HPLC

Presented by-Dr.Siddhartha ghelani

P.G Teacher-Dr.Hemina desai
 Index
• INTRODUCTION
• PRINCIPLE
• INSTRUMENTATON
• CHROMATOGRAMS INTERPRETATION
• APPICATIONS
• ADVANTAGES AND DISADVANTAGES OF OF HPLC
• RECENT ADVANCES
• CASE
 INTRODUCTION
• HPLC stands for “High-performance liquid chromatography”(sometimes
referred to as High-pressure liquid chromatography).

• Is a technique used to separate and identify the components of a mixture

which works by allowing the molecules present in the mixture to distribute
themselves between a stationary and a mobile medium/phase.

• High performance liquid chromatography is a powerful tool in analysis, it

yields high performance and high speed compared to traditional columns
chromatography because of the forcibly pumped mobile phase.
 History
• The word chromatography means "color writing“

• The name Chromatography was coined from the Greek words chromo
(color) and graphy (to write).

• While studying the coloring materials(chlorophyll and carotenoids) in

plant life , a Russian botanist named M.S.Tswett invented
chromatography in 1903.

• Chromatography is such an important technique that two nobel prizes

have been awarded to chromatographers in 1952(for partition
chromatography) and 1980.
 BASICS AND PRINCIPLE of HPLC
• HPLC is a separation technique that involves:
the injection of a small volume of liquid sample into a tube packed with
tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

• where individual components of the sample are moved down the packed
tube (column) with a liquid (mobile phase) forced through the column by
high pressure delivered by a pump.

• These components are separated from one another by the column packing that
involves various chemical and/or physical interactions between their molecules and
the packing particles.
Types of HPLC separation
• Since no two components have the same affinity towards the stationary phase,
the components are separated

• These separated components are detected at the exit of this tube(column) by a

flow-through device (detector) that measures their amount.The output from
the detector is called a chromatogram

• In principle, LC and HPLC work the same way except the speed ,efficiency,
sensitivity and ease of operation of HPLC is vastly superior .
 Mobile phase degassing
• Dissolved gases in the mobile phase can come out of solution and
form bubbles as the pressure changes from the colum entrance to the
exit
-May block flow through the system

• Sparging is used to remove any dissolved gas from mobile phase

-An innert and virtually insoluble gas,such as helium,is forced into the
mobile phase solution and drives out any dissolved gas.

• Degassing may also br achieved by filtering the mobile phase under a

vaccum
 High performance liquid chromatography
HPLC instruments consist of
1.A solvent reservoir -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

-As a sample solution flows through a column with the mobile

phase,the components of that solution migrate according to the
non-covalent interaction of the compound with the column
9
2.Pump:The role of the pump is to force a liquid (called the mobile
phase) through the liquid 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).
-There are several types of pumps used for HPLC analysis, most
commonly used are reciprocating piston pump,syringe pump and
constant pressure pump

3.Injector: The injector serves to introduce the liquid sample into the
flow stream of the mobile phase for analysis. (about 5-
20μL).(Automated/Manual)
4.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 and glass.
-Column packing is usually silica gel because of its particle shape surface
properties , and pore structure give us a good separation
• 5.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
chromatogram.
-various types of detectors are used - Refractive index detector, UV-Visible
detector, Fluorescence detector, Electrochemical detector

• 6.Computer: not only controls all the modules of the HPLC instrument but it
takes the signal from the detector and uses it to determine the time of elution
(retention time) of the sample components (qualitative analysis) and the
amount of sample (quantitative analysis).
-The concentration of each detected component is calculated from the area or
height of the corresponding peak and reported
 Fig : Basic Instrumentation in HPLC

Injector
Solvent Detector
Delivery

Column

Separation

High waste
Solvent pressure collector
Sample
reservoir pump Injection

Data Processor

13
14
 HPLC for hemoglobinopathies.
• Cation-exchange HPLC can be preformed on an automated instrument that
can quantify Hb A2, Hb F, Hb A, Hb S, and Hb C.

• Studies show equivalence or superiority over electrophoresis in terms of

identification of variant haemoglobins and quantification of HbA2 level.

• Negatively charged carboxyl molecules bound to silica make up the cartridge

matrix.

• Positively charge molecules (salt and hemoglobin) bind to the carboxyl

groups.Haemoglobin molecules are bound and displaced by increasing salt
concentration. Haemoglobin variants separate out due to variation in charge.
• The separated fractions pass through a flow cell, where absorbance is measured
at 415 nm and again at 690 nm to reduce background noise.

• Changes in absorbance are monitored over time producing a chromatogram

(absorbance vs. time).

• Each hemoglobin has its own characteristic retention time and is measured from
the time of sample injection into the HPLC to the maximum point of each peak.

• Identification of unknown hemoglobin is achieved through comparison with

known hemoglobin retention times.

• If a peak elutes at a retention time not predetermined, it is labelled as an

unknown.
 Whole blood + Specific hemolysing solution
↓
Mixture made
↓
Automatic injection into the analytical cartridge containing gel
↓
Buffer is poured onto the Hb solution
↓
Hemoglobins move at different speeds
↓
Separated components pass through dual wavelength detectors (Absorbance is measured at 415 nm)
↓
Data is displayed as chromatogram
↓
Converted into peaks as per the retention time
↓
Separated hemoglobins with % displayed
 Checkpoints in the calibrator
1 - Flat baseline –The baseline should be
properly constructed
5
3
2 - Peak profile & shape – Peaks should appear
sharp and symmetrical

3 – Order of peaks – The peaks should follow 6

4
the order F, P2, P3, Ao, A2

4 - Total peak area Should be 1 to 3 million.

5 – Hb A2 and Hb F response- The new

calibration factor should be – 0.7 to 1.3
2

6 - Hb A2 retention time – The retention time

of Hb A2 for the calibrator should be 3.65 + 0.1 1
 Interpretation of chromatograms
• Flat baseline
• Total peak area
• Hb A2 retention time
• Peak profile & shape
• Examine the relative percentages of the hemoglobin fractions found
• Determine whether a variant is present
• Related clinical information
• Consider ethnic origin
• Review the CBC data and Interpret result in conjunction with CBC
• Consider the possibility of more than one hemoglobinopathy being present
Manufacturer assigned windows for Bio-Rad variant high performance liquid
chromatography system
Look for the following :
F – Less than 2%

P2 – changes with the glycemic status,

upto 6 % acceptable

P3 – upto 6 % acceptable ,

A0 – non glycated fraction of Adult

hemoglobin

A2 –normal range 2 to 4 %
 Additional points
• P3 -up to 6% acceptable
-6 to 12% may indicate sample deterioration
-15 to 15% indicate Hb J
• Iron deficiency –Hb A2 found to be slightly lower
• Megaloblastic anemia- Hb A2 found to be higher
• Unknown peak
-May appear any where in the peak table
-More than 1 unknown peak may be seen
-Upto 6% not significant
-If,above 6% -look for the RT for HB identification
 Expected ratios

HbA HbS HbA2 HbF

Hb AS 55-60 40-45 2-3 <1
Hb SS 0 90-95 2-3 5-10
Hb S-α-thal 75 25 2-3 <1
Hb S- β thal major 0 90-95 Inc. 5-10
Hb S- β thal minor 5-30 60-90 Inc. 5-10
Hb S HPFH 0 70-80 2-3 20-30
Hb SC 0 50 2-3 <1
Look for the following :
Typical thalassemia carriers

Hypochromic, Microcytic blood film

Hb A2 > 4.0 %

Hb F < 2.0%
( in some cases Hb F may also be
elevated )
First evaluate age and transfusion
history
If transfusion is involved
report with parental screening

If no transfusion is involved

Look for the following :

Increased NRBC count
Marked variation in shape and size

Hb F elevated upto 90%

Reduced Hb A
Normal or elevated Hb A2
 Hb MCV Hb A2 Hb F Hb S SEVERITY
(g/dl) (fl)
Normal 80 - 90 < 4.0 % <1% 30- 40% Asymptomatic

Look for the following :

Normal indices
Hb S elutes in the S window

For a S trait :
Hb A2 will be normal (however
due to the elution of some
glycated Sickle products the A2
may be elevated in some cases ,
do not consider it as a
compound heterozygous case)

Hb F will be normal
Hb S will be between 30-40%
 Hb MCV Hb A2 Hb F Hb S SEVERITY
(g/dl) (fl)
<12 70-75 <5% > 5% >50% Mild

Hb S elutes in the S window

Look for the following :

For a Hb S homozygous:
Hb A2 will be normal
Hb F will be elevated
Hb S will be > 50%

These values will be variable from

patient to patient and will vary if a
history of blood transfusion is involved
 Hb MCV Hb A2 Hb F Hb S SEVERITY
(g/dl) (fl)
<10 <70 > 5.0 % > 5% >50% Severe

Hb S elutes in the S window

If transfusion is involved
report with parental screening

Look for the following :

Reduced indices

For a Hb S thalassemia:
Hb A2 will be elevated
Hb F will be elevated
Hb S will be > 50%

These values will be variable from

patient to patient and will vary if
there is a history of blood transfusion
 Hb MCV Hb Hb E SEVERITY
(g/dl) (fl) F

Normal 80 - <1% 25-35% Asymptomatic

90

Look for the following :

Hb E elutes in the A2 window

For a Hb E trait :
Hb A2 will be between 25-35%
Hb F will be normal
 Hb MCV Hb F Hb E SEVERITY
(g/dl) (fl)

10-12 65-75 >2% >60% Mild

Look for the following :

Hb E elutes in the A2 window

For a Hb E homozygous:
Hb A2 will be between >60%
Hb F will be between 2-10%

These values will be variable from

patient to patient and will also
vary if there is a history of blood
transfusion
 Hb MCV Hb A2 Hb F Hb D SEVERITY
(g/dl) (fl)
Normal 80 - 90 < 2% <1% 30-45% Asymptomatic

Look for the following :

Normal indices
Hb D elutes in D window

For Hb D trait :
Hb A2 will be normal
Hb F will be normal
Hb D will be between 30-40%
 Hb MCV Hb A2 Hb F Hb D SEVERITY
(g/dl) (fl)
<12 <75 <4% 3-6% >50% Variable
between
mild to
severe

Look for the following :

Normal or reduced indices
Hb D elutes in D window

For a Hb D beta thalassemia :

Hb A2 will be normal or elevated
Hb F will be mildly elevated
Hb D will be between >50%
 Hb MCV Hb A2 Hb F SEVERITY
(g/dl) (fl)
12-14 80-90 <4% <5-30 % Asymptomatic

Look for the following :

Normal indices

For HPFH trait :

Hb A2 will be normal
Hb F will be 5-30%
 Hb MCV Hb A2 Hb F SEVERITY
(g/dl) (fl)
12-14 80-90 10-18% <10% Asymptomatic

Look for the following :

Normal indices

For Hb Lepore trait :

Hb A2 will be 10-18%
Hb F will be normal
The retention time of Hb A2 will be
earlier than normal - 3.45 to
3.6mins
 INTREPRATION OF RESULTS
• Hemoglobin F
–>2-80% Babies
–90-100% Homozygous Hereditary Persistence Fetal Hemoglobin,β0,
δβ0-Thal
–15-40% Heterozygous HPFH
–10-25% SS, Hydroxyurea Treated
–3-10% Homozygous Hemoglobinopathies, Anemias, Leukemias,
Malignancies,
–< 5% β-Thal, Lepore
• Hemoglobin A2
–Increased
• 4.0-7.0% Β-Thalassemia, Sβ+ Thal
• 3.5-4.5% Hb AS, AC, SC, SS, CC
• 6.5-14.0% Hb Lepore
• 25-30% Hb E
–Decreased
• 1.3-1.7% Iron Deficiency, Sideroblastic, Aplastic Anemias
• 1.5-2.3% δ Chain Variant (A2’), α Chain Variant
BIO-RAD A1C-AS CHROMATOGRAM
 Other Applications of HPLC
• To assess blood glucose control in diabetics by HbA1c quantification.

• Diagnosis of lysosomal storage disorders by detection of urine

oligosaccharides.

• Detection of drugs in serum and urine.

• HPLC has been successfully applied to the separation of proteins,

nucleic acids, polysaccharides, plant pigment, pesticides, plant and
animal hormones and complex lipid.
 ADVANTAGES OF HPLC
1.Separations fast and efficient (high resolution power)

2.Continuous monitoring of the column effluent

3. It can be applied to the separation and analysis of very complex mixtures

4. Accurate quantitative measurements.

5. Repetitive and reproducible analysis using the same column.

6. Adsorption, partition, ion exchange and exclusion column separations are excellently
made.
7. HPLC is more versatile than GLC in some respects, because it has the advantage
of not being restricted to volatile and thermally stable solute and the choice of
mobile and stationary phases is much wider in HPLC

8. Both aqueous and non aqueous samples can be analyzed with little or no
sample pre treatment

9. A variety of solvents and column packing are available, providing a

high degree of selectivity for specific analyses.

10. It provides a means for determination of multiple components in a single

analysis.
 DISADVANTAGES
• Cost

• Irreversibly Adsorbed Compounds Are Not Detected

• Co-elution Difficult To Detect

-Hbs may co-elute or may elute before instrument peak integration.

-HbE and HbG Copenhagen co-elute with Hb A2, making quantification impossible
when these variants present.

-The measurement of Hb A2 is complicated in individuals with Hb S because the Hb

A2 is falsely increased by the presence of Hb S adducts.
-Capillary zone Electrophoretic method or micro anion-exchange column
methodology can be used to quantify Hb A2 in the presence of Hb S by
eliminating interference from these adducts.

-Integration errors can result in false decreases in the values obtained,

although this can be minimized by applying known corrections.

-The presence of a structural Hb variant may cause inappropriately high

HbA1c (HbNiigata, HbSherwood Forest, HbRambam, HbRaleigh).
RECENT DEVELOPMENTS
• UPLC – ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY
• HPLC-Chip/MS system
 Contrasting HPLC and UPLC

• In UPLC
-Columns with smaller particles (<1.7 µm)(3 to 10µm in HPLC)
-Mobile phase delivery is done at>15,000psi (up to 6000psi in HPLC)
-Smaller ID tubing (<0.005) and smaller UV detector flow cells (0.5-2µL)
-Smaller system dwell volumes (0.1-0.3µL)(1-3 ml in HPLC)
-Faster detector response/data acquisition for high throughput applications

• UPLC gives faster results with better resolution.

• UPLC uses less of valuable solvents like acetonitratile which lowers cost
• The reduction of solvent use is more environmentally friendly
 HPLC –Chip /MS system
• Is a new revolutionary microfluidic chip-based technology specifically designes
for nanospray LC/MS.

• Most often used for applications with limited sample ammounts or when there
is a need for analysis of trace level components in complex mixtures

Problems of nanospray:
Use of multiple small capillary tubing connections
Frequent cloggings or leaking at the columns and nanospray needle
Solutions by HPLC -Chip /MS system
Integrate all components directly into a reusable biocompatible
polymer HPLC-Chip

HPLC –Chip Cube MS interface module performs

• Solvent and sample delivery to the chip
• High pressure switching of flows and
• Automated chip loading and positioning in the MS source
 case
• The 24 year old patient was admitted to hospital suffering with fever for 10
days and had a history of intermittent jaundice for the past 4–5 years.

• His physical examination showed mild hemolytic facies and a palpable spleen.

• On admission his haemoglobin- 8.4 gm/dl,

MCV -76.6 fl
MCH -19.5 pg,
MCHC -25.5 gm/dl
Retic preparations showed 11% reticulocytes
• The peripheral smear showed presence of microcytes,macrocytes, elliptocytes
and tear drop cells.

• Biochemical investigations showed

Total Bilirubin -5.0 mgs/dl(0.1 to 1.2 mg /dl)
Direct Bilirubin 0.3 mgs/dl(0-0.3 mgs/dl)
Serum LDH 305 U/l (160-450 U/l)
Iron was 150 µg/dl(55-160 µg/dl)
Ferritin 377 ng/ml(12 to 300 ng/ml)
SGOT -35 U/L(5 to 40 U/L)
SGPT -45 U/L,(7-56 U/L)
Alkaline phosphatase -82 U/L and(50-100 U/L)
Haptoglobin-0.25 g/l(0.16-2.2 g/l)
• Based on these findings from the above analyses the hemoglobin variant analysis was initially
conducted on a BioRad D10 HPLC instrument as per standard procedures using the b-Thal
short (BTS) program.
• BioRad D10 HPLC profile showed 2 peaks with Retention time of 0.17 and 0.38 min with area
percentage of 9.8 and 12.9% respectively.HbA2 with RT at 3.29 min was reduced at only 1.0%.
• α Thalassaemia is suggested when eluted proteins are detected at a retention time
between 0 and 1 min. A retention peak less than 1 min on a BTS program of the BioRad
HPLC system is suggestive of Hb Bart’s and HbH .HbA2 in addition, a reduced level of 1.5%
is also indicative of α thalassaemia

• In order to pursue this further,an alkaline hemoglobin electrophoresis at pH 8.6 was

conducted using the HELENA SAS II system. A band which moved ahead of the adult HbA
band was seen highly suggestive of HbH

• A GPO a THAL-IC strip convenient confirmatory tool for screening of a-thalassaemia [5].
The presence of two colour lines, one in the control region and other in that region
confirmed it to be case of α -thalassaemia
• The qualitative confirmation of a-thalassaemia was verified by microscopically
examining slides for HbH inclusions.

• In this case smears prepared from the patient sample were stained with 1% brilliant
cresyl blue (BCB)and kept for observation between 4 and 24 hr.

• Presence of‘‘golf ball’’ cells with classical HbH inclusions confirmed the findings .

• HbH tetramers, when oxidized in vitro, precipitate and hence can be visualized
microscopically and therefore staining unfixed cells with an oxidative dye such as BCB
generate HbH inclusions.
Thank you
• In Hemoglobinopathies
-Method of choice for screening for Hb variants; for quantification of HbA2
+ HbF concentrations and mandatory in neonatal screening.

-Quicker and more sensitive than standard techniques for detecting HbF (in
diagnosis of HPFH and monitoring sickle cell anemia).

-Indeed alkaline gel electrophoresis cannot detect HbF in healthy adults or

those with marginally increased Hb F.

-Can be used to characterize rare haemoglobinopathies not well detected

with other methods (HbRambam).
-Established role in the diagnosis of thalassaemia and haemoglobinopathies,
including with cord blood samples.

-HPLC should be the primary method for detecting variant hemoglobin's and
simultaneous quantitation of HbA2 and HbF.

-This replaces three separate methods:

• Haemoglobin electrophoresis.
• Quantitation of HbA2.
• Quantification of HbF.
 Variables of interpretation
• What is the Retention Factor, Rf ?
• The retention factor, Rf, is a quantitative indication of how far a particular
compound travels in a particular solvent.

• The retention factor, Rf, is defined as

Rf = distance travelled by the solute (X)

distance travelled by the solvent front (Y)

• If the Rf value for the unknown compound is close or the same as the Rf value
for the known compound then the two compounds are most likely similar or
identical.
• What is Retention Time?
• The Retention Time is the time taken by a particular component of the
sample to travel through the stationary phase(column).
• It is characteristic of a particular substance. (for the same column,
temperature, gas flow etc.)

• The area under each peak/ peak height

• indicates the relative quantities.