HOW DOES THE

POLARITY OF ORGANIC
SOLVENTS AFFECT TO
THE PARTITION
COEFFICIENT OF
CAFFEINE BETWEEN THE
ORGANIC SOLVENT AND
WATER?

María del Mar Castro Orta

SEVILLA, 5TH MARCH 2012
Words: 3293
Abstract

The objective of the investigation is to determine polarity index influence on partition

coefficient of caffeine between water and five different organic solvents not miscible with
water at two different temperatures.
In order to achieve this, an aqueous solution of caffeine was mixed with the same volume
of each organic solvent in a closed flask and equilibrium was let to establish, first at room
temperature and later at 80 ºC. Partition coefficient is determined by measuring the variation
of concentration of caffeine in the aqueous phase by UV-VIS spectroscopy.
After doing the investigation, we can state that polarity index of the organic solvents does
affect to the partition coefficient, hence, to its caffeine solubilities. As the polarity index
increase, so does the partition coefficient, which means that caffeine solubility at the organic
solvent increases. However, temperature does not cause a clear effect on the partition
coefficient, it depends on the nature of each organic solvent.

Number of words: 155.

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 INDEX
1. Introduction……………………………………………………………………………………………………………………….3
2. Variables…………………………………………………………………………………………………………………………….5
3. Equipment and procedure…………………………………………………………………………………………………5
3.1. Equipment……………..……………………………………………………………………….………………………….5
3.2. Substances…………………………………………………………………………………………………………………5
3.3. Procedure…………………………………………………………………………………………………………………..5
3.3.1.Spectrum of caffeine…………………………………………………………………………………………..6
3.3.2.Calibration…………………………………………………………………………………………………………..6
3.3.3.Measurements of the concentration of caffeine in different solvents…………….…6
4. Results……………………………………………………………………………………………………………………………….7
4.1. Determination of the suitable wavelength in caffeine spectrum…………………………….…7
4.2. Calibration………………………………………………………………………………………………………………….8
4.3. Experimental measurements……………………………………………………………………………………..9
5. Discussion………………………………………………………………………………………………………………….….…12
5.1. Conclusion………………………………………………………………………………………………………………..12
5.2. Procedure evaluation……………………………………………………………………………………………….12
5.3. Investigation improvements……………………………………………………………………………………..14
6. Bibliography……………………………………………………………………………………………………………………..15

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 1. Introduction

Caffeine is the ordinary name for 1,3,7-trimethylxanthine, with

the molecular structure of figure 1.1(8). Numerous plants naturally
produce this chemical, it occurs in coffee, tea, guarana, kola nuts, maté
and cacao. It has significant effects in human beings because it
stimulates the central nervous system, heart, blood vessels and Figure 1.1.: The
kidneys; it also behaves as a mild diuretic. molecular structure
of caffeine
Pure caffeine is a bitter white powder which has a melting point at 238˚C, it sublimates
at 178˚C at atmospheric pressure. Caffeine is less soluble in organic compounds than in hot
water. Hence, there will be a relation between organic compounds and caffeine solubility.

Polarities of organic compounds may guide the solubility pattern. Polarity depends on
the electronegativity difference between atoms in a bond, the greater the difference, the
more polar the bond; and on the geometry of the whole molecule. This feature must be
analyzed with the solubility in water as immiscible compounds must be used. The data has
been obtained from a website (6) about chemical ecology, which we can see below:

Solvent Polarity Index Solubility in Water (%)

Hexane 0 0.001
Cyclohexane 0.2 0.01
Carbon tetrachloride 1.6 0.08
Benzene 2.7 0.18
Chloroform 4.1 0.815

The variable that I'm going to study is the partition coefficient (kc). Considering a
substance that can be dissolved in two different solvents that cannot be mixed together, we
can find a relationship between the concentrations of this substance in both solvents. This
relationship is expressed by the partition coefficient:

I will study the partition coefficient of caffeine between five different organic solvents
and water. When caffeine solution in water gets in contact with any solvent, surfaces of both
liquids which are in contact interact. This interaction is to transfer a certain amount of caffeine
dissolved in water to the new solvent. The transfer continues until equilibrium is reached,
known as partition equilibrium. Therefore, the following equilibrium represents the reaction
we will work on:

Concentration can be studied by various methods. Gravimetric analysis, for example,

determines quantitatively the amount of analyte based on the mass of a solid. Volumetric
analysis can also provide concentration information of a solution. A known concentration and

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volume of reagent reacts with a solution of analyte to determine concentration, this is also
known as titration. Spectroscopy is a common quantitative method to determine unknown
concentrations, in my case, UV-VIS spectrometer was used; hence, I have needed a deeper
understanding of the analytical technique. Thus, expanding the knowledge on this topic with
the book of Analytical Chemistry (3) has allowed me to understand in greater depth the
function of the UV-VIS spectrometer and data processing explained in later sections.

Organic chemistry had been an important chapter in those years. We had been
working on a deep knowledge in this field, which developed my great interest. Therefore, I
focused my first overview in this area. In addition, caffeine studies and its biological
repercussions attract strongly my attention. Investigating about both paths simultaneously
guided me to different chemical aspects relationing those concepts. The blending between the
investigations about caffeine and the partition coefficient has guided me to the following
research question:

How the polarity of different organic compounds does affects to the partition
coefficient between the caffeine concentration and the caffeine dissolution?

The objective was to find a conclusion about the trend, if any, of the partition
coefficient of caffeine depending on the polarity of organic solvents. Additionally, the variable
has been studied in two different temperatures, obtaining a broader vision of the
investigation.

I have studied caffeine absorbance empirically with the UV-VIS spectrometer,

however, should be checked the results with data. The following graph (12) shows caffeine
absorbance:

Figure 1.2.: Ultraviolet spectrum of the caffeine molecule.

Two absorption maxima can be observed at 205 and 273 nm, but my instruments only
let me to measure the last one. In the results section I will show the caffeine spectrum
obtained in lab. The aim is to identify analytically the exact position of the second peak of the
graph, which will be use in all our measurements.

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 2. Variables

The independent variable is the polarity index of the different organic solvents which
will establish the equilibrium of caffeine concentration with water. Therefore, five solvents will
be used: cyclohexane, carbon tetrachloride, benzene, chloroform and hexane. In addition, the
temperature is an independent variable as well. Two series at different temperatures, room
temperature and 80˚C, using those solvents, will be completed.

The dependent variable is the partition coefficient, Kc, depending on the ratio of
caffeine concentration between water and organic solvents.

Controlled variables should be taken into account. Initial caffeine concentration in

water is 2.5·10-5M, which is essential to maintain it constant at it will be necessary to obtain Kc.
Both water and organic solvents volumes must be equal when they are mixed. In research,
10mL of each liquid will be used. Moreover, organic solvents purity, which is shown in
equipment section, has been supervised as it can vaguely affect results. The peak of the
caffeine absorbance graph determines the suitable wavelength for UV-VIS spectrometer
analysis in order to obtain the corresponding absorbance of each equilibrium sample. Finally,
pressure should be controlled in the entire procedure in order to reduce every possible source
of error.

3. Equipment and procedure

3.1. Equipment
- UV-Visible, Scanning Spectrophotometer (Miton Roy Spectronic 1001 Plus)
- Sample holder
- Plastic pipette
- Pipette
- Pipette bulb
- Volumetric flask
o 1unit - 1L
o 6 units – 50mL
- 5 separatory funnels
- Water bath
- Thermometer
3.2. Substances
- Cyclohexane, purity of 100%, Scharlau.
- Carbon tetrachloride, purity of 100%, Panreac.
- Benzene for analysis, purity of 99%, Panreac.
- Chloroform stabilized with ethanol, purity of 99,0%, Panreac.
- Hexane, mixture of alkanes, Scharlau.
- Acetone
- Distilled H2O
- Caffeine powder

3.3. Procedure
- Prepare a caffeine dissolution 2.5·10-5M in a volumetric flask of 1 liter.
- Operation of spectrophotometer.

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 3.3.1. Spectrum of caffeine
- Pour dissolution of caffeine2.5·10-5 M in one of the sample holder until the
mark that is in it with the plastic pipette.
- In the other sample holder, add distilled water to use as reference in the
scanning spectrophotometer.
- Turn on the scanning spectrophotometer, and notify the distilled water as
reference.
- Measure the absorbance for different wavelengths (λ). Start at 190 nm and
end at 350 nm, measure every 5 nm.
- Look at the maximum absorbance and repeat the analysis for wavelengths
close to it every 2 nm.
- Identify the maximum absorbance; we will work with it during the
investigation.

3.3.2. Calibration

The calibration curve is an analytical chemistry method used to measure the concentration of
a substance in a sample by comparison with a number of elements of known concentration. It
is based on the existence of a mathematical relationship between a character principle
measurable, absorbance spectrophotometry, and the concentration. It will be require the
calibration curve to determine the concentration of caffeine in different solvents through its
absorbance.

- Prepare seven solutions of caffeine with different concentrations, using the

initial solution of 2.5·10-5 M.
o 2.50·10-5 M
o 2.00·10-5 M
o 1.25·10-5 M
o 1.00·10-5 M
o 6.25·10-6 M
o 5.00·10-6 M
o 3.13·10-6 M
- Turn on the scanning spectrophotometer
- Pour distilled water in one of the sample holder.
- Place the distilled water as absorbance 0.
- Measure the absorbance of the 7 solutions.
- Analyze the relation between the concentration and the absorbance by a
mathematical function.

3.3.3. Measurements of the concentration of caffeine in different solvents.

- Clean 5 separatory funnels with acetone and let them air dry.
- Pour 10mL of the caffeine dissolution 2.5·10-5 M in each separatory funnel
using a pipette.
- Pour 10mL of hexane in separatory funnel 1 with a clean pipette.
- Stir it for five minutes.

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 - Repeat the process with the other 4 organic solvents.
- Knowing the density of each solvent (13), we know where the caffeine
dissolution is situated. The absorbance of aqueous phase will be measured as
some organic solvents are slightly miscible in water. Add it to one sample
holder at room temperature (having as reference distilled water in the
scanning spectrophotometer) and measure it absorbance.

Solvent Density (g/cm3)

Hexane 0.655
Cyclohexane 0.779
Carbon tetrachloride 1.595
Benzene 0.878
Chloroform 1.483

- Heat the separatory funnels at 80 ºC in a water bath and a thermometer.

- Pour the caffeine dissolution 2.5·10-5 M of separatory funnel 1 in the sample
holder and measure it absorbance. (Remember that we know the caffeine
dissolution position because the densities of the different solvents.)
- Repeat the previous step with separatory funnels 2, 3, 4 and 5.

4. Results
4.1. Determination of the suitable wavelength in caffeine spectrum.
Finding the wavelength where caffeine has a significantly high absorbance will increase
the accuracy of results, so the peak in the Graph 1 is more sensible to be detected for
followings measurements.

GRAPH 1: Caffeine Absorbance

Caffeine Absorbance
3

2.5

2
Absorbance

1.5

0.5

0
220 240 260 280 300 320 340 360

Wavelength (nm)

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 4.2. Calibration

A calibration curve is necessary in order to obtain caffeine concentration as it

represents the relationship between both variables. Measuring absorbance of several known
concentrations of caffeine in water will give us the graphic relationship.

Absorbance has the spectrometer own error, which is ±0,001 in every sample. As it
error bars are negligible due to the Y axis scale, they will be omitted. Concentration, however,
carries an analyzable error which comes from the method followed. Therefore, each sample
has its own error represented in graph 2.

TABLE 1: Calibration curve

Absorbance
Concentration (M)
(±0.001)
2.50·10-5±5·10-7 2.419
2.00·10-5±7·10-7 1.927
1.25·10-5±5·10-7 1.219
1.00·10-5±5·10-7 1.027
6.3·10-6±4·10-7 0.624
5.0·10-6±4·10-7 0.486
3.1·10-6±3·10-7 0.315

GRAPH 2: Calibration curve

Calibration curve
3

2.5

2
Absorbance

1.5

0.5

0
0.00E+00 5.00E-06 1.00E-05 1.50E-05 2.00E-05 2.50E-05 3.00E-05
Concentration (mol·L-1)

The equation of the calibration curve is:

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Once I reach a mathematical expression relating absorbance and caffeine concentration in
water, it is possible to recognize any caffeine concentration from a known absorbance.

4.3. Experimental measurements

TABLE 2: Solvents’ absorbances

Polarity Absorbance of aqueous phase at Absorbance of aqueous

Solvent
Index room temperature (±0.001) phase at 80 ˚C (±0.001)
Hexane 0 2.418 2.412
Cyclohexane 0.2 2.414 2.410
Carbon
1.6 2.028 1.999
tetrachloride
Benzene 2.7 1.418 1.686
Chloroform 4.1 1.341 1.555

Calculations of the partition coefficient

The ratio between both concentrations of caffeine can be measured by the following
formula:

I determine the ratio between the concentrations in Organic solvent /H2O. Defining
this ratio will indicate the proportion of caffeine concentration in water between different
organic solvents, hence, if Kc>1, the caffeine concentration in the organic solvent is higher than
the one in water; if Kc=1, both concentrations are equals; if Kc<1, the caffeine concentration in
water is higher than the one in organic solvent. In addition, caffeine relative solubilities in
different solvents will be identified due to Kc. Empirically, we measure water absorbance once
reached the following equilibrium:

Previously, we have made the calibration curve showing the relation between
absorbance and concentration in caffeine. Therefore, once the corresponding absorbance is
known, I can obtain its concentration through graph 2.

We need both caffeine concentrations, in water as well as in the organic solvent.

Caffeine concentration in water is already explained as we get it directly from data; however,
as we have used the same volumes of both aqueous and organic phase, and no significant
solubility in water of any of the organic solvent used in the study exists, the concentration in
the organic solvent is obtained through the difference between the initial caffeine
concentration and the new one in water after the equilibrium.

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 TABLE 3: Partition coefficients at room temperature and 80 ˚C.

Concentration of
aqueous phase at Concentration of
Polarity Kc at room
Solvent room aqueous phase Kc at 80˚C
Index temperature
temperature (M x at 80˚C (M x 105)
105)
Hexane 0 2.49±0.02 2.48±0.02 0.006±0.03 0.008±0.03
Cyclohexane 0.2 2.48±0.02 2.48±0.02 0.008±0.03 0.008±0.03
Carbon
1.6 2.08±0.01 2.05±0.01 0.20±0.03 0.22±0.03
tetrachloride
Benzene 2.7 1.46±0.01 1.73±0.01 0.72±0.05 0.45±0.04
Chloroform 4.1 1.38±0.01 1.60±0.01 0.81±0,05 0.56±0,04

The relation between Kc and the polarity index is graphed is Graph 3 at room
temperature, and in Graph 4 at 80 ˚C. In order to observe clearly the relation between both
variables, I have decided to use a logarithm scale in the Y axis.

GRAPH 3: Kc vs Polarity index at room temperature.

Kc vs Polarity index at room temperature

1.00E+00
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

1.00E-01
Partition coefficient

1.00E-02

1.00E-03

1.00E-04
Polarity index

A linear relation between the polarity index and Kc at room temperature can be found:

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GRAPH 4: Kc vs Polarity index at 80˚C.

Kc vs Polarity index at 80 ˚C
1.00E+00
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Partition cofficient

1.00E-01

1.00E-02

1.00E-03
Polarity index

At 80 ˚C, a similar relation is found:

To compare both patterns we should observe both temperatures results in a single graph:

GRAPH 3: Kc vs. polarity index at room temperature and at 80˚C.

Kc vs Polarity index
1.00E+00
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

1.00E-01
Partition cofficient

1.00E-02 80ºC
Room temperature

1.00E-03

1.00E-04
Polarity index

It is not possible to determine a constant temperature influence to partition coefficient as it

varies between organic solvents samples.

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 5. Discussion

5.1. Conclusions

The polarity of the organic solvents chosen affects the partition coefficient. As the
polarity index increases, Kc increases. They are directly proportional because they do represent
an increasing linear relation graphically, represented by a linear function. It is possible to state
an increasing order of solubility of the compounds used:

Hexane<Cyclohexane<Carbon tetrachloride<Benzene<Chloroform<Water

Bearing in mind the partition coefficient expression:

It is possible to deduce the relative caffeine solubility in the organic solvent linked to
its polarity; hence, as the polarity index increases and so does Kc, the caffeine concentration in
the organic solvent increases too, thus rising its solubility qualitatively.

Analyzing temperature variable, partition coefficient at 80 ˚C and room temperature

does not show a clear variation, it depends on the organic solvent. Taking into account the
equilibrium established:

Regarding equilibrium enthalpies, according to le Chatelier’s principle1, increasing the

temperature of the equilibrium will change the position of it to minimize this change. If Kc
increases with temperature the forward rate of equilibrium is endothermic because the
position of equilibrium has been displaced to the right as caffeine concentration in the organic
solvent increases. In contrast, if Kc decreases with temperature the forward rate of equilibrium
is exothermic because the position of equilibrium has been displaced to the left as caffeine
concentration in water raises. Therefore, we obtain the followings equilibrium enthalpies:

Hexane equilibrium is endothermic.

Cyclohexane equilibrium is endothermic.

Carbon tetrachloride equilibrium is endothermic.

Benzene equilibrium is exothermic.

Chloroform equilibrium is exothermic.

Empirically, I did not found an important variation of temperature until equilibrium

was reached, which means that the influence of temperature in the equilibrium enthalpies is
low, hence, it could carry significant error.

5.2. Procedure evaluation

To find out the success of the method designed, it should be analyzed along with the
results obtained and its exactness.

1
“If a change is made to the conditions of a chemical equilibrium, then the position of equilibrium will
readjust so as to minimize the change made.”

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 Regarding the accuracy and precision of results, it is important to begin with the
procedure to determine the suitable wavelength of caffeine absorbance. UV-VIS spectroscopy
is an accurate analytical technique to know the light absorption of a solution; hence, the
results given are considerably correct, taking into account only the measurement error which
is ±0.001. Caffeine spectrum obtainment has been accurate. Comparing my results with the
data2, it highlights the correlation between two graphs. According to this fact, I can check two
things; firstly, the exact wavelength found, and, secondly, the precision of UV-VIS
spectrometer measurements, which will affect to the whole investigation.

Calibration curve absorbance has the same error as the one considered in the caffeine
absorbance spectrum. The possible error of the apparatus is systematic, affecting every
measurement made with it. However, there is no effect on Kc since both ratio concentrations
would carry this error, obtaining an exact coefficient value. Caffeine concentration,
nevertheless, has a calculated error depending on the volume and mass used to prepare the
dissolution. The percentages of error of those concentrations are appreciably low, varying
from 2% to 10%. The equation about absorbance-caffeine concentration obtained is very
accurate as the slope has 1% error, and the constant term error allows (0,0) point belong to
the calibration curve.

Partition coefficient is not affected by the systematic error of the UV-VIS

spectrophotometer as I already explained. If the coefficient depends of both caffeine
concentrations, its error comes from them. Partition coefficient error varies considerably in
each case.

- The first coefficient represents the ratio between the caffeine concentration in
water and hexane. In both temperatures, room temperature and 80 ˚C, Kc is
lower than its error, which shows the importance of its error. This may occur
because of the lowest caffeine concentration in hexane. The absorbance at
room temperature, 1.418±0.001, is in the range in which the initial caffeine
concentration in water does not vary, which means that there has been no
caffeine dissolved in hexane. Therefore, caffeine would not be soluble in
hexane. At 80 ˚C solubility is disturbed, obtaining a lower absorbance; hence,
some caffeine has been dissolved in the organic solvent. Regarding the error, it
comes from the huge difference between both caffeine concentrations. At 80
˚C, Kc is lower than at room temperature, contradicting the line of remaining
solvents, however, as we had noticed their error, it is not representative this
incongruity, so we despise it.

- Caffeine solubility in cyclohexane exposes similarities with the previous

solvent. The error is also higher than the partition coefficient in both
temperatures, and it comes from the same source as the hexane case.
Moreover, both Kc are equal, which may represent invariance under
temperature changes.

- From the analysis of caffeine concentration equilibrium with carbon

tetrachloride until the last organic solvent, the partition coefficient error
decreases. At room temperature, Kc, 5±2, has a 40% error, which is
significantly high. At 80 ˚C, Kc error halved to 20%. I can state that the
temperature does not markedly affect to partition coefficient in carbon
tetrachloride study.

2
PQ:Caffeine Spectrum." MicroSolv, Laboratory HPLC, Separations & Purification Leaders. Web. 19 Feb.
2012. <http://www.microsolvtech.com/hplc/pqkit_10_13.asp>

13
 - Partition coefficient with benzene as the organic solvent, has a value of
1.4±0.2 and 2.3±0.4 at room temperature and 80 ˚C respectively, which means
an error of 12.3% and 17.4%.

- Finally, chloroform studies shows 1.2±0.2 and 1.8±0.3 for each temperature. In
this case, they both have the same error percentage, 16.7%.

A decrease of coefficients errors with the increase in polarity index of the organic
solvent is evident. It may occur because of the growing caffeine solubility in those solvents.

Finally, temperatures should be analyzed. Firstly, working with room temperature is

inaccurate as it is not constant. The whole procedure was around 25 ˚C, however, imprecision
is appreciable. Secondly, at 80 ˚C heat dissipates finding an important agent of error. While I
was adding the dissolution in the sample holder, heat was dissipating in environment and on
contact with colder objects. The positive point is that we do not use temperature data to
obtain any variable; temperature does not affect to the calculation, but it may affect to the
caffeine solubility in organic solvents and, therefore, in the partition coefficient.

5.3. Investigation improvements

The procedure of obtaining concentration values is the one which carries a higher
error, so, improving this method, more accurate results will be obtain. One option is to obtain
caffeine concentration by another method and the average will be a better value. However,
UV-VIS spectrometer is accurate enough to have satisfactory results; moreover, using another
method will spend a time unnecessary for the few improvements we would get.

Another option is to use UV-VIS spectrometer to know the concentration in each of the
solvents, studying each absorbance spectrum instead of doing the math; however, this would
have more procedure errors than the ones I have now.

Temperature variable could be analyzed in several aspects. Temperature dissipation

could be reduced using an isolated sample holder, but taking into account that it should be
made of materials appropriate for the UV-VIS spectrometer.

Moreover, any temperature effect had been stated. If no pattern was found, we
should ask the reason for these results. An investigation guide by temperature as the main
independent variable may be an excellent continuation of my research.

14
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