Calabano, Mark Jasper C.

I-BS Medical Technology

ANALYTICAL SEPARATIONS

Based on the reading material presented, answer the following review questions:

1. Differentiate chromatography and electrophoresis

Chromatography and electrophoresis are powerful analytical techniques that

both separate a sample into its components and provide a means for determining each
component’s concentration. They are very complementary to one another however, these two
analytical techniques separate different types of substances using a different principle.

In chromatography, the mixture is dissolved in a solvent, which we call a

mobile phase, and the sample moves along or through and all over the surface of a
stationary material, which we call the stationary phase. Whereas in electrophoresis, we
learn a mixture that is placed on a some form of a medium between two electrodes (a
positive and a negative electrode) and then the charge particles of the mixture move
towards the electrode opposite to them. So the negatively charged particles move towards
the positively charged particles and vice-versa. In electrophoresis, we are using an
electrical field to separate charged substances as opposed to chromatography, which any
works for uncharged. The key principle of chromatography has two aspects. First one is the
attraction to or the adsorption of the particles to the stationary phase. In stationary phase,
the more it adheres or it is attracted, the slower the rate that these particles move. And the
second is the different solubility in the mobile phase. The more soluble particles in the
mobile phase, the faster it travels. If particles are not soluble at all, then therefore it will not
travel far. Just like the concept of solubility that "like dissolves like". On the other hand,
electrophoresis, explains electric field separating charged particles, molecules, fragments,
ionic current, etc. Electrophoresis is based on the difference of the molecules' electric
charge and size. So, the principle is that, a combination of a large charged molecule with
small electric charged molecule will move the slowest whereas small molecules with high
electric charge will move more quickly. The separation in electrophoresis is based on
moving towards oppositely charged electrodes.

For the stationary phase, in chromatography, we can use solid materials

such as paper, silica plate like aluminium silica plate that is used in thin-layer
chromatography (TLC), a packed column like in high-performance liquid chromatography
(HPLC), or liquid coating on inside an open column like in gas liquid chromatography
(GLC). While in electrophoresis, for the stationary phase, we can use some sort of
absorbent medium like porous paper, a polyacrylamide or agarose gel like in gel
electrophoresis (GE), and also glass capillary that is used in capillary electrophoresis (CE).
In mobile phase, in chromatography, we can use liquid solvents. In some types of
chromatography, solvents can be aqueous and/ or organic and some combinations of polar
or non-polar, depending on what we're trying to separate. In the case of gas-liquid
chromatography, we can have a carrier gas like helium or argon. On the other hand, for
mobile phase, in electrophoresis, we use buffer solutions. The buffer solution controls the
pH of the solution which then, especially for amino acids, dictates the charge on the ions
and also, by containing electrolytes that allow the solution to conduct electricity. So, the
charge can flow and therefore the charged particles of the mixture can move towards the
appropriate electrode. The next important difference about these two techniques is the
substance that can be separated. So, in chromatography, we can use gas-liquid
chromatography (GLC) in separating substances that are volatile which can be turned into
a gas easily. All for things that are non-volatile or are too dangerous to be heating to high
temperatures, we can use high-performance liquid chromatography (HPLC). If we use a
special column like ion- exchange chromatography (IEC), we can separate ions. In
electrophoresis, we can separate mixtures of ions, or charged particles (atoms, molecules
or molecular fragments like in DNA). We can also caused uncharged molecules to become
or be given a charge using chemical reactions to make them appropriate to separate during
electrophoresis. An example of this process is the Micellar Electrokinetic Chromatography,
that is used to surround uncharged substances with charged substances (micelle) that can
then flow in electrophoresis.

2. Discuss one chromatographic method

Gas chromatography is a chromatography technique used for the separation

of volatile compounds. Volatile compounds are the compounds that get easily vaporized at
room temperature. Gas chromatography has several components. Components of gas
chromatography includes the column. The column used in gas chromatography is very long
and arranged in a coil. The column used in gas chromatography are of two types: Packed
column and Capillary column. Packed column can be made up of glass or stainless steel.
The length of this column can vary from 1 to 3 meters and has an internal diameter of 2 to 4
millimeters. The capillary column, on the other hand, is made up of fused quartz. These
columns are very long and have length from 10 to 100 meters. The internal diameter is 0.1
to 1 millimeter. The column is placed in a chamber so that a uniform temperature can be
maintained. The stationary phase with gas chromatography is packed in the inner wall of
the column. The stationary phase is made up of silicon grease or wax which can withstand
a high temperature. The mobile phase used with gas chromatography is usually inert gases
like Helium (He) or unreactive gas such as Nitrogen (N). The mobile phase gas is kept in a
cylinder which is connected with the column y which is a molecular sieve. The molecular
sieve separates the unwanted hydrocarbons, oxygen and water vapor that may interfere
with the test sample during analysis. At the end of the column, there is a detector which
detects the sample. So, how does gas chromatography work? The sample which is to be
separated, is mixed with appropriate volatile solvent such as heptane, acetone or methanol.
Just before the column, there's a septum that allows injection of the sample. The
temperature of the injection region is kept 20 to 50 degree Celsius high, as compared to the
column. This allows rapid volatilization of the sample. Once the sample is volatilized, it
passes down the column, where the separation occurs. During analysis, the temperature of
the column is kept between 150 to 300 degree Celsius. Separation occurs based on the
interaction of molecules between the mobile phase and stationary phase. The less volatile
molecules interact more with the stationary phase, the more it moves slowly while the more
volatile molecules interact more with the mobile phase, the more it moves fast down the
column. Once the separation is completed, the detection is done with a detector attached at
the end of the column. One of the most common detectors used with gas chromatography
is flame-ionization detector (FID). The flame-ionization detector has three inlets. One for
the carrier gas, which comes from the column and the other tube for hydrogen and oxygen.
The igniter ignites hydrogen and oxygen to produce a flame. When the sample molecules
reaches the flame, they gets ionized, and electrons are released. Across the flame, there
are two electrodes, each with a positive and a negative charge. The electrodes detect
electrons generated by the ionization. of the sample. The electrons are detected in the form
of current which is amplified and detected by the computer. When the sample is detected,
the computer gives a peak with respect to the retention time (t t) of the sample. The area
under the peak gives information about the concentration of the sample. If the
concentration is less, the area under the peak will be less and if the concentration is more,
the area under the peak will be more.

3. Make some research about the importance of electrophoresis in laboratory testing

Electrophoresis analysis is a method that is used in clinical labs to create

purified samples of proteins and separate proteins of differing sizes and charges. In clinical
laboratories, electrophoresis is a very important technique because it is useful to do several
analyses such as lipoprotein analysis, protein analysis, and cerebrospinal fluid analysis. It is
also used for the determination of serum microheterogeneities, diagnosis of
haemoglobinopathies and haemoglobin A1c, monitoring of small molecules such as drugs and
steroids, and separation of DNA molecules. In microbiology labs, electrophoresis is an important
technique because it is used to identify microorganisms.

In lipoprotein analysis, free-Sow isotachophoresis (ITP) and capillary ITP (cITP)

has been utilized for many years to study plasma lipoproteins. Isotachophoresis (ITP) is one of
the fundamental electrophoretic separation techniques, where charged constituents are
separated in an electric field due to their differences in their electrophoretic mobilities. In protein
analysis, protein electrophoresis test is used to measure specific proteins in the blood. The test
separates proteins in the blood based on their electrical charge. The protein electrophoresis test
is often used to find abnormal substances called M proteins. The presence of M proteins can be
a sign of a type of cancer called myeloma, or multiple myeloma. Myeloma affects white blood
cells called plasma cells in the bone marrow. Protein electrophoresis also tests for other
proteins and immunoglobulins.  The protein electrophoresis test is also used to diagnose other
conditions affecting the plasma cells. These include Waldenström macroglobulinemia,
monoclonal gammopathy of undetermined significance (MGUS), and primary amyloidosis.
Protein electrophoresis can also be used to help diagnose thyroid problems, diabetes, anemia,
liver diseases, poor nutrition or inability to absorb nutrients and certain autoimmune diseases.
Similar test is the Serum protein electrophoresis that is widely used for the evaluation of
changes in proteins associated with inflammation, liver or kidney diseases as well as for the
detection and identification of paraproteins. In the analysis of cerebrospinal fluid (CSF), proteins
using electrophoretic techniques are particularly useful in the diagnosis and management of
neurological diseases particularly in the detection of immune response within the central
nervous system (CNS). In the determination of serum microheterogeneities, two-dimensional
electrophoresis can be used to study the microheterogeneity of serum glycoproteins in patients
with chronic alcohol abuse as well as in patients with carbohydrate-deficient glycoprotein
syndrome. In the diagnosis of haemoglobinopathies and haemoglobin A1c, isoelectric focusing
has been used to diagnose disorders of haemoglobin synthesis such as sickle cell disease, and
thalassaemias or haemoglobin variants grouped under the term ‘haemoglobinopathies’
Isoelectric focusing provides an excellent haemoglobin separation, with very little band overlap
when bands are measured to 0.1 mm against controls. Isoelectric focusing (IEF) is an
electrophoretic separation method which separates amphoteric molecules such as proteins and
peptides according to their charge as defined by the pKa values of proton-accepting sites within
a molecule. For small molecules drugs, steroids monitoring, capillary electrophoresis of drugs,
as well as their metabolites in almost all biological samples, is used for diagnostic drug
monitoring. Capillary electrophoresis is an analytical technique that separates ions based on
their electrophoretic mobility with the use of an applied voltage. And for the separation of DNA
molecules, agarose gel electrophoresis is most commonly used. Agarose gel electrophoresis is
one of several physical methods for determining the size of DNA.  In the identification of
microorganism, pulsed-field gel electrophoresis (PFGE) is used for typing of bacteria. Pulsed-
field gel electrophoresis (PFGE) is a highly discriminative molecular typing technique that is
used in epidemiological studies worldwide.