Midterm 1 Flashcards
Chromatography def
Resolution of a mixture due to differences in rates at which individual components migrate through a stationary phase under the influence of a mobile phase.
Stationary phase def
Fixed in place
Mobile phase
Moves over or through the stationary phase carrying the analyze with it
Column chromatography def
Separation of solids even if they have similar solubility. When you can’t use recrystallization
Steps in setting up a column
- Plug the column with glass wool
- half-fill the column with pure liquid, solvent
- Add slurry of solid adsorbant in same solvent, silica gel in hexane (solid sinks to the bottom of the column to form an evenly packed column.
- Insert disc of filter paper at the top of the column.
- make sure the silica gel stays wet at all times or cracks may develop
Steps for running a column
- Run off solvent above column, over damp solid.
- Add solution of mixture in same solvent at the top
- Add more solvent to the column
- Allow the solutions of components to emerge
- Collect the solutions o each component in separate flasks.
- The solvent can be removed by distillation
Principle of Column Chromatography def
-Each compound in a mixture has a particular solubility in the solvent and particular tendency to be adsorbed by the solid in the column.
-Readily soluble and not strongly adsorbed move rapidly down the column in the solvent.
- Not so soluble and more strongly adsorbed are held on the column for longer.
- Stationary phase is solid
-Mobile phase is liquid
Separation of solids even if they have similar solubility
Thin Layer Chromatography TLC
- Principle similar to column
- SMaller scale
- Use for identification of components in a mixture
Rf value def
Fraction of distance travelled by each component
Paper Chromatography Principle
Similar to TLC
Uses a rectangular piece of filter paper
Dividing of a solute between two solvents, the eluting solvent (travels up) and water molecules adsorbed on cellulose (filter paper)
More soluble compounds travel faster- largest Rf values
Pro and con of paper chromatography
Pro- works well with compounds that are only slightly soluble in organic solvent but moderately soluble in water, unsuitable for TLC
Con- Takes time, TLC is faster
What are the two types of gas chromatography?
Gas-liquid chromatography (GLC)
Gas-solid Chromatography (GSC)
Both are suitable for separating mixtures of gases, liquids, and volatile solids
GLC properties
Stationary phase- non-volatile liquid (long chain alkene)
Mobile phase- gas (carrier gas- He or N2) that travels through the column containing the stationary phase.
Compounds in a mixture divided between two solvents. one solvent is fixed in position, stationary phase- compare with water in paper chromatography.
GSC properties
Stationary phase- solid (silica gel or alumina)
Similar to column chromatography
Eluting solvent is a gas
Components of a gas chromatograph
-Supply of carrier gas at a constant pressure
- Flow controller to control flow rate of gas
- Rotometer to measure the rate of flow of gas
- Column in a thermostatically controlled column oven
-Detector to determine when each component of mixture is eluted
When is the flow splitter used in Gas Chromatography
Flow splitter arrangement is used when the detector employed measures a change in property of gas stream in presence of analyte molecules
Rotometer
Measure the flow rate of the gas
The height of the float is directly proportional to the flow rate
What does a soap bubble meter do?
measures rate of flow of gas
creates a bubble and watch it go from initial volume to final volume and measures the time. ml/min
Characteristics of an ideal detector in GC
- high analyte sensitivity to detect low concentrations of analyte
- good stability
- good reproducibility
- linear response to analytes
- large range in temperature (25-400)
- Fast response regardless of flow rate
- high reliability and easy to use
- same responses for all analytes
TCD - thermal conductivity detector (katharometer)
- Measures changes in thermal conductivity of emerging gas (carrier gas) when an organic compound is eluted in a stream of carrier gas
Thermal conductivity def
Ability of a substance to transport heat from a hot region to a cold region
Effects of mixing analyte with He in TCD
- Lowers thermal conductovity of gas stream from column
- filament becomes hotter increasing its electrical resistance, which is measured with respect to reference flow by using wheatstone bridge circuit
Wheatstone Bridge
R1 is varied until no current goes from A to C . Under this condition, the bridge is balanced. Then, the electric potential at A = that at C. and the potential difference across R1= that across R2 and R3=R4. R1, R2, and R4 are known and R3 can be determined
R3 = R1 R4/ R2
Flame Ionization Detector (FID)
- Measures concentration of ions in a flame
- At the end of the column:
H2 carrier gas/organics + air -> ions collected on electrode - conc of ions cganges with presence of organics
more organics= higher conc of ions
What is the spectrum output called?
A chromatogram
What can you find from a chromatogram?
- Retention time: characteristics of components and conditions of experiments
- Conditions: rate of flow of gas, temperature, stationary phase (all to be kept fixed)
- Area under peak is directly proportional to the amount of material. Bigger area= more material
Categories of chromatography
-Adsorption chromatography
- Partition chromatography
- Ion-exchange chromatography
-Molecular exclusion chromatography
-Affinity chromatography
Adsorption chromatography def
Examples: GSC, column (elution)
-Involves equilibration of solute between stationary phase (solid) and mobile phase (gas or liquid)
-The oldest form of chromatography
- Solute adsorbed on the surface of solid stationary phase
Partition chromatography def
Examples: GLC, paper
Equilibrium of solute between the stationary phase (liquid) and the mobile phase (liquid or phase).
Solute goes in and out of the liquid stationary phase
Types of ion exchange chromatography
- Anion exchange
- Cation exchange
Anion Exchange
Stationary phase- resin
Cation covalently bonded to the surface of the resin. The anion is attracted to the cation. Anion exchange occurs between the mobile anion and free anions.
Cation exchange
Exchangeable H+ ion can be replaced by Na+, Zn2+. The H+ is replaced by other cations.
Anion covalently bonded to resin, H+ is electrostatically
Other names for Molecular exclusion chromatography
Gel filtration
Gel permeation chromatography (GPC)
Size exclusion chromatography (SEC)
Molecular Exclusion chromatography principles
- separation od molecules based on size
- Small molecules take londer to pass through the column. They go into the pores of the porous gel
Affinity Chromatography Properties
- Very selective
- Separation of a molecule from a complex mixture
Ex. Protein is attached to an antibody covalently bonded to the stationary phase. Bonded protein is dislodged by changing pH or ionic strength.
Other molecules that dont have a matching antibody will wash through
Elution def
Process of passing liquid or gas through a column chromatography
Goes in as eluent, comes out as eluate.
Packed column def
Filled with particles containing stationary phase
Open tubular column
narrow hollow capillary with stationary phase coated on the inside walls
3 types of open tubular columns
- Wall-coated open tubular (WCOT)
- Support-coated open tubular (SCOT)
- Porous- layer open tubular (PLOT)
Advantages of open tubular column
- HIgher resolution
- Shorter analysis time
- Greater sensitivity (low conc needed)
- Lower sample capacity
WCOT, what and where is the stationary phase
Liquid stationary phase on the inside of the wall
Ex of liquid, PEG (polar), PDMS (non-polar)
SCOT, What and where is the stationary phase
Solid support COATED with liquid stationary phase. Better sensitivity and higher capacity that WCOT.
Ex of solid support: diatomite SiO2
Ex of liquid phase: PEG, PDMS
PLOT what and where is the stationary phase
Stationary solid phase particles
Ex solid phase: PDMS, but needs to be high MW to be solid. Good for separating light hydrocarbons.
What happens if you decrease the thickness of stationary phase in open tubular column
- retention time decreases
- sample capacity decreases (need less sample)
- Resolution increases
Why is High Performance Liquid Chromatography important?
Most compounds are not volatile enough for GC
HPLC, how does it work?
High pressure is used to force solvents through colsed (packed) columns
Columns contain very fine particles of stationary phase, therefore high resolution.
Why does small particle size cause high efficiency?
- There’s more uniform flow through the column
- Shorter distance the solute must travel between particles in the mobile phase.
-small particles give high efficiency of separation
-requires very high pressure
What is the typical particle size of the stationary phase
3-10 micrometers
What does large particle size of the stationary phase cause?
- Gives rise to band broadening
- poor resolution
Components of an HPLC equipment
- Solvent delivery system
- Sample injection valve
-High-pressure column - Detector
-Computer to control/ display results - May include an oven to control temperature of column
Properties of an HPLC column
- Expensive
- Guard column and main column contain the same stationary phase
- fine solutes and strongly adsorbed solutes are retained in the guard column
-5-30 cm long with 1-5 mm inner diameter - titanium frits distribute the liquid evenly over the diameter of the column
How to manufacture the Ti frits
- Ti powder with uniform size is compacted using die.
- Compacted Ti powder is sintered (allow the particles to bond) by heating below the MP
- Compact sintered Ti powder with desired porosity and mechanical properties is machined and finished
- Now have Ti frits woth precise dimensions and finished surface. They get cleaned
Advantages of high temp
- Decreases solvent viscosity
- Faster flow of solute (shorter retention time)
- Higher resolution (sharper peaks)
- Less pressure is required
Disadvantages of high temp
- Degrades stationary phase
- Degrades column life-time
Common stationary phase for HPLC
Highly pure microporous particles of silica,
Use silica < pH 8 so that it doesnt dissolve in water
What kind of structure does silica gel have?
Amorphous structure (non crystalline)
Types of silanol groups
Isolated- far apart
Hydrogen-bonded vicinal groups- close together
Geminal-2 OHs on the same Si
Advantages of using silica gel in HPLC
Base-hydrolysis resistant
More rigid, well suited for particles less than 2 micrometers, which must withstand very high pressure.
There are ethylene bridges in place of some oxide bridges between silica atoms
Which is better, common or functionalized silica?
Functionalized: has fewer Si-O-H groups meaning symmetric peaks and shorter retention times. Common silica gives rise to peak tailing giving distorted peaks.
Common polar stationary phases
R group bonded to O2-SiClR(as2)
R=(CH2)3NH2 Amino
R= (CH2)3CN cyano
R= diol
Common nonpolar stationary phase
R= octadecyl, C18 most common
R= octyl
R= phenyl
What pH is HPLC limited to?
2-8
How to prevent hydrolysis below pH 2
Bulky isobutyl/ t-butyl groups protect siloxane bonds from incoming hydronium ions.
Fullerenes def
Allotrope of carbon whose molecules consist of carbons bonded by single and double bonds to form a closed or partially closed mesh
Stationary phase used for separation of fullerenes
zinc(II) tetraphenylporphyrin
(aromatic bowl)
Solutes with the least aromatic character will elute first.
Analyte with more aromaticity stays on the stationary phase.
Results from the chromatogram of fullerenes
C60 and C70 are well-resolved
C76 and C78 are not well resolved
C82 and C84 are not well resolved
Mobile phase used: 25 vol%CS2/ 75vol% toluene
Elution process in HPLC
In adsorption chromatography, solvent molecules compete with solute molecules for sites on the stationary phase.
The solute can be replaced by solvent on the stationary phase.
Gives solvent peaks- impurity
Normal phase chromatography def
HPLC based on acidic/common/bare/unfunctionalized silica
Use a polar stationary phase (diol, amino, cyano) and less polar solvent
Reverse phase chromatography def
More common
Use a nonpolar or weakly polar stationary phase (C18) and polar solvent (CH3CN/ H2O)
Advantages of reverse phase chromatography
- Eliminates peak tailing
Fewer sites of stationary phase can adsorb a solute to cause peak tailing
-Less sensitive to polar impuritues (such as water) in the eluent.
Water wont be retained on the stationary phase
Isocratic elution def
A single solvent or constant solvent mixture is used
Gradient elution def
-Increasing amounts of solvent B are added to solvent A to create a continuous gradient
- Used when one solvent does not elute all components rapidly enough
Pump requirements for HPLC
- Needs to generate pressures up to 400 atm
- Pulse-free output
- Flow rates ranging from 0.1-10 mL/min
- Flow reproducibility of 0.5% or better
- Resistance to corrosion by solvents
What are the two types of mechanical pumps?
- Screw-driven syringe type
- Reciprocating pump (used in modern HPLC)
Characteristics of screw-driven syringe pump
- pulse-free delivery
- Flow readily controlled
- Suffers from lack of capacity (250mL)
- Cannot do gradient elution where the solvent changes
- Old, don’t really have to worry about
Characteristics of reciprocating pump
- More widely used
- Small cylindrical chamber filled and emptied by back and forth motion of piston
- Produces pulse flow (force damper must dampen flow before it goes to the column)
- High output pressure up to 10,000 psi
- Can do gradient elution
Schematic of reciprocating pump
When a cylindrical chamber is filled with solvent, lower check valve is opened and upper valve is closed. When chamber is emptied, lower valve is closed and upper is opened.
How does the flow damper work?
Smoothes out pressure fluctuations caused by the intermittent flow of solvent from the pump.
Consists of a pressure vessel filled with gas, which compresses under the pressure peaks, and expands during low-pressure phases.
Types of sample-injection
- Syringe injection
2.Stop-flow injection - Sampling loop
Syringe-injection
- Non reproducible
- limited pressures <1500 psi
Stop-flow injection
- Solvent flow is momentarily stopped
- Remove fitting at column head
- Inject sample onto head of packing by means of syringe
Sampling loop
- Used in modern HPLC
- Can have interchangeable loops (5-500 microlitres)
- good reproducibility
What are the two positions of the sampling loop
- Load position: lever is turned down, 60 clockwise
1. Rinse loop with mobile phase
2. Sample sizes stay in loop, some mobile phase from previous rinse is displaced by sample volume.
The loop isnt connected to the pump! - Inject position: lever is 60 counter-clockwise
sample in loop is carried to the column by high pressure flow of mobile phase
Common detectors
- Ultraviolet detector: for solutes that absorb UV light
- Refractive index (RI): responds to most solutes
Diode array detector (DAD): scan all wavelength
Schematic of UV detector
Light source: Hg lamp or D, Xe, W lamps with monochromator
Need to set wavelength to where the solute absorbs the maximum.
DAD properties
Linear array of about 1000 Si photodiodes fabricated side by side on a single small Si chip
Lamp Source: D2 and W lamps. Covers UV range and visible range
Dont have to set the wavelength because its an array of diodes.
Advantages of DAD
- Multiwavelength detection
-Better light detection - Gives spectral data of each solute at different wavelengths
- 3D plot of data
RI detector
-Needs a light source, laser or LED light
- Needs specific wavelength
- The 2 diodes male an electric current and the difference between the two is what is measured.
-If there is no sample, the current stays the same.
When there is solute in the sample, there will be refraction and one dipole receives more light.
Parts of the RI detector and what they do
Collimator- collimates the beam of light (makes it more parallel)
Zero glass- directs light to ligh receiver with no loss of light.
Reference compartment- contains the pure mobile phase
Sample compartment- contains mobile phase from the column with presence of solutes
Why is methane used for adjusted retention time?
Methane isnt retained on the column and reaches the detector almost immediately.
adjusted tr= tr-tm
Adjusted retention time def
When sample is spiked woth a little methane because a slight change in flow rate will change the retention time of the unknown. Will then report the adjusted retention time with respect to CH4
Relative retention equation
for any two components
a=tr2/tr1
tr2 is the longer retention time
retention times are adjusted retention times
Relative retention def
The greater the value of a, the greater the separation
independent of flow rate
useful for peak identification when flow rate changes
Capacity factor (k’) equation and factor
k’=tr-tm/tm
adjusted ret time over ret of methane
The longer a component is retained by the column, the greater the capacity factor.
Another way of looking at capacity factor k’
k’= time in stat phase/ time in mobile phase
can also look at it as moles instead of time, get moles from MV
Factors that contribute to efficiency of separation
- Elution time between peaks, the further apart the peaks are, the better separation
- Peak width, the sharper their peaks, the better the separation
Elution volume Vr def
Volume of moile phase required to elute an analyte from the column
Vr= flow rate X ret time
Gaussian profile of a solute
Solute spreads into a Gaussian shape as it moves down the column
The longer a solute spends in the column, the broader the band is
What resolution is needed for quantitative analysis
> 1.5 Baseline is present
Equation for resolution
Resolution= separation between 2 peaks/ average width of two peaks
What does diffusion cause?
band broadening
Diffusion coefficient (D) def
Measures the rate at which a substance moves randomly form a region of high concentration to a region of low concentration m^2/s
Plate height measures what?
column efficiency
Plate height is the constant of proportionality between variance of band and the distance it has travelled.
What is plate height?
height equivalent to a theoretical plate
Not physical plates
Relates width of a band to the distance travelled through the column,
Smaller H-> narrower bandwidth-> better separation
Different solvents have different plate heights due to the different diffusion coefficients
Equation for plate height
H=L/N
What does the van Deemter equation tell us?
Tells us how column and flow rate affect plate height.
Three band broadening mechanisms in Van deemer equation
- Directly proportional to flow rate Cux term
- Inversely proportional to flow rate B/ux term
- Independent to the flow rate A term
- in packed columns, all three terms contribute to band broadening.
- In open tubular column, A=0. Band width decreases, resolution increases.
Longitudinal diffusion
B/Ux term in equation
Solute continuously diffuses away from the concentrated center of its zone. The faster the flow, the less time is spent on the column and the less longitudinal diffusion occurs.
Equilibration time
Cux term in equation
also called mass transfer term.
Finite time available for solute to equlibrate between mobile phase and stationary phase.
Some solute stuck in stationary phase, the remainder of solute moves forward.
Spreading of overall zone of solute.
Term is minimized by using a slow flow rate and by decreasing the thickness of the stationary phase.
Multiple path term
A term
Band spreading from multiple flow paths
The smaller the stationary phase particles, the less serious the problem.
Process is absent in open column
Advantages of open over packed column in gas chromatography
- High resolution with open. Decreased plate height, A=0
- Shorter analysis time, no particles in column to resist flow of mobile phase
- Increased sensitivity
- Lower sample capacity
Partition coefficient K and what affects it
- K increases with increased solubility of solute in the stationary phase
- K decreases when small quantities of solutes are retained more strongly than large quantities
What is the stationary phase used in gas chromatography
Diatomite: hydrated silica
Surface hydroxyl groups form hydrogen bonds with polar solutes, causing serious peak tailing.
What is the name of the reaction of functionalizing diatomite
Silanization
