HPLC is one of the most common analytical methods, ubiquitous in laboratories worldwide. The sheer number of HPLC analyses that are conducted in laboratories every day makes it imperative that the process runs as efficiently and effectively as possible. A small waste of time or solvent on a single run adds up to significant losses over time.
However, like many familiar processes, HPLC is often performed routinely without proper thought about optimizing output. The following MIND MAP provides some mental prompts that should be considered when running a specific HPLC process, or when conducting HPLC analysis in general. Often, the simple adjustment of a standard parameter can lead to huge gains in efficiency and performance.
Invest in new equipment
Any of the components of the HPLC system, or the entire system, may need to be replaced at some time. At this stage, the principal decision is whether to buy a brand new system or component, or a refurbished version. Refurbished equipment can often have similar specifications to newer models but may be available for a greatly reduced capital outlay. Many refurbished models also carry a guarantee.
Upgrade to UHPLC
Transferring from HPLC to UHPLC must be a serious consideration when wishing to increase HPLC output. UHPLC represents the latest technology in liquid chromatography, and interest and use of UHPLC systems has increased rapidly due to its many benefits, including improved throughput, sensitivity and resolution. It uses a smaller particle size than HPLC (about 1-2 μm), and runs at an increased pressure (up to 14,500 psi). By decreasing the particle size of the packing material, the number of theoretical plates can be maximized, making shorter column lengths possible and increasing the range of usable flow rates. Ultimately, the analytical method is shortened without losing separation quality.
Buy new equipment
See June 2010 Lab Manager’s Independent Guide to Purchasing a Liquid Chromatography System.
Buy refurbished equipment
Visit www.LabX.com/HPLC to view hundreds of used and refurbished LC systems.
Instigate user training
HPLC is a complex technique requiring skill and experience to derive the best outcomes. If output is falling below what is expected, consider instigating staff training, particularly if users are new or inexperienced. Staff training is an essential part of GLP compliance. It may also contribute to Continuing Education.
Some companies offer in-house training courses where users are trained on the equipment available. This is a particularly appealing option if several users need to be trained at one time.
Some courses are available online for users to follow at their own convenience. These courses are particularly suited to theoretical training. Considerable HPLC resources are also available online.
Conferences and meetings are a useful way to expose staff to the rigors of HPLC.
In-depth Preventative Maintenance/calibration
Calibration refers to instrument qualification or HPLC performance verification. In most pharmaceutical laboratories, calibration is performed on each HPLC module usually every 6 to 12 months, according to the company’s standard operating procedures. Poor calibration can affect a system’s output.
Flow calibration tool
A digital liquid HPLC flowmeter is a convenient and accurate way to calibrate HPLC pump flow rates. QA/QC software can be purchased for this purpose.
Parts of the system may be rebuilt in order to restore calibration:
- replace valves/fittings
- switch to diaphragm valves
- consider replacing injection ports, tubing, couplings and inlet lines
Find service partner
To keep the HPLC system operating efficiently, it is important to have it serviced regularly. For this you need a service partner you can trust and on whom you can rely. Consider personal recommendations or consult service directories to find such a partner. Always check their credentials and qualifications and decide whether to use a service contract.
Vary experimental conditions
The perfect HPLC run depends on the appropriate selection of components and conditions. Consider varying:
- HPLC column size
- Stationary phase
- Mobile phase
- Solvent gradient
An autosampler provides the means to introduce a sample automatically into the inlets. Automatic insertion provides better reproducibility and time optimization. Different kinds of autosamplers exist. Autosamplers can be classified in relation to sample capacity (auto-injectors vs. autosamplers, where auto-injectors can work a small number of samples), robotic technologies, or analysis techniques.
HPLC technology is fast moving, and the same is true for autosamplers. If your autosampler is more than a few years old, consider investing in a newer model or upgrading your existing model for improved HPLC output.
If you do not currently use an autosampler, this is one simple way to improve HPLC output.
For cost savings, while still enjoying much of the functionality of newer models.
For current models and latest technology.
The column itself is the most fundamental part of the HPLC system. Always consider this component when introducing improvements to the system.
Consider adding an oven, heater or cooler, as appropriate, to improve output.
Clean or replace column
A number of HPLC problems can be attributed to the column itself:
- change in peak retention times
- peak tailing or peak splitting
- mystery peaks
These problems can often be rectified by cleaning or replacing the column, thus improving HPLC output.
If the column is relatively new, try cleaning it to improve output. If this fails, purchase a new column.
If the column is old or has already been cleaned, consider purchasing a new column.
Improve sample preparation
Sample preparation is an essential step in many analyses and can often determine the success of the output. Sample preparation involves a wide variety of processes; the art is in deciding which form of sample preparation is most suitable for a given application. HPLC output may be improved by changing sample preparation to a more suitable technique.
A number of mechanical processes may be used to prepare the samples. These include grinding, sieving and blending (for homogenization); and centrifugation and filtration (including microfiltration and ultrafiltration) to remove solid matter that may disrupt the chromatographic process.
Phase transfer separation
Sample purification can be achieved through a number of phase transfer processes, including distillation, vaporization, drying, dissolution, crystallization and precipitation.
Solvent extraction is a method of purification that separates the desired component from an insoluble impurity.
The most common technique for electroseparation is electrophoresis, although other techniques exist as well. In electrophoresis, particles are separated by the application of a uniform electrical field.
For compounds that do not lend themselves readily to HPLC, a more suitable derivative can first be prepared by means of a chemical reaction. For example, a salt can be prepared by adjusting pH levels.