When investing in an HPLC system, it is important to know enough about what you want it to do, to be sure that what you buy will do the job. This guide is in three parts. First is a walk-through on HPLC systems, listing the choices available with their advantages. Second is a list of other parameters you will need to consider and which may affect your choice. And finally, a list of other things you will need before you can start to do HPLC for real.
If at the end you would like us to recommend a system for you, please call 01634 294001 between 9am and 5pm, Monday to Friday UK time. Alternativley you can email your requirements to [email protected] and we will get back to you with some suggestions.
An Overview of an HPLC System and the choices to be made
HPLC solvent is drawn through a filter into a pump, where it is pumped at a very precise and reproducible flow rate into the HPLC system. From this point on it is at high pressure, typically 1000-3000 psi (equivalent to 70-200 bar). At this stage the sample is introduced via a valve and is swept into the column, where separation occurs (at least that is the idea). As sample components are eluted from the column, they should be detected by the detector and the eluent then goes to waste or is collected by a fraction collector for future use. The electronic signal from the detector is recorded and usually integrated to give quantitative results. So an HPLC system MUST consist of:
- A pump
- An injection valve or autosampler
- A column
- At least one detector
- A computer data system.
It may also need:
- A column heater
- A pulse dampener
- A degasser
- A dynamic or static mixer
- A fraction collector
1) The pump.
Some HPLC methods run isocratically. This means that the same strength eluent is pumped throughout the separation. Hence an isocratic pump does not have to mix solvents, but simply pump a pre-mixed eluent continuously. Other methods require a solvent gradient. In this instance, the system starts with a weak eluent and gradually changes the composition to increase the strength as the run progresses. It is very important to know whether you need an isocratic or gradient pump.
2) Manual injector or autosampler.
A manual injector is always a good idea. Each sample may take 5-50 minutes to analyse, and you need to come back each time to make the next sample injection yourself. An Autosampler may be added at any stage, and is essentially a robot which does the injections for you.
Fixed or variable injection volume Autosampler
For most applications fixed volume is ok. The built in valve has a sample loop which is filled and that loop volume is the injection volume. To change it you have to change the loop.
A variable volume autosampler has a much larger loop and meters in the precise amount of sample with a syringe. This is more useful for research applications, it costs more money and is not an upgrade so if you want it, you must specify it when ordering.
Another important non-upgrade optionis is refridgeration (or heating) of the sample tray. Because the samples sit in the tray for hours, maybe even a day or two, it may be desirable to keep them chilled to prevent degradation. Similarly, some solutions may need to be heated to prevent the sample coming out of solution. Its available, but you must specify when ordering. You cannot usually upgrade later.
3) The column
This is not really part of the HPLC equipment but you will need one to do a separation. If your separation has been done before, you will be able to look up the separation conditions (solvent, column, flow rate, temperature etc) and work from there. Otherwise please call us on 01634 294 001 for advice regarding a suitable column to use.
The most popular type is ultra violet (UV) detection. A lot of samples have a UV chromophore (part of the molecule which absorbs UV light) and hence by tuning the wavelength to a region which is absorbed by the sample it can be detected easily.
UV detectors come in several flavours:
Fixed wavelength: usually 254nm. Set by a combination of lamp and filter. To change wavelength, you change the lamp and filter. It's the cheapest if you will only ever want one wavelength, but very restrictive. Only buy one if you really can't afford anything else.
Variable Wavelength: With these models you can change the wavelength to anything in the UV spectrum (190-400nm). Some detectors use two lamps so you can use the visible spectrum as well (400-800nm). Some detectors use a knob at the front to set the wavelength mechanically. Others have an electronic stepper motor to drive it there automatically. If you need external wavelength control, you must have an electronic wavelength drive.
Programmable Wavelength: This means that the detector can be programmed to change its wavelength during the course of a run. Useful if components do not all absorb at the same wavelength. Each peak can be detected at its lambda max.
Dual wavelength: A dual wavelength detector monitors two wavelengths instead of one. This is significantly more expensive, useful for watching out for impurities which do not show at the wavelength normally used and allows wavelength ratioing to check peak purity. If you need this we offer a 4-channel model (4 wavelengths at once) but you should at least consider buying a diode array detector.
Other features may include Peak Detection, useful if you are recycling the clean parts of the eluent, or to control a fraction collector, or Spectral scanning, which allows you to run the UV spectrum of an unknown compoment for identification purposes.
Refractive Index Detectors: An RI detector is more universal in that it detects everything, but is at least 1000 times less sensitive than UV. It is needed for sugars and many petrochemical products with no UV chromophore. It is relatively easy to use, but peaks can go down as well as up so integration needs special attention. A column oven is essential, as is a degasser,and the detector is totally unsuitable for use with gradients.
More Specialised Detectors
Fluorescence: Very sensitive, but only useful if sample components either fluoresce or can be derivatized to be fluorescent.
Electrochemical: Very sensitive, but species must contain an electroactive functional group (ie part of the molecule which can be oxidised or reduced by an electric current).
Conductivity:Very sensitive. Useful (essential) for detection of ionic species and this type of HPLC is known as an Ion Chromatograph.
5) Data System
To record the retention time of the sample, a chart recorder will do. But to quantify each component you need to integrate. This could be using a stand alone integrator, but in most cases will be with a computer data system. A data system will vary in its resolution (see the 'bit' number, 24 bit is the best), its capacity (how many HPLC's it can work with at the same time and how many detectors on each), its capability (integration functions, reporting functions etc) and its ease of use.
A degasser is nearly always required. If you buy a gradient pump it may be built in, but otherwise you need a separate unit. It contains a vacuum chamber and the solvent flows through a semi-permeable membrane tube which is in the vacuum chamber. Dissolved air is drawn through the walls of the tube leaving the eluent degassed, thereby preventing bubbles from forming in the HPLC.
7) Column Heater
Changes in temperature affect retention times, and we identify our peaks by their retention times. So even if the method specifies working at ambient, it is a good idea to use a column heater to thermosta the system. Some models have a small chamber, which is fine for most analytical methods, but otherwise very restricting. Some heaters have a peltier controlled oven, giving very precise temperature control, and the ability to control at ambient.
Other Parameters to consider when choosing a system
Physical Size: Will it fit on the bench? Some benches are not very deep. Allow space for power cables. Will it stack? ie detector on top of pump etc. Not essential, but it saves space.
Appearance: Better to buy equipment that looks good together. So if a particular detector is needed try and get a pump that looks right with it. See example photos with the equipment we list.
External Control: If you need to be able to set up a Mission Control system where all components are set by a central computer, make sure this is possible. There are advantages (simpler, can include intrument settings such as wavelength and flow rate in the method, only input sample details once for the autosampler and data system) and disadvantages (much more complicated communications, more difficult to fault find, choice of systems is somewhat reduced, much more expensive)
Upgradeable: If you might need gradient elution in the future and cannot afford it now, check there's an upgrade path
Ease of Use: Very important for multi-user labs. Some equipment is much easier to use than others.
Ease of maintenance: Some equipment can be serviced by a monkey with a screwdriver, others requires a rocket scientist. If you service it yourself, it can save loads of money, but check that it is not too complicated. We have a course where you can learn to Service HPLC Equipment.
Cost of Spares: Some equipment can be very cheap to buy (maybe because of big discounts to Universities etc), and seems like a bargain until you find out that parts cost a fortune and the engineer charges over £150 an hour to drive from the other end of the country!
Warranty: 1 year, 3 years, on-site or return to base?
System Compatibility: Does it stack nicely? Does it require long lengths of tubing to connect the column to the autosampler or flow cell? Gradient dwell volume?
IQOQPQ: If you need it, you'll know what it is. But not all equipment suppliers offer it, so you could end up doing it yourself!
Technical Support: How easy is it to get advice? Or Spares? And what happens if you lose the manual?
Finally, what else will you need to make it work?
Tubing: Stainless steel or PEEK, and some PTFE tube
Fittings: To connect the modules to the tubing.
Solvent Reservoirs and Sinter filters
Solvent: HPLC Grade. Check which solvents, and make sure they are all miscible, or you will find out why HPLC is sometimes referred to as High Pressure Liquid Chromatography!
COSHH Assessment: This is a risk assessment. What are the hazards, what precautions can be taken. Hazrds considered should include high pressure, flammable and toxic solvents, UV light, small particle size materials, and waste disposal.
An HPLC Method: This is the separation conditions. If you are not sure, check out our Guide to HPLC Methods.
Training: If you don't have the experience to handle the system, it won't take long before something expensive goes wrong! We have a series of 15 Training Courses to cover most of what you will need, and covering all levels of HPLC experience!