Analysis Flashcards
Describe the method for electrospray ionisation (give the equation)
- Sample is dissolved in a volatile solvent
- Then injected through a fine hypodermic needle to give a fine mist
- Tip of the needle is attached to a positive terminal (high voltage)
- particles ionised by giving a H+ ion from the solvent as they leave the needle forming XH+ ions
- X(g) + H+ –> XH+(g)
Describe the method for electron impact ionisation (give the equation)
- Sample is vaporized
- High energy electron is fired at the sample
- Knocking one electron from each particle forming 1+ ions (called the molecular ion)
- X(g) + e- –> X+(g) + 2e-
What sort of elements is electron gun ionisation used for?
elements with low formula mass (not suitable for biological samples) - lots of fragementation
What elements is electrospray ionisation used for?
elements with higher molecular mass (proteins)
What is electrospray ionisation also called and what rarely happens?
Known as ‘soft’ ionisation and fragmentation rarely occurs
Describe acceleration in a mass spectrometer
- positive ions are accelerated using an electric field
- so all the ions have the same kinetic energy
What does velocity of the ions depend on?
- Mass of each particle (lighter = faster)
How do you find retention factor in chromatography?
Rf = Dspot/Dsolvent
What is chromatography governed by?
IMFs between mobile and stationary phases
explain elution chromatography?
solution of mobile phase with sample injected in, then mobile phase continually added to move it down the column
what is Tm?
retention time when no interactions to the stationary phase
how to find T’R?
T’R = TR-Tm
How do you find relative retention time?
relative retention time = T’R2/T’R1 (different components)
how do you find retention factor for elution chromatography? What value do you want and why?
Rf (K’)= T’R/Tm
K’<1 (goes through too fast) K’>20 (too slow)
you want 1
What are the stationary and mobile phases in Gas-liquid chromatography?
stationary = sticky liquid on inert substrate mobile = gas
How do you find partition coefficient? and factors that affect it?
K = [A]s/[A]m Large K = high affinity for stationary Small K = high affinity for mobile Bigger Mr = bigger K K decreases with temp
What is Vr? and how do you find it?
Vr = volume of mobile phase to make solute to go through the column Vr = retention time x volumetric flowrate
What happens as bands move down a column? How do you decrease this?
band broadening
this decreases efficiency
select conditions so that rate of broadening < rate of separation
advantage/disadvantage of a long column?
- solute separates better but bands broaden more
Explain plate theory in relation to chromatography.
- plate theory supposes that a column contains lots of plates and separate things happen to the sample in each plate
- N (no of plates) = L (column length)/ H (plate height)
- You need a lot of plates for good separation but if the column is too long = band broadening (each plate needs to be as short as possible)
Explain rate theory in relation to chromatography.
- this supposes that the same molecule can travel by different routes (causes band broadening) H = A + B/U + CsU + CmU A = eddy diffusion B = longitudinal diffusion Cs = stationary mass transfer Cm = mobile mass transfer U = linear velocity (L/Tm)
What is band broadening caused by?
band broadening is caused by the same molecule taking different times:
- slow mass transfer between mobile and stationary
- flow distribution of the mobile phase throughout the column, resulting in different longitudinal diffusion
- eddy diffusions
- different paths
How do you find resolution? draw diagram to show what the terms in the equation mean.
Rs = delta z /(Wa/2 +Wb/2)
= delta T/Wav
What is the procedure of GLC? How do you improve accuracy of GLC?
1) Liquid sample introduced through heated injector (vaporising it)
2) Sample is diluted by mobile gas phase
3) components separate due to IMFs
4) different components reach detector at different times
- don’t want too big of a sample (want sample to be injected as a plug of vapour)
- Slow injection of large sample = band broadening
Describe a packed column.
contain finely divided inert support material coated with sticky liquid
Describe capillary column.
WCOT (wall coated open tubular) = stationary phase coated on walls
SCOT (support coated open tubular) = wall lined with powder and then sticky liquid (increased SA)
Compare packed columns and the 2 types of capillary columns
SCOT is less efficient that WCOT but both are more efficient than packed
What are the three types of detectors?
Non-selective = responds to all compounds but not carrier gas Selective = responds to compounds with a common property Specific = responds to a single compound
compare concentration and mass flow detectors.
concentration detector = signal is proportional to the concentration (doesn’t destroy the sample)
Mass flow = signal related to the rate at which the sample flows into the detector (sample often destroyed)
Describe High-performance liquid chromatography
column packed solid with small stationary phase particles and therefore a high pressure is required to pump the mobile phase through (increases separation efficiency)
What are normal phase and reverse phase when you have a non-polar analyte? Which is better for a polar analyte and which is better for a non-polar analyte?
normal phase = polar stationary, non polar mobile (spends more time in the mobile)
Reverse phase = non-polar stationary, polar mobile (more time in stationary)
You want the polarity of solute and stationary phase to be the same
Why is Ulta high vacuum (UHV) used in mass spectrometry?
this is because positive fragments are moved over large distances and need collision-free paths (don’t want N2 etc to interfere)
What is the general principal of MS?
the general principle is to generate ions of a compound by a suitable method and separate those ions by their m/z value and to detect them by their respective m/z and abundance
Draw a general diagram of the different systems of MS?
Sample inlet –> ion source –> mass analyser –> detector/data logger
How does the sample get to UHV?
sample is injected in through a self sealing rubber septum with a molecular leak (0.05mm-0.01mm)
How do you find kinetic energy is MS?
KE of ions = ZeV = 1/2mv^2
Z = distance
e = energy of an electrons
V = voltage of ion slit
Equations for electrical energy?
E = IVT = QV
Disadvantages of electrospray?
not as tolerant to buffers/salts
What happens as the samples leave in electrospray?
Drops evaporate until only the analyte ions remain
Two explanations:
- increased charge density from solvent evaporation causes repulsion to overcome liquid surface tension
- increased charge density causes droplet to divide until single ions
Describe FAB (fast atom bombardment), what is it good for?
- the sample is added to glycerol, forming a monolayer on it (glycerol matrix)
- Then a beam of neutral atoms (argon) of high kinetic energy is fired at the monolayer, ionising it
- It is good for biological samples
Describe SIMS (secondary ion mass spec), what is it good for?
- primary ion beam (Ar+/ Xe+) fired at the sample causing 2+ ions
- not good for biological samples
- good for catalysis
- SIMS can damage the surface of the analyte
Describe MALDI (Matrix-assisted laser detection/ionisation), what is it good for?
- biomolecules are co-crystallised with a solid acid matrix, which is then blasted with a laser
- the laser destroys the matrix and then sample takes the H+
- High tolerance to salts and buffers
- causes singly charged ions, meaning rapid molecular weight determination
- has a high sensitivity
- wastes little sample
How do you calculate time of flight in a TOF MS
t = L/V = L/sqrt(m/2zeV) m/z = (2eVt^2)/L^2
Compare a linear flight tube and a reflection flight tube?
- in a linear flight tube molecules with the same m/z arrive at the detector at different times
- reflection slows down the molecules with the same n/z travelling at different speeds, meaning all ions with the same m/z arrive at the same time
What is a reflection flight tube?
- Has a drift tube and then an ion mirror which repels ions in almost the opposite firection
- How far the reflected ions penetrate into the relector depends on how big its KE is, which slows them down making sure they all reach the detector at the same time
What is a quadrupole MS? Compare the advantages and disadvantages.
- continuous stream of ions
- Has 2 perpendicular electrodes causing ions to spiral
- only ions with the correct m/z will get through
- low cost
- rugged
- lower acceleration voltage
- small
- high scan rate
- but low resolution and low mass range
What is a magnetic sector MS? Compare the advantages and disadvantages. How do you calculate m/z? How do you increase resolution more
- After ions are accelerated they experience a magnetic field which bends them, meaning only ions with the correct m/z will follow the right path and be detected.
- Higher resolution and mass range than quadrupole
- But requires a higher vacuum and scans slower
- to increase resolution use more magnetic fields = more bends
m/z = (B^2r^2e)/2V B = magnetic field (in teslas, 1T = 10^4 Gauss)
How can you tell when two peaks are resolved? Draw diagram. How do you find resolving power?
10% valley resolution
When the height of the valley is less than equal to 10% of the height of the peaks
A = delta m/m –> delta m = peak separation, m= mass of lighter peak
Therefore, Resolving power (R) = 1/A
How do you find the speed of light in a medium?
c' = c/n c = speed of light in vacuum n = refractive index
How transmittance and % transmittance? What is it measured at?
T = P/Po —> %T = (P/Po)x100
Po = power of light in
P = power of light out
it is measured at lambda max (highest absorbance)
How do you find absorbance?
A = log10(Po/P) = -log10(T) = eCl
e = molar extinction coeffiecient c = concentration l = pathlength (cm)
What is the molar extinction coefficient and its units?
a measure of how strongly a species absorbs light at a specific wavelength (includes population of energy levels and the probability of a transition occurring)
mol^-1dm^3cm^-1
How do you plot the beer-lambert law?
A = ecl y = mx y=A x=c m=el no intercept
What would it mean if there is an intercept in a beer-lambert plot?
Due to solvent absorption or fingerprints on the cuvette
What do fingerprint/ smudges on the cuvette cause?
scatters light away from the detector
meaning the detector assumes it was absorbed
What is a chromophore?
Molecule that absorbs the light
What are the two methods used in US vis?
1) measure at a single wavelength (lambda max)
2) measure absorbance , then change wavelength and remeasure etc to get a spectrum of A vs lambda
How do double beam UV vis work?
light fired into blank solvent and then rotating mirror reflects light into solvent and sample
Why do you want an absorbance of 0.3-0.9?
too low absorbance = transmittance ~ 1 (barely any light is absorbed)
high absorbance = small transmittance (most light is absorbed)
How many photons are absorbed in the fundamental, 1st overtone etc in near infrared?
fundamental = 1 photon
1st overtone = 2 photons absirbed
Why do we usually operate with the first overtones?
They have the highest intensity, as it highly unlikely for 3 photons or more to be absorbed
When do combination bands occur?
when more than one photon is absorbed simultaneously
What are the two methods used in near infrared?
1) measure at a single wavelength (lambda max)
2) measure absorbance , then change wavelength and remeasure etc to get a spectrum of A vs lambda
Advantages of near infrared?
- very precise as a laser is being used (monochromatic)
- high resolution
- high energy throughout
- very quick
What is Tm?
the retention time when there are no interactions between the mobile and stationary phases (time for the solvent to reach the detector)
