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The Intricacies of HPLC Sample Preparation

Meticulous sample preparation is necessary for highly sensitive analysis

by Sartorius
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High performance liquid chromatography (HPLC) is a powerful analytical chemistry technique that enables identification and quantification of compounds within a sample. With applications in life sciences, pharmaceutical research, drug development, and quality control, it is a versatile technology capable of generating highly accurate data. Regardless of the application, data quality is largely determined by precise sample preparation. Using the right instruments and techniques can reduce the risk of background noise, false-positive peaks, and even prolong the longevity of the column. With HPLC, results are only as good as the sample preparation.


Poor water quality is one of the largest contributing factors to problems during HPLC runs. Water impurities in the mobile phase can interact with the column and result in poor resolution and sensitivity or unexpected peaks in the chromatogram, commonly referred to as ghost peaks. Using ultrapure water substantially reduces the risk of contamination with organic carbons, microorganisms, endotoxins, RNases, and DNases. Ultrapure water is available for purchase, however using a water purification system in your laboratory is the easiest way to ensure high-quality ultrapure water. Type 1 reagentgrade water that meets ASTM, NCCLS, ISO, and USP standards is the best choice for HPLC applications.


Preparing standards for compound identification and quantification can be a tedious and time-consuming process. Accurately weighing a solid so that a predefined volume of solvent can be used is difficult, and waste due to error can be costly—especially when working with pharmaceuticals. Other factors such as temperature further complicate standard preparation. Temperature fluctuations alter solvent density and contribute to dramatic differences in concentration. Using a balance that enables a weighing accuracy up to several decimal places and automating the process by linking the balance to a solvent dispenser guarantees the highest precision. Using software such as the YAPP16 Dosing Q-App enables automatic calculations and gravimetric monitoring of measured values. Documenting temperature and density of the solvent also ensures highly accurate concentrations, especially when preparing standards on subsequent days. Direct data transfer from the YAPP16 weighing application to the Thermo Scientific™ Chromeleon™ 7.2 chromatography data system (CDS) software is an option for paperless data handling with full data integrity.


The importance of accurate and precise pipetting is too often overlooked and taken for granted. User skill level and experience can have significant implications for sensitive analyses like HPLC. Choosing the right pipette can reduce human error and the associated costs of wasted solvents. Mechanical pipettes with a volume lock offer a simple solution for preventing accidental volume changes, while more sophisticated electronic pipettes eliminate user variability to consistently deliver precise volumes. The pipette tip is also an important detail to consider for HPLC analyses. Low retention tips are best when working with solvents of different viscosities, as they maximize sample recovery. Sample contamination can also occur as a result of leaching from the pipette tip. As such, tips with high chemical resistance preserve sample integrity.


Taking additional care to filter samples prior to analysis improves reliability and can prolong the life of the HPLC column by preventing clogging effects. Syringe filters are a simple, rapid solution for filtering samples prior to column chromatography. As with pipette tips, the solvents used in HPLC necessitate specific filters to prevent leaching and sample contamination. Cellulose membranes are resistant to dimethyl sulfoxide (DMSO), amides, ketones, ester, and ether compounds. For especially harsh solvents, uncoated polyethersulfone (PTFO) membranes prevent leaching and sample contamination. Sample filtration becomes very time consuming when sample number increases. Multi-sample filtration units, such as the Claristep® Filtration System, can process samples without syringes and fast-track sample preparation without sacrificing quality.

HPLC is a valuable technique for life science and biopharmaceutical research as it delivers accurate, reproducible results. This accuracy depends on the integrity of the HPLC sample, and the best way to ensure high quality samples is to start with the best tools. Sartorius products are designed specifically for optimal HPLC sample preparation from start to finish. With solutions for ultrapure water, standard preparation, pipettes, tips, and filters, the HPLC sample preparation line accounts for every detail. Automated solutions and software also help to streamline sample preparation to deliver clean samples for clean results.

The Sartorius Group is a leading international partner of biopharmaceutical research and the industry. With innovative laboratory instruments and consumables, the company concentrates on serving the needs of laboratories performing research and quality control at pharma and biopharma companies and those of academic research institutes. Its broad product portfolio focusing on single-use solutions helps customers to manufacture biotech medications and vaccines safely and efficiently. 

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