how HPLC works - An Overview
how HPLC works - An Overview
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An HPLC normally contains two columns: an analytical column, and that is accountable for the separation, in addition to a guard column that may be positioned before the analytical column to shield it from contamination.
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機械的に高い圧力をかけることによって移動相溶媒を高流速でカラムに通し、これにより分析物が固定相に留まる時間を短くして分離能・検出感度を高くすることを特徴とする。
システムとしてポンプ、インジェクター、ディテクターまでを一貫して製造しているメーカーを挙げる。
A reversed-phase HPLC separation is performed using a cell period of 60% v/v h2o and 40% v/v methanol. What's the cellular stage’s polarity index?
five.1 shows an illustration of a standard HPLC instrument, that has quite a few vital components: reservoirs that shop the cellular stage; a pump for pushing the mobile phase in the system; an injector for introducing the sample; a column for separating the sample into its component areas; and a detector for checking the eluent as it comes off the column. Let’s think about each of such factors.
The interface amongst the HPLC and the mass spectrometer is technically harder than that in the GC–MS due to incompatibility of a liquid cell period Using the mass spectrometer’s high vacuum prerequisite.
順相クロマトグラフィーは高速液体クロマトグラフィーにおいて最初に使われた。固定相に高極性のもの（シリカゲル）を、移動相に低極性のもの（例えばヘキサン、酢酸エチル、クロロホルムなどの有機溶媒）を用いる。分析物はより極性の高いほどより強く固定相と相互作用して溶出が遅くなる。また極性の高い物質の割合が多い移動相ほど溶出が早くなる。順相タイプは近年の逆相タイプの発展とともに使われることが少なくなったが、順相タイプは逆相タイプをはじめとする他の分離モードとは異なった特性を持つため、目的によっては非常に有効なものとなる。例えば、逆相タイプでは分離が困難なトコフェロールの異性体や保持の困難な糖類を容易に相互分析することができ、また主に水を含まない移動相を用いるので、水に難溶の脂溶性ビタミンや加水分解されやすい酸無水物などの化合物の分離に好適である。
Just after loading the sample, the injector is turned into the inject posture, which redirects the cell period throughout the sample loop and on to the column.
This leads to various elution fees for the several elements and results in the separation of the components as they circulation out the column. As compared to column chromatography, HPLC is highly automatic and extremely sensitive.
The stationary section will likely be a click here good aid packed inside a column, Whilst the cell section is often a liquid or a mix of liquids.
Inside a fuel chromatograph the pressure from a compressed gas cylinder is adequate to force the cellular section from the column. Pushing a liquid cell phase by way of a column, nevertheless, requires a terrific offer far more effort, creating pressures in surplus of several hundred atmospheres.
The elution get of solutes in HPLC is ruled by polarity. For a standard-period separation, a solute of decrease polarity spends proportionally fewer time read more during the polar stationary section and elutes in advance of a solute that is definitely a lot more polar. Supplied a selected stationary period, retention moments in typical-phase HPLC are managed by adjusting the cellular stage’s Houses. Such as, Should the resolution concerning two solutes is lousy, switching into a considerably less polar cellular phase retains the solutes around the column for an extended time and gives far more opportunity for his or her separation.
이 검량 곡선을 바탕으로 실제 시료 분석으로 얻은 피크 면적에서 시료 중의 존재량을 산출하여 정량화를 실시합니다.