I love HPLC – High Pressure Liquid Chromatography

HPLC is my favourite analytical technique because it can be applied to pretty much anything soluble. Which includes, well, pretty much everything. This is in contrast to Gas Chromatography (GC), which only works for compounds which are volatile at temperature below about 350C. This is only about 5% of molecules so that’s a fairly restrictive condition, although as us scientific types are jolly clever we’ve worked out cunning ways of changing non-volatile molecules we want to analyse by GC to make them volatile.

Here’s a link to one of the HPLC systems we use in our labs, including some of the applications for this technique. In addition I’d like to present a bit of my history with this technique to provide some examples of what it can achieve.

I used HPLC during my PhD to quantify glutathione ratios in the polychaete worm I was studying. Glutahione ratios are a very useful indicator of oxidative stress as glutathione is the first line of defence against the toxic effects of many metals and is a substrate or cofactor in many antioxidant and other enzymes.

Whilst working on my PhD I also used HPLC to quantify hormones in monkey pooh! This was a quick bit of work to validate a friend’s work looking at social hierarchies in communities of monkeys in zoos. The hormones in their pooh were correlated with their health and with their place in the hierarchy!

Nowadays you tend to find HPLC systems coupled to Mass Spectrometers, harnessing the resolving power of this technology to enhance the capabilities of liquid chromatography. This allows you to identify and quantify many different compounds in very short runs and in very complex matrices such as urine, blood and cell or tissue homogenates. SoAs was lucky enough to acquire an Agilent 6420 triple quadrupole mass spectrometer and an Agilent 1200 series LC stack a couple of years ago and this has become the workhorse of our lab.

During my PhD I first managed to get time on an LC-MS instrument when I was based at Plymouth University, where I worked as Research Assistant on a project characterising the metabolism of a common biocide, 2-hydroxybiphenyl [HBP], in common shore crabs. This was incredibly valuable experience and I was lucky to have an oustanding LC-MS mentor in the form of Dr Claire Redshaw. Claire helped me develop a method to extract HBP and its metabolites from urine we collected from the crabs (that’s a post for another day). The LC-MS allowed us to confirm that the metabolites were mostly sulphate-conjugates of either the parent molecule or of a monooxygenated product of Phase 1 detoxification.

Other pieces of LC-MS work I’ve been involved in or conducted myself include the extraction and quantification of bisphenol A from human urine, profiling of triglycerides in edible oils and in human plasma and the quantification of neonicotinoid pesticides in pollen and honey. I have been working on the latter piece of work for several years now, starting with a postdoc position at Exeter University studying the toxicity of this class of pesticides to bees and continuing now here at AUT.

Hopefully these examples of HPLC and LC-MS applications illustrate why I love the technique so much. One reason which may not be obvious is that LC is a notoriously challenging technique and can be incredibly complex to get right. So much so that it is often referred to as a “Black Art”. I have a favourite joke I tell to all the students when their analysis isn’t working as it should: I ask them how big their chicken was that morning. When they look baffled or alarmed I follow up with the question: “Well, you did sacrifice a chicken this morning, didn’t you?”

LOL!

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Shimadzu LC20 series HPLC

I’m going to start a page for each of my instruments detailing what it does and giving some details of what it gets used for. I’m going to start with our Shimadzu LC20 series High Pressure Liquid Chromatograph [HPLC]. HPLC is my favourite analytical technique and I have more than 10yrs experience getting results from it. 

Here’s a picture of the beast:

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The system is comprised of a quaternary LC pump with integrated degassing; high speed, flushed-path, total-volume sample injection, thermostatted autosampler for up to 70 samples; a venerable Jones Chromatography column heater (HOT!); a twin wavelength UV-visible absorbance detector and a fluorescence detector. The system is connected to a PC running LC Solution. The autosampler and degassing unit were both new this year, replacing functional but less capable units which have been transferred to a secondary system. We now have the ability to run two separate HPLC systems fully automatically.

Apart from our flagship LC-MS this is our most capable and modern LC system and has been responsible for almost all of the HPLC-based research output. Here’s a few of the compounds this system has been used to identify and quantify:

  • the anticancer drug gemcitabine and its metabolites
  • cafffeine & its metabolites in human plasma and urine
  • sex steroids in scallop gonads
  • monosaccharide profiles using phenyl methyl pyrazolone derivatisation
  • methyl anthranilate in grapes

The cutting edge of HPLC technology has moved beyond systems like this one to Ultra-High Pressure Liquid Chromatography (UPLC). However, its great to have a sturdy and capable workhorse system like this one for the more straightforward bits of analysis.