Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.
3. CHROMATOGRAPHY
•Chromatography is a laboratory technique for the separation of a
mixture. The mixture is dissolved in a fluid called the mobile
phase, which carries it through a structure holding another
material called the stationary phase.
• The various constituents of the mixture travel at different
speeds, causing them to separate.
• The separation is based on differential partitioning between the
mobile and stationary phases. Subtle differences in a
compound's partition coefficient result in differential retention on
the stationary phase and thus affect the separation
4. HPLC
•High-performance liquid chromatography (HPLC; formerly
referred to as high-pressure liquid chromatography), is a technique
in analytical chemistry used to separate, identify, and quantify each
component in a mixture.
•It relies on pumps to pass a pressurized liquid solvent containing the
sample mixture through a column filled with a solid adsorbent
material.
• Each component in the sample interacts slightly differently with
the adsorbent material, causing different flow rates for the different
components and leading to the separation of the components as they
flow out of the column.
5. WHY USE HPLC?
•Simultaneous analysis
•High resolution
•Easy to fractionate and purify
•High senstivity
•Not destructive
•Moderate analysis condition
7. Parts of HPLC/Instrumentation
1. Solvent/Mobile Phase
•The mobile phase, or solvent, in HPLC is usually a
mixture of polar and non-polar liquid components
whose respective concentrations are varied
depending on the composition of the sample.
•The solvent is passed through a very narrow bore
column, any contaminants could at worst plug the
column, or at the very least add variability to the
retention times during repeated different trials.
Therefore HPLC solvent must be kept free of
dissolved gases, which could come out of solution
mid-separation, and particulates.
8. 2. Pump
•The HPLC pump drives the solvent and sample through the
column. To reduce variation in the elution,the pump must
maintain a constant, pulse free, flow rate; this is achieved with
multi-piston pumps. The presence of two pistons allows the flow
rate to be controlled by one piston as the other recharges.
•A syringe pump can be used for even greater control of flow rate;
however, the syringe pump is unable to produce as much pressure
as a piston pump, so it cannot be used in all HPLC applications.
9. 3. Column
•In the HPLC column, the components of the sample separate based on
their differing interactions with the column packing.
• If a species interacts more strongly with the stationary phase in the
column, it will spend more time adsorbed to the column's adsorbent and
will therefore have a greater retention time.
•Columns can be packed with solids such as silica or alumina; these
columns are called homogeneous columns.
•If stationary phase in the column is a liquid, the column is deemed a
bonded column. Bonded columns contain a liquid stationary phase bonded
to a solid support, which is again usually silica or alumina.
10.
11. 4. Detector
•The HPLC detector , located at the end of the column, must register
the presence of various components of the sample, but must not detect
the solvent.
For that reason there is no universal detector that works for all
separations.
•A common HPLC detector is a UV absorption detector, as most medium
to large molecules absorb UV radiation.
• Detectors that measure fluorescence and refractive index are also
used for special applications. A relatively new development is the
combination of an HPLC separation with an NMR detector.
•This allows the pure components of the sample to be identified and
quantified by nuclear magnetic resonance after having been separated
by HPLC, in one integrated process.
12. METHOD DEVELOPMENT FOR HPLC OFTEN FOLLOWS THE
SERIES OF STEPS GIVEN AS FOLLOWS:
Information on sample, define separation goals
Need for special HPLC procedure, sample pretreatment, etc.
Choose detector and detector setting
Choose LC method; preliminary run; estimate best separation condition;
Optimize separation condition
Check for problem or requirement for special procedure
Recover purified material
14. There are basically four channels for mobile phase which are as follows:
CHANNEL A: generally contains methanol
CHANNEL B: generally contains water
CHANNEL C: generally contains acetonitrile
CHANNEL D: generally contains buffer as required.
The software used for operating the HPLC is EMPOWER PRO
Different tests carried out in pharmaceutical industries:
•Related substance by HPLC, GC
•Assay by HPLC
•Residual solvent Head space GC
•Classical analysis
•Method development and method validation
•Dissolution study and dissolution profiling
•Tablet disintegration
•Average weight variation
•Content uniformity
15. Advantages
•Speed (analysis can be accomplished in 20 minutes or less),
•Greater Sensitivity (various detectors can be employed),
•Improved Resolution (wide variety of stationary phases),
•Reusable Columns
•Ideal for the substances of low volatility,
•Easy sample recovery, handling and maintenance,
•Instrumentation tends itself to automation and quantization (less time and less labor),
•Precise and Reproducible,
•Calculations are done by integrator itself,
•Capable of handling macromolecules
•Efficient analysis of labile natural products
Disadvantages
•No universal detector
•Less separation efficiency than capillary GC
16. GAS CHROMATOGRAPHY
Gas chromatography (GC), is a common type of chromatography used in
analytic chemistry for separating and analyzing compounds that can be
vaporized without decomposition.
Typical uses of GC include-
•Testing the purity of a particular substance, or
•Separating the different components of a mixture (the relative amounts of
such components can also be determined).
In some situations, GC may help in identifying a compound.
In gas chromatography, the mobile phase is a carrier gas, usually an inert
gas such as helium or an unreactive gas such as nitrogen. The stationary
phase is a microscopic layer of liquid or polymer on an inert solid support,
inside a piece of glass or metal tubing called a column
17.
18. PARTS OF GC MACHINE-
1) CARRIER GAS
The carrier gas must be chemically inert. Commonly used gases include
nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is
often dependant upon the type of detector which is used. The carrier gas
system also contains a molecular sieve to remove water and other
impurities.
2) SAMPLE INJECTION PORT
For optimum column efficiency, the sample should not be too large, and
should be introduced onto the column as a "plug" of vapour - slow
injection of large samples causes band broadening and loss of resolution.
The most common injection method is where a micro syringe is used to
inject sample through a rubber septum into a flash vapouriser port at the
head of the column.
19. 3) COLUMN
The sample is separated into its constituent components in the column.
Columns vary in length and internal diameter depending on the application
type and can be either packed or capillary.
4) DETECTORS
•There are many detectors which can be used in gas chromatography.
•Different detectors will give different types of selectivity.
a) A non-selective detector responds to all compounds except the carrier
gas,
b) A selective detector responds to a range of compounds with a common
physical or chemical property and
c) A specific detector responds to a single chemical compound.
20. The following information gives an indication of the type of sample
(analyte) analyzed by either GC and HPLC and relative strengths and
limitations of each technique.
GC
1. Samples analyzed by GC must be volatile (have a significant vapor
pressure below 250 °C)
2. Most GC analytes are under 500 Da Molecular Weight for volatility
purposes .
3. Highly polar analytes may be less volatile than suspected when
dissolved in a polar solvent or in the presence of other polar species due
to intermolecular forces such as hydrogen bonding.
HPLC
1. HPLC analysis has no volatility issues; however the analyte must be
soluble in the mobile phase.
2. HPLC can analyze samples over a wide polarity range and is able to
analyze ionic samples. Mobile phase components are selected to ensure
sample solubility.
3. HPLC has no real upper molecular weight limit and large proteins of
many thousands of Daltons may be analyzed.
21. DISSOLUTION TESTING
Dissolution is the process by which a solid solute enters a solution. In
the pharmaceutical industry, it may be defined as the amount of drug
substance that goes into solution per unit time under standardized
conditions of liquid/solid
interface, temperature and solvent composition.
Dissolution is considered one of the most important quality control tests
performed on pharmaceutical dosage forms.
Several dissolution apparatuses exist. In United States Pharmacopeia
(USP), there are four dissolution apparatuses standardized and specified.
They are:
• USP Dissolution Apparatus 1 - Basket (37°C)
• USP Dissolution Apparatus 2 - Paddle (37°C)
• USP Dissolution Apparatus 3 - Reciprocating Cylinder (37°C)
• USP Dissolution Apparatus 4 - Flow-Through Cell (37°C)
USP Dissolution Apparatus 2 is the most widely used apparatus among
these four.
23. Different components are:-
1.Dissolution vessels- There is no easy way
to check the uniformity of the vessel. Most
manufacturers provide a certificate of
conformance which should be accepted.
Measuring the centering of the shaft also
helps.
2.Vessel Covers- Vessel covers are an
essential part of the dissolution test. Tests
have shown that a significant amount of
evaporation can occur from media at 37°C
into the atmosphere.
3.Paddle
4.Dissolution Filters- Almost every
dissolution test relies on samples being
taken to produce a result. Filters are used to
trap particles of tablet from the sample and
prevent them getting into the
spectrophotometer.
24. Samples taken during dissolution are analysed using either……
1) UV-Vis Spectrophotometer ,or
2) HPLC (High Performance Liquid Chromatography)
The principle function of the dissolution test may be summarized as
follows:
• Optimization of therapeutic effectiveness during product
development and stability assessment.
• Routine assessment of production quality to ensure uniformity
between production lots.
• Assessment of ‘bioequivalence’, that is to say, production of the
same biological availability from discrete batches of products from
one or different manufacturers.
• Prediction of ‘in-vivo’ availability, i.e. bioavailability (where
applicable).
