Archived Chromatography Technical Tips
By Tony Taylor - Crawford Scientific's Technical Director.
Relating Analyte Properties to HPLC Column Selectivity – on the road to Nirvana
The proliferation of HPLC column stationary phases makes it impossible to screen the whole ‘selectivity landscape’ to choose the best phase for your separation. Therefore most of us use various ‘filters’ such as the academic literature, applications notes, experience, manufacturer’s recommendations and so forth, to narrow down our choices...
Solvent Choice for GC Injection – a critical method variable!
I guess we all have our ‘go to’ solvents for our work in GC, and most of the time this will be based on the availability of the solvent and the chemical nature of our samples. Polar samples (analytes) generally need a more polar solvent (methanol for example) and less polar analytes a less polar solvent (n-hexane or heptane for example). Sometimes we might even use an intermediate polarity solvent when we aren’t sure of the analyte composition, ethyl acetate is a popular choice in this instance.
Drive it like you stole it – getting the most from your GC Quadrupole MS
This time I’m going to be looking at how to get the most from your GCMS (Gas Chromatography – Mass Spectrometry) system, and I intend to keep things as simple as possible, however I also make no apology in the fact that some of the concepts may be beyond your current understanding. My intention here is to explain some of the basics so that you will be able to use your instrumentation to best effect...
Calibration – done (badly?) every day
Where we have a wide expected analyte concentration range within our samples, it’s usual to achieve this using a range of standards of varying but known concentration to build a calibration curve of instrument response against known analyte amount...
Syringe filters – a blessing and a curse!
Many of us use syringe filters to protect our HPLC systems by getting rid of particulate materials which can clog column end frits (filters), block narrow internal diameter tubing and cause damage to valve mechanisms....
Generic Methods – the Potluck supper of analytical chemistry?
As we enter the Generalisation phase of the industrialisation of Analytical Science, we find ourselves striving for the generic in as many areas as possible...
Allowable changes to Pharmacopeial HPLC Methods
Are you taking advantage of the allowable changes to USP methods to speed up or improve the performance of your methods? More importantly - are you falling foul of changes to regulations which restrict changes to gradient USP HPLC methods? Read on to find out more...
Getting the most from Phenyl Stationary Phases for HPLC
I’ve often written about the ‘lazy’ chromatography which has swept through our industry, whereby 0.1% (w/w or w/v!) TFA or Formic acid is used to ‘buffer’ the eluent system well away from the pKa of analyte molecules, leaving most acidic analytes in the ion suppressed form and most basic analytes in the ionised form.
Useful free tools for HPLC method development
I’ve done that thing where I’ve stated a very interesting title - I hope I can deliver something which lives up to it. I dislike it when people ‘overstate’ their talk or poster titles at conferences to draw me in and then don’t deliver against the promise – I’ll let you judge how we go here.
Proper GC Column Installation – The simplest way to improve your Gas Chromatography
Even if your column preparation technique is not ‘catastrophic’, small issues will add to the overall ‘jitter’ of the instrument which may result in sub-optimal performance or even batch failure.
Investigating (and solving) robustness issues in HPLC
Not every method is as we would hope it would be. Some methods come to us in imperfect form and we have to ‘live with them’, whilst others are difficult separations and by necessity need to be developed using close control of several variables such as eluent pH, eluotropic strength, ionic strength etc.
Big molecule analysis for small molecule people
I realise that the separation of biomolecules had been happening for many years, but the expansion and development of protein based therapeutics from that point onwards has seen an avalanche of developments...
Practical HPLC method development screening
I often get asked about the other important aspects of ‘screening’ in HPLC, which include the mobile phase composition, gradient parameters and flow rate – so that’s the theme of this instalment.
Column Selection for HPLC Method Development
I’ve been talking about ‘ToolKit’ method development platforms a lot lately and I have to say I’m not sure we are making the most of this approach. The approaches to method development and especially to column selection in HPLC vary widely and when I ask about column selection criteria I get a range of responses...
Hidden Problems in your LCMS data?
Take a look at the standard curve from an LC-MS determination of Alprazolam from a benzodiazepine screen. The peak area ratio of the analyte against its deuterated analogue is plotted against nominal concentration.
System and Column Volumes in HPLC - we still haven’t got the message!
So modern HPLC systems resemble spacecraft in terms of their technology, designed as they are to operate to the highest efficiencies, compared to traditional systems. However, I still see countless examples where high efficiency columns are used on systems which are not matched and cannot support the highest efficiencies offered by the column.
In defense of Nitrogen as a carrier for capillary GC
There has been much written about the use of nitrogen as a carrier gas for capillary GC. Formerly, to say it wasn’t any good. Latterly to say that it’s pretty good and a better alternative to Helium than hydrogen from a practicality standpoint.
GC Troubleshooting in 20 Pictures (Part 1)
They say a picture paints a thousand words…This month I’ve taken inspiration from a recent webcast, presented at www.chromacademy.com, in which I presented real data from our work that represents some ‘classic’ GC problems.
Simple visual methods to assess HPLC column performance – and get you out of trouble!
A client asked me recently for a quick but ‘as optimized as possible’ separation for some monoclonal antibody (MAb) characterization (digest) samples using LC-MS.
The samples had been prepared and were awaiting analysis...
The Acid Test - More Useful Calculations for HPLC Eluent Preparation
I’ve been through this loop before when investigating a technical transfer issue for a client and set about explaining to him. I used a couple of equations in the explanation which he described at the time as ‘alchemy’, however they are very real and also terribly useful in the lab.
How to Estimate Error in Calibrated Instrument Methods - and why have stopped doing it!
Many of us produce and report precise quantitative measurements on a daily basis. These measurements are often at trace levels or from highly complex matrices, both of which influence the achievable precision associated with the results generated.
The most common mistakes in Solid Phase Extraction
If you use SPE in your work, then most likely it’s very important to the success of your applications and it’s proper implementation will be key to the performance of your analyses.
Why every good analytical chemist also needs to be a statistician
We simply can’t do first rate analytical work without the use of statistics and statistical models.
Troubleshooting retention time issues in reversed phase HPLC
Tony Taylor discusses the relationship between peak height and flow rate in gradient HPLC.
HPLC Troubleshooter – Oh, Larry Got Laid Off...
Tony introduces the CHROMacademy HPLC troubleshooter and explains why it took the Crawford Scientific Team two years to complete the project.
Useful Tips for GC-MS
There are many aspects of analytical science which abound with myth and legend – but gas chromatography – mass spectrometry (GC-MS), and more specifically the electron ionisation (EI) process, stands out as the technique which has given rise to the largest number of ‘urban myths’ and misunderstandings.
HPLC column transfer from Fully Porous to Core-Shell particles in three simple equations
"There are often times in my work when I need to 'mess about' with column dimensions and particle morphologies. For 'mess about' read improve or transfer."
GC Column Conditioning – Stop Wasting Time and Money!
If you ‘do’ Gas Chromatography, then you will have undoubtedly conditioned a capillary GC column ready for use after a period of storage or before first use.
Sweating the Small Stuff
In this issue – I hope to outline some 'small bolts' that are commonly ignored in everyday chromatography and mass spectrometry - instrument settings that I have to come call 'lock and leave' parameters. We can generate data without changing them or paying them any attention, but how much better could our data be if we bothered to optimise them.
Are we scared to properly explore selectivity options in HPLC?
Mixed-mode stationary phases offer unique selectivity for complex HPLC separations which may not be possible with more traditional column chemistries. The use and optimisation of separations on these types of phases is discussed in relation to real world separations.
Troubleshooting HPLC Separation Issues - Are checklists only for pilots?
My colleague (himself an amateur pilot) asked this question recently and I wondered if there were any checklists that I use at work...
So just how well set-up is your UV detector?
Could we be getting better baselines, better sensitivity or better reproducibility from our UV detectors without too much effort? The answer is almost invariably YES - and here's how…
Putting the U into your UHPLC
Column fittings – you have it sorted, right? You have an array of fittings for your HPLC and UHPLC systems which are appropriate, produce zero (or negligible) dead volume and are robust and reliable.
Avoiding the problems associated with HPLC column overload
Much has been written about column overload – but I've seen many instances lately in which overload could be attributed as a causal factor that I wanted to explain how to spot and deal with overload situations.
Robustness in HPLC Eluents
0.1% TFA (aq) / 0.1% TFA in Acetonitrile – the ultimately robust HPLC mobile phase right?
GC Inlet Maintenance... have you really heard it all before?
There are many badly maintained GC Inlets in the world - all leading to analysis which isn't as good as it could be.
Is yours one of them...?
Capillary GC Liner Selection Guide
We thought that a simple 'walk through' guide to liner selection for capillary gas chromatography was a good idea. The Internet is packed with information and recommendations on which type of liner to use, however these sources often lack explanations which practicing chromatographers can relate too. Until now...
Diode Array Detector Settings - 5 minutes to change your chromatography forever
In another role of mine, I deliver webcasts on analytical chemistry for our e-learning site CHROMacademy. Of late we have been running a series called ‘Lock and Leave’, which serves as a reminder to pay attention to those instrument parameters which are often overlooked - you know the type of thing – ‘that’s the best value for this system’, ‘ we always set to that value’ and ‘I have no idea what that does – it’s always at that value’.
The title will already have many of you shouting at your screen – of course we can’t measure quality. But we can measure uncertainty and build a quality system which helps to produce data which are fit for pupose. Surrogates, Internal Standards, Isotopically labelled Standards, External Standards, Calibrants, QC Samples etc. etc.– our working lives are littered with checks to ensure that our instruments are giving us the correct results.
HPLC Filtration - Rocks, Boulders and Sand
I get asked a lot about the value of mobile phase filtration and, especially since the introduction of UHPLC, the various system filters and how best to protect HPLC systems to allow maximum ‘up-time’. I’ve collected together here an assembly of thoughts on all things filtration for HPLC – and how to deal with various nasty’s – from Rocks to Sand...
GC temperature program development
There has been much focus within the industry of late on ways of improving the way in which HPLC method development is carried out – which has led to a more widespread adoption of modelling and optimisation software combined with dedicated method development platforms from the major instrument vendors...
Split Later and the case of the Infinite Dilution Tube
No, this isn’t a new detective novel, in fact its two concepts which were ‘coined’ by my good friend John Hinshaw during a recent CHROMacademy webcast which we presented together on sample introduction for capillary GC. I have to admit to laughing out loud at the term ‘Split Later’ which John proposed as a better term to actually describe spitless injection. It brought home to me that, no matter how long we have been involved with a technique, there is always merit in looking at it from a different angle or seeking better language to describe it...
HPLC Column Selection – Are you barking up the right tree?
How do you choose your HPLC columns? I guess the answer to this varies widely and I’ve seen many strategies being employed including; whatever is on the instrument, first one out of the column drawer, the one that was successful for my last development, from a publication or application, my favourite column from my favourite supplier, we have a set of orthogonal chemistries on our automated column screening system – the list is long...
Real Life GC Column Selection
I’m often asked about the best way to choose GC columns based on analyte or sample characteristics. The full response is of course a very broad topic, and one which is beyond the scope of this short article. However, during a recent consulting exercise I was required to give a very quick summary of the guiding principles behind GC column selection in order to get some results fast. So, I answered the question in the most pragmatic way I could, using the rules of thumb which, I have developed over the past 27 years working with Gas Chromatography. A summary of the response is shared below, I hope it acts as a primer or reminder the next time you need to make a GC column selection decisions on the fly...
5 ways to improve your Split / Splitless Injection technique
1. Understand the process 2. Septa and Septum Purge 3. ...
Buffer choice for HPLC separations
This month we take a look at the important topic of buffer choice for HPLC separations, how to the choose the correct buffer type and concentration as well as how to avoid variability in retention and selectivity...
Retention Shifts in HPLC
The nature of retention time changes in HPLC tends to fall into categories. Firstly, the retention time may ‘drift’ over several injections or several analytical campaigns and secondly, the retention time may suddenly ‘jump’ to a different value between injections or between analytical campaigns (i.e. analyte retention times are very different to when that method was run last)...
Silica for HPLC Stationary Phases - A Five Minute Guide
Most HPLC columns are packed with silica onto which some form of hydrophobic ligand is bonded – these columns form the vast majority of those used for modern reversed phase HPLC...
Sample Diluent Effects in HPLC
Most of us will know that the solvent (diluent) used to prepare HPLC samples can have an effect on HPLC peak shapes. The following discussion highlights some facts, figures, tips and tricks that can help in a practical situation...
Retention Time Variability in HPLC
I’m sure we have all experienced it – that sinking feeling when you realise your analyte retention times have drifted outside the software ‘window’ and you have a pile of chromatograms with no quantitative results. Or you are trying to get that system suitability result to begin your batch of analysis as you really need to get out of the door fast but your retention times just won’t settle down...
Hydrophilic Interaction Liquid Chromatography – HILIC
HILIC Feb 2013
Here’s a short primer on what you need to understand about this separation mode from a practical perspective, using some of the questions that we regularly get asked: When can HILIC chromatography give me an advantage?
HPLC Columns - Pore Sizes and Particle Diameters
The physical characteristics of silica based HPLC columns can affect the performance of the separation almost as much as the bonded phase. This month’s technical tip re-visits some of the lesser known or remembered facts relating to silica particles used for chromatography...
HPLC Column Abuse!
In this months technical tip we answer some often asked questions and dispel some myths regarding HPLC column abuse and reconditioning. Please don’t forget that you can e-mail or call our technical team to get advice and recommendations on column problems - call 01357 522961 and ask for the technical team or e-mail email@example.com
Issues With HPLC Gradient in reverse phase chromatography
We know that gradient and isocratic separations work differently – the separation mechanisms differ greatly between the two forms of chromatography. We also may have examples where gradient methods are not sufficiently reproducible or where our equipment struggles to form a gradient at high percentages of acetonitrile or when running ‘quick’ gradient methods...
Causes of Retention Time Drift in HPLC
We have all faced the situation in which the retention time of our analytes ‘drift’ over time – sometimes to the extent which takes analytes outside their ‘retention window’ within our data processing software – causing analyte peaks to be missed. The phenomenon is often seen over long ‘campaigns’ of analysis and also when installing a new HPLC column, where several ‘priming’ injections might be required to obtain a stable analyte retention time...
Additive Addition in HPLC Eluents
I’m often asked whether HPLC eluent additives should be added to one or both of the constituent mobile phase reservoirs - both organic and aqueous, when mixing gradients online. This is really a matter of reproducibility and robustness within the method...
Polar analyte retention and phase collapse
Why would one want to use a mobile phase containing 100% water? Usually to wash the analyte or old mobile phase from the column (stationary phase surface) or to help retain analytes which elute quickly using mobile phases containing only very small amounts of organic solvent (usually highly polar analytes)...
HPLC Troubleshooting Tips - Selecting Reversed Phase HPLC columns
There are many factors which influence the performance of an HPLC stationary phase, of which the chemical nature of the bonded phase ligand is important but by no means all encompassing in determining the important phase characteristics...
HPLC Troubleshooting Tips - Poor Equilibration in Gradient HPLC
Our technical support centre deals with many issues regarding irreproducibility of retention and selectivity in reversed phase HPLC. Very often, the problem lies in poor equilibration of the HPLC column between injection, which in gradient HPLC can affect the separation selectivity as well as analyte retention...