Analytical techniques in

Biochemistry
Dr. Anand R
Assistant Professor
Department of Biochemistry
Christian Medical College, Vellore
17th March 2016

Objectives
At the end of the lecture, you should be able to
1. Define the principle behind
spectrophotometer/colorimeter
2. List the applications of a spectrophotometer
3. Explain the applications and principle of ion selective
electrodes
4. List the applications of radioisotopes in medical
science
5. Name the commonly used radioisotopes in clinical
medicine
6. Explain the principle, types and applications of
dialysis

What are analytical techniques?

Techniques that help us to answer 2 major
questions
1. Whats there?
2. If present, how much?
Analyte- the substance that is measured

To test: Whats there?

Use of an indicator- simplest
Indicator changes the color of the solution
when a particular analyte is present
E.g. Benedicts test for glucose
The result is YES or NO

What is this data called?

-Qualitative data

To test: How much is there?

Techniques that accurately measure and give
numerical values
Quantitative tests

Why is it needed?
1. Helps is making a diagnosis
2. Helps in monitoring treatment
3. Helps in predicting the outcome

Common analytical techniques

1.
2.
3.
4.
5.

Optical spectroscopy eg. spectrophotometer

Electrochemistry eg. ion selective electrodes
Radioisotopes
Electrophoresis
Chromatography

1. Optical techniques
Those which are based on measurements of
light emitted, transmitted, absorbed, reflected
or scattered
E.g Colorimeter, spectrophotometer

Spectrophotometer
An instrument that measures the amount of
light that is transmitted through a sample
solution

Spectrophotometer

http://chemwiki.ucdavis.edu/@api/deki/files/8472/Spc20Blnk.gif

Two types of spectrometers

1. Ultraviolet spectrophotometers
Uses light in the wavelengths 200-350 nm
2. Visible light spectrum spectrophotometers
Uses light in the wavelengths 400-700 nm

http://cjacks20.edublogs.org/files/2012/10/visspectrum-1c6o7rf.gif

How does a spectrophotometer

work?

But why is the Box Blue?

Blue molecules are blue because they
reflect blue light.

Blue molecules absorb the other

colors of visible light.

 Molecules are whatever color of light that

they do not absorb

Green molecules appear green because

they absorb most wavelengths of visible
light, except the green wavelengths.

 The spectrophotometer measures the amount

of light transmitted through the sample
(Transmittance)

By using a formula transmittance data can be

converted to an absorbance value

Parts of a Spectrophotometer
Outer parts:

Wavelength
Selection
Display

Sample
Holder
Knobs or buttons used to calibrate the
spec to measure the designated molecule.

http://www.flushgordon.info/spectro.gif

Spectrophotometer and colorimeter

Spectrophotometer:
To determine the absorbance of colored or
colorless solutions
Uses specific wavelength Eg 540 nm
Colorimeter:
To determine the absorbance of a colored
solution
Uses a band of wavelength Eg 500-560 nm

Applications of spectrophotometer
1. Qualitative estimation- Identification of an
unknown substance
May be used to identify various classes of
compounds in biological samples.
Eg. Proteins absorb light between 240-280nm

Applications of spectrophotometer
2. Quantitative estimation- Identification of
concentration of the unknown substance
Used in clinical laboratories for measuring
concentrations of different substances
Eg. Concentration of glucose in blood
Concentration of proteins in urine

Applications of spectrophotometer
3. To measure the activity of enzymes
By monitoring the rate of appearance or
disappearance of light absorbing product or
substrate can be measured with the help of
spectrophotometer
Eg. Activity of lactate dehydrogenase in serum

2. Electrochemistry
Methods in which measure change in electric
current or potentials are measured
e.g. Ion selective electrodes

Principle
Two identical electrodes when placed in two
similar solutions (different concentrations)
and are connected by a salt bridge, it gives rise
to a potential difference
The potential between electrodes is related to
the ratio of the two concentrations
If concentration of one solution is kept
constant, concentration of unknown can be
found out

Principle

http://resource.rockyview.ab.ca/t4t/chem30/images/m4/005_voltaic_cell.jpg

Ion-selective electrodes (ISE)

- Also known as indicator electrodes
- Respond directly to the analyte
- Used for direct measurements

- Selectively binds and measures the activity of one

ion

Examples
pH electrode
Calcium (Ca2+) electrode
Chloride (Cl-) electrode

Applications of ion selective electrodes

To measure pH of blood- Arterial blood gas
analysis
To measure concentrations of electrolytes in
serum, plasma or urine- Na+, K+, Cl-, HCO3 To measure concentrations of other charged
ions in plasma- calcium, ammonium, lithium

3. Radioisotopes
Methods which use radioactive elements and
measure change in radioactivity

 Isotopes are elements that have the same

atomic number but different atomic weights
Radioactivity: process by which a nucleus of
an unstable atom loses its energy by emitting
radiation
Molecules with the property- radioisotopes

http://creationwiki.org/images/thumb/8/8a/Radioactiveatom.JPG/350px-Radioactive-atom.JPG

Uses of radioisotopes in medicine

1. Diagnostic purposes (to identify a disease):
Fluorine (18F) is used for positron emission
tomography (PET) scans
Technetium (99mTc) is used for scanning
thyroid, bones, brain and heart

2. Treatment purposes

Iodine (131I) is used for the treatment of thyroid

diseases.
Treatment of cancers (radiotherapy)
3. Radioisotopes are widely used in research 14C,
15N, 2H etc

Advantages
Radioactivity can be easily monitored
Creation of images are easily done, so helps in
visualization
Sensitive and accurate

Disadvantages
Radiation can be harmful to patients and
people working with it

Very long half life

Storage and disposal of radioactive elements
is an important issue. Proper setup is required
to handle radioisotopes

Dialysis

 Diffusion: simply means movement of

substance from a higher concentration to
lower concentration
Osmosis: refers to solvent (water) across a
semipermeable membrane
Dialysis: refers to movement of solutes across
a semipermeable membrane

 Process of separating molecules in

solution based on their differences in
diffusion rates across a semi-permeable
membrane

http://ehumanbiofield.wikispaces.com/file/view/dialysis.jpg/32971865/dialysis.jpg

Types of dialysis in medicine

1. Hemodialysis
The patient's blood is pumped through the
blood compartment of a dialyzer which filters
blood
The cleansed blood is then returned via the
circuit back to the body
2. Peritoneal dialysis
Dialysis fluid is pushed into the patients
abdominal cavity

Dialysis in medicine
1. Used as a treatment in conditions there is
failure of kidney functions
Renal replacement therapy

2. Treatment of poisonings eg. Lithium, aspirin,

alcohol
3. Liver dialysis for liver diseases

Functions of kidneys

Dysfunction

1. Water balance

Fluid retention

2. Sodium balance

Imbalance in the ions

Low sodium
High potassium, magnesium
and phosphate

3. Potassium balance

4. Magnesium, bicarbonate,
phosphate balance
5. Excretion of urea,
Increased blood urea,
creatinine, uric acid
creatinine and uric acid
6. Endocrine functions
Low vitamin D formation

Goals of dialysis
Solute clearance (clearance of accumulated
waste products in body)
Removal of excess water
Does not cure or completely treat renal
failure

Hemodialysis

http://bkfindia.in/files/photo_website/new/DIALYSIS%201.JPG

Peritoneal dialysis

http://www.mmgazette.com/wp-content/uploads/2014/01/peritoneal dialysis.jpg