Medical Grade Polypropylene Tube

Introduction to Solid Phase Extraction Solid Phase Extraction products provide cost-effective, fast and efficient sample clean-up and concentration prior to analysis through gas chromatography (GC), high performance liquid chromatography (HPLC) and/or other analytical methods. How to Use Solid Phase Extraction Products SPE cartridges are comprised of three main components: Clean medical grade polypropylene tubes, clean and porous polyethylene frits, and high quality, reproducible sorbents and/or stationary phases. The cartridges are designed with both manual and automated uses in mind. All of our solid phase extraction syringe cartridges are equipped with male Luer-tips, providing great versatility for connections and processing. Common processing methods include using a syringe plunger to introduce the sample/solvent into the syringe barrel, placing the SPE cartridge into a centrifuge and pulling the sample/solvent through the cartridge by centrifugal force, or boring a hole in a rubber stopper to accommodate the SPE cartridge and placing the stopper in a vacuum flask. Vacuum aspiration is then used to pull the sample/solvent through the cartridge. In a similar application, multiple cartridges may be used to process multiple samples by using an adapter and vacuum manifold hooked up to a vacuum source. Sample throughput may also be increased by using large-reservoir SPE cartridges. Combined with other automation methods, significant gains in lab efficiency may be achieved. The principles of Solid-Phase Extraction are very similar to those of liquid chromatography. However, they are applied differently. In liquid chromatography an appropriate mobile phase is chosen to allow the sample components to continuously interact with the packing bed as they pass through the column. The extent to which the sample components interact with the packing varies due to structural and functional

Porous Polyethylene Frit

Stationary Phase

Porous Polyethylene Frit

Male Luer Tip

group differences, causing them to exit the column at different times, ideally allowing for complete separation of each component. When using Solid-Phase Extraction, initial conditions are chosen which, instead of allowing continual interaction, allow for the complete retention (minimum or no breakthrough) of either the sample compounds or interfering compounds. The solvent is then changed to one which allows for the complete elution (minimal or no interaction) of the pre-adsorbed and desired analytes from the packing bed. Hence the terms “elution” and “extraction” chromatography respectfully are generally used to describe HPLC and SPE techniques. APPROACH TO OPTIMAL EXTRACTION RESULTS 1. Proper physical and chemical characterization of the sample. Factors such as

the polarity of the analytes relative to the matrix, the presence of charged functional groups, solubility, molecular weight, etc. determine how strongly the desired analyte is retained by the packing bed.

2. Selection of proper retention strategy. Two extraction approaches are possible:

First, the interfering compounds may be strongly retained by the packing bed, allowing the desired analyte to be passed through with no retention. Thus the sample is cleaned by the selective retention of the impurities. The second strategy is a reverse of the first. The desired analyte is retained by the packing bed while the interferences are either non-retained, or washed off of the packing bed prior to eluting the analyte. This latter approach is commonly employed when concentration of the sample is desired.

3. Selection of the proper packing type. The different packing types offer various

selectivities that should be exploited to maximize the structural differences between the analytes and sample interferences. Choosing a packing sorbent with the proper selectivity results in the cleanest extract with the highest recovery.

4. Selection of the proper packing bed size. Conversely, poor sample recovery

often occurs when the packing bed size has not been optimized for the sample. A packing bed which is too large yields strong retention and incomplete elution of the desired analytes while a bed which is too small results in incomplete retention (breakthrough) of the desired analytes. Both situations result in poorer than optimal recoveries. (Rule of Thumb: The typical capacity is 1-3% of analyte relative to the packing bed size. This percentage will vary according to the cleanliness of the sample).

5. Selection of suitable conditioning, wash and elution solvents. Attention should

be paid to solvent strength relative to the packing material. (Rule of Thumb: For normal phase packings, the more polar the solvent is, the greater its strength. For reversed phase packings, the less polar the solvent is, the greater its strength.)

With ion exchange packings, the strength is usually related to the pH and the ionic/salt concentration. Buffers should be used to control ionization of potentially charged compounds.

The final conditioning solvents should be “weak solvents” which won’t act as an elution solvent. Wash solvents should remove weakly retained interferences but shouldn’t be strong enough to elute the analyte. Elution solvents should be strong and effective enough to completely elute an analyte in a small volume (1-2 mL). Please refer to the table for comparisons of solvent strengths.

Solvent Strengths for SPE Applications

Normal Phase Weak Solvent Reversed Phase Hexane Water Isooctane Methanol Toluene Isopropyl alcohol Chloroform Acetonitrile Methylene chloride Acetone Tetrahydrofuran Ethyl acetate Ethyl ether Ethyl ether Ethyl acetate Tetrahydrofuran Acetone Methylene chloride Acetonitrile Chloroform Isopropyl alcohol Toluene Methanol

Isooctane Water Strong Solvent Hexane Choosing the Proper Packing Type

Classifications of Solid Phase Extraction sorbents are very similar to that found in liquid chromatography. There are three basic types of sorbents: 1. Reversed Phase. Reversed phase packings include C18 which is the most popular

and widely used reversed phase. In addition, C8, C4, C2, C1, and Phenyl bonded phases are available and offer a variety of selectivities. Generally, non-polar to moderately polar compounds in a polar matrix are retained by reversed-phase sorbents. Reversed phase packings require conditioning with an organic solvent followed by an aqueous solvent prior to use. Elution of moderately polar compounds is often accomplished with mid-polarity solvents while non-polar compounds require less polar solvents.

2. Normal Phase. Silica, Florisil, Amino, Cyano, Diol and Alumina are also available

from Chrom Expert. Normal phase sorbents are usually used to retain polar compounds from non-polar matrices. Conditioning is carried-out with a non-polar solvent. Elution is accomplished with more polar solvents. Compounds with pH

basic functional groups are readily retained by silica. However, very polar compounds (e.g. carbohydrates) can be irreversibly retained on a silica surface. In this case, a bonded and mid-polarity phase such as Diol or Amino is a better choice.

3. Ion Exchange. Strong Anion (SAX) and Strong Cation (SCX) exchangers are

available. Anions and cations are retained on the corresponding resin by exchanging the Anion/Cation in the sample with the Anion/Cation on the resin. Elution is accomplished with high ionic strength buffers (0.1M-0.5M) or by changing the pH of the elution solvent such that the sample compound is no longer charged. These resins are silica based and lose some exchange capacity with increasing organic solvent content in the sample. The nature of the sample and the compound(s) to be isolated will determine the

proper packing type. Aqueous or polar organic matrices generally require reversed phases while non-polar matrices require normal phase. Adding an appropriate solvent to increase or decrease its polarity can often modify the matrix. The analyst must also determine whether the compound of interest is to be retained for later elution or whether the goal is to retain the interfering compounds. For example, a non-polar analyte (e.g. pesticides or drugs) in a polar sample matrix (e.g. water, urine) would indicate that a reversed phase would be the appropriate packing material to choose. A C18 or C8 packing would preferentially retain non-polar compounds, allowing weakly retained polar compounds to be washed off with a polar solvent prior to the final elution with a non-polar solvent.

Chrom Expert Company offers SPE cartridges with a wide variety of sorbent types, weights and column sizes. However, if the desired configurations or phases do not appear in our product list, please contact us to ask about availability. GENERAL EXTRACTION PROTOCOLS

Regardless of the type of phase being used, most SPE protocols will consist of the following steps:

1) CONDITIONING - Wet the sorbent and allow for efficient solvent compatibility

with the incoming sample matrix 2) SAMPLE INTRODUCTION - Effective transfer to the SPE cartridge without

loosing sample to breakthrough ('tight' introduction to sample bed) 3) WASH - Use of weak solvent to cleanse sample matrix of impurities without

affecting the analyte retention with the sorbent 4) ELUTION - Effectively remove the desired analytes from the sorbent bed with

the use of a minimal solvent volume

REVERSED PHASE: 1. Conditioning

A. Rinse packing bed with 3-5mL methanol B. Rinse packing bed with 3-5mL water or

buffer. Don’t let packing bed dry before adding sample.

2. Sample Introduction

A. Apply sample solution to the top of the packing bed. Push or draw the sample through the bed at a flow rate of 1-5mL/minute. Collect sample for analysis if desired compound has passed through the packing bed without being retained.

3. Wash

A. If the desired compound was retained, wash off any weakly retained interfering compound(s) with a polar solvent.

4. Elution

A. Elute desired compound with 1-2mL of a non-polar solvent(s) and collect for analysis.

NORMAL PHASE: 1. Conditioning

A. Rinse packing bed with 3-5mL of a non-polar solvent. Don’t let packing bed dry before adding sample.

2. Sample Introduction

A. Apply sample solution to the top of the packing bed. Push or draw the sample through the bed at a flow rate of 1-5mL/minute. Collect sample for analysis if desired compound has passed through the packing bed without being retained.

3. Wash

A. If the desired compound was retained, wash off any weakly retained interfering compound(s) with a non-polar solvent.

4. Elution

A. Elute desired compound with 1-2mL of a polar solvent(s) and collect for analysis.

ION EXCHANGES: 1. Conditioning

A. Rinse packing bed with 5mL of de-ionized water or low ionic strength buffer (e.g. 0.001M-0.01M).

2. Sample Introduction

A. Apply sample to the top of the packing bed. Push or draw the sample through the bed at a flow rate of 1-2mL/minute. Collect sample for analysis if desired compound has passed through the packing bed without being retained.

3. Wash

A. If the desired compound was retained, wash off any weakly retained interfering compound(s) with de-ionized water or low strength buffer.

4. Elution

A. Elute desired compound with 1-5mL of a high salt concentration solution (e.g. 0.1-0.5M) or change elution buffer pH such that the sample compound is no longer ionized and collect for analysis.

SUMMARY

SPE techniques are simple and effective methods to separate groups of compounds with different characteristics. However this method is not intended nor designed to separate each individual compound. This is why SPE has found its most effective use as a sample clean-up and concentration method prior to further analysis.

The following tables provide product listings for our various SPE cartridges, bulk

sorbents and accessory items.

Tel: 01295 812919Fax: 01295 816717

sales@charltonsci.co.ukwww.charltonsci.co.uk

Charlton Scientific Ltd 2, The Walnuts Main Street, Charlton Oxon, OX17 3DR