A differential refractometer (DRI), or refractive index detector (RI or RID) is a detector that measures the refractive index of an analyte relative to the solvent. They are often used as detectors for high-performance liquid chromatography and size exclusion chromatography. They are considered to be universal detectors because they can detect anything with a refractive index different from the solvent, but they have low sensitivity. [1]
The refractive index increment,, often expressed as mL/g, [2] is the change in a solutions' refractive index vs concentration. A differential refractometer facilitates determining this term. [3] Typical light sources include Helium–neon laser, Argon-ion laser, and Sodium-vapor lamp. [4] There are two compartments or flow cells, one for the sample and the other for the reference solution. [5] [4]
The optical wedge or prism sits after the cells and separates the light coming from the flow cells. [4] The difference in refractive index causes the light paths to reflect at different angles. [6] [7] This difference is magnified by the optical wedge/prism. [8]
A detector that can measure of range of wavelengths, usually a Photodiode array, [4] [8] measures the position of the two light paths. The detector quantifies the angle of refraction, which is proportional to the refractive index.
There exist various brands of differential refractometers. Popular models include:
All refractive index detectors require calibration upon first setting up the instrument as well as periodic quality control. [9] [10] [11] Most manufacturer's recommend calibration with pure water and a sucrose calibration solution of a known refractive index. [12] Once the instrument is in calibration mode, the pure water acts as a zero baseline reading, while the sucrose solution compares its known RI to the output, and the machine is adjusted accordingly. [13]
After the pump has not been used for a while, it is necessary to purge the tubes of any contaminant air that has diffused into the channels. This is typically accomplished with isopropyl alcohol. [10]
Differential refractometers are often used for the analysis of polymer samples in size exclusion chromatography. Other types of information that can be gathered from differential refractometers are:
Since the molecular weight (or extent of polymerization) of a solute will correspond to a specific refractive index increment, the relationship between increasing solute weight and refractive index increment can be plotted to determine the exact molecular weight of an unknown solute. [14]
Increasing addition of solute will alter the solvent's viscosity and polarizability, which cannot be measured by instruments that rely on low viscosity. [14] Since differential refractometer is an external tool, [15] [16] the solvent viscosity does not pose a physical barrier to measurement, making them universal detectors. [17]
The shape of a solute will influence it's induced dipole. [18] This will affect the solvent polarizability, which affects the refractive index. [19]
There are many practical factors that can affect the accuracy of a differential refractometer.
When solutes are added to a solvent, they change the solution's optical density. The size, [20] polarizability [19] and shape and molecular structure [20] of a solute all have effects on the refractive index of a solution. Generally, a Gaussian distribution is observed, although deviations occur. [20]
A controlled temperature is needed to ensure accurate measurements, as temperature affects many properties of a solution. [21] If the temperature changes between measurements, this variance will be reflected in the measured refractive index. [22]
Cauchy's equation and Sellmeier equation describe the effect of wavelength on refractive index of medium.
The use of and results from differential refractometers are valuable in numerous fields of science, with its theory and function applied in various research directions, including drug analysis [23] and nanoparticle tracking. [24]
The nature of refractive indexes allows RIDs to be used in conjunction with additional analytical chemistry instruments. Following the use of other machines, differential refractometers can immediately (further) characterize compounds eluting from chromatographers, spectrometers, and detectors, including:
The molecular mass is the mass of a given molecule. Units of daltons (Da) are often used. Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The derived quantity relative molecular mass is the unitless ratio of the mass of a molecule to the atomic mass constant.
Ultraviolet–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum. Being relatively inexpensive and easily implemented, this methodology is widely used in diverse applied and fundamental applications. The only requirement is that the sample absorb in the UV-Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance (A) or transmittance (%T) or reflectance (%R), and its change with time.
Size-exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their shape, and in some cases size. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc., which have been dissolved into liquid solutions.
Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC), that separates high molecular weight or colloidal analytes on the basis of size or diameter, typically in organic solvents. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964. The proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.
Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential absorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.
In polymer chemistry, the molar mass distribution describes the relationship between the number of moles of each polymer species and the molar mass of that species. In linear polymers, the individual polymer chains rarely have exactly the same degree of polymerization and molar mass, and there is always a distribution around an average value. The molar mass distribution of a polymer may be modified by polymer fractionation.
Surface plasmon resonance (SPR) is a phenomenon that occurs where electrons in a thin metal sheet become excited by light that is directed to the sheet with a particular angle of incidence, and then travel parallel to the sheet. Assuming a constant light source wavelength and that the metal sheet is thin, the angle of incidence that triggers SPR is related to the refractive index of the material and even a small change in the refractive index will cause SPR to not be observed. This makes SPR a possible technique for detecting particular substances (analytes) and SPR biosensors have been developed to detect various important biomarkers.
Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.
Dynamic light scattering (DLS) is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are usually analyzed using the intensity or photon autocorrelation function. In the time domain analysis, the autocorrelation function (ACF) usually decays starting from zero delay time, and faster dynamics due to smaller particles lead to faster decorrelation of scattered intensity trace. It has been shown that the intensity ACF is the Fourier transform of the power spectrum, and therefore the DLS measurements can be equally well performed in the spectral domain. DLS can also be used to probe the behavior of complex fluids such as concentrated polymer solutions.
Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC); however, SFC typically utilizes carbon dioxide as the mobile phase. Therefore, the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state whereby bulk liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. The idea of liquid and gas properties convergence was first envisioned by Giddings.
Static light scattering is a technique in physical chemistry that measures the intensity of the scattered light to obtain the average molecular weight Mw of a macromolecule like a polymer or a protein in solution. Measurement of the scattering intensity at many angles allows calculation of the root mean square radius, also called the radius of gyration Rg. By measuring the scattering intensity for many samples of various concentrations, the second virial coefficient, A2, can be calculated.
Field-flow fractionation, abbreviated FFF, is a separation technique invented by J. Calvin Giddings. The technique is based on separation of colloidal or high molecular weight substances in liquid solutions, flowing through the separation platform, which does not have a stationary phase. It is similar to liquid chromatography, as it works on dilute solutions or suspensions of the solute, carried by a flowing eluent. Separation is achieved by applying a field or cross-flow, perpendicular to the direction of transport of the sample, which is pumped through a long and narrow laminar channel. The field exerts a force on the sample components, concentrating them towards one of the channel walls, which is called accumulation wall. The force interacts with a property of the sample, thereby the separation occurs, in other words, the components show differing "mobilities" under the force exerted by the crossing field. As an example, for the hydraulic, or cross-flow FFF method, the property driving separation is the translational diffusion coefficient or the hydrodynamic size. For a thermal field, it is the ratio of the thermal and the translational diffusion coefficient.
Multi-Angle light scattering describes a technique for measuring the light scattered by a sample into a plurality of angles. It is used for determining both the absolute molar mass and the average size of molecules in solution, by detecting how they scatter light. A collimated beam from a laser source is most often used, in which case the technique can be referred to as multiangle laser light scattering (MALLS). The insertion of the word laser was intended to reassure those used to making light scattering measurements with conventional light sources, such as Hg-arc lamps that low-angle measurements could now be made.
In nonlinear optics z-scan technique is used to measure the non-linear index n2 and the non-linear absorption coefficient Δα via the "closed" and "open" methods, respectively. As nonlinear absorption can affect the measurement of the non-linear index, the open method is typically used in conjunction with the closed method to correct the calculated value. For measuring the real part of the nonlinear refractive index, the z-scan setup is used in its closed-aperture form. In this form, since the nonlinear material reacts like a weak z-dependent lens, the far-field aperture makes it possible to detect the small beam distortions in the original beam. Since the focusing power of this weak nonlinear lens depends on the nonlinear refractive index, it would be possible to extract its value by analyzing the z-dependent data acquired by the detector and by cautiously interpreting them using an appropriate theory. To measure the imaginary part of the nonlinear refractive index, or the nonlinear absorption coefficient, the z-scan setup is used in its open-aperture form. In open-aperture measurements, the far-field aperture is removed and the whole signal is measured by the detector. By measuring the whole signal, the beam small distortions become insignificant and the z-dependent signal variation is due to the nonlinear absorption entirely. Despite its simplicity, in many cases, the original z-scan theory is not completely accurate, e.g. when the investigated sample has inhomogeneous optical nonlinear properties, or when the nonlinear medium response to laser radiation is nonlocal in space. Whenever the laser induced nonlinear response at a certain point of the medium is not solely determined by the laser intensity at that point, but also depends on the laser intensity in the surrounding regions, it will be called a nonlocal nonlinear optical response. Generally, a variety of mechanisms may contribute to the nonlinearity, some of which may be nonlocal. For instance, when the nonlinear medium is dispersed inside a dielectric solution, reorientation of the dipoles as a result of the optical field action is nonlocal in space and changes the electric field experienced by the nonlinear medium. The nonlocal z-scan theory, can be used for systematically analyzing the role of various mechanisms in producing the nonlocal nonlinear response of different materials.
A chromatography detector is a device that detects and quantifies separated compounds as they elute from the chromatographic column. These detectors are integral to various chromatographic techniques, such as gas chromatography, liquid chromatography, and high-performance liquid chromatography, and supercritical fluid chromatography among others. The main function of a chromatography detector is to translate the physical or chemical properties of the analyte molecules into measurable signal, typically electrical signal, that can be displayed as a function of time in a graphical presentation, called a chromatograms. Chromatograms can provide valuable information about the composition and concentration of the components in the sample.
A direct electron ionization liquid chromatography–mass spectrometry interface is a technique for coupling liquid chromatography and mass spectrometry (LC-MS) based on the direct introduction of the liquid effluent into an electron ionization (EI) source. Library searchable mass spectra are generated. Gas-phase EI has many applications for the detection of HPLC amenable compounds showing minimal adverse matrix effects. The direct-EI LC-MS interface provides access to well-characterized electron ionization data for a variety of LC applications and readily interpretable spectra from electronic libraries for environmental, food safety, pharmaceutical, biomedical, and other applications.
Analytical light scattering (ALS), also loosely referred to as SEC-MALS, is the implementation of static light scattering (SLS) and dynamic light scattering (DLS) techniques in an online or flow mode. A typical ALS instrument consists of an HPLC/FPLC chromatography system coupled in-line with appropriate light scattering and refractive index detectors. The advantage of ALS over conventional steady-state light scattering methods is that it allows separation of molecules/macromolecules on a chromatography column prior to analysis with light scattering detectors. Accordingly, ALS enables one to determine hydrodynamic properties of a single monodisperse species as opposed to bulk or average measurements on a sample afforded by conventional light scattering.
An evaporative light scattering detector (ELSD) is a destructive chromatography detector, used in conjunction with high-performance liquid chromatography (HPLC), ultra high-performance liquid chromatography (UHPLC), purification liquid chromatography such as flash or preparative chromatography, countercurrent or centrifugal partition chromatography and supercritical fluid chromatography (SFC). It is commonly used for analysis of compounds that do not absorb UV-VIS radiation significantly, such as sugars, antiviral drugs, antibiotics, fatty acids, lipids, oils, phospholipids, polymers, surfactants, terpenoids and triglycerides.
Wyatt Technology Corporation, or Wyatt Technology, is a developer and manufacturer of instrumentation for the characterization of nanoparticles and macromolecules. Headquartered in Santa Barbara, California, Wyatt Technology was founded in 1981 and incorporated in 1984. Wyatt Technology has developed and introduced instrumentation for the measurement of multiangle light scattering (MALS), quasi-elastic light scattering (QELS), differential refractive index, electrophoresis, viscosity, and composition gradient multiangle light scattering (CG-MALS) for the determination of absolute molecular weights, sizes of nanoparticles, proteins, and polymers within a solution. It was acquired by Waters Corporation in 2023.