Medical Laboratory Training Program

Student Name: Joanna Fields

Course: Biochemistry
Lecturer: Ms. Whittaker

Homework Assignment #2

Techniques used in the Biochemistry Lab

Spectraphotometry

A spectrophotometer consists of two instruments: a spectrometer that produces light of

different colors and a photometer that measures the intensity of the light. These instruments
are arranged so that a liquid in a cuvette can be placed between the spectrometer beam and
the photometer. The photometer measures the amount of light that passes through the tube
and sends a voltage signal to a display device. The display device changes as the amount of light
absorbed by the liquid changes. If the concentration of a substance in a solution is related to the
development of color, then this concentration can be measured by determining how much light
is absorbed at a specific wavelength. For example, hemoglobin appears red because it absorbs
blue and green light more effectively than red light. The absorption of blue or green light is
directly proportional to the concentration of hemoglobin.

Limitations:

 Proper sample preparation is crucial.

 It may not detect low concentrations due to limited sensitivity.
 Overlapping absorption bands can lead to inaccurate results.

Chromatography

Chromatography relies on the distribution or partition coefficient (Kd), which determines how a
compound is distributed between two phases. For two phases A and B, the Kd value remains
constant at a given temperature and is calculated by dividing the concentration in phase A by
the concentration in phase B. In chromatographic systems, there is a stationary phase (solid, gel,
liquid, or solid/liquid mixture) and a mobile phase (liquid or gas) that passes over or through the
Medical Laboratory Training Program
Student Name: Joanna Fields
Course: Biochemistry
Lecturer: Ms. Whittaker

stationary phase after applying the analyte mixture. The analytes continuously move between
the two phases during the separation process, leading to their separation based on differences
in their distribution coefficients.

Limitations:

 Cost and Complexity - Chromatographic instruments and materials can be quite pricey
and trained personnel are needed to effectively operate the equipment.
 Sample Overloading- If you overload the sample, it can lead to errors in separation. To
avoid this problem, proper sample preparation and loading are extremely important.
 Solvent Consumption- Some chromatography methods require a large amount of solvent
to separate analytes. This can be expensive and have a negative impact on the
environment.

Centrifugation

The centrifuge utilizes the sedimentation principle due to gravitational force. The centrifugation
technique uses a centrifugal field to separate particles suspended in a liquid medium. These are
put in the centrifuge’s rotor either in bottles or tubes. Sedimentation is a process whereby
gravity causes suspended particles to separate from fluids. The suspended substance may
consist of powder or clay-like particles.

Limitations:

 High energy usage because it has so many power-consuming features such as

temperature control and rotor spinning.
 Due to their high rotation rates per minute, centrifuges produce noise disruptions. Since
it uses rotations, vibrations are unavoidable, which will cause noise pollution and
prevent its use in some areas.
Medical Laboratory Training Program
Student Name: Joanna Fields
Course: Biochemistry
Lecturer: Ms. Whittaker

 Most often, a backup machine is required to step in when the primary equipment breaks
down.

Microscopy

The most basic type of light microscope is composed of a solitary glass lens set in a metal frame,
essentially functioning as a magnifying glass. In this setup, minimal preparation is needed for
the specimen, which is typically held near the eye by hand. To focus on the area of interest,
adjustments are made by moving the lens and the specimen in relation to each other. Light from
the light source goes through the condenser lens, which is placed under the microscope stage in
an upright microscope (or above the stage in an inverted microscope) in a bracket that can be
adjusted up and down for focusing. The condenser lens focuses light from the light source and
lights up the specimen with parallel beams of light. The human eye serves as the detector, while
the recording tool may be a hand-drawn illustration or a written anecdote.

Limitations:

 Cost: The acquisition, accommodation, and upkeep of microscopes can be quite

expensive.
 Sample Preparation: Extensive preparation of samples is frequently necessary prior to
imaging.
Medical Laboratory Training Program
Student Name: Joanna Fields
Course: Biochemistry
Lecturer: Ms. Whittaker

Enzyme Linked Immunosorbent Assay (ELISA)

There are different types of ELISA tests depending on whether they are checking for antibodies
or antigens. For instance, when checking for HIV, a portion of the virus is attached to a solid
surface like a medical tube or plastic plate. This virus acts as the antigen. A blood sample is then
added to the tube or plate. If the blood sample contains HIV antibodies, they will bind to the
antigen. If there are no HIV antibodies in the blood sample, nothing will happen. If there are no
antibodies to the specific antigen (HIV virus), there will be no color change, indicating a negative
result. The intensity of the color change is proportional to the amount of antibody present.
Therefore, ELISA can determine both the presence of the antibody and its quantity.

Limitations

 ELISA procedures can be time-consuming and complex as there are many steps involved,
like coating, blocking, incubating, and washing, which can be tiring.
 ELISA may not be sensitive enough to detect hard-to-find biomolecules. For instance, it
can be tough to detect microRNAs due to their small size and low levels.
 ELISA only looks at one analyte at a time. It's not good for screening multiple targets at
once.

Polymerase Chain Reaction PCR

The PCR method relies on DNA replication through enzymes. It involves amplifying a small
section of DNA using primer-mediated enzymes. DNA Polymerase creates new DNA strands that
match the template DNA. The DNA polymerase can only add a nucleotide to the existing 3’-OH
group. This is why a primer is necessary. As a result, more nucleotides are added to the 3’ prime
end of the DNA polymerase.
Medical Laboratory Training Program
Student Name: Joanna Fields
Course: Biochemistry
Lecturer: Ms. Whittaker

Limitations

 PCR requires prior knowledge of the target DNA sequence in order to design specific
primers accordingly.
 Errors may arise during replication in PCR due to DNA polymerases, potentially resulting
in mutations within the amplified products.
 The high sensitivity of PCR to contamination means that even a small quantity of
contaminating DNA can produce inaccurate outcomes.

Gel Electrophoresis

When an electric potential is applied, molecules with varying charges will separate due to their
different electrophoretic mobility. Even molecules with the same charge can separate if they
have different sizes, leading to varying frictional forces. Some electrophoresis methods rely on
charge differences for separation, while others focus on differences in molecular size. The
separated samples can be identified through staining with dye or autoradiography for
radiolabeled samples.

Limitations

 Gel electrophoresis requires a large tissue sample for running assays, which can be
challenging for medical analysis, particularly with proteins.
 Electrophoresis is limited in visualizing certain molecules effectively, with DNA and RNA
being somewhat visible but proteins posing a challenge. Despite this limitation, it
provides benefits in separation efficiency, time, and cost.
 Gel electrophoresis can separate proteins based on weight, but the measurements
obtained are only semi-quantitative. For precise mass determination, additional
techniques like mass spectrometry are needed.
Medical Laboratory Training Program
Student Name: Joanna Fields
Course: Biochemistry
Lecturer: Ms. Whittaker
Semiautomatic Analyzer vs Fully Automatic Analyzer

The Semi-Automatic Biochemistry Analyzer necessitates a combination of manual and

automated procedures. Operators are responsible for manually carrying out tasks such as
adding samples and recording results. However, certain stages in the analysis process, such as
colorimetric inhalation, are automated. This type of analyzer is well-suited for laboratories with
moderate testing volumes that can tolerate some level of manual intervention. It strikes a
balance between automation and operator involvement, making it particularly suitable for
specialized tests.

On the other hand, the Fully Automatic Biochemistry Analyzer completes the entire testing
process automatically, eliminating the need for manual intervention. From sample addition to
result reporting, all steps are executed by the instrument itself. This type of analyzer is
characterized by its high throughput and efficiency, making it suitable for laboratories with high
testing volumes. By minimizing the risk of human error and enhancing workflow automation, it
proves to be ideal for routine screening and comprehensive biochemical analysis in large-scale
laboratories.