M AH A N AIM
Time-optimization of a chromatographic
method for monitoring a multistep enzymatic
conversion of epoxides to 1,2-diols
Thesis
Bachelor of Science in Chemistry
March 2014
Bachelor Degree project in Chemistry (15 hp)
Department of Chemistry – BMC
Supervisor: Mikael Widersten and Åsa Janfalk Carlsson
Abstract
The enzymatic conversion of selected epoxides into diols and ketones was monitored by time
optimized reversed phase chromatography. Reaction samples were first analyzed isocratically
to identify the components of different peaks. In the next step the samples were run with a
gradient elution to shorten the retention time between the compounds in the multistep reaction
with retained separation of a good quality.
The use of a 100-2 mm reversed phase C-18 column with an optimized water and acetonitrile
gradient allowed an analysis time of the compounds in the multistep reaction could be
optimized to 20 min, with good resolution. This result represents an improvement compared
to earlier protocols that required 45 min and 74 min respectively.
Svensk sammanfattning
Den enzymatiska omvandlingen av utvalda epoxider till dioler och ketoner övervakades med
en tidsoptimerad reversed phase kromatografi. Reaktionsprover analyserades först sokratiskt
för att identifiera föreningarna med olika toppar. I nästa steg analyserades proverna med en
gradienteluering för att kunna förkorta analystiden mellan föreningarna i multistegsreaktionen
med bibehållen god separation
Användningen av en 100-2 mm reversed phase C-18-kolonn med optimerad vatten- och
acetonitrilgradient tillät att analystiden av föreningarna i multistegsreaktionen kunde
optimeras till 20 min med en god upplösning. Detta resultat är en förbättring jämfört med
tidigare protokoll som krävde 45 respektive 74 min.
2
List of abbreviations
AdhA
Rhodococcus ruber alcohol dehydrogenase A
Diol 1
1-phenyl-1,2-ethanediol
Diol 2
3-phenyl-1,2-propanediol
2,3-EPB
2,3-epoxyprobylbenzene
HPLC
high performance liquid chromatography
Ketone 1
2-hydroxyacetophenone
ketone 2
1-hydroxy-3-phenylpropan-2-one
NAD+
nicotinamide adenine dinucleotide
PDA
photodiode array detector
SO
styrene oxide
StEH1
Solanum tuberosum epoxide hydrolase 1a
3
Table of contents
Abstract ..........................................................................................................................................2
Svensk sammanfattning...................................................................................................................2
List of abbreviations........................................................................................................................3
Table of contents .............................................................................................................................4
Introduction ................................................................................................................................5
Aim of the project ........................................................................................................................5
Materials and Methods ....................................................................................................................7
Equipment ...................................................................................................................................7
Chemicals and materials .............................................................................................................7
Sample preparation .....................................................................................................................7
Gradient experiment....................................................................................................................8
The substrates .............................................................................................................................9
Results .......................................................................................................................................... 11
The isocratic separation of SO, 2,3-EPB and the reaction products of SO.................................. 11
Shortening of the analysis time by gradient elution .................................................................... 12
Discussion .................................................................................................................................... 17
Conclusion .................................................................................................................................... 18
Personal reflection ........................................................................................................................ 18
Acknowledgements ....................................................................................................................... 18
References .................................................................................................................................... 19
Appendix ...................................................................................................................................... 20
4
Introduction
Aim of the project
The main aim of the project was to develop a time optimized analysis of the compounds in the
multistep reaction (Figure 1).
The research project was first run by Professor Mikael Widersten and his coworkers to study
enzymatic conversion of epoxides, using a multistep enzyme approach. As a starting point,
selected epoxides are hydrolyzed by the enzyme epoxide hydrolase 1a from Solanum
tuberosum (StEH1) to form the corresponding diols. These diols are further converted into
ketones by alcohol dehydrogenase A from Rhodococcus ruber (AdhA), (Figure 1).
StEH1
H2O
AdhA
NAD+
Figure 1- Styrene oxide is hydrolyzed by the enzyme StEH1 to form a diol. The diol is then
converted into a ketone by alcohol dehydrogenase AdhA.
The epoxides used here are styrene oxide (SO) and 2,3-epoxypropylbenzene (2,3-EPB). The
hydrolysis of 2,3-EPB cannot be measured by spectrophotometer due to similar absorbance
spectra for the epoxide and the diol. As a Consequence HPLC was used to monitor the
reaction shown in figure 1. In early protocol based on reversed phase chromatography each
sample required 80 minutes. Thus, it is desirable to reduce the analysis time.
The actual separation principle used here was a reversed phase chromatography that employs
a nonpolar stationary phase, in this case a short C-18 column was used. The C-18 name refers
to a stationary phase based on microporous silica particles that are derivatized with 18- carbon
hydrocarbon chains. The requirement was to maintain a sufficiently good resolution, which
5
means not only to obtain as short analysis time as possible, but also to get a good separation
between the compounds.
The packed column efficiency increases as the particles size of the stationary phase decreases.
The experimental factors that could influence the resolution were the flow rate, gradient
elution and the column height.
The resolution is often described by the plate number (N) which corresponds to the number of
discrete distributions that would give the same results. In isocratic elution, the plate number
can be estimated from the peak width (W) and the elution volume (V e) as N = 16 (Ve/W)2.
The height of the theoretical plate is L/N, where the N increases linearly with L, meaning that
the resolution increases with L.
The Van Deemter equation (eq. 1) tells how the flow rate and the column affects the plate
height (H). The term A stands for longitudinal diffusion, the term B for the equilibrium
between the stationary phase and the mobile phase, and the term C is a constant and due to the
properties of the column packing.
𝐻=
𝐴
+ 𝐵𝑣 + 𝐶
𝑣
eq. 1
In packed columns the terms A, B and C all contribute to band broadening. A decrease of the
particle size will normally influence both the B and C terms thus decreasing the plate height
(H), resulting in sharper peaks. [1].
An isocratic elution was used in the experiment to identify the peak of each compound.
Isocratic elution represent the case where the mobile phase composition is unchanged during
the elution process. If one solvent mixture could not give a rapid elution of all components a
gradient elution is often used (Figure 2). Gradient elution provides continuous change of the
composition of the solvent to increase the eluent strength. We designed a stepwise linear
gradient with two different slopes, one until the ketone was eluted and one to elute the
epoxide.
Short analysis time can be obtained in many ways, for example by using the shortest possible
nonpolar C-18-column, changing the flow rate and the composition of the mobile phase, and
by using a gradient elution.
6
Materials and Methods
Equipment
High performance liquid chromatography (HPLC) was carried out using a Chromolith ®
performance RP-18 e column with dimensions of 100x2 mm, using H2O/acetonitrile adjusted
to pH3.0 with formic acid is the mobile phase. The system also consisted of a sampler
(Shimadzu SIL-10AF), pumps (LC 20AD) and detector (Shimadzu SPDM20A photometric
unit). Our experiment was carried out at a flow rate of 0.38 mL/min [2]. The detector was a
photodiode array (PDA).
Chemicals and materials
Styrene oxide, 1-Phenyl-1-2-ethanediol and 2-hydroxyacetophenone were obtained from
Sigma Aldrich. 2,3-epoxyprobylbenzene was obtained from TCI. The epoxide hydrolase
StEH1 was expressed in E.coli transformed by the plasmid pGTacStEH1-5H, that is a pGTacplasmid with a gene for StEH1 that was extended by 5 histidines by the researchers. Alcohol
dehydrogenase AdhA extended by 5 histidines was expressed in E.coli strain BL21-A1. All
other chemicals were of analytical grades.
Sample preparation
Reference sample
The compounds styrene oxide, 1-Phenyl-1-2-ethanediol (diol 1) and 2-hydroxyacetophenone
(ketone 2) were dissolved in H2O to 3 mM concentration. The incubation samples were
prepared as described in appendix 1, Table A1.
The compound 2,3-epoxyprobylbenzene (2,3-EPB) was first mixed with only the enzyme
StEH1 to obtain the corresponding 3-phenyl-1,2-propanediol (diol 2), to identify the peak of
the diol 2. Then 2,3-EPB was mixed with both the enzymes StEH1 and AdhA to obtain the
corresponding 3-phenyl-1,2-propanediol (diol 2) and 1-Hydroxy-3-phenylpropan-2-one
(ketone 2). The compound 2,3-EPB and the enzymes were dissolved in 0.1M sodium
phosphate pH 7.9 to 30 mM concentration. The incubation samples were prepared as
described in appendix 2, Table A5.
7
Gradient experiment
Figure 2- An example of a gradient employed. Gradient elution (dark blue) with acetonitrile
(%) plotted over time (min).
The gradient was increased from 15 % acetonitrile to 30 % acetonitrile between 5 min and 7
min. And then increased to 90 % acetonitrile between 7 min and 20 min and maintained at
90% acetonitrile for 5 min. Between the time 25:00 and 25:01 the gradient decreased from
90% to 15% to end the experiment. The same general method was used to all the experiments
but with different gradients (Table 3).
8
The substrates
1- Separation of reaction products with styrene oxide as a starting substrate
StEH1
H2O
SO
1-Phenyl-1-2-ethanediol (diol 1)
AdhA
NAD+
2-hydroxyacetophenone (ketone 1)
Figure 3- A reaction scheme with SO as starting substrate.
SO, diol 1 and ketone 1 were bought from Sigma Aldrich, the compounds were mixed
together at 3 mM each in H2O. The sample then run in HPLC and a gradient was used to
obtain a shorter analysis time with maintained separation (Table 3 and 4). The experiments in
Table 3 were run in duplicate to document the reproducibility.
9
2- Separation of reaction products with 2,3-epoxypropylbenzene as a starting
substrate
StEH1
H2O
2,3-EPB
3-phenyl-1,2-propanediol
(diol 2)
AdhA
NAD+
1-Hydroxy-3-phenylpropan-2-one
(ketone 2)
Figure 4- A reaction scheme with 2,3-EPB as starting substrate.
2,3-EPB was bought from TCI thus could be analyzed directly whereas diol 2 and ketone 2
were made by the enzymatic reaction in Figure 4. The peaks to the diol 2 and the ketone 2 in
this reaction were identified by preparing two samples, one containing only 2,3-EPB (30 mM)
and StEH1 to form diol 2 [3] and one containing epoxide 2 (3 mM), StEH1; and AdhA to
form the ketone 2 (Table A5). The experiment 3 in table 7 was run in duplicate.
10
Results
The isocratic separation of SO, 2,3-EPB and the reaction products of
SO
In order to develop a time optimized analysis of the compounds SO, 2,3-EPB, diol 1 and
ketone 1 in the multistep reaction, their analysis times in the unoptimized system were needed
to be known. The molecular weight (Mw), concentration, and volume of the compounds are
given in Table A1, Appendix 1. The samples were run isocratically with 15% acetonitrile /
85% H2O at a flow rate of 0.38ml/min (Table 2).
Table 2- The compounds and their retention time
Compounds
Retention time
(min)
SO
2,3-EPB
Diol 1
Ketone 1
23.3
~30
4.9
6.5
When the epoxides where run with the longer HPLC C18-column with dimensions of 25 cm x
4.6 mm, 5 µm, the SO had analysis time of 45 min and 2,3-EPB, 75 min, thus by changing the
column, the analysis times were decreased.
11
Shortening of the analysis time by gradient elution
Separation of the substrate styrene oxide and its products
A mixture of compounds was prepared as follows: 0.36 µl SO (3 mM) was mixed with 30 µl
diol 1 (3 mM) and 30µl ketone 1 (3 mM) in water to a final volume of 1 ml. The sample was
then analyzed by a gradient elution (Figure 2), (Table 3 and 4).
Table 3- The conditions that were used during isocratic elution and gradient elution.
Experiment
Conditions
1
(isocratic
elution)
2
(gradient
elution)
15% acetonitrile / 85% H2O
3
(gradient
elution)
At time 5:00 – 7:00 min the gradient increased from 15% to 30% acetonitrile.
7:00 – 20:00 min the gradient increased to 90% acetonitrile and maintained at it for more
5 min.
25:00-25:01 min the gradient decreased to 15% acetonitrile.
4
(gradient
elution)
At time 00:00 – 15:00 min the gradient increased from 0% to 90% acetonitrile.
15:00 – 20:00 min the gradient was decreased to 15% acetonitrile.
20:00-20:01 min the gradient increased to 90% acetonitrile.
20:01-20:03 min the gradient decreased to 15% acetonitrile
At time 15:00 – 15:01 min the gradient increased from 15% to 30% acetonitrile.
15:01 – 20:00 min the gradient increased to 80% acetonitrile.
20:00-20:01 min the gradient decreased to 0% acetonitrile.
Table 4- The retention time of the compounds with isocratic elution and gradient elution.
Experiment
1
2
3
4
Flow rate
(ml/min)
Ret. time of
diol 1 (min)
0.38
0.40
0.38
0.40
4.9
5.4
4.9
7.3
Ret. time of
ketone 1 (min)
6.5
6.8
6.2
8.2
Ret. time of
SO (min)
23.3
19.2
9.7
10.4
Graph
Figure 5
Figure 6
Figure 7
12
The traces of experiments 2, 3 and 4 with absorption at 220nm (mAU) plotted versus time
(min) are shown in Figures 5, 6 and 7.
19.227/127824327
m AU
Extract-220nm 4nm (1.00)
3000
1500
1000
5.427/16488165
2000
6.832/17277406
2500
500
0
-500
0.0
5.0
10.0
15.0
20.0
25.0
m in
Figure 5- Retention times of, from left to right, diol 1, ketone 1 and SO in experiment 2.
9.696/71354156
m AU
Extract-220nm 4nm (1.00)
2000
1000
500
4.948/20044252
6.251/19098091
1500
0
0.0
5.0
10.0
15.0
20.0
25.0
m in
Figure 6- Retention times of diol 1, ketone 1 and SO in experiment 3.
13
750
10.370/51885187
7.294/22526899
8.239/16808703
m AU
1000 Extract-220nm 4nm (1.00)
500
250
0
-250
0.0
5.0
10.0
15.0
20.0
25.0
m in
Figure 7- Retention times of diol 1, ketone 1 and SO in experiment 4.
The analysis time was decreased from 45 min to 20 min by using a shorter C-18 column for
SO, changing the flow rate and using an optimized gradient.
Separation of 2,3-epoxypropylbenzene and its products
The diol and the ketone formed from 2,3-EPB, Figure 3, are not commercially available, but
could be obtained from the reaction shown in Figure 1.
To obtain the ketone, 1-hydroxy-3-phenylpropan-2-one (ketone 2), 2,3-EPB has to be
incubated with epoxide hydrolase (StEH1) to obtain phenyl-1.2-propanediol (diol 2).
Thereafter, the diol is converted by alcohol dehydrogenase (AdhA) to give ketone 2. Zink is
needed to stabilize AdhA and for the enzyme to function. The co-factor NAD+ is also needed.
To identify the peaks that correspond to the diol and the ketone in this reaction, two samples
were prepared (Table A5). Sample 1 contained only 2,3-EPB (30 mM) and StEH1 to obtain
diol 2, and sample 2 contained 2,3-EPB (3 mM), StEH1, and AdhA to obtain ketone 2. The
retention time of epoxide 2 is approximately 33 min (Table 6).
Samples 1 and 2 were first analyzed with isocratic elution, each sample was run for 100 min
with 15% acetonitrile/85% H2O (Table 6).
Table 6- The retention time of the substrates with isocratic elution.
Compounds
2,3-EPB
Diol 2
Ketone 2
Retention time (min)
32.9
6.7
-
Ketone 2 was not obtained because only a very small amount of it was formed, that did not
give a clearly visible peak. Since no ketone 2 peak was obtained, a gradient optimized for
14
separation of the diol 2 and epoxide 2 only. The mixture was prepared right before the
experiment was run to prevent degradation of 2,3-EPB (Tables 7 and 8).
Table 7- Conditions used during isocratic and gradient elution.
Experiment
1
Conditions
15% acetonitrile / 85% H2O
(isocratic
elution)
2
(gradient
elution)
At time 5:00 – 7:00 min the gradient increased from 15% to 30% acetonitrile.
7:00 – 10:00 min the gradient increased to 80% acetonitrile.
20:00-30:00 min the gradient was increased to 90% acetonitrile.
30:00-30:03 min the gradient was decreased to 15 % acetonitrile.
3
(gradient
elution)
At time 5:00 – 7:00 min the gradient increased from 15% to 30% acetonitrile.
7:00 – 10:00 min the gradient increased to 80% acetonitrile.
25:00-30:00 min the gradient was increased to 90% acetonitrile.
30:00-30:03 min the gradient was decreased to 15 % acetonitrile.
Table 8- Retention times of compounds with isocratic and gradient elution.
Experiment
1
2
3
Flow rate
(ml/min)
Ret. time of 2,3-EPB
(min)
Graph
diol 2 (min)
Ret. time of
0.38
0.38
0.38
6.7
6.2
6.3
33.9
11.8
11.9
Figure 8
Figure 9
Good separation between the diol 2 and 2,3-EPB was obtained, and good reproducibility was
also obtained.
15
m AU
500 Extract-225nm 4nm (1.00)
400
300
6.297/4308599
200
11.893/13082669
Chromatograms of experiment 2 and 3, where peaks are detected at 225 nm (figure 8 and 9).
100
0
-100
0.0
5.0
10.0
15.0
20.0
25.0
m in
m AU
Extract-225nm 4nm (1.00)
6.327/5522478
300
11.935/8659210
Figure 8- Retention times of, from left to right, diol 2 and 2,3-EPB in experiment 2.
200
100
0
-100
0.0
5.0
10.0
15.0
20.0
25.0
m in
Figure 9- Retention times of diol 2 and 2,3-EPB in experiment 3.
2,3-EPB has absorption maximum of 225 nm where also the diol 2 appears clearly. As can be
seen in Figure 8, there is a peak that coelutes with the epoxide peak. It was identified as a
degradation product that was formed in the sample.
The analysis time was decreased from 75 min to 20 min by using the shorter C-18 column for
2,3-EPB, using gradient elution and could not be decreased more without sacrificing the
resolution.
16
Discussion
The HPLC was chosen because many compounds are not volatile enough for gas
chromatography. Since the compounds that we wanted to elute are nonpolar, a nonpolar
stationary phase was used with polar mobile phase and a gradient with less polar compound
[1]. The mobile phase used here consisted of MilliQ-H2O and acetonitrile adjusted to pH 3
with formic acid. Acetonitrile dissolve both ionic and nonpolar compounds and has a high
eluent strength that elutes all compounds rapidly. Acetonitrile was also added to the samples
due to solubility problems [4]. The reason why a shorter non-polar C-18 column was used is
to obtain good separation at shorter time [5].
The gradient was used to elute tight bound compounds, in this case epoxides, faster. Epoxides
are nonpolar compounds why they bound tightly to the nonpolar column. When the
acetonitrile concentration is increased, it elutes the epoxides faster because it has a high eluent
strength. The diols and the ketones are weakly bound to the column because they are polar,
more polar than the epoxides, why they elute faster through the column. 150µl was suitable
for a good detectability [6].
The starting substrate styrene oxide
By using a shorter C-18 column, changing the flow rate and the gradient, the analysis time of
the important compounds could be decreased from 45 min, when a longer C-18 column was
used, to 20 min. When the flow rate was increased, sharper peaks with retained separation
between the compounds were obtained (Figures 5, 6 and 7). The gradient allowed a good
separation for diol 1 and ketone 1 together with a shorter analysis time for SO.
The flow rate could not be increased higher than 0.40 ml/min since the shorter C-18 column
cannot tolerate higher pressure than 180 bar. The results were as expected, by using higher
gradient, as high as 80% or 90% acetonitrile with flow rate of 0.38 ml/min SO was eluted
earlier. The analysis times of the compounds could not be further decreased for this system.
The reproducibility was checked to confirm the results from the first experiments (Table 3).
The results showed good reproducibility for experiment 3 and 4. The reproducibility for
experiment 2 could not be tested due storage problems.
The start substrate 2,3-epoxypropylbenzene
By using the shorter C-18 column and gradient elution, the compounds analysis times could
be decreased. The analysis time was decreased from 80 min, when a longer C-18 column was
used, to 20 min with good separation.
17
Conclusion
A gradient and a shorter nonpolar C18-column with dimensions of 100x2 mm were used to
shorten the analysis times of the compounds in a multistep reaction. As an example, it would
save 42 - 92 h if 100 samples were analyzed. A further advantage of shorter analysis times of
the compounds is that less solvents would be used. Good reproducibility could also be
obtained with the protocol that was used.
Personal reflection
Shortening the retention time of the epoxides, diols and ketones with retained separation is
highly important, since many samples need to be run in HPLC, which is very time consuming.
Reducing the analysis times of the compounds helps the researchers to detect the properties of
the compounds faster and thus they may detect a treatment. This is why this project was
important. To improve the experiment further I would change the flow rate and gradient to
obtain better separation between the compounds. I would also try to obtain the ketone 1Hydroxy-3-phenylpropan-2-one by increasing the concentration of the substrate 2,3-EBP even
more.
Acknowledgements
A big thank You to PhD. Student Åsa Janfalk Carlsson and Prof. Mikael Widersten for
supervision.
18
References
[1] Daniel C. Harris and Michelson Laboratory, “High Performance liquid Chromatography”, in
Quantitative Chemical Analysis, W. H. Freeman and Company, 2010.
[2]. Janfalk Carlsson. Å, Bauer P., Ma H. and Widersten M., “Obtaining optical purity for product
diols in enzyme-catalyzed epoxide hydrolysis: contributions from changes in both enantioand
regioselectivity,” Biochemistry 51, 7627−7637, 2012.
[3] G. Ann, M. Widersten, “Modification of Substrate Specificity Resulting in an Epoxide
Hydrolase with Shifted Enantiopreference for (2,3-Epoxypropyl)benzene,” Volume 11, 14221429, 2010.
[4] Goldberg A.P. , Nowakowska E. , Antle P.E. , “Retention-optimization strategy for the highperformance liquid chromatographic ion-pair separation of samples containing basic compounds,”
Journal of chromatography 316 , 241-260, 1984.
[5] Bahowick J.T. , Synovec E.R. , “Correlation of quantitative analysis Precision to retention
time precision and chromatographic resolution for rapid, short-column analysis,” Anal. Chem.
67, 631-640, 1995.
[6] L.R. Snyder , J.W. Dolan, J.R. Grant, “Gradient elution in high-performance liquid
chromatography : I. Theoretical basis for reversed-phase systems,” Journal of chromatography
Volume 165, 3-30, 1979.
19
Appendix
Appendix 1. The molecular weight (Mw), concentration, and volume of the compounds SO,
diol 1 and ketone 1, see table A1.
Table A1- The Mw, mass, concentration, volume and retention time of the compounds.
Sample
Substance
Physical
state
Mixed
with
Molecoular
weight
(g/mol)
Mass
1
2
SO
1-phenyl-1.2ethanediol
2hydroxyacetophenone
Liquid
Solid
Water
Acetonitrile
120.15
138.16
0.36µl
13.0mg
3
3
1
1
23.3
4.9
Solid
Acetonitrile
136.144
14.4mg
3
1
6.5
3
Conc. Volume Retention
(mM)
(ml)
time
(min)
Acetonitrile was added due to solubility problems.
Appendix 2. The volumes that were used for sample 1 and sample 2, with 2,3-EPB as a start
substrate, see Table A5.
Table A5- The volume of the substrates and enzymes that were used for sample 1 and 2.
Sample
Epoxide 2.3-EPB
(µl)
1
2
4
0.41
StEH1 AdhA NAD+ Zink
(µl)
(µl)
(µl) (µM)
5
1
10
150
1
Buffer
0.1M
Sodium
phosphate
ph 7.9 (µl)
991
837
20
Appendix 3. Some of the gradient experiments that were run with SO styrene oxide as a start
substrate, but failed to give good and shorter retention time. See Table A9 and A10.
Table A9- The conditions that were used during isocratic elution and gradient elution.
Experiment
Conditions
1
15% acetonitrile / 85% H2O (isocratic elution)
2
At time 15:00 – 15:01 min the gradient increased from 0% to 15% acetonitrile.
15:01 – 20:00 min the gradient increased to 80% acetonitrile.
20:00 – 20:03 min the gradient was still at 80% acetonitrile
(gradient elution)
At time 7:00 – 9:00 min the gradient increased from 0% to 15% acetonitrile.
9:00 – 21:00 min the gradient increased to 30% acetonitrile and maintained at it for more
4 min.
25:00-25:01 min the gradient was decreased to 15% acetonitrile.
(gradient elution)
At time 7:00 – 9:00 min the gradient increased from 0% to 15% acetonitrile.
9:00 – 21:00 min the gradient increased to 90% acetonitrile and maintained at it for more
4 min.
25:00-25:01 min the gradient was decreased to 15% acetonitrile.
(gradient elution)
At time 8:00 – 8:01 min the gradient increased from 0% to 20% acetonitrile.
8:01 – 11:00 min the gradient increased to 90% acetonitrile.
11:01-11:03 min the gradient was decreased to 20% acetonitrile.
(gradient elution)
3
4
5
Table A10- The retention time of the compounds with isocratic elution and gradient elution.
Experiment
Flow rate
(ml/min)
Ret. time of
1-Phenyl-1-2ethanediol (min)
Ret. time of
2hydroxyacetophenone
(min)
Ret. time of
SO (min)
1
2
0.38
0.38
4.9
-
6.5
-
23.3
21.3
3
0.38
-
-
-
4
0.38
-
-
12.1
5
0.40
-
-
12.2
Graph
Figure
10
Figure
11
Figure
12
Figure
13
21
The graph of experiments 2, 3 and 4 with absorption at 220nm (mAU) are plotted versus time
(min). The diol 1 and ketone 1 did not have time to form, see figure A10.
21.348/7249473
m AU
60 Extract-220nm ,4nm (1.00)
50
40
10
0
6.548/64192
20
3.162/161421
30
-10
0.0
5.0
10.0
15.0
20.0
25.0
m in
Figure A10- Retention times of, from left to right, diol 1 and SO in experiment 2.
15
10
13.908/161341
20
9.712/730502
6.146/1462687
m AU
25 Extract-220nm 4nm (1.00)
11.745/1352738
The graph of experiment 3 with absorption at 220 nm (mAU) are plotted versus time (min).
The peaks of the diol 1, ketone 1 and SO could either be stuck together or did not have
enough time to form, see figure A11.
28.853/346154
5
0
-5
-10
-15
-20
0.0
5.0
10.0
15.0
20.0
25.0
m in
Figure A11- Retention times of the compounds in experiment 3 are unknown.
22
The graph of experiment 4 with absorption at 220 nm (mAU) are plotted versus time (min).
The diol 1 and ketone 1 did not have enough time to form, see figure A12.
12.172/70329979
m AU
Extract-220nm 4nm (1.00)
2500
2000
6.147/1259633
1000
500
0
-500
0.0
5.0
9.672/698555
1500
10.0
15.0
20.0
25.0
m in
Figure A12- Retention times of the diol 1 and SO in experiment 4.
The graph of experiment 5 with absorption at 220nm (mAU) are plotted versus time (min).
The diol 1 and ketone 1 did not have enough time to form and give a good absorbance. Good
absorbance is 500 mAU, see figure A13.
12.232/34570127
m AU
Extract-220nm 4nm (1.00)
1750
1500
1250
8.996/2489908
750
500
250
0
10.794/3350412
1000
-250
0.0
5.0
10.0
15.0
20.0
25.0
m in
Figure A13- Retention times of the diol 1 and SO in experiment 5.
The reason why these experiments failed may depends on that the gradient was too high that
the diol 1 and ketone 1 did not have enough time to form, or the peaks got stuck together
because of that.
23