Manual to the Advanced Laboratory Course on Analytical Chemistry: CE LIF
1
Capillary Electrophoresis with Laser-Induced
Fluorescence Detection (CE LIF)
Theoretical part
A. Capillary Electrophoresis
Capillary Electrophoresis (CE) is a modern high performance separation method. The
separation is based on different migration of analytes in a capillary over which a high voltage
(typically 10-30 kV) is applied. Typical inner diameters of commonly used capillaries are in the
range of 25-75 µm and the detection is usually on-column.
Capillary zone electrophoresis (CZE) is one of the CE modes that is characterized by the
use of a single background electrolyte (BGE) (isocratic), which enables the generation of a
constant electrical field. The detector response in the end of the capillary (the
electrophoregram), has a characteristic peak profile that is a function of many factors (type of
sample, injection, detection, sorption, mobility differences, etc.). The qualitative characteristics
are related to the migration time of the peaks and the quantitative characteristics are represented
by the peak height or the peak area.
Electrically charged particles are moving in the applied electrical field in a direction
determined by its charge and the field orientation. Positively charged particles are moving
towards the negative pole and negatively charged particles are moving towards the positive pole.
A charged particle is characterized by an electrophoretic mobility  defined as:







Vs
m
.
2
U
L
v
E
v

where E is the intensity of the electrical field, U is the applied voltage, L is the total distance
between the electrodes (the total length of the separation capillary).
The relationship between the mobility and the charge of the particle follows from the
balance between the electric driving force and the retarding friction force. The electric force is
given by the product of the particle charge and electric field intensity; the friction force is given
by the Stokes relationship F = 3μdv, where μ is the liquid viscosity, d is the hydrodynamic
particle diameter and v is the particle velocity. The electrophoretic mobility is conventionally
defined positive for cations and negative for anions.
The electrophoretic mobility is calculated from the migration time tm, which is the time
during which the analyte migrate from the injection end of the capillary to the detector:
Ut
Ll
Et
l
E
v
m
ef
m
ef

where lef is the effective length of the capillary (distance between the injection end of the
capillary and the detector).
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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In the case of commonly used fused silica capillaries, basic electromigration is
influenced by the electroosmotic flow (EOF; Fig. 1). EOF is independent on the charge of the
analytes and the resulting mobility  can be defined as:
where t0
is the migration time of neutral (non-charged ) particles.
Figure 1. Electroosmotic flow
The inner surface of the fused silica capillary carries fixed
negative charges (dissociated silanol groups). In consequence
of the law of electroneutrality conservation, there is an excess of
positively charged ions in the BGE near the inner capillary wall.
Movement of these cations towards the cathode generates a
flow of bulk liquid inside the capillary in this direction.
Many analytes that can be separated by CE has an acid-base character. Because the ionization
equilibrium is established in a much shorter time compared to the electromigration time, all
forms of a certain ion are moving with the same speed in a single zone. The resulting mobility,
the effective mobility, is the sum of the electrophoretic mobilities i of each ion form multiplied
by the respective molar fraction xi:
 iief x 
Analytical chemists usually solve the task how to separate all analytes (or the major
analytes at least) efficiently. The chemists try to optimize the separation, which usually means
finding the conditions at which the differences of the ion mobilities of the analytes are maximal.
The parameters to optimize are: pH, ionic strength and polarity of BGE, voltage and current
during analysis, injection process, etc. Complexation with ligands e.g. crown-ethers or
cyclodextrins or enhanced modes of CE, such as micellar electrokinetic chromatography
(MEKC) can be used to improve separation efficiency. This is especially useful for separation of
neutral compounds (by MEKC, where separation is based on differential distribution into
micelles) or enantiomers (by use of cyclodextrin. Enantiomers interact differentially with
cyclodextrins), which are not separated by conventional CE. Modifiers of capillary walls, such
as organic solvent, gel or linear polymer can be used to suppress sorption of analytes to the
capillary wall and suppress or even reverse the direction of the EOF. For instance, addition of a
cationic surfactant cetyltrimethylamoniumbromid (CTAB) forms a positively charged layer on
the silica surface resulting in a reversed EOF.







0
0
11
ttU
Ll
Ut
Ll
m
ef
m
ef


Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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B. Instrumentation - theory
Capillary zone electrophoresis
The basic instrumental setup for CZE contains a separation capillary, power supply,
detector and device for recording, storing and processing of analytical signals (Fig. 2). The
voltage is typically in the range of 10 – 30 kV and the BGE buffer concentration is in the range
of 10 – 50 mM. The CE analysis is usually performed in a fused silica capillary, and rarely in
Teflon or glass capillaries. Silica capillaries are covered with a thin layer of polyimide on its
outer side to protect the capillary from getting brittle and break. The typical inner diameter (i.d.)
of common capillaries is in the range of 25 – 75 m, and the outer diameter (o.d.) is in the range
of 150 – 400 m. In practice, the length of the separation capillary is in the range 30 – 70 cm
and the volume is a few l.
Figure 2. Scheme of the instrumental setup of capillary electrophoresis
Injection
Injection of a sample into the separation capillary is performed either hydrodynamically
or elektrokinetically.
Hydrodynamic injection is achieved by applying an overpressure on the injection (input)
side of the capillary or using reduced pressure on the detection (output) side of the capillary.
The pressure difference can be accomplished with compressed gas, vacuum pump or by
elevation of the injection vial relatively to the output vial.
Electrokinetic injection is accomplished by the application of an injection voltage for a
short period of time (usually a few seconds). The injected amount of analytes, during the
electrokinetic injection, depends on the EOF as well as on the electromigration of each analyte.
Thus, this type of injection is discriminating: it prefers the ions with higher mobility. If the
conductivity of the sample solution is lower than that of the BGE, the analyte ions are pre-
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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concentrated at the front of the sample plug (a phenomenon called sample stacking). Compared
to the hydrodynamic injection, the electrokinetic injection is usually less reproducible.
Detection
Detection is most often based on absorbance or fluorescence of analyte zones flowing
through the detection window of the capillary. Laser-induced fluorescence (LIF) benefits from
the superior properties of laser light; especially the directional characteristics of the laser beam,
which makes it compatible with on-column capillary detection. The narrow laser beam is
characterized by minimal divergence and hence it can be focused between the inner walls of the
capillary (the width of the focused laser beam is in the range of units or tens of m). Hence, due
to nearly 100% utilization of laser power, even lasers with relative low output (~ a few mW),
can be used for efficient excitation. One limitation of laser applications is the lack of
inexpensive lasers with tunable wavelength. Usually, the only possibility is to select specific
lasers with a discrete wavelength suitable for target application. Common lasers applied in CE
or HPLC and their wavelengths are listed in the following table:
Laser Wavelength (nm)
HeNe 543, 632
Ar+ 488, 514, UV
Diode 400 – 1000
A disadvantage of fluorescence or absorbance detection is that not all analytes contain
chromophores with sufficient fluorescence or absorbance in the common UV-VIS region (180-
800 nm). For these analytes, direct detection is not possible. Other options for such analytes
include derivatization of the analytes with a label containing a suitable chromophore, use of
another detection method (refractometry, amperometry, conductance, MS) or operation in the
indirect detection mode. A disadvantage with the use of fluorescent labeling of proteins is that it
is cumbersome to achieve a reproducible and uniform labeling of the proteins.
C. Instrumentation – setup (Fig. 3)
The instrument is equipped with a DPSS Nd:YAG laser (diode pumped solid state
neodymium-doped yttrium aluminum garnet, 1064 nm) with a frequency doubler emitting light
with the wavelength 532 nm. The laser beam is focused with a quartz lens (with the focal length
of 10 mm) into the center of the capillary held in an adjustable clamp positioned on micrometric
xy stage. Fluorescent radiation is collected with a microscope objective (60×) and directed
through an optical slit (spatial light filtration) towards a photomultiplier tube (PMT). Two filters
are inserted in front of the PMT to assure optical filtration.
Note: If you remove the shielding tube placed between the microscopic objective and
PMT and place a piece of white paper in front of the optical slit, four interfaces (air/outer
capillary wall, inner capillary wall/liquid, liquid/inner capillary wall and outer capillary
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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wall/air) should be visible. The slit should be positioned between the second and the third
interface to transmit the (orange) fluorescence and block scattered (green) light.
The entire optical setup is covered with a light-protective cover (shield). The carousel
with injection and BGE vials and the high voltage electrodes is placed in a protective plastic
cover equipped with a safety switch, which turns off the HV if the protective cover is removed
to protect operators from electrical shocks. The electrical current from the PMT is digitized
with a 16-bit A/D (Analog/Digital) converter in a personal computer. The acquired data (PMT
current as a function of time) are recorded and stored into a file in the ASCII format with a
program written in LabVIEW environment. A simple spreadsheet editor (MS Excel) can be
used for data processing and graphical presentation of the resulting electrophoregrams.
Reaching the lowest detection limits is based on efficient excitation, as well as sensitive
detection and noise reduction. Efficient excitation is accomplished only with proper focusing of
the laser into the center of the capillary. In addition, sensitive detection is based on utilization of
an efficient collector of emitted fluorescent radiation and a detector with great gain (i.e. the
PMT). There is a variety of noise sources, which require application of a range of tools to
suppress noise:
a) scattering of the laser light
- insertion of optical filters and slits in front of the PMT
- insertion of light protective shields into the optical part of the instrument
- black, non-fluorescent painting of the box, shields, and all holders of the optics
b) penetration of daylight into the optical compartment of the instrument
- covering the optical setup with the light protective cover (shield)
- blocking of all optical gaps, openings and slits presented in the cover
c) electrical interference, induction of signal generated with electrical power network
- application of an RC filter for suppression of noise with frequency above ~10 Hz
- integration of signal for the time corresponding to any multiple of the electrical
power line period 20 ms (50 Hz) – proper integration period in the LabVIEW
program.
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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CE
An unmodified fused silica capillary with the effective length of 30 cm and the total
length of 37 cm is inserted into the CE LIF instrument. The inner diameter of the capillary (i.d.)
is 50 m and the outer diameter (o.d.) is 360 m.
Figure 3. Scheme of CELIF instrumentation.
Detector side
vial
Laser
Microscopic objective
on shifting clamp
Slit
-metric
table with
capillary
clamp
High
voltage
supply
Injection vial in
carousel
Shielding cover
Capillary
PhotomultiplierLens
Filters
Shield
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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Figure 4. Description of the CELIF instrument
1 High voltage display
2 Electrical current display
3 High voltage knob (local)
4 Electrical current limiter knob (set approximately on 25 %)
5 High voltage knob for PMT (set on value “650 pcs”)
6 Local/Remote switch (pushed = local/pulled = remote)
7 High voltage switch (local)
8 PMT high voltage switch (local)
9 Laser switch (local)
10 Power (main) switch
11 Carousel with injection vials
12 Protective cover of the carousel
13 Detection side vial
14 Main protective cover (shield)
1 2
3 4 5
6 7 8 9 10
11
13
12
14
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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CE LIF program for instrument control and data recording
The CE LIF program was created in LabVIEW development system. The program is
designed for control of the CE LIF instrument and data acquisition using a 16-bit A/D converter.
Using the program, experiments can be performed in manual control mode (local) or in
automatic control mode (remote). You will operate the CE LIF instrument only in the automatic
control mode (remote), which designed for common use. In the remote mode, the laser, the
PMT, high voltage (for injection and analysis) and the maximum time of the analysis are
controlled by the program. Open the CE LIF program by clicking on the icon “CELIF latest
version” on the desktop of the computer. Before beginning an experiment, enter a name of the
output file (with “.txt” file extension) including directory, total time of analysis (typically 15
minutes) and sampling frequency (typically 4 Hz, maximum is 10 Hz).
Warning: Make sure that the directory exists, or create a new one if it does not exist. (If
the directory does not exist or the entered path is wrong, no data are stored during such
experiment.)
Then, enter the time and the voltage for injection and value for the separation voltage.
Before you run the program, it is advised to clear the display from previously recorded data
(move the cursor of the mouse to the display, click on the right button of the mouse and select
“Data operations » Clear chart” command in the menu).
After entering all parameters of the experiment you can start the CELIF program with
the white arrow (“Run”) placed in the upper left corner of the program’s window. The laser and
the PMT are controlled with the appropriate buttons in the lower left bottom corner of the
program’s window. Injection voltage is switched on by clicking on the button “Injection”, and
the injection voltage is switched off automatically after the entered period of the injection. High
voltage and data recording during the separation are started with the “Start” button; separation is
ended after the entered max. time or manually using the “Stop” button.
WARNING
You may switch on high voltage (HV) only with the plastic protective cover placed over the
sampling carousel with samples!
You may switch on the PMT only with the main protective cover
in place!
During operation with laser beam do not look straight into the laser! Be careful about
reflections
of the laser beam from steel clamps, watch, jewelry, etc.
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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Experimental part
A. Instrumental optimization.
Determination of limit of detection (LOD) of rhodamine 6G.
Before separation experiments, it is necessary to verify the alignment of the CELIF
instrument and check its performance. In this task you should become familiar with the CELIF
instrumentation and verify a proper setup by determination of the LOD for rhodamine 6G.
1. Preparation of solutions
Prepare 100 ml background electrolyte buffer (BGE), 50 mM citric acid in 10 % (v/v)
ethanol (EtOH) and titrate it to pH = 2.5 with a solution of concentrated ammonia.
Fill 2 vials with BGE buffer for the CELIF instrument. Prepare a set of vials with the
calibration samples of rhodamine 6G. Concentrations of samples should be 10-8
M, 10-9
M, 10-
10
M and 10-11
M in BGE buffer.
The stock solution of rhodamine 6G is 2 mM. Dilute the samples gradually in several
steps (e.g. 10x, 100x, 1000x) in small final volumes (100-1000 μl). To improve precision of
pipetting, do not pipette less than 1 μl. Use the small (200 μl) microvials for CELIF samples .
The volume of each sample should not be lower than 150 μl.
Remove air from the BGE and sample solutions in an ultrasonic bath (for 3 minutes).
2. Verification of the proper alignment and function of the CELIF instrument with
determination of the limit of detection (LOD) of rhodamine 6G
Filling of the capillary
Fill the capillary with BGE using the injection syringe with adapter and assure that no
bubbles get into the capillary. Fill from the detection end of the capillary and press gently at
least 3 drops of BGE through the capillary into a small tissue held close to the injection end of
the capillary. Elevate the vial with BGE on the injection side (in carousel) and continue pressing
BGE through the capillary for a few seconds. Quickly but carefully remove the syringe from the
capillary on the detection side and replace it with a vial of BGE. Place the plastic protective
cover on the sampling carousel. Start the CELIF program and set the high voltage to 15 kV.
Run a blank analysis with no injection and observe the progress for a few minutes. If arcing
occurs in the sampling carousel (it produces sharp snappy sound) it is necessary to interrupt the
experiment and clean up the carousel space. If everything is in order, record the value of the
electrical current (in μA) and observe it for ~30 s. In case that the value of the current falls more
than about 10 %, it is necessary to fill the capillary with BGE again. The next step is to observe
the properties of the detection system (laser, PMT). End previous experiment and switch on the
laser and the PMT (do not change the value set on the PMT knob) using the CELIF program.
Record the PMT signal using the CELIF program with the high voltage set to 15 kV. Specify
variation of the PMT signal – from minimum to maximum values of the signal during the blank
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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experiment (without injection of a sample). The optimal value of the variation, from min. to
max., is about 10.
Injection of sample and analysis of Rhodamine 6G
After the capillary is filled with BGE properly, the first sample may be injected. In the
window of the CELIF program write down a name of the output file, the value of the voltage
during separation U = 15 kV, the time of analysis (15 minutes) and the sampling frequency (4
Hz). With the HV switched off, replace the vial of BGE with the vial containing rhodamine 6G
at the lowest concentration (10-10
M) using the sampling carousel (push down, turn and pull up).
Inject the sample into the capillary at 5 kV for 10 s using the CELIF program. Replace the vial
of the sample by the vial of BGE. Then, immediately click on the start button in the CELIF
program and start the analysis. Record the value of the electrical current at start of the analysis
and monitor its value during the entire experiment and check that no significant change occurs
during the experiment. Repeat this experiment with the other rhodamine 6G concentrations in
the set (10-8
M, 10-9
M and 10-10
M) and do not forget entering a new file name for every new
experiment. After you finish all experiments, fill both vials and the capillary with fresh BGE
buffer solution.
Evaluation of results
Process the acquired data using the MS Excel program. Import the ASCII data into a
sheet and construct a graph with all recorded electrophoregrams. In a table, show migration
time, peak height, peak width at half maximum (FWHM) and number of theoretical plates for all
concentrations of the analyte in the set. Construct calibration graphs, i.e. linear function of peak
height and peak area vs. analyte concentration. Estimate a concentration limit of detection
(mol.l-1
):
H
cs
LOD


3
where H is the height of the peak of rhodamine 6G, c is the rhodamine 6G concentration
and s is the standard deviation of the noise signal intensity (use at least 20 values for its
calculation).
0
2
4
6
8
10
12
14
0 10 20 30 40 50 60
the interval
for noise calculation
H
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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3. Questions
1. Two optical filters, selected from the set of filters with transmission spectra shown in Fig.
5a, are placed in front of PMT. The emission spectrum of rhodamine 6G is in Fig. 5b.
Which two filters would you select? Why?
Figure 5a. Transmission spectra of selected optical filters
Figure 5b. Emission spectrum of rhodamine 6G
2. A non-zero value of signal–to-noise could be observed even in the case only water or BGE
was present in the capillary. Explain this observation.
3. Explain the “peak tailing” in the electrophoregrams of rhodamine 6G. How was the peak
tailing suppressed during the experiments? Describe other ways of suppressing peak tailing
in CE.
4. Compare the calculated values of number of theoretical plates in experiments with various
concentrations of rhodamine 6G. Explain the differences.
5. Increased diffusion of the analyte zones is frequently observed at Joule’s heat higher than 1
W per meter of capillary. What power (in the form of Joule’s heat) was produced in the
capillary during your CELIF experiment? Could production of heat in your experiment
cause zone (peak) broadening? Suggest ways of decreasing excessive capillary heating.
6. It is possible that some experiments did not provide results in accordance with your
expectation. Describe the difference from your assumption and try to explain it.
7. Calculate velocity and effective ion mobility of the rhodamine 6G zone during the
separation.
0
20
40
60
80
100
200 300 400 500 600 700 800 900 1000
l (nm)
T(%)
F3
F1
F2
532 nm
0
0,2
0,4
0,6
0,8
1
1,2
200 300 400 500 600 700 800 900 1000
λ (nm)
If(a.u)
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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B. Separation of rhodamine dyes
Flush the capillary with BGE solution for at least 5 minutes and check the current
stability at a voltage of U = 15 kV.
Prepare a sample mixture of rhodamine 6G, rhodamine 123 and rhodamine B with
concentrations of 1 nM, 100 nM and 10 nM, respectively. Use the BGE for sample dilution.
Stock solutions of the rhodamine-based dyes were prepared with dissolution of the dyes
(Sigma-Aldrich) in mixed water/ethanol solvent and concentrations of the dyes in the stock
solutions are:
rhodamine 6G 2 mM
rhodamine 123 300 μM
rhodamine B 1 mM
Figure 6: Structure of the rhodamine dyes
Inject the sample electrokinetically at 5 kV applied for 10 s. Use the BGE: 50 mM
solution of citric acid in 10 % EtOH (v/v) and add concentrated ammonia solution to adjust pH =
2.5. Perform electrophoretic separation at 15 kV. Using MS Excel program construct an
electrophoregram of the separation. After you finish all experiments, fill both the injection and
detector vials as well as the capillary with fresh BGE solution.
O N CH2CH3NHCH2CH3
C O
O
CH2
CH3
CH3
CH3
rhodamin 6G
O N
+ CH2
CH3
CH2
CH3
N
CH2CH3
CH2
CH3
C
OH
O
rhodamin B
O NH2
+
NH2
C O
O
CH3
CH3
CH3
rhodamin 123
Manual to the Advanced Laboratory Course on Analytical Chemistry – CE LIF
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Questions
1. Compare migration times of peaks in this experiment with the migration time of the
rhodamine 6G peak in Experiment A. Which peak in this experiment corresponds to
rhodamine 6G?
2. How many peaks can you observe in the electrophoregram? Is this number different from
your hypothesis? In case it is different provide an explanation.
3. You know the size of all dyes (from its structural formula) and the charge of the dyes at pH ~
2.5. Determine which peaks correspond to rhodamine B and rhodamine 123.
4. Write the migration times, calculated effective migration times and number of theoretical
plates of each analyte into a table.
5. Suggest options to improve separation efficiency (number of theoretical plates).
6. According to the calibration graph obtained in Experiment A calculate the concentration of
rhodamine 6G in the mixed sample and compare it with the expected value.
C. Laboratory report
In laboratory reports, write down the date, all names of participants, and titles of the
experiments. One report for the entire group is sufficient, preferably in electronic format (Word
document). Copy neither the principle nor the instructions from this manual. On the other hand,
write down all experimental conditions (sample preparations, dilutions, injections, capillary
pretreatment, electrical current in capillary, etc.), if your experiments would be reproduced.
Present your results in a clear and well-arranged form (inserted Excel graphs and tables).
Answer all questions stated at the end of every task (experiment).
Finally, review critically the experiments you made. Discuss reproducibility and
sensitivity of the method and importance of the method for practice. We appreciate all
comments and proposals leading to improvement of this laboratory course.