A
Critical Review of Capillary Zone Electrophoresis

 

Introduction  

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Arnes Tiselius showed electrophoresis is capable of separating
proteins in a free solution. The use of a capillary had resolved many of the
disadvantages in electrophoresis. The thin walls of the capillary increased the
surface to volume ratio, this prevented the high voltages from overheating the
electrophoresis environment.1

 

Capillary electrophoresis is an applicable technique for the
quantification and separation of species that are charged. Capillary
electrophoresis only requires a small amount of sample, the method also has the
ability to separate the samples at a fast paste with great efficiency and a
large resolution.2 It is also easy to find experimental conditions that are
sufficient for the separation of ions in a sample.3   

 

The main principle behind capillary electrophoresis is that the
components of a mixture are separated via an electrical field in a confined
narrow tube of a capillary. 4 The electrophoretic mobility of an ion is
dependent upon several factors such as the atomic radius, the overall charge of
the molecule and the viscosity. The particles move at a rate which is
equivalent to the applied electric field. The particles will move faster if the
field strength increases. Only ions with a charge will move, neutral species do
not move.4   

 

Capillary Zone Electrophoresis is a most commonly used technique
for CE. A variety of compounds can be separated efficiently and at a fast rate.
Separation is dependent on electrophoretic mobility. This is corresponding to
the molecules charge and inversely dependent to the radius of the atom and the solvent
viscosity. In CZE there are fused silica capillaries that contain groups of silanol
which are ionised inside the buffer. A stationary and cation layer is created
from the negatively charged silanol ions which attract the cations with a positive
charge. The cation layer moves towards the cathode which has a negative charge.
This creates an electrophoretic flow. Cations that have the greatest charge and
mass proportions are separated first, the cations with the smaller proportions,
species with no charge, anions with small charge to mass proportions and lastly
anions with larger proportions. The rate of the electroosmotic flow can be
controlled by changing the voltage, ionic strength, pH, and thickness of the buffer.4
   

 

A capillary electrophoresis instrumental system consists of a
power supply that is of a high-voltage, a thin capillary tube, a sample
introduction system, an output device and a detector. A few instruments have a
device to control the temperature to make sure the results are reproducible. This
is because as the temperature of the column increases, the electrophoretic
mobility and the viscosity of the solution, which the separation is dependent
on will decrease. An electrode is attached to both sides of the power supply.
The electrodes help the movement of the sample from the anode to the cathode in
the capillary tube by helping to induce the electric field. The capillary is
made out of fused silica as well as occasionally being coated with polyamide.
Both sides of the capillary are placed in a glass tube consisting of the buffer
solution as well as the electrode. The capillary tube must first be washed with
the buffer solution before the sample is run through the column. Most commonly
there is a small window on the cathode side of the capillary to allow for
UV-VISIBLE light to pass through to titre the analyte to measure the analytes
absorbance. The mass to charge ratio of the analyte is also calculated by a
mass spectrum which is constructed by the addition of a photomultiplier tube at
the cathode side of the capillary.4

 

Evaluation
of CZE

 

Quantitative CZE Capillary electrophoresis was compared against
HPLC for the analysis of herbal tea phenolics. Rooibos and honey bush were the
types of herbal tea for analysis. The quantification of phenolics in herbal
teas are most commonly performed by HPLC. CE is a better alternative to HPLC in
terms of operating costs, efficiency and speed. CE is cost effective, optimal
separation of the honey bush and rooibos phenolics was achieved in 21 and 32min
there was also good linearity and repeatability. However, CE is less strong and
less sensitive then HPLC analysis.5

 

Sweeping Capillary Electrophoresis method and cation-selective
exhaustive injection was used for the analysis of chlorpheniramine enantiomers
in rat plasma. Factors affecting the separation and improvement of the
efficiency were explored. A validation was carried out and the method has a
high sensitivity the LOQ was 0.025µ/ml. The RSD was within 8.4%. The method had
an accuracy which was acceptable between 89.2% and 95.0%. The recoveries of the
extraction for the two enantiomers were both greater 72.5%.6

This can be compared to another study that looks at the
stereoselective determination of cetirizine and the studies of the
pharmakinetics in rat plasma. This study used HPLC for the determination of
cetirizine enantiomers in rat plasma. A validation was carried out. The ideal
extraction efficiency was at a pH of 5 and ethylicetate was the solvent used.
Extraction recovery of (+)-cetirizine was 73.2 and (-)-cetrizine was 71.2.7  

 

A study used CZE capillary electrophoresis with a capacitively
coupled contactless conductivity detector for a fast determination of the
components in a pharmaceutical formulation. A fused silica capillary was used
for the separation as well as, an electrolyte for the background. All of the
samples were analysed in one run which was either 2 minutes or less. The
analytes that were analysed were; scopolamine, tramadol, orphenadrine,
promethazine, codeine and paracetamol. The limit of detection for these were
2.5, 0.62, 0.63, 2.5, 1.5 and 1.6µmol/L respectively. A spike and recovery test
was carried out and the recovery values were between 94% and 104%. CE is very
good for analysing pharmaceutical compounds, CE has also been developed to be
used as a coupled contactless conductivity detection. This enabled CE to be
inexpensive, have a quick analysis time and a good level of separation
efficiency. The method also uses a low utilisation of samples and reagents,
making it suitable for the environment therefore it can be a greener analytical
method.8  

This can be compared to a study that used HPLC with a diode array
detection for the examination of caffeine anhydrous, nimesulide, phenylephrine
hydrochloride, cetirizine dihydrochloride and paracetamol in a pharmaceutical
formulation. A kinetex-c18 column that had a gradient elution mobile phase was
made out of a 10mM phosphate buffer of pH 3.3 and acetonitrile. The gradient
elution consisted of three steps. The first step was started with a pH 3.3, 98%
phosphate buffer and 2% acetonitrile. In step 2 until 12min had passed by the
concentration of acetonitrile linearity adapted to 20%. In step 3 the analysis
was finished and the acetonitrile was changed to 2% until 20min. A validation
was then carried out, there were good values of recovery as well as the
excipients not causing any interfering peaks. The correlation coefficients for
all the analytes were greater than 0.9996. The analyses all had linear
calibration curves within the ranges of 5-100, 100-1000, and 10-200mg/ml. A
diode array detector was used to measure the selectivity as well as the
linearity of the method by using a peak purity test. The peaks for the analytes
were not caused by any other analyses making the technique very selectable. The
%RSD values were measured in an intra/inter day assay, indicating that the data
are precise. There was a ±2% acceptance limit. The robustness of the method was
also evaluated, the detection wavelength was changed by ±5 nm, the pH  of the mobile phase was changed by ±0.1% and
the flow rate was changed by ±10%. The method had good robustness.9           

 

MEKC-DAD has been used for the determination of phenolic acids and
flavonoids in tomatoes. HPLC with a UV-visible detection system is usually used
for this or LC-MS. Alternatively CZE or MEKC with a UV detector can be used.
MEKC is able to separate mixtures containing both neutral and ionic analytes.
CZE is unable to do that therefore, this is an advantage for MEKC. A pseudo
stationary phase is created from micelles of a surfactant in MEKC. This allows
the separation of molecules which are neutral because of the various partition
coefficients bounded by the aqueous phase and micellelar phase. MEKC is most
commonly conducted in a non-coated fuzed silica capillaries and sodium dodecyl
is the most commonly used surfactant. Therefore, hydrophobic and solute-micelle
electrostatic interactions, are able to change the differentiation separation
charged of charged solutes via the ionic micelles. With CZE it is difficult to
separate flavonoids therefore the simultaneous separation of flavonoids and
phenolic acids was carrIed out by MEKC. In order for the optimum separation
conditions to be obtained, response surface methodology was used to optimise
the background electrolyte composition. A validation was carried out all of the
polyphenols had excellent lineal models of regression with the r2 values being
greater than 0.995. The % RSD of peak areas and migration times were calculated
as intra and interlay levels. The peak areas had a precision from 1.7 to 3.1%
and the standards had 1.8 to 3.7%. The spiked samples ranged from 0.2 to 0.4%
also 0.1-4.4%. LODs were between 0.8 and 3.8mgkg-1 fw. The recoveries for the
spiked tomatoes were between 77% to 106% for the 12.5mgkg-1 fortication level
and was 77% to 103% for the 50mgkg-  high
level.10

This can be compared to a study that analysed phenolic compounds
using UHPLC coupled to a DAD/ES/-AM-mS. A validation was carried out, r2 values
greater than 0.9990 for all analytes. The LOD’s were between 200 and 900ng g-1
dw and the LOQ were between 400 to 1800ng g-1. A spike and recovery test was
carried out, there were 5 replicates for the higher spiked level and values
ranged from 74% to 100%. The values were 72% and 99% for the values that were
lower. The intermediate precision was less than 11% and the repeatability was
less than 10%.11   

 

A study used CZE method for the examination of kanamycin with a UV
detector. This was compared to LC-Pulsed electrochemical detection. CZE with an
emperometric has been used previously to detect antibiotics but was not as
sensitive. LC-PED separated paramamine and 4-0-(6-AG)DS whilst CZE had
increased selectivity in separating paromamine and 6-0-(3-A-G)DS. This is
because 4-0-(6-AG)DS and paromamine are positional isomers which make them act
the same in the EOF environments. CZE is more convenient compared to LC as LC
contains mobile phases that consist of two or more substances, therefore the
analysis time will be longer. It was 45min for LC and only 15 min for CZE,
making CZE fit for purpose. In the LC-PED method the detector is not very
stable making the UV detector used in CZE more stable and convenient. 12    

 

State of
the Art 

 

There are many types of detectors that can be coupled to CE. These
include DAD, chemiluminescence detectors, UVD’s, laser-induced fluorescence
detectors and mass spectrometry. Silva et al formulated a CE-UVD method for
folic acid as well as vitamins that are water soluble in supplements in food.
The detection limit was 0.28mg.l-1. Due to the small sample volume of CE the
sensitivity of CE is not very good. Therefore, a laser induced fluorescence
detector was utilised for tracing small amounts of folic acid. This will
overcome the low sensitivity limitation. The LIF detector is much more
sensitive than the UVD. Zhao et al. used a CE-LIF method for the identification
of three vitamins and three amino acids. Folic acid was between 0.005 to
0.80µmoll-1 for the linear range. There was a detection limit of 5mmol L-1.
Chemiluminescence (CLD’s) do not need a large amount of light like the above
examples as they are one of the more sensitive detectors. Zhao et al, developed
a CE-CLD method for identification of folic acid in urine, tablets and apple
juice. The method had a LOD of 2.0×10-8 mol L-1 as well as having recoveries
between 94.3 and 105.8%. Therefore, using a CLD detector will increase the
overall sensitivity of the method. Overall CE has advantages of being
inexpensive, high speed and high resolution. On the other hand, its
disadvantages are that it is not very sensitive and has poor reproducibility. 13  

 

With the separation of proteins with CE, it is very important to
have the correct coatings for the column, this will improve the efficiency for
the separation. This is so that the adsorption of proteins on the inner
capillary wall can be reduced and so that the electroosmotic flow (EOF) can be
changed to increase the small variations in EOF. It has crucial that the
capillary coating can withstand high pH’s and any interferences from the
detector. These factors can be applied to permanent coatings but dynamic
coatings may have interference with the detection. There have been developments
in capillary coatings being coated with nanoparticles in order to intensify
reproducibility, separation efficiency and selectivity. Capillary columns filed
with nanoparticles have a large potential in capillary electro chromatography.
This is because of their variety of surface chemistry options and their great
surface to volume ratio. Coupling reagents that are covalent are becoming
increasingly common as they are very gentle on the environment. Capillaries
that are fully coated for analysis of proteins are gaining popularity with
commercial instrumental CE. 14