Mobile phase refractive index

Low sensitivity to sample or mobile phase refractive index change... 

Sensitivity to mobile phase composition changes and pump pulsations is often a reflection of the detector s sensitivity to mobile phase refractive index (Rl). R1 sensitivity is a function of flow cell optical design. As a rule, both density and RI change with mobile phase composition. Stratification develops in the flow cell creating a liquid prism, steering rays to touch or not touch the inner cell bore. Rays that touch the cell bore are partially absorbed and interpreted (erroneously) by the detector as a change in sample absorbance. 

In a more demanding application, the synthesis of various fatty acid alkanolamides was followed by quantitative HPLC using a reversed-phase column and a THF/acetoni-trile/water, pH 2.6, mobile phase. Refractive index detection was used. The methyl ester starting materials were resolved, as were free fatty acids mono-, di-, and triglycerides dialkanolamides and amine esters (154). As a mle, GC or TLC methods provide a more complete characterization of reaction products than does HPLC. 

Another classification of detector is the bulk-property detector, one that measures a change in some overall property of the system of mobile phase plus sample. The most commonly used bulk-property detector is the refractive-index (RI) detector. The RI detector, the closest thing to a universal detector in lc, monitors the difference between the refractive index of the effluent from the column and pure solvent. These detectors are not very good for detection of materials at low concentrations. Moreover, they are sensitive to fluctuations in temperature. 

For acrylate polymers with higher levels of carboxylic acids, THF can be modified by the addition of acids such as acetic, phosphoric, or trifluoroacetic. Levels as high as 10% acetic acid are considered acceptable by most manufacturers for their styrene/DVB columns. If such a modified mobile phase is used, it may need to be premixed rather than generated using a dynamic mixing HPLC pump because on-line mixing often leads to much noisier baselines, particularly when using a refractive index detector. 

Refractive index detectors. These bulk property detectors are based on the change of refractive index of the eluant from the column with respect to pure mobile phase. Although they are widely used, the refractive index detectors suffer from several disadvantages — lack of high sensitivity, lack of suitability for gradient elution, and the need for strict temperature control ( + 0.001 °C) to operate at their highest sensitivity. A pulseless pump, or a reciprocating pump equipped with a pulse dampener, must also be employed. The effect of these limitations may to some extent be overcome by the use of differential systems in which the column eluant is compared with a reference flow of pure mobile phase. The two chief types of RI detector are as follows. 

The deflection refractometer (Fig. 8.4), which measures the deflection of a beam of monochromatic light by a double prism in which the reference and sample cells are separated by a diagonal glass divide. When both cells contain solvent of the same composition, no deflection of the light beam occurs if, however, the composition of the column mobile phase is changed because of the presence of a solute, then the altered refractive index causes the beam to be deflected. The magnitude of this deflection is dependent on the concentration of the solute in the mobile phase. 

The free oil can be determined by an ion exchange HPLC technique. A solution of the sample in ethyl alcohol is analysed by high-performance ion exchange chromatography using a specially prepared ion exchange resin stationary phase, ethanol mobile phase, and differential refractive index detection. 

The dead point is obtained by including in the sample a trace of an unretained solute or, more often, one of the components of the mobile phase. For example, when using a methanol water mixture as the mobile phase, the dead point is obtained from the elution of a pure sample of methanol. The pure methanol can often be monitored, even by a UV detector, as the transient change in refractive index resulting from the methanol is sufficient to cause a disturbance that is detectable. 

If the mixture to be separated contains fairly polar materials, the silica may need to be deactivated by a more polar solvent such as ethyl acetate, propanol or even methanol. As already discussed, polar solutes are avidly adsorbed by silica gel and thus the optimum concentration is likely to be low, e.g. l-4%v/v and consequently, a little difficult to control in a reproducible manner. Ethyl acetate is the most useful moderator as it is significantly less polar than propanol or methanol and thus, more controllable, but unfortunately adsorbs in the UV range and can only be used in the mobile phase at concentrations up to about 5%v/v. Above this concentration the mobile phase may be opaque to the detector and thus, the solutes will not be discernible against the background adsorption of the mobile phase. If a detector such as the refractive index detector is employed then there is no restriction on the concentration of the moderator. Propanol and methanol are transparent in the UV so their presence does not effect the performance of a UV detector. However, their polarity is much greater than that of ethyl acetate and thus, the adjustment of the optimum moderator concentration is more difficult and not easy to reproduce accurately. For more polar mixtures it is better to explore the possibility of a reverse phase (which will be discussed shortly) than attempt to utilize silica gel out of the range of solutes for which it is appropriate. 

The separation was carried out on a TSKgel Amide-80 column 4.6 mm i.d. and 25 cm long with a mobile phase consisting of a 80% acetonitrile 20% water mixture. The flow rate was 1 ml/min and the column was operated at an elevated temperature of 80°C. The saccharides shown were 1/ rhamnose, 2/ fucose, 3/ xylose, 4/ fructose, 5/ mannose, 6/ glucose, 7/ sucrose and 8/ maltose. The analysis was completed in less than 20 minutes. These types of separations including other biomonomers, dimers and polymers are frequently carried out employing refractive index detection. 

This classification is concerned with whether the detector monitors a property of the solute (analyte), e.g. the UV detector, or a change in some property of the solvent (mobile phase) caused by the presence of an analyte, e.g. the refractive index detector. 

Published refractive index data for the mobile phase, polystyrene, polyacrylonitrile, and the two monomers were used to calculate refractive index detector calibrations for the two homopolymers. The published data were used to determine relationship between refractive index increments of monomer and corresponding homopolymer. Chromatographic refractometer calibrations for the two homopelymers were then calculated from experimentally measured calibration data for the two monomers. 

It is important to know the influence of the physicochemical parameters of the mobile phase (dipole moment, dielectric constant, and refractive index) on solvent strength and selectivity. The main interactions in planar chromatography between the molecules of the mobile phases and those of solutes are caused by dispersion forces related to the refractive index, dipole-dipole forces related to the dipole moment, induction forces related to a permanent dipole and an induced one, hydrogen bonding, and dielectric interactions related to the dielectric constant. Solvent strength depends mainly on the dipole moment of the mobile phase, whereas the solvent selectivity depends on the dielectric constant of the mobile phase. 

Detectability may be a significant problem with homologous series of unsaturated compounds, particularly //-alkanes. For these compounds, refractive index detection or evaporative light-scattering, both of which are described elsewhere in the book, may be of use. Indirect photometry is a useful detection scheme for compounds that do not absorb in the UV. Acetone, methylethyl ketone, methyl propyl ketone, methyl isopropyl ketone, methyl isobutyl ketone, and acetophenone are added to an acetonitrile/water mobile phase, generating a negative vacancy peak when the nonchro-mophoric analyte emerges and a positive peak if the ketone is adsorbed and displaced.70 Dodecyl, tetradecyl, cetyl, and stearyl alcohols also have been derivatized with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole and the derivatives separated on Zorbax ODS in a mobile phase of methanol and 2-propanol.71... 

Bulk property detectors function by measuring some bulk physical property of the mobile phase, e.g., thermal conductivity or refractive index. As a bulk property is being measured, the detector responses are very susceptible to changes in the mobile phase composition or temperature these devices cannot be used for gradient elution in LC. They are also very sensitive to the operating conditions of the chromatograph (pressure, flow-rate) [31]. Detectors such as TCD, while approaching universality in detection, suffer from limited sensitivity and inability to characterise eluate species. 

Products were analyzed via Waters Model 515 HPLC Pump fitted with a Waters model 2410 refractive index detector. Separations was performed via an Aminex HP-87H 300mm column at 65°C using 0.005M H2SO4 as the mobile phase. Compounds calibrated for this work included xylitol, arabitol, erythritol, threitol, PG, EG, glycerol, lactate, 1-propanol, 2-propanol, ethanol, methanol, and the butanetriol isomers. Any compounds not visible by RID were not quantified in this work. 

The function of the detector in hplc is to monitor the mobile phase emerging from the column. The output of the detector is an electrical signal that is proportional to some property of the mobile phase and/or the solutes. Refractive index, for example, is a property of both the solutes and the mobile phase. A detector that measures such a property is called a bulk property detector. Alternatively, if the property is possessed essentially by the solute, such as absorption of uv/visible radiation or electrochemical activity, the detector is called a solute property detector. Quite a large number of devices, some of them rather complicated and tempremental, have been used as hplc detectors, but only a few have become generally useful, and we will examine five such types. Before doing this, it is helpful to have an idea of the sort of characteristics that are required of a detector. 

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