UMD CHEM425 Final Review
Is automation critical in CE? Explain.
Yes, automation is critical for CE since repeatable operation is required for precise quantification.
True or False Laser and Plasma Desorption (LD, PD) Methods are often used in time-of-flight (TOF) mass analyzer
True
Unlike in chromatography, N is independent to L in CE, true or false?
True
Why fluorescence measurements are taken in ratio mode?
Sources don't have uniform power at all wavelengths which can bias excitation and emission spectra. Ratio sample/reference signals allow - to remove fluctuations in lamp intensity- normalize for different excitation light intensities with wavelength
What are some modes of CE?
1.Capillary zone electrophoresis (CZE) 2.Micellar electrokinetic chromatography (MEKC) - separation of neutrals 3.Isoelectric focusing (IEF) - separation of amphiprotic species 4.Capillary gel electrophoresis (CGE) -separation on size; particularly useful for proteins 5.Isotachophoresis
What are some applications of fluorescence?
1.Fluorometric determination of a wide range of organic compounds. 2.Tagging biomolecules with fluorophores to probe and image biological processes. 3.Fluorescing chelates - detection for inorganic species:
What are some common sample prep methods of AAS?
-Dry ashing (muffle furnace) - Wet digestion (treatment with hot mineral acids H2SO4, HNO3, HClO4) - Combustion in a closed container to avoid loss of analyte.
Which atomic spectroscopic method is flame more of a factor and why?
- AES because... - At most flame temperatures most atoms are in the ground state. Emission measurements are made on whatever small faction in in the excited state. which absorption and fluorescence measurements are made on the entire sample (100%). - Temperature variations affect absorption and fluorescence indirectly because temperature can affect: atomization efficiency and line broadening (eg. Doppler broadening). But while some temperature control is needed in AASand AFS but it is much more critical in AES.
AFS involves
- The emission of photons induced by resonant absorption - Excited states are populated by light absorption - Both quantitative and qualitative measurements
List AAS Flame Common Fuel and Oxidant Combinations
(Fuel, Oxidant, Temp) - Natural gas, Air, 1700-1900 - Natural gas, Oxygen, 2700, 2800 - Hydrogen Air, 2000-2100 - Hydrogen, Oxygen 2550-2700 - Acetylene, Air,2100-2400 - Acetylene, Oxygen, 3050-3150 - Aceteylne, Nitrous Oxide, 2600-2800
What are Some advs/disavs Flame Atomization?
- Advantages: Very reproducible behavior (1% precision), Easy to optimiz, Usually insignificant matrix interferences - Restrictions: Lower sensitivity due to short residence in flame (~ 10-4 s), Large volumes of sample are needed because a large portion of the sample is lost down to drain, and Sample must be in solution
How is AAS performed?
- Based on Beer-Lambert's law where A<1, and the concentration range is large
Name some emission spectra correction approaches.
- Compare well known spectral output (the output of a standard lamp (eg. NIST) - Most instruments have a reference spectrum stored in computer memory that can be used for instrumental correction.
What are characteristics of discrete Introduction Method: Electrothermal Atomizer?
- Sample volume 0.5-10 μl- Detection limits 10-10-10-13 g- Precision 5-10%- Slow (~several minutes per element) - Advantages- Unusually high sensitivity for small volumes of sample.- Allows analysis of solids - Restriction: Low analytical range (< two orders of magnitude)
What are the effects of EWG and EDG on fluorescence?
- EWG deteriorate or completely destroy fluorescence - COOH lower fluorescence efficiency because it leads to lowest lying n--> pi-start transition
What are some safety precautions of AAS?
- Exhaust system: AAS flames produce large amounts of heat and resulting fumes and vapors may be toxic. Make sure the exhaust is turned ON. - Burners: Keep burners clean and do not allow them to block - UV-radiation: Hazardous radiation is emitted by flames and sources.. Never look directly at any of these.. - If using flammable solvents: Always use solvent with the highest possible flashpoint. Minimize the sample amount and use closed containers. - Gas cylinders have to be located in a cool & well-ventilated areas. - ALWAYS wear appropriate safety glasses..Operate the machine with the door or flame shield closed.
List the desorption ionization sources
- FAB - SIMS - ESI - TSI
What is constant and what varies between with emission and excitation spectra?
- For Emission•λ excitation = Constant; λ emission is varied (For most chromophores, excitation spectrum is similar in shape to absorption spectrum). - For Excitation•λ emission = Constant; λ excitation is varied
What are some characteristics of ICP?
- High stability of the source•low noise. Low background . Freedom from interferences. - Theoretically, all metallic elements can be determined by plasma emission spectrometry. - Applications: clinical analysis: metals in biological fluids (blood, urine) - Pharmaceuticals: traces of catalysts used, traces of poison metals (Cd, Pb). - Forensic science: gunshot powder residue analysis, toxicological examination (Tl). - Industry: trace metal analysis on raw materials, noble metals determination
What are the effects of halogens on fluorescence efficiency?
- Lower fluorescence efficiency observed in molecules containing halogens of higher MW: decreases in sequence fluorine -> iodine.
What are the different types of mass anaylyzer
- Time of flight - Quadrupole mass analyzer (GC and LC) - Magnetic Sector Analyzers - Double Focusing Sperctrometers - Ion Trap Analyzers
Whats the difference in importance of background between absorption and fluorescence?
- In absorption: At low concentrations Po ~ P, we measure a small signal decrease over a high background such that increasing Po does not help. - In fluorescence: The beam is read at 90o and we measure the signal over a very small background such that increasing Po increases signal without increasing the background.
What are some restrictions of AAS?
- Inherent problem: path length and molar absorptivities are not available - Departures from linearity are often encountered (calibration curves are made - Spectral and chemical interference due to sample matrix (standard addition is used to counteract) - Sample has to be introduced into the excitation source in the form of solution(most commonly aqueous), thus not convenient for many materials of interest (soils, animal tissues, plants petroleum products and minerals) - Extensive preliminary treatment is often required to make analyte in a form ready for atomization which is time consuming - Can introduce more error than the spectroscopic measurement itself.
What are common light sources in fluorescence?
- Low Pressure Hg Vapor Lamps - High pressure Xe-lamps
What is AAS used for?
- Most widely used - 68 elements can be determined (most are metals) - not all because not all elements have HCL - Not convenient for many materials of interest (soils, animal tissues, plants petroleum products and minerals) because of aerosol restriction
Define the general procedure of AFS
- Nebulization: Converts sample solution into a mist made up of tiny liquid droplets. - Atomization: Flow of gas carries sample into heated region where sample molecules are broken into free atoms - Desolvation: The solvent is evaporated to produce a molecular aerosol. - Dissociation: Molecules dissociate to produce an atomic gas.Atoms dissociate to produce ions and electrons.
What are some transducer ins Fluorescence?
- PMT - CCD (charged coupled device)
What are the Different types of LC methods?
- Partitioning - Adsorption - Size-exclusion - Ion Exchange - Affinity - Chiral
What are the separation principles for G-L and and G-S chromatography in GC. respectively?
- Partitioning, and adsorption
What are some polar and nonpolar adsorbents?
- Polar: Silica (acidic), alumina (basic), magnesia (very basic) Approximate order of retention (on silica):-COOH> -amides> sulfoxides>RNH2 ~ ROH> ketones ~ aldehydes ~ esters> ethers> sulfides> RX ~ aromatics > olefins > saturated hydrocarbons - Nonpolar: Charcoal, graphite (very rare these days)
What are major requirements of the detectors in LC?
- Short response time independent of flow rate - Small internal volume to reduce zone broadening
What's the difference between single vs double beam instruments?
- Single beam instrument: 100% transmittance adjustment is made by using a blank followed by measurement of transmittance of the sample. - Double beam: The beam from the source is split by a mirrored chopper (Reference beam-Sample beam). The ratio of the beam intensities is then measured/ amplified.The reference beam does not correct for loss of radiant power due to absorption or scatter by the flame
What are the spectral interferences involved in AAS?
- Spectral interferences are due to the overlap of atomic lines with molecular bands, that is absorption or emission of an interfering species that CANNOT be removed by the monochromator. - 1.) overlapping atomic lines, 2.) combustion products (products with broadband absorption and or particles that scatter radiation), 3.) scattering (elements such as Ti, Zr, and W scatter light), and 4.) matrix effect
What are some applications of AFS
- Studies of electronic structure of atoms Quantitative measurements - Particularly useful in determining metals - lubricating oils - metallurgical samples- geological samples - environmental studies (trace metals), - biological samples
What are environmental factors affecting Fluorescence?
- Temperature - Solvent Polarity (Solvents affect fluorescence through their ability to stabilize ground and excited states differently, thus changing the probability and the energy of both absorption and emission)--> solvent relaxation - PH (affect degree of conjugation, leads to resonance structures that change excited state energies) - Impurities - Solvent Viscoscity - Presence of molecular oxygen
What are the different factors affecting line broadening in atomic spectroscopy?
- Temperature - Uncertainty broadening (molecules have finite lifetimes, which leads to uncertainties in the transition times of different states, thus lifetime broadening) - Pressure broadening (collisional broadening which decreases the lifetimes of excited states and thus the range of absorbed or emitted wavelengths). (Collisions lead to broadening ~ 10^2-10^3 larger than the natural linewidth). The effect is greater as the temperature increases. - The doppler effect (Wavelength of radiation emitted or absorbed decreases with the rapid movement of the atom towards the detector; it increases when moving rapidly away from the detector). (Thermal motions leads to randomly distributed shifts). (Produces line broadening ~ 10^2 greater than the natural line width).
What are some characteristics, advantages, and restrictions of AES?
- The intensity of the emitted light is proportional to the number of atoms of the element. - Atoms are thermally excited - Atomization is more complete, occurs in chemically inert environments leading to enhanced lifetimes of the analyte by preventing oxide formation/ - Uses Inductively coupled plasma (ICP): Plasma is much hotter (up to 10,000 K) so atomization is nearly complete. Argon plasma: a conducting gaseous medium containing a significant amount of cations and electrons (Ar+ and e- are the charge carriers). - Advantages: Determination of multiple elements under same excitation conditions Determination of elements forming refractory oxides (W, Zr, U, Nb) and nonmetals (Hal and S). Much large linear range (several orders of magnitude vs. three orders of magnitude max for AAS). Less chemical interferences. Simplest and cheapest. Free of spectral and ionization (chemical) interferences due to high concentration of electrons from ionization of argon atoms compared to that of the sample components. - Restrictions: More expensive equipment needed. Procedures are more complicated than those with AAS. Higher operating cost than AAS.Solutions only
AAS involves...
- The promotions of toms from the ground state to higher energy atomic orbitals via the absorption of light (photons) - Quantitative measurement only
AES involves...
- The relaxation of photons from excited states to the ground state - The thermally population of excited states - Both qualitative and quantitative measurements
What are the different transitions?
- sigma-start - sigma (no fluorescence) - pi-star-pi (fluorescence absrved) - Pi star-n (fluorescence pbserved)
What does the AAS instrumentation consist of?
-Light source: Hallow Cathode Lamp of HCL is made of the same element as the element of interest. can't be white light source, must be monochromatic - Atomizer: Continuous sample introduction into a flame produces a steady-state population of atoms, molecules or ions. Typical temperatures: 1700 -2400oC. vs Discrete (sample is atomized in a short amount of time, so temp is raised very rapidly to evaporate solvent, and has high sensitivity). Types: flame, inductively coupled argon plasma, electrothermal (graphite furnace), electric arc, electric spark). Choice of atomization method depends on the physical and chemical state of the sample and the sample matrix. - Monochromator: after the atomizer, removes interference from other emission lines and flame constituents - Detector: PMT (boots the signal intensity). - Amplifiers: amplifies electric current Readout device: digital
What's the structure of the flame in AAS?
-Primary combustion zone:no thermal equilibrium -thus not useful for flame spectroscopy Interzonal region: useful region b/c free atoms are prevalent - Secondary combustion zone: products are converted into stable oxides and dispersed into the surroundings - Typical temperature profile(air- natural gas). Highest temperature ~ 2.5 cm over the primary combustion zone. Need to align the same part of the flame with the entrance slit for the entire analysis.
What's the range for fluorescence efficiency or quantum yield?
0-1 ?
What is the optimal concentration?
0.05
What are the spectral interferences involved in AAS?
1.) Formation of compounds of Low Volatility - Anion Interference (most common): formation of compounds of low volatility with the analyte and thus reduction of the rate at which the analyte is atomized. Example is the decrease of the absorbance of Ca in the presence of an increasing sulfate or phosphate. Solution (add releasing agent, such as a cation, to tie up the phosphate or use a protective agent such as EDTA). - Cation Interference : For example aluminum interferes in the determination of Mg, apparently as a result of the formation of a heat-stable aluminum/magnesium compound. 2.) Dissociation equilibria - Numerous association and dissociation reactions happen in the flame. At least some of them are reversible (Use another oxidant) Equilibrium involving anions other than oxygen: In the presence of HCl: NaCl ↔ Na + Cl - Vanadium in the presence of Al and Ti (Use fuel-rich flame (low in oxygen) 3.) Ionization Equilibria - Small in combustion mixtures that involve air as the oxidant can be neglected. This is important when O2 or N2O are used as the oxidant (at high temperatures) This results in a significant concentration of free electrons exists. To solve this use an ionization suppressor - a compound that ionizes more readily than the analyte
Name the three plasma atomization methods
1.) Inductively coupled plasma (ICP), intense, brilliante white, nontransparent core- most widely used (for AES, it uses an inductively coupled plasma (plasma torch) to produce excited atoms and ions that emit radiation at wavelengths characteristic of a particular element). Both useful for qualitative and quantitative analysis. 2.) Direct-current plasma AES (DCP) 3.) Microwave plasma AES - least used.
What are some instrumental corrections for spectral interference due to the matrix effect?
1.The Two-Line Correction Method: employs a line from the source as a reference. The decrease in power of the reference line due to scattering or the matrix product of the sample is sued to correct the absorbance of the analyte 2.The Continuum-Source Correction Method: Deuterium lamps provide a source of continuum radiation throughout the UV region. This light as well as the HCL are passed alternatively through the atomizer and the absorbance is then subtracted from that of the analyte beam. This can only be done below 350 nm. 3.Background Correction Based on the Zeeman Effect: the background absorption due to molecular scattering is not affected by the presence of the magnetic field so it can be subtracted. 4.The Source-self Reversal (Smith-Hieftje ): total abs is obtained at low current, and backgnd is obtained at high current, then the total absorbance is corrected.
What is PEEK polymer in LC?
A material that is used to make the column, such as stainless steel, glass, etc.
What are some effects of concentration on fluorescence?
At high concentrations: - There is inner filter effect (optically thick conditions when most of the light is absorbed at the front of the cell) although total fluorescence increases ("primary absorption" at c >0.05). - There may be also inner filter effect through "self-absorption" --- re-absorption of emitted light when absorption and fluorescence bands overlap ("secondary absorption"). - Collisional quenching ("dynamic quenching") - non-radiative energy transfer from the excited species to other molecules increases with c - At low concentrations there is the effect of background noise and dark current
What are the types of atomic spectroscopy?
Atomic absorption spectroscopy, Atomic emission spectroscopy, and Atomic fluorescence spectroscopy
What is an advantage of AFS over AAS
Greater sensitivity of measurements (very low background for fluorescence signal).
What are the difference processes involved in flame atomization?
In a flame atomizer a solution of a sample is nebulized by a flow of gaseous oxidant mixed with gaseous fuel and carried into a flame where atomization occurs. - Desolvation: produces solid molecular aerosol. - Dissociation leads to atomic gas. - Ionization leads to formation of cations and electrons. - Excitation gives atomic, ionic, and molecular emission. - Much higher temperatures are needed for refractory samples. - Importance of a flow rate: need to avoid flashback-gas flow rate has to be higher than the burning velocity.
Name the x and y axis on a size exclusion chromatogram
MW vs. Retention volume/time
How is atomic spectrometry performed?
Molecular species are converted into gaseous phase and then the molecules are degraded into their constituent atoms
Does the excitation of a fluorophore at different wavelengths change the emission profile?
No, it does not change the emission profile, but varies the fluorescence emission intensity. Emission goes in proportion to excitation intensity.
Yes or no, does MALDI produce fragmentation?
No, it is a soft ionization source
Are homologous series well sepatated in LC? Why or why not?
Not well seprated, but can be separated by partitioning. Bc they are so similar
What transition display larger fluorescence efficiency?
Pi-star - pi there's a smaller rate of intersystem crossing, and higher transition probability
In Size-exclusion chromatography, what is the mechanism?
Sieving and NOT partitioning
Define resonance line
The corresponding absorption/emission lines of AAS, AES, and AFS
What's the most popular detector type in LC?
UV-VIS, used ~ 80% of the time
What kind of molecules are ESI and TSI used for?
Very important for large (>10^5 Da) thermally fragile molecules: Peptides, Proteins, Polymers, and Oligonucleotides (Most easily adapted in LC and CE)
Can geometric isomers be separated in LC?
Yes, they are separated very well because of the strength of the surface interaction that depends on steric and dipole interactions
What's the typical line spectra width?
~ 10^-5 nm
