HPLC instrumentation refractive index detector

There are many HPLC detectors that can turn the presence of your compound into an electrical signal to be written on a chart recorder. Time was the refractive index detector was common. Clean eluent, used as a reference, went through one side of the detector, and the eluent with the samples went through the other side. A difference in the refractive index between the sample and reference caused an electrical signal to be generated and sent to a chart recorder. If you ve read the section on gas chromatography and looked ahead at infrared, you shouldn t be surprised to find both a sample and a reference. I did tell you the reference/sample pair is common in instrumentation. 

Molecular weight and polydispersity of the acetone extracted fractions from thermally degraded samples were determined by SEC with a Jasco PU-1580 HPLC pump equipped with two Pigel mixed D columns (Polymer Laboratories UK) connected in series, and a Jasco 830RI refractive index detector. Sample elution was with THF at 1 ml/minute flow rate. The instrument was calibrated with standard polystyrene samples. 

Very often baseline problems are related to detector problems. Many detectors are available for HPLC systems. The most common are fixed and variable wavelength ultraviolet spectrophotometers, refractive index, and conductivity detectors. Electrochemical and fluorescence detectors are less frequently used, as they are more selective. Detector problems fall into two categories electrical and mechanical/optical. The instrument manufacturer should correct electrical problems. Mechanical or optical problems can usually be traced to the flow cell however, improvements in detector cell technology have made them more durable and easier to use. Detector-related problems include leaks, air bubbles, and cell contamination. These usually produce spikes or baseline noise on the chromatograms or decreased sensitivity. Some cells, especially those used in refractive index detectors, are sensitive to flow and pressure variations. Flow rates or backpressures that exceed the manufacturer s recommendation will break the cell window. Old or defective source lamps, as well as incorrect detector rise time, gain, or attenuation settings will reduce sensitivity and peak height. Faulty or reversed cable connections can also be the source of problems. 

Gel permeation chromatograms were obtained using an HPLC Waters 244 Instrument with Zorbax PSM 1000-COS columns and a differential refractive index detector. A 0.25 wt% solution in chloroform solvent was used with a flow of 0.5 ml/min. 

SEC suffers from poor resolution and low sensitivity [5], while GC is limited by the high molecular weight and polar nature of many antioxidants and light stabilisers, which are designed to be reactive and so decompose when exposed to heat [9]. HPLC the most widely used instrumental method also has limitations [10-12]. HPLC lacks a simple sensitive universal detector that is compatible with all liquid mobile phases. UV or fluorescence detectors, which are commonly used, require that additives have a chromophoric moiety, while the universal refractive index detector only functions under isocratic conditions. As a result, Vargo and Olson have coupled HPLC with mass spectrometry (MS) for this type of application by using a moving belt interface [13]. 

Other widely used detectors for HPLC include refractive index (RI), fluorescence and evaporative light-scattering (ELS). The use of Rl and ELS detectors for pantothenic acid analysis in multivitamin dietary supplements has not been reported. The main reason is that the two detectors are not selective and thus cannot resolve pantothenic acid from other components existing in a multivitamin dietary supplement. Although fluorescence detection can be highly selective depending on the application, pantothenic acid does not have fluorescence excitation and emission and so fluorescence detection cannot be used for pantothenic acid analysis unless derivatization methods are applied (Pakin et al. 2004 Takahashi et al. 2009). Derivatization adds more complexity to analytical method and should not be used unless neeessary. For deteetion and quantitation of pantothenic add in multivitamin dietary supplements with HPLC/UHPLC, a highly selective detector such as MS should be the instrument of choice. 

Molecular weights and PDIs were measured by Waters gel permeation chromatography in THF relative to PMMA standards on systems equipped with Waters Model 515 HPLC pump and Waters Model 2414 Refractive Index Detector at 35°C with a flow rate of 1 ml/min. HRMS analyses were performed with Q-TOF YA263 high resolution (Waters Corporation) instruments by +ve mode electrospray ionization. 

After optimization of the correct capillary parameters (ID, OD, Lj), detection at the microscale level became the next major challenge for the survival of CE. Despite the challenges, many of the common HPLC detectors have a CE complement, e.g., absorbance, fluorescence, conductivity, photodiode array, and mass spectroscopy. Small dimensions mean universal detectors such as refractive index cannot be used. A sample of detectors will be discussed. The technical aspects of each detector will not be covered except in relation to the CE instrument. Readers are advised to consult an instrumentation textbook for more details on theory of operation. 

There are excellent HPLC systems available on the market today, yet there is one area of concern with this instrumentation, and this rests with the detection units. Certainly the most widely used detector system employs a low dead-volume micro-ultraviolet detector. This latter unit operates near 200 nm and detects mainly unsaturated linkages in phospholipids (or lipid) samples. Some contribution by carbonyl functions can be expected. This approach is an advantage when the sample under study contains olefinic groups, but will not detect those with saturated side (hydrocarbon) chains. An alternative detector is the refractive index monitor which is often called a universal detector, since it is based on the concept that the refractive index of the solvent changes when a solute is present. The drawback of the latter unit lies in its sensitivity, which is approximately 15- to 20-fold less than that of the ultraviolet monitor. 

Shimadzu has produced the Prominence HPLC, Agilent has developed the 1200 Rapid Resolution HPLC (Figure 3.22), Thermo Scientific has the Accela LC system and Waters has brought out the Acuity UPLC. Jasco s X-LC system allows two systems to fit in the footprint previously occupied by one traditional system. It is also a modular system of detectors and autosamplers, which allows it to be customised. LC Packings has launched the UltiMate 3000, which is a nanoflow LC system for use with columns of 50 im and larger. Modular systems are common now, e.g. Cecil Instruments produces both HPLC and ion chromatography systems and various detectors can be accommodated including UV-Vis, refractive index, conductivity and fluorescence. 

UV detection is used in most chiral analysis by HPLC and other liquid chromatographic modalities. However, some other detectors, such as conductivity, fluorescent and refractive index types, are also used. The choice of detector depends on the properties of the racemic compound to be resolved [41, 144]. Chiroptical detectors, which are based on the principle of polarimetry [145] or circular dichroism [146, 147], are also available. The enantiomer (+)- or (—)-notation is determined by these detectors. Some organochlorine pesticides are not UV-sensitive, and hence they are difficult to detect in liquid chromatography. The detection of these types of pollutant can be achieved by using a mass spectrometry (MS) detector, and therefore LC-MS instruments are now being put on the market for routine use [148, 149]. 

---