15 Jul 2019
Building your HPLC
HPLC vs uHPLC vs Prep
When the need is to isolate and collect a large amount of an analyte from a mixture, a preparatory system is required. They can achieve flow rates much higher than analytical systems but will require much more sample and mobile phase.
The difference between HPLC and uHPLC is the particle size used in the columns. uHPLC columns use sub-2µm particle sizes to improve peak shape but this leads to an increase in backpressure in the system. Pressure limits vary depending on the column employed but in general HPLC columns can reach up to 600 bar whilst uHPLC systems can reach up to 1300 bar. uHPLC systems also require smaller internal volumes to ensure the increased peak performance is not lost.
The benefits and costs of HPLC vs uHPLC could be a blog on its own but to decide between the systems, you need have a clear idea of the needs of your analysis. If high efficiency or rapid run times are required, then a uHPLC is worth the investment but for simpler mixtures with less time constraints, a HPLC is more than capable especially when used with solid core columns.
Isocratic vs Binary vs Quaternary
Isocratic systems have a single pump that is fed from a single bottle. Mobile phases need to be prepared before the analysis and this puts extra emphasis on the analyst to prepare it correctly. The mobile phase composition will not change during the run, so less time is required between runs to ensure the column returns to the correct state between injections.
A binary system can mix 2 solvents during a run to form a gradient and manipulate the selectivity to improve peak shapes, peak capacity and run times. This requires 2 separate pumps, one for each solvent which will increase initial cost and future maintenance. The mixing of the solvents occurs in a high-pressure environment, so the accuracy of the generated gradient is very reproducible.
A quaternary system, contrary to the name, only has a single pump but it can however mix up to 4 solvents during a run. This allows the user to either form more complex separation environments or to run different eluent profiles without the timely procedures of swapping solvent bottles and priming the system. The mixing occurs at low pressure (before the pump) so is not as precise as a binary system.
For routine, simple analysis an isocratic system is suitable for most laboratories where precision and flexibility are not as big a concern. For method development, the quaternary system allows a much more flexible approach as multiple pHs or solvent strengths can be tested in a single sequence. For high throughput laboratories a binary system is more suitable due to the increased precision of gradient formation and reduced dwell volume.
A binary pump will be more expensive to purchase and maintain than an iso/quaternary pump as you have 2 of everything!
Manual vs Auto vs Multi – Samplers
Manual injections have one major advantage over autosamplers, simplicity. Changing the configuration of a manual injector requires little technical expertise and are usually much easier to access than their autosampler counterparts making them more flexible and they are also easier to maintain.
Autosamplers make use of switching valves which allow the rapid infusion of the sample whilst minimising the disruption to the mobile phase flow. The injection is reproducible meaning that the injection volumes are more precise (less RSD in peak areas), quicker (less band broadening) and can be performed without an operator present (overnight running). They can be set up to flush between injections to minimise carry over.
Multi-samplers allow the user to “rack up” sample trays with some systems capable of having over 8000 samples ready for analysis (using 384 well plates). Each rack can be configured for different sample types, vials or well plates.
Autosamplers are commonplace in most HPLC labs due to the considerable improvement in sample delivery and self-cleaning. High throughput labs will invest in multi-samplers especially as run times get shorter, allowing more samples to be analysed per day.
UV vs Fluorescence vs CAD vs MS
UV detectors are the most common detectors used in HPLC and come in different varieties. Variable Wavelength Detectors (VWD) are suitable for linearity and robustness, Diode Array Detectors (DAD) are suitable for looking across the entire UV-Vis spectrum and for applications such as peak purity and peak deconvolution. The sample requirement is that it must contain a chromophore, otherwise it is invisible to the UV detector.
Fluorescence detectors are many orders of magnitude more sensitive than UV detectors due to reduced noise and increased specificity but are limited to samples which contain a fluorophore. If they don’t then it is possible to derivatise the analyte, but this is often a difficult extra step in sample preparation.
If analytes are lacking a chromophore or a fluorophore then Charged Aerosol Detection (CAD) may be applicable. It is a near-universal system that produces a response which is directly proportional to the amount of analyte present which allows it to be used quantitatively without standards.
Mass Spectrometry (MS) is a highly sensitive detector than can provide secondary information about the analyte such as the empirical formula or structural information depending on the type of analyser employed.
UV-Vis detectors are almost universal on HPLC systems with the other detectors used as secondary detectors. Mass spectrometry may be the most sensitive and specific, but it is also the most expensive. The choice of detector will depend on the sample type and analyte properties such as presence of chromophores / fluorophores, volatility, complexity.