3. CHROMATOGRAPHY
• Chromatography is a technique which separates components in a
mixture due to differing time taken for each component to travel
through a stationary phase when carried through it by a mobile
phase.
4. TYPES OF CHROMATOGRAPHY
• Paper chromatography(PC)
• Liquid chromatography(LC)
• Gas chromatography(GC)
• High performance liquid chromatography(HPLC)
5. Some chromatography terms
• Analyte : Substance that is to be separated during chromatography
• Stationary phase: Immobilized phase - which is immobilized on the
support particles or on the inner wall of the column tubing
• Mobile phase
- Phase which moves in a definite direction. (liquid/gas).
- Consists of sample being separated/ analyzed and solvent that
moves sample through column.
• Elluent -Mobile phase leaving the column
6. Basically, all chromatographic systems consist of two phases.
• Mobile phase
-Solvent which carries the analyte
-liquid or gaseous and flows over or through the stationary phase
• Stationary phase
-The substance on which adsorption of the analyte takes place
- Can be a solid, liquid or a solid/liquid mixture which is immobilized
7. HPLC
• A form of modified column chromatography to separate,
identify and quantify the compounds.
• Developed in 1970s.
• Most widely used analytical separation technique.
• For identifying, quantifying and purifying the individual
components of the mixture.
Mikhail tswett
8. Introduction
• A modified column
chromatography
• In column chromatography, a
column is packed with an
adsorbent material like silica,
alumina, cellulose etc and the
mobile phase is passed down the
column because of gravity.
9. • But in HPLC, high pressure
pump is attached to the
column.
• High pressure pump can
generate a high pressure up to
40 MP
10. HIGH PERFORMANCE IN HPLC
• The column is filled with adsorbent material which has a very small
particle size
Decreasing the particle size
Increase the surface area
Increase the number of equilibrations
High performance/ resolution
11. SHORTCOMINGS OF SMALLER PARTICLE SIZE
Decreasing the particle size
Increase the resistance to mobile phase
Back pressure in the column
Damage the stationary phase
Decreasing the eluent flow and resolution
12. Solution of small particle size
• Selection of small particles (5-10µm) that can withstand pressures up
to 40 MPa
• Requirement of high pressure to increase the flow rate
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HIGH PRESSURE LIQUID CHROMATOGRAPHY
13. COMPONENTS OF HPLC
a) COLUMN
Flow rate of mobile phase
through the column is 1-3
ml/min
15. c) MOBILE PHASE
• Mixture of different solvents can be used as mobile phase
• The solvent used depends on type of sample molecule which are to
be separated
• Solvent can be polar or non-polar.
• Kept in solvent reservoir.
• The solvent reservoir is attached to pump which pumps mobile phase
in column with a high pressure
• Just before column, there is a injector which allows introduction of
sample into the column
16.
17. d) DETECTOR
• In order to detect the elluent that is sample coming out of the column
the HPLC column is attached with a detector
• Types of detector:
- UV detector
- IR detector
- Fluorescence detector
- Refractive index detector
- Mass spectrometer
- Electrochemical detector
• Detectors are connected with the computer which collects the
information.
18.
19. Contd…
• Used increasingly as initial diagnostic method in
haemoglobinopathy laboratories with a high workload.
• Both capital and consumable costs are higher than with
haemoglobin electrophoresis, but labour costs are less; overall
costs may be similar.
20. Principle
• HPLC depends on the interaction of charged groups on the ion exchange
material(silica) with charged groups on the haemoglobin molecule.
• Surface of support is modified by carboxyl groups to have a weakly
cationic charge, which allows separation of haemoglobin molecules with
different charges by ion exchange.
• When a haemolysate containing a mixture of haemoglobins is adsorbed
onto the resin, rate of elution of different haemoglobins is determined by
pH and ionic strength of any buffer applied to the column.
21. Contd…
• With automated systems now in use, elution of the charged molecules is
achieved by a continually changing salt gradient; fractions are detected as
they pass through an ultraviolet/visible light detector and are recorded on
an integrating computer system.
• Analysis of the area under these absorption peaks gives the percentage of
the fraction detected.
• The time of elution (retention time) of any normal or variant haemoglobin
present is compared with that of known haemoglobins, providing
quantification of both normal haemoglobins (A, F and A2) and many
variants.
22.
23. Window/ Peak Retention time (Min) Causes/ Variant Haemoglobin
P1 0.63-0.85 Bilirubin, HbH, Hemoglobin Bart’s
F 0.98-1.2 Hemoglobin F
P2 1.24-1.4 Glycated haemoglobin, Haemoglobin Hope
P3 1.4-1.9 Post translationally modified HbA, Hemoglobin J-
Oxford
A0 1.3-3.1 Haemoglobin A
A2 3.3-3.9 Hemoglobin A2, E, Lepore
D 3.9-4.3 Haemoglobin D-Punjab, Haemoglobin G-Philadelphia
S 4.3-4.7 Hemoglobin S, Q-Thailand
C 4.9-5.3 Haemoglobin C, O- Arab
26. APPLICATIONS
a) Pharmaceutical:
- Tablet dissolution of pharmaceutical dosages.
- Shelf-life determinations of pharmaceutical products.
- Identification of counterfeit drug products.
- Pharmaceutical quality control.
27. b) Environmental:
- Phenols in Drinking Water.
- Identification of diphenhydramine in sediment samples.
- Biomonitoring of PAH pollution in high-altitude mountain lakes
through the analysis of fish bile.
- Estrogens in coastal waters - The sewage source.
28. c) Forensics:
• A mobile HPLC apparatus - on-site identification and quantification of
the Drug Ecstasy.
• Identification of anabolic steroids in serum, urine, sweat and hair.
• Forensic analysis of textile dyes.
• Determination of cocaine and metabolites in meconium.
• Simultaneous quantification of psychotherapeutic drugs in human
plasma.
29. d) Food and flavours:
• Ensuring soft drink consistency and quality.
• Analysis of vicinal diketones in beer.
• Sugar analysis in fruit juices.
• Polycyclic aromatic hydrocarbons in vegetables and fruits.
• Trace analysis of military high explosives in agricultural crops.
30. d) Clinical :
• Quantification of DEET in Human Urine.
• Analysis of antibiotics.
• Increased urinary excretion of aquaporin 2 in patients with liver
cirrhosis.
• Detection of endogenous neuropeptides in brain extracellular fluids.
• Screening of Beta-Thalassemia.
38. Pitfalls
• HPLC usually separates haemoglobins A, A2, F, S, C, D-Punjab and G-Philadelphia from each other.
• However, both haemoglobin E and haemoglobin Lepore co-elute with haemoglobin A2 (as other
haemoglobins co-elute with A, S and F).[ Cannot discriminate between HbA2 and HbE].
• Since derivatives of haemoglobin S co-elute with haemoglobin A2, percentages of A2 by this
method are inaccurate in presence of S, and therefore do not have the same significance as
percentage of haemoglobin A2 measured by alternative methods.
• It is important to analyse variants found using second-line techniques, such as a sickle solubility
test, alkaline and acid electrophoresis, or iso-electric focusing.
39. Contd…
• If the quantity of hemoglobin with retention time of HbA2 is higher than
expected, an alternative technique should be applied to confirm its identity.
• Because peak labelled HbA2 can be HbE, Hb Lepore or other hemoglobin that
elutes with HbA2.
40. Advantages:
• In comparison with haemoglobin electrophoresis, HPLC has four
advantages:
1. Analysers are automated and thus require less staff time and permit
processing of large batches.
2. Very small samples (5 μl) are sufficient for analysis; especially useful in
paediatric work.
3. Quantification of normal haemoglobins (including haemoglobin A2) and
variant haemoglobins is available on every sample.
4. Provisional identification of a larger proportion of variant haemoglobins
can be made.
41. TLC Vs HPLC
TLC (Thin layer Chromatography) HPLC
TYPE OF ANALYSIS QUALITATIVE ONLY QUALITATIVE AND QUANTITATIVE
STATIONARY PHASE 2- DIMENSIONAL THIN LAYER PLATE 3- DIMENSIONAL COLUMN
INSTRUMENTATION MINIMAL MUCH WITH MANY ADJUSTABLE
PARAMETERS
SAMPLE APPLICATION SPOTTING
(CAPILLARY)
INJECTION
(RHEODYNE INJECTION)
MOBILE PHASE MOVEMENT CAPILLARY ACTION
(DURING DEVELOPMENT)
HIGH PRESSURE
(SOLVENT DELIVERY)
VISUALIZATION OF RESULTS UV LIGHTBOX ON-LINE DETECTION
(VARIABLE UV/Vis)
FORM OF RESULTS SPOTS, Rf’s
(RETENTION FACTORS)
PEAKS, Rt’s
(RETENTION TIMES)
42. DISTINGUISHED FROM LC (Liquid
Chromatography)
• Operational pressures are significantly higher, while ordinary liquid
chromatography typically relies on the force of gravity to pass the
mobile phase through the column
• HPLC columns are made with smaller adsorbent particles which gives
HPLC superior resolving power when separating mixtures
43. HPLC vs Capillary Electrophoresis
• Capillary electrophoresis is suitable method of quantification of HbA2.
• In contrast to measurements by HPLC, there is no false elevation in
the presence of adjuncts of HbS and no false reduction in the
presence of Hb D-Punjab; accurate measurement is possible in the
presence of Hb E.
• Measurement may, however, be inaccurate in presence of HbC.
44. References
• Bain BJ, Bates I, Laffan MA. Dacie and Lewis Practical
Haematology. 2017 p286-97.
• Corradini D, Eksteen E, Eksteen R, Schoenmakers P, Miller N.
Handbook of HPLC. CRC Press; 1998 Aug 21.
• https://www.bio-rad.com/featured/en/high-performance-liquid-
chromatography
