Standardization of method using HPLC

1. Standardization of method to
quantify carbaryl using HPLC

2. Why to Quantify Carbaryl
 Synergic Effect
Acute toxicity of a carbaryl- piperonyl butoxide mixture
was over 100 times that of carbaryl alone in fishes.
Some carbaryl formulations are found to contain
crystalline silica and this silica is said to have “limited
evidence of carcinogenicity in humans” and “sufficient
evidence of carcinogenicity in animals ” by the
international Agency for Research on cancer

3. Effects of Carbaryl
 Carbaryl studies involved measures of activity of immune
system, decrease in the production of antibodies, decrease in the
resistance to infection by certain diseases in rats and rabbits.
 Researchers found that female beagle dogs fed with carbaryl with
doses less than 1/50 of LD50 had more infant deaths, little pups
and more pups with birth defects as compared to unexposed
mothers .
 A group of scientists led by a toxicologist from the Institute
Nationalde la Recherche Agronomique (France) studied carbaryl
in 2001. They found, in human cells, that carbaryl stimulates the
activity of an enzyme that transforms carbaryl into a compound
that causes damage to DNA.

4. Analysis
 Qualitative Analysis
Qualitative analysis refers to the detection of a particular
compound in the samples. Each compound shows absorbance at
particular wavelength and specific retention time. Obtaining
peak that this particular wavelength and retention time shows
the presence of compound.
 Quantitative Analysis
Quantitative analysis refers to the measurement of the
concentration of compound in the samples. Obtaining peaks in
quantitative analysis has particular height and area which are
proportional to its concentration. A calibration curve is created
using standard samples. The concentration of the compound in
other samples can be concluded using this standard curve.

5. Separation and Quantification
 Chromatography is a separation technique which
mainly used for both separation and quantification of
sample components.
 HPLC (High Performance LC) relies on pumps to pass
a pressurized liquid solvent containing the sample
mixture through a column filled with a solid adsorbent
material.
Instrument used:
The LC-2010HT is a HPLC based on the concept of high-
throughput analysis and automated validation.

7. Features
 The LC-2010HT is comprised of a degassing unit,
quaternary low-pressure gradient unit, pump unit,
mixer, ultra fast autosampler (having the carrying
capacity of 40vials), column oven, and a UV-VIS
detector with a thermostatted flow cell. It has the
latest technology for multiple samples, high-speed
injection and automation.

8. Software
 Analytical conditions can be set using LC solution software (1.24
version).
Two parts involved in analysis:
1.Real time analysis:An analytical method parameters are need to be set in
real time programme, specifically since different compounds have
different condition requirements like temperature, pressure, solvent
flow rate and wavelength.
2.Post run analysis:Integration was done in post-run analysis programme
by measuring the detector response using a suitable method to prepare
a calibration curve using several solutions over a range of
concentrations.
Process
Carbaryl standards prepared in methanol were injected directly to high
through put autosampler. The mobile phase used was on gradient
containing acetonitrile and water (40:60) at the flow rate of 1ml/min
through pump at the isocratic mode.

9. Automated analysis programme
Device start-up
Flow line purging with
mobile phase
Column / system equilibration
Stabilizing baseline
Analysis start
Analysis
Analysis finish
Temp. control off for oven etc.
Degasser and pump halted
Shutdown

10. concentrations
standard Stock µl MeOH µl Total volume µl
1ppm 0.05 499.95 500
2ppm 0.1 499.90 500
3ppm 0.15 499.85 500
5ppm 0.25 499.75 500

11. Chromatogram obtained

12. Standard graph for carbaryl
Y= a x + b Where, a = 9.252955e-007
b = 1.039121
Thus for a given compound whose values of area are
known,its concentration can be obtained by applying
above equation.

13. Area obtained for different
standards
s.no. concentration Area uV Retention time
min.
1 1ppm 339494 4.712
2 2ppm 848480 4.710
3 3ppm 1714996 4.709
4 5ppm 3412327 4.703
This indicates that, the peak area was increased with the increased in
concentration. It has same retention time at a given wavelength i.e.
280 nm, which was specific for carbaryl, and the area obtained
increases significantly with the increase in concentration of a
standard.

14. Future applications of this work
The above standard graph can be used to quantify the
amount of carbaryl in a given samples. It can be used
to study the amount of retention of a toxicant on
exposure in different life forms.
It has typical applications pertain to the quantitative
and/or qualitative analysis of pesticide toxicants in
food composition, natural products, food additives,
flavour and aroma components.

15. Thank You