Teknik Biokimia S1 2010-2011

ACCURACY AND ACCURACY AND PRECISIONPRECISION

Sukarti MoeljopawiroSukarti MoeljopawiroLaboratorium BiokimiaLaboratorium BiokimiaFakultas BiologiFakultas Biologi

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

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

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

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

Glassware

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

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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CHROMATOGRAPHY

Sukarti MoeljopawiroLaboratorium BiokimiaFakultas Biologi

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

ChromatographyChromatography

This technique utilizes differences in the basic physical properties:

● Mass

● Size

● Shape

● Charge

● Adsorption effect

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

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

Gel FiltrationGel Filtration

Gel Filtration Gel Filtration (cont’d)(cont’d)

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.

Adsorption ChromatographyAdsorption Chromatography(cont’d)(cont’d)

Solvent reservoir

Filter paper disc

Adsorbent

Glass wool

Collecting tube

ANION exchanger with exchangeable

counter ions

CATION exchanger with exchangeable

counter ions

Ion Exchange ChromatographyIon Exchange Chromatography

––

–

–

–+

++

+

+ –

+

––

––

–

+

+

+

+

+

Ion Exchange ChromatographyIon Exchange Chromatography (cont’d)(cont’d)

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

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

Ascending ChromatographyAscending Chromatography

Descending ChromatographyDescending Chromatography

Circular ChromatographyCircular Chromatography

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

Thin-layer ChromatographyThin-layer Chromatography(cont’d)(cont’d)

Two DimensionalTwo DimensionalThin-layer ChromatographyThin-layer Chromatography

Two DimensionalTwo DimensionalThin-layer Chromatography Thin-layer Chromatography (cont’d)(cont’d)

Gas ChromatographyGas Chromatography

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

PrinciplesPrinciples

(gas)

STATIONARY PHASESTATIONARY PHASE

Sampleout

Samplein

(solid or heavy liquid coated onto a solid or support system)

MOBILE PHASEMOBILE PHASE


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


Gas chromatography (GC) consists essentially of a gas supply, column and detector


● 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)


● 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)

Schematic Diagram ofSchematic Diagram ofGas ChromatographyGas Chromatography

InstrumentationInstrumentation

● Injection port sample introduction● Manual - Direct Injection

● Automated - Autosampler

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)


● Column

Packed

Capillary


● 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)


● 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

Gas Chromatography SpectraGas Chromatography Spectra

Application of GC and TLCApplication of GC and TLC

Application of GC and TLCApplication of GC and TLC(cont’d)(cont’d)

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Teknik Biokimia S1 2010-2011

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