CHEM 241 Exam 2
Why do large solutes move slower in capillary electrophoresis?
More friction
What is reverse phase chromatography?
Nonpolar stationary phase with a less non polar mobile phase
In gas chromatography, what type of column is used?
OTC or packed columns
What type of column is used in HPLC?
Packed column
Why do OTC result in shorter analysis times?
Particles in a packed column resist flow, so the linear velocity is slower in packed columns
What types of solvents are used in reverse phase chromatography?
Polar; methanol, acetonitrile and water
For a reverse phase HPLC separation with a mobile phase pH 3, which compound would elute first and why? Propanoic acid (CH3CH2COOH, pKa 4.87) or propylamine (CH3CH2CH2NH2, pKa 10.57)
Propylamine because under its pKa it will be protonated and will be charged and charged species will not interact well with the non polar stationary phase
What is a linear range of a GC detector?
Range of concentrations where the peak areas are directly proportional to the concentration of the analyte in the sample.
What is the most common elution method in HPLC?
Reverse phase chromatography
What are ideal detectors to use in HPLC?
Sensitive to low concentrations of many analytes, linear response, insensitive to temperature and solvent changes
What is capillary zone electrophoresis?
Separation is based on differences in electrophoretic mobility.
What is silanol?
SiOH
Rate of migration in capillary electrophoresis depends on what?
Size of solute and the charge of solute
How to decrease Eddy diffusion when using a packed GC column?
Small, uniform particle size, but this will require higher pressure because the smaller the article size, the less space in between particles, so the greater pressure is needed to force the mobile phase through the column
What type of molecules will move faster in capillary electrophoresis?
Smaller molecules and those that have a greater charge
What is the stationary phase in GC?
Solid particles or non-violating liquid
What is isocratic elution?
Solvent or solvents stay constant throughout the entire hPLC
What causes tailing?
Some solute is retained more strongly on the stationary phase
What type of solvents must be used with HPLC?
Special HPLC grade solvents
What is a wall coated open tubular column?
Stationary liquid phase 0.1 to 5 um thick on the wall of the column
What is apparent mobility?
Sum of electrophoretic mobility plus electroosmotic mobility of the ion
What happens if in capillary electrophoresis, the silica is capped with an amino group with a pH of 10?
The amino group will be protonated at a pH 7.4 and it will attract negatively charged groups. This will completely reverse the direction of electroosmosis and the movement will be towards the anode
What is the benefit to using microporous silica as the stationary phase support in HPLC?
The benefit to making it microporous instead of just using solid silica is it significantly increases surface area. With pores running through the silica, you end up with 99% of surface area inside the pores. More surface area=more stationary phase=better separation
In SEC, if we have a Kav greater than 1, what does this mean?
The biomolecule is interacting with something else other than going into the pores...it might be absorption.
In capillary electrophoresis if the silica is capped with an amino group with a pH of 10, where should the detector be?
The detector needs to be near the anode because this is the direction things are flowing now
What is an eluent?
The fluid that is entering the column
What causes electroosmotic flow in capillary electrophoresis?
The fused silica capillary has exposed Si-OH groups that are deprotonated at a pH of 2. The buffer is PBS which has positively charged sodium ions and they build up on the negatively charged wall. These move becasue there is an electric field from the anode to the cathode and this causes the entire mobile phase to move.
Why is it important that detectors are insensitive to temperature changes in HPLC?
The heat and AC can turn on and off
What is the smallest plate height we can get for a given particle?
The lowest your plate height can get is about 2x the stationary phase particle diameter
How does gradient elution alter partition coefficient?
The partition coefficient for all solutes changes based on the solute and stationary phase interacting
In partition chromatography, what determines how much time the solute will spend in each phase?
The polarity it is most like
What is the size of pores in microporous silica?
The pores must be large enough for solvent and solute to fit into them. 6-12 nm pores are typically required.
What is elution?
The process of passing liquid or gas through a chromatography column
What is relative retention/separation factor?
The ratio of adjusted retention times
What is Kovat's Retention Index and why is it useful in GC?
The retention index of a certain chemical compound is its retention time normalized to the retention times of adjacently eluting n-alkanes. While retention times vary with the individual chromatographic system, the derived retention indices are quite independent of these parameters and allow comparing values measured by different analytical laboratories under varying conditions
What is Vr for SEC?
The retention volume
What is extraction?
The separation of solute from 1 phase to another using two immiscible liquids (usually aqueous/organic solvent combos)
Why is it advantageous to use smaller particles?
The smaller the particles, the more particles that can fit and the more particles that are packed within the column, the better we can get separation because there are more attractions at the stationary phase. Decreasing particle size increases the number of theoretical plates, increasing resolution. It increases number of plates because it decreases Eddy diffusion and makes less time for equilibration so it decreases the C term. It also allows the optimum flow rate to be faster because the C term is smaller and we can use detectors with a lower detection limit because the analyte is not diluted
If it is advantageous to resolution to use small particles, why do we not make our particles infinitely small?
The smaller we make particles, the less likely we are to make them uniform, which will cause Eddy diffusion if they are not uniform. The smaller the particles, the greater the pressure needs to be to get a decent flow rate. We can also only make them so small
What are the problems with porous silica beads?
The solute can go straight through and be on the stationary phase for a long time and cause band broadening.
What are the requirements of a UV-Vis spectrometer?
The solute must exhibit absorption in the UV-Vis range and there must be no absorption by solvent
What is temperature programming?
The temperature is raised during the separation to decrease the retention times of later eluting components. If the temperature is low, the less volatile compounds may not elute.
What is Vm in SEC?
The total volume of the column, which includes solvent inside and outside of the gel particles
Why do greater charged molecules move faster in capillary electrophoresis?
There is an increased force
Why is capillary electrophoresis so fast?
There is no A or C term, only the B term to determine plate height, and the B term is reduced by a faster velocity
What are the interactions in affinity chromatography?
They are a combination of hydrophobic, electrostatic, and hydrogen bonding
What are conductometric detectors?
They are an electrochemical detector that measures the conductivity of the solvent
What are potentiometric detectors?
They are electrochemical detectors that monitor changes in the electrochemical potential
What are amperometric detectors?
They are the most common type of electrochemical detector. They apply a known potential and monitor the oxidation and reduction of a redox active species
What are the issues with refractive index detectors?
They are useless in gradient elution because changing the mobile phase will also change the refractive index and you will not be able to tell which change is from the change in solvent or change in the solute, you need a reference cell because it is temperature sensitive, the detection limit is 1000x less than UV-Vis
In normal phase HPLC, what is the behavior of non polar molecules?
They fly off of the column because they have nothing to interact with. There is no separation
In reverse phase HPLC, what is the behavior of polar molecules?
They fly off of the column because they have nothing to interact with. There is no separation
Downsides of thick films of stationary phase in GC
Thick films of stationary phase can shield analytes from the silica surface and reduce tailing, but they can increase bleed (decomposition and evaporation) of the stationary phase at high temperatures
What is affinity chromatography used for?
To isolate a single compound from a complex biological mixture via specific binding of that compound to the stationary phase
To reduce the tendency of stationary phase to bleed at high temperatures, what is done?
To reduce the tendency of stationary phase to bleed at high temperatures, it is usually bonded to the silica surface and cross linked to itself
What is chromotography used for?
To separate compounds of a mixture, to identify components of a mixture, and for preparative purposes...to purify a large quantity of a component of a mixture
What causes overloading?
Too much solute (too high of a concentration) overloads the column. As solute builds up, it gets more soluble in the stationary phase and the stationary phase ends up looking like solute. This means that once the stationary phase starts to look like solutes, other solutes that come by later will not have the same partition coefficient and K goes way down...when solute starts to interact with solute there really is no stationary phase so it just all comes off which is why the peak drops so rapidly
What type of detectors do we use in capillary electrophoresis?
UV-Vis, fluorescence, mass spectrometry, electrochemical
Thermal conductivity detectors are useful for what type of columns?
Useful for packed columns, but less sensitive than other detectors for OTC
How does HPLC work?
Uses high pressure to force solvent through closed columns containing fine particles that give high resolution separation
What is gradient elution?
Using 2 different solvents and increasing the amount of solvent B added to solvent A during the separation to create a continuous gradient
In the electrophoretic mobility equation, what is E?
V/m
How does a refractive index detector work?
Visible light passes through the cell and is directed at a photodiode array. When a solute with a different refractive index than the solvent enters the cell, the beam is deflected and different pixels of the array are irradiated
What happens in gas chromatography?
Volatile analytes are vaporized and transported through the column by a carrier gas
If the solute is larger than the pores in SEC, what is Kav?
Vr=V0 and so Kav=0
If the solute is small and penetrates all of the pores in SEC, what is Kav?
Vr=Vm and Kav=1
What is the most common type of open tubular column in GC?
Wall coated
Why can't we use a very high voltage in capillary electrophoresis?
We can increase the velocity by increasing the voltage, but we can only go so high because: 1. Due to resistance, increasing the voltage generates heat which increases longitudinal diffusion, might denature proteins or alter chemical reactions
Why can't we use OTC in HPLC?
We cannot use open tubular columns in LC because diffusion is 10^4 times slower in liquids than in gases. We use packed columns so the solute does not have to travel very far to encounter the stationary phase, which reduces C term!
How do we determine V0 for SEC?
We need a really large protein that will not go through the pores
With adsorption chromatography, if we want our analyte to come off of the stationary phase what do we do?
We use a stronger solvent to outcompete the analyte
How do we quantify Vm for SEC?
We use something really small that goes through like a radioactive ion
If we are using gradient elution in normal phase HPLC, how would we alter the solvent over time?
We would make it more polar as the experiment went on
In reverse phase chromatography, what type of solvent has a higher eluent strength?
a non polar solvent
What is the adjusted retention time?
additional time required to travel the length of the column beyond that required by solvent (tm). The solvent is unretained and travels through the column the fastest
What are the most common type of electrochemical detectors?
amperometric
What is gas-solid adsorption chromatography?
analyte is adsorbed onto the solid particles of the stationary phase
What is 1 plate height equivalent to?
approximately the length of the column required for 1 equilibration of solute between mobile and stationary phases
What type of solvent is better for an extraction with charged species?
aqueous
How are stationary phases chosen?
based on like dissolves like
For molecular exclusion chromatography, what is the K value for intermediate sized molecules?
between 0 and 1
Capillary electrophoresis is typically carried out in what?
buffer soaked paper or gel
Conductometric detectors measure what type of property?
bulk
In gas chromatography, how is the analyze transported through the column?
by a gaseous mobile phase (carrier gas)
What are the types of capillary electrophoresis?
capillary zone electrophoresis and micellar electrokinetic capillary electrophoresis
What is the order of elution in capillary zone electrophoresis?
cations, neutral, anions
In affinity chromatography, how do we get the solute to come off of the column?
change pH, add surfactant, change the ionic strength of the solution
What is detection limit?
concentration of analyte that gives 3:1 signal to noise ratio
What is electrophoretic mobility?
constant of proportionality between the speed of the ion and electric field strength
What drives separation in capillary electrophoresis?
electric field
What types of analytes can the ECD be used for?
electron capturing solutes
In capillary electrochromatography, what is the mobile phase driven by?
electroosmosis
What gives rise to separation in chromatography?
equilibrium of the solutes between the 2 phases
How is the problem of porous silica beads fixed?
faux porous shell around a solid bead...you still end up with a relatively large surface area, but the analytes cannot go all of the way in.
What is a solid stationary phase in GC?
fine solid particles packed into stainless steel tubing; carbon (carbon black), SiO2 (silica gel), Al2O3 (alumina) and the analyte adsorbs directly on solid particles
In an open tubular column, what are the walls made from?
fused silica
In partition chromatography, what is the mobile phase?
gas
What are the 2 types of gas chromatography?
gas-liquid partition chromatography and gas-solid adsorption chromatography
What is the mobile phase in gas chromatography?
gaseous (He, N2 or H2)...as long as the mobile phase is inert and will not react with the solute the choice is not criticle
What does HETP stand for?
height equivalent to a theoretical plate
In HPLC, what is the mobile phase driven by?
high pressure
Pros of an OTC?
higher resolution, shorter analysis times, greater sensitivity compared to packed columns
What is the general elution problem?
inability of a single isothermal separation to provide adequate separation within a reasonable run time for samples containing compounds of widely different boiling points
How can we decrease longitudinal diffusion of our solute?
increase velocity
In microporous silica, where is the majority of the surface area?
inside the pores
What happens to the refractive index of the mobile phase as a solute passes through it?
it changes the entire refractive index of the solvent
Can gradient elution be used with liquid-liquid chromatography?
it is difficult
What is spiking?
it is used to identify a peak; it compares a certain peak's retention time with that of an authentic sample of the suspected compound. The authentic compound is added to the unknown. If the authentic compound and the unknown are the same, the relative area of one peak will increase
Why is baseline drift a bad thing?
it makes quantification and determination of resolution very difficult
What does a large partition coefficient mean in an extraction?
it means that less solute remains in phase 1
What does it mean if a detector is sensitive?
it responds to a low amount of solute
In ion-exchange chromatography, what is the mobile phase?
liquid
What are the types of chromatography in HPLC?
liquid-liquid partition and liquid-solid adsorption
What is the B term in the Van Deemter equation?
longitudinal diffusion
What is the only source of peak broadening in capillary electrophoresis?
longitudinal diffusion
Below the optimal linear velocity, what is the greatest thing that affects plate height?
longitudinal diffusion broadening
What is a solute property detector?
look at a specific property of an analyte
Above the optimal linear velocity, what is the greatest thing that affects plate height?
mass transfer
What is the C term in the Van Deemter equation?
mass transfer
What is electrophoresis?
migration of ions under the influence of electric field
What does molecular size exclusion separate based on?
molecular size
Whaat type of ions move towards the anode?
negative
What is the charge of a cathode?
negative
What is the limit of detector of UV vis spectrometer detectors?
ng
Can gradient elution be used with size exclusion chromatography?
no
What is an open tubular column?
no packed stationary phase
Is the electron capture detector destructive or non destructive?
non
In normal phase HPLC, which molecules come off of the column first?
non polar and then less polar
In reverse phase HPLC, what types of molecules are separated?
non polar molecules are separated from other non polar
What is a liquid stationary phase in GC?
non-volatile liquid coated on inside of column or on fine solid support
Is the thermal conductivity detector destructive or not?
not
How sensitive is the thermal conductivity detector?
not very
Most common gas chromatography column:
open tubular
What are the 2 types of columns that can be used in chromatography?
open tubular or packed
What type of solvent is better for an extraction with neutral species?
organic
What is a flame ionization detector (FID)?
organic solutes are burned in H2 and hot air producing CH radicals and eventually CHO+. CHO+ ions are collected by a cathode and produces a current as the response. Ion production is proportional to the number of susceptible carbon atoms entering the flame
What types of analytes can flame ionization detectors be used for?
organics (reduced carbons only)
What are the limitations of using silica as a stationary phase support?
pH > 8-9 silica starts dissolving and pH < 2 starts hydrolyzing (bonded stationary phase lost)
In HPLC, what type of column is used?
packed
In adsorption chromatography, what type of column is used?
packed
Which type of chromatography is most like extraction?
partition
In normal phase HPLC, what type of molecules are separated?
polar molecules are separated from other polar molecules
In normal phase chromatography, what type of solvent has a higher eluent strength?
polar solvent
In reverse phase HPLC, what molecules come off of the column first?
polar, then less non polar
What is capillary gel electrophoresis?
polymer network (gel) that entangles larger molecules to a greater extent
What is the charge of an anode?
positive
What type of ions move towards the cathode?
positive
What are HPLC grade solvents?
pre-filtered w/ 0.2 micron filter, extends pump life, reduces column plugging because column plugging can be caused by any particulate in the solvent because the pores in the column are very small, degassed prior to use
What is dynamic range?
range over which detector responds in any manner to changes in analyte concentration
What is a fluorescence detector?
sensitive to naturally fluorescent analyses or to fluorescent derivatives
What is affinity chromatography?
separation based on antibody-antigen or enzyme-substrate interactions.
What is size-exclusion/gel permeation chromatography?
separation based on molecular size
What is adsorption chromatography?
separation based on solute adsorption on surface of stationary phase. Stationary phase has discrete binding places. The analyte can chemically interact with the stationary phase and stick to it and the solvent is competing with analyte for the binding spots on the stationary phase
What is partition chromatography?
separation based on solute dissolving into stationary liquid. The stationary liquid is bound to the silica stationary phase support. Instead of binding to the stationary phase, the solute equilibrates between the mobile and liquid stationary phases and based on the polarity of the phases, the solute will spend more time in 1 phase compared to another
What is micellar electrokinetic capillary electrophoresis?
separation of neutral molecules via partitioning, and so the longer the molecule spends inside of the micelle, the longer its migration time. There is a psuedo stationary phaase so C is not 0
How many detectors do each LC have?
several
The capillaries used in conventional capillary electrophoresis are made of glass and their inner surface bears an immobile negative charge. To what is this charge due and what is its consequence in CE experiments?
silanol (SiOH) and electroosmosis
What is capillary electrochromatography?
similar to HPLC except mobile phase driven by electroosmosis (not high pressure)
In capillary gel electrophoresis, which molecules travel the fastest?
smaller
Why are columns coiled in the oven?
so a greater length can fit because greater length means greater resolution
What is porous layer open tubular column?
solid stationary particles on the inside wall of a column
Amperometric detectors measure what type of property?
solute
Potentiometric detectors measure what type of property?
solute
UV-Vis spectroscopy detectors measure what type of property?
solute properties
Electrophoresis is what type of property?
solute property
In GC, what happens as the column ages?
stationary phase can be lost, surface silanol groups are exposed and tailing can increase
What is the stationary phase in molecular size exclusion?
stationary phase is a cross linked polymer or cross linked gel of controlled pore size
What is gas-liquid partition chromatography?
stationary phase is a non volatile liquid bonded to the inside of the column or to a solid support
Ideal characteristics of a solid support for a liquid stationary phase in GC?
strong, porous, high surface area and inert (non-adsorptive)
What is an eluate?
the fluid emerging from the bottom of the column
What does a large relative retention mean?
the greater the separation between 2 solutes (k2/k1)
What are the first molecules to elute in molecular exclusion chromotography?
the larger molecules that cannot fit into pores
In reverse phase HPLC, which molecules come off of the column last?
the most nonpolar
What removes the friction between the solvent and the walls of the capillary in capillary electrophoresis?
the surface charges
What is retention time?
the time that elapses between injection and arrival of that component to the detector?
In GC, what is the principle determinant of retention?
the volatility of the solutes
What is retention volume?
the volume of mobile phase required to elute a particular solute
What is Vm-Vo in SEC?
the volume of solvent inside of the gel
Why is a large surface area in chromatography a good thing?
this means more stationary phase which means better separation
What is the retention factor/capacity factor?
time solute spends in stationary phase/time solute spends in mobile phase
What type of liquids are used in solvent extractions?
two immiscible liquids (usually aqueous/organic solvent combos)
What types of analytes can a thermal conductivity detector be used for?
universal; responds to all analytes
What are packed GC columns used for?
used for preparative separations or to separate gases that are poorly retained
What are important factors to consider before temperature programming?
variations in solubility, changes in volatility, solute stability problems, flow rate changes, stationary phase stability
What type of stationary phase is silica?
very polar
When doing an extraction, the solute is most soluble in whaat type of solvent?
whatever solvent has a polarity similar to the solute's
In HPLC, what is elution?
when solvent displaces solute from the stationary phase
Ideal characteristics of liquid stationary phase in GC?
wide liquid range (min/max temp limits), stable, low volatility, low viscosity (↑D and ↓RMT)
In an OTC, where does solute interact?
with the walls of the column
Are flame ionization detectors destructive or not?
yes
Can gradient elution be used with adsorption chromatography?
yes
Can gradient elution be used with bonded phase chromatography?
yes
Can gradient elution be used with ion-exchange chromatography?
yes
How does the electron capture detector work (ECD)?
• Gas entering the detector is ionized by high energy electrons (beta particles) emitted from a foil containing radioactive nickel. • Electrons in the plasma formed are attracted to an anode, producing a small current that is maintained at a constant level by variable frequency pulses applied between the cathode and anode • When analytes with a high electron affinity enter the detector, they capture some of the electrons and decrease the conductivity of the plasma and the detector responds by vaarying the frequency of voltage pulses to maintain a constant current • The frequency of pulses is the detector signal
How does a thermal conductivity detector work?
• The carrier gas must have a different thermal conductivity than the analytes. He is often used because it has a really high thermal conductivity. When the analyte emerges, the conductivity of the gas stream decreases, the filament gets hotter, the electrical resistance increases and the voltage across the filament changes. The detector measures this change in voltage. • The thermal conductivity of the elute is measured relative to that of the pure carrier gas
In molecular size exclusion, what is the partition coefficient if the polymer is too large?
0
Theoretically, what are the range of Kav values in SEC?
0 to 1
The partition coefficient of a weak acid HA (Ka=1.44 x 10^-5) between toluene and water is 650. 1. Calculate the formal concentration of HA in the aqueous phase after extracting 75 mL aqueous solution of 0.05 M HA with 25 mL of toluene at pH 2.8 2. Is the extraction of the acid into toluene more or less efficient at pH of 7? Why?
1. 2. Less! q=0.401, more left in water
A molecule with MW=100,000 elutes from a molecular exclusion column in a volume of 28.2 mL. The column has a diameter of 1 cm and a length of 100 cm. It is packed with a gel that has a fractionation range from MW 3,000 to 30,000. 1. At what retention volume would a compound with MW 750 elute? 2. At what retention volume would a compound with MW 45,000 elute? 3. At what retention volume would a compound with Kav=0.6 elute? 4. What is Kav for a molecule with MW of 500? 5. What is Kav for a molecule with MW of 60,000?
1. 78.54 mL 2. 28.2 mL 3. 58.40 mL 4. 1 5. 0
What are the advantages of using gradient elution?
1. Reduced analysis time 2. overall resolution of the mixture improved 3. better peak shape because we don't have a lot of tailing or broadened peaks from being in the column for so long 4. improved sensitivity
Why do we separate compounds?
1. To isolate or concentrate components from a mixture 2. To separate component(s) from other species that would interfere in analysis
What are the 3 characteristics of capillary electrophoresis?
1. Very fast 2. High efficiency 3. especially useful for biopolymers like proteins
Why is capillary electrophoresis so efficient?
1. no particles so no multiple paths (A = 0) 2. no stationary phase so no RMT (C = 0)
If when using a UV-Vis detector, we get a response at 1 mM, at what concentration would we get a response when using a refractive index detector?
1000 mM
What is the Kovat's index for a linear alkane?
100x the number of carbon atoms
What is the linear range of a UV detector?
10^5
What is the linear range of a thermal conductivity detector?
10^5
What is the linear range of flame ionization detectors?
10^7
What is the pKa of silanol?
2
What is the detection limit of flame ionization detectors?
2 pg
w1/2 is equal to what?
2.35σ; width at half peak
A solution in a capillary electrophoresis experiment has an electroosmotic mobility of 4.7 x 10-8 m2/Vs) at pH 4.0. How long will it take a neutral solute to travel 52 cm from the injector to the detector if 27 kV is applied across the 62-cm-long capillary tube at pH 4.0?
2.5 x 102 sec
Blue Dextran 2000 (molecular mass of 2.00 x106) was eluted during gel filtration in a volume of 26.40 mL from a 2.00 x 34.97 cm (diameter x length) column of Sephadex G-50. The fractionation range of G-50 is from 1,500 to 30,000 molecular weight. Radioactive NaCl is eluted at a volume of 109.8 mL. 1. At what retention volume would hemoglobin (molecular mass 64000) be expected? 2. What would the retention volume of a protein be if Kav for the protein is 0? 3. What would the retention volume of a protein be if Kav for the protein is 0.65? 4. What is Kav for a protein with a MW of 1200? 5. If Kav for a protein was 1.3, what could you conclude about this solute?
26.4 mL, 26.4 mL, 80.6 mL, 1, adsorption was happening
When 2 mg/mL pyrene and 2.5 mg/mL perylene were separated by HPLC, relative peaks areas of 590 and 640 cm2 were obtained. A fresh solution was then made by mixing the 5 mg perylene with 1 mL of unknown solution containing pyrene but no perylene. The mixture was diluted to a final volume of 5 mL. HPLC resulted in observed peak areas of 402 and 421 cm2 for pyrene and perylene. Determine [pyrene] in the unknown solution.
4.14 mg/mL
What is the detection limit of a thermal conductivity detector?
400 pg
w is equal to what?
4σ; width at baseline between 2 tangents drawn to the steepest parts of the peak
What is the detection limit of ECD?
5 fg
If our particles are 3 microns in diameter, what is the lowest our plate height can be?
6 microns
A solute with a partition coefficient of 3.00 is extracted from 100.0 mL of phase 1 into phase 2. What volume of phase 2 is needed to extract 95% of the solute in one extraction? What is the total volume of phase 2 needed to remove 95% of the solute in five equal extractions instead?
633 mL 137 mL
Some biomolecules require pores of what size in microporous silica?
> 30 nm
What is a good resolution?
>1
In packed columns, what Van Deemter terms contribute to band broadening?
A B C
How is molecular mass determined in SEC?
A calibration curve is made of logMW vs. elution volume. To figure out the molecular weight of unknowns we can compare the elution volume to that of the standards
What is a faux porous bead?
A faux porous shell around a solid bead core.
What is chromatography?
A method to separate components in a mixtures based on different distribution coefficients between the 2 phases based on the interaction between the solute and the stationary phaase
What is normal phase chromatography?
A polar stationary phase and a less polar or non-polar mobile phase
What type of property is electroosmosis?
A solvent property. Before we even put the solute in there is electroosmosis going on
Indicate the type of chromatography, GC, LC, AC, or CE: Antibody-antigen interactions
AC
Which is the best chromatographic technique for the following analysis: GC, HPLC, AC, CE, GPC Determining the presence of an illegal steroid in blood for which you have a monoclonal antibody
AC
In capillary electrophoresis, what might happen to anions at low pH?
At low pH when electroosmosis is weak, the anion may never reach the detector
In OTC, what Van Deemter terms contribute to band broadening?
B C
What are the disadvantages of gradient elution?
Baseline drift, limits the detectors that we can use because some detectors are sensitive to changes in solvent
How does a UV Vis spectrometer detector work?
Because many solutes absorb UV light, they can create a spectra from an eluting solvent and the spectra can be matched to a library of spectra to identify the compound
Why is the UV-Vis spectroscopy detector zig-zagging?
Because of Beer's Law, A=εbC where b is the path length. If we increase the path length, this can allow the detector to respond to tiny concentrations
What needs to be considered when using an OTC?
Because solute interacts with the walls of the column and in order to give sufficient separation, you need these columns to be longer because the surface area is much less than in a packed column
In capillary electrophoresis, why is the detector placed as close to the anode as possible?
Because we want time for the separation to happen
What type of stationary phase is used in partition chromatography?
Bonded liquid to the stationary phase. There are no binding sites.
Refractive index detectors measure what type of property?
Bulk
Indicate the type of chromatography, GC, LC, AC, or CE: No A or C terms in van Deemter equation
CE
Which is the best chromatographic technique for the following analysis: GC, HPLC, AC, CE, GPC Separating 2 proteins of similar molecular weight and geometry but different overall charge
CE
What is the reaction for a flame ionization detector?
CH + O --> CHO+ + e-
What are the polar stationary phases that are used for HPLC?
CH2)3N2: can be protonated and depronated at different pHs (CH2)3CN: very polar (CH2)2OCH2CH(OH)CH2OH
What are the non polar stationary phases that are used for HPLC?
CH2)7CH3, (CH2)18CH3, (CH2)3Ph
How do we determine the molecular weight in molecular exclusion chromatography?
Compare the Vr of the unknown to that of standards with known molecular weights and a similar strucutre
What is electroosmotic mobility?
Constant of proportionality between electroosmotic velocity and applied field
How can we avoid having resistance to mass transfer cause band broadening?
Decrease velocity, decrease the thickness of the stationary phase, increase temperature, decrease column radius
How does decreasing particle size affect resolution?
Decreasing particle size increases the number of theoretical plates, increasing resolution. A: the smaller the particles are, the more likely you will have uniform paths. C: if you have smaller particles they will be more packed together and the distance that an analyte has to travel to get to the stationary phase is much smaller
What are 2 factors that affect how well 2 components are separated?
Difference in retention time and peak widths
Separation in capillary electrophoresis is based on what?
Differences in migration of charged ions in an electric field in solution
When is a distribution coefficient used?
Distribution coefficient is used in place of the partition coefficient when the species can exist in more than 1 chemical form
What is the A term in the Van Deemter equation?
Eddy Diffusion/multiple paths
Why is plate height decreased when using an open tubular column?
Eddy diffusion cannot occur
What are the causes of band broadening?
Eddy diffusion, longitudinal diffusion, resistance to mass transfer
The migration speed of substituted benzoic acids results from the combined effects of electroosmotic flow and electrophoretic mobility. The voltage applied to a 0.5 m long capillary is 2.5x104 V. The neutral marker carried by electroosmotic flow requires 188s to travel .4 m from the inlet to the detector. Migration times of benzoate and 2-methylbenzoate are 340 s and 371s respectively. What is the electroosmotic velocity and electroosmotic mobility. What are the apparent and electrophoretic mobilities of benzoate and 2-methylbenzoate? If electroosmotic mobility were 3x10-8 m2/Vs, what would be the migration times of the neutral marker and benzoate?
Electroosmotic velocity: ueo=0.00213m/s electroosmotic mobility: 4.26x10-8 m2/vs Apparent mobility of benzoate: 2.35 x 108 m2/vs electrophoretic mobility of benzoate: -1.91x10-8 m2/vs Apparent mobility of 2-methylbenzoate: 2.16x10-89 electrophoretic mobility of 2-methylbenzoate: -2.1x10-8 m2/vs 267s 734s
T/F: For gradient elution in normal phase chromatography, it is better to move from a more polar to less polar mobile phase solvent
False
How are the results in capillary electrophoresis quantified and why?
For quantative analysis, normalized peak areas are required, so peak area divided by migration time. This is because in chromatography each analyte passes through the detector at the same rate, the peak area is proportional to the quantity of the analyte, but in electrophoresis, analytes with different apparent mobilities will pass through the detector at different rates, and the higher the mobility, the less time it will spend in the detector. To correct for time spent in the detector, we must divide by migration time
Why does an OTC create a greater resolution?
For the same length and applied pressure, the linear velocity in an OTC column is much higher, so an OTC can be made 100x longer than a packed column and still have the same linear velocity and pressure drop. If plate height is the same, the longer column provides 100x more theoretical plates, leading to 10x the resolution and band spreading by Eddy diffusion cannot occur
Electron capture detectors are used for what type of chromatography?
GC
Flame ionization detectors are used for what type of chromatography?
GC
Indicate the type of chromatography, GC, LC, AC, or CE: Kovat's Retention index
GC
Indicate the type of chromatography, GC, LC, AC, or CE: Temperature programming
GC
Indicate the type of chromatography, GC, LC, AC, or CE: Thermal conductivity detector
GC
Thermal conductivity detector is used for what type of chromatography?
GC
Which is the best chromatographic technique for the following analysis: GC, HPLC, AC, CE, GPC Separating compounds in automobile exhaust
GC
Which is the best chromatographic technique for the following analysis: GC, HPLC, AC, CE, GPC Determining the molecular weight of a polymer you synthesized
GPC
Write the van Deemter equation for open tubular gas chromatography, including all necessary terms.
H = B/v + Cv H=plate height B=longitudinal diffusion C=mass transfer rate v=linear velocity
What is the most polar mobile phase that can be used in LC?
H2O
What is the Van Deemter equation?
H=A+B/V+Cv
Refractive index detectors are used for what type of chromatography?
HPLC
Which is the best chromatographic technique for the following analysis: GC, HPLC, AC, CE, GPC Identifying a non volatile pollutant in drinking water
HPLC
Amperometric detectors are used for what type of chromatography?
HPLC and CE
Conductometric detectors are used for what type of chromatography?
HPLC and CE
Potentiometric detectors are used for what type of chromatography?
HPLC and CE
UV-Vis Spectroscopy detectors are used for what type of chromatography?
HPLC and CE
What are the carrier gases used in gas chromatography?
He, H2, N2
What does HPLC stand for?
High performance liquid chromatography
Why is it important that HPLC detectors are insensitive to solvent changes?
If we use gradient elution, we do not want background changes
How do we increase velocity in capillary electrophoresis?
Increase the applied voltage
What are the advantages of using faux porous beads over porous beads?
Increases surface area a bit Mass transfer is faster (decreases C) More uniform column packing (decreases A) Partially porous molecules with a total diameter of 2.7 um have a similar Van Deemter curve as a fully porous particle with a diameter of 1.8 um, but the partially porous molecule doesn't require the high pressures
Increasing stationary phase thickness does what in GC?
Increasing stationary phase thickness increases the retention time and increases the resolution of early eluting peaks (see equation for resolution)
Why is tailing bad?
It impacts the ability to resolve 2 different peaks and it is also hard to quantify
In GC, what is accomplished by reducing the diameter of the column?
It increases efficiency by increasing the rate at which solute equilibrates between phases
What is the capillary made of in capillary electrophoresis?
It is a fused silica capillary with aa polyamide coating
What does the Van Deemter/Plate Height Theory say?
It is a measure of column efficiency and it says that separation occurs in discrete stages called plates. can predict column performance based on the following properties, which will all impact resolution: ◦ Phase properties ◦ Solution diffusion ◦ K (partition coefficient) ◦ Phase thickness ◦ Size of the stationary phase ◦ Flow rate ◦ Porosity
What are refractive index detectors?
It is based on the refraction of light as it passes from one media to the next. The presence of a solute changes the refractive index of the solvent
How can we tune the surface of the silica stationary phase support?
It is coated with SiOH groups which can be reacted with different things and the surface can be covalently modified
In HPLC, what is the mobile phase?
It is solvent. There are a lot of different solvents we can use and a lot of variability
If you are looking at a molecular mass calibration graph made from SEC, how do you determine VO?
It is the linear point on the left
How do we calculate V0 for a SEC calibration curve?
It is the volume at which the vertical line starts. Any compound within this vertical area is excluded
What is molecular size exclusion used for?
It is useful in determining size and size range for polymers, proteins, etc
What is desalting?
It is when molecular exclusion chromatography is used to purify macromolecules in biochemistry because salts of low molecular mass or any small molecule can be removed from solutions of larger molecules
What does a linear detector response mean?
It means that the detector response is proportional to concentration
What is the plate theory?
It says that separation occurs in discrete stages/plates (mini-extractions). It says there is a separation every time the solute in the mobile phase interacts with the stationary phase. Assumes a column is mathematically equivalent to a plate column equilibrium established for solute to be between mobile and stationary phase on each plate, but it neglects solute diffusion and flow path
What is a Van Deemter curve?
It shows the plate height as a function of flow rate in mL/minute
Downside of a solid stationary phase in GC?
It will give strong retention of solutes because of the large surface area, which means the peak will not be very sharp
In affinity chromatography, why do we change pH, add a surfactant, or change the ionic strength of the solution...why can't we just want for the antigen to come off?
It would come off after a long time, and the antigens would not all come off at once and we want them to come off at once so we get a sharp peak that we can quantify
Indicate the type of chromatography, GC, LC, AC, or CE: Gradient elution
LC
Indicate the type of chromatography, GC, LC, AC, or CE: Ocadecyl (C18) bonded silica
LC
Indicate the type of chromatography, GC, LC, AC, or CE: Refractive index detector
LC
What type of flow do we get in LC?
Laminar flow because there is some friction
A large partition coefficient means what about how long a solute stays on the column?
Large K means that the solute is on the column for a long time
What is a bulk property detector?
Looks at the overall change of the solvent because the solvent properties changes as an analyte goes through it
What is the downside of packed GC columns?
Lower resolution necause of a high surface area and this gives a lot of places for the solute molecules to interact, so we get a wide peak; Eddy diffusion
What is the disadvantage of using OTC over packed columns?
Lower sample capacity means cannot do preparative work
What is the eluent strength (ε)?
Measure of solvent adsorption on base silica. The more polar the solvent, the greater its eluent strength for adsorption chromotograph with bare silica. The greater the eluent strength, the more rapidly solutes will be eluted from a normal phase column
What is the most common stationary phase support in HPLC?
Microporous silica (SiO2)
What is V0 in SEC?
Mobile phase/void volume. It is the volume of the mobile phase that goes right through. The volume outside of the gel
