Chromatography

- refers to the group of techniques used to separate complex mixtures based on the different degrees to which they interact
with two separate material phases

Basic Components

Mobile Phase Stationary Phase

gas or liquid solid or liquid

which carries the complex mixture – SAMPLE through which the mobile phase flows

moving phase does not move

acidic polar – silica gel

basic polar – alumina

nonpolar – charcoal

Types of Chromatography

Ion Exchange Chromatography

o Solute mixtures are separated by virtue of the magnitude and charge of ionic species
o The stationary phase is a resin, consisting of large polymers of substituted benzene, silicates, or cellulose derivatives, with
charge functional groups.
o The resin is insoluble in water, and the functional groups are immobilized as side chains on resin beads that are used to fill
the chromatographic column
o Changing pH and ionic concentration of the mobile phase allows separation of mixtures of organic and inorganic ions
o https://youtu.be/VOSkyj1dtbc - Ion Exchange

Thin Layer Chromatography

o Is a variant of column chromatography.

o A thin layer of sorbent, such as alumina, silica gel, cellulose, or crosslinked dextran, is uniformly coated on a glass or plastic
plate
o Each sample to be analyzed is applied as a spot near one edge of the plate
o The mobile phase (solvent) is usually placed in a closed container until the atmosphere is saturated with solvent vapor
Retention Factor

o Retention factor is defined as the ratio of distance traveled by solute to distance traveled by solvent.

Dsitancetraveled by solute X
Rf = =
Distancetraveled by solvent Y
High Performance Liquid Chromatography

o Uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution.
o Common HPLC detector is the amperometric or electrochemical detector, which measures electrical current produced when
the analyte of interest is either oxidized or reduced at some fixed potential set between a pair of electrodes.
o https://youtu.be/ZN7euA1fS4Y
Gas Chromatography

o Is used to separate mixtures of compounds that are

volatile or can be made volatile.
o Gas chromatography may be gas-solid chromatography
(GSC), with a solid stationary phase, or gas-liquid
chromatography (GLC), with a nonvolatile liquid stationary
phase
o Is commonly used in clinical laboratories
o https://youtu.be/UycPljfrnWo

Partition Chromatography

o Partition chromatography is a type of chromatography in which the stationary phase is a liquid chemically bonded to the
surface of a solid substrate, whereas the mobile phase is either a liquid or gas.
o The mixture components dissolve in and out of the mobile and stationary phases as the mobile phase moves through the
stationary phase and the separation occurs as a result.
o Method of separation in which the components present in mixture get distributed more likely into two liquid phases be
differences in partition coefficients during the flow of mobile the chromatography column.
o Separation is based on solute partitioning between two liquid phases. (Relative solubility)

Adsorption Chromatography

o As the name implies, the separation mechanism is one of

adsorption.
o The stationary phase consists of finely divided solid
particles packed inside a tube but with no stationary liquid
substance present to function as the stationary phase, as is
the case with partition chromatography
o https://youtu.be/YRFSN9q523M
Size Exclusion Chromatography

o Also called gel permeation chromatography (GPC) or gel filtration chromatography (GFC), is a technique for
separating dissolved species on the basis of their size.
o The stationary phase consists of porous polymer resin particles
o https://youtu.be/L-xIHihOGKs

Paper Chromatography

o makes use of a sheet of paper having the consistency of filter paper (cellulose) for the stationary phase.
o Since such paper is hydrophilic, the stationary phase is actually a thin film of water unintentionally adsorbed on the
surface of the paper
o https://youtu.be/mz_xcNrTK_U

Open Column Chromatography

o Another configuration for chromatography consists of a vertically positioned glass or plastic tube in which the stationary
phase is placed.
o It is typical for this tube to be open at the top (hence the name opencolumn chromatography), to have an inner diameter on
the order of a centimeter or more, and to have a stopcock at the bottom, making it similar to a burette in appearance
o https://youtu.be/2R2iq_XR1IY

Summary of the Different Chromatography Configurations

Configuration Geometry Flow Direction Applicable Types

Adsorption, partition, ion
Paper Planar Ascending, descending, or radial
exchange, size exclusion
Adsorption, partition, ion
Thin Layer Planar Ascending, descending, or radial
exchange, size exclusion
Adsorption, partition, ion
Open Column Column Descending
exchange, size exclusion
GC Column N/A Adsorption, partition
Adsorption, partition, ion
HPLC Column N/A
exchange, size exclusion

Electrophoresis
o Is a separation technique in which mixture components separate based on their differing migration rates in an electric
field.
o A very popular electrophoresis configuration today is the instrumental method called capillary electrophoresis.
o Capillary electrophoresis closely resembles instrumental liquid chromatography.
o https://soe.unipune.ac.in/studymaterial/ashwiniWadegaonkarS elf/BSC%20821%20Ch%205.pdf
 Types of Electrophoresis

Zone Electrophoresis

o https://youtu.be/7F3tzlDP5ps

Paper Electrophoresis

 This technique is useful for the separation of small charged molecules such as amino acids and small proteins.
 It is a form of electrophoresis that is carried out on filter paper. This technique is useful for separation of small
charged molecules such as amino acids and small proteins.
 A strip of filter paper is moistened with buffer and the ends of the strip are immersed into buffer reservoirs containing
the electrodes.
 Whatman filter paper, cellulose acetate filter paper or chromatography paper

Gel Electrophoresis

 Gel electrophoresis is a technique used to separate DNA fragments according to their size.
 DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them
through the gel.
 DNA fragments are negatively charged, so they move towards the positive electrode.
 https://youtu.be/MhJT9yjnl88

Thin Layer Electrophoresis

 Studies can be carried out in thin-layer of silica, kieselguhr, alumina.

 Advantages: Less time consuming and good resolution.
 Application: Widely used in combined electrophoretic-chromatography studies in two-dimensional study of proteins
and nucleic acid hydrolysates.
 Thin-layer electrophoresis appears to be a very convenient method for a preliminary rapid analysis, especially of urine.
 In most cases the deproteinization and desalting of samples are not required, and the procedure can be completed with
photo densitometric analysis.

Cellulose Acetate Electrophoresis

 Is a type of zone electrophoresis that separates molecules in liquid form such as proteins on a membrane or stripe made
of cellulose acetate.

Moving Boundary Electrophoresis

Capillary Electrophoresis

 Is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage.
 The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom's radius.
 https://youtu.be/x7PUqNA0eOA

Isotachophoresis

 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 terms of arrangement of the separation part, ITP could be performed on a capillary isotachophoresis (CITP) or planar
format.
 Has been used for analysis of organic acids in silage, anions in urine and serum, inorganic ions in water, proteins, and
amino acids.

Isoelectric Focusing

 Is a high-resolution technique that can resolve proteins differing in pI by less than 0.05 pH unit and is generally carried
out under nondenaturing conditions, in which antibodies, antigens, and enzymes maintain most of their biochemical
properties.
 Can be used to assess the complexity of protein extracts and to identify components of interest
 Immuno Electrophoresis

 Is defined as the separation and identification of proteins based on differences in electrical charge and reactivity with
antibodies
 Mainly used clinically to determine the blood levels of immunoglobulins, and aids in the diagnosis and evaluation of the
therapeutic response in many disease states affecting the immune system and also in the diagnosis of multiple myeloma

Quality Control
o Is a statistical process used to monitor and evaluate the analytical process that produces patient results.
o For example, when a patient's serum is assayed (tested) for potassium, the test result tells us how much potassium
(concentration) is present in the blood.
o QC in the laboratory involves the systematic monitoring of analytic processes in order to detect analytic errors that occur
during analysis and to ultimately prevent the reporting of incorrect patient test results
o Objectives of Quality Control
 Primary objective: to ensure that result of analysis on patients’ specimen is reliable enough to be applicable in
his/her care without any doubt & does not lead to incorrect clinical interpretation.
 Overall objective: that the result obtained from one lab is comparable to all others using same method with same
degree of significance.

Quality Control in Clinical Laboratory

Hurdles in Quality Control

Process Potential Errors

Test Ordering Inappropriate Test
Handwriting not legible
Wrong patient identification
Special requirements not specified
Cost or delayed order
Specimen Acquisition Incorrect tube or container
Incorrect patient identification
Inadequate volume
Invalid specimen (e.g., hemolyzed or too dilute)
Collected at wrong time
Improper transport conditions
Analytical Measurement Instrument not calibrated correctly
Specimen mix-up
Incorrect volume of specimen
Interfering substance present
Instrument precision problem
Test Reporting Wrong patient identification
Report not posted in chart
Report not legible
Report Delayed
Transcription error
Test Interpretation Interfering substances not recognized
Specificity of test not understood
Precision limitations not recognized
Analytical sensitivity not appropriate
Previous values not available for comparison

Types of QC

Internal

o Daily
o Establishment of reference ranges
o Validation of a new reagent lot and/or shipment
o Following instrument repair
  CAP, CLIA, the Joint Commission
requirement
External
 Must be integrated within routine workload
o Proficiency testing and analyzed by personnel who are running
 Determination of laboratory testing the tests
performance by means of intra-laboratory  Ongoing evaluation of results to correct for
comparisons unacceptable results
 Used to access employee competency