OVERVIEW

-History and introduction of chromatography

-HPLC types and HPLC instrument System
-Principles of working of HPLC
-Advantages and Uses of HPLC
-HPLC Chromatograms
-Chromatography was invented by the Russian botanist Mikhail
Tswett (1872-1919) in the early 1900s.
The technique was first used to separate different plant pigments
from one another.
-Chromatography has developed into a widely used method to
separate various components ofa substance from one another.
Some definations -
•Chromatography : physical method in which separation of
components takes place between two phases-a stationary phase and
a mobile phase
•Stationary phase: The substance on which adsorption of the
analyte (the substance to be separated during chromatography)
takes place. It can be a solid, a gel, or a solid liquid combination
•Mobile phase : solvent which carries the analyte (a liquid or a gas)
Chromatographic techniques are divided into different types based
on:
a) The type of chromatographic bed used
i.e. column chromatography (gas chromatography) and planar
chromatography(paper and thin layer)
b) The physical state of mobile phase
i.e. gas chromatography and liquid chromatography
c) The separation mechanism
i.e. ion-exchange and size exclusion
HPLC is a type of liquid chromatography where the sample is forced
through a column that is packed with a stationary phase
composed of irregularly or spherically shaped particles, a porous
monolithic layer , or a porous membrane by a liquid (mobile phase)
at high pressure.
TYPES OF HPLC BASED ON MODE OF SEPARATION
a. Normal phase chromatography - stationary phase is
polar(hydrophilic) and mobile phase is non-polar (hydrophobic).
b. Reverse phase chromatography- stationary face is non-
polar(hydrophobic) and mobile phase is Polar (hydrophilic).
II.BASED ON PRINCIPLE OF SEPARATION
a.Absorption Chromatography
•In the Absorption Chromatography solute molecules bond directly
to the surface of the stationary phase
•the component which has more affinity towards mobile phase
elutes first & the component which has less affinity towards
stationary phase
elutes later.
No two components have same affinity towards mobile phase &
stationary phase.
b. lon-exchange chromatography
•Ion exchange chromatography is a process that allows the
separation of
ions and polar molecules based on their charge.
•It can be used for almost any kind of charged molecule including
large proteins, small nucleotides and amino acids.
•Retention is based on the attraction between solute ions and
charged sites bound to the stationary phase. Ions of the same charge
are excluded.
Points to remember before
analysis
• The calibrator should be diluted
with 7 ml cold calibrator diluent
The calibrator vial should be
allowed to stand at room
temperature for 5-10 minutes
• 1.5 ml of the calibrator should be
placed in the vials with adapters
having their respective barcodes
and then used for analysis.
Points to remember after
analysis
Once the system is calibrated for
A1c it requires calibration only
when the Buffer 1 can is replaced
with a new one.
•For Hb A2/F/A1c, the calibration
should be performed once in
every 24 hours or when switching
from the A1c program to the dual program.
Acceptance criteria for a
D-10Hb A2/F/A1c,
calibrator
• The calibration should pass
• The Total Area Count of the
chromatogram should be in the
range of 1 million to 4 million
• The peak chapo shoud be sharp.
and symmetrical, there should be
no dogradation peaks at the end
of the run.
c. lon-pair chromatography
•It is a form of chromatography in which ions in solution can be
"paired"
or neutralized and separated as an ion pair on a reversed-phase
column.
d. gel permeation chromatography
•This type of chromatography lacks an attractive interaction
between the
stationary phase and solute.
•The liquid or gaseous phase passes through a porous gel which
separates the molecules according to its size.
e. Affinity Chromatography
•This is the most selective type of chromatography employed. It
utilizes
the specific interaction between one kind of solute molecule and a
second molecule that is immobilized on a stationary phase.
- Automated cation exchange HPLC is
being increasingly used as a the initial
diagnostic method in diagnosis of
thalassemias and
hemoglobinopathies.

HPLC instrumentation includes:

• Reservoir
• Pump
• Injector
• Column
• Detector
• Recorder or data system.
A. Solvent delivery system(mobile phase)
•The mobile phase in HPLC refers to the solvent being continuously
applied to the column or stationary phase
•The mobile phase acts as a carrier to the sample solution
•A sample solution is injected into the mobile phase of an assay
through the injector port
•The chemical interaction of the mobile phase and sample, with the
column , determine the degree of migration and separation of
components contained in the sample
•The solvents or mobile phases used must be passed through the
column at high pressure at about 1000 to 3000 psi. this is because
as the particle size of stationary phase is around 5-10u, so the
resistance to the flow of solvent is high.
B. Pumps
•The role of the pump is to force a liquid (called the mobile phase)
through the liqaid chromatograph at a specific flow rate, expressed
in milliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000 psi
(400-to 600-bar).

-1.Reciprocating piston pumps

-2.Syringe type pump
-3.Constant pressure pump
C. Injector:
•The injector serves to introduce the liquid sample into the flow
stream
of the mobile phase for analysis.
•It is equipped with six port valves so that a samnple can be injected
into
the flow path at continuous pressure
•Typical sample volumes for manual injector are 5-to 20-
microliters(uL).
D. Column
•Considered the heart of the chromatograph" the column's
stationary phase separates the sample components of interest using
various physical
and chemical parameters.
•It is usually made of stainless steel to withstand high pressure
caused by
the pump to move the mobile phase through the column packing
other material include PEEK (poly ether ketone) and glass.
•The small particles inside the column are called the "packing" what
cause the high back pressure at normal flow rates.
•Column packing is usually silica gel because of its particle
shape,surface properties, and pore structure give us a good
separation.
•The dimensions of the
analytical column are usually
straight,
Length(5 ~ 25 cm),
diameter of column(3 ~ 5 mm)
diameter of particle(35 m).
•Other material used include
alumina, a polystyrene-divinyl
benzene synthetic or an ion-exchange resin
E.Detector:
•The detector can detect the individual molecules that elute from
the column and convert the data into an electrical signal
•A detector serves to measure the amount of those molecules
•The detector provides an output to a recorder or computer that
results in the liquid chromatogram
•Detector is selected based on the analyte or the sample under
detection.
Basic detector requirements-
An ideal detector should have the following
properties:
-Low drift and noise level (particularly crucial in
trace analysis).
-High sensitivity.
-Fast response.
-Wide linear dynamic range (this simplifies
quantitation).
-Low dead volume (minimal peak broadening).

F. Data System
• For better accuracy and precision
Routine analysis
- pre-programmed computing integrator
Data station/computer needed for higher control
levels
add automation options
- complex data becomes more feasible.
Retention time: time in minutes from the sample injection to the
maximum point of the elution peak of normal haemoglobin and
abnormal haemoglobins.
Normal Hb types percentage:
In adults :
Hb A:95% to 98%
Hb A2: 2% to 3%
Hb F: 0.8% to 2%
Hb S: 0%
Hb C: 0%
In infants and children:
Hb F (newborn): 50% to 80%
Hb F (6 months): 8%
Hb F (over 6 months): 1% to 2%
Biorad- HPLC (Retention times)
Peak name. Retention time.

Pre integration. <0.63min

F window. HbH, Hb Barts, HbF1
P2 window HbF
0.98-1.20
P3 window HbA1c
1.24-1.40
A0 window HbJ Meerut
1.40-1.90
A2 window HbA
1.93-3.10
D window HbA2, HbLepore, HbD

3.30-3.90 Iran

S window HbD Punjab

3.90-4.30
Unknown Hbs
4.30-4.70
C window HbQ India
4.70-4.90
HbC
4.90-5.30
Advantages of HPLC:
-Automated technique, less time and permits
processing of large batches.
-Very small(0.5ul of blood) samples are
sufficient for analysis.
-Quantification of normal and abnormal
haemoglobin variants can be done on each
sample.
-Highly sensitive and specific.
Uses of HPLC:
-Diagnosis of B thalassemia trait
-Diagnosis of HbDPunjab, HbE, HbQindia
-Pre operative screening of Hbs cases
-Diagnosis of HbH cases
-Diagnosis of rare haemoglobins
-Diagnosis of hereditary persistence of foetal
haemoglobin(HPHF)
-Antenatal screening for thalassemia and sickle cell disease.
Other tests for Hb pathy
• Hb electrophoresis
a) Cellulose actate (alkaline pH- 8.5)
b) Citrate gel (acidic-6)
Capillary elecrophoresis
• Iso electric focussing
• Molecular: PCR, Reverse dot blott
Beta Thalassemia Trait (Hb A2: 4-7%)

Laboratory findings:
Hb is normal or slightly reduced.
The MCV and MCH are reduced (MCV is < 80 fl, MCH< 27 pg) and the
RBC count is elevated.
RDW is normal in thalassemia.
Peripheral smear shows Microcytosis, hypochromia and occasional
target cells

Chromatogram findings:
Cut off of Hb A2, levels for carriers lies between 3.6 and 4.0%. Each
laboratory should establish their own cut off levels.
Borderline Hb A, values (3.6-4.0%) could result due to some mild
Beta thalassemia alleles or co-inheritance of delta thalassemia.
Also, presence of alpha thalassemia can result in normal MCV and
MCH values
Beta Thalassemia Trait (Hb A: 7-9%)

Chromatogram findings:
Elevated Hb A levels for carriers lies between 7 and 9%.
These deletions could be associated with elevated Hb F levels.

Beta Thalassemia Intermedia

Hb F is remarkably variable among patients, ranging from 5 to 100%

based on whether the patient is dependent on transfusion or not.
If the sample contains greater than 16.5% Hb F, the Hb Fmay elute in
the LA1c/ CHb window or A1c window and no Hb F will be reported.
A marked increase of Hb F is seen with a concomitant variable
reduction in hemoglobin A, usually constituting 10-35% of the total
hemoglobin.
Hb A, levels may be reduced, normal or elevated.