Theoretical part Flashcards
Principle of photometry; Warburg optical test-principle and usage
Photometry: Estimation of properties of a solution, e.g. concentration of certain compound, based on absorption of light of a
certain wavelength is called photometry. Amount of light that passed through a sample is called transmittance, formula. Usually measured against a blank. So intensity of light emerging from sample over intensity of light emerging from a blank.
Photometers: Device used to measure absorbance if it can measure at more than one wavelength its a spectra. (source of light, monochromator, sample compartment and detector)
PRINCIPLE of Warburg optical test:
Another interesting situation is reaction of a compound that absorbs light, yielding a product with another absorbing spectrum,
when both the spectrum of reactant and of product widely overlay. Spectra of NAD+ and NADH may serve as example (estimation of these
coenzymes by direct photometry is widely used to measure activity of many enzymes in so called Warburg optical test). Absorption maxima
are apart each from the other so that it is easy to determine concentrations of both forms of the enzyme in this case.Basically it is possible to determine the activity of enzymes based directly or indirectly on the photometry of the absorbance of the reaction mixtures which contain a pyridine coenzyme . This principle has also given a method of marking an “optical test”.
Reversible hydrogenation of nicotinamide adenine dinucleotide (NAD + ) which occurs on the pyridine ring of nicotinamide, resulting in reduced form (NADH + H + ), and is accompanied by significant change in the absorption spectrum. While the oxidized form (NAD + ) having an absorption maximum at 260 nm, the cancellation aromatic character of the pyridine nucleus and the transition in the quinoid form (NADH + H + ), accompanied by the occurrence of another absorption maximum at 340 nm.
The size or change in absorbance at 340 nm is directly proportional to the number of molecules of reduced coenzyme. The conversion of one mole of coenzyme in turn corresponds to a conversion of one mole of substrate, and moreover, the conversion may be monitored in time, ie continuously measure the speed of the reaction using a spectrophotometer .
USAGE: Eg) ALT and GOD reaction.
Chromatographic techniques – principle and usage.
Chromatography is a technique used for separation of a mixture, whose essence is the distribution of components of the sample mixture through the two phases, namely a stationary phase ( stationary phase ) and mobile ( mobile phase ) We separate a sufficient amount of pure substance for further use. Sometimes its hard to cause the abs maxima align and would sum up therefore not able to distinguish so prior measurement they have to be separated from target compound. Compounds to be separated are carried by mobile phase through the stationary and are separated based on their affinity to those phases. So basically complex mixture is loaded onto the stationary phase then eluted using a mobile phase and the analyse having high affinity to the mobile is eluted out first.
Criterias are:
A) For intended use:
Preparative chromatography: One or more compounds are to be isolated from a
complex mixture in a pure form. Usually adapted to process large amount of sample,
to increase yield of target compound.
• Analytical chromatography: Compounds are separated from a mixture just to be
detected/quantified, separated compounds are not collected. Small amounts of sample
are usually processed.
B) Physical state of mobile phase:
Liquid or Gas. Mobile phase is in those states. For gas, has to be at a high temperature to evaporate volatile sample.
C) Arrangement of stationary phase:
Planar or Column, planar is when the stationary is placed on a solid support e.g. TLC and column is when stationary phase is packed in a tube.
D) Phenomenon responsible for separation: Adsorption, ion exchange, affinity, gel permeation
TLC: thin layer of stationary phase (silica gel) coated on a glass, aluminium(used in our lab)
Silica gel is a form of silicon dioxide, silicon is cross linked via oxygen atoms and free OH.
Mobile phase is a mixture of solvents placed in chromo chamber, stationary is placed in, the mobile phase is moved up through the stationary taking components of the mixture with it. Ok so stronger the affinity the faster it moves, then 3/4 mark the RF. How fast they move depends on solubility of the sample and degree of interaction with the stationary phase
Gel filtration:
Separates based on the size of the molecules. Used to purify proteins or other macromolecules from low molecular weight contaminants. Types of gel are hydrophobic, philic, natural synthetic. Most commonly are soft hydrophilic polysaccharides such as sephadex. Polysacc are cross linked with epichlorhydrine to form a 3D net structure The small MW substances can pass through and can be retained inside, whereas the bigger ones pass the beads and elude faster. The bigger the particle the faster it will elude. Elution volume is measured.
Hemoglobin derivatives – principles of detection, significance.
Hb, subunits, HbA function
Oxy (bearing oxygen) and deoxy both in ferrous state, oxygenation changes configuration of Heme Fe and manifests as colour change from dark red venous to bright red arterial
Meth: Ferric ion, sixth coordination bond by H20 instead of oxygen so can’t transport. Reduction is enzymatic (NADH del meth reductase) or non enzymatic (glutathione or ascorbic acid). Methemoglobinemia: Error in the enzyme or poisoning, effects by drugs, high amount of nitrites, Symptoms include cyanosis and hypoxia.
Carbonyl hemoglobin: bound to CO. 250x more affinity, can be reversible if there is an excess of O2. Symptoms include breathlessness, headache vomiting death at 60-80%
Spectro: Hb and derivatives have characteristics abs spectra in the visible range which can be used for analysis and rapid identification.
Oxy: 540, 578nm
Deoxy: 555
Meth:630nm, 500nm
cyanmeth:421, 540, also reaction with tannin, forms a strawberry red ppt, in absence brown grey.
Draw spectro
Quantitative estimation of protein in urine. Types of proteinuria.
Types of proteinuria:
a) Pre renal (high concentration of low molec weight pp, e.g. intravascular hemolysis or crush)
b) Renal (Glom and tubular and mixed)
Glomerular proteinuria results from an increased permeability of the
glomerular membrane for protein. In selective glomerular proteinuria the
negative charge of glomerular membrane is lost, which results in increased
excretion of the middle-size proteins of MW 70,000 – 100,000, such as albumin
and transferrin, while large proteins are preserved. In non-selective glomerular
proteinuria the selectivity with respect to the size is impaired as well, and in
addition to the middle-size proteins also species with MW above 100,000, such
as IgG, penetrate into urine. In glomerular proteinuria the daily loss of protein
usually exceeds 2 g.
- Tubular proteinuria is caused by decreased reabsorption of normally filtrated
proteins. High excretion of low-molecular-weight proteins (microproteins) of
MW 10,000 – 70,000 (2-microglobulin, 1-microglobulin, free Ig light chains),
which are normally reabsorbed in tubules, is found. A common cause is
damage to tubular cells by some nephrotoxic drugs (cytostatics, some
antibiotics) or heavy metals (Hg, Pb, Cd). Tubular proteinuria can also
accompany some prerenal proteinurias (paraproteinuria, myoglobinuria) due to
competition of the pathologic protein and other filtrated proteins for tubular
transport processes. Tubular proteinuria usually results in only moderate loss of
protein, typically 0.3 – 1.5 g/24 hours.
- Mixed proteinuria is a combination of non-selective glomerular and tubular
proteinuria. It is found in advanced stages of kidney diseases as a sign of
damage to all parts of the nephrons.
c) post renal: bleeding tumours and inflammation of urinary tract.
Electrophoresis based on their MW.
2) Quantitative estimation of protein in urine. If a pathologic proteinuria is found, a daily
loss of proteins in urine is measured. The urine is collected for 24 hours, mixed and a
sample is taken in which the protein concentration is measured. The urine protein
concentration in g/l multiplied by volume of urine in liters gives the daily loss of protein.
