Sample Preparation Tools have to be effective!
Nobody likes to invest time into Sample Preparation Tools. However everybody wants to have fast, precise and reproducible analytical results. The importance of Sample Preparation is often under-estimated and ignored. Apropriately chosen techniques will dramatically improve data quality, yield, column, equipment life. This is a land mark in professional chromatography.
- What is the source of your sample ?
- What do you aim to achive with your sample?
Sample Preparation Techniques
1. Dilute and Inject
The sample is diluted with an appropriate solvent and directly injected into LC-MS. It is a fast and low cost strategy and useful only for very lightly contaminated samples e.g. Surface Water Analytics.MS supplier argue that moving to HRMS or uHRMS combined with Dilute & Inject ist the method of choice. Many practitioners reject this claim. They argue that problems can often be solved with simpler MS in combination with sample enrichment. We don’t recommend this strategy because it ignores sample clean up.Matrix contamination can turn out to become very expensive and cumbersome. Solvent volatilisation is like matrix contamination also cumbersome.
2. Precipitate & Inject
The sample is treated with a solvent or reagent that causes physical precipitation of the unwanted contaminats. Its a fast and low cost technique often used for plasma samples.For protein precipitation you may just add acids e.g. TCA 10%(w/v) and HClO4 6%(w/v) or just add a three fold excess of Acetonitrile. Sometimes you may have to add some additional small quantity of Zinc Sulfate to assist precipitation. The mixture is centrifuged and the supernatant injected.
3. Flash and MPLC Chromatography
We offer a broad range of “automated” or “low cost manual Flash Chromatography” systems and expertise. Most chemist in industry use dry packed cartridges or glass cartridges in biochromatography,Cartridges and columns packed with irregular or spherical silica or bonded phase materials. Few users pack their own columns / cartridges, some procure dry packing equipment and most procure sealed professionaly packed cartridges.
Flash Chromatography is mainly used in organic synthesis and medicinal chemistry. In biotechnology uses polymer packed cartridges filled with ion-exchanger and RP material. Discuss this technology with us we offer 20 years experience.
4. Syringe Filters (Sterile / non sterile)
To be used for removal of micro and some nano particles from aqueous or solvent based media.The most popular are 0.45 μm and 0.22 μm porosity filters . Wide range of filter diameters available packed with hydrophilic and hydrophobic membrane filters. Sometimes also used for separating polar apolar solvents.
5. Extraction with Solvents
Very old and traditional method used since many years to extract compounds from plants, plasticiser from polymers, impurities from compounds etc.Materials to be extracted are brought in contact with the extracting solvent either by Percolation or reflux e.g in a Soxhlet or under pressure in a Super Critical Fluid Extractor (SFE).
Most solvents extract unwanted materials that have to be removed downstream.There is a practical book for extraction methods for specific Phytochemical groups. (Laboratory Handbook for the Fractionation of Natural Extracts, Peter J. Houghton, Chapman & Hall, ISBN 0 412 74910 6)
6. Liquid / Liquid extraction (LLE)
Liquid-Liquid Extraction is an old well know method but gained recent popularity due to better understanding of solvent properties.Any solvent could be used but most extract too many different compounds that have to be removed before further processing. Popular is MTBE (methyl – tert – butyl ether) because it is insoluble in water. Extraction is enhanced by manipulating the pH so that the analyte is neutral and soluble in organic solvents. This way it will partition into the organic solvent. The general thought is to move about 2 pH units above or below the pKa. The following rules apply: Bases pH < pKa = R-NH3+ ionised, pH > pKa = R-NH2 unionised. Acids: pH < pKa = R-COOH unionised, pH > pKa = R-COO-ionised. Dont hesitate to ask for help.
7. Supported Liquid Extraction (SLE)
Solid-supported liquid-liquid extraction sometimes also referred to as solid – liquid extraction or SLE. It comprises of a chemically inert, high – surface-area, highly purified, graded diatomaceous earth that serves as a stationary vehicle for the aqueous phase. Water easily adsorbs onto the surface of the diatomaceous earth particles. Diatomaceous earth is packed into cartridges. Aqueous samples such as serum, whole blood and urine, or environmental, food, or consumer products are diluted and pored over the diatomaceous earth. After 10 to 15 minutes distribution and wetting time an immiscible solvent is added to perform the extraction, resulting in a clean extract. SLE offers advantages over standard liquid-liquid extraction methods, including support for automation for high throughput operation. It provides better recoveries and precision by removing issues with emulsions that are often formed when performing liquid-liquid extraction. Because the aqueous sample has been widely dispersed throughout the solid support, the organic solvent has intimate contact with the thin film of aqueous phase and rapid extraction (equilibration) occurs. A phase-separation filter is incorporated into the outlet frit of the of the cartridge to ensure that organic effluents remain uncontaminated by aqueous matrix. SLE is better reproducible than LLE. The entire SLE process is easier to automate than traditional LLE.The 96-well plate SLE format is especially amenable to automation using xyz robotics systems. Pre-packed SLE products are available in all automation formats. In addition, bulk sorbent can be purchased for those who wish to make their own customized SLE device.
8. Solid Phase Extraction (SPE )
SPE is a sample preparation process by which compounds that are dissolved or suspended in a liquid mixture and separated from other compounds in the mixture according to their physical and chemical properties.SPE is fundamentally chromatography but using solid phase extraction materials. SPE is used to isolate analytes of interest from a wide variety of matrices, sample including urine, blood, water, beverages, soil, and animal tissue.The technique is used for concentrating and purifying samples for analysis.
The technique is also used on production scale of biologics e.g. for extracting interesting ingredients from plants that are sold as “Herbal Supplements”. In this context the term of “Adsorption LC is used.
SPE uses the affinity of solutes dissolved or suspended in a liquid (called liquid phase) for a solid (called solid phase) through which the sample is passed to separate the mixture into desired and undesired components. The result is that either the desired analytes of interest or undesired impurities in the sample are retained on the stationary phase.
The portion that passes through the stationary phase is collected or discarded, depending on whether it contains the desired analytes or undesired impurities.If the portion retained on the stationary phase includes the desired analytes, it can then be removed from the stationary phase for collection in an additional step, in which the stationary phase is rinsed with an appropriate eluent. For convenience SPE is sold in professionally packed disposable Cartridges / Kits / Syringes.For production scale application solid phase materials are procured in bulk and packed in large columns to enable back-flushing and reconditioning of columns for long-term use.We offer the most comprehensive product range of polymer, inorganic or silica base stationary phases packed in cartridges, columns or as bulk. We also guide you with relevant know how to assure your success.We have a wide range of organic and inorganic Solid Phase Extraction materials and interesting methods
Stands for (Quick Easy Cheap Effective Rugged Safe).It is a streamlined technique that has been used primarily in pesticide analysis but is now also used in bioanalytics.Some modifications to the original QuEChERS method had to be introduced to ensure efficient extraction of pH dependent compounds, to minimize degradation of susceptible compounds (e.g. base and acid labile pesticides) and to expand the spectrum of matrices covered.
a. The analyst homogenizes the sample in a blender.
b. To the homogenized sample an reagent is added and agitated for one minute. The reagents to be used depends on type of sample to be analysed.
c. The sample is put into a centrifuge tube.
d. The centrifuged sample is put through a clean-up column prior to analysis by Chromatography.
10. Packed Guard Columns for your preferred brand
Many column manufacturers recommend the use of packed guard columns that can be attached to the column to filter “contaminations” before they enter the column. Most manufacturers sell columns packed with relevant materials that can be screwed into the inlet of a column. We assist you to find the appropriately packed guard columns. Mechanically assisted Systems1. Direct Injection with on-line sample preparation system. The sample is applied directly to the LC-MS system which incorporates a sample clean-up/enrichment pre-column / trapping Column and switching valves. First time offered by Spark HollandSample Storage System1. High-Quality Autosampler Glass Vials. Wide range of Autosampler vials and caps to fit all types of Autosamplers2. High-Quality Autosampler Plastic Vials3. Inert Autosampler and Sample Storage Vials. Many analytical data in Trace Analysis may be questionable or simply wrong. Manufacturing glass vials have glass surfaces with different chemical activities. Some of these surfaces may extract key analytes from the sample or will change the chemistry of the sample altogether. We have developed a manufacturing process that produces RSA (Reduced Surface Active) Glass Autosampler or Sample Storage Vials. Every custom synthesiser that tries to reproduce client sample knows that reproducibility of recipes is more than often unsatisfactory. There are the following possible sources of problems: Synthesis and reporting was slapdash and thus not reproducible
The compound may be unstable due to storage in wrong solvents, the influence of light and temperature or due to surface catalytic interaction. Attending the problem will save large sums of money and frustrations!
Future compounds and analytes will become more complex. There are forces in many industries that actively simplify methods and processes. Their ultimate aim is to automate sample preparation through the introduction of Artificial Intelligence (AI) systems. The perpetrator of such strategies wants to reduce labour costs in the analysis and creation of new compounds. (2/3 of costs in drug discovery is wasted!) This strategy is illogical and will not safe money and will destroy the ability to compete. The main causes for the high costs are lack of process reproducibility due to poor sample purity and use of wrong tools. Many large companies have a contracted sole supplier. This eliminates competition and clients pay high prices, get very poor service and lousy delivery lead times. Many people in the supply chain are frustrated and procrastination becomes virulent. The fighting cost can only be done by producing pure raw materials and target compounds. That can only be achieved by using more appropriate technologies and no commodities. However, without motivated people, this is only theory.
High Throughput Bioanalytical Sample Preparation, Methods and Automation Strategies, By David Wells, January 2003, Elsevier, ISBN: 978-0-444-51029-7