What causes baseline drift HPLC?
The primary cause of baseline drift in gradient HPLC is due to changes in the refractive index of the eluent. The common organic solvents used in reversed phase HPLC have a greater UV absorbance (Table 1) than aqueous (water) which results in the rising baseline when monitoring at low wavelengths (~200 nm).
What causes carryover in HPLC?
This carryover is often caused by a mechanical area in the flow path (like the intersection between two surfaces in a valve head where tubing is inserted and the nut is swaged). Hold-up of some sample is progressively diluted upon injecting blanks, causing progressively less carryover with each blank run.
What is HPLC troubleshooting?
In an HPLC system, problems can arise from many sources. First define the problem, then isolate the source. Use Table 1 to determine which component(s) may be causing the trouble. A process of elimination will usually enable you to pinpoint the specific cause and correct the problem.
How is carry over calculated in HPLC?
The percentage of the peak area corresponding to propylparabene in the blank injection 1 does not exceed 0.5% of 10 times the peak area of the propylparabene peak in the chromatogram obtained with the reference solution (b) injected after the blank injection.
What causes high pressure in HPLC?
High back pressure in LC instruments is usually caused by foreign material blocking the flow of mobile phase. Although crimped PEEK or stainless steel tubing will occasionally be the culprit, particulates clogging the system are most often the cause.
What is HPLC calibration?
Know the procedure to calibrate the High Performance Liquid Chromatography ( HPLC) including leakage test, flow rate, reproducibility and linearity, lamp energy and pump pressure drop in Pharmaceutical Quality Control.
How can we reduce carryover?
When analyzing hydrophobic samples, using an organic solvent as the needle wash solution can reduce carryover because it acts to solubilize and wash away the adsorbed sample components. The needle wash solution should be treated like the mobile phase.
What is needle wash?
The typical needle wash process involves forcing deionized (DI) water, solvent, surfactant (soap solution), sodium hypochlorite (NaClO) or other liquid through a needle that is positioned inside a wash station (volume ~10 to 25 mL open-top well; 5 to 50 mL of liquid typically consumed). liquid slurry mixture.
What is RT and RRT in HPLC?
In high pressure liquid chromatography (HPLC), the compound is injected through a column of different sized beads. The amount of time it takes for the compound to pass through the column is the retention time (RT). The relative retention time (RRT) is the comparison of the RT of one compound to another.
What is carryover sample?
“Carry-over” is a term used to describe a type of sample contamination which causes sample peaks to re-appear in later runs which do not actually contain the sample (e.g. blank runs).
What is tailing factor in HPLC?
Peak tailing is the most common chromatographic peak shape distortion. Peak tailing occurs when the peak asymmetry factor (As) is greater than 1.2 — although peaks with As greater than 1.5 are acceptable for many assays.
How do I remove air from my HPLC system?
Turn the handle counterclockwise, about 1/2-turn, to open the valve, and pull back on the plunger to remove air bubbles. Once the air is removed, close the valve and start flow. Flush the system with 100% methanol for a minimum of 30 minutes.
How does a Shimadzu autosampler solve a chromatography problem?
Shimadzu autosamplers offer the unique ability to perform a null-injection, which starts the chromatography run without injecting sample and without rotating the injection valve or high pressure valve of the autosampler. This simple test can help to determine whether the source of the problem is the injection event itself.
How does external rinse work with Shimadzu autosamplers?
External Rinse with Shimadzu Autosamplers This cleans the outer surface of the needle by immersing it and/or actively rinsing it in a rinse port before or after the sample is drawn. There are two rinse ports (immersion and flow). In simplest terms, the immersion rinse port allows you to dip the needle into a rinse solvent (R0).
Is there a problem with the HPLC Shimadzu?
Shimadzus are a bit of a pain for homing due to their sensors. When switched on the HPLC shimadzuu system , initialization failure happening at model number SIL-20A HT / Prominence autosampler with display of SIL ERR PUMP HOME.