Basically, the apparatus required for the liquid chromatographic procedure consists of a pumping system for the mobile phase, a sample injection port, a column, a detector, and a recorder.
A mobile phase component regulator, a thermostat for the column, a pumping system for reaction reagents, and a chemical reaction chamber is also used, if necessary.
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The pumping system serves to deliver the mobile phase and
the reagents into the column and connecting tube at a constant flow rate. The
sample injection port is used to deliver a quantity of the sample to the apparatus
with high reproducibility.
The column is a tube with a smooth interior, made of inert
metal, etc., in which a packing material for liquid chromatography is uniformly
packed. A column with a stationary phase chemically bound on the inside wall
instead of the column packed with the packing material may be used. An ultraviolet
detector is used to detect a property of the samples, which is different from
that of the mobile phase.
The output signal is usually proportional to the
concentration of samples at amounts of less than a few μg.
The recorder is used to record the output signals of the detector. As required,
a data processor may be used as the recorder to record or output the
chromatogram, retention times, or amounts of the components. The mobile phase
component regulator is used to vary the ratio of the mobile phase components in
a stepwise or gradient fashion.
High-Pressure Liquid Chromatography is a method to develop a
mixture injected into a column prepared with a suitable stationary phase by
passing a gas (carrier gas) as a mobile phase through the column, to
separate the mixture into its components by making use of the difference of
retention capacity against the stationary phase and to determine the
components.
This method can be applied to a gaseous of a vaporizable
sample and is used for identification, purity test, and quantitative
determination
Operating Conditions
Column: ODS-C18 steel column, 4.6mm in inside diameter and
150mm in length.
Mobile phase: methanol: water = 80: 20
Flow rate: 1ml/min.
Wavelength: 263.
Procedure of Trichlorocarbanilide Purity Method
Weigh accurately 0.1g of authentic sample in a vessel,
dissolve it with methanol, and dilute to 50ml exactly, through shaking. Pipet
exactly 1ml of the solution to another vessel, and dilute with mobile phase to 25ml
exactly. Taking this as a standard solution.
Weigh accurately 0.1g of sample to be tested in a vessel,
dissolve it with methanol, and dilute it to 50ml exactly, through shaking. Pipet
exactly 1ml of the solution to another vessel, and dilute with mobile phase to 25ml
exactly. Take this as a sample solution.
Adjust the flow rate and wavelength to the values described
in the above operating conditions. Inject 20μl
of standard solution into the column system through the injection port, and separated components are detected by the detector. Read the peak area from the record.
The test is repeated until the relative deviation of the peak areas of the
authentic sample is not more than 1.0%.
Inject exactly 20μl of sample
solution into the column system under the above operating conditions, and read the
peak area from the recorder. Calculate the parity of the test sample according
to the following equation:
Purity of trichlorocarbanilide
A standard M
sample
Purity % = ------------ x ----------- x P x 100
A sample M standard
In the above equation, which
A standard is referred to as the peak area of 3,4,4'- Trichlorocarbanilide in the authentic solution;
A sample is referred to as the peak
area of 3,4,4'- Trichlorocarbanilide in the sample solution;
M standard is referred to as the amount of authentic sample used (g);
M sample is referred to as the amount of sample that is to be tested (g);
P is referred to as the purity of the authentic sample.
Analysing Trichlorocarbanilide (TCC) content in Bar Soaps
The content of dichlorocarbanilide, Tetrachlorocarbanilide,
and Triaryl Biruet are obtained by auto-integration by the computer.
The retention time of dichlorocarbanilide, Triclocarban, Tetrachlorocarbanilide, and Triaryl Biruetare are 3.307, 4.515, 6.665, and 7.382(minute), respectively based on the HPLC method. The retention time may change under different temperatures, but the sequence shouldn't change.
TCC Content Principle
TCC is an antibacterial agent, which is separated from other components in soap and shaving cream by Normal-phase or reverse-phase liquid chromatography detected spectrophotometrically and quantified by comparison with standard TCC. The method can estimate as low as 1 ppm of the TCC component.
Apparatus
a) High-performance liquid chromatography with UV detector of variable wavelength.
b) Column- Lichosorb CN with 5 or 10-micron particle size or equivalent
c) Sample clarification kit.
d) Millipore Filter Apparatus with 0.5-micron filter or equivalent.
Reagents
a) 25% 2- Propanol solution in Hexane
b) Standard TCC.
c) Mobile phase- Put 50 ml of HPLC grade 2 propanol in 500 ml volumetric flask, fill up to the mark with HPLC grade Hexane, close and shake well, assemble Millipore Filter apparatus with 0.5-micron filter, and filter the mobile phase.
Procedure
Standard Preparation - Weigh 40 mg of standard TCC into a 100 ml volumetric flask, dilute with 25% propanol solution in Hexane up to the mark. Add a magnetic stir bar, and stir until it dissolves. Pipette 5 ml of an aliquot in a 50 ml volumetric flask, and dilute with 25% propanol in hexane.
Sample preparation - Weigh accurately about 1.0 g of grated soap into a 100 ml volumetric flask, dilute with 25% reagent grade 2 propanol in Hexane up to the mark.
Add a magnetic stir bar, stir vigorously for at least 1/2 an hour on a magnetic stirrer, allow it to stand for 10 minutes, then filter the supernatant liquid into the test tube with the help of a sample clarification kit. From this clear solution pipette 20 ml into a 50 ml volumetric flask and dilute it with 25% propanol solution in hexane up to the mark.
Chromatography - Equilibrate the column with mobile phase, with a flow rate of 1.0 ml per minute for 15-30 minutes. Set the wavelength at 265 nm and attenuation to 0.02 AUFS. Inject the standard sample - Retention time of TCC is approx. 7.5 minutes.
Inject the sample - From the area of respective components at a particular retention time,
Calculate the actual % of TCC.
Calculation:
Area of sample for TCC Wt. of standard TCC
% TCC = ------------------------------ x ------------------ x 25
Area of standard TCC Wt. of sample