Separation of Peptides and Amino Acids using High Performance Capillary Electrophoresis
Capillary electrophoresis (CE) has been introduced for analysis of charged molecules. It has been introduced as high performance capillary electrophoresis (HPCE), and is a powerful method for separation of nucleic acids, proteins and peptides. In this cha
- PDF / 676,889 Bytes
- 8 Pages / 439.37 x 666.142 pts Page_size
- 40 Downloads / 190 Views
20.1 Introduction Capillary electrophoresis (CE) has been introduced for analysis of charged molecules. It has been introduced as high performance capillary electrophoresis (HPCE), and is a powerful method for separation of nucleic acids, proteins and peptides. In this chapter, we also describe the application of HPCE for separation of PTH amino acids after protein sequencing or protein hydrolysis. The wide range of different separation conditions, including: buffers pH, temperature, ion-forming reagents, micelle-forming reagents, complex reagents, packed capillaries, makes the separation of different biomolecules possible. Capillary electrophoresis was introduced by Jorgerson using a 75-llm fused silica capillary and 30 kV voltage. The new generation of CE equipment allows full automatization of the separation and data evaluation. The advantages of CE are that it has a very short separation time, only nanoliter sample volumes, very high efficiency of the separation and flexibility for detection systems. HPCE is a comparable method to HPLC, but the method optimization is simpler and expenses for eluents and columns are much lower. For optimal separation of biomolecules the following parameters can be optimized: - pH of buffer - temperature - salt concentration - ion strength - capillary coating or packing - additives such as SDS, cyclodextrine, ion pairing reagents, complexing reagents
Principles and Practice Methods in Proteome and Protein Analysis R.M. Kamp, J. J. Calvete, T. Choli-Papadopoulou (Eds.) © Springer-Verlag Berlin Heidelberg 2004
300
Hong Jin and Roza Maria Kamp
The difference between using capillary electrophoresis as opposed to using the conventional method is the use of glass capillaries instead of polyacrylamide slab gels. The separation is performed using a high capacity buffer, which is responsible for the constant pH during the electrophoresis. The buffers usually applied are: - phosphate - citrate - borate - TRIS
-
Depending on buffer additives, different capillary techniques can be used: zone electrophoresis micellar capillary electrophoresis isotachophoresis isoelectric focusing
It is important to overcome the adsorption problems on the capillary surface. This is done by using a buffer which has a pH higher than the isoelectric point and a pKa lower than the silanol groups, and which suppresses the ionization of the capillary surface. The special application of capillary electrophoresis is the use of coated capillaries, e.g. after chemical modification of silanol groups or coating with polymers. For separation of protein and peptides, the techniques usually used are zone electrophoresis, gel electrophoresis and isoelectric focusing electrophoresis. The simplest technique is capillary zone electrophoresis, which separates molecules depending on their mobility, which varies with size and charge of separated molecules. It is important to reduce protein and wall interaction, because of the unspecific binding of molecules. Proteins or peptides ions are separated according to different mob
Data Loading...
