Similar to the SPME–GC desorption/injection system, SPME and HPLC desorption/injection can be combined. The most common way to do this is to use a six-port injection valve (as described in detail in Chapter 3). In SPME–HPLC, the injection loop is replaced by a desorption chamber.
The SPME syringe is introduced into the desorption chamber having the six-port injection valve in the load position. The fiber is positioned and sealed to avoid leakage from the system under pressure. This step is followed by valve turned to inject position with desorption into the mobile phase and subsequent transfer to the analytical column.
9.4.3
SPME Fiber Materials and Extraction Parameters
There are several stationary phases in use for SPME. Table 9.6 lists the most common stationary phases as well as their interaction mode.
Besides the nature of the stationary phase, the thickness of the coating also plays a key role. The thickness varies from 100mm downward to 7mm. The thinner the coating, the shorter the equilibration time needed. Depending on the volatility of the analytes, the SPME procedure can be optimized by choosing the appropriate stationary phase thickness. The more volatile the compound, the thicker the coating.
Low-volatile compounds are extracted using a thin coating.
Factors, other than the already mentioned, affecting the adsorption during SPME are pH, ionic strength, use of water and organic solvents, temperature, agitation, and time.
Table 9.6 Stationary phases for SPME.
SPME stationary phase Interaction
Polydimethylsiloxane (PDMS) Nonpolar to moderate polar
Polyacrylate (PA) Moderate polar to polar
Polydimethylsiloxane-divinylbenzene (PDMS-DVB) Nonpolar to moderate polar
Carbowax-divinylbenzene (CW-DVB) Polar to moderate polar
Polydimethylsiloxane–carboxene Nonpolar to polar
9.4.3.1 pH
Best recoveries are obtained when the analytes are in their uncharged state. This means, as with the other extraction techniques discussed, that the pH of the sample should suppress ionization of basic and acidic compounds. Neutral compounds are not affected by pH. It should be noted that since SPMEfibers can be reused up to 100 times, it is important that the pH range of extraction should be within 2 and 10 (in case of immersion SPME).
9.4.3.2 Ionic Strength
Increasing the ionic strength by adding salt, for instance, NaCl or Na2SO4, decreases the solubility of analytes in the aqueous sample. This salting out effect, caused by the fact that water molecules are attracted to the salt ions and thus less available to solvate analytes, may increase the recovery of analytes. This is especially useful in trace determinations. However, increasing the ionic strength almost always increases the recovery and the effect of salt should therefore always be determined experimentally.
9.4.3.3 Water and Organic Solvents
Sample dilution can improve recoveries in SPME greatly when dealing with complex samples. This is because adsorption of the analyte to matrix components can be reduced, and diffusion can be increased, by a simple dilution step using water.
The use of organic solvents on the contrary should be avoided since they have a negative influence on the distribution of the analyte between the stationary phase and the sample.
9.4.3.4 Temperature
When developing the SPME method for the analyte of interest, temperature is an important factor to optimize. Increasing the temperature leads to a higher diffusion rate and a decreased equilibration time. High temperatures will also lead to increased analyte concentration in the headspace. However, the extrac- tion recoveries decrease, since the distribution of the analyte between the stationary phase and the sample becomes less favorable. Low temperature, on the other hand, will result in long equilibration time (less diffusion), but in higher recovery (favorable distribution). When low detection limits need to be reached, SPME at low temperatures should be chosen. In case of speeding up the extraction procedure, high temperatures are advantageous. Also, higher molecular mass compounds and less volatile compounds are extracted better at higher temperatures.
9.4.3.5 Agitation
The adsorption step in SPME can be accelerated using agitation in order to reduce equilibration times and thus extraction time. Agitation is performed by magnetic stirring, vortex mixing, or sonication. Note that sonication can lead to increased temperature of the sample and thus a change in distribution of the analyte between the stationary phase and the matrix (see above).
9.4 SPME
j
1819.4.3.6 Extraction Time
As mentioned earlier, extraction time should be chosen to ascertain equilibrium. In this case, the best detection limits are reached. In addition, increase in extraction yield is highest in thefirst part of the extraction and becomes less when time evolves.
When sampling using short extraction times, repeatability is often poor, but is improved when extraction times are close to or at equilibrium time. However, when high throughput (i.e., analysis of many samples in a relative short time span) is demanded or when equilibration times are very long, long extraction times are not always chosen. In these cases, extraction of the analytes is carried out under nonequilibrium conditions, which has an effect on the recovery.
9.5
Protein Precipitation
Endogenous and exogenous substances often need to be determined in samples, such as plasma and serum, which are very protein-rich and complex matrices. The protein content usually makes the determination difficult and protein removal is often required, for example, by protein precipitation. Most used protein precipitation agents are organic solvents, salts, and acids. Table 9.7 shows some of the most used precipitants and the amount needed to precipitate 95 and 99% of all proteins. The precipitated proteins are removed by centrifugation, and the supernatant is used for analysis.
When miscible organic solvents are added to aqueous solutions, the dielectric constant (e0) is decreased. This leads to compressed hydrated layers of the proteins, which in its turn allows proteins to interact with each other. The lower the dielectric constant, the more easily this interaction occurs, causing aggregation and precipitation.
Info-box 9.10
A more detailed description of SPME concepts as well as experimental consider- ations can be found in Ref. [3].
Table 9.7 Solvents and solutions used for protein precipitation.
Precipitant Final concentration needed to precipitate
e0 95% protein 99% protein
Acetonitrile 0.65 50% 60%
Ethanol 0.88 60% 75%
Methanol 0.95 60% 75%
Acetone 0.56 50% 60%
10% TCA — 15%
6% HClO4 30% 45%
Saturated (NH4)2SO4 65% —
Acids decrease the pH of the solution and thus influence the charge of the proteins. Proteins have zwitterionic properties due to their acidic and basic moieties.
A low pH will cause the acidic moieties to be uncharged and the basic moieties to be positively charged. Some acids, such as trichloroacetic acid (TCA), will be able to form neutral ion pairs with the basic amino acids resulting in uncharged proteins that can interact, aggregate, and precipitate.
Salts compete for water molecules in the solvation layer around the proteins. The more the salt in solution, the more the water associated with the ions, and thus the decrease in solvation layer. The hydrophobic parts of the proteins then become more exposed causing increased interaction between the proteins, which, in its turn, causes aggregation and precipitation.
Protein precipitation is a very simple sample preparation technique with the disadvantage that the resulting sample, although stripped for proteins, is still very complex and not always compatible with the separation system and thus requires an additional cleanup step. It should be kept in mind that a sample precipitated with the TCA might be too acidic to inject directly into an HPLC system and that a 50%
organic modifier content might cause severe band broadening. In addition, the samples get more diluted, thus lowering the concentration of the analytes. Adjusting the pH can circumvent HPLC incompatibility for the acid-precipitated samples.
Solvent evaporation can lead to both less band broadening and analyte enrichment when protein precipitation with organic solvents is carried out.
9.6
Membrane-Based Sample Preparation Techniques
9.6.1 Microdialysis
The principle of dialysis is based on the free movement of small molecules through a semipermeable membrane (thickness 9–30mm), whereas larger molecules (e.g., proteins) are not able to cross. Small molecules move through diffusion from a high- concentration zone to a low-concentration zone. Scaling down this process to small hollow fiber membranes is referred to as microdialysis. This sample preparation technique can be used to isolate compounds from tissue or other complex samples.
In contrast to most other sample preparation techniques used for biological samples, only the unbound free fraction of the analyte is isolated in this method.
Info-box 9.11
Although protein precipitation is little labor intensive and relatively easy to perform, it should be kept in mind that the resulting supernatant often needs some kind of treatment before injection into the HPLC (such as evaporation of organic solvents to dryness followed by reconstitution, pH adjustment, or precipitation of acidic reagents).
9.6 Membrane-Based Sample Preparation Techniques