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Separations Theory

Introduction to bioanalytical separations

Why Breaking Up Is Hard to Do: The Entropy of Separation Processes

Diffusion: The manifestation of entropy

High Performance Liquid Chromatography

Bioanalytical separations are most commonly performed by high performance liquid chromatography (HPLC). HPLC is one of the most powerful tools in biochemical research due to its ability to separate a wide class of compounds of biological interest raging from simple organic molecules to large biomolecules like proteins and DNA. HPLC's wide use in bioanalytical applications probably results because it can run at or near physiological temperatures avoiding degradation and denaturation of biological molecules. Though HPLC is still generally more expensive and slower than GC, it offers much greater control of separation conditions than does GC, and it has replaced GC in many applications. Instruments that employ a complementary mode of seprataion such as a UV-visible spectrum, like the diode array detector on the Hewlett-Packard 1090 HPLC are called hyphenated instruments, which means that they combine two different analytical techniques. In this case, the two techniques are chromatography and UV-visible spectroscopy. Thus, any compound that elutes from the instrument can be qualitatively identified by its UV-visible spectrum. Quantitative information can be obtained from the peak areas or peak heights. Though HPLC is more difficult to interface to infrared or mass spectrometers due to the difficulty of removing the large solvent background, instruments are now available that allow for these hyphenated techniques (LC-IR, LC-MS).

Problems in the pharmaceutical, forensic and food sciences are often solved by HPLC analyses.- Aqueous based samples like urine and blood work well by this method. The low detection limits possible with some specialty detectors, such as fluorescence or electrochemical (coulometric), have made it possible to analyze very low levels of illicit or prohibited drugs, such as steroids for enhancing athletic performance.- HPLC is often used for quality control in food analysis; colors, additives and natural ingredients need to be determined to insure unifomity and safety of the final products.

Student HPLC Laboratories: references from Journal of Chemical Education


Capillary Electrophoresis

An upcoming bioanalytical technique is capillary electrophoresis. Since its introduction in 1981, capillary zone electrophoresis has rapidly become one of the most important techniques for biomolecule separation. The required instrumentation for capillary electrophoresis is quite simple. The minimal configuration of a CE system is shown in Figure 1.


Fig. 1: A typical capillary electrophoresis instrument.

In a typical capillary electrophoresis experiment, the sample is injected into the capillary column (typically 25-100 µm inner diameter) by hydrostatic pressure (vacuum, positive pressure or siphon) or by electrophoretic injection by applying a voltage to the sample solution for a given period of time. The sample migrates through the capillary column due to its charge (electrophoretic mobility) or due to the solution transport from the charge on the inner wall of the capillary column (electroosmosis). The charge on the inner wall is due to the ionizable silica groups (pKa ~ 2) on the inner wall of the fused silica capillary column. The sample is detected as it migrates through the capillary by one of a number of different detectors. In this experiment we will use a UV-Vis detector that is similar to those used in HPLC, except that the optics have been modified to better focus light into the center of the narrow diameter capillary column.

The observed electrophoretic mobility of a compound through the column is a vector sum of the electrophoretic migration and the electroosmotic flow. Thus in order to calculate the electrophoretic mobility the two vectors must be added, as in equation 1.

µobs = µeof + µep (1)

The direction of electroosmotic flow is toward the cathode (negative electrode) because the walls of the capillary have a fixed negative charged above ~pH 2 due to the deprotonated silica groups and the mobile carriers are positive ions. The direction of the electrophoretic flow depends on the charge of the compound relative to the polarity of the system. The velocity of the electroosmotic flow depends on the pH of the buffer and the concentration and type of ions in the buffer. Generally, as the concentration of a buffer decreases, the potential right at the capillary wall (due to the electric double layer) increases due to uneven pairing between mobile and fixed charges. This wall potential is called the zeta potential. The electroosmotic flow is directly proportional to the zeta potential. For this experiment, we will run with the cathode at the detector end so that electroosmosis is always favoring flow toward the detector. The direction of the electrophoretic flow will depend on the charge on the protein, which will vary with pH. However, the electroosmotic flow is also a function of pH. At low pH the inner capillary walls are less ionized than at high pH. Therefore, we expect to have a decrease in electroosmotic flow with a decrease in pH.

Student CE Laboratories: references from Journal of Chemical Education


Links to cool CE Websites:

ce and cec information

Harry's CE Pages


Ion Exchange Chromatography

Another common method of separation is ion-exchange chromatography. Like thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), ion exchange chromatography (IEC) employs the same principle techniques of stationary and mobile phases to separate different compounds by electrostatic force. IEC is very useful in the separation and purification of peptides and nucleosides so it is a very important procedure in bioanalytical research.


Polyacrylamide Gel Electrophoresis Tutorial


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