Exam 3 Flashcards
Chromatography
Separation of a mixture of compounds based on how each compound interacts a stationary/mobile phase
Why is there a detector @the end of a chromatograph
If there is a detector at the end of a chromatogram, an instrument response can be plotted in order to display when analytes begin to leave the instrument
Elution
Analytes are “pushed” through a column by addition of a solvent (the eluent)
Mobile phase
Moves through the stationary phase of a column or on the surface of a chromatographic plate
Stationary phase
The solid or liquid that is fixed in place in a chromatographic instrument for the mobile phase to move through
Distribution constant (k)
Ratio of concentration of analyte in the stationary phase to analyze in the mobile phase @the point of equilibrium between the two phases
Retention time
The amount of time between injection of the analyze and its “peak” at the other end of the column
Retention factor (k’)
The algebraic relationship between the constant of an analyze and the volumes of the stationary and mobile phases
Volumetric flow rate
The volume of fluid passing through the column per unit of time
Linear flow velocity
Volumetric flow rate over cross-sectional area of a column
Selectivity factor
Ratio of the distribution constants of two analyses with the more strongly retained species over the less strongly retained species
Plate height (H)
Relationship between variance of a chromatographic peak over length of the column
Column resolution
Relationship between the width of the peak for two species and the distance between the peaks
Longitudinal diffusion
Source of band broadening where a solute diffuses both up and down the column
General elution problem
Describes a situation in which the retention time of two species are so vastly different that a good chromatographic peak for once species results in a severe loss of resolution for the other either in the form of band broadening or extreme narrowing
Factors affecting zone broadening
Linear velocity of mobile phase; diffusion coefficient; retention factor; diameter of packing particles; thickness of liquid coating on stationary phase
Major differences between gas-liquid and gas-solid chromatography
Partition vs adsorption; liquid stationary phase us solid; liquid can use higher concentrations
Differences between liquid-liquid and liquid-solid chromatography
Liquid-liquid is done under higher pressure and has smaller packing of materials
Variables that affect selectivity factor
Retention factor of the analytes and therefore retention times of the analytes; anything that affects retention time
How to manipulate retention factor of a solute
Alter the respective volumes of the stationary and mobile phases until an ideal ratio is reached.
How to determine theoretical plate height
Multiply 16 by (retention time over peak width) squared
Two methods for improving resolution
Decreasing peak width and increasing interval between peaks
Why does the minimum in a plot of plate height vs flow rate occur @lower flow rates in Lc
Longitudinal diffusion is a large contributor to h at lower flow rates and longitudinal diffusion occurs much faster in gas chromatography
Gradient elution
A method of liquid chromatography in which the composition of the mobilephase is changed continuously in order to optimize separations
Variables that lead to zone separations
Column length increases; variation in mobile phase composition; optimizing column temperature; changes in pH of the mobile phase; stationary phase species that selectively complexes certain analyses
Effect of introducing sample to column too slowly
Band broadening
Main problem with using retention time as indicator of presence of a particular analyze
Some analyses may have the same retention time
Discuss each of the terms of the Van Deemter equation and what effect flow rate has on each
H= theoretical plate height
A= the multiple flow path term, also known as eddy diffusion
B=longitudinal diffusion; the higher the mobile phase velocity, the narrower the peaks
C=mass transfer; the slower the mobile phase, the narrower the peaks
Mu= mobile phase velocity
Minimizing each term in the Van deemter equation
A= make particle size as homogeneous as possible and make the diameter of the packing material as small as possible B= make mobile phase velocity as large as possible, decrease the diffusion coefficient of the mobile phase C= make mobile phase velocity as small as possible, decrease film thickness and particle size; increase diffusion coefficients of stationary/mobile phases
Variables that affect migration rates of solutes
Retention time of the stationary and mobile phases and the distribution constant of the analyze
How can the separation of two non-separated peaks be improved?
Reduce zone broadening; change the relative solubility of the two compounds relative to one-another within the stationary/mobil phases
What is temperature programming in GC and why is it used?
The temperature of the column is increased over time.it is particularly usefulfor the separation of compounds that have widely varying boiling points as the compounds with low boiling points will be separated early on with good resolution and the compounds with higher boiling points will also be separated with good resolution as the temperature of the column increases
Retention volume
V-retention = t-retention X volumetric flow rate
Corrected retention volume
Corrected retention volume adds the variable j, the pressure correction factor
Specific retention volume
Is specific to the chromatographic method being used. Includes as variables the volume of an unretained analyze, the mass of the stationary phase, and the temperature of the column
Operating principle: thermal conductivity detector
Defects decreases in thermal conductivity of the helium or hydrogen carrier gas due to the presence of analyze molecules
Operating principle: atomic emission detector
Based on the intensity of specific species’ atomic emission lines. Atomization is achieved by passing the eluent through an energetic microwave field
Operating principle: thermionic detector
Used primarily for analyses that contain phosphorous/nitrogen; detects ion currents produced when the analyze is passed through a hydrogen flame and then over a rubidium-silicate bead
Operating principle: electron capture detector
Detects changes in a standing ion current by analyze molecules; electronegative functional groups are particularly effective
Operating principle: flame photometric detector
Detects radiation emitted by sulfur/phosphorous containing molecules as they are passed through a low-temperature hydrogen/air flame
Operating principle: flame ionization defector
Detects current produced by ions and electrons resulting from passing the analyze through a small air/hydrogen flame
Advantage/disadvantage: thermal conductivity detector
Advantage: widely applicable, large linear range, simple, nondestructive
Disadvantage: low sensitivity
Advantage/disadvantage: atomic emission detector
Advantage: selective, large linear range, high sensitivity, widely applicable
Disadvantage: destructive, expensive
Advantage/disadvantage; thermionic detector
Advantages: high sensitive for compounds containing N/P, good linear range
Disadvantages: destructive, not widely applicable
Advantage/disadvantage: electron capture detector
Advantages: high sensitivity, selectivity, nondestructive
Disadvantages: potentially non-linear response limited response range
Advantage disadvantage: flame photometric defector
Advantages: selectivity, good sensitivity
Disadvantages: destructive, not widely applicable
Advantage/disadvantage: flame ionization defector
Advantages: widely applicable, large linear range, good sensitivity, low noise, simple, easy to use
Disadvantages: destructive
Difference between total ion chromatogram and mass chromatogram
Total ion is obtained by summing each mass spectrum and plotting v time; Mass is obtained by monitoring one m/z ratio and plotting it v time
Why is combining gas chromatography and mass spectrometry so powerful?
Mass spec tells the experimenter what analyze species is being elated; allows for more specific id of species
Most used packing material in GC columns
Diatomaceous earths w/ diameters from 250 to 170 nm or 170 to 149 nm
What are megabore open tubular column, why are they used?
Have larger inside diameter than normal open tubular columns; used because that
Differences between WCOT and SCOT columns
WCOT: wall coated open-tubular; inner wall is coated w/ thin layer of stationary
SCOT: support coated open-tubular; inner wall is coated w/ thin layer of support material (diatomaceous earths)
What are the advantages of fused silica columns
Less reactive toward analyses; more durable
Properties of a stationary phase liquid for gas chromatography
Low volatility, thermal stability, chemically inert,provide suitable K and alpha values for analyze separation
Effect of stationary phase film thickness on gas chromatograph
Less thick = more faster analyze movement = less band broadening
Why are gas chromatographic stationary phases often bonded and cross-linked? What do these terms mean?
Bonding/cross-linking provides thermal stability and a more “permanent” stationary phase
Bonding: the stationary phase is bound in a single lager to the packing surface chemically
Cross-linking: treats the stationary phase with a reagent, increasing cross-links between the molecules of the stationary phase
Variables that increase band broadening/band separation in GLC
Band broadening: very high or low flow rates, large packing particles,thick layers of stationary phase, and slow injection rates
Band separation: enhanced by ensuring k lies between 1 and 10, small packing, limiting stationary phase for thin coatings, and rapid injection
How would the following affect plate height?
A increasing the mass of the stationary phase relative to packing mass
B decreasing the rate of sample injection
C increasing the injection port temperature
D increasing the flow rate
E reducing packing particle size
F decreasing column temperature
A increases plate height
Bband broadening; increase in plate height
C decrease in plate height
D depends
E decrease in plate weight
F increase in plate weight * in most cases *
What kind of mixtures are separated by gas-solid chromatography?
Permanent gases and low-boiling hydrocarbons
Why is gas-liquid more prevalent than gas-solid?
Gas-solid have a tendency for analyses to form complexes with the stationary phase while gas-liquid sees this problem significantly less frequently
What is the purpose of the injection port in gas chromatography and what does the port liner do?
The component used to introduce the liquid sample, heat it to the gaseous phase, and allow the carrier gas to carry the analyze into the column; the glass liner protects the analyze from ionization
List the components of the injection port & describe the purpose of each
Liner: provides a known area for flash vaporization of the analyze
Syringe: used to introduce a known volume of analyze
Injection port: where the sample is introduced
Heated metal block: heats the chamber for flash vaporization
Septum purge outlet; allows out any decomposed material from the septum
Split outlet: allows excess sample to be ejected
Rubber septum: self-sealing area for the syringe to be inserted without loss of analyze
Optimum temperature of injection port
50° Celsius above the highest boiling point among the analyses
Properties of ideal immobilized liquid stationary phase for gas chromatography
Low volatility, thermal stability, chemically inert, k’ and alpha values fall within suitable limit
Factors to be considered for temperature programming method and how to determine conditions
Take into account the boiling point of each analyze
Steps:
1. Determine initial temperature and time based on best separation of first few peaks
2. Do the same for the last few peaks
3.determine experimentally the best temperature ramp conditions for separation of the analyze
Ideal use of gas-liquid chromatography
Species that are somewhat volatile and thermally stable
Ideal use of liquid adsorption chromatography
Non-polar low to moderate mass organic species; particularly isometric organics
Ideal use of liquid-liquid partition chromatography
Molecular species that are non-volatile or thermally unstable
Ideal use of reversed-phase partition chromatography
Most low to moderate mass organics that are non-volatile or thermally unstable
Ideal use of ion-exchange chromatography
Substances that are ionic or can be derivitized into ions
Ideal use of gel permeation chromatography
High mass compounds that are soluble in non-polar solvents
Ideal use of gas-solid chromatography
Low mass non-polar gaseous speeds
Ideal use of gel filtration chromatography
High mass hydrophilic compounds
Ideal use of ion pair chromatography
Small organic and inorganic compounds
3 methods for improving resolution of partition chromatography
Adjustment of ka and kb by employing an optimized multi-component mobile phase
Variation of the composition of the solvent to make alpha larger
Employ a different packing to increase alpha
Ways to manipulate retention factor of partition chromatography
Make use of a two (or more) component solvent system and vary the ratio of the parts
How can selectivity be manipulated in gas chromatography
Vary the column packing
How can selectivity factor be manipulated in liquid chromatography
Vary the packing size, or the chemical composition of the mobile phase
Isocratic elution
Solvent composition is held constant throughout the elation
Gradient elution
Two or more solvents are used in the mobile phase and their ratio is varied throughout the elation
Reversed-phase packing
Non-polar packing used in partition chromatography; paired with a polar mobile phase
Normal-phase packing
Stationary phase is polar and mobile phase is non-polar
Ion chromatography
Stationary phase is an ion-exchange resin; normally uses conductivity defector
Sparging
Removing dissolved gasses from a solution by sweeping the liquid with a stream of fine bubbles of inert gas with low solubility
What is a guard column?
Short column before the injection port through which the Mobil phase flows; composition is similar to analytical column, but the particles are larger to avoid pressure drops; removes particulates from the mobile phase and saturates the mobile phase with the stationary phase
How are normal-phase partition chromatography and adsorption chromatography similar?
In both cases, the stationary phase is polar and the mobile phase is non-polar
Fundamental difference between adsorption and partition chromatography
Adsorption separates based on adsorption equilibrium and partition separates based on distribution equilibrium between two immiscible liquids
Fundamental difference between ion-exchange and size-exclusion chromatography
Size exclusion separates based on size and, to a lesser degree, shape of the molecule while ion-exchange separates based on ion-exchange reactions between the stationary phase and the analyze
What species can be separated by HPLC and not gas chromatography
Non-volatile and thermally unstable compounds
Describe the various pumps used in HPLC and their advantages/disadvantages
Reciprocating pumps: involve back-and-forth motion of a piston that fills and empties a cylindrical chamber; advantages: small internal volume,high pressure output, flow rates are independent of viscosity and back-pressure; disadvantages: damping of pulsed output possible
Screw-driven syringe pump: motor driven screw moves piston; advantages: pulse free and variable rate of delivery; disadvantages: limited capacity
Pneumatic pump: vessel that can be pressurized by compressed gas; advantages: simple, cheap, pulse-free; disadvantages: limited capacity,pump rates depend on solvent viscosity
Differences between single-column and supressor-column chromatography
In suppressor-column chromatography, non-ionic compounds are elated through a separate “suppressor” column
Why aren’t many of the gas chromatography defectors used for HPLC
A gas chromatography detector needs to be able to detect analyses in the gaseous phase. HPLC analyses leave the column in a liquid phase, so any defector that only works for gas couldn’t be used for HPLC
Solvent reservoir
Container used to hold the mobile phase; allows us to change the composition of the mobile phase throughout the run
Degassing system
Removes dissolved oxygen/nitrogen which can cause band broadening and interfere w the detector;
Ideal pump system
Mix solvents & vary polarity of the mobile phase during run; “unlimited” reseservoir; pressures up to 6000 psi; variable flow rates from 0.1 to 10 ml/min; flow reproducibility of 0.5% or better; resistance to corrosion; pulse-free output
Gradient controller
Allows experimenter to program a “gradient” elution
Sample injection system
sample loop and valve system for syringe allows insertion of syringe, deposit of sample, and removal of the syringe. Then a value is flipped and the sample is allowed into the column
Effect of particle size on column efficiency
Large particles = band broadening; small particles may not allow flow
Purpose & method of end-capping
Done to cover charged O molecules on the surface of the stationary phase, preserving the hydrophobicity of the stationary phase. Smaller functional group is introduced after the stationary phase in order to “cap off” any o not bound to the stationary phase.
Size exclusion chromatography
Exclusion limit: anything above maximum size particles that can fit through the pores
Permeation limit: anything too small that gets “trapped” in the pores
Selective permeation region:anything between above
