Refractive index (RI) is a property of all liquids and solids through which light can pass. As the light passes through the material, the beam is refracted and the extent of this refraction is measured by the Refractive Index. Air has a Refractive Index of 1.0, water of about 1.3 and glass of 1.5. Hence when a light beam passes from a material of one RI to another with a higher RI, we can observe the change in direction of the beam caused by the increase in RI. A common everyday example is to observe a spoon or glass rod in a beaker of water. The rod appears to bend as it enters the water.
Since all eluents and dissolved samples have a refractive index, this is the most universal method of detection available in HPLC. The detector measures a change in refractive index, so the reference cell is filled with the eluent, and the detector subtracts the reading from the reference cell from that of the sample cell. However the change which is measured is very much smaller than the example given above, typically of the order of 0.001 RI units.
Ease of use. An RI detector is the easiest of all the detectors to use. There is no wavelength or voltage to set or other parameters to optimise. Just flush the reference cell for 30 minutes and the detector is ready for use.
Purging the Reference Cell. When the detector is turned on, the reference cell must be flushed for at least 30 minutes, preferably longer. This is normally done by pressing the PURGE button. The flow path through the detector will normally just pass through the sample cell and to waste. Pressing the PURGE button operates a valve which diverts the flow coming from the sample cell to go through the reference cell as well before going to waste. Once this process is complete, pressing the PURGE button again will isolate the reference cell, and flow from the sample cell goes directly to waste. The reason that such a long time is required is that the reference cell needs time to reach the temperature of the eluent, since it has a relatively high mass compared to the eluent flow rate. Purging should be repeated periodically, or when the baseline starts to drift.
Degassing. Air has a MUCH lower RI than any sample or eluent, so it is really important that there is no air in the eluent flow. Degassing is vital for eluents which may contain dissolved air.
Sensitivity. RI detectors are at least 1000 times less sensitive than UV detectors, which are in turn much less sensitive than Fluorescence or Electrochemical detection. An RI detector is also about 100 times less sensitive than an ELSD. So the peaks will be much smaller than with other types of detectors unless a higher sample concentration is used.
Positive and negative peaks. Since sample components could have a higher or lower RI than the solvent used as eluent, peaks can be both positive and negative with RI detection. This can give problems with a data system, and so if possible it is best to choose an eluent which has a higher or lower RI than most if not all of the sample components to be analysed.
The problem with a data system is that it does not interpret a negative deviation as a peak. However it cannot accept such a sharp change as just part of the baseline, so it attempts to create a baseline which runs under the peak like a rubber band. Adjacent peaks are then integrated down to this false baseline, resulting in vastly inflated integration results. The solution is to mark the negative peak region as such using a time window in the data system. Within this window the signal is inverted and all peaks occurring within that time window are thus shown as negative peaks and correctly integrated. Care must be taken with the time window, so that no positive peaks elute during that window. Otherwise the same problem occurs but in reverse!
Not suitable for Gradient Elution Even a small change in solvent composition causes baseline drift. Running a gradient causes a massive change in RI (at the sensitivity range at which the detector is used) which thus causes the baseline to drift rapidly off scale. So an RI detector cannot be used when running a gradient.
Not usable with a Gradient Pump mixing isocratic solvent on line. All eluent must be premixed off-line, otherwise baseline noise is severe. A gradient pump premixing an isocratic eluent generates a flow consisting of alternating small slugs of each pure eluent component, often not properly mixed. The refractive index change observed when these arrive at the flow cell is significant, and usually makes such an approach impractical, and pre-mixing the eluent becomes a necessity.
A column oven is essential. Any tiny change in temperature causes baseline drift. Temperature must be very precisely controlled, preferably to 0.1oC. Ideally the column heater should pulse the heater, making the pulses longer or shorter to control temperature to match the rate of heat loss. Any column heater which uses a thermostat will give a wavy baseline using an RI detector.
Lamp Life RI detectors normally use white light, from a tungsten lamp or an LED. Either way, the lamps are inexpensive and last a very long time. The LA2000 detector sold by Laserchrom has a lamp life of 50,000 hours, which equates to 5 years using it 24/7, or 30 years if used Monday-Friday 9-5! So if you want to leave it on when not in use, with the solvent recycling to the eluent bottle and the purge button pressed, feel free! It will then be ready to go as soon as you come in!
Applications. Typical applications are sugars or industrial polymers with no UV chromophore. In particular an RI detector is used with GPC applications.