I. ABSTRACT (ENGLISH)
The experimental determination of the yield of pyrethrins from the Chrysanthemum cinerariaefolium flower is usually carried out with Chromatographic Techniques. A lot of methods about this have been reported over the years [Z-M. ChertY.H. Wang (1996)]. These include HPLC [13 -22], GC [22-26] and SFC [B. Wenclawiak, A. Otterbach (2000) methods. Because of the need for only analyzing the pyrethrins (not reporting for the individual six pyrethrins but analyzing for total pyrethrins), GC was selected as a method of choice.
The yield reported from literature usually ranges from 0.91 - 1.30% of the dry weight [Kolak et al., 1999], 0.60 - 0.79% [Bakarić (2005)], 0.75 - 1.04% [Bhat (1995)],
1.80 - 2.50% [Morris et al. (2005), Bhat and Menary, (1984); Fulton, (1999)], 0.50 - 2.0% [Kiriamiti et al. (2003)], and 0.90 - 1.50% [Pandita and Sharma (1990)].
However, Casida and Quistad (1995) in their book: Pyrethrum Flowers: Production, Chemistry, Toxicology and Uses (pp123-193), states that it is possible to obtain pyrethrin yield of 3.0% or even more.
We obtained, with hexane extraction in a water bath at controlled temperatures and vigorous stirring (with three magnetic stirrers at a speed of 30rpm); pyrethrin yields varying from 0.85 - 3.76% of the dry weight. To our knowledge, this is the first time to report of pyrethrins yield above 3% envisaged by Casida and Quistad.
Supercritical Fluid Extraction, Supercritical Carbon Dioxide, Pyrethrins, Solvent extraction, Extraction Yield, Gas Chromatography, Pyrethrin concrete, Crude Hexane Extract.
III. ABBREVIATIONS AND SYMBOLS
GC Gas Chromatography
HPLC High Performance Liquid Chromatography
SFC Supercritical Fluid Chromatography
SCF Supercritical Fluid
SCFs Supercritical Fluids
SFE Supercritical Fluid Extraction
SC-CO2 Supercritical Carbon Dioxide
CO2 Carbon Dioxide
et al et alii (and others)
CHE crude hexane extracts
BC Before Christ
PYI pyrethrin 1
PYII pyrethrin 2
C1 cinerin 1
C2 cinerin 2
J1 Jasmolin 1
J2 Jasmolin 2
P1 pyrethrin 1
P2 Pyrethrin 2
A1 area of pyrethrins 1
A2 area of pyrethrins 2
WHO world health organization
Pc critical pressure
Tc critical Temperature
Cp critical point
oC degree centigrade
MPa mega Pascal
FID flame ionization detector
n-hexane normal hexane
/min per minute
tR retention time
k' Retention factor
R2 Pearson correlation coefficient
LOQ limit of quantification
LOL limit of linearity
LOD limit of detection
BOD beyond limit of detection
IS internal standard
k response factor
f relative response factor
µm micro meters
µL micro liters
rpm revolutions per minute
1.0. CHAPTER 1: Introduction and Literature Review
1.1.0 Supercritical Fluids (SCF)
When a fluid is forced to a pressure and temperature above its critical point (Fig. 1), it becomes a supercritical fluid. Under these conditions, various properties of the fluid are placed between those of a gas and those of a liquid.
The supercritical state of a fluid is thus defined as one whose liquid and gas are indistinguishable from each other, or one in which the fluid is compressible (i.e. behaves as a gas) while having a density similar to a liquid and, therefore, similar solvating power. Their low viscosity and relatively high diffusivity gives them better transport properties than liquids, can diffuses easily through solid materials and hence faster and better extraction yields. The most important properties of a SCF are its density, viscosity, diffusivity, heat capacity and thermal conductivity. Higher densities of SCFs contribute to greater solubilization of compounds, while low viscosities enable Easy penetration into solids and facilitate flow with fewer hindrances. Manipulating the temperature and pressure above the critical points affects the properties of SCFs and enhances their ability to penetrate and extract targeted molecules from the source materials . Since density is directly related to solubility, by altering the extraction pressure, the solvent strength of the fluid can be modified to exhibit desirable transport properties that enhance its adaptability as a solvent for liquid extraction processes. The density of a SCF is closer to that of liquids and its viscosity is comparable with gases, hence high diffusivity and faster dissolution of solute particles (the diffusivity of SCFs is ~10-4 cm2 s-1 while that of liquid solvents is ~10-5 cm2 s-1). This has contributed to the increasing use of SCF as solvents for extraction purposes.
1.2.0 Supercritical Fluid Extraction (SFE)
Supercritical Fluid Extraction (SFE) is a separation technology that uses supercritical fluid as the solvent. Every fluid is characterized by a critical point, which is defined in terms of the critical temperature and critical pressure. Fluids cannot be liquefied above the critical temperature regardless of the pressure applied, but may reach a density close to the liquid state as mentioned earlier.
The consumer and public awareness of the health, environmental and safety issues emanating from organic solvents use in chemical processes and above all, the possibility of contaminating the final products with the solvent are forces to reckon with in recent times. This has driven the chemical industry looking for the best separation technologies to obtain natural compounds from high purity and healthy products that are of excellent quality . The high cost of organic solvents, environmental regulations, and new requirements in the medical fields, for all time purer and highly valuable products have also revitalized the need for the development of new but clean technologies for products processing .
1.3.0 Supercritical Carbon Dioxide (SC-CO2)
SFE with Carbon dioxide (CO2), for this and many reasons is most unique and is able to save both time and money while retaining an overall extraction precision and accuracy.
CO2, therefore, compressed to pressures above its critical pressure ; isothermally shows effective solubility powers in the region of its critical temperature [5-6].
Though a lot of SCFs can be adapted as solvents, CO2 is by far, the most extensively used due to its non-toxic, inert and non-flammable nature. It is also inexpensive and is generally environmentally accepted substance .
Biological products are often thermally labile, lipophilic, and non-volatile and as such required to be kept and processed at around room temperatures. CO2 has a critical temperature of 31oC which makes it particularly an attractive medium for this task. Other fluids show critical temperatures in the vicinity of critical state but are often difficult to handle and to obtain in pure state, may be toxic, explosive or ecologically unsafe. For such logical reasons, SFE using CO2 has emerged as an attractive unit operation for processing biomaterials.
However, its limitations include the difficulty of extracting polar analytes, owing to the non-polar character of CO2, the different recoveries obtained from spiked and natural samples, and the frequent need for clean-up steps after extraction. Poor thermodynamic description of Supercritical (SC) solvent-solute mixtures, high capital cost for its extraction processes and an almost absence of engineering data to facilitate scale-up and design are also the prime factors that limits the use in industrial and commercial scales. For an excellent engineering design requires reliable data on the transport of a given biomaterial into the SC-CO2; such as the thermodynamic properties, fluid-liquid or fluid-solid equilibrium data of the biomaterial in the region of the temperatures and pressures where processing is technically and economically viable. Also, the measurements for the biomaterial plus SC-CO2 mixture in this condition, any mass transfer limitations for the bulk material into the SC solvent and the selectivity for the desired chemical species with the rest of the solubles in the biomaterial is paramount. Nonetheless, SFE with CO2 has great potential in the field of biomaterial processing as evidenced by the many papers published and the communications presented at the recent symposia on Supercritical fluid Technology . CO2 is a good solvent for extracting lipid-soluble compounds and enables a high level of recovery . CO2 is supercritical above 31.10C and 7.38 MPa, which makes it an ideal solvent for extracting thermally sensitive materials such as pyrethrins.
1.4.0 Set-Up and Principle
A Pilot plant equipped with two fractionation cells. (1) CO2 pump; (2) modifier pump; (3) solid samples extraction cell; (4) fractionation cell 1; (5) fractionation cell 2; (6) valve.
A fluid (CO2) is brought to a specific pressure-temperature combination, which allows it to attain supercritical solvent properties for the selective extraction of active ingredients from the sample matrix of a biomaterial (in this case Pyrethrum). The sample is exposed to the SC- CO2 under controlled conditions; time, temperature, and pressure that allow dissolution of the active ingredients (Pyrethrins) from the sample in the SCF. The dissolved active ingredients will then be separated from the supercritical solvent by a significant drop in solution pressure . Several guiding principles can be utilized to effect the extraction of these ingredients, particularly the quantitative extraction. This ideal extraction method would afford total recovery and high purity of the isolated desired ingredients (Pyrethrins). Due to the inherent variability in density, chemical composition etc, many substances that can be extracted by SFE, modification of the extraction conditions; specifically temperatures, pressures, and extraction time, may be necessary to obtain maximum extraction yield.
In addition to being used for total active ingredient (Pyrethrins) quantification, the pressure-temperature-time variables in this case would be manipulated to allow selective extraction of minute quantities of polar or non-polar analyses from the Pyrethrum sample matrices. This will help attain the optimum extraction time for the process.
Pyrethrum flowers come from the Chrysanthemum genus. Due to the size, shape, and colour of the petals; and the daisy-like appearance, they are often called “painted daisies” or “painted ladies.” Other names given to it are Buhach, Chrysanthemum Cinerariaefolium, Ofirmotox, Insect Powder, Dalmatian Insect Flowers, and Parexan.
According to Visiani (1842-1852), it was first recorded in Dalmatia . Other writers [Bakarić P. (2005)] believe that the Pharmacist Antun Drobac (1810-1882) from Croatia was the first to prove its insecticidal activity .
Yet there are claims that it was first identified to possess insecticide properties in Asia around 1800 or about 300 B.C.[ Jeanne Roberts]; and that the Crushed and powdered plants were used as insecticides by the Chinese as early as 1000 BC.
The Pyrethrum contains about 1-2% pyrethrins by dry weight, but approximately 94% of the total yield is in the seeds of the flower [Casida J.E., Quistad G.B. (1995)] .
From literature  [Coomber H.E. (1948)], the chemical structures of the active ingredients, pyrethrin I and pyrethrin II was identified in 1924 by a German chemist Herman Staudinger and a Croatian scientist Lavoslav Ružička.
Kenya is the world's main producer today, producing more than 70% of the global supply [Casida (1973)]. [15-17]
Pyrethrins are the natural active ingredients of the chrysanthemum. Pyrethrin I, cinerin I and jasmolin I are esters of the chrysanthemic acid, and cinerin II, pyrethrin II, and jasmolin II are esters of the pyrethric acid. The three chrysanthemic acid esters are referred to as pyrethrins I (PYI), and the pyrethric acid esters as pyrethrins II (PYII) [Essig K., Zhao Z. J. (2001)]. Pyrethrins I, though insoluble in water, are soluble in some hydrocarbons and organic solvents [WHO (1975)].
According to Todd et al. (2003), they are non-volatile at ambient temperatures, non-toxic to mammals and other worm-blooded animals; and highly unstable in light (photodegradable) [Chen and Casida, (1969)], biodegradable [WHO (1975)], but toxic to aquatic animals. They are mainly used for biological crop protection and as domestic insecticides; and are the major formulations of synthetic pyrethroids.
Pyrethrins when used in sufficient amounts are very effective in killing many insects.
Although pyrethrins are soluble in a number of organic solvents such as hexane, acetone, benzene, petroleum ether, methanol, chlorinated hydrocarbons, etc; other considerations as practical, economic and environmental concerns limit the use of many of these solvents. This reduces the options to just a few.
One of the qualities of Hexane in the extraction of pyrethrins is that it can dissolve the active ingredients effectively without dissolving all the other natural contaminants (pigments, waxes, fatty acids, etc), which are present and must be removed. Removing it from the concrete is also possible at lower temperatures, which limits degradation due to prolonged heating. Its low boiling point is also an added quality. Again, it can be recovered for recycling and reduces the weight of the concrete. Above all, it is inexpensive, accessible and environmentally friendly. It is non-toxic, non-corrosive, non-reactive, and non-flammable.
1.7.0 Pyrethrin Extracts
Although pyrethrins are soluble in a number of organic solvents such as hexane, acetone, benzene, petroleum ether, methanol, chlorinated hydrocarbons, etc; other considerations as practical, economic and environmental concerns limit the use of many of these solvents. This reduces the options to just a few.
Normal Hexane (n-hexane) is the solvent for the extractions. One of the qualities of Hexane in the extraction of pyrethrins is that it can dissolve the active ingredients effectively without dissolving all the other natural contaminants (pigments, waxes, fatty acids, etc), which are present and must be removed. Removing it from the concrete is also possible at lower temperatures, which limits degradation due to prolonged heating. Its low boiling point is also an added quality. Again, it can be recovered for recycling and reduces the weight of the concrete. Above all, it is inexpensive, accessible and environmentally friendly. It is non-toxic, non-corrosive, non-reactive, and non-flammable.
The Hexane is heated above ambient temperature, considering its boiling point (pyrethrum is 170 oC - 200 oC)  as the upper limit; for efficient extraction but raising the temperature has an effect. It promotes and aids degradation of the active ingredients. The concentration of pyrethrins in the concrete is expected to be 30 ± 10% by weight with contaminants. Organic compounds that have lower molecular weights (ester, ether, etc) are soluble in liquid carbon dioxide. Some constituents of pyrethrum are partially soluble in liquid CO2, while others are not. Fatty acids, alkanes, triterpenols, Water, are those slightly soluble yet inorganic salts, amino acids, sugars, carotenoids, and fruit acids are insoluble [Marc Sims, (1981)]. Pyrethrins are very soluble in liquid CO2 due to the lone ketone and at least one or ester in its molecules. The rest of the molecule is hydrocarbon.
Normally, GC analysis of the Pyrethrin components is difficult because Pyrethrin I and II undergo thermal isomerization to form isopyrethrin I and II at temperatures above 200oC [24-27]. These temperatures can neither be avoided in split or splitless injection systems nor in the elution from capillary columns. This brings about a transition of the isopyrethrins continuously and lead to improper integration of Pyrethrin I and II the peaks.
The use of very short thin film columns combined with an on-column injection system17 will reduce this thermal conversion. Yet even with such columns the capacity and the separation performance will be insufficient. It also means that the natural Pyrethrins will appear together but the overall detection of the total amount of the pyrethrins is therefore feasible.
Pyrethrum is very important due to these key properties:
1.8.1 Action: It attacks rapidly, the nervous system of the insects providing knockdown and killing effects eventually.
1.8.2 Immunity: In fact, there are beliefs that insects developing resistance to Pyrethrum is not practicable due to the complex nature of its structure
1.8.3. Toxicity: For a long time, it has proven to be safe for humans and other warm blooded animals but some claim it is toxic for cats [PetEducation.com], but toxic to aquatic animals even at as 2 parts per trillion [Bhanoo, Sindya (2010)] Green Inc. Energy, Environment, and the Bottom Line. New York Times, http://greeninc.blogs.nytimes.com
1.8.4 Activity: It has a vast spectrum of activity and can be used against any insect species. This is because of its closely related group of compounds (PYI and PYII).
1.8.5 Repellency and inhibition/jamming: Pyrethrum is also used to repel insects in food and grain during storage and personal protection. Beside this, it is also used to inhibit insect's biting efficiency [ ] or jam their biting ability
1.8.6 Flushing: It is considered to have the greatest flushing action than any insecticide. It disturbs and flashes out the insects in their hiding outs.
1.8.7 Environment: Pyrethrum is photodegradable and as such is environmentally friendly (half-life of 12 days in soil) [www.ehow.com]. It also decomposes in air and relatively high temperatures and therefore presents no hazards due to persistence [NPTN Fact Sheet]. National pesticide telecommunications network
Pyrethroids are man-made (synthetic) but chemically stabilized form of natural pyrethrins. Their structures are adapted and resemble that of pyrethrins and as such have similar activities. They are altered to improve their stability and potency.There are two kinds. Type I include tetramethrin, Allethrin, bioresmethrin, resmethrin and permethrin. Some type II pyrethroids are cyfluthrin, cypermethrin, deltamethrin, fenvalerate, cyphenothrin, and fluvalinate. They are persistent compounds (cypermethrin, permethrin and deltamethrin) and are resistant to degradation by air and light and therefore, are appropriate for use in wide applications, but they have higher significant mammalian toxicities (Morgan, 1989).
Despite the potency and safety of pyrethrum, it has few limitations. Some insects are able to recover from the knockdown effect. Again, because it breaks down in air and sunlight, it looses its effectiveness quickly in outdoor use. These are combated by treating pyrethrum extract with a liquid called Synergist. This has the ability to protect the pyrethrum from breaking down in the insect's system. Small quantities of pyrethrum are mixed with these chemicals to effectively and efficiently control insect populations. The most popular of there are MGK-264 and Piperonyl butoxide.
The purpose of this experiment is to determine, compare the efficiency and provide a method by which pyrethrins are obtained in an appreciably pure and at the same time stable form (yet economical) from pyrethrum flowers by extracting
1) with an organic solvent (n-hexane) in a Soxhlet extraction and finally obtaining the pyrethrins from the concrete using sc.CO2 (proposed method); and
2) Directly with SC.CO2 and finally dissolving the concrete in organic solvents (methanol, petroleum ether and n-hexane) to obtain the pyrethrins (Factory method).
Grounded chrysanthemum cinerariaefolium, Hexane, CO2 SFE grade
Soxhlet apparatus, burner, and flask, filter paper
1.8.9. SFE apparatus
A self built apparatus with the following parts will be used: the pump, cylinder, oven, flow valves, pressure gauges, and thermometers.
1.9.0. Normal (Organic) Solvent Extractions
1.9.1. Water bath
Extraction of the pyrethrins from 100g of the grounded pyrethrum flowers with hexane would be conducted in a water bath (YUHUA, DF-101S) in batches at temperatures of 35oC, 40 oC, 45 oC, 50 oC, 60 oC and 70 oC in 3hrs, 4hrs, 5hrs, 6hrs and 7hrs; in a 1000mL round bottomed flask. Agitation would be achieved by stirring vigorously with three big size magnetic stirrers at a speed of 20rpm. The hexane would then be removed with a rotary evapourator (YUHUA, RE-2000B) at a temperature of 25 oC and at a speed of 180rpm; to obtain the pyrethrin concrete (20mL) also called Crude Hexane Extract (CHE).
1.9.2. Chromatographic Conditions
A Gas Chromatogram with flame ionization detection (FID); Agilent…., HP-5 capillary Column, 30mm × 0.25mm id., 0.25um ﬁlm thickness would be used. Each run required about 50mins. The instrument would be calibrated with multiple-point standard additions calibration method using the six individual standard samples to be prepared. The peak area of each component in the sample solutions would be fitted within the linear range of the standard. The split/split less injector, in the ratio 20:1, would be kept at 250 ◦C. Nitrogen would be the carrier gas at a ﬂow rate of 1.6µL/min with an injection volume of 0.1µL.
The temperature program would start at 180 oC, kept for 11 minutes, heated at 10◦C/ min to 200 ◦C, kept for 8 minutes; heated to 210 ◦C at 10 ◦C/min, kept for 18 minutes, then heated to 245°C at 30°C/min, staying at this temperature for 4 minutes.
1.9.3. SFE Conditions
The carrier gas will be Hexane with a constant flow rate of 2mL/min, pressure would be between 13-25Mpa. The temperature program would be from 35-45oC. Contact time would be within 3hrs to 6hrs.
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2.0. CHAPTER 2: Report on GC analysis and Organic (Normal) Extractions
The objective of this section of the experiment was to establish standard curves, by gas chromatographic techniques; for pyrethrins 1 (PYI) and pyrethrins 2 (PYII); the two groups of the six essential ingredients (Cinerin 1, Jasmolin 1, Pyrethrin 1; and Cinerin 2, Jasmolin 2, Pyrethrin 2) of the chrysanthemum (with the standard sample provided by the company), and to determine the percentage yields (and global yield) after Hexane (normal) extractions.
In analytical chemistry, the accuracy of quantitative measurements of the constituents of samples, using standard samples of known composition usually requires calibration. It is usually, but not automatically, done with samples and standards dissolved in appropriate solvents. This is due to the ease of preparing and diluting accurately, mixture of standard samples. Several standard solutions are prepared and analysed or measured, a line or curve is drawn (fit) to the data points and the obtained equation is used to translate values from the unknowns into corresponding concentrations. It has the advantage that random errors in the preparation and readings of standard solutions are averaged over many standards. Again, non-linearity can be seen and eliminated by fitting into the linear sensitivity range by dilution.
Yet still, this can be compensated for by using non-linear curve fitting methods.
It is usually, but not limited to a first-order of measured fit signal (area) on the y-axis against concentration on the x-axis. The model equation is:
y (signal) = m (slope) * x (concentration) + c (intercept)…………………… (1)
It is the most common and straightforward method, but its main drawback is that it cannot compensate for non-linearity. A minimum of two data points are needed to construct the curve. The concentration, x of the unknown sample is given by
x = (y-c)/m……………………………………….. (2)
Where y is the measuredsignal, m is the slope and c is the intercept from the curve (straight line fit). The value of c is zero if the curve is forced through the origin: then
x = y/m……………………………………….. (3)
2.3.0 Gas Chromatography
Gas Chromatography (GC) is a means by which separations, quantifications and identifications of analytes of a given solution or mixture is done.
The essentials required for this method are an injection port where samples are placed. There is also a column on where separations of the components are done; a carrier gas whose flow is regulated to carry the samples all along the instrument, and a detector for the identification of analytes as well as a data processor.
By this tool, a sample is brought to the vapour form and a carrier gas sends the sample into a column. The carrier gas should be inert; chemically. The choice is often governed by the type of detector which is used. With a gas-liquid chromatography, the column is normally packed with a solid stationary phase. Once the sample moves along the column, the analytes that interact strongly with the phase spend more time in the stationary and the moving gas phases, hence will require more time to travel along the column.
There are generally two types of columns: packed and capillary (sometimes called open tubular). Packed columns are 1.5 - 10m in length and have about 2 - 4mm internal diameter. Capillary columns are coated with liquid stationary phases or lined with thin layer which adsorb the stationary phase. They are more efficient than packed columns.
After exiting the column the analytes once separated are detected by a detector and their response recorded for analysis.
The time from injection of a sample to the time an analyte is detected is defined as
Retention time tR. The boiling point of the sample is vital in determining retention time. Those with higher volatility (lower boiling points) tend to have shorter retention times as they spend more time in moving from the gas phase. Each analyte (component) will have a different retention time.
Retention factor, k', (or capacity factor) is a term used to describe the travel rate of an analyte on a column. If the retention factor is less than one, elution will be quick such that to determine accurately the retention time is difficult; otherwise elution takes a very long time. The retention factor for an analyte is usually between one and five. For optimum efficiency, the volume must not be very large, and must be put in the column as a vapour "plug". The most common injection method is the use of a micro syringe to inject the sample. For effective separations, peaks must symmetrical, sharp and hence no band broadening and trailing must be present.
The greatest constraint of gas phase chromatography is the vaporization of the solid and liquid samples on the column in the gaseous state. This limits its use usually to the study of thermo stable and sufficiently volatile compounds (1) （www.chromatographyonline.com）
2.4.0 Determination of the Standard/Calibration Curve
A calibration curve provides the relationship between a signal produced by an instrument and the concentration of the analytes being measured. Different analytes produces different and unique signals. The measurements (signals) of an analyte of unknown sample are commuted, from the standard curve into concentrations.
Quantitative analysis with GC is based on comparative methods. The Concept is that the sample with the analyte and a standard Sample that has the same concentration of this analyte will produce similar results, using an instrument with the same conditions. Several standards of known are prepared and their concentrations calculated.
Then a standard curve is established from the values of the analytical result (in this case; area) as a function of analyte concentration. This standard curve is then used to find the concentration of an unknown sample. Usually, the abscissa (x-axis) corresponds to the concentration and ordinate (y-axis) of the signal result (area).
2.4.1. Regression analysis
The import of this analysis is to provide an equation that relates the instrument results to the concentrations used, such that with a given result the corresponding unknown concentration can be determined. The model of the function is y = f (x), defining y. The errors in calculating the concentrations would be acceptable (less) if the signal of the unknown are in the range (middle) of the signals of the standards.
2.4.2. Simple linear regression
Once the results of the detectors are linear as a function of the measured variable, then the goal now is to obtain the parameters of a straight line of best fits. The least squares regression line, which reduces the sum of the square or the error of the data points; is represented by the linear equation,
y = mx + c……………………………………………………………………….. (4)
x is the independent variable, and y is the dependent variable. The term c is the y-intercept or regression constant (c is the value when x = 0), and the term, m; is the slope (sensitivity) or regression coefficient. The Pearson correlation coefficient R2 gives a measure of the reliability of the linear relationship between the x and y values. If R2 = 1, the linear relationship between x and y exists and is exact. Values of R2 close to 1 indicate excellent linear reliability. If the correlation coefficient is far away from 1, the relationship is less reliable.
A straight line suggests that the errors in y follow the law of normal distribution and usually, the experimental error is considered to affect the y value only but not the x value (concentration recorded).
If the response of any unknown falls outside the range of the standard, then additional work is required. Likewise if it falls below the Limit of Detection, then the sample needs to be concentrated and must be diluted if it lies above the Limit of Linearity.
2.4.3 Calibration Methods
There are about three methods for the determination of the standard/calibration curve. These are explained below.
22.214.171.124 External standard method
This method involves the comparison of two chromatograms obtained successively but with the same control conditions.
The first chromatogram is acquired from a standard solution (reference solution) of known concentration in a solvent, for which a known volume is injected and the corresponding area in the chromatogram is measured. The second chromatogram results from the injection of the same volume of the sample in a solution containing an unknown concentration of the compound to be measured. Since the same volumes of both samples are injected, the ratio of the areas is proportional to the ratio of the concentrations which depend upon the masses injected.
For precision, several solutions of varying concentrations are used in order to create a calibration curve.
126.96.36.199 Internal standard method
With internal standardization, a second compound, often related to the analyte but never found in the sample, is added at a known concentration to every sample and calibrator. It is the ratio of the analyte to internal standard that is the critical measurement in an internally standardized method. The calibration curve data are generated by injecting calibration samples of different concentrations, that all contain the same concentration of internal standard. The ratio of the analyte area to the internal standard area is calculated and plotted as the y-value against the concentration of the calibrator in x-axis. This compensates for any imprecision resulting from the injected volumes, which is the main drawback of the External standard method. This method relies on the relative response factor of each compound to be measured against the standard. It requires two chromatograms, one to calculate the relative response factors of the compounds of interest, and the other for analysis. The idea of relative response factors arises because the detector does not respond to each analyte in a mixture the same way. The areas of peaks could directly be used, if this was so; to obtain the total composition. This is done by dividing the peak area of each by the total area of the peaks. Hence, every peak area should be multiplied by an appropriate factor known as the response factor, (k) to compensate that. The compensated areas are then what are used to calculate the total mixture composition. Each response factor is then ratioed to that of a chosen component and this is termed relative response factor (f).
The relative response factors then enable the determination of the total composition of any unknown mixture of similar components.
The regression equation is rearranged as in equation (3), which allows calculation of the unknown concentration.
188.8.131.52. Method of Standard Additions
Usually, in both the external and internal standard methods, matrix-based standards are prepared. This implies that the standard to be used for the calibration must not have any traces of the analyte. By this, reduces the likely signal stifling or interference of the matrix. To be exact, a blank matrix sample is analysed to ensure that no interfering peaks exist. Unfortunately sometimes, it is not possible to have an analyte without interfering peaks (free matrix). In such cases, this method is employed. A series of standards are obtained in several different concentrations. The standards are so added to portions of the sample. Several concentrations of the reference sample may be prepared (if available), without an internal standard. Thereafter, the samples are assayed and the corresponding results appropriately plotted.
2.5.0 How to determine an unknown concentration
Determination of the unknown concentration of any sample can be done in two ways:
2.5.1. Graphically: Once the signal of the unknown is obtained, a horizontal line is drawn from the signal on the y axis (0.068) to meet the calibration curve and then a vertical line drawn straight down to the concentration on the x-axis (shown with blue arrows). The value at this point (estimated as 0.32M) is the concentration of the unknown sample as shown below.
2.5.2. Mathematically: The equation of the calibration curve is fitted to the data, and solved for concentration as a function of the signal (y). Then, the signal for each unknown is substituted into this equation and the corresponding unknown concentration calculated for. This gives more accurate concentration values compared with the graphical method.
The fit equation is as in equation (1) and it is expressed mathematically as in equation (4) above. Solving equation (4) for Concentration (x) yields either equation (2) or (3) depending on whether the fit is forced through zero or intercept at c. i.e.
x = (y - c)/m………………………………………… (2)
Or x = y/m……………………………………………… (3)
x is the unknown concentration to be calculated.
2.6.0 GC Analysis
My first problem was to determine a set of experimental analysis conditions that give a better separation of the six analytes in a pure solution prepared from the standard sample （provided by the company）, in a reasonable time frame. Several conditions were tested until this particular one below chosen. Identification of the individual peaks was based on the similarities between the peaks produced and those from literature (B.W. Wenclawiak et al, 1997) with different conditions though.
Analyses with these conditions were repeated three times for accuracy and reproducibility.
Six standard solutions prepared were injected and chromatograms obtained. Replicate injections of each solution were made for precision and accuracy. The peaks from these chromatograms were compared with those found in literature for the identification of the individual analytes and their corresponding retention times noted. After this, the peak areas for PYI and PYII of all the six solutions were calculated by the software and recorded. A plot of these peak areas and the concentrations calculated earlier gave the standard curve for the analysis.
2.7.0 Experimental Procedure
2.7.1 Chemicals and Reagents
Hexane: 【110-54-3】，C6H14， 97.0%（86.18M）
Ethanol：【64-17-5】， C2H5OH，99.7% (46.07M)
Filter papers (7cm and 15cm):
All the solvents were analytical-reagent grade. Hexane and Ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd and used without any pre-treatment.
2.7.2 Equipment and Apparatus
The grounded chrysanthemum (light green with a characteristic smell) was provided by the company in ………as well as two samples of the pyrethrum concrete (yellowish in colour). The first sample contained 50% of pyrethrins (i .e. 29.5% of pyrethrins 1 referred to as PYI and 20.5% of pyrethrins 2, called PYII); and the second one had 85.15%, comprising of 46.33% PYI and 38.82% PYII.
The conditions finally chosen as the best, after a series of conditions tried was:
The split/split less injector, in the split ratio 20:1, was kept at 250 ◦C. Nitrogen was used as carrier gas at a ﬂow rate of 1.6µL/min. The injection volume was of 0.1 µL.
The temperature program was started at 180 ◦C kept for 11 minutes, heated at 10◦C/ min to 200 ◦C, kept for 8 minutes; heated to 210 ◦C at 10 ◦C/min, kept for 18 minutes, then heated to 245°C at 30°C/min, staying at this temperature for 4minutes.
The chromatographic analysis was performed in a gas chromatograph with an FID detector, Agilent GC, HP-5 Column, 30mm × 0.25mm id., 0.25 µm ﬁlm thicknesses. This column was chosen because it gives the best resolution, identiﬁcation and quantiﬁcation for products containing OH and C=O. (Rosana, Vanessa, 2003; Analytica Chimica Acta 505 (2004) 223-226).
2.10.0 Standard Curve
Once the conditions were established, 2g of the PY concrete was transferred into a 100mL volumetric flask and Ethanol was filled to the mark and shook to mix. Knowing the mass, the concentration of the PY solution (20mg/mL) was then calculated using the relation:
Concentration (mg/mL) = mass (mg)/Volume (mL) ………………………. (5)
The concentration of PYI (9.266mg/mL) and PYII (7.764mg/mL) was calculated keeping in mind the % of each group in the sample provided (i.e. 46.33% and 38.82% respectively).
Six standard aliquots; 1mL, 2mL, 4mL, 8mL, 16mL and 32mL of this PY solution was then transferred into a 50mL flask each and diluted with Ethanol again to the mark and mixed.
The concentration of each standard portion and the PYI and PYII concentrations were then calculated appropriately.
Pure PY sol
Table 1: Concentrations of each standard portion calculated
With a micro syringe, 0.1µL of each of these solutions were injected into the GC for analysis. The elution times and the corresponding peak areas were noted and recorded. See the table below.
Table 2: Elution times and peak areas of analytes in standard sample
The concentrations and the peak areas are then tabulated to construct the overall standard curves for PYI and PYII respectively.
Table 3: concentration and corresponding peak areas
The Pearson correlation coefficient R2 in each of the curves is about 0.99. From literature（chromatography online）, this indicates the measure of the reliability of the linear relationship between the x (concentrations) and y (peak areas) values. Therefore, the standard curves could be used for the determination of corresponding unknown concentrations given their peak areas.
3.0 CHAPTER 3: Organic (Normal) solvent Extraction
3.1.0 Objective and Procedure
The main objective of this extraction process is to obtain a light coloured Product with a high recovery rate of the six pyrethrin active ingredients [Kiriamiti et al. 2003].
Extraction essential components with an organic solvent is the simplest, commonest and most importantly, economic technique in modern Chemical industry （Wikipedia）.
The desired samples are submerged completely and agitated in an organic solvent. This agitation (with or without heat) helps to dissolve the desired compounds needed. Hexane, dimethyl ether, methanol, ethanol are some of the most common organic solvents used for these kinds of extractions.
However, not only the desired components are extracted during this process. Other soluble substances (waxes and pigments) that are hydrophobic are also extracted. The solvent is removed from the extract by vacuum processing at lower temperature, for re-use. The process can last for hours or weeks, or even more. After the solvent removal, the waxy thick mass left is the concrete. This is composed of essential oils and other oil soluble (lipophilic) materials (Wikipedia). The concrete is too thick (viscous), coupled with the presents of undesired components; to be used directly. A further treatment, usually with another solvent that only dissolve the desired compounds from the concrete is necessary. This solvent is again removed leaving behind the absolute (substance).
3.2.0 Sample Preparation
For all chemical analyses, the analyte to be measured must be in a sufficient quantity and in a suitable form for the GC analysis. Usually, samples require pretreatment. This has an influence on the end result. Sample preparation is therefore an essential step in analysis just as measurements. Then appropriate extraction methods are employed.
A 100g of the grounded chrysanthemum material containing the desired analytes was weighed out and placed inside a 500mL round bottom flask and a bottle of normal Hexane (as extraction solvent) was poured in to submerge the sample completely.
This was then transferred into a water bath. The set up was then equipped with a condenser, to condensate the liquid vapour and connected to water source.
The temperature program was set and the system was heated at various temperatures (40 oC, 50 oC, 60oC and 70 oC) each at times 5hrs and 7hrs.
Magnetic stirrers were used to maintain equal distribution of heat and solvent and the rotation was set at 20rpm in each case. Each solution was filtered（filter paper-7cm）with the aid of a rotary evapourator after the set time and the extracted solid discarded. The filtered solution was condensed to 10ml each with a rotary evapourator to remove the solvent.
This system has the advantage that the solvent is repeatedly recycled and also the temperature can be controlled (since the sample is thermo labile).
Each concentrated sample was thereafter, filtered (0.45µm) and 0.1µL of each analysed in the GC. The results are below.
Table 4: results for 5hrs analysis
Table 5: results for 7hrs analysis
The average elution time for each analyte was calculated within each time frame.
Table 6: average retention times
With the peak areas from Table 4, the concentrations and yields for PYI and PYII within these times was calculated as well.
Table 7: concentrations of PYI and PYII
Ratio (PYI : PYII)
Table 8: yields and ratio for PYI and PYII
The areas of PYI and PYII for the various temperatures from the analysis (Tables 4 and 5), gave higher concentrations (Table 7). These exceeded the range set for the standard. The range for PYI is 9.27mg/ml and that of PYII is 7.79mg/ml. Yet the lowest concentrations for PYI and PYII (Table 7) are 211.27mg/ml and 141.52mg/ml respectively. The total PY concentration in the standard (range) is 17.03mg/ml and the highest PY concentration from the analysis (extractions) is 142.32mg/ml (since PYI and PYII have the same volume, their concentrations could be added).
Therefore, and for accurate results, these concentrations should be diluted (mix with more solvent) to fit into the range before proceeding with the analysis. This can be done by ways:
1) Finding the Dilution Factor. This in a way will tell how many times the initial volume (before the analysis) should be diluted to fit into the range. For such cases, the concentrations gotten from the reading on standard curve should be multiplied by the dilution factor. Therefore, the dilution factor,
Df = final concentration/initial concentration …………………… (6)
The final concentration is 1423.19mg/ml and the initial concentration is 17.03mg/ml
Df = 1423.19(mg/ml)/17.03(mg/ml)
Df = 83.56958
This show a that the initial concentrated volume of 10ml should be multiplied almost 84 (i.e. about 850ml) times. This is too much solvent to use, hence not economical.
1) By taking a portion (aliquot) of the concentrated concrete and diluting it with an amount of solvent. The concentration of the concentrated concrete, Cc = 142.32mg/ml and that of the diluted concrete is Cd. The volume of the concentrated concrete taken is Vc and that of the dilution targeted is Vd
Using the dilution equation
Cc * Vc = Cd * Vd…………………………………………………………….. (7),
the diluted concentration, Cd can be calculated.
Cc = 1432.19mg/mL, if Vc = 1mL and Vd = 50mL. Cd = ?
Cd = 1432.19 x 1 / 50 (mg/mL)
This new diluted concentration falls within the range of 17.03mg/mL
Therefore, after concentrating the extract to 10mL, 1mL aliquot is moved into a 50mL flask and solvent topped to the mark before the GC analysis.
2) Eliminating the use of the rotary evapourator. Since a bottle of Hexane (500mL) is used for the extraction each time, it is not necessary to concentrate the solution after filtration but top up with some hexane to 500mL (some hexane will escape during the extraction process) mark before the GC analysis.
3.3.0 Optimum Extraction Temperature
Table 8 shows the extraction temperatures and the corresponding yields between 5hrs and 7hrs. The result in this case suggests that the optimum temperature is at 40oC. This is because pyrethrins are thermo labile and therefore degrade after 40oC [E. Stahl, 1998; C. Gourdon, 2002; W.H.T.Pan, 1994]. At 40oC, targeted PY components are extracted more but after this temperature (with the increase) more undesirable components are extracted at the expense of the pyrethrins components which decompose. Again, at 40oC ，5hrs gave a better yield than 7hrs. This suggests that with prolong heating, even at a safer extraction temperature (40oC), the PY yield is affected negatively.
Therefore, an investigation into the optimum time and yield at this temperature (40oC) was done and the results, by fitting into the concentration range this time, before GC analysis are below:
Table 9: Concentrations at various times at 40oC
The results show that the concentrations are fitted into the range such that all the concentrations less than the maximum range set (17.03). Even more, they are within half of the range. This is important because the errors in the concentrations will be minimal if the signal (area) from the unknown lies in the middle of the signals (areas) of all the standards (chromatography online).
Ratio (PYI: PYII)
Table 10: Yields and ratio of PYI and PYII
3.4.0 Results and Discussions
The percentage yields I obtained are not out of place comparing with literature. In some cases, the yield of PY varies from 0.91 to 1.30% of the dry weight [Kolak et al., 1999; Casida and Quistad, 1995]. According to Bakarić (2005), the yield is between 0.60 - 0.79%. Bhat (1995) reported content ranging from 0.75 to 1.04%. However, Morris et al. (2005), reported yields of approximately 1.80 to 2.50%. Still according to Kiriamiti et al. (2003) the yield ranges between 0.50 and 2.0% while Pandita and Sharma (1990) gave yields varying from 0.90 to 1.50%. Above all Casida and Quistad (1995) states that it is possible to obtain pyrethrin yield of 3.0% or more. Therefore, the yield from my analysis of 0.85 to 3.76% conforms to literature.
From this analysis, the optimum extraction conditions with Hexane are at 40oC in 4 hours (yield 3.76) but is this the real optimum extraction conditions (especially the temperature)? Since PY does not decompose between 20oC and 40oC, is it possible to have the optimum temperature at 25oC, 30oC or even 35oC?
With this in mind, a further investigation was carried out in the same time frame (4hrs) beginning with 30oC such that if the result gave more yield than the one at 40oC, then the next would be 25oC and possibly 20oC. On the other hand, if the result gave fewer yields then the next would be 35oC and possibly 45oC. This would confirm the optimum conditions for the extraction process.
2) chemical analysis book
5) B.W. Wenclawiak et al, 1997
6) Rosana, Vanessa, 2003; Analytica Chimica Acta 505 (2004) 223-226
7) Kolak et al., 1999; Casida and Quistad, 1995
8) Bakarić (2005)
9) Bhat (1995)
10) Morris et al. (2005)
11) Kiriamiti et al. (2003)
12) Pandita and Sharma (1990)
13) Casida and Quistad (1995)
14) ‘Journal of chemical education' vol. 75, no. 9, September 1998
Pyrethrins are well separated on polar columns but their analysis with a conventional (normal) column takes about 1 h. This is unacceptable but with a short OV-1 column, analysis time is reduced and thermal decomposition is also drastically limited; since the residence time is shorter and the elution temperature of pyrethrins I and II is lower. [Recommendation]
A cold-on-column injection system and a high constant flow rate can further reduce pyrethrin degradation and elution temperatures and as a consequence their isomerization. [Recommend]