C3110 Final Flashcards
Mobile Phase
The phase in which the solute moves through the column
Eluent
Fluid entering column
Eluate
Fluid leaving the column
Stationary phase
Determines the amount of time the solute spends in the column
2 Main types of columns
- Packed
- Open Tubular
What are the 5 mechanisms of separation?
- Adsorption
- Partition
- Ion-exchange
- Size exclusion
- Affinity
Definition of retention time
Time between injection onto the column and when the component reaches the detector
Definition of retention volume
Volume of mobile phase reuired to elute a particular solute from the column
Dead time
Time required for an unretained solute to travel through the column
Volume flow rate
How many mL of solvent per minute travel through the column
Linear Flow rate
how many cm are travelled in 1 min by the solvent
What do we keep constant in terms of flow rate when changing column size?
Linear flow rate
Adjusted retention time
Time required for a solute to elute beyond the time for the solvent
Relative retention equation
a= tr2’ / tr1’
What value of alpha gives better separation?
Higher
Retention factor equation
k= (tr-tm) / tm
2 factors that contribute to how well compounds are separated
- Difference in elution time between peaks (farther apart = better separation)
- Peak broadness (wider peaks = poorer separation)
Column Resolution
how far apart two bands are relative to their widths (ability of the column to separate 2 solutes)
Plate Height
proportionality constant between variance of a band and distance travelled; length of column required for one equlibration
What is the plate height/number relation?
N=L/H
Does smaller or larger plate height give better resolution?
Smaller
Is a band narrow or wider for lower number of theoretical plates?
Wider
In the Purnell resolution equation, how are resolution and plate number related?
Resolution is proprtional to the square root of N
If the column length is increased by a factor of 4, how much does the resolution increase?
Factor of 2
What are the 2 main sources of extra column broadening?
- Variation due to injection or detection
- Variance due to connective tubing
What equation describes on column broadening?
Van Deemter Eqn
Describe what vD eqn terms are present in packed, OT, and capillary columns
Packed: A, B, C
OT: B and C
Capillary: B
Describe longitudinal diffusion
Solute molecules diffuse away from the concentrated center of a band in both directions due to a concentration gradient. Inversely proportional to flow rate. Greater for gases than liquids.
Describe finite equilibration time
The solute must diffuse from the mobile phase to the surface of the sp for equlibration to occur. The time required depends on the distance the solute must travel to reach sp (and inversely on how fast it diffuses)
Describe the multiple flow paths term
Some flow paths through a packed column are longer than others.
What are the advantages of OT columns? (3)
- Higher resolution
- Shorter analysis time
- Increased sensitivity
Disadvantages of OT columns (1)
Lower sample capacity
Fronting
Too much solute has been applited (or too concentrated) and the solute becomes more soluble in sp, eventually resembling sp.
Tailing
Small quantities of solute are retained more strongly than large quantities. Leads to a long tail of gradually decreasing concentration
Separation Process in GC
Gaseous analyte transported through a column by inert mp (carrier gas)
2 Main types of GC Separation
- Partition
- Adsorption
Common carrier gases
He, N2, or H2
Column Oven
Provides temperature control for the column
Importance of Temp Control in GC
Column must be hot enough to provide sufficient vapour P for analytes to be eluted in a reasonable time
In what type of chromatography are long, narrow OT columns made of fused silica coated with polyimide used?
GC
Wall Coated OT
Liquid sp on inner wall of the column
Support coated OT
Solid particles coated with liquid sp attached to inner wall of column
Kovats Retention Index for a linear alkane
100 times the number of carbon atoms
Temperature Programming
Temperature of the column is raised during separation to increase analyte vapor pressure and decrease RT of late-eluting compounds
Lower Temp Limits
Isothermal T limit at which column can be kept for a long time
Upper Temp Limit
Column should only be exposed for a few minutes at the end of a T run
What is a consequence of using really high temperature in GC?
Sp bleeding
3 Types of Injection into OT columns
- Split Injections
- Splitless Injections
- On-column Injections
Split Injection
Used for high concentrations of analyte. High T. Mixing chamber to promote vaporization and mixing of analytes. A large portion of the sample is vented (only 0.2-2% of sample goes to column). Can be inaccurate because split ratio is not reproducible.
Splitless Injection
Trace analysis. Same port used as split injection. No mixing chamber. A large volume of dilute solution is injected slowly. Around 80% of the sample is applied to the column.
Solvent Trapping
Initial column T is 40C below boiling point of solvent, which condenses. As solutes become trapped in a narrow band at the head of the column, detection occurs.
Cold Trapping
Column T is 150C lower than the boiling points of analytes. Solvent and low boiling solutes are quickly eluted. High boiling solutes trapped in a thin band at the head of the column. T is increased to initiate chromatography.
On Column Injection
Sensitive compounds that decompose above their boiling points. Solution injected directly to the column without entering a hot port. Initial column T is low enough to condense solutes in a narrow zone. Warming initiates chromatography.
List a couple common GC detectors (8)
- Thermal Conductivity
- Flame Ionization
- Electron capture
- Flame photometric
- Nitrogen-phosphorus
- Sulfur chemiluminescence
- Photoionization
- Mass Spec
Briefly describe thermal conductivity detection in GC
Common in the past, simple and universal. Not very sensitive.
Describe thermal conductivity detection (the process).
Eluate from column flows over a hot filament (tungsten-rhenium). H2 and He have thermal conductivities about 6-10 times that of organics. A drop in eluates thermal conductivity causes the filament to heat and electrical resistance to increase, changing the voltage across the filament. This is measured by the detector.
Advantages of Thermal Conductivity
Simple
Large linear range
Responds to organics and inorganics
Nondestructive to sample
Responds to everything but carrier gas
Disadvantages of Thermal Conductivity
Low sensitivity
Won’t work well for complex mixtures
Describe flame ionization detection (process)
Eluate is burned in a mixture of H2 and air. Carbon atoms produce CH radicals, which further produce CHO+ ions and electrons in the flame. Ion production is proportional to number of carbons entering the flame. Current changes in presence of analyte.
Advantages of FID (4)
- Insensitive toward noncombustible gases
- Good for samples contaminated with water/oxides
- Good detection limits (100 times better than thermal conductivity)
- High linear response
Disadvantages of FID (2)
- Destructive to sample
- Requires additional gases and controllers
Describe electron capture detection
Sample eluate from column is passed over a radioactive beta emitter, usually 63Ni. An electron from the emitter causes ionization of the carrier gas which produces a cloud of electrons. With no analyte, there is a constant current between electrode. Current decreases when analyte with EN FGs enter and capture electrons.
What is electron capture detection used for typically?
Environmental samples, anything with halogens
What detector is also called an alkali flame detector?
Nitrogen-phosphorus
Which detector measures optical emission from selected elemetns?
Flame photometric
Which detector uses a vacuum UV source to ionize aromatic and unsaturated compounds?
Photoionization
Sample Prep Processes (4)
- Extracting analyte from complex matrix
- Preconcentrating dilute analytes
- Removing or masking interfering species
- Derivatizing analytes into a more convenient or easily detectable form
Solid Phase Extraction
Sorbent is loaded into a cartridge and sample is loaded onto the phase, retaining analytes that strongly interact with the phase.
Solid-phase mixroextraction
Extracts compounds from liquids, air, or sludge without solvent.
Fused silica fiber coated with sp is attached to a syringe. A large volume of solution is extracted; mass extracted is proportional to concentration of analyte in solution.
Which sample prep method uses a magnetic stir bar to absorb hydrophobic analytes?
Stir-bar sorptive extraction
Purge and Trap
Method for removing volatile analytes from liquids or solids, concentrating the analytes, and introducing them into GC. Goal is to remove 100% analyte from sample.
Thermal Desorption
Method for relasing volatile compounds from solid samples.
Order of decisions in method development (GC), (5)
- Goal of analysis
- Sample Prep
- Detector
- Column
- Injection
Typical length and inner diameter of fused-silica capillary tubes
50cm
25-75um
Principle of CE
Different ions have different mobilities and move through the column at different speeds.
What is the only source of band broadening in CE?
Longitudinal diffusion
Describe electrophoresis
When an ion with charge q is placed in an electric field E, the force on the ion is qE. The ion reaches a steady speed when the accelerating force equals the frictional force.
Electrophoretic mobility
Proportionality constant between the speed of the ion and the electric field strength
Describe electroosmosis (EOF)
Inside wall of a fused-silica capillary is covered with silanol, which have negative charge above pH 3. A tightly adsorbed layer of cations partially neutralizes the negative layer. Remaining charge is neutralized by mobile cations in the diffuse part of the double layer in the solution near the wall. Excess cations in the diffuse part are attracted to the cathode and impart net momentum, called EOF
What is the contrast between EOF and hydrodynamic flow?
In hydrodynamic flow, the velocity flow is fastest at the center and slower at the walls due to friction. This is not the case in EOF.
Apparent mobility
Sum of electrophoretic mobility and electroosmotic mobility
True or false: smaller ions have smaller electrophoretic mobility
False
True or false: At neutral or high pH, electroosmosis transports anions to cathode because electroosmosis is faster than electrophoresis.
True
What is the number of theoretical plates proportional to in CE?
Apparent mobility, applied electric field, and distance to the detector.
At a fixed voltage, EMF is strongest in shorter or longer capillary?
Shorter
2 Methods of Sample Injection in CE
- Hydrodynamic injection (P)
- Electrokinetic Inject (electric field)
What is BGE and how does it change during CE runs?
Background electrolyte
pH will be altered during electrophoresis, potentially forming a gradient and effecting migration times. BGE must be replaced after every run to avoid this.
How do we reverse the direction of EOF?
Add a cationic surfactant
What is Micellar electrokinetic chromatography?
Used to separate neutral species. Uses a pseudostationary phase in the form of a micellar phase.
Describe the process of MEKC
In the absence of micelles, neutral analytes reach detector at the same time. Negative micelles, injected into sample, reach detector at a specific time. If a neutral molecule equilibrates between solution and micelle, the migration time is increased, since the micelles migrate slower.
What term does MEKC contain that CE does not in the vD eqn?
Mass transfer, C
How can we use MEKC to separate 2 chiral molecules?
ADD BGE modifiers, such as cyclodextrins
Name 5 common CE detectors (briefly describe each)
- UV; poor sensitivty and background must have low absorption
- Fluorescence; high dynamic range
- Contactless conductivity detection; electrodes outside capillary that measures conductivity differences
- Amperometric detection; sensitive to analytes that oxidize or reduce
- Electrospray MS; low detection and gives structural info
Advantages of CE compares to chromatography (4)
- High resolution
- Low waste (Green)
- Simple equipment
- Low sample consumption
Disadvantages of CE (4)
- Higher detection limits (insensitive)
- Irreproducibility
- Insolubility of analyes in BGE
- Challenging scale up
What is a real world advance of CE?
Lab on a chip devices
Zipchip
What are the 6 components of HPLC?
- Autosampler
- Solvent delivery system
- Sample injection valve
- High-P chromatography column
- Detector
- Computer
Why do smaller particles give better resolution in HPLC? (2)
- More uniform flow reduces A term
- Less distance the solute must diffuse reduces C term
What do smaller particles give in HPLC? (4)
- Higher plate number
- Shorter run time
- Lower detection limits
- Higher P
Describe HPLC column sp
Filled with sp of highly pure, spherical, microporous particles of silica.
Guard Column
Protects the main chromatographic phase in the column
Most common bonded phase
C18
RPLC
sp is nonpolar/weakly polar, solvent is polar. Less polar solvent is a stronger mp.
NPLC
Polar sp and less polar solvent. More polar solvent has higher strength.
Eluent strength
Measure of the solvent adsorption E on bare silica; value of pentane defined as 0
What is HILIC and what is it used for?
HILIC is hydrophilic interaction chromatography. Used to separate molecules too polar to be retained by RPLC.
Sparging System
Removes dissolved gases by sweeping them out of solution with fine bubbles of an inert gas such as He.
Why is sparging necessary in LC?
Dissolved oxygen can absorb UV and interfere with UV detection. Other gases can cause issues for the column, pumps, and detectors.
What is the process to clean a column before storage (LC)?
- Replace aqueous buffer with water and wash with 5-10 mobile phase volumes.
- Wash with 10-20 volumes of strong eluent.
- Store column with solvent to inhibit bacterial growth
Why would a material with a high UV cutoff not be used very often as a solvent with UV detection?
A high UV cutoff results in very many materials absorbing UV and thus is not very sensitive.
Describe the general elution problem and what is used as a solution.
The general elution problem is that one set of conditions is not suitable to separate a complex mixture of analytes in a reasonable amount of time. Gradient elution is a solution.
Gradient Elution
Uses a continuous change of solvent composition to increase the mobile phase strength.
How do spectrophotometric detectors work?
A photodiode array records the spectrum of each solute as it is eluted. Spectra can be matched with library spectra to identify the analyte.
Fluorescence Detection
Eluate is excited with a laser and fluorescence at longer wavelengths is measured. Up to 100 times more sensitive than UV detectors, but few analytes fluoresce.
Electrochemical Detector
Responds to analytes that can be reduced or oxidized. Potential is controlled with respect to a reference electrode, while current is measured between working electrode and auxillary electrode.
Refractive Index Detection
Responds to almost every solute. Detection limits 1000 times poorer than UV. When a solute with a different refractive index than the mobile phase enters the cell, a beam of light is deflected. Useless for trace analysis and gradient elution.
Common methods of LC-MS interface
- ESI
- APCI
What does a performance qualification sample (internal standard) tell us?
If there is a problem with the run, the internal standard indicates whether or not the issue is with the chromatography of the analyte or with the instrument.
