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Despite very careful planning, HPLC experiments can come upon various concerns. In this area, we'll talk about a lot of the common complications it's possible you'll deal with, such as baseline drift, peak broadening, and retention time shifts, together with useful troubleshooting strategies to solve them:
. HPLC separation of a combination of flavonoids with UV/Vis detection at 360 nm and, from the inset, at 260 nm. The choice of wavelength influences Every analyte’s signal.
Lowering the amount of acetonitrile and rising the level of water within the cellular will increase retention situations, providing much more time and energy to impact a separation.
. Solvent triangle for optimizing a reversed-section HPLC separation. The 3 blue circles demonstrate cellular phases consisting of an natural solvent and h2o.
Peak locations: The region under Just about every peak while in the chromatogram is proportional to the amount of analyte existing, letting for quantification.
Knowledge Investigation software is essential for interpreting the data acquired within the detector. The computer software displays the chromatogram, which is a plot of detector signal compared to time. Essential facts details involve:
The working tension inside of an HPLC is sufficiently high that we cannot inject the sample in the mobile period by inserting a syringe by way of a septum, as is feasible in gas chromatography. Rather, we inject the sample utilizing a loop injector
The information acquisition system documents and processes the signals within the detector, enabling for that creation of chromatograms along with the quantification of compounds.
Maximize or minimize the ionization point out of analytes, impacting their affinity with the check here stationary section.
When the cellular section’s pH is adequately acidic, the solutes are present as neutral weak acids which might be additional soluble during the stationary section and take more time to elute. Because the weak acid solutes do not need identical p
In loop injection, a defined quantity of sample is loaded into a loop. The injector valve then switches, directing the sample on to the head on the column, where it's carried from the cell phase.
A reversed-section HPLC separation is completed using a mobile period of sixty% v/v water and 40% v/v methanol. Exactly what is the cell phase’s polarity index?
An HPLC ordinarily includes two columns: an analytical website column, that's answerable for the separation, as well as a guard column that's placed ahead of the analytical column to guard it from contamination.