Development of CE-based Methods for the
Analysis of Technical Surfactant Samples
Bachelor Project, BSc Chemistry
B.W.J. Pirok
Summary of Contents
Abstract ....................................................................................................................................... 3
1 Introduction ............................................................................................................................. 4
2 Capillary Electrophoresis ........................................................................................................... 5
2.1 Principles ...................................................................................................................................... 5
2.2 Electro-osmosis ............................................................................................................................ 6
2.3 Ionic Mobilities ............................................................................................................................. 7
3 Detection ................................................................................................................................. 9
3.1 UV Absorption Detection .............................................................................................................. 9
3.2 Indirect Detection........................................................................................................................ 10
3.3 Time of Flight Mass Spectroscopy Detection .............................................................................. 11
4 Experimental .......................................................................................................................... 13
4.1 Apparatus ................................................................................................................................... 13
4.2 Chemicals.................................................................................................................................... 13
4.3 Procedures .................................................................................................................................. 13
4.4 Methods ..................................................................................................................................... 14
5 Results ................................................................................................................................... 15
5.1 UV Absorption Detection............................................................................................................ 15
5.2 Indirect Detection ....................................................................................................................... 17
5.3 Time of Flight Mass Spectrometry Detection ............................................................................. 20
5.4 Conclusions ................................................................................................................................. 22
6 Discussion .............................................................................................................................. 23
Achknowledgements ......................................................................................................................... 23
References ................................................................................................................................. 24
ATTACHMENTS........................................................................................................................... 25
Sample 93 .......................................................................................................................................... 26
Sample A1 .......................................................................................................................................... 27
Sample D3.......................................................................................................................................... 29
Sample H1.......................................................................................................................................... 31
Sample J1 ........................................................................................................................................... 33
Sample W0 ........................................................................................................................................ 36
2
Abstract
Technical anionic surfactant samples were separated by capillary electrophoresis using, time-of-flight
mass spectroscopy and direct and indirect UV detection as analysis technique. Fifteen out of sixteen
samples provided were divided into five classes based on experimental data. One sample was found
to be unique. Six samples were found to be alcohol propoxylate based, ten samples were olefin
based. Disulfates were also found for some of the alcohol propoxylate samples. Samples D3, D4 and
DD were found to be unique. A molecular structure was proposed for components found in alcohol
propoxylate samples.
3
1 Introduction
The goal of the project was to develop methods, based on capillary electrophoresis, for the
characterisation of technical surfactant samples. Sixteen different samples were provided by Shell
Amsterdam. The samples were alcohol propoxylate sulfate and olefin sulfonate products with
different alkyl chain lengths, with distributions varying from about C12 to C30. For most of the
samples an active material percentage value was provided.
Differences in viscosity, colour and active material concentrations were observed and used to divide
the samples in groups showing similar external characteristics. The information provided indicated
that the samples were expected to contain surfactants that contained no chromophoric groups. As
such, the surfactants were not expected to absorb UV light. Together with the absence of context
information regarding the samples, this complicated the development of a method for analysis.
This thesis will summarize the project’s results. First, the theoretical background will be explained, by
giving an overview of the principles of capillary electrophoresis, followed by an overview of the
different detection methods and the possibilities for their application for the analysis of the samples.
Second, the experimental section will specify the developed methods used to analyse and prepare
the samples, including other relevant data. Next, the results will be presented for all investigated
detection techniques, followed by a discussion in which the complications presented by the
characteristics of the samples and their consequences for method development will be addressed.
4
2 Capillary Electrophoresis
2.1 Principles
Electrophoresis is the migration of charged particles in a solution under influence of an electric field.
The size and/or charge determine the velocities at which different particles migrate, which is the
basic principle of all electrophoretic separation methods.1
The electrostatic force 𝐹 exerted on a particle 𝑖 in a solution is proportional to the net charge of the
particle 𝑞𝑖 and the electric field strength 𝐸 in the solution:
𝐹 = 𝑞𝑖 ∙ 𝐸
The direction of the electrostatic force is to the electrode with an opposite
charge than that of the particle itself. The influence of the electrostatic
force causes a charged particle to accelerate and to start migrating, whilst
being opposed by viscous forces in the solution, which increase
proportional with the velocity 𝑣𝑖 of the particle. The Stokes equation gives
the viscous force for a spherical particle
Figure 1 – Basic CE setup
𝐹 = 6𝜋𝜂 ∙ 𝑟𝑖 ∙ 𝑣𝑖
where 𝜂 is the viscosity of the solution and 𝑟𝑖 the effective radius of the particle. The opposing forces
cancel each other out after a very short acceleration time. The particle then moves with a constant
velocity through the solution:
𝑣𝑖 =
𝑞𝑖 ∙ 𝐸
6𝜋𝜂 ∙ 𝑟𝑖
Different ions can be separated when having different charge, radius or a different charge/size ratio.
In order to easily compare experimental data obtained with different field strengths, the ionic
mobility 𝜇𝑖 has been defined as:
𝜇𝑖 =
𝑣𝑖
𝐸
with a dimension of m2 V-1 s-1. When combining the above two equations it follows that the mobility
of a spherical particle can be written as:
𝜇𝑖 =
𝑞𝑖
6𝜋𝜂 ∙ 𝑟𝑖
As such it is clear that different ions can be separated when they
differ in charge or radius or when their ratio differs. For mobility
the radius of the moving particle in aqueous solution, including
its hydration shell, determines the mobility of the particle. The
Ion
H+
Li+
K+
Mobility (m2 V-1 s-1)
362
40
76
mobility of a K+ ion is for example larger than the much smaller Table 1 – Mobilities of some inorganic ions
Li+ (Table 1), as it binds more water molecules. The much higher in water at infinite dilution.
mobility of a H+ ion, however, has little to do with the relative
5
small size of a bare proton as its hydrated shell is much larger compared to other simple inorganic
ions in aqueous solution. In fact the proton has interactions in the form of hydrogen bonds with
surrounding water molecules. Therefore simple rearrangements in the electronic structure
throughout the solution may transport protons and since there is no real mass transport involved,
the transport may take place at higher velocities relative to other simple inorganic ions.
Ionic Field Strength
The equations addressed thus far for mobilities of ions are only valid in very dilute solutions, where
the ionic strength approaches zero. In CE, a finite salt concentration will be presented as background
electrolyte (BGE) in the solution. In an electrolyte solution an ion will be surrounded by other ions,
which together form a diffuse cloud of ions oppositely charged to the surrounded or central ion, so
that they neutralize the charge of the central ion.2
When increasing the ionic strength of the solution, this cloud will be much denser. As the diffuse ion
cloud has a charge opposite to that of the central ion, it will migrate in the opposite direction during
the electrophoresis process. This creates an extra viscous force exerted on the central ion, which
becomes stronger as the ionic field strength of the solution increases and is called the
electrophoretic effect. In addition, when the central ion and the diffuse ionic cloud have moved in
opposite directions, they will attract each other again due to electrostatic differences and the cloud
will rearrange itself again around the central ion. This is called the relaxation effect. Both the
electrophoretic and relaxation effect work in the same direction and the mobility of an ion decreases
as the ionic strength of a solution is increased.3 This effect is larger for multiple-charged ions,
changing the ionic strength of the BGE therefore may help to separate overlapping zones of
differently charged analytes.
2.2 Electro-osmosis
In electrophoresis an aqueous solution of electrolytes is used and in contact with the wall of the
capillary. (Fig. 2) The fused silica wall of a capillary contains negatively charged SiO- groups. This
causes a charge separation between the wall and the solution as the electrolyte solution is positive,
due to the electro neutrality principle. This excess of positive ions is electrostatically attracted by the
negative wall.
Figure 2 – Schematic of the capillary wall, showing the principles of electro-osmosis. (Reproduced from W.Th. Kok, 1999)
6
Applying voltage between the ends of the capillary results in the electric field to exert a force on the
excess positive charge present in the solution, close to the wall, driving the solution towards the
negative electrode. The viscous forces in the thin layer of solution near the wall will counteract the
electrostatic force, rendering a constant flow of the solution and is called electro-osmosis. This
principle is an important feature of CE as the separation is performed in free solution.
Since the velocity of this electro-osmotic flow (EOF) is proportional to the field strength, the electroosmotic mobility 𝜇𝑒𝑜 can be defined in a way that is similar to the definition of the ionic mobility. The
electro-osmotic mobility depends on the charge density on the wall, which is determined mainly by
the wall material and the pH of the solution.
While present in a background electrolyte buffer that migrates towards the negative pole, anionic
analytes will migrate toward the positive pole. Therefore, the electro-osmotic mobility is expected to
be higher than that of the anionic analytes present in the technical samples.
When introducing a sample at the positive end of the capillary, with the detector at the negative end,
the solutes may be determined in a single run. From the net velocity of an ion, as obtained from the
length of the capillary up to the detector position 𝐿𝑑 and the migration time 𝑡𝑖 , the apparent
mobility 𝝁𝒂𝒑𝒑,𝒊 can be calculated:
𝜇𝑎𝑝𝑝,𝑖 =
𝐿𝑑
𝑡𝑖 ∙ 𝐸
It is important to note that as the electrostatic force acts only on the outer layer of the solution, with
its excess of positive ions, nearly the entire solution moves with the same speed, except for a very
thin layer of a few nm close to the wall. This implies that the electro-osmotic flow does not cause
zone broadening.
When comparing electropherograms of two different runs of the same sample, the mobilities of the
electro-osmotic flow and the analytes may differ because of slight changes in the surface of the wall
of the capillary. Through subtraction of the electro-osmotic mobility from the apparent mobility of
particle 𝑖, the effective mobility of the particle may be obtained:
𝜇𝑒𝑓𝑓,𝑖 = 𝜇𝑎𝑝𝑝,𝑖 − 𝜇𝑒𝑜
2.3 Ionic Mobilities
For the design of a CE method it is useful to estimate the expected ionic mobilities of the analytes on
forehand. Any differences in observed values compared to those expected may then be addressed
and will ensure that supposedly known or provided information will be regarded with a critical eye.
When the equivalent ionic conductivity of particle 𝑖 is known, the ionic mobility 𝜇𝑖 may easily be
calculated using the Faraday constant 𝐹 through:
𝜇𝑖 =
𝜆0,𝑖
𝐹
For electropherogram interpretation it is useful to be aware of the different parameters that may
influence the mobility. The remainder of this paragraph will be address these parameters.
7
Of course the mobility of an ion is influenced by its charge. An ions mobility therefore is expected to
be proportional to its charge number z, but generally the mobility of a multiple-charged ion is less
than z times that of a singly-charged ion of similar size. This effect is also observed for compounds
that may have a different charge depending on the pH of the solution and is stronger for multiplecharged ions that have the charges located on the same functional group. Furthermore a higher ionic
strength of the solution will increase this observed effect as the retardation effect, mentioned in the
previous paragraph, will be stronger for higher charged compounds.
It is clear, from the equation for the mobility of a spherical particle from paragraph 2.1, that the
mobility is inversely proportional to its molecular weight. So a larger molecular weight will render a
lower mobility of the ion. For the provided technical samples, distributions in chain length to the
present surfactants are expected. A longer chain length equals a higher molecular mass and thus a
lower mobility of the ion.
In practice molecules with the same charge and similar molecular weight may often easily be
separated. Indeed, it follows that the decisive influence of the mobility of an ion is a result from its
chemical structure. This influence is hard to predict, but several factors may be of importance.
First of all, the type of charged group may be of importance. Despite their higher molecular weight,
sulfonic acids and sulphates have higher ionic mobilities than the corresponding carboxylic acids.
Cations often have a higher mobility than anions with the same molecular weight. Secondly, the
substitution of extra alkyl groups in the structure decreases the mobility of an ion similarly to that
expected of the increase in molecular weight. Third, the substitution of a hydroxyl group may be very
small or even render an increase of the ionic mobility instead. Finally, hydrophobic groups will render
a smaller decrease in ionic mobility when substituted, compared substitution of a methyl group.
8
3 Detection
3.1 UV Absorption Detection
The measurement of the absorption of light in the UV region is the standard method of detection for
capillary electrophoresis. The detection may be performed directly on-column and no physical
contact between the detection system and capillary is needed which reduces zone broadening. This
absence of contact with the separation medium also allows for high voltages to be used during
separation, as the medium is not in contact with the sensitive equipment. The capillaries used are
made from fused silica, a quartz-like material which is transparent for light with wavelengths down to
190nm. This makes the capillary itself a perfect optical cell. For most samples a colour in the range of
yellow to brown was observed, which indicated that they should show absorption in the UV
spectrum.1
The detector produces a signal, the absorbance 𝐴, that is obtained through the molar absorptivity of
the analyte 𝜀, its concentration 𝑐 and the effective length of the light path through the solution 𝐿𝑒𝑓𝑓 :
𝐴 = 𝜀 ∙ 𝑐 ∙ 𝐿𝑒𝑓𝑓
Following from this definition, the sensitivity (𝐴/𝑐) for a particular compound is proportional to 𝐿𝑒𝑓𝑓 .
Since the light is measured in a direction perpendicular to the capillary axis, 𝐿𝑒𝑓𝑓 can be no more
than the capillary diameter. This explains why UV absorption measurements with CE have limits of
detection that are approximately two orders of magnitude higher than in HPLC. Yet this may be
compensated by using a less low wavelength, where many compounds show high absorptions.
Finally, it is important to note that the UV detection is performed on an optical cell that has a
cylindrical shape.4 Illumination of the cell by a parallel beam of light with a certain width (𝑤𝑏 ) results
in a varying length of light over the width of the beam. Aside from the capillary inside diameter, the
effective light path length 𝐿𝑒𝑓𝑓 depends also on the ratio between the light beam width and the
capillary inside diameter. When 𝑤𝑏 is exactly as wide as the capillary inside diameter, the path length
varies from 0 at the top and the bottom, to 𝑑𝑐 in the middle of the capillary cross-section. When the
light beam is wider than the capillary inner diameter, part of the light will simply pass the capillary
undisturbed by the absorbance in the solution. This will decrease the sensitivity of detection since
the signal produced by the UV detector is based on a comparison of the intensity 𝐼 of the light beam
passing the optical cell with a reference value 𝐼0 .5 The ratio of 𝐼 and 𝐼0 is translated electronically into
the signal 𝐴:
𝐴 = − log
𝐼
𝐼0
The diode-array detector (DAD) allows a part of the spectrum where no absorbance is expected to be
used for reference. Using this, when due to insufficient focusing of the beam a part of the light then
passes the capillary undisturbed, the amount of light absorbed by compounds in the solution is not
affected. Yet, relative to the total intensity, the change of 𝐼 by absorption will be smaller and the
lower change of the ratio between 𝐼 and 𝐼0 will then be translated into a lower signal.6
Even though the target surfactants in the technical samples are not expected to contain any
functional groups, a colour could be physically observed and hinted at the presence of absorbing
9
material. This could be unreacted material used to create the surfactants, but also could have been
an unexpected feature of the surfactant molecules. Henceforth, UV absorption measurements were
taken of all sixteen samples.
3.2 Indirect Detection
An attractive feature of CE was the possibility to apply indirect detection on analysis of the
surfactants, as they were expected to show have absorption themselves.7 For indirect detection, an
ionic compound with a high UV absorbance is used as one of the constituents of the background
electrolyte solution and functions as monitoring ion.8 Using a monitoring ion results in an
electropherogram with a background absorption. When an analyte in such a solution passes through
the detector, the local concentration of the monitoring ion will be reduced, resulting in a decrease of
absorption. As such indirect detection is universal and non-selective.
In order to make a founded choice of the operating conditions, the transfer ratio for a specific
analyte-BGE combination should be considered.9 This ratio may be defined as:
𝑇𝑅 = −
∆𝑐𝑎
𝑐𝑖
Here, ∆𝑐𝑎 is the change in concentration of the monitoring species by the analyte concentration 𝑐𝑖 in
a zone. The minus sign indicates that the changes in absorption are expressed as decrease in the
baseline signal. Since the changes of 𝑐𝑎 are being monitored, the transfer ratio is a measure of the
sensitivity that will be obtained.
In indirect detection, the quantitative reliability for low analyte concentrations is often not so much
limited by detector noise but rather by instabilities of the baseline. Instabilities as baseline drifts and
shifts and the appearance of unexpected often broad peaks in the electropherogram may interfere
with the recognition and integration of the analyte peaks. These baseline instabilities are often
repeatable and seem to be unrelated to the injected sample. Even injecting no sample at all may
results in the same baseline pattern after switching on the high voltage source.
These baseline instabilities can be explained through temperature differences over the length of the
capillary.10 The disturbance of the monitoring ion concentration visible on the electropherogram can
come from both ends of the capillary to the detection window. It is impossible to eliminate all the
thermal nodes through with these disturbances could occur. Yet the number of disturbances coming
is related to the number of components in the BGE composition. These disturbances will from now
on be referred as system peaks. To keep the number of system peaks low and improve the selectivity
of the separation, the number of ionic components should be kept to a minimum.
To analyse the technical samples using indirect UV detection, sorbate and benzoate were
investigated as monitoring ions and each used during measurement of all sixteen samples.
In order to investigate the identity of observed phenomena on electropherograms obtained, the
samples were spiked with standard compounds. These standard compounds were a sulfonate and
sulfate like the surfactants, but have a fixed and, more importantly, known alkyl chain length. The
electropherograms of the standards could then be cross-referenced with those of the samples,
providing information about the identity of observed phenomena. Lauryl sulfate (C12) and hexane
sulfonate (C6) were used to spike the samples.
10
3.3 Time of Flight Mass Spectroscopy Detection
To be able to obtain more information on the technical samples, a time of flight mass spectrometer
(TOF-MS) was coupled with the CE instrument using an interface with the electrospray (ESI)
ionization ion source where the sample is introduced into the TOF analyser.
Electrospray Ionization
Electrospray is produced by applying a strong electric field, under atmospheric pressure, to a liquid
passing through a capillary tube with a weak flux. By applying a potential difference of 4 kV between
this capillary and the counter electrode, the electric field is obtained. This field induces a charge
accumulation at the liquid surface located at the end of the capillary, and explodes to form highly
charged droplets.
A gas injected coaxially at low flow rate allows the dispersion of the spray to be limited in space.
These droplets then pass either through a curtain of heated inert gas, most often nitrogen. At onset
voltage the pressure is higher than the surface tension, the shape of the drop changes at once to a
Taylor cone and small droplets are released, which divide and explode, producing the spray. This
spray in turn consists out of very small highly charged droplets that continue to lose solvent and
accumulate charge. When the electric field on their surface becomes large enough, desorption of
ions from the surface occurs. Because of limitations from the electrochemical process, the
electrospray current is limited. This process occurs at the probe tip and is sensitive to concentration
rather than to total amount of sample.
Time of Flight Mass Spectroscopy
To couple the continuous ion beam from the
ESI with the TOF analyser, orthogonal
acceleration was used (Fig. 3). As the sample
was continuously ionized in the ion source, the
parallel beam passes an RF octapole ion guide
that transports the ions efficiently to the
orthogonal accelerator. This not only focuses
the ions from the source but also controls the
kinetic energy of these ions by collisional
cooling. The resulting beam is directed to the
orthogonal accelerator and continuously fills
the first stage of the ion accelerator in the
space between a plate (depicted in figure 3 as
a red line) and a grid (purple) at 0 V. Initially,
the voltage on the plate is 0 V like the grid. So
the region is field free and ions continue to
Figure 3 – Simplified schematic of a TOF-MS instrument.
move in their original direction. Then an
injection pulse voltage of 𝑉𝑖𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛 is applied to the plate. The ions between the plate and the grid
are pushed by the resulting electric field in a direction orthogonal to their original trajectories and
start flying to the analyser. After passing through the grid the ions are accelerated more towards a
second grid (blue) which is at a voltage of 𝑉𝑇𝑂𝐹 . Then the ions enter the field-free drift region at a
potential of 𝑉𝑇𝑂𝐹 where the TOF mass separation occurs.
11
Once all ions moved to the field-free region through the second grid, the voltage on the plate is
restored to 0 V and ions from the ion source will begin to fill the space between the plate and the
first grid again. Thus during the time that the ions continue their flight in the field-free region, the
orthogonal accelerator is refilled with new ions. One flight cycle will end when the ion with the
highest m/z reaches the detector. Another flight cycle will begin by reapplying a pulse voltage to the
plate. As such the distance travelled by the ion beam between two successive injection pulses in the
first stage of the orthogonal accelerator is the same as the distance travelled by the ion in the flight
tube between the orthogonal accelerator aperture and the detector.
It should be noted that the time required for the ion beam to fill the orthogonal accelerator is lower
than the time for sampled ions to fly to the detector. Therefore a part of the ions produced in the
source are not pushed to the flight tube and are lost in the first stage of the orthogonal accelerator.
Reflectron
The reflectron is located behind the field-free region opposed to the ion source. The detector is
located on the source side of the ion mirror and captures the arrival of ions reflection by the
reflectron. This reflectron corrects the kinetic energy dispersion of the ions leaving the source with
the same m/z ratio. Ions with more kinetic energy and hence more velocity will penetrate the
reflectron more deeply than ions with lower kinetic energy. Consequently, faster ions will spend
more time in the reflectron and will reach the detector at the same time than slower ions with the
same m/z. Although the reflectron increases the flight path without increasing the dimensions of the
mass spectrometer, this positive effect on the resolution is of lower interest than its capability to
correct the initial kinetic energy dispersion. However, the reflectron increases the mass resolution at
the expense of sensitivity and introduces a mass range limit.
Coupling of a CE instrument
In CE, the flow rate of the buffer in the capillary, caused by the
generation of the electro-osmotic flow, is very low, on the
order of several hundred nl/min. For this reason, in order to
conduct stable CE/MS analysis, the system is devised so that a
sheath liquid is sent from outside the capillary to supplement
the flow rate (Fig. 4). Furthermore, the sheath liquid serves to
electrically connect the CE outlet to the ground potential at
the sprayer, similar to the function of the outlet buffer in a CEonly system. During these experiments a LC pump with a
splitter was used to obtain a sheath flow of 0.4ul/min.
Figure 4 – Schematic of the spray needle used
to couple the CE instrument with the TOF-MS.
12
4 Experimental
4.1 Apparatus
Two Agilent G1600AX HP 3D CE systems were used throughout the experiment, both with a standard
setup using an UV detector for analysis. One for direct UV detection and one for indirect UV
detection. At a later stage the CE instrument used for direct UV detection was coupled to a Agilent
G1969A LC/MSD TOF instrument. The TOF instrument had an electrospray ionization ion source
installed. An Agilent 1100 Series Isocratic Pump and Degasser were used to provide the sheath liquid
at a constant flow (Fig. 5). The isocratic pump had an 1:100 splitter installed to be able to produce
sheath flows on the order of µl/min.
4.2 Chemicals
Background Electrolyte
For direct detection, a 5 mM ammonium acetate solution was used as BGE. For indirect detection a
solution of 5 mM borax and 10 mM sorbate was used as BGE, where sorbate functioned as
monitoring ion. Both solutions were made using methanol/water (8:2) as solvent. For CE-TOF-MS
analysis the BGE consisted of 5 mM ammonium acetate also using methanol/water (8:2) as solvent.
Capillary
Fused silica capillary with an internal diameter of 75 µm was used during all analysis. For direct and
indirect UV detection the capillary had a total length of 50 cm, with a length to the detector of 41.5
cm. For CE-TOF-MS analysis the
Original
Code % AM Type
Viscosity Colour
Class
capillary had a total length of 80 973
93
35,4
OS
Low
Yellow
W
cm, with a length of 21.5 cm to A771
A1
30,5
APS
High
Colorless
D3
72,4
OS
Very Low Light Brown
D
the UV detector of the CE Drum 3
Drum 4
D4
72,4
OS
Very Low Light Brown
D
instrument and the full 80 cm to
Drum D
DD
28,0
OS
Very Low Creme
D
the TOF-MS instrument.
Drum Q
DQ
33,0
OS
Low
Yellow
W
Used Chemicals
All chemicals used were obtained
from standard supplies.
Samples
Sixteen samples were provided
for analysis. Details have been
layed out in table 2.
H771
H771-2
J13131
J13131-2
J771
WFE10
#1
#5
#7
#11
H1
H2
J1
J2
J7
W0
X1
X5
X7
X11
30,8
26,0
34,7
28,1
28,9
35,5
-
APS
APS
APS
APS
APS
OS
OS
OS
OS
OS
High
High
High
High
High
Low
Very Low
Very Low
Very Low
Very Low
Colorless
Colorless
Colorless
Colorless
Colorless
Brown
Light Yellow
Yellow
Brown
Dark Brown
H
H
J
J
J
W
X
X
X
X
Table 2 – Detailed information on provided technical samples. (APS = Alcohol
propoxylate sulfate, OS = olefin sulfonate)
4.3 Procedures
Preconditioning
Since onward analysis revealed that the capillaries could clot due to some of the samples a special
preconditioning program was executed at the start of each day. The program consisted of flushing
the capillary first 15 minutes with acetonitrile, followed by 15 minutes of 0.1M sodium hydroxide
solution and finally 15 minutes with water.
13
Sample Preparation
Since for 12 samples the active material (AM) concentration was provided (Table 2), an amount of
these sample was added to 10 mL water/methanol (8:2) until an AM concentration of 30% was
reached. For the remaining four samples the AM concentration was estimated based on their
observed densities, after which they were dissolved to a obtain an AM concentration similar to 30%.
4.4 Methods
Direct and indirect UV Absorption Detection Measurements
Each measurement run started with a preconditioning program where the capillary was flushed with
acetonitrile for 2 min, 0.1M sodium hydroxide solution for 2 minutes and BGE solution for 2 minutes.
Next, the sample was injected by a pressure of 30 mbar/4 sec after which the run started by applying
a voltage of 30 kV during the acquisition time of 30 min. The CE was set to run electrically in the
positive mode. The cassette was kept at a temperature of 25 oC during the entire time. Wavelengths
of 200nm and 254 were observed during each run with a response time of 0.2sec.
CE-TOF-MS Measurements
On the CE side of the system: Each measurement run started with a preconditioning program where
the capillary was flushed with BGE for 4 min. Next, the sample was injected by a pressure of 30
mbar/3 sec after which the run started by applying a voltage of 30 kV during the acquisition time of
30 min. The CE was set to run electrically in the positive mode. The cassette was kept at a
temperature of 25 oC during the entire time. Wavelengths of 200nm and 254 were observed during
each run with a response time of 0.2sec. The isocratic pump was set to provide a flowrate of 0.4
ml/min, with the 1:100 splitter this resulted in a flow of 4µl/min sheath flow. The TOF-MS instrument
had its ion polarity set to negative. The gas temperature was set to 300 oC. The drying gas ran at a
flow of 7 l/min with the nebulizer at 15 psig. A voltage of 4 kV was applied to the capillary and the
fragmentor had a voltage of 200 V.
Figure 5 – Schematic of the setup used to couple the CE instrument with a TOF-MS instrument.
14
5 Results
Electropherograms were successfully obtained in both direct and indirect mode for each of the
samples. In order to identify possible system phenomena a blank was taken for each mode as well.
System and standard phenomena were compared with the sample electropherograms using ionic
mobility calculations for each of them.
During sample preparation a notable difference in solubility and viscosity of the samples was
observed. Samples H1, H2, A1, J1, J2 and J7 were much easier to dissolve in water than the remaining
ten samples. Furthermore, no colour was observed for the six samples mentioned, whereas the
remaining 10 were all showing colours in the range of crème-yellow to yellow-brown.
These observations indicated that the properties of the six samples mentioned featured notable
difference compared to those of the remaining ten.
5.1 UV Absorption Detection
Experimental data taken in the direct mode revealed a close relation between certain samples, which
allowed classification. For samples D3, D4 and DD a near perfect correlation was observed, both in
peaks and intensity of the peaks (Fig. 6a). Mobility calculations (paragraph 2.2) performed on the
largest peak visible on each of the three electropherograms confirmed that they were all the same
and the samples were grouped as Class D. Indeed, a large shift can be observed for the green line
which represents sample DD. This shift, however, is also visible for the electro-osmotic flow peak. As
explained in paragraph 2.1, to compare a peak observed on one electropherogram with one
observed on another, one must compare the mobilities for both peaks. These mobilities are first
corrected by the mobility of the electro-osmotic flow.
Figure 6 – Overlays of electropherograms taken using direct UV detection at 200nm. a) Samples D3 (blue), D4 (red) and
DD (green) at 200 nm. b) Samples 93 (blue), DQ (red) and W0 (green). S stands for system peak, EOF for acetone,
representing the peak of the electro-osmotic flow.
15
The electropherograms taken for samples 93, DQ and W0 showed many similarities (Fig. 6b), which
had no correlation with electropherograms taken from other samples. The peak marked with x
proved to be not reproducible and was in fact an artefact. It can be seen that the intensity of the
peaks observed for sample W0 (green) are slightly higher than those for the other two samples.
Slight differences in peak shapes can be observed, but mobility calculations confirm that the relative
locations are nearly identical. These samples were grouped and will from now on be referred to as
class W.
Direct UV detection performed on samples J1, J2 and J7 revealed a gathering of individual
components to be observed for all samples (Fig. 7a). Highest intensities were observed for sample J1,
whereas intensities were somewhat lower for sample J2 and J7. The spike observed for sample J2 at t
= 5.6 was not reproducible on later runs and included here as the runs were all taken on the same
sequence. A notable difference of the electropherograms of these three samples compared to those
of the previous six was that the intensities of the observed peaks were significantly lower. This
supports the preliminary observation that the properties of these samples were notable different
than those of the previous two classes. The three samples were grouped as class J.
Samples H1, H2 and A1 proved to be difficult to analyse using CE. Large baseline instabilities were
observed on repeated measurements of all three samples (Fig. 7b), with odd phenomena to be
observed for some of the runs. The significant absorption for sample A1 around t = 6.7, was
consistently observed whereas the electropherograms of samples H1 and H2 did not show this
increase. The broad peak observed on the electropherogram of sample H2 is the same as the small
broad peak for sample H1, yet not as intense as it is for H2. Samples H1 and H2 correlated quite well
and were grouped as class H.
Figuur 7 – Overlays of electropherograms taken using direct UV detection at 200nm. a) Samples J1 (blue), J2 (red) and J7
(green) at 200 nm. b) Samples H1 (red), A1 (blue) and H2 (green).
16
Direct UV detection analysis of samples X1, X5, X7 and X11 revealed little useful information. The
electropherograms correlated very well. Yet due to the absence of information regarding active
material concentrations it was not possible to compare peak intensities.
5.2 Indirect Detection
Both sorbate and benzoate were investigated as monitoring ions on standard compounds.
Comparison of the obtained electropherograms showed that the performance of benzoate was poor
compared to that of sorbate, as such sorbate was used to perform indirect detection on all sixteen
samples.
To be able to get an indication on the molecular size of sample compounds, several compounds of
similar structure as the expected sample compounds, were investigated to be used as standard
reference. Indirect UV detection measurements were performed for hexane sulfate, lauryl sulfate
and tetradecyl sulfonate (Fig. 8). These compounds have an alkyl chain length of respectively six (C6),
twelve (C12) and fourteen (C14) carbon atoms. Tetradecyl sulfonate turned out to perform poorly as
standard compound and was discarded. Hexane sulfate and lauryl sulfate were used to spike the
sampled to be measured with indirect UV detection. Comparison with the electropherograms
obtained from analysis of a blank run of the background electrolyte buffer allowed identification of
the system peaks.
Figure 8 – Electropherogram obtained by indirect UV detection performed on hexane sulfate, lauryl sulfate in 5 mM
sodium tetraborate decahydrate and 10 mM sorbate as BGE buffer at 220nm.
For each class one sample was selected to represent the other samples of the class. Each of these
samples was spiked with the two standard compounds, after which indirect UV analysis was
performed. For every obtained electropherogram, two peaks were identified as the standard
compound through comparison of effective mobility data from the electropherograms of the
standards and analysed sample.
An electropherogram of sample D4 spiked with the two standard compounds was obtained using
indirect UV detection. Two peaks could successfully be identified as standard compound. Irregular
peak shapes complicated phenomena classification as baseline instability, system peak or analyte
peak. A wide peak was observed from t = 16.5 until t = 20.5 and identified as the peak at t = 6.5 on
the direct UV detection electropherogram of sample D4 (Fig. 9). The shape of this peak was
interpreted as being caused by the absorption of compounds reaching a concentration at the UV
detector that was higher than that of the monitoring ion.
17
Figure 9 – Indirect UV detection electropherogram of Sample D4 in 5 mM sodium tetraborate decahydrate and 10 mM
sorbate as BGE buffer at 220nm. Phenomena denoted S are system peaks.
Indirect UV detection analysis of sample X7 provided little information as no clear specific
phenomena could be observed, though standard compounds were correlated to two observed
phenomena. (Fig. 10a) The broad peak observed at t = 15.5 was identified to be the same as peak
observed on the direct UV detection electropherogram, similarly to sample D4.
For sample W0 a broad valley was observed at t = 9.7 on the indirect UV detection electropherogram (Fig. 10b). This valley was also observed for samples 93 and DQ, supporting the earlier
observation that samples 93, DQ and W0 belonged to the same class of samples.
Figure 10 – Indirect UV detection electropherogram of a) sample X7 and b) sample W0 in 5 mM sodium tetraborate
decahydrate and 10 mM sorbate as BGE buffer at 220nm. Phenomena denoted S are system peaks.
18
Similar to the direct UV detection analysis of sample H2, the indirect UV detection electropherogram
showed a gathering of individual components. (Fig. 11) Standard compound peaks were successfully
identified. The location of the remaining peaks relative to those of the standard compounds
suggested that most peaks represented surfactant ions with an alkyl chain length being larger than
12 carbon atoms. The electropherogram showed that surfactants with chain lengths smaller than 12
carbon atoms appeared to separate better than those with longer chain length. The valley observed
around t = 10 consisted out of multiple phenomena that merged into each other. One could also say
that they did not separate well. At the same time information provided indicated that the minimum
amount of carbon atoms expected in the variable alkyl chain would be 12. Whereas peaks were
observed that would suggest an amount smaller than 6. The electropherogram interpretation was
not consistent with information provided.
A gathering of individual components was also observed for the electropherogram of indirect UV
detection analysis of sample J2. Compared to the electropherogram of sample H2, the number of
component phenomena observed was low. Also the intensities of the phenomena observed for
sample J2 were lower than for sample H2.
For these two sample classes, the electropherograms taken by indirect UV detection revealed that
the area of C6 valley, which represented an amount smaller than 0.2mg ml-1 hexane sulfonate, was
much larger than the area of the individual component valleys added up, while the sample
concentration after dilution of a technical sample with a factor required to obtain an AM of 30%, was
12 mg ml-1. Hence, the observed surfactants did not account for the majority of the sample and thus
were not strongly present in the pure sample.
Figure 11 – Indirect UV detection electropherogram of a) sample H2 and b) sample J2 in 5 mM sodium tetraborate
decahydrate and 10 mM sorbate as BGE buffer at 220nm. Phenomena denoted S are system peaks.
19
5.3 Time of Flight Mass Spectrometry Detection
A TOF-MS instrument was successfully coupled to the CE instrument. In this setup, the length to the
UV detector was merely 21.5 cm. So when the sample zone reached the UV detector, the sample had
not enough time yet to separate. As such electropherograms obtained through the UV detector
were only used as diagnostic tool. Total ion current (TIC) chromatograms were obtained through
TOF-MS analysis of the samples. Each of them showed a peak at t=4, whilst the UV detector showed
that the EOF should have been appearing around t=5.6.
This meant that the observed peak at t=4 represented compounds that were faster than the EOF and
thus must have been positively charged. The gap between each observed peak on the mass spectrum
of these compound (Fig. 14b) was 82 m/z. This value corresponded with sodium acetate. Based on
this information the observed phenomenon was classified as a system peak representing positively
charged ion clusters, consisting of sodium acetate, as a result of the use of ammonium acetate as
BGE.
Class D and W
Through analysis of the samples of class D, a TIC chromatogram was obtained that correlated well to
the electropherogram obtained by direct UV detection analysis (Fig. 12). Mobility calculations
allowed the conclusion that peak 4 in fact was the same as the large peak observed on earlier UV
electropherograms. The peak was found to be consisting mainly of a compound with mass 377,27.
TOF-MS analysis of class D samples showed that abundances and compounds found for all the
samples were nearly identical. This information allowed the conclusion that all of the class D samples
were nearly identical.
Figure 12 – TIC chromatogram of sample D3.
Samples 93, DQ and W0 were also found to be very similar based on information obtained through
TOF-MS analysis. Some of the compounds found, having masses of 322,25 and 349,25 were also
found during analysis of class D samples. The samples were found to be very similar in components
observed on mass spectra, but their abundances were different.
Class X
As with UV detection, lack of information regarding
active material concentrations hindered accurate
abundance comparison of samples within the X
class. Multiple gathering of individual components
were observed for all the class X samples with
similar masses (Fig. 13).
Figure 13 – Mass spectrum of an observed phenomenon
on the TIC chromatogram of sample X5.
20
Class H and J
A TIC chromatogram and mass spectra were
obtained through mass spectrometry detection
applied on sample H1 (Fig. 14a). A high abundance
phenomenon was observed from t = 8-8.5. The
mass spectrum of this domain revealed a
distribution being present (Fig. 14c). The larger
gaps between the peaks represented a difference Figure 15 – Proposed molecular structure of a part of the
of 58m/z, whereas the smaller gaps observed had surfactant molecule observed in distribution of sample H1.
Presumably, the alkyl chain has a length of 12 or 13 carbon
a difference of 14m/z. The information provided atoms (n = 12,13) and 0 to 10 propoxy groups (m = 0 – 10).
indicated that sample H1 was a propoxylate
product, a propoxy group has the mass of 58, explaining the observed distribution. The smaller gaps
observed represented the addition or loss of a methyl group. The information pointed at a molecule
(Fig. 15), consisting out of an alkyl chain of 12 or 13 carbon atoms and 0 to 10 propoxy groups.
However, the intensities observed for an alkyl chain of 13 carbon atoms was found to be an odd
number. Furthermore, this distribution was observed only for the broad peak on the TIC
chromatogram. It was not observed on the mass spectra for the other peaks.
Figure 14 – a) TIC chromatogram of sample H1. b) Mass spectrum of peak 1. c) Mass spectrum of peak 2. Area in green
was used to substract background spectrum from peak spectra.
21
Analysis of the other peak shaped phenomena on the TIC revealed that peaks 3, 4 and 5 represented
a compounds with a rounded m/z value of 291, 262 and 233 respectively. The difference between
every following peak was constantly 29 m/z, exactly half of 58. This pattern was continued for the
remaining peaks and hinted at the presence of a second distribution being present of the same
compounds but with a second sulphate group attached to the molecule. When an ion is doubly
charged the m/z value halves, thus gaps of half of the original mass value were expected. This would
also explain why the doubly charged ions separate better than the singly charged ions from peak 2.
Furthermore, it explained why there were valleys appearing on the indirect UV detection
electropherogram near the hexane sulfate valley. As these valleys represented in fact disulfates,
which are, being doubly charged, not of similar structure as hexane sulfate.
Similarly, the separated compounds were also found for sample J1. The number of separated
components and phenomena observed for sample J1 was, however, higher than for sample H1. Also
the distribution found for sample J1 was present only at a higher mass range, indicating a different
alkyl chain length. Sample J7 was notably different from J1 and J2: additional compound peaks were
observed in the mass spectra that were also found for sample H1 and H2.
Finally, sample A1 also showed the distribution with gaps of 58 and 14 m/z. A difference of 58
indicated a propoxy component, whereas 14 indicated a methyl component. This confirmed that
samples A1, H1, H2, J1, J2 and J7 were in fact the alcohol propoxylate products.
5.4 Conclusions
Experimental data allowed classification for fifteen of the sixteen samples into five distinct classes,
two alcohol based, three olefin based. Samples H1 and H2 were very similar. Sample A1 was found to
be unique, however, the combined distribution observed for samples H1 and H2, with gaps of 14
(methyl) and 58 (propoxy) was found for sample A1 too. This distribution was not found for class J
samples. Class J samples had a distribution in the mass range of 700-900, with only gaps of 58
(propoxy), not 14 (methyl). Samples J1 and J2 were found to be very similar, sample J7 was notably
different and contained compounds that were also found for samples H1 and H2. Samples A1, H1,
H2, J1, J2 and J7 were all found to be alcohol propoxylate products. Class D, W and X samples were
found to be olefin products. Sample D3, D4 and DD were found to be identical. Minor differences in
compound abundances were found between samples 93, DQ and W0. No distributions were
observed.
The performance of CE in general was decent. Compounds and samples were successfully separated
using developed methods, with disulfates naturally separating better than monosulfates. The
presence of absorbing material hindered the use indirect UV detection.
22
6 Discussion
With three different techniques of detection applied on the sixteen technical samples, three different
types of information were obtained. This information was found to be difficult to relate as specified
target information to be used for comparison were not always very clear.
The low amount of background information complicated the analysis and interpretation of obtained
data. Where indirect UV detection would have been ideal for analysis of these samples based on
information provided, too much absorbing material of unknown identity was found to be significantly
complicating analysis. Different monitoring ions were not found to be successful in overcoming this
problem.
Analysis by mass spectrometry was found to be more successful. Some components were identified,
as for example in sample H1 (paragraph 5.3). Most components, however, have not entirely been
identified and more investigation is required for identification. Data obtained for class X samples
indicated the presence of a great number of components that were not observed for the other
twelve samples, and require more investigation.
Achknowledgements
Thanks go out to prof. dr. ir. P. Schoenmakers for his insights in interpretation of some of the data,
and prof. dr. S. van der Wal, for his help with the TOF-MS instrument. However, special thanks go to
dr. W. Th. Kok, who guided me in this project. His insights and academic skills proved to be very
helpful during critical moments of this project. A good mentor and teacher was found in him.
23
References
(1) Kok, W. Capillary electrophoresis: Instrumentation and operation. Chromatographia 2000, 51, S5S89.
(2) Harned, H. S.; Owen, B. B. In The Physical Chemistry of Electrolytic Solutions; Reinhold Publ.Corp.:
New York, 1958; .
(3) FRIEDL, W.; REIJENGA, J.; KENNDLER, E. Ionic-Strength and Charge Number Correction for
Mobilities of Multivalent Organic-Anions in Capillary Electrophoresis. J. Chromatogr. A 1995, 709,
163-170.
(4) Hjerten, S. Free zone electrophoresis. Chromatogr. Rev. 1967, 9, 122-219.
(5) Xu, X.; Kok, W.; Poppe, H. Change of pH in electrophoretic zones as a cause of peak deformation.
J. Chromatogr. A 1996, 742, 211-227.
(6) Bruin, G. J. M.; Stegeman, G.; Vanasten, A. C.; Xu, X.; Kraak, J. C.; Poppe, H. Optimization and
Evaluation of the Performance of Arrangements for Uv Detection in High-Resolution Separations
using Fused-Silica Capillaries. J. Chromatogr. 1991, 559, 163-181.
(7) Yeung, E. S.; Kuhr, W. G. Indirect Detection Methods for Capillary Separations. Anal. Chem. 1991,
63, A275-&.
(8) Foret, F.; Fanali, S.; Ossicini, L.; Bocek, P. Indirect Photometric Detection in Capillary Zone
Electrophoresis. J. Chromatogr. 1989, 470, 299-308.
(9) Xiong, X.; Li, S. F. Y. Design of background electrolytes for indirect photometric detections based
on a model of sample zone absorption in capillary electrophoresis. Journal of Chromatography a
1999, 835, 169-185.
(10) Xu, X.; Kok, W. T.; Poppe, H. Noise and baseline disturbances in indirect UV detection in capillary
electrophoresis. Journal of Chromatography a 1997, 786, 333-345.
24
ATTACHMENTS
25
Sample 93
Total Ion Current Chromatogram
Mass Spectrum (Area 1) // EOF
Mass Spectrum (Area 2)
Mass Spectrum (Area 3)
26
Sample A1
Total Ion Current Chromatogram
Mass Spectrum (Area 1) // EOF
Mass Spectrum (Area 2)
Mass Spectrum (Area 3)
27
Mass Spectrum (Area 4)
Mass Spectrum (Area 5)
Mass Spectrum (Area 6)
Mass Spectrum (Area 7)
Mass Spectrum (Area 8)
Mass Spectrum (Area 9)
Mass Spectrum (Area 10)
28
Sample D3
Total Ion Current Chromatogram
Mass Spectrum (Area 1) // EOF
Mass Spectrum (Area 2)
29
Mass Spectrum (Area 3)
Mass Spectrum (Area 4)
30
Sample H1
Total Ion Current Chromatogram
Mass Spectrum (Area 1) // EOF
Mass Spectrum (Area 2)
Mass Spectrum (Area 3)
Mass Spectrum (Area 4)
31
Mass Spectrum (Area 5)
Mass Spectrum (Area 6)
Mass Spectrum (Area 7)
Mass Spectrum (Area 8)
32
Sample J1
Total Ion Current Chromatogram
Mass Spectrum (Area 1) // EOF
Mass Spectrum (Area 2)
Mass Spectrum (Area 3)
33
Mass Spectrum (Area 4)
Mass Spectrum (Area 5)
Mass Spectrum (Area 6)
Mass Spectrum (Area 7)
Mass Spectrum (Area 8)
Mass Spectrum (Area 9)
34
Mass Spectrum (Area 10)
Mass Spectrum (Area 11)
Mass Spectrum (Area 12)
Mass Spectrum (Area 13)
35
Sample W0
Total Ion Current Chromatogram
Mass Spectrum (Area 1) // EOF
Mass Spectrum (Area 2)
Mass Spectrum (Area 3)
36