Lecture 9-10 Flashcards
ESI stand for? How does it work?
Electrospray ionization.
Liquid in metal capillary - taylor cone - fine filament of liquid - generate to smaller “+” droplet (relatively large droplet - smaller droplet - unstable droplet - gas-phase ion) inlet to mass spectrometer.
LC definition?
LC creates a problem, what is it?
Solution?
Liquid chromatography
LC creates a huge volume of gas (as solvent evaporizes)
MS only works in vacuum, we need to remove the gas after LC prior to introduction into the MS-> need ‘interface’
How does the improvement of MS work?
____ ____ ____ at ____ outlet combined with ____ flow of N2 creates a fine aerosol of ____ ____.
There is little ____.
Strong electric field, nebulizer, coaxial
charged particles
fragmentation
Pneeumatically assisted electrospary interface for MS, for proteins, common to find multiply charged ions, ____, ____ and sometimes ____ or ____.
[M+nH]^n+
[M+nNa]^n+
[M+nNH4]^n+
____ is the key to identify and quantify one/more components from complex mixtures.
Separation
Solvent extraction: transfer of a ____ from one phase to another, to ____ or ____ desired analyte(s).
solute
isolate
concentrate
Solution that use for extraction of aqueous solution with an immiscible organic solvent:
- lighter than water: ____, ____ and ____.
- heavier than water: ____, ____ and ____.
diethylether, toluene, hexane
chloroform, dichloromethane, carbontetrachloride
Effect of pH on extraction
if solute is ___ or ___ -> molecular form and charge may change with pH.
____ ____more soluble in aqueous solution;
____ and ____ ones more soluble in organic solvent.
acid base
Charged species
Neutral hydrophobic
Extraction with a ____ ____: separate metal ions from each other: selectively complex one ion with an ____ ____ and extract the complex into an ____ solvent.
metal chelator
organic ligand
organic
Each ____ can react with many different metal ions, but selectivity can be achieved by controlling ___ -> select ___ where K ____ for one metal and ____ for others.
ligand pH
pH large small
Partition chromatography: a technique to separate ____ ____ by repeated ____ by two ____ ____.
similar substances
extraction
immiscible liquids
How does chromatography work?
Chromatography operates on same principle as extraction, but one phase held in place while other moves past it.
What is mobile phase?
What is stationary phase?
What is elution?
Mobile: solvent (liquid or gas)
Stationary: column phase
Elution: process of passing liquid/gas through a chromatography column
The idea behind chromatography?
Separate a mixture into its components based on partitioning(分隔，分开) of compounds.
Types of chromatography:
Adsorption chromatography (Tswett)
Partition chromatography (Martin and Synge)
Ion-exchange chromatography (Adam and Holmes)
Size exclusion chromatography (Porath)
Affinity chromatography
Method of adsorption chromatography:
Solute adsorbed on surface of solid particles: the stronger solute molecules adsorbed -> the slower it travels.
Method of partition chromatography:
Liquid stationary phase bonded to solid surface (SiO2) solute partitions between stationary “liquid” and mobile phase (a gas in gas chrmatography).
Method of ion-exchange chromatography:
Anions (-SO3-) or cations (-N(CH3)3+) covalently attached to stationary phase (“resin”), mobile phase = liquid
Mobile anions held near cations that are covalently attached to stationary phase.
Anion-exchange resin: only anions can be attracted to it.
Method of Size exclusion chromatography:
Separates molecules according to their size, larger ones elute first followed by smaller ones.
Method of affinity chromatography:
Most elective kind of chromatography: employs specific interactions between one kind of solute molecule and a second one that is covalently attached (immobilized) to stationary phase.
Speed of mobile phase passing through chromatography column:
- Volume flow rate (volume solvent/unit time; ml/min)
- Linear flow rate (distance of solvent travelled/unit time)
What’s chromatogram?
A graph showing detector response over elution time.
What’s tr, tm and tr’?
What’s relative retention (alpha)? What’s k?
The ____ a compound retained by column (k=tr’/km), the ___ its k = ____ of column dimensions.
tr = tm + tr’ tr: retention time
tm: unretained solute tr’: adjusted retention time
alpha - separation factor
k - for each peak -> calculate retention (capacity) factor
longer larger independent
Efficiency of separation: 2 factors determine how well compounds separated:
- the farther apart the better their separation
- the wider the peaks the poor their separation
What’s resolution?
The ____ solute resides in column, the ____ the band/peak.
Inflection point has? The width of the band/peak?
Solute moving through column -> spreads into a Gaussian shape.
longer broader
steepest portion of curve
breadth
Net transport of solute from region of ____ concentration to ____ concentration. By ____ ____ of molecules.
high low
random movement
What measures the rate at which molecules move from region with high concentration to one with low concentration? ____ is proportional to diffusion coefficient.
Diffusion in liquid __ times ____ than in gases.
Macromolecules diffuse ____ times ____ than small molecules.
Diffusion coefficient
Flux = J = -D dc/dx unit: mol/m^2*s
10^4 slower
10-100 slower
What’s plate height?
The ____ plate height, the ____ the bandwidth. GC ___mm, HPLC ___ um, CE ___ um.
H: A measure of column efficiency. H = sima^2/x (x: travel distance) smaller narrower 0.1-1.0 10 <1
Ability of column to separate components is improved by ____ ____ ____ -> more “____ ____” = N
decreasing plate height theoretical plates N (dimensionless) N=L/H L=column length H=plate height
Why bands spread?
Band of solute spreads as it travels through column and emerges at detector with std.dev.sima, each individual mechanism that contributes to broadening produces a std.dev.sima.
Broadening outside the column: Solute _____ be applied in an ____ thin zone -> band has ____ width before it enters column.
cannot
infinitesimally(极小)
finite
Plate height equation, also known as ____:
H ____ to variance of chromatographic band ____.
There are band-broadening mechanisms that are:___, ___ and ___.
A, B and C constants for a given column and stationary phase.
Van Deemter equation proportional H=sigma^2/x
H = A + B/ux + Cux
-A: Multiple paths (proportional to flow-rate)
-B: Longitudinal diffusion (inversely proportional to flow-rate)
-C: Equilibration time (independent of flow-rate)
Explain A, B and C in van Deemter equation.
The ____ the linear flow, the ____ complete equilibrium; the ____ broadening.
A: arises from multiple effects for which theory murky. B: zone of solute travel to right, band becomes broader. C: comes from finite time required for solute to equilibrate between mobile and stationary phase.
slower more less
What’s the mobile phase and stationary phase for GC? What’s the disadvantage?
Mobile: gas; Stationary: usually a nonvolatile liquid, but sometimes solid. Analyte: gas or volatile liquid. 1. Only ~20% of known compounds can be detected by gas chromatography. 2. If we have O2 in our gas, may oxidize the compound inside the mixture.
How does gas-solid adsorption chromatography work?
Gas-solid adsorption chromatography: analyte absorbed directly on solid particles of stationary stage.
Column -> hot enough to provide sufficient vapour.
