크로마토그래피 원리의 큰 틀도 마찬가지로 두 상에 대한 분배 차이를 이용하여 분석물을 분리, 정제할 수 있습니다. 다만 크로마토그래피에서 두 개의 상은 하나는 고정하고 다른 하나는 일정 방향으로 이동시켜 사용합니다.
최상의 결과를 위해서는 올바른 시약을 사용함으로써 피크 대칭성을 개선할 수 있습니다.
. HPLC separation of a combination of flavonoids with UV/Vis detection at 360 nm and, during the inset, at 260 nm. The choice of wavelength impacts Just about every analyte’s signal.
Transforming the cellular period’s composition as being the separation progresses is a person Option to this problem. For just a reversed-phase separation we use an initial cellular stage that is certainly more polar. Given that the separation progresses, we alter the composition of cellular section so that it gets considerably less polar (see Figure twelve.5.6
Next, a number of the compounds inside the serum may perhaps take in way too strongly on the stationary period, degrading the column’s performance. At last, Though an HPLC can independent and review sophisticated mixtures, an Assessment is tough if the volume of constituents exceeds the column’s peak ability.
5.1 exhibits an illustration of a standard HPLC instrument, which has quite a few important elements: reservoirs that retail store the mobile phase; a pump for pushing the cellular section in the system; an injector for introducing the sample; a column for separating the sample into its part parts; in addition to a detector for monitoring the eluent as it arrives from the column. Let’s look at Every of such parts.
各種の高速液体クロマトグラフィーの項目にある違いは、カラムの違いである事が多いため、装置はそのままでカラムの変更で行える場合が有る。ただし、誤って不適当な溶媒を通すとカラムを破損することがあるため、切り替えを行う際には注意が必要である。
The functioning stress in an HPLC is sufficiently high that we are unable to inject the sample into the mobile phase by inserting a syringe through a septum, as is achievable in gasoline chromatography. Rather, we inject the sample using a loop injector
The determine under exhibits the calibration curve and calibration equation for your set of external expectations. Substituting the sample’s here peak space to the calibration equation presents the concentration of caffeine during the sample as 94.4 mg/L.
移動相としては、カラムや装置に悪影響を与えない範囲で各種の溶媒が使用される。水や塩類の水溶液、アルコール類、アセトニトリル、ジクロロメタン、トリフルオロ酢酸などが用いられる。相溶性のある（互いに混じり合う）溶媒を混合して使用する場合が多い。
. Solvent triangle for optimizing a reversed-section HPLC separation. The three blue circles demonstrate cellular phases consisting of the natural and organic solvent and water.
, for instance, displays retention situations for four weak acids in two cell phases with approximately equivalent values for (P^ key ). Although the get of elution is similar for both equally cell phases, each solute’s retention time is impacted in a different way by the choice of organic solvent.
. A person trouble using an isocratic elution is usually that an proper cell section here energy for resolving early-eluting solutes may perhaps bring about unacceptably extended retention occasions for late-eluting solutes. Optimizing the cell period for late-eluting solutes, Alternatively, may possibly supply an insufficient separation of early-eluting solutes.
Decreasing the level of acetonitrile and increasing the level of drinking water while in the cellular will boost retention instances, supplying more time and energy to result a separation.