Chemistry Techniques

Question 1

Seperation techniques

Answer 1

Chromatography
Electrophoresis

Question 2

Detection techniques

Answer 2

colorimetric: spectrophotometry immunoassay: radioimmunoasssay
enzyme-linked immunoassay

Question 3

What is the purpose of separation techniques

Answer 3

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

Question 4

Stationary phase of chromatography

Answer 4

solid
liquid

Question 5

Mobile phase in chromatography

Answer 5

Liquid
Gas

Question 6

Types of chromatography

Answer 6

 planar chromatography
 column chromatography

Question 7

KD

Answer 7

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

Question 8

Thin layer chromatography

Answer 8

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

Question 9

Phenylketonuria

Answer 9

 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

Question 10

Column chromatography

Answer 10

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

Question 11

Ion exchange chromatography

Answer 11

-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

Question 12

Gel exclusion chromatography

Answer 12

-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

Question 13

Affinity chromatography

Answer 13

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

Question 14

Diabetes Mellitus

Answer 14

 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

Question 15

Electrophoresis

Answer 15

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

Question 16

Factors affecting electrophoresis

Answer 16

net charge on molecule
size of molecule
voltage of electric field

Question 17

Types of support medium :inert

Answer 17

Cellulose acetate:

 providesphysicalsupport
 doesnotabsorbproteins
 separation based on charge density

Question 18

Types of support medium :porous

Answer 18

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

Question 19

Procedure of electrophoresis

Answer 19

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

Question 20

Multiple myeloma

Answer 20

 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

Question 21

Principle spectrophotometry

Answer 21

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)

Question 22

Colorimetric methods requirements

Answer 22

 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

Question 23

Advantages of colorimetric methods

Answer 23

 speed
 lowcost
 automation
 wideapplication

Question 24

Renal disease

Answer 24

 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

Question 25

Using colorimeters

Answer 25

1: switch on at least 10 minutes before use to allow instrument to warm up and stabilise
2: set the correct wavelength
3: use the same cuvette for blank, standards and samples
4: do not overfill cuvette ie more than 2/3 the way up
5: gently wipe outside of cuvette to clean and dry

1: switch on at least 10 minutes before use to allow instrument to warm up and stabilise
2: set the correct wavelength
3: use the same cuvette for blank, standards and samples
4: do not overfill cuvette ie more than 2/3 the way up
5: gently wipe outside of cuvette to clean and dry
11: if method obeys Beers law then use linear part of graph to determine unknowns drawing line of best fit
12: if values for unknowns do not fall on linear part of graph then dilute original solutions, repeat analysis and multiply new unknowns by dilution factor