Graph 1: The capacityfactor as a function of the percentage methanol
Graph 2: the calibration series of piperine:
Optimal separation-conditions for the separation of piperine and piperonal.
The extent of separation with HPLC is strongly related with the used eluent.
Before we start the qualitative determination of piperine and piperonal we have to determine the optimal separation-condition by measuring the retention times of the components using a different methanol-water ratio.
Components with a predominant apolar character will have a greater affinity with the apolar eluent. Since pure methanol is more apolar than pure distilled water, piperine and piperonal will dissolve better in methanol. The retention times of these components will be shorter when the percentage methanol in the eluent is increased.
This will result in a smaller capacity factor k.
The methanolratio in the eluent f and the capacity factor k are related as follows:
(14)
This expression can be linearised to
(15)
In which:
k0
= the capacity factor of pure water
ß = constant specific for the combination sample
component/column packing
f = methanol ratio in the eluent
By measuring the retention times by different methanol ratio en plotting the logarithmic value of k against the methanol ratio, a linear function will be formed.
By choosing the eluent composition one should constantly choose between de quality of the separation and the length of the analysis. If a high methanol percentage is used, the time of analysis will be very short, but the quality of separation will be far from optimal.
Back to introduction:
Calibration points:
Table 22: linear regression of the calibration series:
| Calibration | N: |
6 |
|||
Measurement |
x |
y |
x^2 |
y^2 |
x*y |
1 |
1,0000E-05 |
1,5823E-02 |
1,0000E-10 |
2,5037E-04 |
1,5823E-07 |
2 |
2,0000E-05 |
2,9879E-02 |
4,0000E-10 |
8,9275E-04 |
5,9758E-07 |
3 |
4,0000E-05 |
7,3280E-02 |
1,6000E-09 |
5,3700E-03 |
2,9312E-06 |
4 |
6,0000E-05 |
1,0430E-01 |
3,6000E-09 |
1,0878E-02 |
6,2580E-06 |
5 |
8,0000E-05 |
1,3723E-01 |
6,4000E-09 |
1,8832E-02 |
1,0978E-05 |
6 |
1,0000E-04 |
1,7623E-01 |
1,0000E-08 |
3,1057E-02 |
1,7623E-05 |
som: |
3,1000E-04 |
5,3674E-01 |
2,2100E-08 |
6,7281E-02 |
3,8546E-05 |
gem.: |
5,1667E-05 |
8,9457E-02 |
|||
Sxx: |
6,0833E-09 |
a0: |
-2,3942E-03 |
sa0: |
2,4376E-03 |
Syy: |
1,9265E-02 |
a1: |
1,7778E+03 |
sa1: |
4,0165E+01 |
Sxy: |
1,0815E-05 |
rho: |
9,9898E-01 |
sr: |
3,1327E-03 |
Black pepper:
Table 23: linear regression of black pepper
Measurement |
yc |
N: |
3 |
1 |
5,7973E-02 |
yc aver.: |
5,8341E-02 |
2 |
5,8289E-02 |
Xc: |
3,4164E-05 |
3 |
5,8760E-02 |
Sxc: |
1,3073E-06 |
4 |
95%conf: |
3,6342E-06 |
White pepper:
Table 24: linear regression of white pepper
| Measurement | yc |
N: |
3 |
1 |
6,6281E-02 |
yc aver.: |
6,7794E-02 |
2 |
6,9144E-02 |
Xc: |
3,9481E-05 |
3 |
6,7956E-02 |
Sxc: |
1,2761E-06 |
4 |
95%conf: |
3,5475E-06 |
Green pepper:
Table 25: linear regression of green pepper
| Measurement | yc |
N: |
3 |
1 |
1,1023E-01 |
yc aver.: |
1,0714E-01 |
2 |
1,0808E-01 |
Xc: |
6,1615E-05 |
3 |
1,0312E-01 |
Sxc: |
1,2661E-06 |
4 |
95%conf: |
3,5199E-06 |
Our first idea was comparing the percentages of both piperine and piperonal in the different pepper species, using HPLC. Another compound in pepper, piperidine could not be measured since this compound is not UV-absorbing.
Detecting piperine in the samples wasn’t a problem, since the retention time was acceptable and there weren’t any other peaks around the piperine peak detected.
However, detecting piperonal with HPLC was more difficult. First of all, the amount of piperonal in pepper is sufficiently lower than the amount of piperine. Without diluting the extract after centrifuging there was still a very small peak discovered (see results part two).
Another problem was the retention time of piperonal and other pepper compounds. More peaks were detected in the time interval around the retention time of piperonal, by which the separation was quite bad.
We tried to avoid this problem by measuring at a different wavelength (312 nm instead of 231 nm), but that didn’t change a lot.
We probably could have solved the problem by using a lower MeOH-water ratio in the eluent or by using more pepper in the sample. The latter, however, would have affected the recovery and therefore the accuracy of the measurement. There was no time to find the optimal separation conditions for piperonal and very low values of MeOH in the eluent would result in a very long retention time, so we decided to try to detect both compounds with MECC.
We didn’t get any results with MECC, using a TRIS/SDS buffer (pH 8.2).
Since MECC didn’t work out, we tried CZE.
The pKa of piperine is 12.2, and therefore piperine must act as a strong cation at low pH-values, which means the compound has a high mobility at low pH.
Using a PVA/CTA buffer pH 5.85, we expected a peak, but again no results were found.
We don’t have a good explanation for the results we got using MECC and CZE. It could be that there was something wrong with the settings or the buffer, since theoretically there should have been a peak.
Looking at the results, we noticed very low absorbance in both MECC and CZE, which also indicates that the method (especially in CZE) was right, but that other factors caused the failures.
After trying MECC and CZE we decided to perform an organoleptic research, since there was no more time to try other ways detecting piperine and piperonal.
What is the scoville heat scale?
Every pepper contains a certain amount of capsaicinoides, which cause the burning taste
in your mouth or throat. The most important of those capsaicinoides is capsaicin.
The scoville heat scale was set up in the mid 1920’s by the pharmacologist Wilbur
Scoville, a pharmacologist. His test was fairly simple. He had a panel of five persons.
They analysed a solution made of exact weights of dried chili peppers dissolved in alcohol
and diluted with sugar water. The pungency of those peppers were measured in multiples of
one hundred Scoville units. However, this method is subjective. So, with use of HPLC a new
method has been developed.
The HPLC measures the amount of capsaicin in the pepper. Every ppm of capsaicin is equal to 15 Scoville Heat Scale units.Pure capsaicin has a scoville heat score of 16,000,000. Here are some more examples of peppers and their scores.
Table 26: Scoville heat index
| Scoville heat scale Pepperspecies |
| 600,000 World record red savino habanero |
| 100,000 -300,000 Habanero, Scotch bonnet; Jamaico hots; Bahamian |
| 50,000 -100,000 Santaka; Chiltepin; Thai; |
| 30,000 -50,000 Aji; Cayenne; Tabasco; Piquin; Rocoto |
| 15,000 -30,000 Chile de arbol |
| 5,000 -15,000 Yellow wax, Serrano; New Mexico |
| 2,500 -5,000 Jalapeno; Mirasol |
| 1,500 -2,500 Sandia; cascabel |
| 1,000 -1,500 Ancho, Pasilla, Espanola |
| 500 -1000 NuMex Big Jim |
| 100 Cherry |
| 0 Bell |
Chemical properties of piperine and piperonal
Piperine
Formula C17H19NO3
Molecular weight 285,34 g/mole
Structure
Melting point 130° C (16)
pKa (18° C) 12.22
Solubility in water (18° C) 40 mg/l
Isostereomers isopiperine, chavicine and isochavicine
Piperine is isolated from pepper, in which it causes the pungency. It’s also
responsible for the pungency in brandy.
Piperine is often used in insecticide and peppersprays.
Piperonal
Formula C8H6O3
Molecular weight 150,13 g/mole
(17)
Boiling point 263° C
Piperonal is often used in perfumery, cherry- and vanillaflavours and in organic syntheses.
Syntheses of piperine
In 1882 Rugenheimer synthesised piperine by condensing piperinic acid chloride with
piperidine.
In 1894 Ladenburg and Scholtz synthesised piperine by total synthesis of piperinic acid.
Piperinic acid was obtained by condensation of piperonal with acetaldehyde and then
treatment with sodium acetate and acetic anhydride.
Treatment of piperinic acid with thionyl chloride and piperidine formed the piperinic
acid piperidine, also called piperine.
The Ladenburg and Scholtz synthesis is shown underneath.
(18)
Ladenburg and Scholtz
Graph 3: individual results organoleptic research:
Notice that there is a logarithmic scale instead of a linear. This is because the quantity of ppb tasted by
Het doel van dit experiment is de hoeveelheid piperine en piperonal te bepalen in zwarte, witte en groene peper met gebruikmaking van HPLC. We zijn eerst begonnen met het bepalen van de optimale scheidingscondities van piperine en piperonal voor de HPLC. Hieruit bleek dat de scheiding piperine voldoende was bij een methanol-water eluens, met een methanolgehalte van 70 volumeprocent.
Vervolgens hebben we de recovery van piperine in de verschillende peperextracten bepaald. Deze bleek voor zwarte, witte en groene peper respectievelijk 92%, 96% en 89% te zijn.
Met behulp van een ijkreeks en deze recovery is de hoeveelheid piperine in de pepersoorten bepaald. De resultaten waren als volgt:
| Monster | Piperinegehalte (g/g) | 95% B.I. (g/g) |
| Zwarte peper | 0,0202 | 0,0019 |
| White pepper | 0,0239 | 0,0021 |
| Groene peper | 0,0383 | 0,0032 |
De hoeveelheid piperonal kon niet met behulp van HPLC bepaald worden, noch met MECC en CZE.
Verder hebben we een organoleptisch onderzoek uitgevoerd waarin we een elftal mensen sterk verdunde peper- en sambaloplossingen hebben laten proeven. Hieruit bleek dat capsaicine een hetere uitwerking heeft in sambal dan piperine in peper. Van de drie geteste pepers blijkt piperine in zwarte peper de heetste uitwerking te hebben.
Pepper was first treasured by the ancient Egyptians in the jungles of India, centuries before Christ.
They used spices for various things including medicinal, cosmetic and even embalming purposes. From the moment that Alexander the Great conquered Egypt, spices came to be known in larger regions and land routes to the sources of spices were established. Pepper and other spices were already mentioned in Sanskrit, Brahminic texts and the works of Greek historians. Also excavations in the Indus Valley show that spices were well known in India.
In 400 B.C. Hippocrates wrote of its medicinal benefits for heart and kidney. It was also used as a powder or tincture, as a local irritant or liniment, or as a gargle.
By the time of Christ, pepper was worth its weight in gold (70 cents per pound) and was often offered in exchange for it. Sea routes between Rome and Egypt and the coast of India were established during the first century A.D. For centuries, the Islamic Arabs, travelling the Arabian Spice routes from the East, mainly controlled the pepper trade. In 408 A.D., Alaric, the ruthless Visigoth ruler, demanded a ransom of 3,000 pounds of black pepper as his forces lay siege to Rome.
Cities as Alexandria and Constantinople became centres of the spice trade and after the fall of the Byzantine Empire, Venice and Genoa grew rich by dominating the European trade in pepper and other spices from the far East.
In the Middle Ages pepper became more important. Spice merchants came to be known as "Pepperers" in England, "Poivriers" in France and "Pfeffersacke" in Germany. Eventually, pepper became so expensive in Europe that rents and taxes were paid in peppercorns and that dockmen unloading pepper from ships were forbidden to have pockets in their clothing.
The Venetian monopoly was not broken before Vasco da Gama found a route around Cape of Good Hope to the East.
Also Columbus made dangerous voyages establishing new trade routes to the West Coast of India.
Columbus, born in 1451 in Genoa became an experienced sailor and navigator, and his brother Bartholomew was a mapmaker. They were convinced they could make a lot of money by sailing across the ocean to the Far East. Since this would be a very expensive trip, they started to find sponsors in the big cities of Europe. Queen Isabella and King Ferdinand of Spain, jealous of the route that Vasco da Gamma of Portugal had found, financed their voyage.
He left at August 2, 1492. He landed at what is now Cuba, or what he thought was Japan, on October 29. He arrived again in Lisboa on March 4th 1493, without pepper but with the discovery of America.
In the 16th and 18th century, countries like England, Spain and Holland got involved into several wars, fighting for their trading position in spices. At the end of the 18th century, the monopoly of spice trade was broken when the United States of America got involved. Cities as Singapore, Calicut, New York, Hamburg and London became new centres of spice trade. Spice trade grew more and more but spices were still meant for the rich people. Even Yale University was also founded on pepper fortunes.
In the last two centuries the production grew due to the application of modern agriculture techniques.
And today, pepper remains the most important spice in the world, accounting for more than a fourth of worldwide spice trade.
