Hplc

HPLC
Submitted
To:
Sayema Khanum
Senior Lecturer
Department Of
Pharmacy,
SEU.
Submitted By:
MD. Rashed
Mahmud
Nilufa Haque Chaity
Nusrat Jahan
Sarmin Akter

HPLC
HPLC is an abbreviation for-High Performance Liquid
Chromatography(It has also been referred to as High Pressure
LC)
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.
 These separated components are detected at the exit of this
tube (column) by a flow-through device (detector) that measures
their amount. An output from this detector liquidis
chromatogramcalled
a liquid chromatogram.

Stationary phases
 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.
 Reverse phase
-β, β '-Oxydipropionitrile
-Carbowax.
-Glycols (ethylene, diethylene),
-Cyanoethylsilicone
 Normal phase
-Squalane
-Zipax-HCP
-Cyanoethylsilicone

Mobile Phase
 The mobile phase carries the sample molecules along whereas
the stationary phase interacts with them, the stronger the
interaction the slower their movement and this differential
interaction causes them to separate out and elute at different
times.
 Normal phase chromatography
-hexane
-heptane
-aromatic solvents(e.g. benzene, xylene)
 Reverse phase chromatography
-Water and alcohol-water mixtures
-acetonitrile and
-acetonitrile-water mixtures

Parameters used in HPLC
1.Theoritical Plate
2.Retention Time
3.Retention Volume
4.Seperation Factor
6.Resolution
5.Tailing Factor etc.

1.Theoritical Plate
A theoretical plate in many separation Processes, is a
hypothetical zone or stage in which two phases, such as the
liquid and vapor phases of a substance, establish an
equilibrium with each other.
Such equilibrium stagesmay also be referred to as an
equilibrium stage, ideal stage, or a theoretical tray.
The performance of many separation processes depends
on having a series of equilibrium stages and is enhanced by
providing more such stages.
In other words, having more theoretical plates increases the
efficacy of the separation process be it either a distillation,
absorption, chromatographic, Adsorption or similar process.

2.Retention time (RT)
In a chromatogram, different peaks correspond to different
components of the separated mixture.
Time elapsed between sample introduction and maximum of
response, it is the characteristic time it takes for a particular
analyte to pass through the system.
Time taken for the analyte to
travel from the column inlet to
the point of
detection(maximum peak)

Retention time (RT)
 Different compounds have different retention
times. For a particular compound, the retention
time will vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a
way of identifying compounds.

3.Retention volume:
Retention volume is the volume of carrier gas
required to elute 50% of the component from the
column. It is the product of retention time and flow
rate.
Retention volume = Retention time × flow rate.
4.Seperation factor:
Separation factor is the ratio of partition
coefficient of the two components to be separated.
S=Ka/Kb=(tb-to)/(ta-to)

Where ,
to = Retention time of unretained substance
Ka, Kb = Partition coefficients of a, b
ta, tb = Retention time of substance a, b
If there is a big difference in partition coefficient between
2 compounds, the peaks are far apart and the separation factor is
more. If the partition coefficient of 2 compounds are similar, then
the peaks are closer and the separation factor is less.
5.Resolution:
Resolution is the measure of extent of separation of 2
components and the base line separation achieved.
Rs = 2 (Rt1-Rt2) / w1+w2

6.Tailing Factor
The tailing factor is a measure
of peak tailing.
It is defined as the distance
from the front slope of the peak to
the back slope divided by twice
the distance from the center line
of the peak to the front slope, with
all measurements made at 5% of
the maximum peak height.
The tailing factor of a peak will
typically be similar to
the asymmetry factor for the
same peak, but the two values
cannot be directly converted.

Application:
Chemical Separations
The extent or degree of separation is mostly determined by the
choice of stationary phase and mobile phase.
Purification
Purification is defined as the process of separating or extracting the
target compound from a mixture of compounds.Each compound
showed a characteristic peak under certain chromatographic
conditions.
Identification
Generally assay of compounds are carried using HPLC. The
parameters of this assay should be such that a clean peak of the
known sample is observed from the chromatograph. The identifying
peak should have a reasonable retention time and should be well
separated from extraneous peaks at the detection levels which the
assay will be performed.

Pharmaceutical applications
 Tablet dissolution study of pharmaceutical dosages form.
 Shelf-life determinations of pharmaceutical products
 Identification of active ingredients of dosage forms
 Pharmaceutical quality control
Environmental applications
 Detection of phenolic compounds in drinking Water
 Identification of diphenhydramine in sedimented samples
 Bio-monitoring of pollutant
Forensics
 Quantification of the drug in biological samples.
 Identification of anabolic steroids in serum, urine, sweat, and hair
 Forensic analysis of textile dyes.
Clinical
 Quantification of ions in human urine analysis of antibiotics in blood
plasma.
 Estimation of bilirubin and bilivirdin in blood plasma in case of hepatic
disorders.
 Detection of endogenous neuropeptides in extracellular fluids of
brain.

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

Disadvantages of HPLC
 Cost
 Complexity-There is complexity of usage as well as low
sensitivity for some compounds.
 Low sensitivity for some compounds
 Irreversibly adsorbed compounds notdetected
 Coelution difficult to detect

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