HPLC

Making Size Exclusion Chromatography columns

drchrispook , , , , ,

My summer student, Kalita, has been digesting oligosaccharides, derivatising them and injecting them into the mass spectrometers in an effort to derive structural information from these complex molecules. We had hoped to use acrylamide gel electrophoresis to visualise the performance of our digests, in the way of Pomin et al (2005).

Screenshot from 2016-03-03 20:30:41.png

This figure from the paper shows the effect of their hydrolysis technique upon the molecular weight of the oligomer. Note the banding patterns resulting from selective hydrolysis of certain glycosidic bonds. This produces a regular reduction in size of the fragments. We wanted to use this feature to produce polymeric fragments in the <10kDa size rage. These would be amenable to LC-MS/MS, as in Lang et al (2014), allowing us to infer the sequence, functionalisation and bonding of the monomers within the oligomer.

As it turned out our acrylamide gels got lost somewhere amidst The Great Bureaucracy and so, with time running out we cast around for alternate technologies. Enter Yang, et al (2009), who used a similar technique in their paper, but also deployed Size Exclusion Chromatography to illustrate the size-class of fragments produced.

Screenshot from 2016-03-03 21:11:31.png

The thing is we didn’t have any GPC or SEC columns.  😦

 

So we decided to try making our own!  😀

 

Fortunately or chemical store had a shelf of old bottles of dextran and other GPC or ion-exchange substrates. We dug up a protocol from an MSc thesis by Wilfred Mak in which he’d used an anion exchange substrate to determine the molecular weight of intact sulfated fucan oligosaccharides, rifled through the stores to find some substrates that looked about right and away we went!

We started out with a biuret:

IMG_20160302_172848.jpg

At the bottom, hidden by the blue compression screw, is a plug of deactivated glass wool with a few mL of sand on top of that and then the white dextran gel. This was the first addition of substrate and settling. After topping it up we have a column of about 40cm length. This type of column is purely gravity-fed. You add sample and running buffer at the top and wait for the head of fluid to pass through the column, collecting fractions through the tap at the bottom. This can take hours.

While Kalita was putting this together I was looking at some of the old silica particle LC columns I had and wondering if I might dismantle them, remove the packing and repack them with the dextran to give a real, high-pressure column. This could be plumbed into one of our conventional LC setups, allowing us to push samples through at a faster rate and giving the option of automated sample injection, data and fraction collection. I had something of a brain wave and realised that I had some Swagelok fittings which would allow me to fit a piece of 1/4″ polypropylene air line with pressure-tight caps and LC fittings at either end to fulfil exactly that function. A couple of hours later Kalita and I were the proud parents of monstrous creation on the left!

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The white tube held between the two clamps on the left hand retort is the air line packed with hydrated dextran. The line at the top comes from the Shimadzu LC pump on the right, which is pumping Tris buffer through the column to settle the packing material. We can get a flow of 2 mL/min through the column with a back pressure of about 5 bar. Plenty for LC!

For now our creation is parked until we can get round to doing something cool with it on Monday but watch this space to see the outcome. Our intention is to add an autosampler to the front for sample injection, a Refractive Index Detector and maybe even an electrochemical detector on the outflow to detect what came off the column and possibly even a fraction collector for downstream LC-MS/MS analysis of the fractions! Fun!

Our first goal is to validate the SEC function by injecting a range of proteins stained with Bradford Reagent. We can also try some di- and tri-saccharides along with our oligo digests.

 

References cited

Lang et al (2014). Applications of Mass Spectrometry to Structural Analysis of
Marine Oligosaccharides. Mar. Drugs 2014, 12, 4005-4030
doi:10.3390/md12074005

Pomin et al (2005). Mild acid hydrolysis of sulfated fucans: a selective 2-desulfation reaction and an alternative approach for preparing tailored sulfated oligosaccharides. Glycobiology vol. 15 no. 12 pp. 1376–1385, 2005
doi:10.1093/glycob/cwj030

Yang et al (2009). Mechanism of mild acid hydrolysis of galactan polysaccharides with highly ordered disaccharide repeats leading to a complete series of exclusively odd-numbered oligosaccharides. FEBS Journal 276 (2009) 2125–2137
doi:10.1111/j.1742-4658.2009.06947.x

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liquid chromatography of photosynthetic pigments

drchrispook , , , , ,

I’m developing a method for quantifying photosynthetic pigments with liquid chromatography. I started out just mashing up a couple of leaves from some plants in the teaching lab. A begonia leaf and a tobacco leaf. Here’s the begonia leaf before grinding:

begonia leaf pigment extraction 2

And after grinding in acetone:

begonia leaf pigment extraction

I centrifuged the liquid to remove particulates and diluted some of the supernatant in a little more acetone as that bright green colour was probably too concentrated to inject directly.

I initially tried using the diode array detector and the mass spectrometer in tandem so that I could confirm identities of DAD peaks with the mass spectrum but I think the sensitivity of the mass spec was too low. The pigments absorb light pretty strongly, as you might expect, so you don’t need to inject high concentrations to get a signal from an absorbance detector. So I gave up using the mass spec and just looked for peaks in the DAD. Initially I just infused them straight through the DAD without using an LC column so that I could confirm that I could see peaks at the right wavelengths. Plant pigments fall into several different categories. I was expecting to see chlorophylls, which are common to all plants, as well as some xanthophylls and/or carotenoids. We happened to have some carotene in our chemical store so I prepared a standard dissolved in acetone to inject as a reference.

The direct injections worked well with large peaks visible at 440nm (xanthophylls/carotenoids) and 640nm wavelengths (chlorophyll). The carotene showed up very nicely at 440nm, as expected, with nothing visible at 640nm. Next I wanted to see how many different pigments I could separate but I wanted to do it really quickly, without setting up a full LC gradient. So I ended up trying just a Phenomenex C8 guard column in a holder instead of an actual analytical LC column and set a 1 minute gradient from 20 to 100% isopropanol. I had to use isopropanol, instead of the more usual acetonitrile or methanol, because these pigments are quite hydrophobic. This seemed to work well so I stretched the gradient out to 6 minutes and ran them again (yes, I ended up running a full gradient after all but it was worth building up to it). As you can see from my quick-and-dirty method it actually worked beautifully:

pigments HPLC C8 guard column

This rather busy chromatogram shows several different plots. The two relevant ones are the pink one (440nm) and the black one just above it (640nm). The peaks in these plots represent different pigments in their respective classes. There’s five xanthophylls or carotenoids and at least three chlorophyll peaks (the last one is two peaks coeluting). The last of the pink peaks happens to coelute perfectly with my carotene standard and they shared the same absorbance spectra. Each pigment tends to have a unique absorbance spectra corresponding to its role in absorbing different wavelengths of light. The plot below shows the five xanthophyll/carotenoid peaks and their respective absorbance spectra.

pigments HPLC C8 guard column - xantho & caro peak spectra

All in all a pretty successful day’s HPLC! I’m looking forward to applying this to some marine benthic sediments tomorrow to see what sort of pigments are present in the microphytobenthos. I’m also now well set up to investigate the pigments in the giant springtails I mentioned recently.

A useful paper for background and huge amounts of detail about pigment HPLC is Bidigaire et al, (2005):

Bidigare, R. R., L. Van Heukelem and C. C. Trees. 2005. Analysis of algal pigments by high-performance liquid chromatography. In: Algal Culturing Techniques (R. A. Andersen, Ed.), Academic Press, New York, pp. 327-345.

Research Update

drchrispook , , , , , , , ,

Having meant to write several posts about exciting things that have gone on in the past couple of weeks I am now faced with combining them all, for efficiency’s sake, into another “research update”.

This week I have received an enquiry about analysing pigments and toxins found in the colourful tips of New Zealand giant springtails, Holacanthella. This could be a really cool little piece of analysis if it works. I’m going to have to head out into the Waitakeres to poke some lumps of rotting wood in the hope of finding some of these punk woodlice to play with.

After a visit from Don MacLeod of the NZ Beekeeper’s Association last week I am testing out some more extractions of neonicotinoid pesticides. A new paper was published this week in Environmental Chemistry documenting the occurrence of these pesticides in pollen and honey from hives across the US. The paper was written by Alex Chensheng Lu et al (2015), who kindly shared a copy with me. The paper reports that, during the Summer months, several neonicotinoids were present in pollen at concentrations of several ng/g; concentrations that may be acutely toxic to bees (Laycock et al 2012). The authors discovered measurable concentrations of at least one neonicotinoid pesticide in >70% of honey and pollen samples. I am hoping I can repeat their analysis in New Zealand samples to see if we have a similar issue here.

I have also been developing a method for the quantification of bile acids by LC-MS, which is causing me headaches as certain compounds won’t stay in solution (lithocholic acid, I’m looking at you), some seem to have a very low response in the instrument and others are playing hide-and-seek! The solution, as ever, is a bigger chicken.

I have also been plotting further awesome research plans for the future, submitting an application for a Summer studentship to get someone to look at polysaccharide structures, again with LC-MS. My life seems to revolve around the instrument sometimes but this week has not been all about the liquid phase. I have also been emailing around the results of some test analyses I conducted using methyl chloroformate derivatisation and GC-MS to try and expand the use of this very nice little method within the school. Consequently I found myself preparing samples of mangrove leaf extract, lamb and wagyu beef, fermented mussel liquor and hydrolysed beef protein. I was meant to have a go at some polyamines for another of the PhD students but I forgot their sample. Doh.

Apart from this mass-spectrometry-based fun I may have a student looking to measure total triacylglycerol content and fatty acid profiles of fish oocytes at some point. I’ve also had a really awesome kick-off meeting for my new PhD student, who is going to be studying plant phenology. We are kicking about ideas for the acquisition and installation of a phenocam.

Its been a crazy busy week!

Refs

Chensheng (Alex) Lu, Chi-Hsuan Chang, Lin Tao and Mei Chen (2015). Distributions of neonicotinoid insecticides in the Commonwealth of Massachusetts: a temporal and spatial variation analysis for pollen and honey samples. Environmental Chemistry – http://dx.doi.org/10.1071/EN15064

Laycock I, Lenthall KM, Barratt AT, Cresswell JE (2012). Effects of imidacloprid, a neonicotinoid pesticide, on reproduction in worker bumble bees (Bombus terrestris). Ecotoxicology 21(7):1937-45. http://link.springer.com/article/10.1007%2Fs10646-012-0927-y

research update

drchrispook , , ,

I’ve been meaning to write a post for a couple of weeks detailing the state of play of my current analytical projects but I’ve been awfully busy helping my MSc student get the last of her LC-MS data on cherry anthocyanins and phenolics. We managed it in the end and she has a lovely result so I spent last week broadening my focus again and picking up some of the projects I’d had to put aside. Most of last week I also spent trying to derivatise reducing sugars for a collaboration with some lovely people from the Human Potential Centre at AUT’s Millenium Institute. This is a really exciting capability as sugars are notoriously hard targets to analyse chromatographically. We currently use a gas chromatographic method which involves a quite lengthy and fiddly acetylation procedure but I’ve found a compound that works really nicely for liquid chromatography with a relatively simple derivatisation procedure. There’s also lots of potential to push the analysis into larger oligosaccharides and glycans, with at least two exciting projects already on the table. I’m hoping this might result in two publications, the first being method development and the second an application.

I’ve also started developing a quantitative method for bile acids and have been approached to quantify lignin-derived phenolic compounds in sediment and plant material from estuarine habitats across the South Pacific. Very exotic! At some point I’d like to get some more method development done on neonicotinoid pesticides as I’ve developed a very cool and powerful extraction method which may solve the recovery and sensitivity problems. Again, definite options to present this as a method development and validation paper so no details yet.

😉

I love HPLC – High Pressure Liquid Chromatography

drchrispook , , , ,

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!

Shimadzu LC20 series HPLC

drchrispook , , ,

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:

image

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.