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الكيمياء الصناعية | HPLC : Apparatus |
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date: 10-2-2020 |
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Specialized apparatus is required for an HPLC separation because of the high pressures and low tolerances under which the separation occurs. If the results are to be reproducible, then the conditions of the separation must also be reproducible. Thus HPLC equipment must be of high quality; it is therefore expensive.
The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample. As the solvent is passed through a very narrow bore column, any contaminants could at worst plug the column, or at the very least add variability to the retention times during repeated different trials. Therefore HPLC solvent must be kept free of dissolved gases, which could come out of solution mid-separation, and particulates.
In the HPLC column, the components of the sample separate based on their differing interactions with the column packing. If a species interacts more strongly with the stationary phase in the column, it will spend more time adsorbed to the column's adsorbent and will therefore have a greater retention time. Columns can be packed with solids such as silica or alumina; these columns are called homogeneous columns. If stationary phase in the column is a liquid, the column is deemed a bonded column. Bonded columns contain a liquid stationary phase bonded to a sold support, which is again usually silica or alumina. The value of the constant C described in the van Deemter equation is proportional, in HPLC, to the diameter of the particles that constitute the column's packing material.
The HPLC pump drives the solvent and sample through the column. To reduce variation in the elution, the pump must maintain a constant, pulse free, flow rate; this is achieved with multi-piston pumps. The presence of two pistons allows the flow rate to be controlled by one piston as the other recharges. A syringe pump can be used for even greater control of flow rate; however, the syringe pump is unable to produce as much pressure as a piston pump, so it cannot be used in all HPLC applications.
The HPLC detector, located at the end of the column, must register the presence of various components of the sample, but must not detect the solvent. For that reason there is no universal detector that works for all separations. A common HPLC detector is a UV absorption detector, as most medium to large molecules absorb UV radiation. Detectors that measure fluorescence and refractive index are also used for special applications. A relatively new development is the combination of an HPLC separation with an NMR detector. This allows the pure components of the sample to be identified and quantified by nuclear magnetic resonance after having been separated by HPLC, in one integrated process.
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دراسة يابانية لتقليل مخاطر أمراض المواليد منخفضي الوزن
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اكتشاف أكبر مرجان في العالم قبالة سواحل جزر سليمان
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اتحاد كليات الطب الملكية البريطانية يشيد بالمستوى العلمي لطلبة جامعة العميد وبيئتها التعليمية
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