Liquid Chromatography Blog

Just Enough Sample Preparation

Sample preparation is an integral part to successful chromatographic analysis. Selecting the right sample preparation technique is dependent upon...

  • Analytes(s)
  • LOD, LOQ
  • Sample matrix
  • Analytical instrumentation
  • Sample size

Sample preparation is believed to be a very time consuming and error prone step in the chromatographic process, therefore it is often ignored or eliminated.

Sample Prep Solutions #2: ‘Just Enough’ Sample Preparation

   Dilute and shoot is an example of not incorporating sample preparation. The advantage of dilute and shoot is that it is very quick, only requiring the dilution of the sample; therefore, a simple pipette to a fully automated instrument can accomplish this task. Dilute and shoot, at least in my world, is not a true sample preparation technique. The definition of sample preparation is chemical separations and extraction methods to prepare a sample for analytical measurement of compounds. Dilute and shoot only dilutes the matrix; it does not remove any interferences. In time, interferences will still build up in the system inlet and column causing chromatographic problems such as carryover, loss of sensitivity and ion suppression. Many feel it is just easier to replace a column than incorporate sample preparation, but it is not that simple. Due to sample matrix differences whether they are biological (plasma, urine, saliva, post-mortem), food (fruit, vegetable, meat, processed), or environmental (soil, water, waste and biota) problems manifest themselves, and will be unpredictable. Additional sporadic peaks, co-eluting peaks, increase in background interferences and lower area counts all lead to substantial increases in data analysis time required by the analyst to manually reprocess all of the data. Troubleshooting and cleaning the system is a time consuming process; the system will be down, which means that sample analysis will be delayed, costing the lab time and money. Implementing simple ‘just enough’ sample preparation techniques will drastically minimize the problems related to excluding or bypassing sample preparation.

   ‘Just enough’ sample preparation is a descriptive term for simple techniques such as filtration, protein precipitation, supported liquid extraction, extraction partitioning for toxicology screening and QuEChERS. These ‘just enough’ sample preparation techniques are gaining more acceptance even for complex samples because of the improved sensitivity and selectivity of mass spectrometers and improving separations with UHPLC. All ‘just enough’ sample preparation techniques have the following features in common:

  • Extremely easy to use
  • Little to no method development
  • Minimal time required 
  • Very reproducible results
  • Low RSDs
  • Multi-suite extraction

   These sample preparation techniques remove a significant portion of the matrix interferences but as the name suggests not all interferences, so it is very important to evaluate recoveries based on a matrix matched calibration curve. In addition, evaluate the amount of co-extractants gravimetrically in order to determine the amount of matrix removed by the method. Let’s take a brief look at some ‘just enough’ techniques.

Sample Prep Solutions #2: ‘Just Enough’ Sample Preparation-2

In general filtering the sample prior to analysis is considered to be proper sample ‘hygiene’. Sample filtration is a mechanical process in which particles are retained and, therefore, eliminated from the sample by a membrane or frit/membrane material. This can be accomplished by syringe filters, filter vials, filter vacuum design and for high throughput samples, filter plates. Sample filtration has become more important with UHPLC columns than with standard HPLC columns composed of particle sizes greater than 2 micron. UHPLC columns are packed with sub-2 micron particles that require the need for a smaller pore size frit, 0.5 micron. To avoid clogging of the frit; filtering samples with a 0.2 micron filter prior to analysis is very advantageous. Column life time is extended, in addition to less wear and tear on critical moving parts of the injection valve and clogging capillary tubing.

Sample Prep Solutions #2: ‘Just Enough’ Sample Preparation-3

Protein Precipitation
Protein precipitation can be facilitated by the addition of salts, low pH and metals but the addition of organic solvent namely acetonitrile or methanol is often preferred. The principle is to precipitate a large amount of unwanted proteins while eliminating analyte-protein interactions, which in turn makes the analytes soluble and chemically stable. The organic solvent (2 to 3 volumes relative to the sample volume) is mixed with the proteinaceous sample followed by centrifugation or filtration. In many cases this is all the sample preparation that is required for analysis. Protein precipitation plates/columns allow the entire procedure to be completed within the well; the filtrate is then passed through a filter/membrane and collected. There are several manufacturers that offer protein precipitation plates and columns and are definitively worth investigating over centrifugation. The plates/columns are five times faster, do not require a transfer step, increase reproducibility, achieve greater protein removal (as much as 99%), and while filtering the sample at the same time (Figure 1). However, the filtrate from protein crashed samples will still contain unwanted salts and lipids, especially from human samples; where lipids can be a major contributor to ion suppression and matrix build-up within the analysis system. Salts are usually not of major concern in reversed-phase chromatography conditions.

Sample Prep Solutions #2: ‘Just Enough’ Sample Preparation-4

Advanced Precipitation
Advanced precipitation plates incorporate protein precipitation membrane with lipid depletion chemistry, also known as lipid removal plates. The lipid depletion chemistry can range from a hydrophobic to metal-coated silica. The same in well mixing of the organic solvent with the biological sample as described with a protein precipitation plate applies. As the crashed sample passes through the protein precipitation membrane the sample comes in contact with the lipid depletion material. The lipid depletion material has high affinity for the lipids, therefore, they never pass through and are eliminated from the sample eluent. The advanced precipitation plate removes well over 95% of the lipids found in the sample, in addition to the proteins (Figure 2).

   The protein and lipid depletion plates/columns can be used manually, semi-automated or in fully automated systems quite easily. Removal of the proteins and lipid will allow one to reach much lower LOD and LOQs for the analytes of interest and significantly reduce maintenance issues, especially related to dilute and shoot.

   The next three ‘just enough’ techniques are based on liquid-liquid extraction (LLE) LLE is also known as solvent extraction and partitioning. It is a well established method to separate compounds based on their relative solubilities in two different immiscible solvents, usually water and an organic solvent. LLE is advantageous with its short method development time and relatively low cost; however it has several limitations; time consuming, large volumes of organic, variable results, special glassware, difficult to automate and formation of emulsions. In order to overcome these limitations, modifications and use of materials such as diatomaceous earth or salts with an organic have been employed, and are described below.


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