Chromatography Flashcards
Which Van Deemter equation is used in open tubular GC?
Golay’s
(ingen A term)
Which Van Deemter equation is used in packed column LC?
Giddings
(A term is included)
What is A, B and C in the Van Deemter equation?
A is the multiple paths term
B is longitudinal diffusion
C is resistance to mass transfer
What is the B term affected by?
flow rate and mobile phase speed
Do we want to minimise or maximise the A, B and C term of the Van Deemter equation?
We want to minimise it because it causes band broadening
What is the C term in the Van Deemter equation affected by?
stationary phase thickness
Is there an A term in the Van Deemter curve when using open tubular columns?
No
How does bead diameter of the packed bed affect the A term of the Van Deemter equation?
We want a uniformly packed bed to minimise the number of multiple paths and thereby minimise A. Smaller bead diameter gives fewer irregulations and therefore fewer paths
Is the B term of the Van Deemter equation relevant even in CE?
Yes
How do you decrease the diffusion (B term in Van Deemter)?
Increasing the flow rate will decrease the residence time which will decrease the diffusion
How does temperature affect B?
When the temperature increases the kinetic energy increases and the diffusion increases. To minimise B we want to minimise temperature.
How does the analyte’s diffusion coefficient and molar mass affect B?
A higher molar weight and larger size gives less diffusion and therefore decreases B and decreases H and increases N
What is the C term in the Van Deemter equation?
Resistance to mass transfer
Do we want to maximise or minimise the number of partitioning events?
We want to maximise them because it allows for the analytes X and Y to separate
How does the length of time spent in the column affect the C term (resistance to mass transfer) in Van Deemter?
The longer time spend the more partitioning events and therefore the smaller C which is good for minimising plate height and maximising plate number N
How does the linear velocity affect the C term of the Van Deemter curve?
A lower linear velocity means longer residence time which is good for allowing for more partitioning events and therefore decreasing plate height and increasing plate number
How is plate height (H) and plate number (N) related?
N = L / H
A smaller plate height gives a larger plate number
How does the temperature affect C in the Van Deemter equation?
Increasing the temperature increases the rate of partitioning and therefore the number of partitioning events go up which decreases plate height and increases plate number. So high temp is good for minimising C (but bad for minimising B)
How does the film thickness affect C term in Van Deemter in open tubular columns?
If film thickness is low it is easier for solutes to equilibrate between mobile phase and stationary phase which decreases H and increases N
Which term, B or C is more significant (for causing band broadening) at high flow rates?
C
Which term, B or C is more significant (for causing band broadening) at low flow rates?
B
How do we find the optimal flow rate (linear velocity)?
Where plate height is at its minimum
Give an example of some kinetic properties
Diffusion
Mass transfer resistance
Multiple flow paths
Can flow rate and temperature affect kinetic properties?
Yes
Give examples of thermodynamic properties
Intermolecular solutions
Solubility
Which experimental variables affect the plate number?
Linear velocity
Packing structure (particle size and size distribution, porosity, pore structure)
Viscosity
Temperature
Column length
What is KD?
The distribution constant, how the analyte distributes between organic and aqueous phase
What are the requirements for GC?
Volatile compounds (can be derivatised to make more polar)
Small molecules
Thermo stable compounds
What is effective volatility?
P = gamma * P0
What are the three steps of analyte motion in GC?
Step 1: irregular analyte motion in gas phase
Step 2: Analyte motion in the stationary phase
Step 3: at the film surface the analyte pulls itself free or reenters film
What is an equation of the retention factor that includes the distribution constant and the SP and MP volume?
k = KD * Vs/Vm
Which law do ideal solutions follow in GC?
Raoults law
P = X * P0
What is gamma equal to in an ideal solution in GC?
gamma = 1
What happens if gamma is smaller or larger than 1 in GC?
gamma > 1 the analyte “doesn’t like to be with the stationary phase” and wants to “escape” so makes more vapour pressure (effective volatility increases)
gamma < 1 the analyte interacts better with stationary phase than with itself and therefore less vapour pressure (effective volatility decreases)
What does the gamma factor in effective volatility depend on in GC?
The properties of the analyte and the liquid stationary phase
How much is the retention factor reduced by if the temperature is increased by 30 degrees in GC?
50 %
How is selectivity expressed in terms of effective volatility in GC?
alpha = k2/k1 = KD2/KD1 = gamma1p01/gamma2p02
Because KD is proportional to the inverse effective volatility KD = 1/gamma*p
What different types of bonding happens between analytes and stationary phase in GC?
Pi-pi
Dispersion
Dipole
Which is the most common stationary phase used in GC?
Silicon oil, very non-polar. Can add phenol groups instead of CH3 to get pi-pi interactions. Can add CN groups and then it becomes more polar.
How is the stationary phase attached to the capillary wall?
Capillary wall is silica and the silica groups are bound with covalent bonds.
How do you optimise N (plate number)?
Using the Van Deemter equation and the plate height curve
What is Snyder’s separation triangle used for?
Optimising the separation factor, to determine mobile phase composition
Which are the ideal values of resolution, retention factor, separation factor and
plate number, respectively?
Rs larger than 1.5
k between 1 and 5
alpha above 1
N as large as possible
How can the retention factor be optimised?
Retentivity in LC is increased by increasing the interaction forces between analyte and
stationary phase, either by using a more retentive stationary phase or by reducing the elution strength of the mobile phase.
Retentivity in GC for open tubular columns is increased by either reduced effective
volatility, i.e. by reduced temperature or by increased volume phase ratio Vs/Vm = f(df /dc)
How can you optimise selectivity, alpha?
If alpha =1 then it means that the selectivity of the chosen two-phase
system (incl. temperature settings) is too poor, meaning that there is no difference in KD-
values of the observed analyte pair/s. To improve the selectivity of the system the
interaction chemistry of one of the phases must or temperature must be changed.
In LC: To change selectivity, the stationary phase ligand or the strong solvent in the
mobile phase mixture must be replaced. For dissociable species (both analytes, stationary
phase ligands and mobile phase components), also the effect of the mobile phase pH is
significant.
In GC: The selectivity of the stationary phase film or the column temperature must be
changed
How do you optimise plate number, N?
This is
best controlled by reduction of H (zone spreading mechanisms) as N=L/H.
In LC (packed column) generally by reduced particle size.
In GC (open tubular column) by optimized velocity, which depends on the carrier gas
identity (Dm) and inner diameter of the column. Reduced column diameter and/or film
thickness also lowers H and increases N. Note that the latter also affects retentivity and
column capacity
Does split injection lead to broad or narrow peaks?
Narrow
Is split or splitless injection more preferable?
Split because narrower peaks and limits the risk of adverse reactions (degradation, adsorption or breakdown because of long time in high temp), but it requires high concentration
When should splitless injection be used in GC?
If your analyte concentration is very low you need to use splitless since you can’t “afford” to dilute the sample further during injection. Then you need a much slower flow rate. This will lead to increased diffusion which result in broader peaks (zone broadening).
What is the septum purge valve used for in GC?
It is used to get rid off septum debris and remove excess sample vapour
What is a common split ratio in GC?
Between 5:1 to 500:1
What is a good standard carrier gas flow into the column in GC?
1 ml/min
What is the difference between on-column and splitless injection?
Splitless injection is injected into an injection chamber. On-column is injected directly into the column.
One might prefer on-column if the analytes are decomposing at temperatures close to their boiling point, then you want to avoid analytes spending too long time in chamber (it spends long when you use splitless)
How much micro liters of gas will 1 micro liter of liquid turn into?
500 micro liters of gas
How much liquid sample is usually injected in GC?
1 micro liter
What is the difference between split and splitless injection in GC?
In split injection the split chamber is open, which means that most of the sample (solute) will not go into the column
When should you use splitless injection in GC?
When you have low concentration or trace levels of analyte
When is on-column injection best to use in GC?
Best for thermally unstable solutes (analytes) in high boiling solvents (higher boiling points) and best for quantitative analysis
Is there a purge flow in on-column injection?
No
What is solvent trapping in GC?
The initial column temperature set 40 degrees below boiling point of the solvent which condenses. Analytes are then trapped in the plug of solvent. Chromatography is then initiated by raising the temperature to vaporise the trapped solvent. This leads to sharp chromatographic peaks.
What is cold trapping in GC?
The initial column temperature is 150 degrees lower than the analyte of interest.
Solvent and low boiling components are eluted rapidly but but high boiling components remain in a band at beginning of the column.
An injector can be either ___ sensitive or ____ sensitive. Fill in
Concentration sensitive
Mass sensitive
What is an FID detector?
Flame ionisation detector
Which detector is best to use when you need high sensitivity and increased detectability?
It depends on the analytes you have. If you have analytes containing halogens or nitro then use a detector for that (electron capture detector). If you have hydrocarbons use FID.
What is a TCD detector?
Thermal conductivity detector
How does the thermal conductivity detector work?
It measures the difference in heat conductivity between pure carrier gas and analyte-containing carrier gas
What is an ECD detector?
Electron capture detector
How does an electron capture detector work?
Analytes with high electron affinity absorb electrons which decreases the measurable current
What type of analytes is electron capture detector selective for?
Halogens, nitro compounds, organo-metallic compounds
How does nitrogen phosphorous detector work?
analytes containing N and P interact with the metal ions formed in the plasma and they produce electrons which produces a measurable current
How does the flame ionisation detector work?
Combusts hydrocarbons and the number of hydrogen forming ionic species is propertional to the concentration of its analyte and its molecular size. Longer carbon chain will give higher peak (and elute later)
When doing quantitative analysis, what is it based on, on the chromatogram?
Either the area or height of the chromatographic peak. Area is preferred.
What is the TID detector?
Thermionic Ionization Detector
How does the Thermionic Ionization Detector work?
he Thermionic Ionization Detector is similar in design to the FID and NPD. The electrically heated thermionic bead (TID bead) is positioned so that the column effluent contacts the hot bead surface.
Analyte molecules containing NO2 (nitro) functional groups such as TNT (trinitrotoluene) undergo a catalytic surface chemical reaction. The resulting ions are attracted to a collector electrode, amplified, and output to the data system.
The TID is extremely selective, having little or no response to most aromatic and aliphatic hydrocarbons. The TID also responds to chlorinated phenols such as pentachlorophenol (PCP) at slightly less sensitivity.
What is WCOT short for?
Wall coated open tubular
Which is the main requirement for a separation system to be defined as chromatography?
Mobile phase and stationary phase. CE and FFF not defined as chromatography
Describe two reasons for peak tailing
Overloading (injecting too much sample, too high concentration)
Too good binding (hydrogen bonds can cause tailing)
Describe multiple flow paths (A) in Van Deemter and how to reduce it
Analytes can take different paths in the packed bed. Reduced by having smaller beads in the packed bed to have more uniformity.
Is it thermodynamic or kinetic properties that mostly affect k and alpha?
Thermodynamic
Is it thermodynamic or kinetic properties that mostly affect N?
It is kinetic properties because kinetics strives towards equilibrium
If the composition of the chromatographic system is changed (MP or SP), will it affect k?
Yes, through affecting KD
k = KD Vs/Vm
Can Rs be increased by increasing k?
Yes up to k = 5
Rs = 1/4 * sqrt(N) * alpha-1/alpha * k/(k+1)
If the chemical composition is changed by a change of a phase component, will alpha be changed?
Yes
alpha = KD1/KD2
Would a change in the particle size of the packed bed affect the kinetics and thereby N?
Yes
Does column length, inner diameter or film thickness affect N? How does it affect resolution?
If column length increases it will increase efficiency which will also increase resolution. If inner diameter decreases it will increase resolution.
Does column length, inner diameter or film thickness affect k? How does it affect resolution?
k increases when inner diameter decreases which increases resolution. If film thickness increases it increases k which increases resolution.
What is N a function of?
Column length and inner diameter
What is k a function of?
Inner diameter and film thickness
What is alpha a function of?
Film thickness and temperature
What is the UV Cutoff and why do I need to know it?
Every solvent has its specific absorbance cutoff wavelength. Below this wavelength the solvent itself absorbs the light. When choosing a solvent be aware of its cutoff and where your desired analytes will absorb. If the wavelengths are close, choose a different solvent. The list below displays the UV cutoff wavelengths of common solvents.
Is cold trapping needed to give sharp peaks in splitless injection in GC?
Yes
Do the analytes interact with the mobile phase in GC?
No, it is an inert gas!
Is it more common in LC or GC to use packed columns?
LC
Do we sometimes use packed columns in GC? If yes when?
Sometimes, when KD is very low. KD depends on volatility. You solve a very low KD by having a very large stationary phase volume (using packed columns)
Does retentivity depend on both analyte properties and column properties? How about N and alpha?
Yes, k depends on
both analyte and
column properties. N only on column properties and alpha only on analyte properties
Should a compound you use in GC be thermostable?
Yes
What is a suppressor column used in an ion conductivity detector?
The suppressor method (EPA Method 300.0 for example) changes the eluent composition to one with a lower electrical conductivity. Solutions with aqueous sodium carbonate or sodium hydroxide are used. The suppressor is attached between the column outlet and the detector. The suppressor exchanges all the cations for hydrogen ions. Because hydrogen ions have a higher equivalent electrical conductivity, the signal is increased. Thus, the sensitivity of suppressed conductivity is higher.
Is acetonitrile a polar or non-polar mobile phase?
Non-polar
