Chemistry Techniques

Chromatography Electrophoresis

colorimetric: spectrophotometry immunoassay: radioimmunoasssay enzyme-linked immunoassay

 to separate out analyte of interest from a mixture  to check purity of analyte of interest

solid liquid

Liquid Gas

 planar chromatography  column chromatography

Partition or distribution coefficient (Kd): concentration in mobile phase = Kd concentration in stationary phase

Principle: differences in adsorption of analytes Procedure:sampleapplication ,development,detection Advantages:speed,cost,simplicity Disadvantages:not quantitative

 inherited absence of phenylalanine hydroxylase  inability to convert phenylalanine to tyrosine  phenylalanine therefore accumulates and is toxic to developing brain  lack of tyrosine results in lack of thyroid hormones, catecholamines and melanin  high serum phenylalanine detected by TLC used for screening of PKU

Principle: mobile phase passes through stationary phase and analytes separated separation based on differences in charge, size or shape separated components leave column in eluant and collected Procedure: sample application running of column sample collection

-Stationary phase is a cation exchange resin -sample cations displace mobile counterions and are retained on the column -anionic/neutral solutes are not retained -flow of mobile phase

-stationary phase is a microporous gel -small molecules have access to the whole matrix taking longest to pass through the column -medium sized molecules may have access to the larger pores -large molecules are totally excluded from the pores,passing rapidly through the column

Complemmatery molecule bound by specific interaction with ligand -flow of mobile phase -all other solutes are eluted

 defective production or action of insulin causes hyperglycaemia  hyperglycaemia responsible for acute symptoms  hyperglycaemia responsible for chronic complications  hyperglycaemia causes excessive glycation of haemoglobin  measurement of glycated haemoglobin by column chromatography used to monitor treatment of diabetes mellitus

Separation due to differences in migration of charged molecules in response to an applied electric field Electrophoretic mobility (μ) is determined by charge to mass ratio: μ = qE r q = net molecular charge r = molecular radius E = field strength

net charge on molecule size of molecule voltage of electric field

Cellulose acetate:  providesphysicalsupport  doesnotabsorbproteins  separation based on charge density

Agarose:  gelwithporesofvariablesize  separation based on molecular size  good resolution

 sampleapplication  electrophoresis of sample using kit  staining of separated components

 malignant proliferation of plasma cells in bone marrow  cells from a single clone make identical immunoglobulins  immunoglobulins detected as a discrete paraprotein band on electrophoresis used for diagnosis of multiple myeloma

Molecules in solution absorb light of a characteristic wavelength Beer-Lambert Law: absorption of light by molecules in solution is related to their concentration and length of light path through absorbing medium: A = Ecl A = absorbance E = molar absorption coefficient (Lmol-1 cm-1) c = concentration (mol/L or g/L) l = light path (usually 1 cm)

 analyte should absorb light of specific wavelength or be converted to substance that can  pureanalyterequiredforstandards  wavelength of light at which there is maximum absorbance is determined  relationship between absorbance and concentration must obey Beer-Lambert law

 speed  lowcost  automation  wideapplication

 associated with a decline in functioning nephrons  decreased removal of waste products  decreased removal of creatinine causes build-up of serum creatinine  creatinine reacted with chemical reagents eg picrate to give coloured products that absorb light  serum creatinine measurements useful in prognosis of renal failure