Sample Preparation Techniques

Sample preparation techniques in chromatography

Advanced chromatography methods require a step called sample preparation to enhance separation efficiency and sensitivity.

 

Sample preparation can be anything from simple dilutions to advanced peptide cleavage with enzymatic reactions, and often involves cleaning your sample before analysis to remove any interfering sample matrix or contaminants.

 

For example, samples contaminated with unnecessary matrices, like plasma in blood can cause ion suppression during MS detection or signals from unwanted compounds in optical and charged aerosol detectors.

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Removing interfering matrices is critical to the success of your separation efficiency, peak resolution, and data quality. Simply put, a cleaner sample gives you clearer, more exact results.

Analytical benefits of sample preparation

Proper sample preparation and cleanup are essential to the success of your chromatographic analysis.

 

Choosing the best technique can save you time, reduce the likelihood of repeating an analysis due to poor-quality data, and extend the lifetime of your instruments and consumables.
 

Greater method sensitivity due to concentrating target analytes

  • More accurate quantitation and lower limits of detection

  • Better method precision and easier data processing

 

More robust instrument and consumables performance

  • Fewer system contaminants in the flow path

  • Autosampler syringes are less likely to block

  • Less detector maintenance and repairs

  • Longer column lifetimes

 

Improved selectivity of your detector

  • Better solvent compatibility with the analytical technique

  • Cuts down interference from matrix and particulates

  • Reduced ion suppression and protein binding

Sample preparation techniques from simple to most advanced

Dilution

Dilution is simply adding more liquid of any form to your sample. You may want to do this to lower the organic strength of your injection solvent in cases where your HPLC method gives early eluting peaks with poor peak shape. You may also want to use dilution when the concentration of your sample is too high for detection and quantitation. Dilutions help ensure your sample is within a measurable range, especially when dealing with highly concentrated samples or early eluting chromatographic peaks

 

Filtration

Filtration is a must-do when the sample matrix consists of small particulates that will clog your column and give you a low column lifetime. Always filter your samples if you do not perform any other sample preparation, especially for more sensitive systems such as low-flow HPLC and UHPLC.

Protein precipitation

Protein precipitation is a fast and effortless way to remove unwanted proteins in your blood or plasma sample. The technique involves adding about 1:1 organic solvent to the sample and then waiting for the proteins to precipitate from the solution. Protein precipitations are often performed using a 96-well plate, which allows for miniaturization of the procedure, reduced solvent use, greater reproducibility, and amenable to high-throughput processing.

Derivatization

In other cases, analytes cannot retain onto an HPLC column or become sufficiently volatile for GC analysis so, derivatization is a solution to alter the molecule slightly to provide the desired physical attributes. Derivatization is most common in GC applications and at times for HPLC applications where a molecule needs to better retain on a particular stationary phase or become visible for optical detectors.

 

Liquid-liquid extraction

Liquid-liquid extraction (LLE) is a sample preparation technique to separate analytes between two immiscible solvents, typically an organic solvent and water. The solvent with a higher affinity for the target analytes selectively self-extracts from the sample.

 

Solid-supported liquid extraction

Solid-supported liquid extraction (SLE) is an advanced technique that enhances traditional LLE extractions by adding a solid, porous material to support the liquid phase. The sample is loaded onto the solid support, and the analytes of interest are extracted as the liquid adheres to the surface material. SLE reduces solvent consumption and minimizes sample dilution compared to LLE, making your methods more efficient and robust. Typically used to give cleaner and more efficient extractions required for pharmaceuticals and environmental testing.

QuEChERS

QuEChERS stands for Quick, Easy, Cheap, Effective, Rugged, and Safe. The method was developed to test pesticide residues in food and soil. This technique simplifies your extraction and cleanup processes for complex matrices, making your method more efficient.

QuEChERS involves homogenizing your sample and then mixing the homogenate with salt and an organic solvent. The mixture is shaken to extract the pesticides into the organic layer. The organic layer is removed and analyzed by LC or GC-MS.

Solid phase extraction

Solid phase extraction (SPE) is a method used to separate and purify analytes from complex mixtures, such as biological fluids, environmental samples, or chemical mixtures.

 

SPE selectively retains your target analytes based on chemical properties and works by passing a liquid sample through a solid adsorbent material. After this retention, the target compounds are eluted from the solid phase with a solvent, leaving unwanted materials behind.

The top analytical advantages of SPE are:

  • Cleaner samples: SPE allows for high selectivity by choosing a sorbent that specifically interacts with your analyte, giving cleaner samples.

  • High sensitivity: SPE helps you concentrate trace amounts of analytes to improve the detection limits in subsequent analyses and is more effective at removing matrix interferences in LC and LC-MS analyses.

  • Automation: SPE is easy to automate and greatly enhances sample throughput and reproducibility for high-volume processing.

SPE is a highly effective and straightforward technique for cleaning and concentrating your samples.

 

Common applications include pharmaceutical, environmental, and food safety testing.

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Enzymatic digestion

Enzymatic digestions performed on proteins and other large biomolecules effectively cut the macromolecules into smaller more manageable pieces, such as peptides, which are easier to analyze. We can effectively map how a protein looks based on the cut-out sections.  There is a plethora of different enzymes, that all cut the proteins at various cleaving sites.

  • Trypsin cleaves the C-terminal side of lysine and arginine amino acid residues and is a recommended starting choice.

  • Chymotrypsin cleaves the peptide bonds formed by aromatic residues such as tyrosine, phenylalanine, and tryptophan.

  • Pepsin preferentially cleaves hydrophobic, preferably aromatic residues in P1 and P1’ positions, and also cleaves at the carboxyl side of phenylalanine and leucine and the carboxyl side of glutamic acid residues.

  • Proteinase K cleaves the peptide bond next to the carboxyl group of aliphatic and aromatic amino acids with a blocked alpha amino group.
Enzymatic Digestion Protein

For Research Use Only. Not for use in diagnostic procedures.