solute with at least two _______ alkanes having retention times that bracket that of the solute.
normal
Analytes always elute in the (increasing/decreasing) order of Kovats index.
increasing
What is the most common carrier gas used with most detectors?
He
List the carrier gases in order of increasing optimal flow rate.
N2 < He < H2
Why does H2 and He give better resolution (smaller plate height) than N2 at high flow rates?
becuase solutes diffuse more rapidly which decreases mass transfer (Cux)
List the carrier gases in order of decreasing diffusion coeffcients.
**rentention factor, k, is the only variable that effects the rate of mass transfer, therefore is the diffusion coeff.**
H2 > He > N2
What is a guard column used for in gas chromtography?
accumulates nonvolatile substances that would contaminate the chromatorgraphy column
Label and define compenents of the air sandwhich Injection
word bank:
(Sample, solvent, 1st air, 2nd air, 3rd air)
1st air (far left): prevents the sample from evaporating
Sample: Analyte being injected
2nd air: prevents the sample and the solvent from mixing.
Solvent: used to wash the sample off.
3rd air: used to wash the solvent off.
Name the three different injection types used into open tubular columns.
- Split Injection
- Splitless Injection
- On- column Injection
When is it preferred to use the method of Spilt injection over the others?
- when analytes of interest constitute >0.1 % of the sample
- Small sample size volume ( ≤1 µL)
About how fast should samples be injected in a split injection?
< 1 s
Describe the process a sample goes through within the instrument when injected by split injection.
- sample is injected rapidly through the septum into the evaporation zone, which is kept at high temperature to promote fast evaporation. Septum purge also occurs to remove excess sample vapor.
- carrier gas sweeps sample to the mixing chamber where vaporization and mixing occur.
- Sample then travels to the split vent where a small fraction of vapor enters the chromatography column and the rest goes to waste
In split injection, what is the sample that does not reach the column reffered to as.
and what does it range from.
split ratio
50:1 to 600:1
When is it preferred to use the method of Spiltless injection over the others?
- analytes that are less than 0.01% of the sample
- best for trace levels of high-boiling solutes in low-boiling solvents
Describe the process a sample goes through within the instrument when injected by splitless injection.
- large sample (~2µL) of dilute solution in a low-boiling point solvent is injected slowly (~2s) with split vent closed
- 80 % of sample is used applied to the column, and little fractionation occurs.
How does splitless injection differ from split injection.
- spliless injection does not use a mixting chamber
- Injection volume is larger (~2µL)
- injection time is longer (~2 sec)
- amount of sample applied to column is larger (80% of sample)
For splitless injection technique, solvent trapping, temperature at the column head is set ______0 C (above/below) the boilng point of the _______.
40; below; solvent
Explain how solvent trapping works in splitless injection.
- solvent condenses at the beginning of the column, then solutes catch up with the condensed solvent and become trapped in the solvent band.
- Chromatography is initiated by raising the column temperature to evaporate and vaporize the solutes and solvent
**Note: without solvent trapping, there would not be as sharp chromotographic peaks.**
For splitless injection technique, cold trapping, temperature at the column head is set ______0 C (above/below) the boilng point of the _______.
150 ; below; analyte
Explain how cold trapping works in splitless injection.
- Solvent and low-boiling components are eluted rapidly, but high boilng analytes remain in a narrow band at the beginning of the column.
- Chromatography is initiated by rasing the temoerature and allowing analyte to evaporate.
When is it preferred to use the method of On-Column Injection over the others?
- best for thermally unstable solutes and high-boiling solvents;
- best for quantitative analysis
Describe the process a sample goes through within the instrument when injected by on-column injection
soultion is injected directly into the column, without going through a hot injector.
What characteristics does on-column injection have that differs from the other two types of injections?
- No mixing chamber
- direct injection into the column
- very little loss of solute
When using the method of on-column injection, the initial oven temperature is kept (low/high) in order to trap all solutes in narrow band.
low
List the detectors used for gas chromatography?
- Thermal Conductivity Detector (TCD)
- Flame Ionization Dectector (FID)
- Electron Capture Detector (ECD)
- Mass Spectrometer
Which carrier gas gives the lowest detection limit with thermal conductivity detectors?
H2
He
What does a Thermal Conductivity Dectector do?
measures the ability of a substance to transport heat from a hot region to a cold region by measuring the conductance in relation to temperature.
Any _______ that is mixed with the carrier gas (highers/lowers) the conductivity of the gas stream, (increasing/decreasing) the temperature of the detector lock.
analyte; lowers; increasing
With thermal conductivity detectors, as conductivity goes down, resistance goes ________
up
What is the relationship between the sensitivity of the thermal conductivity detector and the flow rate.
sensitivity of thermal conductivity detector is inversely proportional to the flow rate.
What are the advantages of using a thermal conductivity detector?
(5 advantages)
- Simple and Universal
- Detects organic and inorganic compounds
- Non-constructive
- Allows detection of solutes after detection
- Passes solutes to a second detector
Whats are the drawbacks of using a thermal conductvity detector?
(2 drawbacks)
- Responds to impurities, decomposed staionary phase, or air that leaks into the system
- low sensitivity
How does a Flame Ionization Detector work.
write the representative equation.
eluate is burned in a mixture of H2 and air. Carbon atoms from organic compunds produce ions in the flame.
CH + O —-> CHO+ + e-
In a flame ionization detector, a __________ monitors the amount of ions from the reaction
collector
What type of carbons cannot produce ions and electrons in the flame
carbonyl and carboxyl carbons
What is the most widley used detector in GC?
Flame Ionization Detector
List the advantages of using a Flame Ionization Detector.
- Mass Sensitive
- Independent of flow rate
- 100~1000 fold better than TCD
- Most widely used detector in GC
List the Drawbacks of using a Flame Ionization Detector.
- Insensitive to non-hydrocarbons and non-combustible gases (H2O, CO2 , SO)
How does a Electron Capture Detector (ECD) work?
Electrons travel between a 63Ni electrode and a collector electrode. Analytes with electron capturing ability pass through the cell and decrease the flow of electrons.
In an electron capture detector, what is the relationship between the change in current and analyte concentration?
the change in current is proportional to the analyte concentration.
List the advantages of an electron capture detector?
- Sensitive to halogens, nitriles, carboxyl, and nitro compounds
- Non-destructive
List the drawbacks of using an Electron Capture Detector.
- A radioactive source is used
- Analytes must be able to capture electrons
List the other detectors
- Nitrogen-Phosphorous Detector (NPD)
- Flame photometric detector
- Photoionization detector
- Sulfur/Nitrogen chemiluminescence detector
What does a Mass Spectrometer do?
converts eluting analytes into gas phase ions, forms a molecular ion and breaks molecular ions into fragments which are then eluted based on thier mass to charge ratio
Which GC detector is best for detecting organic compounds?
Flame Ionization Detector
Which detector is best for detecting compunds with electronegative groups?
Electron Capture Detector
Which detector is best for detecting compounds containing N and/or P?
Nitrogen-Phosphorus Detector
Define Sample Preparation
Transforming the sample into a form that is suitable for analysis.
Name the four different techniques of sample preparation applicable to GC
**that were discussed in class**
- headspace analysis
- Purge and trap
- Thermal Desorption
- Solid-Phase MicroExtraction (SPME)
Describe Headspace analysis
- the sample phase is introduced into a vial and the vial is sealed.
- components diffuse into the gas phase until headspace has reached a state of equilibrium.
- The sample is taken from the headspace with a gas sample syringe
Describe Purge and Trap
bubble purge He gas is mixed with a heated sample to evaporate analytes, and are captured on an adsorbant column.
**extracts 100% of analyte from sample**
Describe Solid-Phase Microextraction
extracts compounds from liquids or air without using any solvent by being absorbed on a coated fiber
**only extracts a portion of analyte from sample**
What five topics do you consider when choosing a proper method development for GC?
**in a specific order**
- Goals of analysis
- Sample Preparation
- Detector
- Column
- Injection
In liquid chromatography, the efficiency of a packed column increases as the size of the stationary particles (increases/decreases).
decreases
List the chromatographic process
- Sample Injection
- Sample Transportation
- Column separation
- Analyte detection
- Analyte exiting
Describe how a 6-port injection valve in the load position works.
Sample loop is filled with sample solution by applying pressure on it, excess sample is delivered to waste, and solvent is continously pumped through the column.
How much sample can a sample loop hold?
2 to 1000 µL
Describe how a 6-port injection valve in the Inject position works.
from the load position, the valve is rotated 600 clockwise and the solvent pushes the analyte solution in the sample loop into the column
What is a guard column?
a short column that is placed in front of the main column and used to remove particle and contaminants from the solvent.
What is the most common stationary phase used in LC?
silica
Name the 5 types of HPLC techniques
- Partition Chromatography
- Adorption Chromatography
- Ion-Exchange Chromatography
- Size Exclusion Chromatography
- Affinity Chromatography
normal-phase chromatography uses a (polar/nonpolar) stationary phase
polar
In normal-phase chromatography, (most/least) polar component is eluted first.
least
In normal phase chromatography, the (more/less) polar solvent has higher eluting strength
more
In normal-phase chromatography, inreasing the polarity of the solvent (increases/decreases) the elution time.
decreases
Reverse-phase chromatography uses a (polar/non-polar) stationary phase.
non-polar
In Reverse-phase chromatography, the (most/least) polar component is eluted first.
most
In Reverse-phase chromatography, increasing the polarity of the mobile phase (increases/decreases) the elution time.
increases
What are the requirements of a solvent to act as a mobile phase in HPLC.
- Must be HPLC grade
- must prevent contamination and damages to columns.
what are the two types of programmed elution techniques used for HPLC?
- Isocratic Elution
- Gradient Elution
What is Isocratic Elution?
a HPLC elution technique performed with a single solvent or constant composition solvent mixture
What is Gradient Elution?
- One or more solvents
- continously changing in solvent composition
- Fast elution for late-eluting species
- Re-equilibrate at the beginning of each one
In HPLC, What are the different treatments that can be used on sample to ensure no contamination
- Sonication
- Filtration
- Sparging
What is sonication?
Removes bubbles from sample
what is filtration?
Removes particles from sample
What is Sparging?
Removes O2 by an introduction of an inert gas
(purging with gas)
List optimization steps used to achieve optimal separation in HPLC.
1) Acetonitrile and water
2) Methanol and water
3) Tetrahydrofuran and water
List the suggested order of steps to try separating two closely spaced peaks in HPLC.
- Change the solvent strength
- change the temperature
- change the pH (in small steps)
- use a different solvent
- use a different kind of stationary phase
What is the desired Rs?
>1.5
what range of k (retention factor) value is an adequate separation in a reasonable time?
0.5~20
what is ion-exchange chromatography?
charged stationary phase attracts solute ions
