How to prevent contamination by high performance (HPLC) or capillary (CapLC) LC

How to prevent contamination by high performance liquid chromatography ( HPLC ) or capillary liquid chromatography ( CapLC )

Recommendations for minimizing or reducing background contaminants: All solvents and acids, bases, buffer salts are chemicals, none of which are 100% pure. Therefore, there is always some chemical background in LC/MS. In order to improve data quality, it is worth considering to minimize interference and background noise. Here we give you some small suggestions for your reference.

1. Solvents and additives

a. Use the highest purity reagent – HPLC grade or higher purity.

i. Best to use Fisher Optima grade

1. Some HPLC grade reagents contain polyethylene glycol (PEG) compounds that are not detected by UV detectors.

2. Some HPLC grade reagents contain iron ions (Fe ⁺³ )

Ii. Water may be a major source of pollutants

3. The online pure water system (Milli-Q) must be maintained. 18 M ohms of water are not completely free of organic contaminants.

4. Bottled water is not necessarily better than Milli-Q's water, because it may accumulate contaminants after the bottled water is turned on.

5. It is not recommended to store water in plastic containers.

6. Further purify the water by passing it through a clean _C18 column to remove organic material, which can be carried out online via a high pressure binary gradient and a _C18 guard column.

b. Additives such as acetic acid may contain large amounts of iron. 100 ppb sounds like a very low concentration, but 100 ppb = 100 ng/L or 100 pg/mL.

i. The adduct of iron formed by acetic acid results in a strong ESI ⁺ mass spectrum.

Ii. Formic acid has been shown to contain less iron ions.

c. Adding the lowest concentration of additives can reduce chemical background noise, increase signal-to-noise ratio, and sensitivity.

2. Containers for sample and solvent

a. Bottle and lid, sample extraction plate:

i.Waters vials are quality controlled and other brands may not be as clean.

Ii. A vial cap with a gasket may contaminate the autosampler due to the presence of plastic or adhesive.

Iii. The sample extraction plate is made of plastic and may be released by a plasticizer (eg, dioctyl phthalate contaminant).

Iv. The cover of the sample extraction plate glued to the metal foil may have a loss of adhesive material.

b. Glassware

i. After washing with ordinary detergent, there may be detergent residue (polyethylene glycol compounds) and other "sticky" substances on the glassware.

Ii. Flush with reagents of the mobile phase quality to be used.

c. Plastic containers or pipes:

Do not store solvents or water in plasticizers (because they are like dioctyl phthalate contaminants).

3. Hand cream

a. Containing polyethylene glycol compounds, oils, vitamins, and the like.

b. Wear gloves when handling HPLC parts that can contact the mobile phase or sample.

4. Sample and sample matrix

a. The sample matrix may contain salts and other undesired substances. When preparing a needle cleaning solution, the solubility of the matrix must be considered.

i. Inorganic salts are insoluble in solutions of high organic ratios, avoid using buffers containing Na ⁺ , K ⁺ , PO ₄ ^-3 because they are non-volatile. Buffers containing NH ₄ ⁺ acetate or formic acid are volatile and compatible with MS.

Ii. Proteins (eg, in tissue, blood, or serum samples) will precipitate in high organic phase solutions (>40%), which will block the injector and tubing and will adsorb analytes or contaminants.

b. The chemicals used to prepare the sample (eg, detergent, salt) can produce large, undesirable ions. The way the sample is purified and enriched needs to be coordinated with LC/MS.

c. The solubility of the analyte may be a problem. If the sample does not dissolve in the high organic phase solution, some of the analytes may precipitate in the mobile phase in the mobile phase injected into the initial low organic solvent and contaminate the injector or column, then proceed to the next gradient. They may dissolve again when they are.

d. Samples with very high concentrations at the time of injection may contaminate the injector. This situation is likely to occur when looking at 0.1% of the impurities in the sample, because it is necessary to inject enough samples to view.

5. Column

a. The column can behave like a filter. Particles, precipitated proteins, and the like all stay on the column head and are slowly washed down, which gradually increases the background ions.

b. The C ₁₈ analytical column or the Traping desalting column will capture hydrophobic compounds from water and will slowly enrich them.

i. When eluted by an enriched compound, it may appear as a distinct peak or in a similar tailing pattern throughout the chromatogram.

Ii. Trace enrichment can result in an increase in the amount of contaminants from the solvent or HPLC system.

Iii. Long-term equilibrium C ₁₈ columns will enrich more contaminants.

Iv. Flushing the column with an isocratic mobile phase with a low organic phase concentration will also enrich the contaminants. The column should be periodically cleaned with a solution of a high organic phase.

6. PEEK tubing and quartz capillary tubing

a. PEEK is a plastic polymer. Many hydrophobic compounds (such as steroids) may adsorb on them, and when they elute, they form contaminants.

b. The quartz capillary path has a surface with reactive ions, and certain substances (such as some proteins and peptides) adhere to the quartz. Any substance that adheres to the surface of the glass will adhere to the quartz capillary. When they are eluted, they will form contaminants.

7. HPLC System – Clean and test for contaminants prior to use.