Basic Theory and Introduction
Separation Science has grown to become one of the most useful scientific tools. Used extensively in diagnostic and
clinical science, Separation Science is relied upon by scientists, physicians, law enforcement officials and the
general public to provide quantified information about our health, our food, our environment, our products we use
and in almost every aspect of our modern society.
Separation Scientists work in many different disciplines, some of these include:
Different samples and sample matrices present distinct challenges to the separation scientist and therefore there
are different techniques to separate and quantify and then qualify these samples. Smaller and smaller sample
availability presents further challenges to analysis.
- Analytical Chemistry
- Forensic Science
- Food Science
- Clinical Science
- Medical Research and Production
- Pharmaceutical and Nutraceutical Science
- Other Disciplines
Phase Partitioning and Extraction, Chromatography and Electrophoresis are the main techniques today used by
separation scientists. Each technique has its own place and value in the “tool box” of those presented
with the challenge of determining the quantity of each component of a sample.
Chromatography - High Selectivity
The tool that is most used by these scientists is Chromatography. The high selectivity of chromatography and
the large body of knowledge in this field makes it the first choice of separation scientists. The mechanisms of
separation makes it ideal for small molecules such as pharmaceuticals, chemical analysis, toxicological analysis and
many others. Simple automation, high reliability and robustness makes this technique highly coveted.
Electrophoresis Grows into Analytical
Electrophoresis uses a completely different mechanism of separation. This has many advantages to separation
scientists. Until the advent of capillary electrophoresis (CE) and capillary Electrochromatography (CEC),
electrophoresis could not be automated, was not precise enough for analysis of small molecules and was limited to
uses in proteins and other BioMolecules.
CE and CEC has changed that. Both techniques can be easily automated and are very precise (as precise as
HPLC). Robust and reliable, separation scientists now use electrophoresis as a technique for small and
Electrophoresis is a completely different mechanism of separation from chromatography. Should be used for the
What Is HPCE?
- Non Chromatographic. Provides different information. Should be used with Chromatography.
- Small sample size required. Provides analysis of samples not possible before.
- Low Cost. Cost of CE and CEC is realized in the purchase and disposal costs of solvents used in
- Ideal for Compounds that present costly and time consuming challenges in chromatography (proteins,
peptides, nucleic acids, basic drugs, chiral compounds)
- Easy to develop methods of analysis.
The term, “High Performance Capillary Electrophoresis”, HPCE* is an umbrella for many sub-techniques such
* The acronym “HPCE” (High Performance Capillary Electrophoresis) was appointed to Capillary
Electrophoresis and is somewhat misleading in that the term is very similar to HPLC. This term was given to
Capillary Electrophoresis by chromatographers but it is not similar to HPLC in the separation mechanism. The results
may or may not be the same as in HPLC and this should be considered valuable by the separation scientists when they
understand what CE is providing. More information about a sample can be gained.
- cIEF, Capillary Iso-Electric Focusing
- MEKC, Micellar Electro-Kinetic Chromatography
- CZE, Capillary Zone Electrophoresis (also known as “Free Zone” or CE)
- CGE, Capillary Gel Electrophoresis
- CITP, Capillary Isotachophoresis
- OTCEC, Open Tubular Capillary Electrochromatography
- CEC, Capillary Electro-Chromatography
Electrophoretic separations utilize the following terms compared to its “counterpart” in HPLC.|
EOF, Electro Osmotic Flow
Flow Rate (loosely related)
Basic Theory of Capillary Electrophoresis:
Electrophoresis gets its name from the process whereby the movement of ions is produced under the influence of an
applied voltage across a field that the ions exist. It is well known that opposites attract and in electrophoresis,
ions of opposite charge to electrodes on either end of the voltage, will migrate toward that electrode. Simply
stated, ions that are negatively charged will move or migrate toward the positively charged electrode and vice versa
for the positively charged ions. This is the basis for which CE and CEC operate.
Rate of Migration
What makes CE a powerful tool in separation science is the phenomena whereby every ion
will migrate at different rates. This difference is based on its quantity of charge compared to its relative
hydrodynamic size. Hydrodynamic size very closely relates to the mass of the molecule. It is commonly called the
“Charge to Mass Ratio”. A simpler term that is used to describe the molecules affinity for its opposite electrode is
commonly called “Electrophoretic Mobility”. These mobilities can be exploited for incredible separations of very
closely structured molecules.
The actual mobility of an ion takes into account the environment that the ion exists in during the separation
process. For example, electrophoretic mobility will differ from actual mobility when viscosity changes and of course
the amount of voltage that is applied. In simpler terms, this makes the attraction much greater or weaker to the
opposite charged electrode. The more voltage, the more attraction and greater the speed.
Electro-Osmotic Flow (EOF)
CE has incredible efficiency or ability to separate similarly structured
compounds. This is due to EOF. When a voltage is applied across a tube filled with an electrolyte solution (a
solution that conducts electricity), the solution begins to move toward the cathode. This is not similar to the
chromatographic pump, but it does provide the flow of materials past a detector like the pump in HPLC. This should
not be confused with electrophoretic mobility described above. It is a separate phenomenon and is exploited in CE
for maximum flexibility in separation power. Both EOF and Electrophoretic mobility can occur at the same time
working in opposite directions to provide greater resolution.
Technical descriptions of how and why EOF occurs in a capillary is well documented and the reader of this primer
should visit our web page dedicated to EOF.
Plug Flow in CE
In a capillary, the charge from electrode to electrode is conducted by the buffer system (ions in a water solution)
or what is better described as the Back Ground Electrolyte (BGE). Ions of the BGE conducts electricity and provide
the current needed in CE. This current is evenly distributed over the entire capillary diameter and the phenomenon
of EOF occurs (evenly). The water molecules in the BGE also naturally move toward the cathode in a very even manner.
The electro osmotic flow that occurs is called a “plug flow” because of its plug like shape. Due to this very even
flow and flat front, extremely high Theoretical Plate Counts are observed in CE. The diagram below shows the
difference between the Plug Flow of CE and the Parabolic Flow of pressure induced flow as in HPLC.
In the first diagram the EOF has a flat front due to the water molecules moving evenly toward the cathode. You can
think of this in terms of them being pulled by an equal force.
In the second diagram, the parabolic flow depicts the shape of the flow due to the friction from the column walls.
Pressure is applied evenly across the diameter of the column but resistance to flow occurs at the walls and a “drag”
occurs. This provides a “leading edge” of separated molecules reducing the number of theoretical plates that can be
CE Instrument Schematic
A typical CE instrument uses the following components to achieve both EOF and Electrophoretic Mobility and therefore
- Cathode (Negatively Charged Electrode)
- Anode (Positively Charged Electrode)
- Power Supply to generate Voltage/Current
- Catholyte (Buffer Solution at the Cathode End)
- Anolyte (Buffer Solution at the Anodic End)
- Capillary (25mm to 100mm ID)
- A Detection Method
- Data Acquisition Method
Samples are introduced into the capillary for separation by two different methods. Both having advantages and
disadvantages; Electrokinetic injection and Hydrodynamic (Vacuum or Pressure) Injection.
Electrokinetic injection works when the capillary is placed into the catholyte on one end and into the anolyte
(containing the sample to be analyzed) on the other end. When a voltage is applied, the EOF moves from the tip of
the capillary to the end of the capillary. A siphoning effect occurs, dragging a representative sample into the
capillary. Also, ions begin moving into the capillary from the buffer solution due to electrophoretic mobility as
part of the sample loading. This can be an advantage when trying to analyze small concentrations of these ions.
These injections usually last for 1-5 seconds.
Hydrodynamic injection works when a pressure is applied at one end of the capillary or a vacuum is applied. The
pressure differential between the two opposite sides of the capillary will make liquid move into the capillary.
Temperature and therefore viscosity plays a major role in reproducibility in both injections so it is important to
Always Use a Water Plug
After injection the capillary injection end is moved into a sample vial containing CE grade water. A “water plug” is
injected in the same manner that the sample was injected. Then the capillary is moved into a different anolyte
solution that does not contain the sample. Voltage is applied across the capillary and the separation takes place as
the separated samples move (electrophoretically and with EOF) past the detector.