Chromatography and Spectroscopy Flashcards
Use of chromatography
Used to seperate a mixture into its different components.
TLC stationary and mobile phase
Mobile- liquid solvent
Stationary- thin layer of solid adsorbent substance (silica SiO2 or alumina Al2O3) coated on inert solid support.
How does seperation work
Adsorption meaning
A solid stationary phase separates by relative adsorption
A liquid stationary phase separates by relative solubility
(tlc is always adsorption as it is always a solid stationary phase)
The attraction between the adsorbant layer and the components in the mixture.
Different components in mixture adsorb differently to surface of stationary phase.
Describe movement of molecule that is highly adsorbed
- Smaller distance travelled
- Move slowly
(The stronger a component reacts with the stationary phase, the less distance it travels)
Factors affecting movement up plate
Polarity*
Temperature
Solvent
Solid adsorbant coating
- Adsorbent layer is polar, so if component is polar, it can form hydrogen bonds with stationary phase, increasing adsorbance.
Carrying out TLC
1) Take a TLC plate and using a pencil to draw a base line (2cm above bottom)
2) Use a capillary tube and drop a small volume of sample onto base line
3) Prepare chromatography tank- solvent in a beaker with watch glass placed on top
4) place TLC plate into beaker with watch glass on top. Base line must be above solvent level
5) Allows solvent to rise up plate then mark solvent front with a pencil.
Compounds can then be identified using an Rf value
Chromatography paper sprayed with developing agent e.g., ninhydrin, produces a deep blue or purple stain, which makes it easier to identify spots.
Ways to improve chromatography practical
Wear plastic gloves to prevent contamination of hands to plate
pencil line –solvent doesnt interact/ dissolve pencil line
tiny drop – prevents spread of components and overlap
Depth of solvent– if the solvent is above base line it will dissolve/wash the sample spots off the plate.
lid– to prevent evaporation of toxic solvent
dry in a fume cupboard as the solvent is toxic
UV lamp used if the spots are colourless and not visible
Rf value (Retention factor)
- What it shows
- How to calculate
- Limitations
Component identified by comparing its Rf value to known Rf values for same solvent and absorbant system.
Rf value: Distance moves by component / distance moved by solvent front
Similar molecules have very similar Rf values- inaccurate
If spots merge, difficult to tell where one ends and one starts
Use of gas chromatography
Used to seperate and identify volatile organic compounds in a mixture
Gas chromatography stationary and mobile phase
Stationary - thin layer of liquid or solid absorbed onto inert solid support
Mobile phase- inert carrier gas (He,Ne)
Retention time definition
Time for component to travel (through capillary column) from injection to detector of chromatograph
Seperation in gas chromatography: solid vs liquid stationary phase
Solid stationary phase: seperation by adsorption
Liquid stationary phase: seperation by relative solubility
Gas chromatography with a solid stationary phase
Components with strong adsorption dont travel as far/fast and have greater retention time
Gas chromatography with liquid stationary phase
Components with greater relative solubility (dissolve more) have longer retention time.
“like dissolve like”
Liquid Stationary phase dissolves most similar
e.g.
Liquid alkane stationary phase dissolves propane (alkane) most readily as london forces form between them, slowing component
How does gas chromatography work
- Sample injected into gas chromatograph.
- Mobile inert carrier gas carries it through the capillary column which contains the liquid/solid stationary phase absorbed onto the solid support.
- components slow down as they interact with the liquid stationary phase
- more soluble, the slower it moves, the longer the retention time
Limitations of gas chromatography
- some chemicals have same r.t so can’t tell them apart
- unknown substances don’t have reference peaks
THEREFORE, often used alongside mass spectroscopy
TLC and Gas chromatography stationary and mobile phase
TLC
mobile- liquid solvent
Stationary- layer of solid adsorbent (silicon) on inert solid support
Gas
mobile- inert carrier gas (Ne, He)
stationary- h.b.p liquid absorbed or solid on inert solid support
Chromatogram and chromatogrph difference
Chromatogram- the data from the experiment
Chromatograph- the equiptment
Factors affecting retention time
- solubility (greater solubility, greater retention)
- Boiling point (Greater boiling point, greater retention)
- Temperature of instrument (greater temperature, shorter retention)
Faster as a gas than liquid, so if it has a high boiling point, will spend less time as gas, so slower retention.
The higher the instrument temp, more are gases, so shorter retention time.
Interpreting a gas Chromatogram- it shows 2 things
Retention time (identifies components by comparing to retention times of known components)
Peak integrations (tells concentration of components in sample)
Finding concentration of component through integration
Concentration found by comparing peak integration with values from standard solutions of component.
(compare using a external calibration curve)
1) Use serial dilutions of different known concentrations of compound being investigated
2) Obtain gas chromatogram and calculate integration of each peak
3) Plot graph of concentration (x) against peak area (y)
4) Obtain gas chromatogram of compound being investigated and compare with graph to find concentration.
Qualitative analysis for organic functional groups (chemical test and observation):
Alkene
Haloalkane
Carbonyl
Aldehyde
Primary & secondary alcohol, aldehyde
Carboxylic acid
Phenol
Alkene
- Bromine water drop-wise
- Orange to colourless
Haloalkane
- Add AgNO3 and ethanol, warm to 50 degrees in water bath
- chloroalkane white slowest
- bromoalkane cream
- iodoalkane yellow fastest
Carbonyl
- 2,4-DNP (dinitrophenylhydrazine)
- yellow/orange precipitate
Aldehyde
- Add Tollen’s reagent and warm
- Silver mirror
1,2 alcohol and aldehyde
- add potassium dichromate, H2SO4 and warm in water bath
-Orange to green
Carboxylic acid
- Add aq NaCO3
- effervescence
Phenol
- Add NaOH and a carbonate
- solid will dissolve with NaOH (colourless solution) and nothing happens with carbonate (only reacts with strong acids)
Use of NMR spectroscopy
Used to identify structure of a compound
What is nuclear spin
Why does hydrogen and carbon have a nuclear spin.
If a nucleus has an odd number of nucleons (protons & neutrons), the nucleus gains magnetic moment and has a nuclear spin (change in orientation)
Hydrogen- 1 proton
Carbon- although has 6 protons and 6 neutrons, 1% of carbons are C13, which have 7 neutrons.
What is resonance
(nuclear magnetic resonance)
When an external strong magnetic field and strong radio frequency radiation is applied,
the nucleus absorbs the radio waves, increases energy level and rapidly flips between the 2 spin states.
Nuclei that spin in the direction of the magnetic field have a lower energy.
Use of magnetic field and radio frequency radiation
- Magnetic field creates different energy levels.
- When RF radiation at the right frequency is applied, resonance occurs, allowing the nuclei to absorb energy and flip to a higher energy state.
Resonance and nuclear spin diff explanation
Nuclear Spin: Just a property of nuclei w odd no. , with no energy absorbed.
Resonance: Short process of absorption of energy to switch between different spin states in presence of magnetic field and r.f radiation.
What is a chemical shift (δ)
How much the field has shifted away from the field of TMS
What is TMS and why is it used
TMS (tetramethylsilane) (CH3)4Si
Used as a standard reference peak which other chemical shifts are measured against. Helps calibrate the spectrum.
TMS is used because:
*its signal is away from all the others
*it only gives one signal
*gives strong signal so only a small amount needed
*it is non-toxic
*it is inert
*it has a low boiling point so can be removed from sample easily
What does NMR measure
The amount of radiowave energy absorbed to resonate the nuclei (flip the spin states of nuclei)
The amount of energy a nucleus absorbs depends on the environment that it’s in.
Solvent used
CDCl3 Deuterated chloroform (polar)
CCl4 (non-polar) good solvent for non polar substances
Deuterated solvents are used as they don’t interfere with spectrum and produce no NMR signal.
Deuterium is an isotope of hydrogen (2H)
- produces no NMR signal.
- H1 replaced with H2 (deuterium)
- which has 1 proton & 1 neutron
X and Y axis on graph of spectroscopy
x axis- δ chemical shift in parts per million (ppm)
y axis- absorption
Running the spectrum
always remember:
- sample
- solvent (deutereum CDCl3, D2O, CCl4)
- reference (TMS)
- Dissolve sample in solvent and place in NMR tube with small amount of TMS
- Tube placed in spectrometer and spun (evens out imperfections in magnetic field within sample)
- Spectrometer is zeroed against TMS standard
- Sample given pulse of radiation containing range of radio frequencies and provided a constant magnetic field
- Any absorptions of energy during resonance are detected and diplayed on computer screen.
- After analysis, sample can be recovered by evaporation of solvent
When carbon atoms are positioned symmetrically (have same chemical environment)
They absorb radiation at same chemical shift.
How does polarity affect chemical shift
In polar environments, electronegative atoms draw electron density away, shifting it to a higher chemical shift due to deshielding from magnetic field of nuclei.
What factors may move a peak outside of the range of the spectrometer
Solvent, concentration, substituents
Integration values tell us
Relative number of H in each environment
Splitting of proton NMR is called
What causes splitting
Spin-spin coupling or spin-spin splitting
Proton’s spin in one environment creates a small magnetic field which interacts with the magnetic field/spin state of protons in an adjacent environment.
Shorter: Caused when a proton’s spin interacts with spin states of protons from different environments.
Splitting patterns pascals triangle
Names of splitting patterns
1
1 1
1 2 1
1 3 3 1
singlet
doublet
triplet
quartet
quintet/pentet
sextet
heptet/septet
(anything more than a singlet is a multiplet- but only write it after quintet)
Problems with -OH and -NH protons
- Chemical shift occurs over a range of values
- Broad signals
- no splitting pattern
What are OH and NH known as
Why are OH and NH signals so broad
OH and NH protons known as labile (readily changeable)
Rapid proton exchange with spall traces of H2O in solvent
How to identify -OH and -NH protons
1) A proton NMR spectrum is run as normal
2) Small volume of deuterium oxide is added, the mixture is shaken and second spectrum is run
3) In second spectrum, OH peak disappears.
Deuterium will exchange and replace OH and NH protons
CH3OH + D2O –> CH3OD + HOD
Protons bonded to electronegative atoms are able to hydrogen bond to the deuterium oxide. This weakens OH or NH bond, allowing exchange.
What does spectroscopy identify
Bonds and functional group
What does mass spectroscopy identify
- Mr of molecule
- Fragments of molecule
