ANAL 315
Level of transferrin different between age groups, sex can be an
-> indicator of health status
Why is it important that emission spec have stable flame
Emission intensity proportional to population of excited state which is proportional to temperature
different carrier gases have different van Deemter Curve mininmum which means
Have different Optimal velocity (where H is lowest)
Chemical interference doesn't occur in ______________
ICP (argon = inert)
What does the Boltzmann distribution describe about temp and atoms in state
Increase the temperature -> greater fraction of atoms in excited state 10º increase = 4% excited state increase
Interference in measuring Fe content of transferrin in blood by spectrophotometry:
Interference due to copper (also reacts with ferrozine) • ROLE OF MASKING AGENT ---> Neocuproine or thiourea added to complex Cu+ (lower absorbance to different wavelength)
Partition chromatography
Martin and Syngne Similar to extraction where solute partitions between liquid stationary phase and mobile phase
Photomultiplier tube vs photodiode array
PMT can only quantify analyte (measure at one specific wavelength) PHOTODIODE ARRAY can record an entire spectrum to qualitatively identify analyte
selectivity
ability to distinguish analyte from other species in sample (avoided interference)
strong cationic exchangers are strong _______ while strong anionic exchangers are strong _______
acid (dont protonate and loose negative charge at low pH base (dont deprotonate and loose positive chage at high pH)
acid digestion or fusion first
acid digestion
longer GC column means ____________ and ____________
better separation increase analysis time
What happens when • Molecule absorb energy of photon
energy of molecule increases (electron promoted to excited state) Molecule emits photon -> energy lowered (electron back to ground state)
irradiance
energy per second, per unit area of light beam
example of affinity chromatorgraphy interactions
enxyme-substrate antibodies-antigen receptor-hormone
efficiency of a column is a measure of
how well the separation is
What is transferrin
iron transport protein that delivers iron to every organ
define internal standard
known amount of compound [different from analyte] is added to unknown. Signal from analyte is compared with signal from internal standard to determine quantity of analyte.
smaller plate height = _________ the band
narrower
frequency
oscillations per second
smaller particle size means higher ______ higher _______ shorter________ lower __________
plate number pressure optimal run time (shorter distance to diffuse) detection limit (peaks sharper)
prone to forming stable oxides, that interfere with the formation of atoms (diminish mass transfer)
refractory elements
ion exchange chromatography
retention of solute based on their attraction to charged sites bound to stationary phase
Increase thickness of stationary phase increases the ________ and __________ of _______________
retention time resolution k<5 (early eluting peaks)
separation of labile proteins best use
size exclusion chromatography
Stationary phase thickness can reduce _______, but increase _________
tailing (shield analyte from silica surface) bleed (decomposition and evaporation of stationary phase)
The colour of a substance is the wavelength that was ______
transmitted
transmitance
transmitted light/incidence light
IF the most snesitive wavelength of the element of interest is in the 193nm range, what should be done?
use argon/hydrogen flame use less sensitive wavelength
how to prevent formation of refractory elements
use hotter flame (acetylene nitrous oxide) combo of fuel/oxidizer`
hydrophobic interaction chromatography require
water soubility
spectrophotometer detector is a function of __________. What is the implication of this
wavelength Must calibrate -> 100% transmittance control must be readjusted each time the wavelength is changed
How temp affects atomic spectroscopy
- extent to which atom in ground/excited/ionized state -degree to which sample breaks down (FAAS and GFAAS)
UPLC
1-5-2um particle size revolution came from development of very high pressure system allowed for increased resolution and decreased run time = less mobile phase required
Steps in atomization in GFAAS
1. Dry - heat to remove solvent 2. Ash - remove matrix/destroy organic matter 3. atomize - generate atom 4. clean -remove remaining residue Temperature higher in each step
How does a Photomultiplier tube (PMT) work
1. Photon hit photosensitive surface -> emit electrons 2. Electrons strike dynode (positive) with acceleration -> knock more electrons 3. More and more electrons knocked off
Measuring Fe content of transferrin in blood by spectrophotometry:
1. Reduce Fe 3+ (released from transferrin) -> Fe 2+ • Add acid as reducing agents - hydroxylamine hydrochloride (NH3OH1Cl2), thioglycolic acid, or ascorbic acid 2. Precipitate the protein and centrifuge -> Fe 2+ in solution • Trichloroacetic acid • Light scattered by precipitate protein mistaken for absorbance =3. Add ferrozine to form purple complex (with buffer)
How does a photodiode array work
1. White light (with all wavelengths) pass through the sample 2. Light enters a polychromator - disperses the light into its component wavelengths and directs the light at the diode array 3. Each diode receives a different wavelength, and all wavelengths are measured simultaneously 4. Spectrum generated
how to gauge accuracy and precision in the QA process
1. blanks, calibration checks, quality control samples, spike recoveries 2. replicate samples/portions of samples
What to do about isobaric interference
1. use a high resolution ICP-MS to resolve species (differentiate between smaller m/q difference) 2. use dynamic reaction cell (DRC) to remove ions - reactive gas such as NH3, N2O that react with either the interfering Ar ion or measurement species -> change size can be revmoved or better measured (electric field is configured to select lower and upper masses of ions to pass through the cell)
distinct isotopic peaks of Cl and Br
1/3 and 1:1
property of laser: extremely bright Coherent plane polarized
10^13 time brighter than sun all waves in phase electric field oscillates in one plane
diffusion in liquids is _____ times ______ than in gases
10^4 slower
ionization energy of Ar is _________. Which is ________ than all elements except He, Ne, F
15.8 electron volt higher (can ionize other molecules)
most common width for HPLC columns nowadays
2.1mm
only ______ of known organic compounds are volatile
20%
What do the three terms in the van Deemter equation represent?
3 different band-broadening mechanisms that are affected differently by flow rate
how to prevent microbial degradation of hydrocarbons
5% HNO3
How does hollow cathode lamps work
500V applied to gas (some element we want to analyte) gas ionized -> positive ions accelerate toward cathode cations stike cathode and metal sputter into vapour gaseous atoms collide with high energy electrons to emit photons
splitless injection used if analytes are ______ of sample
<0.01% (trace)
GC column using MS as detector diameter must be ________ or else would overload the vacuum system (pump cannot keep up).
<0.32mm
residence time of flame vs furance
<1s several s
Chromophore
= part of molecule that absorbed light
split injection used if analytes are ______ of sample
>0.1% (minor)
ICP-MS cannot tolerate ______ of salt. Why and what can be done
>1%. very small aperature. use ion exhange chromatography using carboxylic acid resin that can quantatitively bind to ions at >6pH
for quantitative analysis, resolution of _______ is called baseline resolution and is highly desirable
>1.5
we chose a peak with retention factor of _____ to measure plate height for a column
>5
silica particles cannot be used at pH _____ unless using ethylene bridged silica
>8 (dissolution and SF bleed)
absorption spectrum measures how
A depends on wavelength
What is measured in FAAS
Absorbance of the anlyte A=log(I_0 / I_T) transmitted light after passing through sample is compared with the emission of the HCL
What did Mikhail Tswett discover
Adsorption chromatography isolated plant pigments easily dissolved in ligroin, but not from plant leaves - dissolving power of ligroin for pigment lower than adsorption force of pigement to tissue used different adsorbents -> separation
Adsorption chromatography
Analyte interact with (adsorb onto) the surface of stationary particle (solid). Mobile phase competes with analyte for adsorption on station phase (elution) can distinguish between molecules with different functional groups ( Not be able to separate molecules with same functional group, tail group differs in length )
Chromatography carried out to separate and identify/measure components of a mixture is called _______________ and uses___________ columns
Analytical purposes narrow
the _______ _____________ of a laser beam is very small which means it has very little deviation (illuminates very small target area)
Angular divergence
___________________ during GFAAS atomization is halted because
Ar purging Increase residence time -> increase signal (avoid analyte blow out of furnace)
how to reduce bleeding
Arylene stationary phases have increased thermal stability use the thinnest possible stationary phase and the narrowest and shortest column that provides adequate separation purest carrier gas
At what wavelength to measure absorbance for spectroscopy and why
At max absorbance of molecule • greatest sensitivity • curve flatter -> little variation from Monochromatic drift (set wavelength moves around a little bit) Most precise when A~ 0.3-2
What are the 3 types of atomic spectroscopy
Atomic Absorption Atomic emission Atomic fluorescence
What is signal averaging
Average measurements on top of each other (increasing number of reading improves quality)
most common bonded stationary phase
C18 (octadecyl)
CCD vs PMT
CCD higher signal-to-noise ratio than PMT
carbohydrates can complex with _____ if their OH groups are arragned _____________
Ca2+ axial-equatorial-axial
Precursor of spectrophotometry
Colorimetry -> use eyes to compare colour of two solutions Dilute unknown mixture until same colour as standard mixture (known conc.)
flame photometric detector
Compounds are burned in a hydrogen-air flame emission measured and is measured by PMT SULFUR AND PHOSPHOROUS containing
different solutes passing through same column have different plate heights because they have different ___
D (diffusion coeffcient)
spectrophotometer light source for UV range
Deuterium
what is laser ablation
Direct laser light at material Energy of laser - so intense that chemical matter vaporized (abated) Vapour flushed into ICP-MS - analyze chemical matter
on column injection
Directly inject analyte into column initial column temperature is set 40ºC below the boiling point of the solvent (condense, to trap solute in solvent) form sharper peaks
ion excahnger place in eelctrolyte solutions with conc. of electrolyte greater outisde than inside. ions with same charge as resin will be excluded
Donnan-equilibrium
height in flame at which max atomic absorption/emission depends on:
Element fuel/oxidizer flow rate nebulization speed
Why are some spectrometers purged with Ar or N2?
Exclude O2, allowing ultraviolet wavelengths in the 100-200-nm range [for halogens, P, S, and N] to be observed. Energy levels of halogen atoms (F, Cl, Br, I)+ P, S, and N are so high that they emit ultraviolet radiation below 200 nm. However O2 absorbs radiation below 200nm (interferes)
What is a charge coupled device (CCD)
Extremely sensitive (Lower DL) solid state detector that convert light to photo generated charge and store in a 2D array (pixels) Electron stored depends on intensity of light
DL order of different atomic spec techniques
FAAS, > ICP-ES (ultrasonic nebulizer) > GAAS > ICP-MS
how does injection differ in HPLC and GC
GC: inject liquid, moelcule VAPOURIZED HPLC: must dissolve sample in solvent (liquid) **solvent changes retention times (may have mobile phase strength stronger than mobile phase itself)
what is Atomization
Generate analyte into gaseous atoms in ground state
Why is the Hollow cathode lamp more superior to the light source used to produced white light Kirchoff and Bunsen used?
Gives a much narrower emission. More sensitive, need less analyte to observe difference in absorption due to analyte
_________ is to HPLC as _____ to GC
Gradient elution temperature programming
What is Transverse platform in GFAAS
Graphite tube is semi-circular platform ---> nearly uniform temperature over whole furnace reduces memory effect
van Deemter equation
H ≈ A + B/Ux +CUx
_____ gas has fastest Optimal velocity
H2
Component of Flame atomic absorbance spectrometer (FAAS)
HCL (contain same element as being analyzed) flame (air/acetylene or nitrous-oxide/acetylene)- sample is introduce into flame as aerosol by nebulizer (transforms liquid solution in to sample droplets) monochromator to selectr wavelength detector (PMT CCD)
What is the dominant form of atomic spec used today
ICP-AES no map required intensity of emission proportional to analyte concentration in sample
what is the purpose of matrix modifier
Increase volatility of matrix Reduces volatility of analyte
Kirchhoff and Bunsen experiment
Introduced table salt into a flame, a dark line observed at 589nm Na atoms in the salt absorbed light at that wavelength Wavelength of light at 589nm has energy to push electron in Na to higher energy state (absorb light) -> excite Na atom to higher state give off energy of wavelength as it falls back to ground state (emission, but not what was observed)
chemical ionization
Ionization source filled with reagent gas (CH4, isobutane, NH3) Energetic electron convert CH4 ---> CH5+ or C2H5+ These act as proton donors - protonate analyte molecule (soft ionization) DOESN'T CAUSE FRAGMENTATION
Why is GC more preferable to HPLC
Less expensive generate less waste low pressure faster analysis dont have to worry about mobile phase viscosity size of particle (OT) Don;'t have to dissolve in solvent
Explain how single beam spectrophotometer works
Light source -> monochromator -> blank / sample (alternates) ->detector measures irradiance
affinity chromatography
MOST SELECTIVE chromatography type separate based on specific interaction between one kind of solute and another kind that is immobilized on stationary phase ie. antibody and protein
_________ can have a huge effect on the yield of ions generated in ICP. As a result:
Matrix Different ionization of standard vs unknown = error in quantification Calibration standard must have same matrix as unknown (in standard addition) OR if using internal standard , must choose on that has nearly the same ionization energy as analyte
What is a spectrophotometer detector
Measure intensity of light/ photons and transforms into electric signal
How to increase volatility of matrix in Analysis of seawater
NH4NO3 (ammonium nitrate) increases NaCl volatility (forms compounds with lower BP)
how does evaporative light scattering detector work
Nebulizer (eluent mixed with N2 (g)) -> droplet -> heated drift tube -> evaporate (solvent gone) -> particles of dry analyte -> laser shine -> register if particle reflect light
what holds Fe3+ in solution (makes it soluble) in spectrophotometric titration
Nitrilotriacetate
why must carrier gas in GC be low in oxygen
O2 can degrade stationary phase via oxidation
_________is the constant of proportionality between the variance, σ^2, of the band and the distance it has traveled, x
Plate height, H
________ protect GC column with support and protection from atmospheric moisture
Polyimide coating
Chromatography carried out to purify large quantity of component of a mixture is called _______________ and uses___________ columns
Preparative purposes wider
What is a Hollow cathode lamp
Produces emission of wavelength associated to atom (of interest) to produce narrow lines of correct frequency Contains vapor of same element being analyzed
photodiode array
Records entire spectrum at once in a fraction of a second -different wavelengths reach different parts of the detector array. All wavelengths are recorded simultaneously
Feature of the Double beam scanning spectrophotometer
Rotating mirror that chops the light from the monochromator, allowing it to through sample then immediately do through blank (don't have to manually swap sample and blank)
Examples of actions in the specifications process
Sampling requirements Accuracy and precision Rate of false results Selective/specificity Sensitivity Calibration check Standard Operating Procedure (SOP)
spectrophotometer light source for IR range
Silicon carbide globar (1500K)
how are Adsorption and Partition Chromatography fundamentally different
Solute attach to silica, solvent displace solute Solute partition between stationary and mobile phase
describe bonded stationary phase
Stationary particle with silanol particle attached which has R group attached (different characteristic -> selectivity )
example of strong and weak cationic exchangers
Sulfate - very high pKa (can use even at low pH condition) Carboxyl group - low pKA (will protonate at <4)
Problem with Single- Beam Spectrophotometer
Switch between blank, sample, blank, sample - Not good for rapid kinetic measurements When you want to change wavelength, must re-measure blank Light source and detector fluctuates (Light source may vary with intensity)
Spectroscopy vs spectrometry
Theoretical - science of studying interaction between matter and readiated energy practical application - method used to acquire quantitative measurement of the spectrum
spectrophotometer light source for visible and near-infrared range
Tungsten (filament near 3000K)
selected reaction monitoring
Used specific chemistry to select for ion First select precursor ion for compound of interest Fragment to produce prominent ion
Atomic emission spectroscopy does not require ___________. Implication is that an ICP-AES allows for _________________ analysis
a lamp multi-element (dont need different lamps to create emissions to compare absorbance, etc)
What does resolution mean in terms of grating
ability to separate two closely spaced wavelengths greater the resolution, the smaller is the difference between two wavelengths that can be distinguished from each other
In order for compound to be quantifies by spectrophotometry, it must ________ and ___________
absorb light be distinguishable from others (Nothing else in solution that absorb at that wavelength)
What property of flames in FAAS affects the measurement at specific wavelength / choosing of flame type
absorb light with differing degrees at different wavelenghs
ICP cannot be used for atomic __________ spectroscopy
absorption
broadening caused by many individual mechanism --> variance is __________
additive
retention factor/capacity
adjusted retention time relative to elution time of mobile phase peak specific - used to reproduce results as is independent of column dimension (as K is larger)
normal phase chromatography
adsorption chromatography
_____ chromatography is ideal for separating isomers
adsorption. steric factors affect interaction
ionization interference is especially a problem for
alkali metals becuase they have low ionization potentials
ion exchangers resins are often made of
amorphous organic polymers start with monmer, incorporate charge into polyer by using derivatives that are charged
ionization interference
analyte atoms ionized which decreases conc. of ground state atoms ions have different energy levels from ground state atoms (different signal)
what is separation anxiety
analyte containing different types of compounds (polar and non-polar), not sure what stationary phase to use
If temp too high in ash step in GFAAS
analyte escape with matrix
liquid extraction
analyte is dissolved in a solvent that does not necessarily dissolve the entire sample and does not decompose the analyte
steps to obtain mass spectrum
analyte molecule -> gaseous species (no "put into gas phase" ionize speicies accelerate ions using electric field separate ions according to m/z ratio detection Vary magnetic field strength -> spectrum
What are calibration check
analyze solutions formulated to contain known concentrations of analyte periodic checks for large samples to ensure instrument works properly Check solutions should be different from ones used to prepare original calibration curve
Ion-exchange chromatography
anion or cation covalenthly attached to stationary phase (insert into polymer). ion separation based on interaction with charged stationary phase
cationic exchangers are _______. At ______ pH, they can be __________, thus loosing their charge. Strong cationic exchangers have ________ pka values and thus are not ________ at ________ pH
anions low protonated high protonated low
spectrophotometric titration in determining apo-Tf conc.
apo-TF (transferin without Fe) is colourless titrated with Fe3+ to form red complex Titration: start-out near zero, add solution increase absorption, due to formation of chromophore Past endpoint -> will not generate anymore chromosphere -> level off absorption • but Still increase due to colour of nitrilotriacetate
_________of a chromatographic peak is used for quantification
area
EI of _________ compounds usually give significant intensity for M+.
aromatic extremely stable molecules (don't fragment)
Inductively Coupled Plasma
atomizing sample by using the high energy of electrons generated by ionization of Ar gas using magnetic field
What is the memory effect in atomic spectroscopy
atoms from previous run creates interference at the next run (due to condensation then vapourization)
precision of GFAAS enhanced by
autosampler
column effiency
band broadening
width of radiation selected by the slit is called the
bandwidth
hydrophilic interaction chromatrography
bare silica (silanol group) bonded with zwitterion ion -> can bind anion and cation use ELSD
______ silica is to __________ chromatography as _______ stationary phase is to ________ chromatography
bare, adsorption bonded, partition
most intense peak in mass spectrum = ___________ peak
base Peak intensities all relative to base peak (normalized)
why are electronic absorption bands broad?
because vibration and rotational transitions also occur
low noise spectrometer
cancel noise generated by variation in light source light spits and hits both reference and sample cell and both irradiance detected Variation in light source Both V_ref and V_sam affected simultaneously Difference between two detector signal -> should be zero if no sample absorption (any value due to sample ) Basically used the same light
Source of interference in GFAAS
carbide formation memory effect chemicals in matrix
what is the gaseous mobile phase in GC called. What are examples
carrier gas He, N2, H2
anionic exchangers are _______. At ______ pH, they can be __________, thus loosing their charge. Strong anionic exchangers have ________ pka values and thus are not ________ at ________ pH
cations high deprotonated low deprotonated high
How could pH affect extraction
changing molecular form and charge neutral to charged by deprotonation/protonation charged in aqueous phase neutral in organic phase
Random unknown... how can electron ionization and chemical ionization give information?
chemical ionization - find the molecular mass (M+. ) Electron ionization - fragments provide clues to the structure of an unknown
chemical interference
chemical reaction that lowers extent of atomization of analyte -oxides/refractory elements (formation of calcium oxide due to SO42- and PO43-) reduce by increasing T to breakdown oxides
organic solvents heavier than water
chloroform dichrolomethane carbontetrachloride
plate height is a measure of
column efficiency
flame ionization detector
combust eluate using hydrogen/air flame from GC column generate ion that hits collector ions proportional to the # of susceptible carbon atoms entering the flame. lower detection limit compared to thermal conductivity detetector HYDROCARBONS
benefit of hydride, but why its not often used
complete elimination of interference separation and enrichment of analyte only a handful of elements can form hydride
flux is proportional to _______ and _______
concentration gradient diffusion coefficient amount of molecules moving depends on how strong the gradient is and ability of molecule to difusse
absorbance is proportional to ___________
concentration of analyte path length molar absorption/extinction coefficient (characteristic of substance measuring)
partition / distribution coefficient
concentration of solute in each of 2 phases affected by Nature of components of phase 1 and 2 Molecular structure of solute
what is higher measurement than true value attributed to
contamination
gradiant elution
continuous change of solvent COMPOSITION to increase eluent strength
_______ plate height = ________ variance
decrease decrease
Plate height decrease with particle size decrease in HPLC
decrease A term - more uniform flow decrease C term - less distance solute must diffuse (equilibrate faster)
what is the significance of ultrasonic nebulizer
decrease concentration of analyte needed (greater mass transfer) therefore much lower detection limit vibrating crystal generate mist -> condensed Create dry solids particles injected into plasma
ablity of colum to separate components is improved by _________ plate height, which means ______ theoretical plates
decreasing more
What did Isaac Newton in 1772
degree by which light refracted was a property of rays NOT prism. White light was not pure, rather composed of mixture of rays of different colour ---- SPECTRUM
what does a chromatogram show
detector response over elution time
What is the evaporative light scattering detector
detector that evaporates analyte and laser is shone on it and light reflection off particle is registered
What is Atomic Spectroscopy
determine elemental composition of a substance vapourize samples at higher temperature to decompose into atoms. Concentration of atoms in vapour measured by emission or absorption characteristic wavelengths of radiation
organic solvents lighter than water
diethylether toluene hexane
Explain differences in colours of objects
different substances have different properties that cause them to have varying absoprtion spectrums (absorbs different amount of different wavelengths)
heterogeneous vs homogeneous
different/same composition from place to place
what is the main cause of band broadening
diffusion net transport of a solute from a region of high concentration to a region of low concentration caused by the random movement of molecules
3 important difference between HPLC and GC
diffusion coefficient smaller in HPLC (diffuse slower in liquid so slower) mobile phase much more viscose in HPLC (high flow resistance) lower mobile phase compressibility in HPLC (can use high pressure)
What is fusion
dissolve inorganic materials that do not dissolve in acid through a hot, molten, inorganic "flux" • Very aggressive technique Impurities introduced by large mass of solid reagent
If temp too high in dry step in GFAAS
droplet sputter and throw analyte on wall (lose sample)
define monochromator
dsiperses light into its component wavelenths and selects narrow band of wavelength
Define interference
effect that changes signal while analyte concentration remains unchanged
electron capture detector
electrons generated that produce current when hits anode high electron affinity molecules capture electrons and decrease current current loss is measured ELECTRONEGATIVE COMPOUNDS: Halogens, nitrates and conjugated carbonyl very sensitive
M+1 peak in MS gives info on _________________
elemental composition (element exist in forms of different isotopes )
Bleeding of column causes
elevated background signals, reducing the signal-to-noise ratio for analyte potentially contaminating the detector.
isocratic elution and the isocratic condition
elution with single solvent • can elute either early-eluting or late-eluting peaks (different) polarity of analyte Similar to GC separating polar and non-polar compounds
any material that can ______ light of a certain wavelength will also _______ light of that wavelength
emit absorb
Define matrix
everything in sample not the analyte
thermal conductivity detector
filament with applied current pure carrier gas (He and H2) have highest thermal conductivity analyte decrease conductivity of gas stream -> increse resistence -> voltage change measured least sensitive UNIVERSAL
Packed GC column
fine solid particles that are coated with liquid stationary phase (partition) or solid itself is the stationary phase (adsoprtion)
C term in the van Deemter equation
finite equilibrium time between phases SLOWER FLOW RATE = smaller C term contribution to H more time for equilibrium to be reached (Analyte molecule won't "lag" while the mobile phase moves on due to less time available for mass transfer that causes "smearing" Based on relativity of mass transfer compared to mobile phase velocity
Ways to atomize sample
flame (FAAS) electrically heated graphite furance (GFAAS) inductively coupled plasma (ICP) heated quartz (hydride technique)
in FAAS, _________ emit light that must be subtracted from total signal
flames
In HPLC, Pressure directly proportional to ______ and ______, inversely related
flow rate and length of column particle size
spectrophotometric titration
follow course of titration by UV-Vis spectroscopy -> can tell us when equivalence point reached (Monitor changes in absorbance during titration)
significance of 70eV electron kinetic energy in ionization
fragmentation likely to occur (MUCH higher than ionization energy of molecules) lower IE = less fragmentation (greater chance of observing M+.) BUT 70eV used because it gives reproducible fragmentation that can be compared with the library
Why is the photodiode array ESPECIALLY Useful in LC
full spectrum of compound can be record as it emerges from the column Qualitatively identify compounds separated on chromatography column
partition chromatography vs adsorption chromatography in GC
gas-liquid: liquid stationary phase bonded into column or on solid support (analyte partition between stationary and mobile phase gas-solid: analyte adsorbed directly on solid particle
________ are used in ion exchange chromatorgrphy for large molecule (protein and nucleic acid)
gels (electrophoresis)
How is furance used to atomize sample
graphite tube which holds sample is heated electrically. atomic cloud is formed
What is L'vov Platformin GFAAS
heat platform by radiation from furnace wall analyte doesn't vaporize until wall reaches constant temperature Delay of temp equilibrating (don't lose sample to cooler ends)
Matrix modifer icrease BP of analytes which means
hgiher ashing temperature can be used without losing analyte
______ salt content will cause proteins to salt out of solution
high withdraw water from protein -> Exposed hydrophobic pockets of proteins come together and crash out of solution
how does electron ionization occur in MS
high energy electron is accelerated (70eV) and bombard (neutral) analyte molecules small fraction of molecule absorb (12-15eV) energy energy for ionization M+. generated
drawback of small particle size
high pressure (expensive) frictional heating • Force mobile phase through particle bed -> heat generated • Difference in temperature resulting in different diffusion -> band broadening)
Porous layer open-tubular columns
high-surface-area porous solid particles adhering to the column wall. The highly retentive surface area allows for retention of small molecules (He, Ar, O2, N2, CH4, and CO)
ion exchangers fabour binding of ions of ________ charge, _______ hydrated radius, and ___________ polarizability
higher decreased increased
separation of carboxylic acids by cation exchanger with 0.006N H2SO4 as mobile phase. carboxylic acids with ______ pKa will be retained longer than ________pKA
higher pKa (more acidc, easier to be deprotonated, interact with cationic exchanger)
Gc column must be _____ enough to provide sufficient ___________ for elution of analytes
hot vapour pressure
resolving power of MS
how close two peaks in mass spectrum can be spaced and still be resolved (ability to separate 2 peaks of similar mass)
What does the van Deemter equation describe
how column and flow rate affect plate height
two factors that determine how well compounds separated on column
how far apart peaks are (difference in elution time) how wide the peaks are
what is residence time
how long atoms in ground state reside in optical path
hydrophobic interaction chromatography
hydrophobic pore (phenyl or alkyl groups attached to agarose gel) in which protein enter decreasing salt content will increase increase the solubility of proteins in water and elute them from the column.
increasing temperature in GC ________ analyte vapour pressure which _________retention time for late eluting components (elute faster, sharper peaks)
increase decrease
How does increased temperature affect AA and AE?
increase excited state pop AA = atoms in ground state, therefore AA signal not affect AE = atoms in excited state [emission intensity prop. to pop of excited state]. increase temp would increase AE signal
effect of using transversely heated GF
increase signal due to greater mass transfer reduces memory effect
how does one increase ionic strength in ion exchange chromatorgrphy
increase the concentration of the mobile phase (displace solutes that are strong and stronger)
How does ICP atomization work
induction coil connected to generator. Magnetic field generated High purity Ar gas fed through and is ionized free electrons collide with atoms -> transfer energy to entire gas (maintaining a temperature of 6 000 to 10 000 K) to cause atmoization nebulizer introduces sample to plasma torch
why CO2 used as supercritical fluid
inexpensive eliminates need for costly disposal
__________ used in ion exchange chromatorgrphy with harsh conditions (high temp, pH)
inorganic ion exchangers
gradient elution in ion exchange chromatorgrphy occurs by changing _______ or _______
ionic strength (of mobile phase) changing pH
components of mass spec
ionization source (accelerate high energy e-) electric field - accelerate ions vacuum pump - ion don't bump into oxygen/nitrogen in the air (no ion deflection) magnetic field that deflect ion in circular path
Quadrupole mass spec
ions enter quadrupole filter -contains 2 parallel metal rods (electric field between) in which constant and RF oscillating voltage applied ions oscillate back and forth -> only those with correct M/Z can pass through exit slit (stable trajectory)
Double focusing mass spec
ions pass through ELECTRIC SECTOR (parallel positive and negative) before passing through magnetic sector electric sector only permits a narrow range of ion kinetic energies (select for) to pass through decreases kinetic energy variation resolving power of 10^5
in Size exclusion chromatography ________ particles elute quicker
large
Superficially porous particle
layer of porous silica over solid silica core solute doesn't have to fully diffuse into particle, so particle can be larger pressure doesnt have to be so high but still achieve good separation and fast run time because analyte diffuse over shorter distance (smaller C term)
plate height from the theory of distillation means
length of column required for one equilibration
in reverse phase chromatography, ______ polar the solvent, the higher eluting strength
less
How is the UPLC more cost efficient
less mobile phase use less waste less analyst time
benefit and cost of operating HPLC at higher temperature
less pressure required higher flow rate and shorter run time (productivity) degradation of column
Choosing the liquid stationary phase
like dissolves like - since its partition chromatography
B term in the van Deemter equation
longitudinal diffusion - Peak gets wider as it longitudinally moves along column FAST FLOW RATE = smaller B term contribution to H
characteristics of packed GC columns
lower resolution (A term), broader peaks greater sample capacity
How to improve quality of spectrophotometry data (increase signal:noise ratio)
lower temperature (reduce white noise) signal averaging - Average measurements on top of each other (increasing number of reading improves quality) Beam chopping - using rotating mirror to alternatively send light through sample and reference cell use a low noise spectrophotometer
technique for studying masses of atoms/molecules and fragments of molesule
mass spec
__________ is the physical movement of solute form one phase to another that causes __________ of solute
mass transfer partitioning
corrected absorbance in calibration curves
measured absorbance - blank absorbance
define atomic absorption
measurement of the absorption of optical radiation by ATOMS in the gaseous state
Instrument DL or method DL higher
method DL
most common HPLC stationary phase support
microporous silica particles (Silicon and oxygen bonds, silanol groups) Porous ->>> higher surface area means quicker establishment of equilibria for adsorption
capillary electrophoresis
migration of ions under influence of electric field voltage separates components of solution inside fused silica capillary different solutes have different motility and migrate at different speeds
sum of atomic masses
molecular mass
example of ion exclusion
moleules with the same charge as resion will be excluded
Lasers are ______chromatic
mono
________ grooves in grating, the better the resolution
more
in normal phase chromatography, ______ polar the solvent, the higher eluting strength
more
Sample adsorb onto silanol group if interacts __________ than solvent
more strongly
Why FAAS has smaller linear range than ICP
more uniform heating of plasma and no self-absorption of flame
Why is visible region more relevant than uv for spectrophotometry
most samples absorb light in UV region (inconclusive and no fingerprint
Describe ICP-MS
multielemental analysis where analyte elements in Ar plasma (atomized) can be ionized by collisions with Ar+, exxited Ar atoms, or energetic electrons. The plasma is directed into mass spec which separates and measures ions based on their mass-charge ratio. ICP generates the atom, which can be ionize (as oppose to ion of molecule)
A term in the van Deemter equation
multiple flow paths - Broadening simply due to different paths molecules can take based on Eddy diffusion independent of flow rate minimize A contribution by making articles in column are more homogenous (smaller difference in sizes), negligible in open columns
smaller the plate height = ________ the band width
narrower
__________ GC column provide better separation but require __________ and have _____________
narrower higher pressure less sample capacity
cation exchanger contain bound ________ charged functional groups while anion exchangers contain bound ____________ charged functional groups
negatively positively
implication of ionic liquid
newest type of stationary phase for GC melt at RT (in liquid phase) and stable (low volatility) at high temperature
Oritrap mass spec
no magnetic or RF field 2 outer electrodes electrically isolated and central electrode potential differences cause ions to oscillate back and forth between 2 halves oscillating frequency prop. to inverse of square root of m/z
integer mass of most abundant isotope of each constituent atoms
nominal mass
____ phase chromatography is very sensitive to water because
normal/adsorption water is very polar -> solute interact more strongly to mobile phase and won't be adsorped on stationary phase
what is flux
number of moles crossing each square meter per second
What is isobaric interference
occurs in ICP - MS Ar forms different ions in plasma with elements in the sample being analyzed result in ions with similar mass-charge ratio as ions of interest
perferred for quantitative purposes and for sample that decompose above BP
on column injection
____________ columns are most frequently used as stationary phase in GC
open tubular (fused silica)
Spectral Interference
overlap of analyte signal with signals due to other elements/molecules in the sample or with signals due to the flame or furnace -memory effect
physical basis of chromatography: relative retention of 2 solutes proportional to the ratio of their __________________________
partition coefficients
what is stimulated emission
photon with energy state that spans two states can stimulate excited molecule to emit a photon. When the emitted photon falls, it stikes another exicted molecule wthat can be emitted with same phase and polarzation as indicent photon (one photon stimulates emission of many others if population inversion
ion pair chromatography used for
polar and ionic species that are not polar enough to be separated by adsorption chromatography but not non-polar enough due to charge better retain molecules based ion exchange (especially molecules that have similar structure)
A necessary condition of lasing is ___________ ____________. Define it
population inversion higher energy state has greater population than lower energy state in lasing medium (longer E2 wavelength than E1)
Two things GFAAS can do that FAAS cannot
preconcentration analyze solid samples
packed GC columns usualy used for
preparatory separation isolate large volume of volatile compound (require more stationary phase)
hydrophobic interaction chromatography principally used for
protein purification
Coupling Gc with MS
provide qualitative analysis - identify compound via m/z spectrum provide quantative analysis-measuring the relative intensities of the mass spectrum
application of ion exchange chromatography
purify water (Eliminate cation and anion using ion exchanger organic synthesis - pass through column, ion exchange occurs. Elutes the desired product
lower DL techniques for spec means greater ________ required
purity (more sensitive so more noise from contaminants)
In GFAAS, how to prevent elements forming carbides with graphite of tube
pyrolytically deposited carbon tubes ( seals porous graphite, smoother surface)
Beer's Law requires that linewidth of _________________ be ____________ than the linewidth of ________________________. Why
radiation source narrower atomic sample vapour measured absorbance wont be proportional to sample concentration [Radiation source becomes part of the measured absorbance]
what does the diffusion coefficient measure
rate at which molecules move randomly from a region of high concentration to a region of low concentration
relative retention/separation factor
ratio of adjusted retention time between two solutes • Fairly independent of flow rate -> used to identify peaks when flow rate changes
Non-oxidizing acids (HCl, HBr, HF, dilute: H2SO4, HNO3, HClO4) dissolve metals by ______ reaction
redox Metals with -VE reduction potential should dissolve (protonated)
when light absorbed by sample the irradiance of the beam of light is _________
reduced
light bending by prism is called ________. light bending by grating is called
refraction diffraction
How is ion exchange chromatography used in analysis of trace metals in the ocean
remove salt from sample (won't clog aperture for ICP MS) use resin with carboxylic acid at pH >6, resin quantitatively binds desired metals (while alkali and alkaline earth metals are weakly bound) selectivity for certain metals nitric acid can elute all metals removed salt as well as preconcentration
________ are used in ion exchange chromatorgrphy for small molecule (<500MW)
resin
How to use ion exchanger chromotagraphy to analyze trace metals in ocean
resin (CA) bind certain metals quantatively at >6 pH
_____ and ____ can be used to measure # of theoretical plates (to calculate efficiency)
retention time and width of a single solute
how to calculate resolution of peaks in chromatogram
retention time divided by average width of peaks: Peak width at half height (50% of intensity) Measure at bottom(4σ)
adjusted retention time
retention time of solute (Tr) - time takes for mobile phase to travel through column (Tm)
Someone used water as mobile phase and nothing showed up on detect ... Why is that?
reverse chromatography -> water too non-polar not strong enough elute analyte
ion exchange is a _______ and __________ process
reversible stoichiometric
Molecular processes when light absorbed by molecules: • Microwave - • Infrared - • UV/Vis - • X-ray -
rotation of molecules vibration of molecules electronic excitation bond breaking and ionization
Purpose of Alternating air pockets in GC injection
sample not immediately vapourized
Total Ion Chromatogram
scans for full range of compounds present
selective ion monitoring
select for particular m/z increased sensitivity -> much lower detection limit looking at particular analyte
how to selectively bind ligand to particular metal ions
select pH where K large for particular metal
How does extraction with metal chelator work
selectively complex metal ion with an organic ligand (chelator) and extract complex into organic solvent
the greater ___________ of a spectrophotometer detector, the greater the __________ produced by incident irradiance
sensitivity current/voltage
Size exclusion chromatography
separate particles based on size POROUS GEL = stationary phase Inject different size Small molecule diffuse into the pores then out and elute therefore large molecules elute quicker
why does capillary electrophoresis have muhc higher resolution compared to LC
separation driven entirely by current.... no particle which means no A or C term
suppressed-ion chromatography
separation of ions use strong acid (HCl) or strong base (KOH) to elute however, use thermal conductivity detector -> BUT eluent has very high conductivity (higher than ions, therefore, only see one line) use suppressor column after separator to pump in OH- to neutralize H+ or H+ to neutralize OH- (water has low conductivity) analyze pond water
How is chromatography similar/different from extraction
separation technique utilized interaction of solute with different phases chromatography - one phase is held in place while other moves past it
define sample prep
series of steps that convert laboratory sample to a form suitable for analysis and removing substances that interfere with analysis
heated quartz cell (hydride technique)
similar setup as FAAS HCL flame optical system but has a hydride generation system and no nebulizer hydride generation system - aspirate liquid sample -Mix liquid sample with sodium borohydride and HCl -Create a volatile hydride of the analyte -Flow that gaseous hydride into the optical cell
__________ chromatography is a very gentle method. Why
size exclusion no direct attractive interaction (just diffusion)
macromolecules diffuse _______ slower than small moleucles examples
slower. sucrose< glycine < RNase < serum albumin
What is dark current (spectrophotometry) _______ temperature = greater dark current
small response in absence of light (spontaneous emission of electrons from cathode of PMT)
_________ the particle size in HPLC columns means better separation but require _________ pressure
smaller higher
HPLC is restricted to analytes that are ________
soluble
What is matrix effect
something in the matrix affecting response to analyte
why must mass spec operate under vacuum
species in air doesn't cause deflection of ions
What is a Photomultiplier tube (PMT)
spectrophotometer detector that captures light, produce electrons, amplified electrons, and creates a electric signal Can measure extremely low intensities since every photon produces 10^6 electrons greater analyte concentration = greater light intensity
Gradient elution is used for
speeding up late eluting peaks eluting peaks that need strength solvents
quantitative analysis can be inaccurate when using split injection because
split ratio not reproducible from run to run
HPLC column uses _____ column to ___________ that can be easily clogged with dust (from analyte or mobile phase), so __________ or _________ used
stainless steel withstand high pressure mobile phase filtered use guard column that an be replaced
_________ ______________ can be used to componensate for different types of interference
standard addition
column aging can cause ________ loss
stationary phase loss
how to reduce tailing
stationary phase usually bonded (covalently attached) to the silica surface and covalently cross-linked to itself. thinker stationary phase silanization - block silanol groups with non-polar groups
consequence of stationary phase loss
tailing peaks (exposed silanol groups on stationary phase bind polar solutes strongly) bleeding especially at high temp
What is acid digestion done in
teflon-line bomb
What is the issue of Longitudinal platform in GFAAS
temperature gradient (hotter at center compared to ends) Atomic cloud condense at cooler end of tube results in: • less analyte at measuring stage • Condensed atoms at end vaporize during next sample run -> interference at the next run (memory effect)
atomic emission vs atomic fluorescence
the emission of wavelength from excited atoms when they return to the ground state that is identical to the wavelength that was absorbed by atoms in the ground state In atomic fluorescence the energetic level from which the excited atom returns to the ground state is lower and therefore the wavelength is different than the atomic emission.
natural abundance of isotopes allows one to calculate for the _______ of M+1 peak
theoretical intensity (as a percent relative to M+. )
universal GC/HPLC detector
thermal conductivity
define extraction
trasfer solute from one phase to another to isolate or concentration desired analyte(s)
ways to do qualitative analysis using GC
use MS as a detector to look at M/Z ratio and compare with spectral library spiking -> authentic compound (predicted) added to unknown sample and analyze chromatogram • if right compound suspected, peak area should increase **identification firmer with several column/stationary phase
What is done to increase equilibrating rate of stationary and mobile phase in GC and HPLC
use narrower OT columns use packed columns
solid phase micro-extraction
use thin fiber with film of stationary phase that is exposed to sample to extract analyte
what is temperature programing
used in GC packed GC columns
ion pair chromatography
uses reverse phase HPLC column instead of ion exchanger coloumn anionic surfactant (hexanesulfonate) added to mobile phase that lodges in the hydrophobic stationary phase, transforming the stationary phase into an ion exchanger when analyte passes through, retention mechanism is a mixture of reversed-phase (neutral part) and ion-exchange interactions (cation with the anionic surfactant
time of flight mass spec
using time require for ions to travel from source to detector as a way of separating ions of different M/Z reflectron ensure kinetic energy the same If ions have the same kinetic energy, but different masses, lighter ions travel faster than heavier ions. In its simplest incarnation, the time-of-flight mass spectrometer is just a long (|1 m), straight, evacuated tube with the source at one end and the detector at the other end. Ions expelled from the source drift to the detector in order of increasing mass, because the lighter ones travel faster.
spectrophotometer noise attributed to
variation in light intensity sources independ of light source sources proprotional to photo generated current
limitations of FAAS (2)
very small mass transfer (5%) requires large amount of sample (min 1-2mL) flame emits and absorbs light
When molecule absorbs light which has sufficient energy to cause electronic transition (UV-Vis), ___________ and __________ transitions also occur
vibration rotational
Types of cuvettes appropriate for different spectroscopy
vis light - Glass cuvets UV light - Quartz cuvets (glass absorbs UV light) IR light - mix sample with 1% KBr, ground, pressed (600 bar) into translucent pellet
GC only works for analytes that are _________
volatile (can be evaportaed intact at higher temperature)
two ways of quantifying speed of mobile phase passing through chromatography column
volume flow rate (volume of solvent/unit time) linear flow rate (distance solvent travel/unit time)
Why such a difference in measurement of magnesium in blood serum
washed/unwashed polyethylene bottles
benefit of Double beam scanning spectrophotometer
• Automatically correct for drift (of light source intensity and detector response) • Make several measurements per second
Basic requirements of spectrophotometer:
• Continuous light source • Monochromator • Cell containing sample (path length "d" which light passes through) • Light detector measures irradiance of emergent light
drawback of Gradient elution
• End of run -> column equilibrate with different mobile phase at beginning • New run -> must equilibrate back to starting mobile phase
Why is Fluorescence easier to measure compared to absorption:
• Fluorescence is more sensitive than absorption because we can observe a weak fluorescence signal above a dark background. • In absorption, we are looking for small differences between large amounts of light reaching the detector.
advantanges of ICP
• High temperature (2x hot as combustion flame) -higher excited-state population -more complete ionization • stable, inert Argon environment decrease interference from flame (no oxygen, no interference from oxide formation) Simultaneous multi-element analysis (ICP-AES) stable plasma [emission spec relied on stable flame -> emission intensity depend on temperature of flame]
open tubular column in GC offer:
• Higher resolution - no particles in there (eliminate A term) • Short analysis time - higher flow rate (low resistance) • Greater sensitivity - higher resolution (A term) so detect less analyte • Lower sample capacity ( inject less because of small diameter)
How are CID and CCD different
• In a CCD detector, each pixel must be read one at a time in row-by-row order • Each pixel of a CID detector can be read individually at any time.
Beer's Law (a ∝ C) only works well with
• Monochromatic radiation • Dilute solutions (<0.01) - solute molecules influence one another • Absorbing species not participating in concentration-dependent equilibrium
why is GFAAS superior to FAAS
• Much less sample (5-500uL) needed than FAAS (1-2mL) • Lower DL due to longer residence time atomic cloud confined by graphite tube, while sample is flushed away in flame
how does solid phase micro-extraction work
• Needle of fused silica fiber with stationary liquid-phase coating (C18-silica) • Insert into sample, stir with magnetic stir bar • Analyte enrich on needle tip (chemical equilibrium reached) • Pre-concentrated analyte on tip • Retract needle out of vessel, inject into GC-MS system High temperature condition - analyte desorb
Why is purging with Argon in GFAAS necessary
• Prevent oxidation of graphite during ashing • remove any volatile material
Why supercritical fluid useful as HPLC mobile phase
• Reduces waste of organic solvents (greener) • Low viscosity of supercritical permits faster flow (high diffusion of solutes in SC fliud) • SC fluid can dissolve non-volatile solutes
How does ICP-AES perform multi-elemental analysis
• a collimating mirror makes light rays parallel, light disperse by grating Dispersed radiation lands on a charge injection device (CID) detector
difference between Atomic Absorption, emission, fluorescence spectroscopy
• measure light transmitted: HCL with Fe (example) cathode bombarded with energetic Ne+/Ar+ ions -> excite Fe -> vaporize -> emit light with same frequency absorbed by Fe analyte in flame -> compare intensity of light due to absorption • NO LAMP REQUIRED: collisions in hot plasma promote atoms in sample to excited electronic states from whcih they emit photons to return to lower energy states MUCH MORE SENSTITIVE THAN ABSORPTION Atoms in flame irradiated by a laser to promote to higher electronic state -> fluoresce to return to ground state
types of noise in electrical instrument
• random fluctuation of electrons in a device • drift of light source/flame (spectrophotometer/atomic spectroscopy) due to slow changes of components with temperature
how to monitor column performance
• retention factor of a standard • the number of plates peak asymmetry
each "line" in the SRM is take from at least ____ measurements
2
minimum detectable concentration
3s/m 3 standard deviations (from measurements of n replicate samples) divided by slope of linear calibration curve
analyte concentrations in sample: MAJOR, MINOR, TRACE
>1.0%, 0.1%-1.0%, <0.1%
Horwitz Trumpet
Coefficient of variation of mean values between different labs increase as analyte concentration decrease as concentrations of analyte decrease, the interlab precision become poorer Horwitz Trumpet = inevitable
types of precision (4)
Instrument/injection - reproducibility when same quantity of one sample is repeatedly introduced (>10X) into and instrument. Includes injected quantity and variation of instrument response Intra-assay - same person performing analysis of different aliquots of homogeneous material on same day with same equipment intermediate - assay performed by different people in same lab on different instruments on different days Interlab - assay performed by different people in different labs
Wet ashing
Microwave digestion with acid in Teflon bomb H2SO4 and HNO3 added to an organic substance in a glass digestion tube fi tted with the reflux cap cycles of oxidation and dissolution
What does it mean to construct a calibration curve
Response of procedure evaluated for known quantities of each analyte (standardize) so the response to an unknown quantity of each analyte can be interpreted
What is the SRM
Standard Reference Material are samples sold by national measurement institutes containing known quantities of analytes) to test accuracy of analytical procedures (quality of material certified with painstaking care using at least two different methods)
Define quality Assurance
Steps taken to verify accuracy and precision of analytical results what we do to get right answer for purpose
What does 3s/m means in practical terms
When signal 3 times the noise, the signal is measurable, but too small for accurate measurement
false negative vs false positive
YES or NO to whatever is being measured drinking water below limit: (+) exceed limit (-) below limit
define robustness
ability of analytical method to be unaffected by small, deliberate changes in operating parameters (injection volume, detector wavelength, room temp, solvent content percent, pH etc)
method specificity
ability of analytical method to distinguish analyte from everything else in the sample ie. must achieve baseline separation on chromatogram (cefotaxime with impurities)
what is standard addition
adding known quantities of analyte to unknown sample increase in signal allows us to deduce quantity of original unknown done by plotting detector response by concentration of analyte added and extrapolating to obtain x-intercept
important to maintain integrity of sample and analyte concentration during sample transport and storage by avoiding
contamination analyte loss analyte degradation
how to avoid volatilization
put lid on containers use ice chest to maintain temperature <10ºC
Define specifications
(part of QA process) statement of how good the numbers need to be and precautions required for analytical procedure
Define use objectives
(part of QA process) states purpose for which results will be used -> affects kind of data collected, way collected, how data used.
method of least squares
(used in context of calibration curve) fit best line through data points - linearize data By minimizing squares of deviations (Δy) Do some fancy math to obtain m and b, plug into equation to get best fit line
process of fusion
1. Finely powdered unknown is mixed with 2-20x its mass of solid flux (Na2CO3, K2S2O7, Na2O2) 2. fusion (melting) is carried out in a platinum-gold alloy crucible at 300-1200ºC in a furnace or over a burner. 3. Molten flux is poured into beakers containing 10 wt% aqueous HNO3 to dissolve the product.
measurements of accuracy (nearness to truth)
1. analyze appropriate SRM (that contains certifies concentrations of analytes in realistic material/ same matrix 2. compare results from 2 different analytical methods 3. SPIKING. Analyze BLANK sample with known addition of analyte (with same matrix) -> 3 "levels" of 3 replicates 4. Standard addition of analyte to UNKNOWN (cannot prepare blank with same matrix as unknown) SPIKING = most common since SRM and and 2nd analytical method unavailable.
method of least squares assumptions
1. same standard deviations for all data (standard made using the same method) 2. error in y values > error in x values (concentration error should be less than measured response values)
test of linearity
1. square of correlation (R^2 value) 2. y-intercept of the calibration curve should be close to 0 (<2% of response for target value)
lower limit of quantification (LOQ)
10s/m Smallest amount that can be measured with reasonable accuracy
What is Assessment in the QA process
Documentation and records of procedure compare data with specification •Verify final results meet use objectives
What is recovery of fortification/spike recovery
Ensure that response to analyte not affected by something in the matrix (matrix effect). Also ensure correct qualitative identification of analyte (selectivity) known amount of analyte added to sample to test if response expected from calibration curve adding spike of 5uL to 10uL reading should give 15uL (if not, something in matrix is interfering with analysis) -> Only small volume of concentrated spike added to avoid significant volume change of sample/matrix
What acid is used for silicates (has toxins, can't use glass)
HF
Components of the Quality Assurance process
Use Objectives Specifications Assessment
In dissolving with acid, _________ species formed by ______ _______________ will be lost from hot acids is open vessels (also metal halides)
Volatile anion protonation
derivatization
analyte chemically modified to make easier to separate or detect aldehyde/ketone + 2,4 DNP = derivative that can be detected using UV at specific wavelength
what is lower measurement than true value attributed to
analyte loss: -adsorption of metal on glass - volatilization
Analyte
component to be measured
reporting limit
concentration below which regulations say that given analyte is reported as "not detected" at least 5-10X DL
define range
concentration interval over which linearity, accuracy and precision are all acceptable
how to make sure measurement falls within linear range
calibration curve cover larger range dilute sample
sensitivity
capability of responding reliably and measurable to changes in analyte concentration (must have lower detection limit of method than concentration measured
Condition of prepared standard in standard addition
concentration standards to only small volumes added and sample matrix is not appreciably altered
Absorbance reading from blank from:
colour of reagents impurities in reagents interfering species
Types of sample prep (5)
dissolving the sample extracting analyte from a complex matrix concentrating dilute analyte to a level that can be measured chemically converting analyte into a detectable form removing or masking interfering species.
What are Quality control samples
eliminate bias from analyst performing calibration check (who know the concentration of check samples) samples of known conc. issued to analyst as unknown (by manager, agency)
3 important purposes of liquid extraction problem of liquid extraction
extract analyte remove matrix concentration analyte emulsion formation
describe stages in sampling/sample prep lot representative bulk sample homogeneous sample aliquot
homogeneous sample-> by grinding and mixing thoroughly aliquot -> small potions used for analysis
linearity
how straight is the calibration curve -> indicates how proportional is the response to the quantity of analyte (implications = how accurately quantify using the line)
define precision
how well replicates agree with one another (small 2 value)
acid and fusion is a dissolving method used for __________ ___________
inorganic materials
purpose of dissolving samples for analysis
lab sample has the same composition as the bulk sample ***entire lab sample must be dissolved***
What does high quality acid mean
least amount of residue (minimize metal contamination)
Quality of mortar and pestles
less expensive = more porous and easily scratched (contamination) shouldn't be made of same material as sample (lose some fractions of mortar and pestle) agate < boron carbide
dynamic range
measurable response but not linear relationship between response and known quantity
What was discovered from the International Measurement Evaluation Program (Belgium)
no guarantee results were reliable from "accredited" laboratories (very few were measured within bounds) national institute measurements = much more accurate
the standard deviation a measure of ____ in a blank or small signal
noise or random variation
Types of blanks
o Method blank - contain all components except analyte taken through all steps of analytical procedure o Reagent blank - not subjected to all sample prep (less complete) o Field blank - similar to method blank, but expose to site of sampling (analyte picked up by field conditions) -> more complete
Preparing calibration curve
o Prepare standard solutions (at least 6) covering range of concentrations expected for unknowns and prepare blank samples o Subtract the average blank measurement from each standard measurement o Make graph of correct measurement v.s known standard quantity o Use method of least squares to plot best straight line - find the slope, intercept and uncertainties
Al forms protective oxide coat on inorganic materials that _______ ____________ .
prevents dissolution
define method validation
process for proving analytical method acceptable for purpose
define sampling
process of selecting a sufficient, representative bulk sample from a lot (total material from which sample taken)
What assumption is internal standard method based on
relative response of detector to analyte and standard constant over range of conditions
Instrument DL vs method DL
replicate measurements (≥7) aliquots from one sample preparing replicate samples (≥7) and analyzing each one
Linear range of calibration curve
response proportional to known quantity
What type of samples is standard addition used for and why
sample composition is unknown and complex (no way to construct a calibration curve) and affects analytical signal (strong matrix effect) all samples in same matrix in standard addition
Chain of custody
signed as sample pass off and check sample is in right condition
define detection limit
smallest quantity of analyte that is significantly different from the blank
how to verify linear response of analyte to standard
spike solutions of analyte with known standard amounts see if detector response to each component similar to not (RF value)
internal standard vs standard addition
standard SAME substance as analyte standard DIFFERENT substance from analyte
coefficient of variation/ relative s
standard deviation divided by the mean X 100%
Supercritical fluid extraction
uses a supercritical fluid (often CO2)as the extraction solvent
solid extraction
uses small volume of chromatography stationary phase (C18 silica) to isolate analytes from a sample and remove much of the sample matrix Nickle IMAC analyte through -> weak solvent to elute weakly bound solute -> stronger solvent to elute desired analyte important for "sample clean up" in analysis of serum protein
solid-supported liquid-liquid extraction
way to reduce solvent use in liquid-liquid extraction no emulsions formed aqueous sample (urine, plasma) is suspended in a microporous medium (diatomaceous) through which organic solvent is passed to extract analytes
Dispersive L-L-extraction
way to reduce solvent use in liquid-liquid extraction small volume of extraction solvent + larger volume of disperser solvent (miscible to both phases, lowers energy)-> create emulsion ->mass transfer of two phases due to large interfacial SA
Standard Operating Procedure (SOP):
what steps will be taken and how will be carried out - must be rigorously followed to prevent false results due to "shortcuts" Reagent gone bad Maintenance of instruments Fridge temperature
For what type of analysis is internal standard method used
when instrument response varies slightly from run to run (chromatography) - flow rate different -quantity of sample different (small injection volume for chromatography) - sample loss can occur during prep (same fraction of standard/unknown lost)
signal detection limit
y_blank + 3s (mean value of blank measured n times, plus the standard deviation from measurements of n replicate samples)
What does it mean to have a procedure that produces detection limit of ~99% chance of being greater than blank
~1% of false positive (sample containing no analyte giving signal greater than detection limit)
components of method validation
• Method ofspecificity • Linearity • Accuracy • Precision • Range • Limit of detection • Limit of quantification • Robustness
dry ashing
• Microwaved induce combustion (analysis of halogens in coal, metals in plant matter) 500mg coal in ash paper, in vessel with 6M NH4NO3 and solution at bottom, vessels capped and pressurized (20 bar), microwave initiated combustion -> high temp -> halides released and dissolved in solution -> measured by ion chromatography
Criteria for acceptable range (in method validation)
• correlation coefficient > 0.995% • spike recovery 100 ± 2% • inter-laboratory precision = ± 3%
stages/types of sample dissolving
• drying solid at 110ºC to remove moisture • mild conditions (grinding) • acid digestion or fusion if mild does not work combustion/dry ashing or oxidation with liquid reagents (wet ashing) for organic material
Raw data treated data results
•Individual measurements (Readings from buret, absorbance) •conc derived from apply calibration procedure to raw data •reported after apply stats - with STD, mean, confidence interval
