HPLCs are becoming a standard requirement in almost every lab. We purchased the Waters HPLC in our department for the multiple detection features it offers – in addition to be able to detect and quantify the UV-active compounds, this HPLC is extremely helpful for detection of carbohydrates which are generally UV-transparent.
The instrument contains a dual pump system that can take very high pressures (up to 6000 psi). A wide variety of solvents can be used in the two pumps, but we mainly use a combination of different buffers (prepared in water) and sometimes organic solvents such as acetonitrile and methanol. The injection volume can vary from 5 ul to 5 ml depending on the column used. We use the small volume loop when we are analyzing compounds on an analytical HPLC column, and a larger loop (we have a 2 ml loop) is used when the preparative column for separating and purifying compounds is being utilized. Flow rates can vary from 0.5 ml/min to 10 ml/min, depending on the column.
The instrument has the advantage of being connected to either a refractive index (RI) detector or a dual wavelength absorbance detector. In our laboratory, we have the RI detector which enables us to detect sugars. This RI detector can be used for the detection of other non-UV absorbing compounds as well. A temperature controlled flow cell is available in the RI detector. The LED in the RI detector can be set at 880 nm or 690 nm as required. The LED has a very long lifetime, unlike standard lamps. The detector has an internal cuvette with a 10 ul volume and can take pressures up to 100 psi. The detector should be kept in a cool room – the cuvette temperature is constant and can only be set above the ambient temperature – hence, the ambient temperature of the room should be as low as possible.
An autosampler is also available from the manufacturer; it can be programmed to specifically inject various samples during different run programs. The instrument also offers the advantage of heating or cooling the sample before injection with a heating/cooling module. A similar module is also provided for the column so that the temperature of the sample can be maintained while the run is in progress. We, however, do not currently use this module.
All parts of the instrument are controlled by the Breeze™ software. The software is very easy to understand. The software allows protocols to be written and saved, such that the run can be controlled and monitored at different time points. The software also allows for the run to be monitored in real time. A report from the run can be generated easily. The View Data option available in the software can be used to process peaks in order to obtain retention times and peak volumes. Alternately, the raw data can be readily exported in ASCII format and processed using your choice of software. We find this feature very useful for obtaining digitized plots of various runs. This feature is not present in several other instruments.
A detailed manual accompanies the instrument. Any queries are also addressed immediately through the online help center.