Bio Analysis Flashcards
What is quantitative analysis
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Disease
• Dysregulation of the cell cycle is common during tumorigenesis (cancer display uncontrolled growth)
• Increased levels of CDK molecules and cyclins are sometimes found in human tumours, such breast cancer and brain tumours
• Inhibition of certain CDKs has been shown to inhibit tumour cell growth, induce apoptosis and cause tumour regressions in animal models
• CDKs are potential targets for new anticancer therapy
Analytical chemistry
a branch of chemistry that deals with the identification of compounds and mixtures (qualitative analysis) or the determination of the proportions of the constituents (quantitative analysis): techniques commonly used are titration, precipitation, spectroscopy, chromatography, etc. 1
Biochemical analysis:
characterisation of biological components within a sample using appropriate laboratory techniques
Qualitative Analysis
indicate whether a particular substance (analyte) is present above a threshold level
Marquis test
presence of alkaloids
Trinder spot test
presence of salicylates (aspirin)
hCG
human chorionic gonadotrophin
Quantitative Analysis:
determines the amount of a particular analyte present in the sample (e.g. the concentration of a drug in blood serum).
Accuracy
Closeness of a measured or derived data value to its true value
Precision
Closeness of values with repeated measurements
Sensitivity
• ability to detect small amounts of analyte in a sample
• OR the percentage of patients with the disease that will be correctly
identified as disease positive
Specificity:
• ability to detect only the analyte of interest in a sample
• OR the percentage of patients without the disease that receive a negative result
Why measure biological molecules?
Medicine:
• Blood hormone levels • Enzyme activity
• Drug levels
• Metabolites
• Clinical trials
Toxicology:
• Xenobiotics
• Drugs / metabolites
• Pharmacokinetics
• Anti-doping testing in sports
Forensics:
• Xenobiotics
• Drugs / metabolites
• Environmental toxins
• Other toxic compounds
Typical analytical scheme
Sample
Sub-sample
Extraction ; the test substance or remove the interfering substance before analysis can proceed
Separation; Separation procedures depend on physical or chemical characteristic of the compound
Detection
Result
Interpretation
Separation technique for polar molecules that are volatile,soluble,and absorbable
Gas-liquid chromatography Liquid-liquid chromatography
Liquid-gas chromatography
Separation technique for ionic molecules that are charged
Ion-exchange chromatography
Electrophoresis
Separating technique for molecules with a mass that have the physical property are diffusion and sedimentation
Gel filtration chromatography
Dialysis Ultracentrifugation
Separating technique for molecules with a shape that have the physical property of ligand binding
Affinity chromatography
Chromatography
-developed by a Russian botanist
-Technique that allows the resolution of a mixture of compounds as a consequence of the different rates at which they move through a stationary phase, under the influence of a mobile phase
Planar chromatography
The stationary phase is supported on a flat plate or in the fibres of a paper.
The mobile phase moves through the stationary phase by capillary action or by gravity.
• Paper chromatography
• Thin layer chromatography (TLC)
Column Chromatography:
The stationary phase is held in a tube through which the mobile phase is forced either by pressure or by gravity.
• Simple column chromatography
• High pressure liquid chromatography (HPLC) • Gas chromatography (GC)
Thin layer chromatography
-TLC plate (stationary phase): a sheet of glass, metal, or plastic coated with a thin layer of a solid adsorbent (usually silica or alumina)
-Samples are “spotted” at the base of the sheet and dried.
-Sheet is placed in the tank containing a shallow layer of solvent (mobile phase)
-Substances reacting to the stationary phase more strongly will have retarded migration.
Rf
distance travelled by substance /distance travelled by solvent
Gel Filtration (Column) Chromatography
-Separates proteins, peptides and oligonucleotides on the basis of size
-The stationary phase (gel) consists of beads (e.g. Sephadex)
-The mobile phase (buffer) is used to elute the analytes from the gel matrix based on their size
Gel Filtration Chromatography: the Column
Gel matrix: Sephadex (separation Pharmacia dextran) a cross-linked dextran gel
Parts of the gel filtration column in chromatography
Vo: volume required to elute molecules bigger than the pore size of the column gel
Vt=Vs+VI +Vo
VI= volume of solvent in the pores
Ve: elution volume of a particular solute
Partition coefficient: Kav =(Ve-Vo)/(Vt-Vo)
Partition coefficient:
Kav =(Ve-Vo)/(Vt-Vo)
Gel Filtration Chromatography
Relative molecular weight (Mr) of analyte can be estimated by plotting the partition coefficient Kav against the logarithm of the molecular weight of standard protein samples
Typical analytical scheme 2
Sample
Sub-sample
Extraction
Separation
Detection ; Liquid chromatography detectors: • UV / visible
• Fluorescence
• Electrochemical
Result Interpretation
Detection and Analysis
Spectroscopy
Measures the absorption and emission of electromagnetic radiation by atoms and molecules in solution
Isaac Newton (1666):
uses a glass prism to split sunlight into a monochrome spectrum
Io
intensity of light striking a substance
I
intensity of light transmitted through the substance
Spectroscopy: Beer-Lambert law
-Describes the absorbance of monochromatic light as it passes through a solution
-log10(io/I)=A=ECL
-I/io=10 -ecl.
l = the distance travelled by the light through the substance [in m]
ε = molar extinction coefficient of the substance c = concentration of the substance [mol/l]
A= absorbance
A=ecl Absorbance is linearly related to the concentration of the analyte
With known ε and l the concentration of a substance can be estimated using a standard plot – Abs against concentration
How do spectrophotometers work
-light source
-collimator(lens)
-monochromator(prism or grating|)
-wavelength selector
-sample collation in a cuvette
-detector (photocell)
-digital display or meter
