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Trichloroacetic acid is used as a caustic on the skin or mucous membranes to
treat local lesions and for the treatment of various dermatological disease such as
warts. Trichloroacetic acid has been determined in water using liquid-liquid
extraction, conversion to its methyl ester and gas chromatography with electron
capture detector. In this study trichloroacetic acid was determined using High
Performance Liquid Chromatography (HPLC) with UV detector. The
determination of trichloroacetic acid using HPLC with UV detector shows a good
result. The results indicate that trichloroacetic acid can be determine using HPLC
with UV detector.
Keywords : Trichloroacetic acid, HPLC.
Trichloroacetic acid (Figure 1) (C2HCl3O2) was created in the 1830 and used
in humans for the first time in 1926, is composed of carbon, chlorine, oxygen and
hydrogen (Ricardo et al., 2012). Trichloroacetic acid is produced on an industrial
scale by chlorination of acetic acid or chloroacetic acid at 140-160C. It is
prepared by the reaction of chlorine with acetic acid in the presence of a suitable
Trichloroacetic acid is a sharp odor, colorless to white crystalline solid. It has
melting point 57 to 58C. It has boiling point 196 - 197C. It soluble in 0.1 parts
of water. The pKa of trichloroacetic acid is 0.66.
It is used as a caustic on the skin or mucous membranes to treat local lesions
and for the treatment of various dermatological disease such as warts. It produces
a denaturalization, precipitation and destruction of the lesions due to chemical
coagulation of the affected tissue (Ricardo et al., 2012). Application of TCA to

the skin causes precipitation of proteins and coagulative necrosis of cells in the
epidermis and necrosis of collagen in the papillary to upper reticular dermis. Over
several days the necrotic layers slough and the skin reepithelializes from the
adnexal structures that were spared from chemical damage. Dermal collagen
remodeling after chemical peel may continue for several months. Many
investigators have observed that the clinical effects of TCA were due to both a
reorganization in dermal structural elements and an increase in dermal volume as
a result of an increase in collagen content, glycosaminoglycan, and elastin (Lee,
et al., 2002). It is also used as a precipitant of protein in the chemical analysis of
body fluids and tissue extract.

Figure 1


High performance liquid chromatography (HPLC) is one of the most widely

used analytical techniques in industry, it is used to separate and analyze
compounds through the mass-transfer of analytes between stationary and liquid
mobile phases. The components are first dissolved in solvent and then forced to
flow through a column under high pressure.
HPLC is a separation technique that can be used for the analysis of organic
molecules and ions. HPLC is based on mechanisms of adsorption, partition and
ion exchange, depending on the type of stationary phase used. HPLC involves a
solid stationary phase, normally packed inside a stainless-steel column, and a
liquid mobile phase. Separation of the components of a solution results from the
difference in the relative distribution ratios of the solutes between the two phases.

HPLC can be used to assess the purity and/or determine the content of many
pharmaceutical substances. It can also be used to determine enantiomeric
composition, using suitably modified mobile phases or chiral stationary phases.
Individual separation mechanisms of adsorption, partition and ion exchange
rarely occur in isolation since several principles act to a certain degree
HPLC instrumentation includes a pump, injector, column, detector and
integrator or acquisition and display system. The heart of the system is the
column where separation occurs. Since the stationary phase may be composed of
micron-sized porous particles, a high-pressure pump is required to move the
mobile phase through the column (Kupiec, 2004).
The schematic of an HPLC instrument typically includes a sampler by which
the sample mixture is injected into the HPLC, one or more mechanical pumps for
pushing liquid through a tubing system, a separation column, a digital analyte
detector (e.g. a UV/Vis, or a photodiode array (PDA)) for qualitative or
quantitative analysis of the separation, and a digital microprocessor for
controlling the HPLC components (and user software). The components of HPLC
is shown in Figure 2.
HPLC can be divided into two broad categories, normal phase and reversed
phase. The normal phase (NP) chromatography has a polar stationary phase and a
non polar mobile phase where the analyte are retained by the interaction of its
polar functional group on the surface of the packing. Therefore the least polar
analyte will elute first followed by the more polar analyte. The normal phase
chromatography is useful in the separation of analytes with low to intermediate
polarity and high solubility in low polarity solvents. Water-soluble analytes are
usually not good candidates for normal-phase chromatography.

Figure 2 HPLC Components

The reversed phase (RP) chromatography has a non-polar stationary phase

and an aqueous polar mobile phase. The analytes are attracted to the surface by
their non-polar functional groups. The more polar analyte elutes from the
reversed phase column first followed by analytes in decreasing order of polarity.
The reversed phase chromatography is useful for the separation of compounds
having intermediate to high polarity.
A reversed-phase HPLC column that is end-capped has gone through a
secondary bonding step to cover unreacted silanols on the silica surface. Endcapped packing materials eliminate unpredictable secondary interactions. Basic
analytes tend to produce asymmetric tailed peaks on non end-capped columns,
requiring the addition of modifiers to the mobile phase. Non end-capped
materials exhibit different selectivity than end-capped columns. This selectivity

difference can enhance separations of polar analytes by controlling the secondary

silanol interactions.
There are many types of stationary phases used in HPLC including
unmodified silica, alumina or porous graphite, used in normal-phase
chromatography, where separation is based on differences in adsorption; a variety
of chemically modified supports prepared from polymers, silica, or porous
graphite, used in reversed-phase HPLC, where separation is based principally on
partition of the molecules between the mobile phase and the stationary phase;
resins or polymers with acid or basic groups, used in ion-exchange
chromatography, where separation is based on competition between the ions to be
separated and those in the mobile phase; porous silica or polymers. The Surface
of Reversed Phase Stationary Phase is shown in Figure 3.
The choice of mobile phases is based on the desired retention behaviour and
the physicochemical properties of the analyte as well as the type of detector
chosen. For normal-phase HPLC using unmodified stationary phases lipophilic
solvents should be employed. The presence of water in the mobile phase must be
avoided as this will reduce the efficiency of the stationary phase. In reversedphase HPLC aqueous mobile phases, with and without organic modifiers, are
The mobile phase should be filtered through suitable membrane-type filters
to remove particles or undissolved material. Multicomponent mobile phases
should be prepared by measuring the required volumes (unless masses are
specified) of the individual components, followed by manual or mechanical
mixing. Alternatively, the solvents may be delivered by the individual pumps or
proportioning valves of the liquid chromatograph and mixed according to the
desired proportion. Solvents are normally degassed by sparging with helium or by
means of sonification before pumping to avoid the formation of gas bubbles in
the detector cell.

Figure 3 The Surface of Reversed Phase Stationary Phase

The system suitability test represents an integral part of the method and is
used to ensure the adequate performance of the chosen chromatographic system.
Efficiency, capacity factor, peak-to-valley ratio, resolution factor, relative
retention and symmetry factor are the parameters that are normally used in
assessing the column performance.














electrochemical detector, Mass spectrometer detector, evaporative light scattering

detector. UV-Vis detectors are typical in many laboratories as theyUV detectors
are the most commonly used detectors because they can be used to analyse a
range of organic compounds and are relatively simple to use. A cross section of a
UV flow cell is shown in Figure 4.

Figure 4 U-Shaped geometry flow cell

Chemicals and Reagents
Trichloroacetic acid (reagent grade), HPLC grade water was prepared by
purifying demineralized water in a Milli-Q filtration system (Millipore, Bedford,
MA)., methanol (HPLC grade) was obtained from Merck (Darmstadt, Germany),
phosphoric acid (reagent grade).
High Performance Liquid Chromatography Agilent Technologies (USA)
1100 Series, consisted of a quartenary pump G1311A, a degasser G1379 A, a
Rheodyne model 7725i manual injector with 20 l sample loop G1328B, Diode
Array Detector G1315B, Column Compartement G1316A. Econofilter Nylon 0.2
m Agilent Technologies, Inc. (Germany). Volumetric flask 25 mL Iwaki Pyrex.
Syringe 50 L Agilent Technologies. Laboratory bottle 500 mL Schott Duran
made in Germany. Analytical weight AB204S Mettler Toledo. Analyrical Weight
XS 205 Dual Range FACT. Stainless-steel spatula. Beaker glass 500 mL Iwaki
Pyrex. Funnel glass 250 mL Whatmann made in Germany. Filtering Flask 1 L

schott made in Germany.

Mobile phase --- Prepare a filtered and degassed mixture of this solution
water, methanol, and phosphoric acid (75:25:0.1)
Standard preparation --- Dilute trichloroacetic acid in water to give a
concentration of 0.7 to 90 g/mL
Assay preparation --- sample tube are opened and the front and back section
of each tube are placed in separate 1 mL insets in 4 mL vials. Each section is
desorbed with 1 mL of deionized water. The vial are sealed immediately anf
allowed to desorb for 30 minutes with occasional shaking.
Chromatographic system --- The liquid chromatography is equipped with
229 nm detector and 150x4.6 mm column that contain 5m packing L1. The flow
rate is about

1 mL per minute. The mobile phase used are mixture of water :

methanol : phosphoric acid (75 : 25 : 0.1). Chromatograph the standard

preparation, and record the peak responses as directed for procedure: The
Resolution, R, not less than 2, the column efficiency calculated from clindamycin
peak is not less than 4000, the tailing factor for the clindamycin peak is not less
than 0.8 and not more than 1.2, and relative standard deviation for the
clindamycin peak is not more than 1 %, capacity factor not less than 1.5.
Procedure --- Separately inject equal volumes of the Standard preparation
and the Assay preparation into the chromatograph, record the chromatograms for
a period of time that is about twice the retention time of the trichloroacetic acid
peak, and measure the trichloroacetic acid peak areas.
System suitability test --- Inject 7 times Standard solution, then record the
concentrations, retention time (RT), capacity factor (K), tailing, efficiency
column (TP), and resolution (Rs). Make sure that each parameters have SDR not
more than 2 %.


Figure 4 Chromatogram of Trichloroacetic Acid

The determination of trichloroacetic acid using HPLC with UV detector
shows a good result. HPLC was selected because it is one of the most widely use
instrumentation in pharmaceutical industries that can qualify and quantify
analyte, time saving, and sensitive.
The trichloroacetic acid, which used as a caustic on the skin or mucous
membranes to treat local lesions and for the treatment of various dermatological
disease such as warts, can be determine using HLPC with UV detector. The peak
of trichloroacetic acid looks good, not tailing.
The results indicate that trichloroacetic acid can be determine using HPLC
with UV detector.

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