# Standard Curves By Gas Chromatographic Techniques Biology Essay

**Published:** **Last Edited:**

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

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.

## Background

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, mixtures of samples and standards in solution form. 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 readings of the unknown samples into concentrations.Â The method has the advantage that random errors in the preparation and readings of standard solutions are averaged over many standards. Again, non-linearity can be detected and avoided by diluting into the linear sensitivity range. Yet still, this can be compensated for by using non-linear curve fitting methods. It is usually, but not limited to a first-order (straight line) fit of measured signal (area) on the y-axis against concentration on the x-axis. The model equation is:

## y (signal) = m (slope) * x (concentration) + c (intercept)â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ (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)/mâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. (2)

Where y is the measuredÂ signal, 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/mâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. (3)

## Gas Chromatography

Gas chromatography (GC) is a means by which separation, identification and quantification of components in a mixture or a solution is done.

The essentials required for this method are an injection port through which samples are loaded, a "column" on which the components are separated, a regulated flow of a carrier gas which carries the sample through the instrument, a detector for the identification of analytes, and a data processor.

Figure 1: A schematic of a GC (Wikipedia)

By this tool, a sample is brought to the vapour form and a carrier gas sends the sample into a column. The carrier gas must be chemically inert. 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/ open tubular. Packed columns contain a finely divided, inert, solid support material. They are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. Capillary columns have an internal diameter of a few tenths of a millimeter which are coated with liquid stationary phases or lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. 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.

Figure 2: retention time (chemical analysis by Scoog & West)

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 so fast that accurate determination of 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 should not be too large, and should be introduced onto the column as a "plug" of vapour. The most common injection method is the use of a micro syringe to inject the sample.

To obtain optimal separations, sharp, symmetrical chromatographic peaks must be obtained. This means that band broadening must be limited.

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) ï¼ˆwww.chromatographyonline.comï¼‰

GC is still the method of first choice for the analysis of pyrethrins [Z. Cheng, Y. Wang I.J. Cromatogr. A 754 (1996) 367-395]

## 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 signals. When an unknown sample is measured, the signal from the analyte is converted into concentration using the calibration curve.

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. A series of standards of known concentrations are measured.

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).

## 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.

## 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 + câ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. (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).

Figure 3: A calibration curve plot showing limit of quantification (LOQ), limit of detection (LOD), (BOD) beyond Limit of Detection, limit of linearity (LOL) and dynamic range,ï¼Œ Sourceï¼šwww.wikipedia.com

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.

## Calibration Methods

There are about three methods for the determination of the standard/calibration curve. These are explained below.

## 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.

## 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 is not equally sensitive to each component in a mixture. The peak areas could be used directly, if this was so; to give the percentage composition of the mixture (by dividing the area of each peak by the total area under all of the peaks).

Therefore, each peak area must be multiplied by a suitable factor (called the response factor, k) to correct this. The corrected areas are then used for the calculation of the percentage composition of the mixture. Each response factor is then ratioed to that of a chosen component and this is termed relative response factor (f).

These relative response factors can then be used to determine the percent composition of an unknown mixture of the same components.

The regression equation is rearranged as in equation (3), which allows calculation of the unknown concentration.

## Method of Standard Additions

Usually, in both the external and internal standard methods matrix-based standards are prepared. This implies that the calibration standard is prepared in a solution that represents the sample extract in all ways except the presence of the analyte. By this, the likelihood of signal suppression or enhancement by the matrix is reduced. To be exact, a blank matrix sample is run to confirm that there are no interfering peaks present in the matrix.

Unfortunately sometimes, it is impossible to obtain an analyte-free matrix or one without interfering peaks. In such a case, the method of standard additions can be used to determine the concentration of the analyte in an unknown sample. A series of standards is prepared at several concentrations. The standards are then added to aliquots of the sample. Series of concentrations of a reference sample may be prepared (if it is available), in this case without an internal standard. Next, the samples are analyzed and the results plotted.

## y = mx + c, c = 0

## y = mx,

## x = y/457.59

## x = concentration (mg/mL)

Line of best fit

Figure 4: An example of a standard curve

## How to determine an unknown concentration

Determination of the unknown concentration of any sample can be done in two ways:

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.

Figure 5: graphical determination of concentrationï¼ˆadopted from journal of chemical educationï¼‰

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)/mâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ (2)

Or x = y/mâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ (3)

x is the unknown concentration to be calculated.

## GC Analysis

My first task was to experimentally determine a set of analysis conditions that yield a good separation of the six analytes in a pure solution prepared from the standard sample ï¼ˆprovided by the companyï¼‰, 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.

## Experimental Procedure

## Chemicals and Reagents

Hexane: ã€110-54-3ã€‘ï¼ŒC6H14ï¼Œ 97.0%ï¼ˆ86.18Mï¼‰

Ethanolï¼šã€64-17-5ã€‘ï¼Œ C2H5OHï¼Œ99.7% (46.07M)

Ethyl dodecanoate:

Filter papers (7cm and 15cm):

Finel:

filters (0.45um):

Syringes (1mL):

All the solvents were analytical-reagent grade. Hexane and Ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd and used without any pre-treatment.

## Equipment and Apparatus

## Samples

The grounded chrysanthemum (light green with a characteristic smell) was provided by the company in â€¦â€¦â€¦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.

## Conditions

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 â-¦C. Nitrogen was used as carrier gas at a ï¬‚ow rate of 1.6ÂµL/min. The injection volume was of 0.1 ÂµL.

The temperature program was started at 180 â-¦C kept for 11 minutes, heated at 10â-¦C/ min to 200 â-¦C, kept for 8 minutes; heated to 210 â-¦C at 10 â-¦C/min, kept for 18 minutes, then heated to 245Â°C at 30Â°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 Ã- 0.25mm id., 0.25um ï¬lm thickness. This column was chosen because it gives the best resolution, identiï¬cation and quantiï¬cation for products containing OH and C=O. (Rosana, Vanessa, 2003; Analytica Chimica Acta 505 (2004) 223-226).

## 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) â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦. (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

PY conc.(mg/ml)

PYI conc.(mg/ml)

PYII conc.(mg/ml)

pure sol

20

9.266

7.764

1ml

0.4

0.1853

0.1553

2ml

0.8

0.3706

0.3106

4ml

1.6

0.7413

0.6211

8ml

3.2

1.4826

1.2411

16ml

6.4

2.9651

2.4845

32ml

12.8

5.9302

4.969

Table 1: Concentrations of each standard portion calculated

With a micro syringe, 0.1ÂµL 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.

## Comp.

## 1ml

## 2ml

## 4ml

## 8ml

## 16ml

## 32ml

Elution

(min)

Peak area

Elution

(min)

Peak area

Elution

(min)

Peak area

Elution

(min)

Peak area

Elution

(min)

Peak area

Elution

(min)

Peak area

Cinerin1

19.86

20.01

19.87

42.79

19.87

81.28

19.87

174.29

19.88

260.04

19.91

618.90

Jasmolin1

23.11

11.94

23.11

12.86

23.11

24.18

23.11

58.79

23.12

87.79

23.13

209.17

Pyrethrin1

24.27

56.51

24.29

121.83

24.29

227.91

24.31

487.30

24.34

722.69

24.43

1711.29

Cinerin2

38.44

24.09

38.46

37.92

38.47

75.19

38.48

161.23

38.51

238.44

38.57

581.32

Jasmolin2

42.06

19.06

42.07

29.42

42.06

48.35

42.07

114.08

42.07

161.40

42.09

390.64

Pyrethrin2

42.91

4.22

42.91

3.60

42.91

8.09

42.91

18.14

42.92

26.26

42.96

57.89

Table 2: Elution times and peak areas of analytes in standard sample

With these, a calibration curve can thus be drawn for each component.

Figure 7: Calibration curves for C1, J1 and P1

Figure 8: Calibration curves for C2, J2 and P2

The concentrations and the peak areas are then tabulated to construct the overall standard curves for PYI and PYII respectively.

Soln.

PY(mg/ml)

PYI(mg/ml)

A1

PYII (mg/ml)

A2

pure sol

20

9.266

4436.5566

7.764

1790.7451

1ml

0.4

0.1853

88.4592

0.1553

47.3710

2ml

0.8

0.3706

177.4739

0.3106

70.9374

4ml

1.6

0.7413

333.3663

0.6211

131.6275

8ml

3.2

1.4826

720.3725

1.2411

293.4468

16ml

6.4

2.9651

1070.5243

2.4845

426.1003

32ml

12.8

5.9302

2539.3593

4.969

1029.8515

Table 3: concentration and corresponding peak areas

Figure 9: Pyrethrins 1 Standard Curve

Figure 10: Pyrethrins 2 Standard Curve

The Pearson correlation coefficient R2 in each of the curves is about 0.99. From literatureï¼ˆchromatography onlineï¼‰, 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.

## Organic (Normal) solvent Extraction

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 ï¼ˆWikipediaï¼‰.

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).

## 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.

A magnetic stirrer was use to maintain equal distribution of heat and solvent and the rotation was set at 50rpm in each case. Each solution was filteredï¼ˆfilter paper-7cmï¼‰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 (0.45um) and 0.1uL of each analysed in the GC. The results are below.

## Cp

## Art

## 40Â°C

## (5Hrs)

## 50Â°C

## (5Hrs)

## 60Â°C

## (5Hrs)

## 70Â°C

## (5Hrs)

t

A

t

A

t

A

t

A

C1

19.91

20.88

23270.3

20.24

5483.71

20.25

5039.21

20.30

5921.06

J1

23.13

23.80

10711.0

23.33

3043.60

23.33

2753.30

23.39

3258.92

P1

24.43

25.44

19810.3

24.86

8830.62

24.87

7989.68

24.95

8956.26

C2

38.57

39.55

4121.32

39.16

3328.70

39.19

3132.16

39.35

3767.63

J2

42.09

42.289

1125.67

42.23

2075.81

42.25

1956.50

42.29

2505.47

P2

42.96

43.237

215.407

43.18

329.54

43.20

304.55

43.25

1030.98

T'tl

191.09

190.27

389.82

193.01

23091.95

193.09

21175.4

193.53

25440.32

Table 4: results for 5hrs analysis

## Cp

## Art

## 40Â°C

## (7Hrs)

## 50Â°C

## (7Hrs)

## 60Â°C

## (7Hrs)

## 70Â°C

## (7Hrs)

t

A

t

A

t

A

t

A

C1

19.91

20.19

4175.84

20.13

3015.38

20.16

3069.10

20.96

21666.50

J1

23.13

23.31

2341.50

23.27

1642.98

23.32

1651.68

23.87

10253.00

P1

24.43

24.80

6795.98

24.72

5034.69

24.76

4946.84

25.51

18905.40

C2

38.57

39.08

2675.11

38.98

1841.87

39.06

1896.36

39.73

3905.71

J2

42.09

42.21

1638.08

42.18

1097.48

42.22

1206.58

42.34

1616.78

P2

42.96

43.16

284.72

43.11

182.52

43.15

187.01

43.28

167.43

Ttl

191.09

192.75

17911.23

192.39

12814.92

192.7

12957.57

195.69

56514.82

Table 5: results for 7hrs analysis

The average elution time for each analyte was calculated within each time frame.

component

Standard sample

At 5hrs

At 7hrs

Cinerin 1

19.91

20.43

20.36

Jasmolin 1

23.13

23.46

23.44

Pyrethrin 1

24.43

25.03

24.95

Cinerin 2

38.57

39.31

39.21

Jasmolin 2

42.09

42.26

42.24

Pyrethrin 2

42.96

43.22

43.18

Total

191.09

193.71

193.38

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.

## Concentrations (mg/ml)

Num

Comp

## Pyret

Stand

## 5hrs

## 7hrs

40

50

60

70

40

50

60

70

1

C1

PYI

## 9.27

1175.54

379.33

344.90

396.34

290.94

211.83

211.27

1110.71

2

J1

3

P1

4

C2

PYII

## 7.76

247.65

259.97

244.51

331.15

208.46

141.52

149.16

257.96

5

J2

6

P2

Total

PY

17.03

1423.19

639.30

589.41

727.49

499.40

353.35

360.43

1368.67

Table 7: concentrations of PYI and PYII

## Yields

Num

Comp

## Pyreth

Stand

## 5hrs

## 7hrs

40

50

60

70

40

50

60

70

1

C1

PYI

## 0.46

1.18

0.38

0.34

0.40

0.29

0.21

0.21

1.11

2

J1

3

P1

4

C2

PYII

## 0.38

0.25

0.26

0.24

0.33

0.21

0.14

0.15

0.26

5

J2

6

P2

Total

PY

0.85

1.42

0.64

0.59

0.73

0.50

0.35

0.36

1.37

Ratio (PYI:PYII)

1.21

4.75

1.46

1.41

1.20

1.38

1.50

1.40

4.27

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:

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 these samples, the concentrations obtained from the standard curve when analyzing the results must be multiplied by the dilution factor. The dilution factor,

## Df = final concentration/initial concentration â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ (6)

The final concentration is 1423.19mg/ml and the initial concentration is 17.03mg/ml

Therefore,

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.

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 * Vdâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. (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)

## = 14.3219mg/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.

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.

## 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 ï¼Œ5hrs 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:

Num

Comp

## Pyret

Stand

## 40oC

3hrs

4hrs

5hrs

6hrs

1

C1

PYI

## 9.27

4.23

5.72

3.77

3.59

2

J1

3

P1

4

C2

PYII

## 7.76

1.03

1.81

0.83

0.70

5

J2

6

P2

Total

PY

17.03

5.26

7.53

4.60

4.29

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).

Num

Comp

## Pyreth

Stand

## 40oC

3hrs

4hrs

5hrs

6hrs

1

C1

PYI

## 0.46

2.12

2.86

1.88

1.80

2

J1

3

P1

4

C2

PYII

## 0.38

0.52

0.90

0.41

0.35

5

J2

6

P2

Total

PY

0.85

2.64

3.76

2.29

2.15

Ratio (PYI: PYII)

1.21

4.10

3.16

4.56

5.15

Table 10: Yields and ratio of PYI and PYII

Figure 11: graph of PY yield

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 BakariÄ‡ (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.

The next step is to establish the optimum extraction conditions for the Supercritical fluid extraction of the PY crude extract.