Student Notes:

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Student Notes --Fused silica is the most popular
capillary material. It is chemically and
electrically Inert, flexible,robust and
inexpensive. Teflon is also utilized, but it is
difficult to obtain with homogenous diameters,
exhibit sample adsorption problems and has poor
heat transfer.
--Fused silica columns with internal diameters
(i.d) ranging from 10-200 mm with a range of
outer diameters (o.d) are available. 25-75 mm
i.d and 350-400 mm o.d are typical. Effective
lengths range from as short as 10 cm for
gel-filled capillaries to 80-100 cm for complex
samples.Most commonly, 50-75 cm effective length
are used.
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In a typical CE experimental procedure the
following steps are needed
a) Flush cycle Capillary is flushed with 1N
NaOH and/or 0.1 N NaOH for 30-60 min
b) Buffer cycle Capillary is flushed with
running buffer (e.g., 25-50 mM NaH2PO4 (pH
2-4), Na2HPO4 (pH 6-8), Na2B4O7 (pH 8-10) and so
on.
c) Sample Cycle Removal of the buffer vial
with the a sample vial and loading the sample
on the capillary using either pressure, vacuum or
voltage
d) Run Cycle Application of the separation
voltage (1-30 kV)
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Student Notes --One of the most important factor
leading to good reproducibility is
capillary conditioning. Maintaining a
reproducible capillary surface is important.
The most reproducible conditions are encountered
when no conditioning other than with buffer is
employed. However, adsorption of sample to the
surface and changes in EOF often do not allow
this.
--Base conditioning to remove adsorbates and
refresh the surface by deprotonation of the
silanol groups is most commonly employed. A
typical wash method includes flushing a new
capillary with IN NaOH and then buffer. Before
each analysis, only the last two steps are
performed.
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Role of Capillary Thermostating
Student Notes Effective control of capillary
temperature is important for reproducible
operation. Temperature regulation to or -0.1 0C
is necessary due to the strong viscosity
dependence of sample injection and migration
time. As mentioned earlier, the two approaches
generally used are to bathe the capillary in a
(i) high velocity air stream, or (ii) in a liquid.
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High Voltage Power Supply
Up to 30 kV and current of 200-300 mamps Should
have polarity switching ability
Student Notes --In CE a DC power supply is used
to apply voltage upto about 30 kV and
current levels of 200-300 mA. Stable regulation
of voltage is required to maintain high migration
time reproducibility. The power supply shoould
have the capability to switch polarity.
---Under normal conditions, the EOF is in the
direction of the cathode. In this case, the
injection is made at the anode. If the EOF is
reduced, reversed, or if gels are used it may be
necessary to reverse the polarity of the
electrodes that is to switch the cathode to the
injection end. Polarity switching must be
performed at the power supply.
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Student Notes --In CE (i.e., CZE) selectivity
can most readily be altered through changes
in running buffer pH or through the use of buffer
additives such as surfactants or chiral selectors
are especially useful. These buffer ions are
generally large and can be used in high
concentrations without generating significant
currents.
--A potential disadvantage of these buffers is
their strong UV absorbance. Matching the buffer
ion mobility to the solute mobility is important
for minimizing peak shape distortions. Buffer
ions can also be used to complex with solutes
and alter selectivity. Tetraborate is used to
improve the separation of catechols and
carbohydrates.
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Commonly used buffers
Student Notes The sensitivity of EOF to pH
requires the use of buffers that maintain
constant pH values. Effective buffer systems have
a range of approximately two pH units centered
around the pKa.
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Buffer Replenishment for Improved Migration Time
Reproducibility
Student Notes Buffer replinshment improves
migration time reproducibility. Replenish the
buffer after every 3-5 runs. The number of runs
between replinshment is dependent upon the vial
volume, buffer capacity, and the current
generated.
--If using indirect detection buffers, consider
buffer replenishment between every run. These
buffers have a low buffer capacity.
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Reproducibility of rhGH Peptide Map
Student Notes The rhGH peptides map shown above
is an excellent example of CE reproducibility.
The capillary conditioning was done for 3 min
with 0.1N phosphoric acid, then for 10 minutes
with buffer. The buffer replinshment was
performed for every run. Note also that the
current was 135 mA. This value is higher than
recommended, but it works.
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Surfactants as Buffer Additives
Surfactants are among the most widely used buffer
additives in CE. Anionic, cationic,
zwitterionic, or non-ionic surfacntants can be
used. At concentration below the critical
micelle concentration (CMC) monomer surfactant
molecules act as a solubilizing agents for
hydrophobic solutes, or as ion-paring reagents
for hydrophilic solutes or simply as wall
modifiers. In addition, many surfactants
adsorb to the capillary wall, modifying EOF and
also limiting potential solute adsorption.
Depending on the surfactant charge and CMC, EOF
can be increased or reversed. EOF increased can
occure with anionic surfactants. On the other
hand cationic surfactants when used above CMC can
reversed EOF (e.g., CTAB)
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Bonded or Adhered Phases Type Comment Silylati
on Coupling -Numerous usable functional
groups (Si-O-Si-R) -Simple to prepare, -Stable
pH 4-7 -Limited long term stability
Direct Si-C coupling -Si-C bond eliminates need
for silyation, -pH 2-4, -Difficult to
prepare
Adsorbed polymers -Poor long term
stability Cellulose, PEG, PVA -pH 2-4,
Relatively hydrophobic
Adsorbed Crosslinked -Reverse EOF Polyethylenimin
e -Useful for basic proteins,
stable at pH 7
GC phases -Hydrolytically unstable PEG,
Phenylmethylsilicones
LC phases -Can increase protein adsorption
A number of permanent wall modifications are
described. Notably silation is followed by
deactivation with a suitable functional group.
Deactivation can be accomplished with varied
species such as polyacrylamide, arylpentafluoryl
groups or polysaccharides. Depending on the
deactivation EOF can be eliminated or reversed.
Deactivation with polyacrylamide or
polyethyleneglycol eliminates EOF. Deactivation
with cationic groups reverses the EOF
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Student Notes From basic theory, it is expected
that Macromolecules such as proteins Would
Yield very high efficiencies due to their Low
diffusion coefficients. It has been found
however, specificially for proteins, that
interaction with the capillary surface generally
reduces the efficiency.These interactions can be
ionic and or hydrophobic in nature
Capillary Wall Modification
with
Capillary wall modification is an Alternative to
limit solute adsorption, Two fundamental
approaches have Been taken (1) permanent
modification by covalently bonded or
physically adhered phases, and (2) dynamic
deactivation using running buffer additives.
without
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CYCLODEXTRINS SELECTORS AS BUFFER ADDITIVES
Chiral analysis by CZE usually involves the
additon of a chiral selector to the buffer. These
selectors can be cyclodextrins, crown ethers,
bile salts, or copp(II) aspartate Complex
Selectivity can be tuned by adjusting the type
and concentration of chiral additive and also may
be due to the addition of modifiers.
Cylcodextrinss are the most widely used chiral
selector.CDs are nonioni cyclic oligosaccharides
consisiting of six,Seven, eight glucose units.
CD has a shape of hollow truncated cone with a
cavity Diameter determined by the number of
glucose units.The cavity is relatively
hydrophobic. While the external surface is
hydrophilic. The cicumference contain secondary
hydroxyl grou, Chiral selectivity results from
the inclusion of a hydrophobic portion of the
solute in the cavity and also from H-bonding to
the chiral hydroxyl moities
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Dynamic Deactivation Methods
--Addition of modifiers to the running buffer is
an alternative to the bonded or adhered phases.
An advantage of dynamic coatings is stability.
Since the modifier is in the buffer, the coating
is continously regenerated and permanent
stability is not required. As with covalent
coatings, additives can interact with the wall
and alter charge and hydrophobicity. These
modifiers are both easier to implement and
optimize because they are prepared by simple
dissolution of the modifier in the buffer.
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Poor Peak Shape Use of Coated Capillaries
Student Notes Often, samples can interact with
the capillary wall resulting in a loss of
efficiency and resolution. Commercially
available coated capillaries such as the PVA
coated capillary shown here can eliminate
adsorption and tailing. Some commercially
available coated capillaries include PVA from
HP, BioRad linear polyacrylamide, Blackman
neutral coating, HP-CEP capillary.
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Effect of Ionic Strength (Case of 50 mm i.d
capillary)
Student Notes Increasing ionic strength of the
buffer decreases EOF (increase migration time).
More concentrated buffers generate more heat due
to greater conductivity when voltage is applied.

Increasing ionic strength also affects the
viscosity of the medium. The viscosity depends
on temperature and so there is also dependence on
the capillary diameter.
With 50-um i.d capillary column, the migration
time lengthens as the buffer concentration is
increased. Because solute ions are always
surrounded by double layer of ions of opposite
charge, the migration of the ions (in this case
positively charged ions) are in direction
opposite to that of the solute.

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Hence, increasing the concentration of the buffer
reduces the mobility due to increase drag caused
by the countermigration of the more densely
packed counterions.

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Effect of Ionic Strength (Case of 75 mm i.d
capillary)
Using 75 mm i.d capillary, the solute migration
time first increase as expected but then they
decrease. This decrease is the consequence of the
significant effects of joule heating at high
buffer concentrations.
Note, as well the impact of buffer concentration
on peak width. Sharper peaks width at
higher concentration are due to stacking.
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Effect of Ionic Strength
--The highest mobility is obtained with the
smallest cation. However, high mobility may
decrease neighboring solute resolution so care
must be taken in chosing the cation. The buffer
anion may also affects the mobility of the EOF,
although the trends are less apparent.
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Effect of Temperature
As the temperature increases migration time
always decreases because of the reduced
viscosity of the BGE,which increases the EOF.
There is no significant change in resolution
and selectivity (in this particular example)
elevated temperature can be used to speed up the
separations (provided that the resolution is not
sacrificed).
When secondary equlibria is involved (e.g., in
chiral separations), subambient temperature are
often used to improve resolution and selectivity
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N mVl 2DL
Rs ¼ (N)1/2 Dm m
--The efficiency of separation is directly
proportional ot the capillary length provided
that the field strength is kept constant. The
limitation here is applied voltage. Most
instruments produce a max of 30 kV. The above
figure shows that once the capillary reaches a
certain length, no further gains in efficiency is
realized.
--Based on the equations derived the resolution
depends on the square root of the capillary
length. Increasing the capillary length beyond
the limits imposed by the maximum
voltage lengthens the separation time (due to low
field strength) without any substantial
improvement in resolution (since diffusion
occurs resulting in band broadening).
In the above figure N is linear with capillary
length until 30 kV is reached and the resolution
increase proportional to the square root of the
number of plates
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Tailing can result from a poorly cut
capillary due to injection related peak
dispersion. If the capillary is not cut squarely,
a concentration gradient can occur
upon injections which highlights the
importance of properly cut capillary.
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METHODS OF SAMPLE INJECTION
The two most common methods for CE are
hydrodynamic injection and electrokinetic
injection.
Hydrodynamic injection is the most widely used
method. Three different type of hydrodynamic
injection include (a) pressure, (b) vacuum, (c)
gravity.
Pressure Pressure injection involves application
of pressure at the injection end of the
capillary.
Vacuum Involves application of vacuum (negative
pressure) at the exit end of the capillary.
Gravity Involves siphoning action, which
is obtained by elevating the injection
reservoir relative to the exit reservoir.
With hydrodynamic injection, the quantity of
sample loaded is nearly independent of the sample
matrix.
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METHODS OF SAMPLE INJECTION (Contd)
Electrokinetic or electromigration injection is
performed by replacing the injection end
reservoir with the sample vial and applying the
voltage.
In electrokinetic injection, the analyte enters
the capillary by both migration and by pumping
action of the EOF.
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METHODS OF SAMPLE INJECTION (Contd)
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Effect of Injection size
Injection size can severly limit N values but can
improve sensitivity of detection.
A 100 nL injection represents about 10 of the
capillary volume. Compared with 5 nL injection
the limit of detection is improved by a factor of
13 (while 20-fold) was expected. For 10 nL
injection improvement was only 8-fold (10-fold
expected).
Clearly, the limit of detection can be improved
at the expense of electrophoretic resolution and
efficiency