FLAME PHOTOMETRY

Flame convert el. to be measured atomic vapor reversible transformation bet ground & excited electronic energy states optical signal (measured)

Atomic forms in flame: (atoms)

1) 2)

Ground state el state;e- havent absorbed heat from flame

A. Atomic Emission Flame Emission Photometry / Spectro - Prin: % of atoms temporary excited state by flames heat energy
released light (specific for atomic specie) emitted light focused by lens/mirror monochromator (transmit radiant energy from atoms) photodetector (generates electrical signal proportional to conc. of atoms in flame)

Excited e- absorbed heat energy higher electronic energy level (Excited State) 3) Ionized atoms lose e- (high flame temp) Components: 1) Fuel source (gases used): - Diff. is in temp produced & sensitivity it provides

Plot of standard readings against conc. calibration curve from w/c unknown can be read (direct or w/ IS) - Interference: (source of error) 1. Lipid 2. proteins

B.

Atomic Absorption AA SPECTROSCOPY (AAS) measure conc. of an el. : detecting absorption of EMR by atoms Prin: hollow cathode lamp (use el as cathode) produce

- Purpose of flame in FES: 1. Breaks chemical bonds produce atoms

2. Energy source atoms to be able to enter excited state - Ways by w/c energy is related to envi: 1. Collision w/ other atoms 2. Heat loss 3. Transfer to other atoms 4. Emission of radiant energy - Light emission by atom as it returns to ground state

a.

Mixture of He & O2 produce hot temp, commonly used b. Natural gas (combine w/ air or O2) c. Acetylene d. Propane Components: 1) Atomizer / Burner Pre-Mix Design Burner

monochromatic light (for el) beamed thru long flame into w/c is aspirated soln. to be analyzed heat energy dissociates mol & converts components atoms GS atoms of same el (as in cathode cup) absorb own resonance lines amt of light absorbed reaches monochr. (passes only WL close to resonance lines of el) transmitted light detector dec. in transmitted light measured Sample analyzed must contain metal in atomic state Most atoms (99.998%) remain in GState at flame temp Free atoms at GS can absorb radiation at narrow band path width (0.001 0.01 nm) Components: 1. Hollow cathode lamp light source

- Break

up soln into droplets evaporate solvent

Na & Ks (easily excitable alkali metals) valence e- (unstable in

atoms w/c absorb heat energy from flame excited 3. Monochromator interference filter Select & isolate WL from interfering light before it goes to 4

excited) changes from high electronic low energy elec. State give up excess energy to envi. light (can have diff. WL & energy levels) K violet light (767 nm) Na yellow light (589 nm) Red light (761 nm)

Continuous Emission light of various emission given off from burned non-ionic mat Interference Filter preferred made to pass very narrow WL range yet still permit max amt of line emission to pass thru 4 4. Detector: Photomultiplier Generates electrical signal L conc of atoms in flame 3 (1 for each el.) simultaneous readouts of Na & K conc

- Has gas-tight chamber w/ anode & cylindrical cathode (hollow,

constructed from metal to be determined) - Separate for each el

- Alloy

- Measurement of light emitted by excited atom (at specific WL) diff.

for each el. WL for measurement depends on selection of spectral line of strong enough intensity to provide sensitivity Constant & controlled conditions: light intensity of WL L no. of atoms emitting energy L conc. of subs. of interest - Essential parts of FP: 1. Atomizer sys create fine spray of salt-containing soln. & feed spray into burner If using internal standard method gravitational effect reduced to negligible factor (fast feeding atomizer not used) some vapor directly fed to burner base w/o passing chamber

used to make Multi-El Cathode several metals incorporated to same extent

5. Readout sys converts elec. signal sent by 4 usable form

emission intensity of unknown . emission intensity of internal standard Na: Li or K: Li or Li: Ca ratio compared to standard solns. 6. Internal standard sys achieve stability & compensate for changes in photometer signal due to problems in instrument Major problems causing changes in photometer signal: 1) Fluctuating flame temp 2) Variations in photometer uptake 3) Nebulizer performance 4) Atomization efficiency 5) Flame parameter Char. Of Good IS: El used as IS must produce light of diff. WL from other analytes (Na & K) 2) Amt of energy to excite IS must be close to amt needed to excite el measured 3) Emission lines far apart to be separated by monochrom. 4) IS not normally found in soln. being analyzed 7. Standards High, Medium, Low Run completely, immediately after all unknowns are done Rerun completely significant drift Conc. =

- Filled w/ inert gas (Ar, Neon, He) at low pressure filter gas Ne-filled reddish orange glow Ar-filled blue-purple glow - Lamps illuminated (voltage is applied) filter gas atoms are
ionized strike cathode w/ high velocity metal atoms sputtered from inner surface of cath cath filled w/ atomic vapor atoms in vapor electronic excitation by collision w/ ions & e- excited metal atoms emit radiation as it loses energy (return to GS)

- Light

emitted: specific WL/resonance line for metal in cath

filter installed before air regulator remove dirt, oil & moisture (detrimental to smooth performance)

(Ca-emission line: 422.7 nm most used for Ca analysis)

a.

2. Mechanical

Upfeed - Air blown across top of capillary tube

(bottoms immersed in soln.) vacuum (fluids sucked up into tubing) dispersed into fine spray in air stream - Small amt. of soln - Not subject to hydrostatic pressure variations (as in gravity feed atomizers) b. Gravity feed funnel w/c tapers to fine capillary - Air blown across tip of capillary disperses soln. into spray

1)

- D: level of soln. descends atomization rate changes 2. Burner sys where nebulization & atomization of soln happens

rotating chopper light detector (distinguish bet. light beam by cath lamp & by excited atoms in flame) - Bet light & flame - 2 light signals from flame: a. Alternating signal from cath. lamp b. Direct signal from FE 3. Burner parts: a. Nebulizer sample soln. aspirated or reduced to fine spray b. Premix chamber where mists mixed w/ oxidant & fuel (Acetylene)

- Where large droplets are trapped & drained off thru baffles
only fine droplets reach flame

c.

Burner Head long, narrow slit w/c produce

elongated flame inc. sensitivity

- Gases flow thru 10cm-long slit on this produce thin flame

curtain where light from cath lamp passes thru greatly enhancing absorption of light by GS atoms in flame 4. Monochromator Prism / Defraction Grating w/ narrow BP - Isolate desired lines by blocking light of WL diff from desired resonance from reaching detector

V. sensistive, used to quantitate porphyrins, catecholamines, Vit, steroids, pharmaceuticals Separates essential light signal from extraneous light signal Common problems in FPhotometry & Remedies:

6.

Run STD & control sera frequently check on various aspects of sys of instrument

1.

Location if near source of variable light (window): varying

intensity of stray light striking photocells cause reading to drift - Drafts (windows, fans, AC units) cause erratic performance

ELECTROPHORESIS (EP) used to separate mixture of charged solutes by migration in an electrical field

- Excessive 2.

- Protect detector from excessive light from FE

- Filters out extraneous light 5. Detector: Photomultiplier

activity in vicinity instability in readings (esp. when theres no closed burner sys)

- Cations (+) cathode (-) electrode - Anions (-) anode (+) electrode
Zone EP migration of macromolecules (serum protein) - Migration in porous medium

Clogged atomizer sensitivity loss & gross changes in Std reading

- Converts

light from CL (not absorbed in flame) electric

current amplified to read-out device - Amplifier: recognize only pulsed signal from source lamp & rejects steady light from flame Interferences: A. Chemical

- Poke particles / air bubbles out by pipet probe - Atomizer funnel, when not in use covered w/ paper cup (prevent
dirt from falling to it)

Iontophoresis migration of small ions

3.

Chipped atomizer atomization related / stopped /no sensitivity Dirty chamber clean w/ flexible brush or by soaking in strong acid

- some

anions form refractory cmpd w/ Ca incompletely dissociated in

4. 5.

flame absorption cant occur depression of photometric signals

Electrophoretogram result of Zone EP - Consists of sharply separated zones of macromolecules Ampholytes MW 300-600 - Capable of expressing net (+) charge, net (-) charge or net charge of 0 (small polyanions & polycations) > Immobile zwitterions Isoelectric pt (PI) AA exhibits net charge 0 at diff. pH Electroendosmosis / Endosmosis relating to induced charges in medium

- CaPO4 complexes: PO4 interference in Ca determination

- Methods to control: 1. Use special type high-temp burner dissociate refractory cmpds. (Nitrous oxide-acetylene flame) 2. Add cation to displace Ca from PO4

Dirty glass chimney sensitivity loss wash chimney periodically & windows leading to instrum. should be kept clean 6. Improper flame adjustment burner fitted w/ orifice (accdg. to gas)

- Movement of buffer ions & solvents relative to the fixed support

charged buffer affect migration of ions

- city

gas used gas regulator for maintenance of steady flame (demands on gas lines vary t/o day) may drift & w/ bottled gases erratic flame, strong odor

Lanthanum: preferred forms tighter complex w/

PO4 Lanthanum PO4 ppt 1st as aerosol evaporates leave Ca to form more easily dissociated cmpd free neutral Ca atoms can absorb Ca-light from cath lamp 3. Use Chelating agents prevent rxns.w/c lead to formation of refractory cmpds - EDTA, Citric A., Glycerol relieve PO4 interference on Ca B. Ionization

- flame not adjusted & controlled erratic performance readings 7. 8. 9.

Dirt in gas or Air Lines yellow flecks of light in flame when no Dirty burner red/orange glow in flame unstable Static electricity some instruments grounded soln. is atomized remedy: Filter in lines performance burner grid & orifice should be washed occasionally - Erratic movements of galvanometer correlate w/ touch of instrum by operator - R: connect chassis of instrum to cold water pipe w/ wire

Wick flow flowing of buffer into membrane to replace H2O lost thru evaporation; Support medium in buffer Densitometry measure of density of light passing thru fractions Clearing membrane is made transparent to visible light

- atoms in flame excited (instead of being dissociated) emit energy of

WL as that being measured

- strongly temp-dependent (minimized w/ low-temp flames)

- Mechanisms to control:

1.

10.
Add excess of easily ionized subs w/c absorbs most

of flame energy subs of interest wont be excited 2. Operate flame at low temp C. Matrix

Electrical / Electronic marked dec. in sensitivity weak photocell - Check line voltage w/ voltmeter or install stabilizer

- Spare tubes (have shelf life) kept on hand change faulty ones
w/ minimum loss of operating time 11. Contamination glassware, stopper, H2O & std soln guard against Na or K contamination Precautions before prep of Sample & STDs: 1. Glasswares clean & rinsed well w/ deionized H2O - Polyethylene bottles / Borosilicate glass storage of soln

Components of EP:

- i.e.

enhancement of light absorption by organic solvents (instead of

1. Support medium holds sample; provide path for migration of protein - i.e. paper, cellulose acetate, agarose-gel film

aqueous) atom absorbs 2-5 times more energy

- light absorption C.

- some 2.

- formation of solids from sample droplets as solvent is evaporated in flame Atomic Fluorescence Photofluorometry Photometric measurement of fluorescence Fluorescent absorbs violet light & emits light to longer visible WL Similar principles w/ colorimetry (diff: emitted light is measured at right angles to incident light) Use as analytic tool for cmpds that can be made to fluoresce

need hydration before sample is put (pre-treated) after hydration: both ends should make contact w/ buffer for passage of electric current Buffer soln. w/ specific ionic strength & molality

2.

Use disposable plastic cups for sample handling contamination 3. Flush & clean periodically aspiration line & burner 4. Pipetting of sample & diluents accurate & reproducible 5. Maintain proper pressures of gas & air

- Keep system hydrated continuous flow of current 3. Electrophoretic chamber where support medium is attached - Has partitions (+) & (-) electrodes

- Wiring

chamber allow passage of electric current into 1 buffer

chamber support medium 2nd buffer chamber

4. 5.

Driving force / Electric power / power supply converting

Neutral (agarose, polyacrylamide) no endosmosis

5. Electrophoretic temp result from poor electrical supply

alternating light current into direct current (AC DC) - w/ regulator stable electric field throughout EP / analysis Sample common: serum proteins - Urine, CSF pre-concentrated (by centrifugation or filtration) only small amt. of protein

- Resistance produce heat evaporation of buffer (H2O) inc.

ionic strength (more concentrated) ionic cloud migration

Relationship of 1st 3 factors to the rate of mobility * (migration) = Q . k(constant) x r x n TP = 7.5 gm%

Distance migrated % Total Protein Albumin 44 4.4cm 48.9 1 14 1.4cm 15.6 2 10 1 cm 11.1 7 0.7cm 7.8 15 1.5cm 16.67 9 cm 100 Electrophoretic support media & techniques A. Frontal EP Saw Lines Area Patients value (from Goldberg refractometer / Total Solids meter) 3.67 1.17 0.83 0.58 1.25 7.5

- Application: at cathode end (pH of buffer is basic (-) particles) 6.

Detecting system densitometer - not part of EP - For quantitating diff. protein fractions

- Globulin protein fractions 1, 2, ,

Factors that determine rate of migration:

General procedure: A. EP 1. Hydration of support medium (paper & cellulose-acetate)

- Pre-heated: agarose & polyacrylamide

2. Application of sample

1.

Net charge of particle (Q) any charged particle will migrate - Buffer for EP = pH 8.6 - AA: side chains w/ electric charge at certain pH - 5 serum proteins have pI a. Albumin = pH 4.6 b. 1 glob = pH 5 c. = 6.5

3. 4.

Turn on power supply separation of particles Complete separation support medium put in a fixative

d. = 7
e. = 7.6

- pH is pI (-) - pH is pI (+)
2. Size & shape of particle (r) - Ionic radius of particle - Molecular shape (globular, fibrous) determine flow - Smaller faster migration 3. Strength of electric field / viscosity of buffer (n)

- Ionic strength / conc.- determine migration distance - ionic cloud faster migration distance
- Protein fraction at diff. levels accdg. to migration distance

4.

Chemical & physical properties of support medium

B. Identification 1. Protein fractions stained w/ mat. w/c produces visible bands of color on support medium Traditional dyes for non-specific proteins (stains all proteins except low-level proteins): - Coomassie blue - Amido black - Ponceau S Excellent for low level proteins: Silver staining 2. Other techniques for ID: a. Enzyme reaxn stain enzyme of interest b. Immunofixation stain immunoglobulin c. Quantitation a) Elution time consuming 1) Cut EP strip 2) Elute each fraction into soln (0.1M NaOH) put in test tube (stain on strip transfers to NaOH) 3) Take A reading of each fraction 4) Sum up total A readings 5) Compute for % of each protein fraction

1.

Moving Boundary Technique developed by Arne Tiselius (1930)

- Sole type; No support medium; Very delicate

- Use U shaped tube - Needs complex optical system (Scheileren sys) - Expensive, complicated, time consuming B. Zone EP 1. Paper EP in 1950 - Support medium: filter paper strip (earliest SM) A: fragile, no uniform pore size 2. Starch gel EP SM: starch gel A: greater pore uniformity D: fragile, larger sample required, unable to store result permanently, difficult preparation of gel 3. Thin layer EP procedure diff. from EP - SM: Kieselgur / Silica gel - Buffer: volatile solvent (Butanol & Acetone) 4. Cellulose Acetate EP in 1957; Standard Method - SM: cellulose acetate

- Maybe passive w/ little or no physical interaction w/ proteins 1)

b)

Densitometry method of choice; based on colorimetry

- commercial apparatus marketed in early 1960s

A: uniform pore size, faster separation, less sample, stronger, can be cleared D: major extremely brittle when dried w/ enzyme clearing soln remove developed colored band

Stain band strip is placed in holder & slowly moved thru beam of light 2) As light interacts w/ dye bound to specific fraction, an amt of light is absorbed 3) Readout from sys. - trace w/c relates amt of light absorption to location of protein fractions

5.

Agarose Gel EP AG derivative of agar

- Major factor of separation: electric charge

4)

Data further analyzed electronically provide % distribution of each fraction Light source filter EP strip detector recorder Principle of Densitometry:

- Sample requirement: serum: < 1 L

urine & CSF: larger - Buffer: dilute barbital - Stain: Protein: amido black CSF: Coomassie blue A: electrically neutral 6. High resolution Protein EP - SM: Agarose gel - Modifications: w/ cooling system buffer w/ Ca lactate at pH 8.6

- Function (other than support) : 1. Affect migration 2. Play impt. role in separation process (sieve/sifter separation based on size) 3. Affect charge of system separation based on net charge

(-) charge of buffer (paper, cellulose-acetate) endosmosis

- Permit detection of 12-15 diff. protein fractions

- Accepted approach to routine fractionation in clin. lab

3.

Heat & Voltage Effects more voltage = faster migration, but current

7.

Polyacrylamide Gel EP gel composed of acrylamide polymer

- Major factor of separation: molecular size

- Better resoln. than 5, but difficult prep. of gel

- Used for separation of lipoproteins, Hb & some isoenzymes

- current temp: Proteins denatured (labile, if 50C or higher) Mobilities of fractions enhanced each band in a
unexpected location (than at lower temp)

diff. &

8.

Isoelectric focusing diff. approach to EP

- Special molecules incorporated into sys stabilize pH at given pt. in separating medium

- Concentrate on pI of particles, make use of carrier ampholytes detect

proteins

Solvent evaporation n distortion & tailing of bands - Cooling device regulate temp reproducibility & optimal patterns devt
enhanced 4. Staining & Scanning

- Indiv. Proteins move in electrical field till pH = pI no charge cease

movement - Used in study of: a. Immunoglobulins b. Abno proteins in CSF (patients w/ multiple sclerosis) c. Isoenzymes (ALP isoenzymes) Routine EP Isoelectric Focusing Separation condition Constant pH (8.6) pH gradient (3.5-9) SM CA, AR, polyacrylam. AR, polyacrylam. Gel Separation factor Q, r pI Narrow zones Vol. & starting area not Application (Cathode side) critical Not critical, Conc. Critical concentrating effect Counteracted by Diffusion Reduces resolution focusing effect Cooling. 10C Temp Rm temp Salt soln. Not critical Detrimental 9. 2-Dimensional Protein Fractionation - Synthesis & modification of 1) IEF (8) & 2) PAGE (7) - 2 dimensions: 1st proteins separated on basis of pI in tube gel 2nd result fractionation slab further treatment by PAGE - Can examine most of thousands of proteins in body fluids - Patterns usually detected w/ Silver Staining - Theoretical resoln. over 10,000 proteins possible - Use of sophisticated computer-driven scanner capable of: Examine complex patterns Store data Pattern comparison to quantitate materials Detect presence or absence of specific proteins - Used as research tool 10. ImmunoEP EP + immunochem. rxns in agar - detect monoclonal immunoglobulins

- Stain conc. & rxn must be selected allow devt of all bands
- Background supported by SM impt when proteins are quantitated by densitometric scanning Significant proteins in EP Fractions Prealbumin Albumin 1 globulin 2 globulin globulin globulin Prealbumin Albumin 1 antitrypsin, lipoproteins Ceruplasmin (combine w/ H2O), haptoglobulin, 2 macroglobulin, lipoproteins Lipoproteins, transferrin, hemopexin, complement sys. Immunoglobulins

1.

Problems associated w/ protein & measurement Buffer conc. & Ionic Strength affect amt. of heat build-up

- Dilute buffer results into diffusion

- Conc. buffer shorter zones of separation 2. Electroendosmosis prevented thru electrically neutral SM
- To avoid distortion of patterns & achieve optimum separation: minimal separation