Chemical Analysis Exam 3
What does derivatization do if used prior to analysis of a chemical?
1) increase the volatility and decrease the polarity of compounds 2) reduce thermal degradation of samples by increasing their thermal stability 3) increase detector response 4) improve separation and reduce tailing
What is the equation for eddy diffusion
H = 2 xd where x = packing constant and d =diameter of the stationary phase particle smaller H, greater N => more efficient
What is the equation for longitudinal diffusion?
H = 2yD/u y = obstruction factor and D the molecular diffusion coefficient
What is the number of theoretical plates N in CE?
N = uV/2D
Combining both the two effects for migration velocity, at pH greater than 2, what happens to a cation, anion, and a neutral compound?
cations migrate towards cathode; anions migrate toward anode; neutrals flow towards cathode
What are some characteristics of packed GC columns?
column is coated with a thin film of liquid; easy to make and use; long lengths are impractical due to pressure drop
What is it called when there is a constant temperature through the entire run?
isothermal elution
What are the different injection ports for GC?
split, splitless, and on-column injection
What is it called if the temperature changes during the course of the run?
temperature gradient
What is an advantage of GC ?
1) Requires only a very small sample size with minimum sample preparation requirements 2) Good at separating complex volatile mixtures into their components 3) Very high precision 4) Results are rapidly obtained from like 1-30 minutes 5) Only instrument with the sensitivity to detect volatile organic mixtures of low concentrations 6) instrumentation not very complex
1) What are different adsorbents for stationary phase of TLC and PC? 2) What are different solvents used to flow through these adsorbents?
1) SiO2, polyamide, alumina, cellulose 2) who knows
What is the procedure for performing electrophoresis?
1) a strip of filter paper is moistened with a buffer solution of the desired pH 2) ends are fixed to dip in buffer solutions in two troughs fitted with electrodes 3) electric field of 20 volts/cm is established 4) the charged particles of sample migrate along the strip towards the respective electors of opposite polarity, according to charges, sizes and interactions with the solid matrix 5) group of particles migrate as a separate band 6) electrophoresis carried out for 16-18 hours 7) separated proteins are tied to a solid support using a fixative such as acetone or methanol 8) proteins are stained to make them visible 9) proteins appear as bands
What are the two different types of electrochemical cells? and what are they?
1) galvanic cells: chemical action used to produce electrical energy 2) electrolytic cells : consumes electricity from an external source to produce chemical action
What are he different types of open tubular columns?
1) liquid phase 2) solid support coated with liquid phase 3) coated beads (uncoated beads are better for low weight gases)
How can you make sure the gas is pure?
1) molecular sieve trap 2) hydrocarbon trap 3) oxygen trap
What are the four methods used to quantify chemicals in GC?
1) normalizing peak areas 2) internal standard 3) external standards 4) standard addition
How can you improve column efficiency?
1) raising temperature during the separation by gradient, which increases the solute vapor while decreasing retention time
Electroanalytical techniques can provide vital characterization info such as:
1) rates 2) extent of adsorption 3) stoichiometry and equilibrium constants for chemical reactions
What are the four stages in TLC?
1) sample application 2) development 3_ visualization 4) interpretation
What will increase the number of theoretical plates?
1) smaller stationary phase particle diameter 2) higher temperatures 3) decreasing viscosity 4) decreasing stationary phase thickness 5) increase mobile phase flow rate
The degree of band broadening due to longitudinal diffusion depends on:
1) the diffusion of the solute 2) the flow-rate of the solute through the column
Electrochemical processes are oxidation-reduction reactions in which:
1) the energy released by a spontaneous reaction is converted to electricity 2) electrical energy is used to cause a non-spontaneous reaction to occur
The degree of band broadening due to stationary phase mass transfer depends on:
1) the retention and diffusion of the solute 2) the flow rate of the solute through the column 3) kinetics of interaction between the solute and the stationary phase
The degree of broad-banding due to eddy diffusion and mobile phase mass transfer depends mainly on:
1) the size of the packing material 2) the diffusion rate of the solute
The degree of band broadening due to stagnant mobile phase mass transfer depends on:
1) the size, shape, and pore structure of the packing material 2) the diffusion and retention of the solute 3) the flow-rate of the solute through the column
What are 3 common GC detectors?
1) thermal conductivity detector 2) flame ionization detector 3) electron capture detector
Compounds A and B are to be separated using GC. If A spends more time in the column, what does that tell you about its interaction with the stationary phase as compared to B?
Compounds A and B can interact with the stationary phase of the column through intermolecular forces. A must interact more strongly with the stationary liquid phase and is retained. B spends more time in the gas phase, and advances more rapidly through the column. Typically, components with similar polarity elute in order of volatility
What is usually done to interpret the results of TLC?
Determination of the retention factors for each spot; use Rf's of reference spots to identify other components
Because you cannot measure the flow rate within the column, it is measured as it exits the column. What is the equation that is used to calculate the average flow rate using either soap bubbles or a rotameter?
F = Fmeasured (Tc/T)(p-Pwater/P) where Tc = column temp, T = temperature at flow meter apparatus, P = ambient pressure at column outlet, P water = vapor pressure of water at temperaure **term with P is unnecessary if using a rotameter**
What is miniaturized GC?
GC on a chip
What is the equation for mobile phase mass transfer?
H = f(k') d^2u/D where u = the mobile phase linear velocity in cm/sec D = diffusion coefficient of the analyte in the mobile phase
What is the equation for stationary phase mass transfer?
H = f(k')d^2u/D where d = stationary phase thickness (cm) and D = solute diffusion coefficient of the analyte in the stationary phase
What happens/causes the separation in TLC?
Individual components move up at different rates depending on intermolecular forces between the component and the silica gel stationary phase and the component and the mobile phase
What kind of stationary phase is SiO2? What happens when either polar or nonpolar analytes interact with it?
It is very polar; it is capable of strong dipole-dipole and H-bond donating and accepting interactions with the analyte; more polar analytes interact more strongly with the stationary phase and move very slowly up the plate
What should TLC be set up like?
Jar has a top; plate does not touch filter paper; filter paper is completely moistened by solvent; solvent front travels up side by capillary action; spot must be above solvent level
Is longitudinal diffusion most important in GC or LC?
LC; due to the relatively low solute diffusivity in liquids, where D = 0.1 in gas phase and D = 0.00001 in solution
What are two different types of GC columns?
Packed columns and Capillary columns
What does the development of the TLC plate entail?
Placing TLC plate in developing chamber, developing solution is drawn up the plate by capillary action, removing the TLC plate when solvent reaches top line (developing solution is the mobile phase)
What are some characteristics that should be present for the carrier gas?
Purpose of the gas is to carry volatile components through the column. It should be inert towards sample and stationary phase, pressures at about 10-60 psi and hen flow rates between 10-100 mL/min. Carrier gas does not impact analyte retention, but it does influence column efficiency N and thus resolution.
How is a sample introduced into a GC column?
Sample injection system; using a microsyringe to inject a liquid or gaseous sample through a whole that is self-sealing the sample port is usually about 50 C above the boiling point of the least volatile component of the sample
How does the split less injection work?
Split vent is closed
What is the capacity factor? and how is HPLC related to TLC
The Ef value is related to the capacity factor k' by k' = (1-Rf)/Rf, and knowing k' allows you to calculate the selectivity or separation factor a
What kind of mobile phase is typically used? What happens when either polar or nonpolar analytes interact with it?
The mobile phase is usually relatively nonpolar; it is capable of interacting with analytes by stronger Lond forces; nonpolar analytes interact less with the silica gel and more strongly with the mobile phase, moving up the TLC plate.
What the two types of Planar Chromatography?
Thin layer chromatography (TLC) and Paper chromatography (PC)
What are some detectors that may be used for capillary electrophoresis?
UV/vis, fluorescence detector (laser induced fluorescence), MS
How should tubing size and sample size be in order to maximize N?
Use short lengths of tubing and narrow bore; increases in the amount of sample introduced decreases the efficiency (N goes down with the more solute introduced)
What is sample application?
Using a capillary to spot the sample on a starting line of the plate (sample must be dissolved in MeOH)
What is the equation for specific retention volume?
Vg = (Vr-Vm)/(Ms) * 273/Tcolumn
What can you do if the spots being separated are colorless?
Visualization if spots under a UV 254 lamp (fluoresce green) or visualization using spray reagents (general unspecified color)
Equation for the relationship between retention time and volume in GC
Vr = tr*F Vm=tm*F where F is the average volumetric flow rate within the column; V and t are retention volumes and times, and R and M refer to species that are retained and not retained
What is the separation factor for GC? How is efficiency calculated?
a = Vg2/Vg1 Efficiency N is calculated using same equations as in HPLC
What is mobile phase mass transfer?
a process of peak broadening caused by different flow profiles within channels or between particles of the support in the column a solute in the center of the channel moves more quickly than solute at the edges
What is eddy diffusion?
a process that leads to peak band broadening due to the presence of multiple flow paths through a packed column as solute molecules travel through the column, some arrive at the end sooner than others because of a different path traveled around the particles in the column
What is an electrochemical cell?
a simple arrangement of two conductors that are immersed in a suitable electrolyte solution that can yield electricity
What is gel electrophoresis?
a technique used for the separation of DNA, RNA, or protein molecules according to their size and electrical charge using an electric current applied to a gel matrix
What are some characteristics of polyacrylamide gel?
acrylamide monomers are typically used; form long chains; used to separate most proteins and small oligonucleotides because of small pores; vertical stability; neurotoxic; staining done after pouring the gel
What is electrochemical analysis and what are some of the methods it includes?
analysis based on the behavior of the sample solution when it is part of an electrochemical cell; methods include: potentiometric methods voltammetric methods coulometric methods conductimetric methods
What is stagnant mobile phase mass transfer?
band broadening due to differences in the rate of diffusion of the solute molecules between the mobile phase outside the pores of the support (flowing mobile phase) to the mobile phase within the pores of the support (stagnant mobile phase)
What is longitudinal diffusion?
band broadening due to the diffusion of the solute along the length of the column in the flowing mobile phase
Why do peaks broaden?
band width is proportional to the diffusion coefficient of the molecule in the solvent and its elution time 1) eddy diffusion 2) mobile phase mass transfer 3) stagnant mobile phase mass transfer 4) stationary phase mass transfer 5) longitudinal diffusion
What is stationary phase mass transfer?
band-broadening due to the movement of solute between the stagnant phase and the stationary phase different solute molecules spend different lengths of time in the stationary phase, and thus spend different amounts of time on the column
What are some disadvantages of CE?
cannot do preparative scale separations; low concentrations and large volumes; sticky compounds; reproducibility
What is meant by band broadening?
components in a mixture typically broaden as they travel through a chromatographic column; a broad band means that the solute equilibrates with a longer section of the column than does a narrow band
What are salt bridges and liquid junctions?
connection between a separate indicator and reference electrode in an electrochemical cell which allows the passage of ions but does not permit the solutions to mix
Elution strength
considered to be equivalent to polarity; a more strongly eluting solvent will move all analytes to a greater extend than a less polar, weakly elution solvent
What occurs at the anode?
electrode at which oxidation occurs
What occurs at the cathode?
electrode at which reduction occurs
What are some characteristics of open GC columns
fused silica capillary tubing; thin film coating of liquid phase; tube is open so very low resistance to flow; longer lengths are possible, and still high resolution can be achieved
What is a gel? What are the different types of gels?
gel s a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target; agarose gel, polyacrylamide gel
What is the electroosmotic flow in capillary electrophoresis?
goes form + to -; so, anions typically sim agains the push towards the negatively charge anion; thus cations will be the first to come out
What is high speed GC?
good for separation of the so called critical pair conditions optimized for the separation of the most difficult to separate pair of components
What are some general characteristics of electrochemical analysis?
high degree of sensitivity, high selectivity, good accuracy, inexpensive
What are some characteristics of agarose gel?
highly purified uncharged polysaccharide; used to separate large macromolecules; fragile because of the formation of weak hydrogen bonds; direction of migration is towards the positive electrode; no vertical stability; nontoxic; staining can be done before pouring the gel
What does the Van Deemter Equation tell us?
how the column and flow rate affect the plate height H = A + B/u +Cu where u = linear velocity, H = total plate height of the column, A = constant representing eddy diffusion, B = constant representing longitudinal diffusion, C = constant representing stagnant mobile phase. The smaller the height plate, the narrower chromatographic band, the better the separation.
what does electrophoretic capacity refer to? what about electroosmosis?
if it is positive, it will be pulled further to the right; the greater the charge, the greater the pull; electroosmosis refers to the size of the molecule - the larger the molecule, the first amongst them to elute
List how both TLC and PC important techniques
important techniques for identification and separation of mixtures of primarily organic compounds; they are useful in: 1) the identification of components of a mixture, 2) following the course of a reaction, 3) analyzing fractions collected during purification, 4) analyzing the purity of a compound, 5) a scouting technique for HPLC
The absorption strength of compounds________with ________polarity of functional groups.
increases; increasing i.e. alkanes, halides, ethers are weakly absorbed while amides and carboxylic acids are strongly absorbed.
How must the components to be separated be in terms of volatility, boiling point, and stability?
l1) larger molecules are not volatile enough for GC 2) boiling points lower than 250 are best 3) thermal stability is required 4) the structure of the analyze could be changed in order to increase volatility, which is known as derivitization
How does the split mode work with sample injection?
most of the vaporized sample goes to split vent
Is resolution dependent on the length of the column?
no
What are some advantages of CE?
offers new selectivity, an alternative to HPL; high separation efficiency, fast separations, easily coupled to MS
What does efficiency depend on for GC?
on carrier gas; some gases have broadening dominated by longitudinal diffusion with low gas velocity, while others have broadening dominated by finite rate of mass transfer
What are the three different types of electrophoresis?
paper electrophoresis, gel electrophoresis, capillary electrophoresis
Advantages and disadvantages in relation to HPLC?
requires smaller sample; high resolution, accurate quantification drawbacks: limited to volatile samples, samples may require preparation, requires spectroscopy
What is the Rf factor? How do you interpret it?
retardation factor; the distance the center spot moved divided by the distance the solvent front moved Rf= (distance spot moved)/(distance solvent moved) Rf value is not a physical constant, and can only be used to compare spots on the same sheet; substances with the same Rf may be identical, but substances who's Rf does not match are not identical
What does thin layer chromatography do?
separates dried liquid samples with a liquid solvent (mobile phasE) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)
What does paper chromatography do?
separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)
How does size separation occur with DNA and electrophoresis?
size based separation due to interaction of DNA molecules with entangled polymer strands; gel is not attached to capillary wall; polymer length and concentration determine the separation characteristics
What is the junction potential?
small potential at the interface between two electrolytic solutions that differ in composition
How can you separate components with a wide range of boiling points?
start at low oven temperature and increasing the temperature over time to elute the high-boiling point components
What is micellar electrokinetic capillary chromatography used for?
surfactants are added to the buffer solution to form micelles; negatively charged opposes asymmetric flow; separation is based on a differential partition between the micelle and the solvents; very good at separating mixtures containing both ionic and neutral species; if compounds bind to the micelle, they have a longer retention time.
What is capillary gel electrophoresis?
the adaptation of traditional gel electrophoresis into the capillary using polymers in solution
What does the visualization of TLC results entail?
the farthest the solvent traveled on the plate is marked as the solvent front; allow solvent to evaporate from the surface of TLC plate; view results under UV light, mark spots with pencil while viewing
How is gas chromatography different from liquid chromatography? What parameters are useful for GC to use and why?
the mobile phase is a gas rather than a solvent; to take into account the effects of pressure and temperature in GC, it is useful to use retention volumes rather than the retention times
What is resolution in terms of TLC? How can you improve Rfs?
the separation between two analytes on a chromatogram Rs = (distance between center spots)/(average distance diameter of spots) if Rs is greater than 1.0, the analytes are considered resolved. You can improve Rfs by changing the elution strength of the solvent, changing the polarity of the solvent, decrease the diameter of the analyte spots by applying less concentrated spots during application
What does the migration and separation depend on in electrophoresis?
the superposition of solute's own inherent electrophoretic mobility and electroosmotic flow;
What is the heigh equivalent of a theoretical plate (HETP) useful for?
to compare efficiencies of columns with different lengths: H = L/N
What is capillary electrophoresis? How can the charges on the walls be changed?
tubes that provide a better thermal cross-section of the sample within the tube; charges can change depending on the pH
What is the equation for electrophoretic mobility?
u = q/(6pinr) where q is charge on ion, n is viscosity, and r = ion radius
What is the equation for migration velocity?
v= uE = uV/L where, v is migration rate, u is electrophoretic mobility, E is field strength, V = applied voltage, and L = length of capillary
What is a disproportionation reaction?
when an element is simultaneously oxidized and reduced
What is a benefit and drawback of a packed column? How is an open tubular column more useful?
you have greater sample capacity; broad peaks, longer retention times, and less resolution; you can improve resolution by using small uniform particle sizes; open tubular columns allow you to separate more components over a period of time compared to a packed column
Why do you need to have corrected retention volumes?
you have to take into consideration the pressure drop correction factor j; corrected retention volume: Vr = j*tr*F, where j = 3/2 *[(Pinlet/Poutlet)^2-1]/[(Pinlet/Poutlet)^2-1]
