Sample 15

Dr. Verdi, Gary

I am a retired doctor who has more than 25 years’ experience in the profession. My qualifications are MB, BS., LRCP., MRCS., DRCOG., MRCGP. and Dip Sport Med. I have been an examiner for Nursing Board examinations. I have been a postgraduate tutor and undergraduate lecturer at two UK Midland universities. I write on a broad spectrum of medical, nursing and general science-based issues. I have a particular interest in sports medicine and sports-related subjects. I have a considerable working knowledge of the political and practical issues within the NHS, as well as issues on a global scale. I have personally researched, authored and published four internationally respected and frequently cited academic papers. I am happy to tackle assignments from undergraduate to PhD level. I have a wide experience of academic writing and only use the most up-to-date references unless there is a good reason for using older ones. I actually enjoy writing and turning out good-quality, highly referenced work.

Sample (Copyright of the following sample belongs to Dr Verdi, Gary)

Discuss the advantages and disadvantages of capillary electrophoresis (CE), compared with other high resolution separation techniques, for the detection and analysis of EACH of the following types of a molecule of biological significance:

  • Proteins
  • DNA
  • Steroids or non-polar molecules
  • Chiral molecules

Introduction

Capillary Electrophoresis
The process of capillary electrophoresis is actually a generic term which covers a number of related techniques that essentially rely on a high-voltage electric field being applied over a solution which is held in a capillary tube. It is primarily used to separate a number of soluble compounds, usually biological compounds.(Simó et al. 2004). Other variants of this technique include:-

  • Capillary zone electrophoresis (CZE) (Gijbels et al.2004)
  • Capillary Gel Electrophoresis (CGE) (Tsai et al. 2004)
  • Capillary Isoelectric Focusing (CIEF) (Gao et al. 2004)
  • Isotachophoresis (ITP) (Zhang et al. 2004)
  • Electrokinetic Chromatography (EKC) (Marsh et al 2004)
  • Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) (Akbay et al 2005)
  • Micro Emulsion Electrokinetic Chromatography (MEEKC) (Altria et al 2004)
  • Non-Aqueous Capillary Electrophoresis (NACE) (Altria et al 1998)
  • Capillary Electrochromatography (CEC) (Mangelings et al.2004)

Capillary zone electrophoresis (CZE) is the simplest form of capillary electrophoresis (sometimes referred to as free solution capillary electrophoresis (FSCE). Its ability to separate molecules accurately and consistently relies on a constant field strength across the capillary and the pH of the buffer solution and then ultimately on the charge/mass ratio of the molecules (after Gijbels et al. 2004).

Capillary Gel Electrophoresis (CGE)is the simplest form of capillary electrophoresis (sometimes referred to as free solution capillary electrophoresis (FSCE). Its ability to separate molecules accurately and consistently relies on a constant field strength across the capillary and the pH of the buffer solution and then ultimately on the charge/mass ratio of the molecules (after Gijbels et al. 2004).

Capillary Isoelectric Focusing (CIEF) relies on a graduated pH buffer gradient to provide an isoelectric point where the net charge on the molecule is zero. Different molecules will migrate to different points in the gradient (after Gao et al. 2004).

Isotachophoresis (ITP) This is a variant of CIEF which is primarily used as a concentration technique by assembling specific molecules into small focused zones (after Zhang et al. 2004).

Electrokinetic Chromatography (EKC). This is discussed in more detail under the heading of chiral separation. It relies on the differential interactions of the enantiomers with compounds such as the cyclodextrins (after Marsh et al. 2004).

Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) This is also discussed later in the section on non-polar molecules as it relies on the formation of micelles to produce differential charges on non-polar molecules (after Akbay et al. 2005).

Micro Emulsion Electrokinetic Chromatography (MEEKC) This has a prime use in the separation of water-soluble and insoluble molecules and has a major use in the pharmaceutical industry (after Altria et al. 2004).

Non-Aqueous Capillary Electrophoresis (NACE) is a similar process which is useful in the separation of compounds that are insoluble in water as it relies on the use mainly of organic solvents (after Altria et al. 1998).

Capillary Electrochromatography (CEC) is another variant which gives very high levels of efficiency in separation. It currently tends to be used to concentrate samples prior to separation by CZE (after Mangelings et al.2004).

In modern usage, the technique of capillary electrophoresis can be applied to many different technical applications by modifying the basic underlying principle to fit the specific requirements of the task. It is commonly to be found in processes such as drug or chemical manufacture where it is a prime tool for ensuring purity in the manufacturing process (Altria K.D & Elder D. 2004).

Another common use is in the diagnosis of biological disease processes where specific protein (or other) fractions need careful separation in serum urine or other tissue fluid samples. It has the practical advantage over many other separation techniques in that, being capillary based, it can be done on minuscule sample quantities, it is easily (and ideally )adapted for automation and (after capital expenditure on equipment) is comparatively cheap as it uses very small amounts of reagent (Kaiser et al 2004).

The actual theory behind the process is comparatively simple. The speed of movement of an ion in a solution is a reflection of its charge and the effect of the potential difference from the electric field that is acting on it. It experiences motor forces from the electric field and retardant forces from the friction or resistance in the medium that it is suspended in. Equally, the larger the charge on the ion, the faster it will move in a given electric field. As these factors are subtly different for different particle classes, this gives a useful mechanism for potential separation.

The typical voltages employed are in the region of 10-30 kV across a capillary with a bore diameter of about 25-100mu this gives rise to operational currents in the region of 10-100 microamps.

There is a complicating factor called electro-osmotic flow which is caused by an interaction between the (usually) negatively charged walls of the capillary and the positive ions in solution. This phenomenon is pH dependent and can be manipulated to assist in the particular separation process required (Krull et al. 2000).

Proteins
The clinical applications of this process fill many books and are the subject of a great many papers. The ability to identify particular proteins can be critical to both the diagnosis of many disease processes and sometimes also the staging of the disease process. Usually it is the identification of polypeptide fragments that is clinically important but whole proteins can also be identified by this method. Kaiser et al. (2004) describe a separation process where the methods of capillary electrophoresis are combined with a mass spectrometer to give a particularly sensitive indication of the levels of polypeptides in body fluids.

The problems associated with electro-osmotic flow are particularly noted with protein and polypeptide assays. Soga et al. (2004) offer a novel solution to enhance the assay by coating the walls of the capillary with a polymer solution prior to the assay and this appears to reduce the EOF effect.

DNA
DNA separation is best done by gel electrophoresis. It is generally used for the separation of the large and very large group of proteins. This is basically a more mechanical variant of electrophoresis, as the DNA is encouraged to move across a potential difference but through a gel substrate which causes a “friction” dependent on the size of the molecule.

In broad terms therefore, it is the size and shape of the molecule that is exploited in this form of separation rather than the strength of charge. This is a variant of the older form of gel slab. The slab method suffered from the heating effects of passing a strong charge through the slab. If the charge was too high, it heated the slab with the risk of melting the gel and denaturing the protein. Reducing the charge meant that the process was slowed down.

With the capillary form of gel electrophoresis, the heat generated is far more easily dissipated because of the relatively greater surface area. This means that higher voltages can be safely employed which make for faster separation times. It should also be noted that in the gel slab method, different gel matrices could be used which were appropriate for different sized molecules. Agarose was a favourite for DNA as its “sieve size” was comparatively large.

Smaller protein molecules were more appropriately separated using gels with smaller pores. With capillary electrophoresis the gel matrix used is not always solid and can be liquid. The capillary format effectively stabilises the physical integrity of the matrix. This has the great advantage that, in the automated forms of the system a fresh gel matrix can be used for each assay.

In the context of the question, these methods are considerably easier and less time-consuming than the older methods employed for DNA fractionation and identification (Plus/Minus system, chain degradation and didyoxy chain termination).

Steroids or non-polar molecules
Steroids, as a group, are hugely clinically important in many biological mechanisms. Their identification and assay is also therefore very important. The immediate difficulty is that they are, as a class, usually comparatively water insoluble and electrostatically neutral. At first sight, this does not make them good candidates for capillary electrophoresis.

It should be obvious from all that has been discussed thus far that capillary electrophoresis is a separation method for charged molecules. Many biological molecules do not have a net electrostatic charge and therefore require some form of modification in order to make them amenable to this type of separation process.

As we have already commented, steroids, as a group, tend not to have a net charge. This is accommodated and adapted to the technique of capillary electrophoresis by the use of a procedure called Micellar electrokinetic capillary chromatography (MECC). This was developed in the mid 80s by Terabe (et al. 1985). This involves using a high pH electrolyte buffer solute and surfactants. The net effect of this is that the surfactant will spontaneously begin to form micelles with a hydrophobic core which effectively “protects” the electrochemically neutral molecules.

The outer, hydrophilic shell of the micelle has a heavy net negative charge and is therefore amenable to capillary electrophoresis processes. It follows from what we have said that the selectivity of this method is primarily determined by the choice and concentration of the particular surfactant – sodium dodecylsulfate being the most commonly used in everyday practice. The high pH levels employed are responsible for the generation of a large EOF (see above) which is a major factor in this separation process.

Chiral molecules
Chiral molecules (literally hand-like), are those molecules that are a non-symmetrical form of enatiomer compounds. In plain terms, they have a unique symmetry which is similar, but not identical to, the mirror image of the molecule. They will also have the characteristic feature that they will also possess a stereogenic centre carbon atom (a carbon atom with four non-equivalent groups bonded to it).

This chirality has been found to be biologically important, as the different chiral forms of biologically active compounds may have different actions (Scriba G 2003).

It is becoming progressively more important to be able to separate and identify chiral molecules. The current method of choice seems to be the capillary zone method of electrophoresis (CZE). It is both simple to use and can be extremely versatile. In practical terms the capillary only holds buffer solution and the target molecules. Its major drawback is that while it is very efficient at separating changes (cation and anion) particles, it has no effect (apart from the EOF) on electrically neutral particles (Rudaz et al. 2003).

Chirals can be separated by gas chromatography, but these are both technically difficult and expensive to perform. The capillary zone electrophoresis is both technically easy, highly efficient and cheap. Chiral differentiation is often augmented by the use of chiral selectors (which can be of many varieties – perhaps cyclodextrins are the commonest) which bind the hydroxyl groups of the chiral molecule. Other chiral selectors include both natural and synthetic chiral micelles, crown ethers, some proteins and polysaccharides (Amini 2001).

Conclusions
In this essay we have discussed the various advantages and disadvantages of the techniques related to capillary electrophoresis. It is a technique which is expanding and evolving at an enormous rate and this must therefore reflect both its versatility and its use in the modern biochemistry lab. Different variants of the technique have been discussed with their intrinsic suitability for different types of molecular separation.

References
Akbay, Syed A. A. Rizvi, and Shahab A. Shamsi, 2005 Simultaneous Enantioseparatoin and Tandem UV-MS Detection of Eight Beta-Blockers in Micellar Electrokinetic Chromatography Using a Chiral Molecular Micelle Analytical Chemistry, 77,1672-1683 (2005)

Altria 1999 Overview of capillary electrophoresis and capillary electrochromatography J Chromatogr A 856: 1999 443-463

Altria K.D., Broderick M., Donegan S., Power J., 2004 The use of novel water–in-oil microemulsions in microemulsion electrokinetic chromatography, Electrophoresis 25 2004 645-652

Altria K.D & Elder D. 2004 Overview of the status and applications of capillary electrophoresis to the analysis of small molecules Journal of Chromatography Vol.1023 No. 1, Jan 2004. Ppg 1-14

Altria KD, Wallberg M and Westerlund D, 1998 Separation of a range of cations by non-aqueous capillary electrophoresis using indirect and direct detection J.Chromatogr.B, 714 1998 99-104,

Amini A 2001 Recent developments in chiral capillary electrophoresis and applications of this technique to pharmaceutical and biomedical analysis. Electrophoresis. 2001 Sep;22(15):3107-30.

Gao & Liu 2004 Cross-Linked Polyacrylamide Coating for Capillary Isoelectric Focusing Anal. Chem., 76 (24), 7179 -7186, 2004

Gijbels, Coster & Bossuyt 2004 Interference by Gelatin-Based Plasma Substitutes in Capillary Zone Electrophoresis Clinical Chemistry. 2004;50:1473-1475.)

Kaiser , Wittke , IJust , Krebs , Bartel , Fliser , Mischak , & Weissinger 2004 Capillary electrophoresis coupled to mass spectrometer for automated and robust polypeptide determination in body fluids for clinical use Electrophoresis Vol 25, Issue 13 Ppg 2044-55

Krull, Stevenson, Mistry, and Swartz. 2000 Capillary Electro-Chromatography and Pressurized Flow Capillary Electro-Chromatography: An Introduction. New York: HNB Publishing, 2000,

Mangelings, C. Perrin, D.L. Massart, M. Maftouh, S. Eeltink, W.Th. Kok, P.J. Schoenmakers and Y. Vander Heyden 2004 Optimisation of the chlorthalidone chiral separation by capillary electrochromatography Analytica Chimica Acta 509 (2004) 11-19.

Marsh A, Clark B, Broderick M, Power J, Donegan S, Altria K 2004 Recent Advances in Microemulsion Electrokinetic Chromatography, Electrophoresis, 25 2004 3970-3908

Rudaz, Calleri, Geiser, Cherkaoui, Prat, & Veuthey 2003 Infinite enantiomeric resolution of basic compounds using highly sulfated cyclodextrin as chiral selector in capillary electrophoresis Electrophoresis 24, 15 (August, 2003) pp 2633-2641

Scriba G 2003 Pharmaceutical and biomedical applications of chiral capillary electrophoresis and capillary electrochromatography: An update Electrophoresis 24, 15 (August, 2003) pp 2409-2421

Simó , Elvira , González . San Román , Barbas , & Cifuentes 2004 Capillary electrophoresis-mass spectrometry of basic proteins using a new physically adsorbed polymer coating. Some applications in food analysis Electrophoresis Vol 25, Issue 13 Ppg 2056-6

Soga, Kakazu, Robert , Tomita & Nishioka 2004 Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry Elecrophoresis . Vol 25 Issue 13 Ppg 1964-72

Terabe S, Otsuka K and Ando T, 1985 Electrokinetic chromatography with micellar solution and open-tubular capillary, Anal. Chem., 57 (1985) 834-841

Tsai, Loughran, Susuki & Karube 2004 Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip. Electrophoresis. 2004 Feb;25(3):494-501

Zhang, Matsunaga & Saku 2004 Associations Among Plasma Lipoprotein Subfractions as Characterized by Analytical Capillary Isotachophoresis, Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:e144.