Model PL-ELS 2100 Detector (Polymer Laboratories. Ltd. Church Stretton,
Shropshire, UK). The experimental conditions are indicated in the figure captions.
Application Note AN 110-01-C
Combining OPLC with ELSD for Universal Analysis
A
Optimum performance laminar chromatographyTM (OPLC) can be coupled with Evaporative Light Scattering detection (ELSD) to provide an analytical methodology which is especially well suited for scouting work and the analysis of unknowns. bstract OPLC is superb for separating the compounds in the sample as the progress of the separation can be monitored, and ELSD is ideal as a detection method since all compounds in the sample will provide a response without having to form a derivative.
Introduction
Experimental OPLC separations were performed using the OPLC 50 Chromatograph (Bionisis SA, Le Plessis Robinson, France). ELSD detection was performed using a Model PL-ELS 2100 Detector (Polymer Laboratories Ltd. Church Stretton, Shropshire, UK). The experimental conditions are indicated in the figure captions.
Results The separation of carbohydrates is presented in Figure 1.
In recent years, evaporative light scattering detectors (ELSD) have become a popular tool for chromatographic detection. ELSD is capable of detecting essentially all compounds in the sample (i.e. the compounds need not have a chromophore, fluorophore, electroactive group, etc.); in addition, ELSD is very sensitive and allows for the use of gradients. An important benefit of coupling OPLC with ELSD is the ability to readily view the overall progress of the separation on the sorbent bed during the separation to ensure that all components of the sample have been eluted. All compounds in the sample can be accounted for when OPLC-ELSD is employed; if the analyst does not see a peak (or a smaller peak than expected) on the detector input, he/she can quickly view the sorbent bed to determine the progress of the separation and change the nature of the mobile phase if required. This can be done by UV lamp inspection or, if necessary, by spraying with a derivatizing agent. If needs be, using sulfuric acid at 120°C will reveal almost every time anything present on the HTSorbTM. In contrast, when HPLC is used to separate the sample, the chromatographer does not know the total progress of the separation as he/she cannot view the status of the sample on the sorbent bed. In this application note we describe the use of OPLC with an ELSD to provide a chromatographic system that can be used for the analysis of a mixture of carbohydrates and a mixture of fatty acids. These compounds can be readily detected via OPLC-ELSD without the need for derivatization.
BIONISIS
Figure 1: Separation of Carbohydrates. Stationary Phase HTSorbTM Si (BSLA011, Bionisis). Mobile Phase: CH3CN:H2O, Linear Gradient: 0-1.3 min – (95:5), 1.3-2.6 min – (90:10), 2.6-15.6 min (80:20) to end of chromatogram. Flow Rate 0.7 mL/min Sample 20 µL 1: Xylose 0.3 mg/mL, 2: Arabinose 0.3 mg/mL, 3: Fructose 0.3 mg/mL, 4: Galactose 0.3 mg/mL, 5: Sucrose 0.15 mg/mL, 6: Maltose 0.3 mg/mL, 7: Lactose 0.3 mg/mL. Detection Conditions: N2 Flow = 1.5 L/min, T(nebulizer) = 50oC, T(evaporator) = 75oC.
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Optimum Performance Laminar Chromatography (OPLC) is a powerful chromatographic technique that is commonly used to separate complex samples using a pressurized sorbent bed since it provides excellent resolution. When OPLC is used, the presence of solutes in the mobile phase can be determined by a variety of detectors by monitoring a physical property of the eluent (e.g. absorbance, fluorescence, electroactivity, etc.). If the compound(s) of interest does (do) not absorb, fluoresce, etc., either a derivative must be formed (which can be a tedious process) or a universal detector such as a refractive index detector (which provides poor sensitivity and does not allow for the use of gradients) must be used.
The separation of fatty acids is presented in Figure 2.
Discussion The combination of OPLC and ELSD provides the analyst with the ability to monitor the overall progress of the separation. OPLC allows for viewing the sorbent bed and ELSD provides for detection of all eluted compounds. The separation can be visually observed as well as monitored by the detector, if a component of the sample is not eluted, or is poorly eluted, the mobile phase can be readily altered to ensure that all compounds are eluted. In contrast, when HPLC is used, the un-eluted sample cannot be visually observed, and the analyst cannot determine if the entire sample has been separated. The benefit of ELSD is that it is a universal detector, and essentially all compounds that are eluted can be observed. This is especially important when the sample is of unknown origin, since a priori the analyst does not know what derivative to form. An additional benefit of OPLC-ELSD is that gradients can be employed; other “universal” detectors such as refractive index detectors prohibit the use of gradients.
Figure 2: Separation of Fatty Acids . Stationary Phase HTSorbTM RP-18 (BSLA112, Bionisis). Mobile Phase: CH3CN (+0.1% TFA):H2O (+0.1% TFA) 90:10, Flow rate 0.6 mL/min.
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Sample 5 µL. 1: Linoleic Acid 1.1 mg/mL, 2 Palmitic Acid 2.5 mg/mL, 3: Stearic Acid, 2.5 mg/mL, 4 Arachnidic Acid 1.9 mg/mL. Detection Conditions: N2 Flow = 1.6 L/min, T(nebulizer) = 30oC, T(evaporator) = 35oC.
BIONISIS
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