II – High-Pressure Liquid

Daniel Abegg – Nicolas Calo – Emvuli Mazamay – Pedro Surriabre

Universit´ede Gen`eve, Science II, Laboratoire 144 – Groupe 4

December 29, 2008

Summary

In this experiment we have determined for a mixture of vanilla compounds the best separation is given with a flow rate of 2 ml/min. By working with this flow rate we made calibrations curves of those compounds at different concentration and have quantified the amount of vanillin commercials (Migros and Coop) vanilla pod. The found amount is around 1.1% which is less then the value expected in vanilla pods.

Introduction

HPLC is a method used in chemistry to analyze solutions components. It is based on affinity and interactions between analytes of a liquid mobile phase and the stationary phase of a chromatography column. There are two types of stationary phases: the normal ones which retain only polar molecules and the reverse ones, those are made of long alkyl chains on silanols groups which allow the retention of apolar groups. An UV detector is used to obtain the dead time and the retention times. The aim of this experiment is to observe a HPLC in reverse phase and to analyze the 4 main metabolites of vanilla (vanilic acid, vanillin, and hydroxyl benzaldehyde and hydroxyl benzoic acid) in a vanilla pod. After preparing different solutions of the 4 substances above in a mix of acetonitrile and water (50 : 50) making them pass through the column at diverse flows, we calculate their different chromatographic parameters. Those data will be used to find the concentration of the 4 molecules in a solution of vanilla pod.

1 . ABC D

O OH O O O OH .

O O

OH OH OH OH

Vanillic acid Vanillin 4−hydroxy−benzaldehyde 4−hydroxy−benzoic acid 260nm 230nm 280nm 260nm

Figure 1: Vanillic acid, Vanillin, 4-hydroxy-benzaldehyde and 4-hydroxy-benzoic acid are four vanilla compound

Methodology

The solvent used is a 50 : 50 mix of water and acetonitrile. 4 solutions containing 3 mg in 25 ml are prepared. The three first are made of vanilic acid, vanilin , 4-hydroxy - benzaldehyde and the last one is a mixture of all of them with 4-hydroxy-benzoic acid. Using a syringe, the column is cleaned with the standard eluant solution and with the solvent. Then the column is filled with the sample. The operation is repeated for different flows for the mix.

By diluting the mix, with different factor, we make calibrations curves to determine the concentration of those species in commercial vanilla pod.

Results

Elution order

ABCD mass mg 3.5 3.0 3.1 – massmix mg 3.3 3.0 3.2 3.3 Table 1: Mass of the compound A, B and C for the order and the mass of the four compound in the mix for the determination of the flow rate. Those a dissolved into 25 ml of water

ABC tr min 1.333 1.858 1.558

Table 2: Retention time for A, B and C alone with a flow of 3 ml/min.

2 The mix was also tested at 3 ml/min (table3) before the determination of the flow rate.

The elution order is the following:

DACB

Determination of the flow rate

flow ml/min tM trAW1/2A trBW1/2B trCW1/2C trDW1/2D 0.7 2.733 6.292 0.633 8.942 0.539 6.975 0.525 5.350 0.682 1.0 1.975 4.833 0.483 6.867 0.412 5.367 0.383 4.083 0.527 2.0 0.950 2.142 0.231 3.033 0.179 2.375 0.159 1.825 0.216 3.0 0.633 1.442 0.168 2.042 0.152 1.600 0.145 1.233 0.150 4.0 0.450 1.017 0.132 1.442 0.133 1.125 0.116 0.875 0.104 5.0 0.358 0.792 0.106 1.108 0.115 0.867 0.109 0.683 0.075

Table 3: Raw data form vanilla compounds (Vanillic acid, Vanillin, 4-hydroxy- benzaldehyde and 4-hydroxy-benzoic acid) to be used to determined the best flow rate to analyse commercial vanilla. The retention time are in minutes and the width at the half height are in %

With those values we calculate the capacity factor (k’), the selectivity factor (α), the number of plate and the resolution (Rs).

t − t k’ = r M tM k’A αA-D = k’D ! t N = 5.54 r W 1 √ 2 N · (α − 1) · k’D RsA-D = 4 α (1 + k’D) L 10 cm H = = N N

3 k’ A k’ D αA-D RSA-D NB H[µm] 1.302 0.958 1.360 0.598 1525 65.6 1.447 1.067 1.356 0.618 1539 65.0 1.255 0.921 1.362 0.634 1591 62.9 1.278 0.948 1.348 0.608 1000 100.0 1.260 0.944 1.334 0.602 651 153.6 1.212 0.908 1.335 0.640 514 194.4 Table 4: Calculation of the resolution, plate number and plate height

Determination of flow rate Efficiency of the chromatography column 0.65 200 original modified 0.64

150

0.63 D D m − − µ A A 0.62 100 s s H R R

0.61

50

0.6

0.59 0 0 1 2 3 4 5 6 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 flow ml/min flow ml/min

Figure 2: The best flow rate for the separa- Figure 3: Efficiency of the chromatography tion of vanilla compound is 2 ml/min. column (Van Deemter curve)

The modified plot (figure2) only affects the last point. The difference is that we change the area at half-width (table3) from 0.075 to 0.08 ( ∼ 6%) because the computer had some problem with the calculation.

As we can see, the higher resolution is obtained with a flow rate of 2 ml/min. For the rest of the experiment, we’ll use that value to have the best separation we can in the chromatogram.

Analysis of vanillin in vanilla pods

Migros Coop mass mg 310.9 330 Table 5: Mass of the Migros and Coop vanilla pods witch will be dissolved to 25 ml.

4 Calibration lines

A AreaA B AreaB C AreaC D AreaD stock sol. 0.132 64.2 0.12 101.4 0.128 138.6 0.132 112.1 dilated 2x 0.066 32.2 0.06 51.0 0.064 69.0 0.066 57.1 dilated 5x 0.026 12.9 0.02 20.5 0.026 27.3 0.026 23.0 dilated 10x 0.013 6.8 0.01 11.0 0.013 14.8 0.013 12.1 −2 −2 −3 Migros1 hour 1.02 10 5.2 0.158 133.0 1.06 10 11.7 3.44 10 3.9 −2 −2 −3 Migros2 hours 1.10 10 5.6 0.163 137.4 1.08 10 11.9 5.10 10 5.3 −2 −2 −2 Coop1 hour 1.29 10 6.5 0.135 114.2 2.75 10 29.9 6.45 10 55.4 −2 −2 −2 Coop2 hours 1.12 10 5.7 0.135 113.8 2.70 10 29.4 6.51 10 55.9 Table 6: Calibrations lines and raw results from the analysis of vanilla compound in vanilla pods. The compound (A, B, C and D) are in mg/ml and the area in mV/min.

Calibrations lines for the analysis of vanilla compounds 140 A(x)= 484x + 0.3

B(x)= 839x + 0.7 120 C(x)= 1079x + 0.3

D(x)= 843x + 1 100

80

60 Area mV/min

40

20

0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 Mass concentration mg/ml

Figure 4: Calibrations lines for the analysis of commercial vanilla (Migros and Coop)

Migros Coop mass mg 3.94 4.07 3.38 3.37 mini mg 310.9 310.9 331.5 331.5 mean mg 4.01 3.38 st deviation 0.09 0.01 vanilin vanilla pods % 1.29 1.02 Table 7: Concentration of vanillin (compound B) in commercial vanilla pods from Migros and Coop

5 Discussion

When we look at the different compounds, we can see that the D one is the most polar (it has a carboxylic which can be deprotonnated and an alcohol group), that the vanillin is the most apolar and the A is slightly more polar than the C. As we are in inverse phase, we can predict that the more polar the specie is, the shorter it will take to go through the column because of its unfavorable interactions with the stationary phase. The order of elution we got (D A C B) shows well that the polarity of the compounds is the key factor for the elution time.

As we can see, the amount of vanillin in the two samples is about the same (around 1.1%). This is twice less than the amount written in the protocol but because vanilla is a plant, we can assume this amount depends of its growth conditions. It can also change with the variety of vanilla and the quality of the maturation process. An other way to explain this is that during the preparation of the samples because, when we crushed the vanilla, we had some losses we couldn’t avoid.

The detection of the different species is done at their maximum absorption wavelength in order to have the best sensibility. Like this, it will be easier to get the elution time and to do the integration from the peaks. The peaks have a Gaussian form because the equation of diffusion also involves a Gaus- sian equation. When we increase the flow rate, the plate heigth is almost stable until we reach 2 ml/min (figure3) and then increase very quickly. It is not surprinsing if we look at the theorical Van Deemter curve.

Figure 5: Coubre de Van Deemter th´eorique[2]

From 0.7 to 2 ml/min, the peaks of all compounds are about the same on the chro- matogramm (it makes sense because the plate height doesn’t change much) but after this flow, they begin to spread and overlap with each other due increasing plate height.

If UV/VIS detection wasn’t possible, we could use mass spectrometry or a refraction index detector to detect and quantify the different compounds that are in the mix.

6 Conclusion

In conclusion, a flow rate of 2 ml/min gives the best separation for those compounds. The founds values for vanillin in the commercials pods is less the expected value but this can be explained by factors like the maturation of the pods or the loss in the extraction process.

References

[1] M. Borkovec. Lab journal of analytical chemistry II: High-Pressure Liquid Chro- matography, 2008.

[2] Wikipedia. Van Deemter equation. http://en.wikipedia.org/wiki/Van_Deemter_ equation 13.11.2008.

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