Introduction

The refractive index detector is a bulk property detector. A bulk property detector responds to some physical property of the total column eluent and not some specific property of the solute. Bulk property detectors have an inherently limited sensitivity, irrespective of the instrumental technique used. Consider a hypothetical bulk property detector that is to monitor the density of the column eluent. Assume it is required to detect the concentration of a dense material, such as carbon tetrachloride (specific gravity 1.595), at a level of 1 |g mL"1 in «-heptane (specific gravity 0.684).

This situation will be particularly favourable for this hypothetical detector, as the solute to be sensed exhibits a large difference in density from that of the solvent. Let the change in density resulting from the presence of the solute at a concentration of 10"6 g mL"1 be Ad. It follows that, to a first approximation:

where d1 is the density of the solute, carbon tetrachloride, d2 is the density of the mobile phase, «-heptane, and XS is the concentration of the solute to be detected.

Thus, for the example given:

Ad"

Now the coefficient of cubical expansion of n-heptane is approximately 1.6 x 10~3 per °C. Thus, the temperature, AT, that would produce a change in density equivalent to the presence of carbon tetrachloride at a concentration of 10_6gmL_1 can be calculated.

It follows that:

AT-5Z1XE: oc

Assuming that a concentration of one part per million carbon tetrachloride is just detectable (it provides a signal-to-noise ratio of 2), then the temperature fluctuations must be maintained below 1.8 x 10"4°C to achieve this sensitivity. In practice, such temperature stability would be extremely difficult to maintain and thus the temperature control can severely limit the sensitivity obtainable from such a detector. Even the heat of adsorption and desorption of the solute to and from the stationary phase can easily result in temperature changes of this order of magnitude. The density of the contents of the cell will also change with pressure and, if there is a significant pressure drop across the cell, with flow rate. These restrictions apply to all bulk property detectors and so all bulk property detectors will have a limited sensitivity determined by the stability of the ambient conditions. This limit of detection is probably around 10"6 g mL"1.

The refractive index detector was one of the first online detectors to be developed and was described by Tiselius and Claesson in 1942. It was also one of the first online liquid chromatography (LC) detectors to be made commercially for general use. The refractive index detector is probably the least sensitive of the commonly used LC detectors. Its major disadvantage (as already discussed) is its sensitivity to changes in ambient conditions, such as temperature, pressure and flow rate. Another handicap is that it cannot be used for gradient elution, due to the continuous change in mobile-phase refractive index that results from the change in solvent composition. Nevertheless, as the refractive index detector has a universal response, it can be extremely useful for detecting those compounds that are nonionic, do not absorb in the UV and do not fluoresce (e.g. aliphatic alcohols, fatty acids, ethers, etc.).

When a monochromatic ray of light passes from one isotropic medium, A, to another, B, it changes its velocity and direction. The change in direction is called the refraction, and the relationship between the angle of incidence and the angle of refraction is given by Snell's law:

where i is the angle of incident light in medium A, r is the angle of refractive light in medium B, nA is the refractive index of medium A, nB is the refractive index of medium B and nB is the refractive index of medium B relative to that of medium A.

Refractive index is a dimensionless constant that normally decreases with increasing temperature; values given in the literature are usually quoted at 20°C or 25°C, the actual measurement taken as the mean value for the two sodium lines. If a cell takes the form of a hollow prism through which the mobile phase flows, a ray of light passing through the prism will be deviated from its original path. If the light is focused on to a photocell the output will change as the refractive index of the mobile phase in the cell changes. This method of monitoring refractive index is called the angle of deviation method and has been used by a number of manufacturers in their detector design.

The modern refractive index detector is the result of considerable research which has been extended by the research and development laboratories of many instrument companies. A diagram of a simple refractive index detector based on the angle of deviation method measurement is shown in Figure 1. A beam of light from an incandescent lamp passes through an optical mask that confines the beam to the region of the cell. The lens collimates the light beam, which passes through both the sample and reference cells to a plane mirror. The mirror reflects the beam back through the sample and reference cells to a lens, which focuses it on to a photocell.

The location of the beam, rather than its intensity, is determined by the angular deflection of the beam caused by the difference in refractive index between the contents of the two cells. As the beam changes its position of focus on the photoelectric cell, the output changes and the resulting difference signal is electronically modified to provide a signal proportional to the concentration of solute in the sample cell.

An alternative method of refractive index measurement, the Fresnel method, has also been used in the design of commercially fabricated detectors. The two different systems provide comparable performance with respect to sensitivity and linearity, and mostly differ in the manufacturing techniques used to construct the instruments. The relationship between the reflectance from an interface between two transparent media, and their respective refractive indices, is given by Fresnel's equation:

sin2(i — r) tan2(i — r) sin2(i + r) tan2(i + r)

where R is the ratio of the intensity of the reflected light to that of the incident light and the other symbols have the meanings previously assigned to them. Now:

where n1 is the refractive index of medium 1 and n2 is the refractive index of medium 2.

Consequently, if medium 2 represents the liquid eluted from the column, then any change in n2 will result in a change in R and thus the measurement of R could determine changes in n2 resulting from the presence of a solute. Conlon utilized this principle to develop a practical refractive index detector. His device, now obsolete, illustrates the principle of the Fresnel method very simply (Figure 2).

The sensing element consists of a rod prism sealed into a tube through which the solvent flows. The rod prism is made from a glass rod 6.8 mm in diameter

Figure 1 The refractive index detector based on the angle of deviation method of measurement. (Courtesy of Waters Chromatography.)
Figure 2 Early detector based on the Fresnel method of refractive index measurement.

and 10 cm long, bent to the correct optical angle (just a little less than the critical angle) and an optical flat is ground on the apex of the bend, as shown in Figure 2. The optical flat is then sealed into the window of a suitable tube that acts as a flow-through cell. The photocell is arranged to be one arm of a Wheat-stone bridge and a reference photocell (not shown) monitoring light direct from the cell is situated in another arm of the bridge.

This detector was never manufactured as it had too large a cell volume and limited sensitivity. However, it was one of the first refractive index detectors to work on the Fresnel principle. A commercial refractive index detector that works on this principle is shown in Figure 3.

Light from a tungsten lamp is directed, through an infrared filter to prevent heating the cell, to a magnifying assembly that also splits the beam into two. The two beams are focused through the sample and reference cells respectively. Light refracted from the mobile phase/prism surface passes through the prism assembly and is then focused on two photocells. The prism assembly also reflects light to a user port where the surface of the prism can be observed. The output from the two photocells is electronically processed and either passed to a potentiometric recorder or a computer data acquisition system.

The range of refractive index covered by the instrument for a given prism is limited and consequently three different prisms are made available to cover the refractive index ranges of 1.35-1.4, 1.41-1.44 and 1.40-1.55 respectively. An example of the separation of a series of polystyrene standards monitored by the detector is shown in Figure 4. The separation was carried out by size exclusion on a column packed with 5 |im particles operated at a flow rate of 0.8 mL min"1.

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