Frequently Asked Questions: Sample Preparation
- What size SPE cartridge do I need?
- What is the composition of the SPE cartridges and frits?
- Is conditioning necessary for SPE cartridges?
- What SPE cartridge parts and accessories does Restek offer, and how are they used together?
- Are all of the necessary components included with the Resprep SPE manifold kits?
- How are SPE cartridges processed?
- What vacuum is suggested when using the SPE manifold?
- Can you suggest an SPE procedure for my matrix and the extraction of _______?
- What parts are included in the ASE cell end-caps?
- What ASE models does Restek support?
- Where can I find a QuEChERS method for my application?
- Can I use centrifuge tubes more than one time?
- What size syringe filters do I need?
- Which syringe filters are compatible with my solvents or matrix?
- Are Restek syringe filters autoclavable?
- What syringe filter should I use to filter nonaqueous streams (organic solvents)?
- How do I filter my sample using syringe filters?
- Can I get a free sample pack of syringe filters?
- What solvents are compatible with the filter vials?
- What drugs and compounds are compatible with the filter vials?
- What needle depth is used on the autosamplers?
- What are the total fill and dead volumes for the filter vials?
- What size are the filter vials?
- Are free sample packs available?
- Can I use solvents other than acetonitrile?
- Can I use acidified organic solvents?
- What should the organic solvent:sample ratio be for effective precipitation?
- How long should I vortex to mix?
- Is dilution necessary?
- Does it matter what filtration device I use?
- Do I have to use all the wells on a single plate at the same time?
- Does this product have an expiration date?
- How long can Resprep PPT3 96-well plates hold the solvent and sample without dripping?
Solid Phase Extraction (SPE)
Accelerated Solvent Extraction (ASE)
Thomson SINGLE StEP Filter Vials
Resprep PPT3 96-Well Plates
What size SPE cartridge do I need?
Cartridge size should be selected based on sample volume, while the bed weight of the packing is determined by the compound load (analytes of interest and interferences). Choose the SPE cartridge that is best for your application based on the following table.
100 mL - 1 L
If the sample volume exceeds the capacity of the SPE cartridge being used, then a larger, empty SPE cartridge can be used along with a connector to hold the additional volume. Alternatively, the Resprep Sample Delivery System can be used.
What is the composition of the SPE cartridges and frits?
In general, all solid phase extraction (SPE) products are made of an empty cartridge, two frits (top frit and bottom frit), and sorbent media that is held between the frits as shown in the figure below. Materials for the cartridges are usually high-quality polypropylene, but some variations (e.g., glass) may be available. Frits are typically porous polyethylene, but in some SPE products PTFE or metal mesh may be used for specific applications. The sorbent media is where the separation of target compounds and interferences occurs through different modes of interaction between the sorbent media and your sample. The sorbent media is often called a “phase,”’ and it can be bonded or unbonded silica, polymeric materials, or various carbon materials.
Is conditioning necessary for SPE cartridges?
Yes. Detailed instructions for conditioning SPE cartridges can be found here. All SPE cartridges should be conditioned to wet and settle the bed, activate the packing materials, and remove any residual process materials (e.g., fines).
As a general guide:
• Use 1 to 2 column volumes of the conditioning solution(s) recommended in the table below.
• Use a high flow rate.
• Do not allow the packing bed to go dry. The packing bed should be wet before adding sample.
Characteristics of Reversed and Normal Phase SPE
packing and analytes are nonpolar
polar, often aqueous
a) methanol, then water or buffer (same as sample), or
b) final extraction solvent, then water-miscible solvent, then water or buffer
nonpolar solvent or mixed solvent solution
silica, Florisil, carbon
packing and analytes are polar
nonpolar, often an organic solvent
fresh solvent (same as sample)
What SPE cartridge parts and accessories does Restek offer, and how are they used together?
Restek carries many accessories for SPE cartridges. To assure compatibility, simply match sizes across parts when ordering. For example, if 6 mL SPE cartridges are used, then 6 mL SPE accessories are needed, so part numbers 26012 (empty cartridge), 26018 (frits), 26003 (tube caps), and 26007 (connectors) are the correct items to choose.
The empty tubes are just that, empty SPE cartridges. To assemble, the frits should be securely placed at the bottom of each empty SPE cartridge. The tube caps are then used to cap the tops of the SPE cartridges. Connectors are used to join accessories and cartridges. The tops of the connectors all have a female luer fitting that will accept any accessory that has a male luer fitting, such as NORM-JECT syringes, syringe filters, and SPE tubes (full or empty). The female luer end caps (cat.# 26000) are universal and fit all male luer tips. This means the female luer end caps are compatible with all Restek SPE tubes, as well as with NORM-JECT syringes and syringe filters.
Are all of the necessary components included with the Resprep SPE manifold kits?
Yes, please refer to the exploded view diagram below for all the parts that are included in the kit and their relative positions in the manifold. Other manifold replacement parts are also available and can be purchased separately.
Resprep Manifold Replacement Parts
How are SPE cartridges processed?
Cartridges may be processed by any of the techniques shown below.
A. Vacuum Manifold
The most common way to process SPE cartridges is to use an SPE vacuum manifold along with a vacuum source, such as a house vacuum or an oil-free vacuum/pressure station. If you are using a well plate SPE product, a vacuum manifold specifically designed for well plates, such as the Resprep VM-96 vacuum manifold, can be used to process your SPE well plate.
B. Positive Pressure
The positive pressure technique uses an appropriate syringe (with a male luer fitting) along with the correct size SPE cartridge connector. An electronically controlled or air-actuated positive pressure system can also be used to process either a well plate or multiple cartridges at once.
C. Vacuum-Sidearm Flask
A vacuum-sidearm flask, along with a water faucet for aspiration, takes advantage of the Venturi effect. A standard vacuum source can also be used.
What vacuum is suggested when using the SPE manifold?
Any vacuum up to 20 inches of Hg would be acceptable. Do not exceed 20 inches of Hg. Complete SPE manifold instructions can be found here and you can find a recommended vacuum pump for SPE here and here.
Can you suggest an SPE procedure for my matrix and the extraction of _______?
Identifying the most appropriate SPE procedure is very dependent on the matrix and the target compounds. Our wide range of technical resources will help you find a procedure that is suitable for your specific analytes and matrix. If you still have questions, please contact our technical support team and we will provide a recommendation for your application.
What parts are included in the ASE cell end-caps?
A frit, washer, PTFE O-ring, threaded insert, and snap ring are all included with each ASE cell end-cap. Restek also offers a more comprehensive line of ASE replacement parts.
What ASE models does Restek support?
Where can I find a QuEChERS method for my application?
QuEChERS, which stands for “Quick, Easy, Cheap, Effective, Rugged, and Safe,” is an increasingly popular approach to sample preparation for multiresidue pesticides analysis of a variety of food matrices. It is simpler and more streamlined than traditional liquid-liquid extractions and even easier than SPE. Three simple steps are involved with all QuEChERS methods: sample homogenization, extraction, and cleanup using dSPE tubes.
QuEChERS sample preparation for many analyses can be done by using the Original Unbuffered Method, which is the simplest procedure. Additionally, several buffered methods exist, which can improve recoveries for pH-sensitive pesticides. The most widely adopted buffered methods are EN 15662, Mini-Multiresidue, and AOAC 2007.01. In addition to these frequently used methods, you can quickly find a method for a particular analysis using our extensive bibliography.
Download Restek’s QuEChERS guide for an illustration of method steps, example applications, and a selection guide that shows exactly which dSPE tubes and sorbents are needed for each of the primary QuEChERS methods, including dSPE tubes for generic or universal use. In addition, validation and proficiency data for the QuEChERS method are available for a wide variety of pesticides in several common food matrices at www.quechers.com. Restek's ChromaBLOGraphy is also a good source for a variety of QuEChERS applications. Use "QuEChERS" as a keyword to search for relevant blog articles.
Can I use centrifuge tubes more than one time?
Only FEP (fluorinated ethylene propylene) centrifuge tubes are designed for multiple use. Polypropylene centrifuge tubes are recommended for one-time use only.
What size syringe filters do I need?
Which syringe filters are compatible with my solvents or matrix?
Are Restek syringe filters autoclavable?
What syringe filter should I use to filter nonaqueous streams (organic solvents)?
How do I filter my sample using syringe filters?
Can I get a free sample pack of syringe filters?
What solvents are compatible with the filter vials?
What drugs and compounds are compatible with the filter vials?
What needle depth is used on the autosamplers?
What are the total fill and dead volumes for the filter vials?
What size are the filter vials?
Are free sample packs available?
Can I use solvents other than acetonitrile?
Can I use acidified organic solvents?
What should the organic solvent:sample ratio be for effective precipitation?
How long should I vortex to mix?
Is dilution necessary?
Does it matter what filtration device I use?
Do I have to use all the wells on a single plate at the same time?
Does this product have an expiration date?
How long can Resprep PPT3 96-well plates hold the solvent and sample without dripping?
Solid Phase Extraction (SPE)
Accelerated Solvent Extraction (ASE)
Syringe filters are sized according to inner diameter. Choose the correct size based on the volume of your sample using the table below.
4 mm ID
13 mm ID
25 mm ID
30 mm ID
Please use the Syringe Filter Membrane Selection Guide (below) and the Syringe Filter Solvent Compatibility Chart. Best practices dictate verifying compatibility prior to use.
Syringe Filter Membrane Selection Guide
hydrophilic, low protein binding
bases, HPLC solvents, alcohols, aromatic hydrocarbons
acids, aggressive halogenated hydrocarbons, proteins
hydrophilic, low protein binding, fast flow rates
filtration of butters & culture media
organic solvents, acids, alcohols, bases, aromatics
aqueous samples without pre-wetting (to avoid high backpressure)
hydrophilic, low protein binding
bases, esters, ethers, ketones
Hydrophilic applications: cellulose acetate, nylon, PES, PVDF
Yes, they are autoclavable to 121 °C for 15 minutes (up to 75 psi).
PTFE syringe filters, which are hydrophobic in nature, should be used to filter nonaqueous (organic) solvents. Higher backpressures will result from using PTFE filters with aqueous streams, which may prevent the stream from passing through the filter.
NORM-JECT plastic syringes are recommended for sample syringe filtering. To filter your sample, simply attach the appropriate syringe filter to the NORM-JECT syringe and depress the plunger, capturing the effluent in a collection vial. Be sure to choose the correct volume syringe for the sample volume you wish to filter.
Please note that NORM-JECT plastic syringes should not be used with any solvents or chemicals that are incompatible with polypropylene or high-density polyethylene (e.g., aliphatic hydrocarbons, aromatic hydrocarbons, aqua regia, carbon tetrachloride, methylene chloride, nitric acid, tetrachloroethylene, trichloroethylene, and xylenes). Use a glass-barreled syringe for any incompatible solvents.
Yes, contact Restek’s customer service team to request a free sample pack. Please note there is a limit of one free pack per customer.
Thomson Single StEP Filter Vials
Most solvents and mobile phases used in liquid chromatography are also compatible with Thomson SINGLE StEP filter vials. Use the Solvent Compatibility Chart for Filter Vials as a guide.
Many common drugs and compounds of interest in clinical/toxicology or drinking water samples are compatible with Thomson SINGLE StEP filter vials. Use the chart on page 2 of our Thomson Single StEP Filter Vial brochure as a guide.
Set the needle depth to 5 mm above the bottom of the shell (outer) vial.
The total fill volume is 450 µL up to the fill line, and the total dead volume is 120 µL.
The filter vials are 12 mm x 32 mm and fit most standard autosamplers.
Yes, just add "-248" to any filter vial catalog#.
Resprep PPT3 96-Well Plate
Yes, you can use a variety of solvents. Resprep PPT3 96-well plates are made of high-quality polypropylene housing and solvent-resistant membrane materials. Many standard solvents such as methanol, dichloromethane, etc., can be used, but different solvents will produce different protein precipitation results. Acetonitrile is recommended because it is readily available in most laboratories, and it provides strong protein precipitation capability for samples that contain high levels of protein (e.g., plasma).
Yes. Acetonitrile with 0.1–1% formic acid is often used to improve protein precipitation because it disrupts binding between the protein and the protein-bound analytes. Acidified acetonitrile also is widely used in LC-MS/MS analysis with an electron spray ionization (ESI) source.
Typically, a 3:1 ratio of organic solvent to sample is used. Adding more organic solvent may produce better protein precipitation, but the additional solvent should be calculated into the dilution factor. The analyte concentration in the final filtrate will be diluted by the actual solvent:sample ratio that is used.
To ensure proper mixing, we recommend vortexing for 0.5–2 min at a minimum speed of 2,000 rpm. The mixing time will depend on solvent volume, sample volume, and sample type. If protein removal is incomplete, increase the mixing time and/or the vortex speed (up to 3,000 rpm).
Theoretically, if a 3:1 solvent:sample ratio is used for protein precipitation during the filtration steps, then the final filtrate will be approximately 75% acetonitrile. If the initial liquid chromatography mobile phase composition is not close to 75% organic solvent or if poor chromatographic peak shapes are observed for early eluting compounds, we recommend diluting the filtrate in the collection plate with water or aqueous mobile phase to more closely match the initial mobile phase composition. If further dilution is not suitable for your application due to limited instrument sensitivity, evaporation followed by reconstitution with your initial mobile phase can be performed. This approach can be used to obtain a suitable concentration while avoiding peak shape issues that can be caused by unmatched sample solvent and initial mobile phase.
No. Resprep PPT3 96-well plates are compatible with vacuum manifolds, positive pressure manifolds, and centrifuges. You may choose whichever method is available in your laboratory.
No. You may use some of wells on the plate and then use the unused wells later.
Resprep PPT3 96-well plates do not have an expiration date. Customers usually store them for a few months to a few years for inventory purposes.
No dripping for a minimum of 12 hours is guaranteed as shown on the certificate of analysis. Our experiments demonstrate 24 hours without dripping, using both acetonitrile and dichloromethane.