Analytical Chem - Chem 245 - Exam 2

Front 1

Amperometry

Back 1

-non-equilibrium electrochemistry
-monitor current at constant applied voltage -- at potential which limiting current will occur

Front 2

Voltammetry

Back 2

-non-equilibrium electrochemistry
-monitor current at varying applied voltage -- at Eappl

Front 3

Electrolytic Cells

Back 3

-apply external voltage to pair of electrodes to force rxn to occur
-Eappl = Ew - Eref - iR

Front 4

Anodic Current

Back 4

negative current

Front 5

Cathodic Current

Back 5

positive current

Front 6

Limiting current

Back 6

-maximum current that can be achieved in given electrolytic cell
-diffusion limiting

Front 7

Decomposition Potential

Back 7

-potential at which current is zero

Front 8

E1/2

Back 8

-potential at which 1/2 limiting current occurs
-usually close to the Eo value for Ox + ne- <--> Red
if reaction reversible
-however if electron kinetics are slow then E1/2 < Eo -- diffusion dependent

Front 9

Diffusion in Amperometry

Back 9

-the closer the Ox species to the electrode the higher the [Red] will be
-increased electrode area and charge also leads to increased [Red]

Front 10

Mediator

Back 10

-mediating species can be used instead of normal reaction to measure concentrations due to chemical limitations of reactants
-ie replacing O2 with Ferrocenium derivative in glucose sensors

Front 11

Three electrode configuration

Back 11

-in amperometry/voltametry
-used when current flowing through reference electrode would change the potential of that electrode
-in three electrode config no current passes through reference

Front 12

Matrix Effect

Back 12

-sensitivity towards analyte in question altered by other species in sample -- other components change the effective electrode area by adhering to electrode surface
-corrected by standard addition

Front 13

Standard Addition Technique

Back 13

1. obtain signal Ix
2. spike sample with known amount and obtain new signal (Ix + Is)
3. use ratio of concentrations to intensities to solve for [x]

Front 14

Polarography

Back 14

-type of voltammetry
-uses DME (dropping mercury electrode)
-Hg drop functions as electrode - new drop = fresh electrode
-no adsorption of species in sample

Front 15

Stripping Voltammetry

Back 15

-most sensitive of all voltammetric techniques
-use thin Hg film over large carbon electrode
1. apply negative voltage to Hg
-solid metals dissolve into Hg
2. stir solution and rest
3. scan positive voltage
-the result is different metal ions stripping out of Hg at different voltages

Front 16

Electrogravimetry

Back 16

-based on plating metal onto electrode and weighing the electrode

Front 17

Coulometry

Back 17

-measures total current flow over time
-as analyte consumed, conc decreases and limiting current decreases as well
-need sample to be pure for analyte (100% current efficiency) --> limiting factor

Front 18

Absorption Spectroscopy

Back 18

-light source shines light at specific λ through sample and measures light that comes out
-absorbance

Front 19

Emission Spectroscopy

Back 19

-measures light emitted by sample after addition of heat
-also measures bioluminescence (no heat added)
-no light source

Front 20

Fluorescence Spectroscopy

Back 20

-light added at specific wavelength
-light excites sample and emits light of varying λ
-detector set at 90 angle to avoid contamination from light source
-no nephelometry

Front 21

Nephelometry

Back 21

-particle scattering by sample
-light is not absorbed it passes through sample (scatters)
-bac sample spec in lipuma lab is like this

Front 22

Molecular Photon Emission

Back 22

Emitted light is lower energy (high λ) then light absorbed

Front 23

Molecular Fluorescence Spec Setup

Back 23

1. Light Source
- Tungsten = vis
- Deuterium = UV
2. Excitation monochromator
- selects λ1 = λmax absorbance
3. Sample
4. Emission monochromator (90 degree angle)
- selects λ2 = λmax fluorescence
5. Detector

Front 24

Red Shift

Back 24

-phenomenon of energy loss from fluorescence
-photons lose energy due to vibrational and non-radiative energy
- λ2 > λ1 in terms of wavelength --> the red shift
- property unique to molecules and acts as a fingerprint due to its specificity

Front 25

Fluorescence applications

Back 25

-most are quantitative
-exploit low detection limits
-use quenching
-chemiluminescence
-used in immunoassays (labeled antibody)

Front 26

Chemiluminescence

Back 26

-light emits as a side product of a chemical reaction
-no light source needed

Front 27

Fluorescin

Back 27

-common fluorescent indicator
-binds AAs and can therefore be used to label proteins

Front 28

Quenching

Back 28

-indirect method of fluorescent detection
-O2 is a known quencher
-relax excited molecules without emitting light
ie FRET

Front 29

FRET

Back 29

-quenching method that relies on close proximity of quencher to fluorescing molecule
-quencher absorbs at the λ that fluorescing molecule emits

Front 30

Flow Cytometry

Back 30

-cells labeled with fluorescent tags
-apparatus setup so one cell passes through detection area at a time at predetermined rate
-can count number of cells by the number of fluorescent peaks -- histogram
-different cells have different emission spectra (diff number of fluorophores per cell, diff types of fluorphores emitting at diff λs)

Front 31

Optical Atomic Spec

Back 31

-Two types-
1. Atomic Absorption
2. Atomic Emission
-primarily measure metallic activity (toxic metals)

Front 32

Atomic Aborption

Back 32

-flame vaporizes element and light shown through sample
-light passes through to monochromator and then detector

Front 33

Hollow Cathode Lamp

Back 33

-used only for AA as a light source
-emits narrow λ range of light - specific for element of choice
-done by placing element in cathode of lamp and using high voltage to ionize gas
-excited ions strike cathode surface emitting light
-monochromator set to same λ

Front 34

Atomic Emission

Back 34

-flame heats and vaporizes sample
-light emitted by excited gas phase atoms of sample into monochromator and then detector

Front 35

Atomization via Flame

Back 35

-aspirate sample using nebulizer
-sample then heated by flame and atomized
-high temperature works best for emission and low temperature works best for absorption

Front 36

Boltzmann Distribution

Back 36

-equation that shows how T effects emission techniques much more than absorption (negligible change in aa)
-lower T results in clearer spectra but less emission

Front 37

Flames

Back 37

-T dependant on oxidant used
-Acetylene/O2 is the hottest and used for emission
-Acetylene/Air is the coolest and used for AA
-in AA light shined through the side to maximize 'b' in the Beer-Lambert Law

Front 38

Graphite Furnace

Back 38

-second way to atomize atoms -- more efficient than flame
-used exclusively for AA
-uses a graphite tube heated by an external power source
1. sample dried - low T
2. sample ashed - low T
3. sample atomized - high T
-light is shined through tube containing atomized sample

Front 39

ICP-E(mission)S(spectroscopy)

Back 39

-uses ionized Ar gas and oscillating magnetic field to cause a hot plasma of ionized gas
-most sensitive for emission
-much hotter than any flame

Front 40

ICP-M(ass)S(pectroscopy)

Back 40

-most sensitive of all atomic methods
-ionize a small fraction of sample (using same magnetic coil as in ES)
-these ions are accelerated into a mass spec where ions with different mass/charge ratios are separated and detected
-each element has its own detection limit -- the lower the better

Front 41

Spectral Interference

Back 41

due to overlapping bands for emission or absorption
-solved by choosing a different λ

Front 42

Chemical Interference

Back 42

due to species in sample that may hinder atomization -- causes false low value
-solved by using releasing agent to protect atom of interest or sequester species responsible for interference

Front 43

Ionization Interference

Back 43

due to other ionizable species in sample releasing electrons into sample and causing error

Front 44

Glycosylated Hemoglobin

Back 44

-test for diabetes
-reference method based on cation exchange separation
-glucose permeates RBC if in blood plasma for long time -- glycosylates hemoglobin

Front 45

Raman Spectroscopy

Back 45

-excite molecules with powerful external radiation source -- can us any λ as long as it doesn't correspond to electronic transition
-gives information about vibrational energy levels of molecules
-few photons emitted due to low energies - therefore intense light source needed (laser)
-Raman spectra can be used to report on tissue quality -- healthy v. damaged tissue

Front 46

Chromatography Basics

Back 46

-separation of solutes through a column containing two miscible phases: mobile and stationary phase
-Based on interactions between mobile phase solutes and stationary phase species
-More interactions = slower movement = later signal

Front 47

Adsorption Chromatography

Back 47

-solute adsorb onto surface of stationary phase
-inert particles

Front 48

Partition Chromatography

Back 48

-polymer or liquid support coating of solid stationary phase provides binding for solutes in mobile phase
-coated particles

Front 49

Hybrid Adsorption/Partition Chromatography

Back 49

-adsorption chromatography with thin monolayer
-polar particle with hydrophobic monolayer -- reversed phase packing materials
-polar molecules wash out first (don't interact with monolayer)

Front 50

Ion-Exchange Chromatography

Back 50

-charged functional groups adhered to solid stationary phase bind to ions in solution
-particles coated with charged molecules
-electrostatic interactions

Front 51

Molecular Exclusion Chromatography

Back 51

-stationary phase contains pores where smaller molecules can enter
-porous particles
-smaller molecules come out last

Front 52

Affinity Chromatography

Back 52

-stationary phase contains molecules with selective interactions for analyte
-particles coated with selective proteins (aka antibody)
-immunoassays

Front 53

Chromatogram

Back 53

-detector signal (y) v. time (x)
-peaks represent one molecule each -- peaks should appear gaussian

Front 54

Tm

Back 54

- void time
- time for mobile phase to pass through column
-called "air peak" or "dead time" in GC

Front 55

Tr

Back 55

- time for solute to exit column

Front 56

Adjusted Retention Time

Back 56

Tr-Tm

Front 57

Equation of Chromatography

Back 57

Vr = Vm + (k)Vs
-Vm = volume of mobile phase
-Vs = volume of stationary phase
-Vr = retention volume

Vr = F(Tr)... F=flow rate
Vm = F(Tm)

Front 58

Resolution

Back 58

-higher = better
-dependant on large peak separation and small band widths

Front 59

Theoretical Plates

Back 59

-concept used to describe column effectiveness
-a theoretical plate is essentially one unit of equilibration between mobile phase solute and stationary phase -- imaginary sections of column
-the greater the number of theoretical plates (N) the better the column

Front 60

Longitudinal Diffusion

Back 60

-band spreading
-important in GC because gas diffuses much quicker than liquid
-greater diffusion means wider bands -- less efficient column
-decreases with higher flow rate (less time in column)

Front 61

Mass Transfer Kinetics

Back 61

-how quick the solute interacts with the stationary phase
-dependant on film thickness, particle size, and velocity of mobile phase
-a higher value means a wider band - mass transfer lags behind mobile phase speed
-faster mobile phase, bigger particles, and thicker film means a wider band

Front 62

Eddy Diffusion

Back 62

-depends on the path the solute takes through the packing column
-only found in particle columns
-larger particles = greater diversity of paths = higher diffusion

Front 63

Mobile phase speed (u) v. Plate Height (h)

Back 63

-at really low u, h is high (longitudinal diffusion)
-at really high u, h increases (finite mass transfer)
-find the "sweet spot" of intermediate u to minimize h
-determined experimentally

Front 64

Capillary Columns

Back 64

-aka 'open tubular'
-no particles (packing) hence no Eddy diffusion
-consists of extremely long tube with very small radius
-stationary phase coats the inside of the tubing
-greater length = greater N
-low back pressure
-small sample size due to small tubing radius

Front 65

Capillary Column v Packed Column

Back 65

-capillary chromatogram has more and higher peaks than a packed column
-has lower A and C term than packed column
-has lower back pressure than packed column

Front 66

Overloading

Back 66

-[solute] is too high
-peak is non-gaussian -- rear of peak abruptly dips to baseline

Front 67

Tailing

Back 67

-stationary phase contains some sites with increased interaction with solute
-peak is non-gaussian -- rear flares out and is wider than gaussian peak

Front 68

GC mobile phase

Back 68

-inert gas usually H2 or N2

Front 69

GC stationary phase

Back 69

-solid/liquid
-packed or capillary

Front 70

Packed GC

Back 70

- GS -- packed column particles
- GL -- liquid/polymer coated particles

Front 71

Capilary GC

Back 71

-glass column wall or hydrocarbon solvent covalently bound to wall

Front 72

GC overview

Back 72

-sample must be volatile
1. small needle containing volatile liquid or gas injected into injector
2. constant gas flow pushes sample into column
3. detector determines elution and represents as peak

Front 73

Injection

Back 73

-poor reproducibility (usually use internal standard technique)
-1-10 ul volumes used
-injector chamber preheated to temp >50

Front 74

Polydimethylsiloxane

Back 74

-all-purpose GC stationary phase
-non-polar coating added to quartz column
-steps
1. coat with DMCS
2. add alcohol
3. add more DMCS to create film
-substitute methyls with phenyls to make column more non-polar

Front 75

Polyethylene Glycol

Back 75

-polar GC stationary phase
-great for acids and alcohols

Front 76

Polydiphenylsiloxane

Back 76

-more non-polar stationary phase of GC column

Front 77

Trifluoroporpyl siloxane coating

Back 77

-non-polar GC coating

Front 78

GC Elution

Back 78

-based on boiling point (vapor pressure) and solvation (miscibility) of analytes
-lower b.p. tends to elute first
-polarity however may cause species to elute much later

Front 79

GC Temperature

Back 79

-too low = really long experiment times
-too high = poor peak resolution
-linear temperature ramp is a solution -- linearly raise temperature to both shorten time and maintain resolution

Front 80

Thermal Conductivity Detector

Back 80

-sensitive to inorganics and hydrocarbons
-carrier gas is H2 or He
-as species elute the temp of the wire changes -- wire resistance changes
-needs calibration
-0.3 ng detection limit

Front 81

Flame Ionization Detector

Back 81

-most common detector
-good for hydrocarbons
-analyte burned in flame
-ions produced are detected as current on collector electrode
-4 pg detection limit

Front 82

Electron Capture Detector

Back 82

-uses beta particle decay to ionize carrier gas
-free electrons ionize analyte which registers as current drop
-detection limit in fg

Front 83

GC and LC Mass Spec

Back 83

-GC - leak small amt of carrier gas into mass spec
-LC - use electrospray ionization source
-gain retention time elution as well as mass spec data for one sample -- tool to further show species presence in sample

Front 84

HPLC

Back 84

-high pressure liquid chromatography
-use very small particles
-high pressure needed to pass solution through limited void space
-small void space ensures mass transfer

Front 85

HPLC packing

Back 85

-porous silica particle
-surface modified with monolayers (can be non-polar)

Front 86

Normal Phase LC

Back 86

-polar stationary phase and non-polar mobile phase
-non-polar elute first

Front 87

Reversed Phase LC

Back 87

-non-polar stationary phase (silica coated with non-polar monolayer)
-polar mobile phase
-polar elute first

Front 88

Mobile v Stationary Phase

Back 88

-mp alters sp structure based on polarity
-polarity of mp controls LC peak separation -- like temp in GC

Front 89

Particle Size

Back 89

-very important in LC
-smaller particle size = smaller C term = less slope = greater N = greater resolution

Front 90

Mobile Phase Reservoir

Back 90

-HPLC component
-attached to pump

Front 91

Pump

Back 91

-uses high pressure to force liquid through small particle column
-pushes liquid into injection valve

Front 92

Injection Valve

Back 92

-injects reproducible volume of mp into column
1. loads sample into loop in excess (loop filled)
2. valve links MP and pushes entire loop volume into column
-uses 25-100 ul volume

Front 93

Guard Column

Back 93

-small length of column to protect actual column from species in sample which may irreversibly bind SP

Front 94

UV-Vis Absorption Detector

Back 94

-most common
-light source shines light into eluant (monochromator may be used)
-coupled with diode array to obtain full spectrum of each solute
-detection limit: 1 uM - 1nM (solute dependent)
-MP must not absorb strongly

Front 95

Fluorescence Detectors

Back 95

-excite sample with xenon lamp or laser
-monochromator used before or after sample
-detection limit: 1 pM- 1 nM

Front 96

Electrochemical Detector

Back 96

-amperometric most common
-three electrodes (reference, counter, and working)
-detection limit: 0.1 to 1 nM
-used to detect neurotransmitters and "native" electrochemistry

Front 97

Ion Exchange LC

Back 97

-HPLC method
-SP is charged and MP contains ions - LC based on these interactions
-larger ions and more charged ions are retained longer (ionic radius is what matters)
-use polystyrene-based columns (not silica)

Front 98

Cation Exchange Column

Back 98

-requires fixed concentration of given cation in MP - usually by using acidic pH of mobile phase
-SP usually sulfonated

Front 99

Suppressor Column

Back 99

-in ion exchange LC
-added column after separation column to neutralize charged MP species and limit background conductivity
-sample ions measured at low conductivity

Front 100

Ion-Pairing Chromatography

Back 100

-uses regular reversed phase HPLC column
-add surfactant to MP to coat SP with ion-exchange sites
-used to separate organic ions

Front 101

LC MP Composition

Back 101

-low conc = long elution times
-high conc = poor resolution
-use gradient elution to solve problem

Front 102

Gradient Elution

Back 102

-changing MP composition during separation (increasing strength) can decrease elution times but maintain resolution
-in ion-exchange LC increasing the MP ion concentration will have the same effect

Front 103

Molecular Exclusion Chromatography

Back 103

-used to separate and purify proteins and polymers
-separation based on size/shape/MW
-porous silica or polystyrene particles 'trap' smaller species
-smallest come out last

Front 104

Affinity Chromatography

Back 104

-used to isolate or pre-concentrate one specific compound
-SP possesses bound molecules with affinity for compound -- undesired solutes washed off column
-change in pH or ionic strength of MP allows elution of compound to then occur

Front 105

Electrophoresis

Back 105

-separation by migration in an electric field
-based on size and charge

Front 106

Electrophoretic Mobility

Back 106

μ = zF/f... F = faradays constant, f = hydrodynamic radius
U = (μ)(E)... E = electric field, U = velocity (cm/s)

Front 107

PAGE

Back 107

-polyacrylamide gel electrophoresis
-polyacrylamide = cross-linked polymer -- sieves molecules using size
-used for protein (+SDS) and DNA

Front 108

Coomasie Blue

Back 108

Protein PAGE stain

Front 109

Albumin

Back 109

-2nd highest concentrated protein in blood (hemoglobin first)
-maintains blood vessel integrity and can also act as a carrier protein

Front 110

Alpha-1-globulins

Back 110

-high-density lipoprotein (HDL)
-"good" cholesterol

Front 111

Alpha-2-globulins

Back 111

-includes haptoglobin - binds hemoglobin

Front 112

Beta-globulin proteins

Back 112

-carrier proteins (ie Fe)
-help fight infection

Front 113

Gamma Globulin

Back 113

-antibodies

Front 114

MGUS

Back 114

-autoimmune disorder
-predominantly found in men
-patients produce very high concentrations of antibody with unknown fxn
-possible precursor to cancer (multiple myeloma)

Front 115

SDS

Back 115

-sodium dodecyl sulfate
-surfactant that adds -ve charge to protein peptide backbone
-gives all protein same mobility in electric field

Front 116

Capillary Electrophoresis

Back 116

-allows for much higher voltages due to excellent heat dissipation (low current)
-therefore faster separations
-silica capillary flows from +ve to -ve
-dangerously high voltages used (100-300 V/cm)
-no A or C terms due to electroosmotic flow

Front 117

Electroosmotic flow

Back 117

-flow due to electrochemical properties
-consistent throughout tubing - unlike pump flow (laminar flow)
-higher number of theoretical plates

Front 118

Electrophoresis Migration Order

Back 118

- Cations first -- higher charge and smaller size first of these
- Neutrals second -- all migrate together
- Anions last -- higher charge and smaller size come last

Front 119

Capillary Electrophoresis Detection Limits

Back 119

UV-Vis = 1 uM or 1 fmol
Fluorescence = 1 pM - 1 uM
Electrochemical = 1-10 nM
MS = 0.1 to 1 uM

Front 120

MEKC

Back 120

-Micellar Electrokinetic Chromatography
-Negatively charged surfactant (SDS) used to form micelles with neutrally charged species in center
-Separates out neutrally charged species based on lipophilicity -- the more micelles bound the more negative the complex and the later it elutes
-Used for separating out steroids