Chemistry

Department

CHM 4373
Assignment topic
0
HPLC M.D & Application
Instrumentation of Capillary Electrophoresis

Instructor: Mam Sumaira Saleem

Group-3 Members
Usman Ghani-62 Sana Basheer-77 Section: CF3-17
Sajjad Ur Rehman-52 Kinza Lateef-74 Submission Date: 4th Feb
Mudassir Shabbir-73 Ifra Ramzan-76 Due Date: 4th Feb
Talha Fareed-75 Soha Israr-55
Faseehullah-70 Mohsina Irshad-69
 Electrophoresis:

Electrophoresis is one of the most important techniques for molecular separation because this
powerful technique is reasonably easy and inexpensive. Electrophoresis can be one-dimensional (1D)
meaning one plane of separation or two-dimensional (2D) meaning two planes of separation. One-
dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two-
dimensional separation of proteins is used for finger printing, proteomics studies, etc. Broadly, different
types of electrophoresis are categorized as follows:

I. Slab electrophoresis
II. Capillary electrophoresis

Capillary electrophoresis

Capillary electrophoresis is a more sensitive technique, it can detect samples as small as

few nL. Besides being more sensitive, this separation technique has high-speed and high
resolution. Instead of staining of the samples as in slab electrophoresis, there is one quantitative
1 Instrumentation of Capillary Electrophoresis
detector at the end of the capillary. The capillary can also be filled with a gel, which eliminates
the electroosmotic flow (vide infra). Thus, the separation is accomplished as in conventional gel
electrophoresis but the capillary allows higher resolution, greater sensitivity and on-line
detection.

Instrumentation:

A buffer filled fused silica capillary that is typically 10 to 100 µm in internal diameter
and 40 to 100 cm long, extends between two buffer reservoirs that also hold platinum
electrodes.

Sample introduction is performed at one end and the detection at the other. The polarity
of the high-voltage power supply can be as indicated in Fig. 1 or can be reversed to allow rapid
separation of anions. Although the instrumentation is conceptually simple, there are significant
experimental difficulties in the sample introduction and detection due to the very small volume
involved. Since the volume of a normal capillary is 4-5 µL, injection and detection volumes must
be of the order of a few nano litres or less.
 Fig 1. Schematic of Capillary Electrophoresis

Capillary Cross-Section:

The illustration below shows a cross-section through a typical capillary tube. Most capillary
2 Instrumentation of Capillary Electrophoresis
tubes are made from fused silica coated with a 15–35 μm layer of polyimide to give it
mechanical strength. The inner diameter is typically 25–75 μm—smaller than the internal
diameter of a capillary GC column—with an outer diameter of 200–375 μm.

Capillary intersection
 The capillary column’s narrow opening and the thickness of its walls are important. When an
electric field is applied to the buffer solution within the capillary, current flows through the
capillary. This current leads to the release of heat—what we call Joule heating. The amount of
heat released is proportional to the capillary’s radius and the magnitude of the electrical field.
Joule heating is a problem because it changes the buffer solution’s viscosity, with the solution at
the center of the capillary being less viscous than that near the capillary walls. Because a solute’s
electrophoretic mobility depends on viscosity, solute species in the center of the capillary
migrate at a faster rate than those near the capillary walls. The result is an additional source of
band broadening that degrades the separation. Capillaries with smaller inner diameters generate
less Joule heating, and capillaries with larger outer diameters are more effective at dissipating the
heat.

Flow in Silica Capillary:

When a high potential is applied across a capillary tube made of silica containing buffer solution,
the positively-charged ions migrate towards the negative electrode and carry solvent molecules
3 in the same direction. As shown in Fig. 2, the electric Instrumentation
double layer is of Capillary Electrophoresis
developed on the interface
of silica and solution. The surface of the silicate glass capillary contains negatively-charged
functional groups (formed due

Fig. 2: A charge distribution in capillary during electroosmotic flow

 to the presence of Si-O-H) and they attract positively charged counterions. This overall solvent
movement is called electroosmotic flow. During a separation, the uncharged molecules move at
the same velocity as the electroosmotic flow (with very little separation). The positively-charged
ions move faster and negatively-charged ions move slower.

Sample Introduction

The most common sample introduction methods are as follows:

• Electrokinetic injection
• Pressure injection

With electrokinetic injection, one end of the capillary and its electrodes are removed
from their buffer compartment and placed in a small cup containing the sample. A potential is
then applied for a period of time causing the sample to enter the capillary by a combination of
ionic migration and electroosmotic flow. The capillary end and electrode are then placed back
into the regular buffer solution for the duration of the separation. This injection technique
discriminates by injecting larger amounts of more mobile ions relative to the slower moving
4 Instrumentation of Capillary Electrophoresis
ions.

With pressure injection, the sample introduction end of the capillary is also placed
momentarily into a small cup containing the sample and a pressure difference is then used to
drive the sample solution into the capillary. The pressure difference can come from applying a
vacuum at the detector end by pressurizing the sample or by elevating the sample end. Pressure
injection does not discriminate due to ion mobility but cannot be used in gel field capillary. For
both the injection procedures, the volume injected is controlled by the duration of the injection.
Injections of 5-50 nL are common, and the injection of volumes below 100 pL has been reported.
For a buffer with density and viscosity near the values for water, a height differential of 5 cm for
10 s injects about 6 nL with a 75 µm inside diameter capillary. Microinjection tips constructed
from capillaries pulled down to very small diameters allow sampling from picolitres
environments such as single cells or substructures within single cells. This technique has been
employed to study amino acids and neurotransmitters from single cells.

Detection:
 There are several ways one can detect the samples after capillary electrophoresis. Examples are
absorbance methods, fluorescence methods, mass spectrometry, conductivity measurements,
potentiometry, amperometry, radiometry, electrochemical detection, etc.

Most common detectors are UV/Vis detectors. Whenever possible, detection is done “on-column”
before the solutes elute from the capillary tube and additional band broadening occurs.

Examples of UV/Vis detectors are shown here. Because absorbance is directly proportional to path
length, the capillary tubing’s small diameter leads to signals that are smaller than those obtained in
HPLC. The z-shaped system in (a) and the multiple reflections in (b) provide for a longer pathlength.

5 Instrumentation of Capillary Electrophoresis

Citation:
 Fundamentals of Analytical Chemistry 9th E by Donald M. West, F. James Holler, and
Stanley R. Crouch 

 Analytical Chemistry, 7th E by Gary D. Christian, Kevin A. Schug, and Purnendu Dasgupta

 Quantitative Chemical Analysis 10th E Daniel C. Harris Charles A. Lucy

  Modern Analytical Chemistry 1st E Book by David Harvey
 https://community.asdlib.org/imageandvideoexchangeforum/2013/08/04/uvvis-
detectors-for-capillary-electrophoresis/
 https://www.sciencedirect.com/science/article/abs/pii/S0003267016307784

6 Instrumentation of Capillary Electrophoresis