As with my article on a related theme, can LC/LC-MS ever replace immunoassays, I have to admit that I have a history and fondness for both gel electrophoresis and liquid chromatography. They each perform the same function in that they separate compounds, but that is about where the similarities stop. I will try to give a balanced and honest overview of both techniques strengths and weaknesses and my opinion on whether we will be saying goodbye to gel electrophoresis in the not too distant future.
Believe it or not, gel electrophoresis is the younger of the two techniques with its first description using sucrose in the 1930s. However it really began to gain prominence with emergence of disc electrophoresis in the 1950s and the publication of Lammeli’s seminal paper on SDS-PAGE (polyacrylamide gel electrophoresis) in 1970 which most separation people are familiar with. In polayacrylamide gel electrophoresis, charged large molecules, such as proteins, are basically ‘sieved’ approximately based on size through the polyacrylamide matrix, with the smallest macromolecules travelling fastest to give the separation and it is polyacrylamide gel electrophoresis which will form the basis of this article.
Gel Electrophoresis Strengths
There is no doubt that gel electrophoresis is a powerful separation technique with many advantages:
Inexpensive – Gel electrophoresis equipment is relatively inexpensive with a gel tank and power supply costing only a few hundred dollars/euros and will last many years. The cost per gel can also be very low if making your own gels and buffers and you can run multiple samples per gel.
Simple – It’s a relatively straightforward and well characterised technique, the only laborious part is the pouring of the gels, however the invention of long-life precast gels has overcome this, although it will add a little more to the cost.
Detection flexibility and sensitivity –Post electrophoresis you do have many options for detection including colorimetric and fluorescent total stains, and specific detection with mass spectrometry and western blotting for proteins and Southern blotting for DNA. Many of these detection options such as western blotting and fluorescence do also offer high levels of sensitivity.
Speed – Depending on your choices, gel electrophoresis can be very fast. In the past, a typical gel would take about 60 minutes to run, but advances in gel chemistry have now reduced this to about 20-30 minutes. Western blotting has also seen dramatic reductions in the time required to produce the blot. If you also consider that you can run multiple samples per gel then the time per sample is low.
Gel Electrophoresis Weaknesses
Limited to macromolecules – Gel electrophoresis is only suitable for macromolecules such as proteins and DNA/RNA.
Resolving power – The number of different macromolecules that can be clearly resolved is somewhat limited, even when using 2-D gel electrophoresis and/or bigger, longer gels. This is also related to the next point.
Separation options – You really only have two options with gel electrophoresis; mass and isoelectric point.
Precision and reproducibility – It can be difficult to get the same band occurring, at the same position in the gel repeatedly. Using precast gels and ready-made buffers definitely improves the situation as you would expect, but there are many different variables which are harder to control such as temperature, equipment consistency, water quality, etc.
2-D gel electrophoresis – While 1-D gel electrophoresis is straightforward and relatively quick to perform, 2-D gel electrophoresis does require a great deal of skill and experience to perform and takes considerably longer.
Mass spectrometry compatibility – Macromolecules separated by gel electrophoresis can be analysed by mass spectrometry to identify and quantify them, particularly proteins, however the big hurdle is removing the macromolecule from the gel itself and getting it into the right condition for mass spectrometry. This can be time consuming.
The first description of chromatography using a liquid adsorption column was by the Russian-Italian scientist Mikhail Tsvet in 1901, with many subsequent flavours and improvements of this original concept since then. Liquid chromatography can be divided in to two broad areas; preparative and analytical liquid chromatography. With preparative chromatography the main application is to purify and obtain your molecule(s) of choice from your sample with good yields. In analytical chromatography the focus is more to separate your compounds with high resolution to identify the components within the sample and is typically associated with higher pressures or HPLC. Analytical liquid chromatography is thus the direct competitor to gel electrophoresis and is the focus of this section of the article.
Liquid Chromatography Strengths
High resolution – With high pressures, small particle sizes and long columns it is possible to produce fantastic resolution and clearly resolved peaks of mixtures of hundreds of compounds across a broad dynamic range.
Broad analyte range – Liquid chromatography is not just limited to macromolecules. Many different column chemistries and mobile phases can be used to separate a wide range of compounds, ions and macromolecules.
Automated – Typically a HPLC system is fitted with an autosampler so after loading the samples and programming the software, the user can walk away and the instrument can run all the samples automatically. To put this in to context, the Thermo Scientific™ Vanquish™ UHPLC system with the charger module can run nearly 9000 samples unattended.
Flexible separation and detection options – Unlike gel electrophoresis, you are not limited to only two properties of mass and isoelectric point to utilise in separation. With liquid chromatography there are many more options to form the basis of separation including hydrophobicity, polarity, size, affinity etc. You also have great flexibility in detection options as well including UV, fluorescence, charged aerosol and mass spectrometry. With liquid chromatography though you don’t have to worry about extracting your molecules of interest from the gel.
Reproducible – Using a high precision, automated instrument allows for the control of multiple variables and as such very precise and reproducible separations across hundreds of samples.
Liquid Chromatography Weaknesses
It’s not all plain sailing for liquid chromatography when it’s compared to gel electrophoresis though:
Equipment costs – A good UHPLC instrument that delivers the resolution and reproducibility required costs considerably more than a gel tank and power supply. You also have to consider maintenance costs. However, the columns are relatively inexpensive considering the amount of use you can obtain from them if looked after.
Training – There needs to be some investment in training on the instrument and software. Most people come into a lab equipped with knowledge of how to run a gel, but fewer with knowledge of operating a HPLC instrument.
What’s the future for gel electrophoresis and liquid chromatography?
I’d like to start by saying that I think both techniques have a future as I see them as more complimentary than competing techniques. There will always be a place for gel electrophoresis as people need a quick, simple and inexpensive method of visualising macromolecules. Also with western blotting it removes the requirement for expensive and time consuming mass spectrometry for many applications. However, when scientists need more than a simple, low-resolution separation coupled to indirect detection, for example when they need more throughput and automation, increased resolution and reproducibility or the ability to detect and quantify large numbers of proteins in the same experiment then liquid chromatography will be more applicable. I do see this need as a major driving force so an increasing role for liquid chromatography can be envisaged, sometimes coupled to mass spectrometry.
If I look at the field of proteomics, then I can already see this happening. At the start of the century, proteomics was dominated by 2-D gel electrophoresis as the separation mechanism of choice. However, researchers required more resolving power and speed to see more deeply into the proteome in a timely fashion and to be able to couple easily to mass spectrometry to identify unknown proteins. This resulted in a shift to liquid chromatography which is now the norm and 2-D gel electrophoresis more the exception.
What about Capillary Electrophoresis?
I’ve talked in some depth about gel electrophoresis and liquid chromatography, but there is another technique that has the potential to marry together some of the best attributes of both and that is capillary electrophoresis. Its appears to have come in and out of fashion over the years and did have some technical limitations, but due to its heavy involvement in DNA sequencing the technology has evolved and has the potential to be a third complementary technique to gel electrophoresis and liquid chromatography. A topic for another article potentially?
In summary, I see these techniques as complimentary and having a role to play in separation science in the future, although with a gradual shift towards liquid chromatography (and potentially capillary electrophoresis). Gel electrophoresis is unlikely to become extinct though as I see the pouring and running of a gel as a rite of passage for every science student! Are you really a scientist if you have never run a gel?