unit one instruments
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Analytical techniques
UNIT 1
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Self Study
Principle of the followinginstruments/techniques and identify specificanalytes that are measured by each
instrument: Flourometry
Turbidimetry
Nephelometry Chemiluminescence
Chromotography (HPLC; GLC & TLC)
Elisa Prof T. Matsha 2
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Lecture Outline
Photometry & Spectrophotometry Mass spectrophotometry
Electrochemistry (Nersnt equation) Electrophoresis
Osmometry
Enzyme kinetics
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Photometry &Spectrophotometry (1)
Measurement of light intensity
Light as other forms of electromagneticradiation make characteristic patterns
(waves) as they travel through space Wavelength distance between two peaks
(high points) as the light travels in a wavelike manner
http://id.wikipedia.org/wiki/Berkas:Wavelength.pnghttp://id.wikipedia.org/wiki/Berkas:Wavelength.png -
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Photometry &
Spectrophotometry (2)
Each wave has a certain shape and
length depending on the frequency ofthe waves. Frequency of a wave is inversely proportional
to the wavelength
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Photometry &Spectrophotometry (3)
Previously, photometric instruments measured lightintensity independently of wavelength
Sunlight mixture of spectrum of radiant energy atdifferent wavelengths (rainbow) human eye recognizes itas white
Modern Instruments can isolate a narrow wavelengthrange of the spectrum for measurements
Filters filter photometers Prisms or gratings - spectrophotometers
http://www.lpi.usra.edu/education/fieldtrips/2005/activities/ir_spectrum/images/emspectrum.jpg -
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Determinations in the ClinicalLaboratory are based on measurementof radiant energy
Emitted Partially reflected Transmitted
Absorbed Photometers light emitted Spectrophotometers - absorbed
Photometry &Spectrophotometry (4)
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SPECTROPHOTOMETER
Used to measure to concentrationsof substances
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SPECTROPHOTOMETER cont.
A combination of a spectrometer & a photometerSpectrometer produces light of any selectedPhotometer measures light intensity
A cuvette with liquid is placed between the 2.
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SPECTROPHOTOMETER cont.
The amount of light passing through the cuvetteis measured by the photometer.
The photometer delivers a voltage signalto a display device.The signal changes as the amount of lightabsorbed by the liquid changes
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SPECTROPHOTOMETER cont.
Lightabsorbed
Transmitted
light
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SPECTROPHOTOMETER cont.
Light absorbed
Units: Absorbance (A) or Optical density (OD)
(logarithmic scale)
Transmitted light - % transmission(Arithmetic scale)
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SPECTROPHOTOMETER cont.
Obeys Beers law:
If a solute absorbs light of a particular ,
the absorbance is directly proportional tothe concentration of substance in solution.
I0A = log = clI
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Beers Law (1)
Beers law The intensity of a solution when viewed through monochromatic light
(single wavelength, e.g. 600nm) is directly proportional to theconcentration of the substance through which the light passes.
Lamberts law
- When monochromatic light passes through a transparent medium, therate decrease in intensity with the thickness of the medium isproportional to the intensity of light
Beer-Lambert law (also known as Beers law)- When monochromatic light passes through a coloured solution the
amount of light transmitted decreases exponentially with the increasein concentration of the solution through which the light passes orsimply; absorbance is directly related to concentration if the light pathstay constant
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Beers Law (2)
Absorbance Absorption is a process in which incident radiated
energy is retained without reflection or transmissionon passing through a medium
Therefore for ray to be absorbed it must have thesame frequency as a rotational or vibrationalfrequency in the atom or molecule it strikes
Transmittance The ratio of transmitted energy to the amount ofincident energy is called transmittance.
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USE OF ASPECTROPHOTOMETER
Blank
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Beers Law (3)
Some of Io is either reflected by surface of the cell orabsorbed by solvent or cell wall , Io < Is
Percent transmittance %T = Io / Is X 100 For some applications in optics it might be useful to see
transmittance values as percent transmittance values. Allintensities will be scaled to fit an interval between 0 and 100percent transmittance.
Focus interest eliminate factors use blank Blank absence of compound of interest but same solvent. No
light absorbed %T = 100%
Add compound of interest serially %T varies inversely andlogarithmically with concentration
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Clinical Example
1. Blank Reading reagent buffer without serum
Read the blank Most light transmitted & small amount absored bycuvette, solvent or reflected from detector
Set instrument abitrarily at 100%T (A = 0)2. Sample Reading Reagent buffer + serum Difference amount light passed blank vs. sample due
to presence of compound measured % T Sample beam signal X 100
Blank beam signal
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USE OF ASPECTROPHOTOMETER
1. Switch on2.Set wavelength3.Use after 30 min
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USE OF ASPECTROPHOTOMETER
4. Calibratea. Insert blank
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SPECTROPHOTOMETER cont.
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USE OF ASPECTROPHOTOMETER
5. Set to 0
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USE OF ASPECTROPHOTOMETER
4. Insert sample &read
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Beers Law (4)
Absorbance is more convenient to usebecause it is directly proportional toconcentration.
Amount of light absorbed particularwavelength depends:1. Molecules and ions present2. [ ]
3. pH4. Temperature
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Standard Curve
Unknown [ ] isdetermined from acalibration curve orstandard curve
Standards of knownconcentration
Plot on graph linearcurve
B2MG Concentration (g/ml)
Absorbance(450nm)
0 0.046
0.625 0.385
1.25 0.723
2.5 1.241
5 2.199
10 3.094
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SpectrophotometicInstruments
Measure light transmitted by solution
Mathematically converted absorbance
Determine [ ] light absorbing substance
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Spectrophotometer (2)
Light Source Provides radiant energy
Visible light (350 - 700nm) tungsten light bulb
To increase lifetime iodine or bromide is added -tungsten-iodide lamp UV region (165 -360nm) low pressure mercury-vapor
lamp, emits discontinuous spectrum Hydrogen & deuterium lamps low. Deuterium-
discharge more stable than hydrogen Mercury & xenon high pressure
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Monochromator - isolate radiant energy of desired wavelength butexcludes others
Monochromator
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Filters
To obtain monochromatic light useTypes1. coloured-glass filter
Transmistts energy over a wide range of wavelength Not precise Simple & inexpensive
2. Interference filters Pass very narrow range wavelength Efficient
http://images.google.co.za/imgres?imgurl=http://www.tufts.edu/as/tampl/projects/micro_rs/monochromator4.jpg&imgrefurl=http://www.tufts.edu/as/tampl/projects/micro_rs/setup.html&h=307&w=345&sz=19&hl=en&start=1&tbnid=8dA7BUCxxYBM5M:&tbnh=107&tbnw=120&prev=/images%3Fq%3Dmonochromator%26gbv%3D2%26svnum%3D10%26hl%3Den%26sa%3DGhttp://images.google.co.za/imgres?imgurl=http://www.tufts.edu/as/tampl/projects/micro_rs/monochromator4.jpg&imgrefurl=http://www.tufts.edu/as/tampl/projects/micro_rs/setup.html&h=307&w=345&sz=19&hl=en&start=1&tbnid=8dA7BUCxxYBM5M:&tbnh=107&tbnw=120&prev=/images%3Fq%3Dmonochromator%26gbv%3D2%26svnum%3D10%26hl%3Den%26sa%3DG -
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Prisms
Type of monochromator Separates white light to continuous spectrum
by refraction, i.e. shorter wavelengths arerefracted or bent.
Consequently nonlinear spectrum with longerwavelengths closer together, but
Suitable narrow-bandwith portion of the
spectrum
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Separation of light to differentwavelengths
Most commonly used monochromators
Consists of parallel grooves etchedpolished surface
Diffraction gratings
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Cuvettes
Cuvette/Sample/Absorptioncell:
Holds sample& provides
con stant l ight path
Round / square
Light path must be kept
constant
Otherwise A no t C
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Round
Difficult constant light path Not uniform Etched for constant positionSquare/rectangular
Plane-parallel optical surface, constant light path Less error Most common Plastic cells inexpensive, but designed for single use
application
Good clarity for both UV and visible light Problems etching by solvents, temp. deformations,
cleaning Quartz cuvettesexpensive, UV range
Cuvette types
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QC: Cuvettes
Scratched opt ical surface scatter light
Do not touchon optical surface
Wipeoptical surface tissue before use
Insert correct orientation spectrophotometer
Clean immediatelyafter use (DO NOT soak)
(mild detergent & rinse deionised water)
Drainupside down to dry
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Convert transmitted radiant energy equivalent amount electrical energy
Photodetector
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Photodetector types
Pho tocell / barr ier-layer cell
=> least expensive
=> film light-sensitive material (eg. selenium /
iron / silver)=> require no external voltage
=> rely internal e- transfer produce current
=> wide bandpass instruments
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Phototube
=> also photosensitive material=> e- generated from light energy=> outside voltage required
h l i li b
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photomultiplier tube
=> detects & amplifies radiant energy=> e- attracted series anodes / dynodes=> each (+) voltage=> generate 2ndary e-=> multiple cascade => amplification=> thus 200x more sensitive than phototube=> narrow bandpass instruments
wavelength scanner instruments
double-beam spectrophotometers
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DOUBLE BEAM
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DOUBLE-BEAMSPECTROPHOTOMETER
Automatic correctionsample A &reference A
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QA/QC Spec.
Wavelength accuracy Stray light scratched and dust particles Linearity
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Spec in Clin lab
General chemistryanalytes, e.g. glucose
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Mass spectrophotometer
MS used to:
Identify unknown compounds
[ ] of known substances
Molecular structure
Chemical composition of both in-& organic materia In clinical chemistry:
Drug metabolism
Drug abuse (steroids use in sport)
Damage to DNA
Metabolic disorders in infants
Research, e.g. search for unique proteins in specimen for use as
diagnostic or therapeutic targets
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Mass spec basic components
Sample converted into
ions or molecules by
thermal or electrical
energy
The ions in a gaseousmedium are accelerated
into the mass analyzer
where they are separated
into species, such thatdifferent species of ions
strike the detector at
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ATOMIC ABSORPTION
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ATOMIC ABSORPTIONSPECTROPHOTOMETER
Measure [ ]. by detecting A ofelectromagneticradiationby atoms
(not molecules)
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Components
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Light Source
Hallow-cathode lamp Consists of evacuated gas-tight chamber +
anode
Cathode Inert gas(argon / helium)=> voltage applied=> filler gas ionised
=> ions excite metal atoms=> light energyemitted
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Sample Cell
Flame
Why? Sample must contain reducedmetal in atomic vaporised state
Done via heat of flame break chemicalbonds unexcited atoms
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AAS vs SPEC
Light source passes through sample
Note, atoms though bonds are broken ground state (unexcited)
Light source excites atoms returns toground state emits energy = absorbedlight
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Chopper
Aim: measure light absorbed by atoms
Need to distinguish between lightemitted by light source and excitedatoms
Hence, the chopper
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Monochromator
Also used to protect the photodetectorfrom excessive light from flameemissions
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Adv:Disadvantages
ADV:=> very sensitive & precise
DISADV: onlymeasure elementsthat exist atomic state
eg. Mg2+, Mn2+, Cu2+, Pb2+ Flame not dissociate samples into free atoms,ie. PO4
2- interfere Ca2+
analysis (forms CaPO4complex)
Overcome adding cations compete with Ca for P
Ionisation atoms upon dissociation by flame,overcome reducing flame temp.
Matrix interference,eg. atoms in organic solvents enhanced lightabsorption. Overcome pretreat sample (extraction)
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Fluorometry
Measure concentration of solution thatcontain fluorescing molecules
Principle: is based on an energy exchange
process that occurs when valence shellelectrons absorb EMR, become excited andreturn to an energy level lower than theiroriginal level. The lifetime of an excited state
is about 10-9 to 10-6 seconds and the lightemitted fro a single excited state is calledfluorescence
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Monochromator
Primary filter selects the wavelength Secondary filter as in AAS protects the
photodetector from radiant energy
emitted by flourescing molecules insample Spectrofluorometer filters are
replaced with prisms or gratings
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Advantages
Specificity- because selection optimalwavelength for both absorption &fluorescence
Sensitivity- 1000x more sensitive thanspectrophotometry
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Disadvantages
Sensitiveto environmental changes pHchanges
=> affects availability e-
Temperaturechanges=> affects probability loss energy viacollision (rather than fluorescence)
Contaminating chemicals / change solvent=> change structure molecules
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QC Fluorometry
Any fluorescence as result ofenvironmental changes
Quenching
QC: extreme care mandatory=> analytical technique
=> instrument maintenance
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Chemiluminescence
Chemiluminescence differs from flourescence in thatemission of light is created from a chemical orelectrochemical reaction (rxn) and not from EMRstimulation of electrons.
It involves the oxidation of an organic compound suchas luminol, in the presence of a catalyst such as anenzyme, metal ions (though not always)
The excited products formed during the oxidation
rxn produce chemiluminescence on return to thesinglet state that can be measured by a luminometer.
ADVANTAGES
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ADVANTAGESCHEMILUMINESCENCE
Subpicomolar detection limits
Speed rapid
Ease of use, ie. one-step procedure
Simple instrumentation
Increased sensitivity over flourescence
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TURBIDITY & NEPHELOMETRY
Techniques used to measuring the [ ] of a solutionthat contains particles too large for absorptionspectoscopy.
Nephelometry measurement of light scattered by a
particulate solution. Commonly used for antibody-antigen rxns.TURBIDIMETRY measurement of the reduction inlight transmission caused by particle formation.
Applications: microbiology bacterial growth in brothcultures; hematology clot formation in coagulationanalysers.
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QC
Reagents free particles
Cuvettes no scratches Sample handlingcritical because particles
tend aggregate & settle out solution
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Electrochemistry
Measurement of current of voltagegenerated by the activity of specificions
Clinical chemistry: potentiometry;coulometry; voltammetry; andamperometry
All use eithergalvanic electrochemicalcellOR electrolytic cell
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GALVANIC & ELECTROLYTIC
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GALVANIC & ELECTROLYTICCELLS
Electrochemical cell consists of:
Two half-cells and a salt bridge(textbook figure)
Electrodes (cathode & anode) immersed2 beakers salt solution
If only 1 beaker then solution = saltbridge
ION-SELECTIVE
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ION-SELECTIVEELECTRODES (ISE)
Potentiometric method pH electrodes
Sensitive to individuals ions measuredirect electrical potential due toactivity of free ions
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pH Electrodes
Components Indicator Electrode
consists of a silver wirecoated with AgCl in
0.1mmol/L HCl
All above place in into atube containing specialglass membrane tip sensitive to H+ only.
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pH METER
Measures the acidity of a solution.
pH = - log aH+
pH = - log [H+]
AH+ = hydrogen ion activity
[H+] in moles / of solution
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pH METER
2 electrodes measure voltage
1 electrode is in a liquid with fixed acidityreference electrode
Other electrode responds to acidity of thesolution sensing electrode
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pH METER
A voltmeter measures the differencebetween the voltage of the electrodesA meter converts this into pH
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Indicator Electrode
pH electrode into test solution=> movement H+ near tip electrode=> produce potential difference
=> between internal & test solution=> measured as pH by voltmeter
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Reference electrode
Most commonly used - calomel electrode Calomel is a paste of mercurous chloride &
potassium chloride
In electrolyte solution KCl it is in directcontact with metallic mercury All reference electrodes must generate a
stable electrical potential
[ ] of electrolyte must constant &temperature stable voltage
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NERNST EQUATION
Electromotive force generated because H+ atglass tip=> described by Nernst equation (self study)
THUS temperature H+ activity Set temperature-compensation knob
pH COMBINATION
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pH COMBINATIONELECTRODE
Indicator + reference electrodecombine in one small probe
Consists of: internal reference
electrode=> Ag/AgCl OR=> Hg/Hg2Cl2
Sealed into narrow glass cylinder withpH-sensitive glass tip
E E
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pH METER
Combination probe contains both electrodes
H l d
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QC pH electrode
Balance the system with the electrodesin a buffer whose pH is 7.0
Replace buffer with one of different
pH, usually 4.0 or 10DONT touch glass bulb with your fingers.Rinse with distilled water
Keep within ambient temperature rangeAvoid air bubblesKeep clean of deposit
GAS-SENSING
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GAS SENSINGELECTRODES
Similar pH electrodes but separated fromsolution bygas-permeable hydrophobicmembrane
BUT designed detect specific gases insolutions
eg. CO2 (PCO2 electrode)eg. O2 (Clark electrode)eg. NH
3
(NH3
gas electrode)
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Electrophoresis
Migration charged solutesin electricalfield
In clin lab protein serum, urine,
CSF Lately, Nucleic acids
C
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Components
Electrical power => driving force Support medium
Buffer
Sample
Detecting system
SUPPORT MATERIAL
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SUPPORT MATERIAL
All gels transparent Scanned densitometer Dried permanent record
Cellulose acetate Agarose gel Polyacrylamide gel Starch gel
PRINCIPLE
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PRINCIPLE
Charged particles migrate to oppositecharged electrode
Velocity of migrationcontrolled by=> particle net charge(directly )=> particle size & shape(inversely )=> strength electrical field=> chemical & physical properties supporting
medium=> temperature
P d
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Procedure
Support with gel placed inelectrophoresis chamber
Chamber filled buffer- contact both
ends support/gel Samplesapplied to gel Apply constant voltage / current
specific time - Electrophoresis
D t ti
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Detection
Support/gel in fixative / dried F: prevent diffusion sample
Stainwith appropriate dye aidvisualisation & quantitation
NOTE: dye uptake sample conc.
Excess dye washed away Drygel (permanent record)
QC l t h i
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QC electrophoresis
Operate either constant current / constantvoltage - constant currentpreferred
Why? Current flows through medium heat is
produced Results increased agitation of dissolved
solutes increased current increased heat &buffer evaporation ionic strength of buffer
increased current
QC b ff
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QCbuffer
Affects charge ampholytes (e.g Protein)1 => pH &2 => ionic strength of buffer
Ampholyte net charge either (+) / (-)
if buffer more acidic than pI ampholyte=> ampholyte binds H+
=> ie. (+) charge=> migrate cathode (-)
if buffer more basic than pI ampholyte
=> ampholyte loses H+=> ie. (-) charge=> migrate anode (+)
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DETECTION &
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DE E ION &QUANTITATION
Separated protein fractions stained visualise- UV light (nucleic acids)
Quantitation:densitometer
=> each band = peak=> surface area of peak = % of total
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Ch m t h
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Chromatography
Refers to a group of techniques used toseparate complex mixtures on the basisof different physical interactions
between the individual compound andstationary phase of the system
C mp n nts
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Components
Mobile phase=> gas / liquidF: carry sample
Stationary phase => solid/liquid
F: mobile phase flows
Column: F: hold stationary phase &separated components
MODES OF SEPARATION
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MODES OF SEPARATION
Adsorption chromatography Partition chromatography
Steric exclusion chromatography
Ion-exchange chromatography
Adsorption chromatography
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Adsorption chromatography
Liquid-solidchromatography Competition sample & mobile phasefor
adsorptive sites solid stationary phase High affinity molecules retained longer Stationary phase:(a) acidic polar (eg. silicagel) (b) basic polar (eg. alumina)(c) nonpolar (eg. charcoal)
Partition chromotography
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Partition chromotography
Liquid-liquidchromatography Separation solute basis relative solubility
nonpolar (organic) solvent & polar (aqueous)solvent
Molecules polar & nonpolar groups in aqueoussolution added to immiscible organic solvent
Vigorous shaking -two phases separate
Chloroform method DNA extraction
Steric Exclusion
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Steric Exclusion
Liquid-solidchromatography Separate solute molecules basis of size& shape
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Ion-exchange
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gchromatography
Solute mixtures separated by charge Stationary phase is a resin consistingof large polymers with charged
functional groups Cation exchange resin,anion exchangeresin or mixed bed exchange resin
Resin is insoluble in water
USES OF ION-EXCHANGE
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CHROMATOGRAPHY Removeinterfering substancesfrom
solution
Concentratedilute ion solutions
Separatemixtures charged molecules(eg. amino acids)
Chromotographic procedures
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Chromotographic procedures
1. THIN-LAYER CHROMATOGRAPHY (TLC) Variant column chromatography Thin layer sorbenteg. alumina, silica gel, cellulose, cross-linked
dextran=> uniformly coated glass / plastic plate
Sample applied near bottom edge plate Mobile phase / solventin closed container until atmosphere
saturated solvent vapour Bottom edge plate in solvent NOTE: samples NOT immersed solvent
solvent migrates up thin layer=> capillary action=> sample molecules dissolved=> ie. carries sample molecules
TLC
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TLC
Separation depends sorbent & solvent(1) adsorption(2) partition(3) steric exclusion
(4) ion-exchange Solvent close top => plate removed & dried Sample Rfcompared standards Rf- Rf=>
retention factor Rf = distance sample component
total distance solvent front Method semiquantitativescreening test
HPLC
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HPLC
Improved TLC Components: Pumpforces mobile phase
through column much greater velocity thangravity-flow
Column: stationary phase packed into longstainless steel column
Fine & uniform packing=> high resolution separation=> requires pressure to force mobile phasethrough
Components HPLC
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Components HPLC
Sample injector- small syringe introduce sample intopath
Mobile phase carries sample through column
Detector- monitor eluateas leaves column produce
electronic signal conc. each component Spectrophotometersmost common
Recorder- record detector signal versus time mobilephase ie. from time sample injection
Graph => chromatogram
Chromatograph
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Chromatograph
Identify compounds=> comparedretention timeto standard retentiontimes (BUT only identical conditions)
Determine conc. each compound=> peak area conc. compound
GAS CHROMATOGRAPHY
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GAS CHROMATOGRAPHY
Separate mixtures volatilecompounds / made volatile Similar HPLC except mobile phase = gas Thus samples partitioned between
gaseous mobile phase & liquid stationary phase
Carrier gas=> nitrogen / helium / argon, selectiondepends on type of detector used Samplemust be injected asgas OR Temperatureof the injection port must be above
boiling point of the components to vaporise sampleupon injection
SCINTILLATION COUNTER
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SCINTILLATION COUNTER
Radioimmunoassay used to measuretrace concentrations of hormones ordrugs
Detect radioactive signals Development of non-isotopic
immunoassay diminished use
Osmometry
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Osmometry
Measure conc. solute particles in solution Refers to measurement of osmolality of an aqueous
solution such as serum, plasma or urine 4 physical properties solution change as number
dissolved particles in solvent: collectively they arecalled colligative properties of the solution becausethey can be related to each other and to theosmolality(1) osmotic pressure; (2) vapour pressure; (3)
boiling point; (4) freezing point Thus, osmometry is based on measuring changes inthe colligative properties and the freezing-pointdepression is the most commonly used in clin
FREEZING-POINT
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OSMOMETER Consists of a sample refrigerated chamber containing a stirrer
and a thermistor (temperature sensing device)
Sample is supercooled below its freezing point in a chamberusually containing ethylene glycol
The stirrer is used to agitate the sample in order to initiate
freezing As the ice crystals form, heat is released from the solution,
which at some point reaches an equilibrium with the rate of heatremoved by the colder temp. of the sample chamber
The equilibrium temp is known as the freezing point and is
detected by the thermistor osmolality of the sample and isexpressed as milliosmoles per kilogram of water (mOsm/kg)
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Enzyme Action:
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Induced Fit Model
Enzyme structure flexible, not rigid
Enzyme and active site adjust shape to bind
substrate
Increases range of substrate specificity
Shape changes also improve catalysis duringreaction
Enzyme Action:
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Induced Fit Model
E + S ES complexE + P
SP
P
SS
Learning Check E1
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Learning Check E1
A. The active site is(1) the enzyme(2) a section of the enzyme(3) the substrate
B. In the induced fit model, the shape of
the enzyme when substrate binds(1) Stays the same(2) adapts to the shape of thesubstrate
Factors Affecting Enzyme
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Action: Substrate
Increasing substrate
concentration increases the
rate of reaction (enzyme
concentration is constant)
Maximum activity reachedwhen all of enzyme
combines with substrate
First order kinetics rxn
rate a [substrate]
Zero order kinetics
rxndepends on [enzyme]
Michael is-Menten constant (Km )
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Michael is Menten constant (Km )
1913 Michaelis & Mentenhypothesised role [S]
S binds free E at low [S] (ie. more E than S)
reaction rate steadily as more S added
thus reaction rate [S]=> f i rst-order kinet ics
eventually E saturated with S
thus maximum reaction velocity
as P formed free E immediately combines excess free S=> zero-order k inet ics
thus reaction rate depends [E]
Factors Affecting EnzymeT
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Action: Temperature
Little activity at low temperature Rate increases with temperature
- Movement of molecules
- Rate of intermolecular collusion- Energy for rxn
Most active at optimum temperatures(usually 37C in humans)
Activity lost with denaturation at hightemperatures
Factors Affecting EnzymeA i T
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Action: Temperature low temp. (eg. refrigeration / freezing)
=> enzymes reversible inactive (specimens for enzymeanalysis frozen or refrig.)=> some enzymes NOT frozen (activity lost)=> avoid repeated freeze-thaw (denature)
control temp. lab=> accurate 0.1C
labs choose enzyme analysis=> 25C=> 30C
=> 37C(most common)NOTE: reference ranges vary...
Factors Affecting EnzymeA i
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Action
Optimum temperature
ReactionRate
Low High
Temperature
Factors Affecting Enzyme
A i H
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Action: pH
Maximum activity at optimum pH
R groups of amino acids have proper charge
Tertiary structure of enzyme is correct
Narrow range of activity
Most lose activity in low or high pH
Factors Affecting Enzyme Action
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Factors Affecting Enzyme Action
Reaction
Rate
Optimum pH
3 5 7 9 11
pH
Enzyme Inhibition
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Enzyme Inhibition
Inhibitors
cause a loss of catalytic activity
Change the protein structure of an enzyme May be competitive or noncompetitive
Some effects are irreversible
Competitive Inhibition
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Competitive Inhibition
A competitive inhibitor Has a structure similar to substrate
Occupies active site
Competes with substrate for activesite
Has effect reversed by increasing
substrate concentration
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Learning Check E2
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Learning Check E2
Identify each statement as describing an
inhibitor that is
(1) Competitive (2) Noncompetitive
A. Increasing substrate reverses inhibition
B. Binds to enzyme, not active site
C. Structure is similar to substrate
D. Inhibition is not reversed with substrate
Solution E2
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Solution E2
Identify each statement as describing an
inhibitor that is
(1) Competitive (2) Noncompetitive
A. 1 Increasing substrate reverses inhibition
B. 2 Binds to enzyme, not active site
C. 1 Structure is similar to substrate
D 2 Inhibition is not reversed with substrate