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