Frequently Asked Questions: Sample Preparation
- How are Resprep PLR phospholipid removal SPE products different from similar products on the market?
- 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?
- What is included with Resprep Manifolds and are there replacement parts?
- 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 _______?
- When should I use CarboPrep Plus cartridges instead of Florisil cartridges for the cleanup of samples being analyzed for organochlorine pesticides (e.g., EPA Method 8081)?
- What is the difference between CarboPrep and CarboPrep Plus cartridges?
- Will CarboPrep Plus cartridges clean up sulfur?
- Why are there 95 mg of treated carbon in CarboPrep Plus cartridges?
- Where can I look up a certificate for my CarboPrep Plus cartridges?
- What is the shelf life of an unopened package of CarboPrep Plus cartridges?
- How should I store unused CarboPrep Plus cartridges?
- How are CarboPrep Plus cartridges a more economical choice for our lab overall?
- The retention of 2,4,5-trichlorophenol is sometimes used as a measure of Florisil cartridge performance. Can I use the same measure to validate CarboPrep Plus cartridges?
- Can I get a free sample of CarboPrep Plus cartridges to try for my lab?
- What parts are included in the ASE cell end-caps?
- What ASE models does Restek support?
- What ASE parts from Restek are similar to my Dionex parts?
- 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?
- Are Restek SPME fibers compatible with a Sigma manual holder?
- Are Sigma SPME fibers compatible with a Restek manual holder?
- Are Restek SPME Arrows compatible with a Sigma holder?
- How do I tell the difference between 1.1 mm and 1.5 mm diameter SPME Arrows?
- How does SLE work?
- When might SLE be a better option than SPE or liquid-liquid extraction?
- What types of samples and analytes can be processed with SLE?
Solid Phase Extraction (SPE)
CarboPrep Plus Cartridges
Accelerated Solvent Extraction (ASE)
Thomson SINGLE StEP Filter Vials
Resprep PPT3 96-Well Plates
Solid Phase Microextraction (SPME)
Supported Liquid Extraction (SLE)
How are Resprep PLR phospholipid removal SPE products different from similar products on the market?
The performance of Resprep PLR SPE products is similar to other commercially available phospholipid removal products, but the novel co-sintered design results in a stabilized sorbent, a high degree of manufacturing control, and excellent uniformity from well to well, plate to plate, and cartridge to cartridge. The proprietary sorbent used in Resprep PLR products is immobilized in a rigid, porous polymeric structure, which is not susceptible to problems that packed beds of loose sorbent can experience, such as channeling.
Solid Phase Extraction (SPE)
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 and HENKE-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 and HENKE-JECT syringes and syringe filters.
What is included with Resprep Manifolds and are there replacement parts??
Components included with Resprep Quick-Replace (12- or 24-port) and Resprep VM-96 vacuum manifold are listed below.
Resprep Quick-Replace (12- or 24-port) vacuum manifold components:
- Cover with 12 flow control valves & gasket
- Glass basin with vacuum gauge & valve assembly
- Collection rack (base, 3 support rods, center plate, 10 mm test tube plate, 12 clips)
- Plate for 16 mm test tubes
- 12 test tubes (10 x 75 mm)
- 12 liner guides (stainless steel)
- 100 quick-replace disposable liners (PTFE)
Resprep VM-96 vacuum manifold components:
- Top unit with glass observation window (constructed of aluminum with stainless-steel support around the glass window)
- Bottom unit (aluminum) with four rubber feet
- Precision height adaptor (aluminum)
- Vacuum gauge
- Valve block assembly
- a. Vacuum attachment port (with on/off valve)
- b. Vacuum control valve
- Shim pack
- a. Two 2 mm aluminum shims
- b. One 6 mm polypropylene shim
- c. Two 11 mm polypropylene shims
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 a Chemker or Rocker 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.
CarboPrep Plus Cartridges
When should I use CarboPrep Plus cartridges instead of Florisil cartridges for the cleanup of samples being analyzed for organochlorine pesticides (e.g., EPA Method 8081)?
Florisil cartridges are a long-standing choice for the cleanup of organochlorine pesticide samples, and they are still a good choice when you are confident that your samples are relatively clean and free of low-volatility compounds that can be deposited in inlets and on the heads of columns. The ability of Florisil adsorbent to retain highly polar compounds also makes it ideal if your sample contains polar matrix components. However, if you know or suspect that your samples contain relatively nonpolar matrix contaminants, or if your samples are highly colored, CarboPrep Plus cartridges will do a better job of removing compounds that will contaminate inlets and result in premature failure of calibration check standards.
What is the difference between CarboPrep and CarboPrep Plus cartridges?
CarboPrep material is available in two different published surface areas: CarboPrep 90 adsorbent, which has an approximately 90 m2/g surface area and CarboPrep 200 adsorbent, which has approximately 200 m2/g surface area. These are both considered general-purpose products. In contrast, CarboPrep Plus cartridges use a carbon adsorbent with a proprietary surface area that has been specifically optimized for the cleanup of organochlorine pesticide extracts.
Will CarboPrep Plus cartridges clean up sulfur?
No. For the cleanup of sulfur, we recommend using granulated activated copper (cat.# 26136).
Why are there 95 mg of treated carbon in CarboPrep Plus cartridges?
This mass was carefully chosen because it provides the best performance for the analysis of organochlorine pesticides in soils and water while still using the same solvents, solvent volumes, and equipment that are used for a typical Florisil cleanup.
Where can I look up a certificate for my CarboPrep Plus cartridges?
What is the shelf life of an unopened package of CarboPrep Plus cartridges?
How should I store unused CarboPrep Plus cartridges?
CarboPrep Plus SPE cartridges are manufactured in a strictly controlled environment and housed in superior contamination-resistant packaging. Once the original packaging has been opened, the cartridges should be used immediately; if this is not possible, we strongly recommend that any unused cartridges be stored in a clean, closed environment, such as in a desiccator that is free of organic volatiles.
How are CarboPrep Plus cartridges a more economical choice for our lab overall?
The cost of instrument downtime, delayed or late data generation, and unplanned consumables replacement overtime are beyond simple expense calculations. CarboPrep Plus cartridges can reduce downtime for maintenance and increase the lifetime of continuing calibration checks by removing the nonvolatile sample matrix compounds that contaminate inlets and columns and cause calibration check standards to fail prematurely.
The retention of 2,4,5-trichlorophenol is sometimes used as a measure of Florisil cartridge performance. Can I use the same measure to validate CarboPrep Plus cartridges?
No. Even though CarboPrep Plus cartridges have been carefully designed to permit excellent recoveries of organochlorine pesticides, the adsorbent material does have a different selectivity than Florisil material. That different selectivity is what makes it so good at stopping the relatively nonvolatile compounds in sample matrices that can contaminate inlets and columns. However, that difference also means certain traditional gauges of Florisil cartridge quality (e.g., the retention 2,4,5-trichlorophenol) cannot be used to validate CarboPrep Plus cartridges.
Can I get a free sample of CarboPrep Plus cartridges to try for my lab?
Accelerated Solvent Extraction (ASE)
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?
What ASE parts from Restek are similar to my Dionex parts?
Please see individual ASE replacement parts on our website or our convenient ASE replacement part cross-reference. We guarantee the compatibility of Restek and Dionex ASE parts only between entirely assembled caps and bodies. For example, if a Restek cap is assembled using all Restek parts, then it is compatible with a Dionex body. However, if a Restek cap has a Dionex insert (or vice versa), we cannot guarantee compatibility.
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.
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?
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
Which syringe filters are compatible with my solvents or matrix?
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
Are Restek syringe filters autoclavable?
Yes, they are autoclavable to 121 °C for 15 minutes (up to 75 psi).
What syringe filter should I use to filter nonaqueous streams (organic solvents)?
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.
How do I filter my sample using syringe filters?
NORM-JECT and HENKE-JECT plastic syringes are recommended for sample syringe filtering. To filter your sample, simply attach the appropriate syringe filter to the NORM-JECT and HENKE-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 and HENKE-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.
Can I get a free sample pack of syringe filters?
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
What solvents are compatible with the 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.
What drugs and compounds are compatible with the filter vials?
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.
What needle depth is used on the autosamplers?
Set the needle depth to 5 mm above the bottom of the shell (outer) vial.
What are the total fill and dead volumes for the filter vials?
The total fill volume is 450 µL up to the fill line, and the total dead volume is 120 µL.
What size are the filter vials?
The filter vials are 12 mm x 32 mm and fit most standard autosamplers.
Are free sample packs available?
Yes, just add "-248" to any filter vial catalog#.
Resprep PPT3 96-Well Plate
Can I use solvents other than acetonitrile?
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).
Can I use acidified organic solvents?
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.
What should the organic solvent:sample ratio be for effective precipitation?
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.
How long should I vortex to mix?
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).
Is dilution necessary?
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.
Does it matter what filtration device I use?
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.
Do I have to use all the wells on a single plate at the same time?
No. You may use some of wells on the plate and then use the unused wells later.
Does this product have an expiration date?
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.
How long can Resprep PPT3 96-well plates hold the solvent and sample without dripping?
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.
Solid Phase Microextraction (SPME)
Are Restek SPME fibers compatible with a Sigma manual holder?
Are Sigma SPME fibers compatible with a Restek manual holder?
Are Restek SPME Arrows compatible with a Sigma holder?
How do I tell the difference between 1.1 mm and 1.5 mm diameter SPME Arrows?
The 1.1 mm SPME Arrow has one ring around the hub, whereas the 1.5 mm SPME Arrow has two rings around the hub.
Supported Liquid Extraction (SLE)
How does SLE work?
SLE is essentially a scaled-down liquid-liquid extraction without the solvent waste and time-consuming, variation-introducing shaking steps. In SLE cleanup, samples are diluted in an aqueous solution and loaded onto a cartridge or 96-well plate containing a diatomaceous support material. Simply load the sample, wait 5 minutes for the water to fully disperse through the high surface area of the support, then elute your target compounds with a nonpolar solvent. The matrix materials remain in the polar aqueous phase absorbed in the support material while the analytes of interest partition cleanly into the nonpolar extraction solvent. It’s ideal for neutral (nonionizable) compounds and can also be used for acids and bases following a simple pH adjustment for charge suppression.
When might SLE be a better option than SPE or liquid-liquid extraction?
If your sample matrix needs some cleanup, but does not require the intensive power of SPE or liquid-liquid extraction, we recommend comparing SLE to your current method. If your results have improved relative to dilute-and-shoot, adopting SLE will be advantageous; but, if your samples still require additional cleanup, then SPE is recommended.
Evaluating whether SLE cleanup will work for your application is simple. If your analytes are neutral compounds, simply dilute in an aqueous solution and load them onto a Resprep SLE cartridge or 96-well plate. Wait 5 minutes, then elute in a nonpolar solvent. If you are analyzing acids or bases, pretreat the samples by adjusting the pH relative to the analytes’ pKa values. By changing the pH, you suppress the molecular charges, which increases partitioning into the extraction solvent.
Details on pretreatment pH adjustment and extraction solvent recommendations are available in our Resprep SLE instruction sheet.
What types of samples and analytes can be processed with SLE?
For many compounds, excellent analytical results can be obtained with a quick and easy SLE cleanup. As shown in this comparison, accurate and precise results—with performance matching a leading supplier—were obtained for acidic, basic, and neutral analytes following a simple Resprep SLE cleanup of plasma samples.
Experimental detail: Gender-pooled and K2EDTA-treated human plasma samples were fortified at concentrations ranging from 0.0125 µg/mL to 2.18 µg/mL. Acidic compound aliquots were diluted in 2% formic acid (aqueous) while basic compound aliquots were diluted in 5% ammonium hydroxide (aqueous). 200 µL of diluted sample (1:1 dilution with pretreatment solutions) were loaded onto a 200 mg Resprep SLE well plate. After 5 min, the samples were eluted with 1 ml of 50:50 dichloromethane:ethyl acetate. 100 µL 50 mM HCl was added to the basic pretreated wells; then, all wells were blown down to dryness and reconstituted in 1:1 methanol:water. Recovery values were determined using matrix-matched calibration. N≥36 wells for each treatment; 4.7–14.4 %RSD across all compounds.