teknik biokimia s1 2010-2011
TRANSCRIPT
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ACCURACY AND ACCURACY AND PRECISIONPRECISION
Sukarti MoeljopawiroSukarti MoeljopawiroLaboratorium BiokimiaLaboratorium BiokimiaFakultas BiologiFakultas Biologi
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Accuracy and Precision
● Accuracy: May be defined as the degree of conformity to the truth
● Precision:● The degree of agreement between replicate
experiments
● Precision does not mean accuracy, since measurements may be highly precise but inaccurate due to a faulty instrument
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Type of errors
● Errors may be:● Random● Careless● Inaccurate instruments
● Random error which are individually unpredictable
● Errors can arise from careless experiments work:● Using apparatus wrongly● Do not read the manufacture’s instructions● Using broken instruments
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Standard and blank
● To obtain a value as accurate as possible from an estimation errors must be reduced to a minimum
● This can be done by:● Careful working● Using standard solution● Using a blank
● Standard solution:● Should be included in all measurements● Should be treated in an identical manner to the fluids
under investigation● Function: provides a useful check on the accuracy of a
method
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Standard and blank (cont’d)
● Blank solution:● Should be included in any measurement● The same volume of distilled water replaces the
substance to be estimated● Should be treated in exactly the same way as the test
and standard● Function: provides a useful check on the reagents
purity
● The function of standard and blank: to correct the obtained value
Several blanks and controls need to be used when working with enzymes
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Glassware
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Macam Glassware● Pipet gondok (Volume pipette)● Pipet ukur (Graduated pipette)● Pipet tetes (Pasteur pipette)● Pipet mikro
● Labu godog (Digestion flask)● Labu ukur (Volumetric flask) ● Labu pemisah
● Gelas piala (Beaker glass) ● Botol timbang● Gelas arloji● Gelas ukur (Measuring cylinder)● Erlenmeyer (Erlenmeyer flask)
● Buret makro● Buret mikro● Buret semimikro
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Cleaning glassware
● Grease rag soaked in chloroform or benzene and soaking overnight in chromic acid
● Very dirty apparatus soaking in a mixture of concentrated nitric and sulphuric acids
● After rinsing in tap water followed by several rinses in distilled water
● Normal glassware dried in an oven● Volumetric glassware rinsed with
alcohol then dried warm air
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Terima kasih Terima kasih ……Terima kasih Terima kasih ……
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CHROMATOGRAPHY
Sukarti MoeljopawiroLaboratorium BiokimiaFakultas Biologi
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Separation of Compounds ofSeparation of Compounds ofBiological OriginBiological Origin
Using:● Extremes of temperature● Extremes of pH● Organic solvents● Oxidizing agents● Reducing agentshave to be avoided
Extreme physical conditions may:● Irreversibly change the structure of the
molecules● Destroy any biological activity
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ChromatographyChromatography
This technique utilizes differences in the basic physical properties:
● Mass
● Size
● Shape
● Charge
● Adsorption effect
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Chromatography Chromatography (cont’d)(cont’d)
A three component system is involved:
● The mixture to be separated (liquid)
● A stationary phase (column or film)
Solid play an active part in the separation process
● The mobile phase
Liquid (in most case) provides a medium for the molecules to move through
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Kinds of ChromatographyKinds of Chromatography
● Gel Filtration
● Adsorption Chromatography
● Ion Exchange Chromatography
● Partition Chromatography● Paper Chromatography
● Thin-layer Chromatography
● Gas Chromatography● Gas-liquid Chromatography
● Gas-solid Chromatography
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Gel FiltrationGel Filtration
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Gel Filtration Gel Filtration (cont’d)(cont’d)
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Adsorption ChromatographyAdsorption Chromatography
A and C : Association-dissociation phenomenaB : Solvation of soluteD and E : Interaction with adsorbent
Solute
Adsorbent
Solute Solvent SolventA B C
D E
Adsorption (chem.) : The taking up of one substance at the surface of another
Absorption (chem.) : Penetration of a substance into the body of another
Adsorbent (chem.) : The substance, either solid or liquid, on whose surface adsorption of another
substance takes placeChamber Dictionary of Science and Technology. Chambers Edinburgh.
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Adsorption ChromatographyAdsorption Chromatography(cont’d)(cont’d)
Solvent reservoir
Filter paper disc
Adsorbent
Glass wool
Collecting tube
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ANION exchanger with exchangeable
counter ions
CATION exchanger with exchangeable
counter ions
Ion Exchange ChromatographyIon Exchange Chromatography
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–
–
–+
++
+
+ –
+
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–
+
+
+
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Ion Exchange ChromatographyIon Exchange Chromatography (cont’d)(cont’d)
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Partition ChromatographyPartition Chromatography
Two kinds of Partition Chromatography:● Paper Chromatography
● Thin-layer Chromatography (TLC)
Partition Chromatography for the compounds that are soluble in both water and organic solvents
Adsorption Chromatography for the compounds that are readily soluble in organic liquid but sparingly soluble in water
Ion Exchange Chromatography for ionizable water soluble compounds
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Paper ChromatographyPaper Chromatography
Based on direction of solvent flow:● Ascending Chromatography● Descending Chromatography● Circular Chromatography
Rf = Distance of migration of X
Distance moved by the solvent
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Ascending ChromatographyAscending Chromatography
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Descending ChromatographyDescending Chromatography
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Circular ChromatographyCircular Chromatography
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Thin-layer ChromatographyThin-layer Chromatography
● This method is very rapid (many separations can be completed under an hour)
● The spots are very compact (so it is possible to detect compounds at low concentration)
● Compounds separation is much better than paper chromatography
● Separated compounds can be detected using corrosive sprays at high temperature
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Thin-layer ChromatographyThin-layer Chromatography(cont’d)(cont’d)
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Thin-layer ChromatographyThin-layer Chromatography(cont’d)(cont’d)
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Two DimensionalTwo DimensionalThin-layer ChromatographyThin-layer Chromatography
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Two DimensionalTwo DimensionalThin-layer Chromatography Thin-layer Chromatography (cont’d)(cont’d)
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Gas ChromatographyGas Chromatography
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Gas Chromatography Gas Chromatography (cont’d)(cont’d)
● This method was first described by James and Martein (1952), and has been developed very rapidly
● This is the best method for separation of biological compounds
● Advantages of GC:● Very good separation● Time (analysis is short)● Small sample is needed (picogram)● Good detection system● Quantitatively analyzed
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PrinciplesPrinciples
(gas)
STATIONARY PHASESTATIONARY PHASE
Sampleout
Samplein
(solid or heavy liquid coated onto a solid or support system)
MOBILE PHASEMOBILE PHASE
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Gas Chromatography Gas Chromatography (cont’d)(cont’d)
Gas chromatography (GC) is a preferred method, only applicable to volatile substances
In GC mobile phase is gas, stationary phase could be:
Solid Gas Solid Chomatography (GSC)
Liquid Gas Liquid Chomatography (GLC)
In GLC, solid support is coated a liquid
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Gas Chromatography Gas Chromatography (cont’d)(cont’d)
Gas chromatography (GC) consists essentially of a gas supply, column and detector
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Gas Chromatography Gas Chromatography (cont’d)(cont’d)
● Gas supply consist of:● Cylinder of high purity gas under high
pressure● Gas could be nitrogen, helium, etc.
● Pressure regulation device● Flow regulation device● Flow measuring device
● Column:● Glass/stainless steel● Containing solid support (GSC)● Solid support is coated a liquid (GLC)
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Gas Chromatography Gas Chromatography (cont’d)(cont’d)
● Detector is some device which generates a change in electrical signal in response to the solute as it comes off the column
● Most detectors require electronic amplification of the signal (electrometer)
● Kinds of detectors:● Flame ionization detector (FID)
● Nitrogen phosphorus detector (NPD)
● Electron capture detector (ECD)
● Flame photometric detector (FPD)
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Schematic Diagram ofSchematic Diagram ofGas ChromatographyGas Chromatography
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InstrumentationInstrumentation
● Injection port sample introduction● Manual - Direct Injection
● Automated - Autosampler
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Instrumentation Instrumentation (cont’d)(cont’d)
● Oven Temperature Control● Isothermal
● Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
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Instrumentation Instrumentation (cont’d)(cont’d)
● Column
Packed
Capillary
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Instrumentation Instrumentation (cont’d)(cont’d)
● Detector● Destructive
● Mass Spectral (CI/EI)
● Flame Ionization (FID)
● Nitrogen-Phosphorus (NPD)
● Flame Photometric (FPD)
● Electrolytic Conductivity (Hall/ELCD)
● Non-destructive● Thermal Conductivity (TCD)
● Electron Capture (ECD)
● Photo Ionization (PID)
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Instrumentation Instrumentation (cont’d)(cont’d)
● Detector● Biological detector
● Gypsy moth to detect Gypsy moth’s hormone● Human at the end of column detecting separated
aromas
● Normal detectors have been mentioned
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Gas Chromatography SpectraGas Chromatography Spectra
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Application of GC and TLCApplication of GC and TLC
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Application of GC and TLCApplication of GC and TLC(cont’d)(cont’d)
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… … Terima kasih …Terima kasih …