Romain Laverriere Tatiana Pachova HPLC

Transcription

1 Romain Laverriere Tatiana Pachova HPLC

2 Table of content: 1 Abstract Introduction Methodology Results Peak identification Determination of the optimum flow rate Sample analysis Discussion Questions Conclusion References

3 1 Abstract The order of elution of the molecules present in vanilla was determined to be, from first to last: 4- hydroxy-benzoic acid (D), vanillic acid (A), 4-hydroxy-benzaldehyde (C) and vanillin (B). The optimal flow-rate for those compounds was determined to be.4 ml/min by using a least squares fit to the Van-Deemter equation. The content of the compounds in a real vanilla stick sample was found: Table 1: Sum up of the results A B C D c [mg/l] content in vanilla [%].46% 1.64%.16%.12% 2 Introduction The high performance liquid chromatography (HPLC) is based on the differences in adsorption affinities of the compounds. Therefore the retention time is different for each chemical. The main interest of the HPLC is that a good resolution is obtained with reasonably rapid separation times. In our experiment, we proceed with an inverse chromatography. The mobile phase is a mixture of water and acetonitrile and is therefore polar. The stationary phase consists of 5 µm porous silica particles with a functionalised surface and is apolar. So, the polar compounds elute faster than the apolar ones because they are more solvated and interact less with the stationary phase. 3 Methodology The methodology given by the protocol [1] was followed. nly one commercial vanilla sample was analysed. 4 Results The presence of four compounds was first identified and their concentration was further determined in the vanilla sample. The compounds are further referred to as A, B, C and D. A B C D H H H H H H Vanillic acid (4-hydroxy-3-methoxy-benzoic acid) 26nm Vanillin 4-hydroxy-benzaldehyde 2nm 28nm Figure 1: Structure of the vanilla compounds 4-hydroxy-benzoic acid 26nm 3

4 4.1 Peak identification The chromatogram obtained when running an HPLC presents several peaks corresponding to different compounds. In order to identify each peak, three solutions mixing respectively (A, B, C, D), (A and B) and finally (C and D) were made. From the structure, it is easy to conclude that A is more polar than B and that D is more polar than C. Therefore, based on the comparison of polarity and on the peaks of the chromatograms, we see that the compounds elute from first to last in the following order: D>A>C>B. For each compound the retention time, number of plates, height of an equivalent plate and resolution were determined. The number of plates for a peak i is given by: N! = t! w!! with t i : the retention time and w i : the peak width at half-height. The plate height is given by: H! = L N! with L=15 cm the length of the column. The resolution factor between two peaks is given by: t!!! t! R!,!!! = w!!! + w! The results obtained for the compounds A, B, C and D at the fastest flow of 1.8 ml/min are: Table 2: Analysis of the chromatogram at 1.8 ml/min flow-rate Name t [min] w N H [um] R t D t-d 12.5 A D-A.96 C A-C 5.12 B C-B 1.92 This flow-rate is unfortunately too fast to have a good enough resolution. In fact the resolution factor between the peaks D and A is too low (<1.5). So it is necessary to test slower flow rates to find the optimum one. 4.2 Determination of the optimum flow rate First, the flow velocity u is determined using the formula: u = L = 1 = mm/min t!.992 Then, the porosity of the column is calculated by: ε = Φ πr! = !! u using information provided by the chromatogram at the fastest flow-rate (1.8 ml/min). 4

5 Then, the ideal flow-rate is defined for each compound, by performing a non-linear least squares fit to the van Deemter equation and finding the minimum of the function: H! = A! Φ + B! + C! Φ dh! dφ = A! Φ! + C! And the following equation is resolved for each compound: dh! dφ = Φ = A! C! Since ε and r are constant the calculations were done with the flow rate rather than the flow velocity. H (D) [um] A = ± 2.57 B =1.197 ± 1.5 C =2.889 ± 6.69 H (A) [um] A = ± 2.11 B = ± 8.58 C =15.8 ± H (C) [um] A =2.256 ± 1.14 B = ± 4.62 C = ± 2.96 H (B) [um] A =2.815 ± 1.4 B =12.16 ± 4.23 C = ± Figure 2: Least squares fit of plates height to the flux using the Van Deemter equation

6 Table 3: Number of plates and plates height for different flow-rates Flux (Φ) [ml/min] N D H D [um] N A H A [um] N C H C [um] N B H B [um] Table 4: Determination of optimal flow-rate for each compound Coefficient D A C B A B C Φ opt [ml/min] Therefore the average optimal flow-rate is.4 ml/min. 4.3 Sample analysis First, calibration curves for each of the four vanilla compounds were made: Compound A Compound B y = x R² = y = x R² = Compound C Compound D y = x R² = y = x R² = Figure 3: Calibration curves for the compounds 6

7 The concentration of each compound in the vanilla sample was therefore defined: Table 5: Content of each compound in the vanilla sample A B C D c [mg/l] error c vanilla [mg/g] content in vanilla [%].46% 1.64%.16%.12% 5 Discussion Even though the optimal flow-rate was determined to be.4 ml/min, an actual flow-rate of.8 ml/min was used to make the measurements a little faster. Using the ideal flow-rate would make the results more precise, but in our case, samples weren t diluted enough. In fact the calibration curves for the compounds were too high. 6 Questions Discuss the changes in the chromatogram does one expect when one varies the ratio of acetonitrile to water. Does one expect changes in retention times, number of plates, or equivalent height? If the ratio of acetonitrile to water is modified, it means that the polarity of the mobile phase is modified. Therefore if there is a bigger proportion of acetonitrile, it means that the mobile phase is more polar and the polar compounds will elute faster, because their affinity to the apolar stationary phase will decrease. The retention times are therefore changed, as well as the number of plates and the equivalent heights. Propose how the resolution could be improved. Resolution can be improved (without changing the column) by playing on the polarity of the mobile phase and by choosing the best flow-rate. 7 Conclusion In this experiment, the analysis of compounds was done using the HPLC. The results were satisfactory, even though using more adapted dilutions would have probably made the results better. 8 References [1] TP_HighPerformcanceLiquidChromatography, Travaux pratiques de chimie analytique II pour chimistes, M. Borkovec, 212 [2] M. Borkovec, Error Analysis Introduction, Travaux pratiques de chimie analytique II pour chimistes, 212 7