SPE Method Development

SPE is Chromatography

Keep in mind that solid-phase extraction has the same fundamental basis as HPLC. Any knowledge

of the chromatographic behavior of the analytes of interest, and of other matrix components, can

help in choosing the proper sorbent and eluents. If, for example, you know that certain

chromatographic conditions provide excellent separation of your analyte from interferences, then

you may choose a similar SPE sorbent and solvent combination. Similarly, if you are trying to

remove an interference that coelutes in HPLC, then you know a priori that similar SPE conditions

will not be successful.

General Elution Protocols

There are two general strategies for isolating and cleaning up sample components of interest:

adsorb matrix interferences while components of interest pass through the cartridge unretained.

adsorb components of interest while matrix interferences pass through the cartridge unretained.

The first strategy is usually chosen when the desired sample component is present in high

concentration. When components of interest are present at low levels, or multiple components of

widely differing polarities need to be isolated, then the second strategy is generally employed.

Trace enrichment of compounds present at extremely low levels and concentration of dilute

samples are also achieved by the second strategy.

Steps of a Solid-Phase Extraction Procedure

The following section describes the steps involved in a complete solid-phase extraction procedure.

In many applications, one or more of the steps, listed below and subsequently described by general

examples, can be omitted, thereby simplifying the procedure. The procedures illustrated here use

samples containing dyes so that separations may be easily visualized. Keep in mind that most

samples contain colorless components that require some type of detector or test to locate them in

the collected fractions. Use the following information as a guideline in the development of your

own procedure or when modifying procedures published in the literature.

1. Pretreatment of the sample

2. Conditioning of the cartridge

3. Loading the sample

4. Elution of the fractions

Principal Separation Modes in Solid-Phase Extraction [SPE]

Normal-Phase Chromatography

This mode is classically used to separate neutral organic compounds whose chemical nature

ranges from hydrophobic to moderately polar.

To perform normal-phase chromatography with SPE cartridges, use a step gradient of nonpolar

solvents with a polar packing material.

1. Condition the cartridge with six to ten hold-up volumes of non-polar solvent, usually the same

solvent in which the sample is dissolved.

2. Load the sample solution onto the cartridge bed.

3. Elute unwanted components with a non-polar solvent.

4. Elute the first component of interest with a more polar solvent.

5. Elute remaining components of interest with progressively more polar [stronger] solvents.

6. When you recover all of your components, discard the used cartridge in a safe and appropriate

manner.

This procedure is illustrated in the figure below for a sample containing a mixture of three neutral,

relatively non-polar organic dyes [yellow, red, and blue] that appears black when initially loaded

onto the cartridge bed.

Illustration of a General Elution Protocol for Normal-Phase Chromatography on SPE Cartridges

(Silica, Florisil, Alumina, Diol, CN, NH2)

Reversed-Phase Chromatography

Because of the multiplicity of aqueous samples spanning a breadth of applications from

environmental water to fruits and vegetables, from beverages to biological fluids, reversed-phase

chromatography has become the predominant mode of SPE.

To perform reversed-phase chromatography with SPE cartridges, use a gradient of strongly to

weakly polar solvents [from weak to strong solvent elution strength] with a non-polar packing

material.

1. Solvate the silica-bonded phase or polymer packing with six to ten hold-up volumes of methanol

or acetonitrile. Flush the cartridge with six to ten hold-up volumes of water or buffer. Do not allow

the cartridge to dry out [unless using HLB].

2. Load the sample dissolved in a strongly polar [weak] solvent [typically water].

3. Elute unwanted components with a strongly polar solvent.

4. Elute weakly retained components of interest with a less polar solvent.

5. Elute more tightly bound components with progressively more non-polar [stronger] solvents.

6. When you recover all the components of interest, discard the used cartridge in a safe and

appropriate manner.

This procedure is illustrated in the figure below for a sample of an aqueous grape drink containing

two polar food dyes [red and blue], as well as sugar and artificial flavor [but no real grape juice!].

As prepared, this drink appears light purple in a glass, since the dye concentration is dilute. When

a portion is loaded onto a prepared SPE cartridge, the strongly retained dyes become concentrated

near the inlet in a dark purple band.

Illustration of a General Elution Protocol for Reversed-Phase Chromatography on SPE Cartridges

(C18, tC18, C8, CN, Diol, HLB, Porapak RDX, NH2)

Ion-Exchange Chromatography

Compounds that are ionic or ionizable are often best isolated using some form of ion-exchange

chromatography. This separation mode is orthogonal to the more widely used normal-phase and

reversed-phase modes and provides a powerful, selective second dimension to sample preparation

protocols.

Illustration of the Two Major Types of Phases—Anion and Cation Exchange—

and How They Selectively Attract and Retain Molecules of Opposite Charge

To perform ion-exchange chromatography with SPE cartridges, use a gradient of pH or ionic

strength with an ion exchange packing material.

1. Condition the cartridge with six to ten hold-up volumes of deionized water or weak buffer.

2. Load the sample dissolved in a solution of deionized water or buffer.

3. Elute unwanted, weakly bound components with a weak buffer.

4. Elute the first component of interest with a stronger buffer (change the pH or ionic strength).

5. Elute other components with progressively stronger buffers.

6. When you recover all of your components, discard the used cartridge in an appropriate manner.

This procedure is illustrated in the figure below for a sample of an aqueous mixture of two ionic

dyes with different pKa values. When loaded onto the cartridge, both are strongly retained, and the

combination of blue and yellow components appears as a green band near the inlet.

Illustration of General Elution Protocol for Ion-Exchange Chromatography on SPE Cartridges

(NH2, Accell™ Plus QMA, Accell Plus CM, SCX, SAX, WCX, WAX)

Cation and anion exchangers are further categorized as either weak or strong exchangers,

depending upon the type of ionic group on their surface. Strong cation exchangers possess an

acidic surface moiety such as a sulfonic acid that is always ionized [negatively charged] over the

whole pH range. Weak cation exchangers possess an acidic surface moiety such as a carboxylic

acid that is negatively charged at high pH but neutral at low pH. Similarly, strong anion exchangers

typically bear quaternary ammonium groups that are always positively charged, while weak anion

exchangers possess primary, secondary, or tertiary amine groups that may be positively charged

at low pH but neutral at high pH.

Use the following table as a guideline to choose the appropriate SPE ion-exchange cartridge type

for your particular analyte.

Mixed-mode ion exchange chromatography combines the use of reversed-phase and ion-exchange

modes into a single protocol on a single SPE cartridge. It can be used to isolate and separate

neutral, acidic, and basic compounds from a single complex matrix. An ideal mixed-mode SPE

sorbent substrate remains water-wettable while exhibiting strong reversed-phase retention of

hydrophobic compounds. On its surface are ion-exchange functionalities of one of the four general

types just described above. Intermediate washes with organic solvent mixtures of appropriate

elution strength may be used to isolate neutral compounds [including ionizable analytes in their

neutral state]. Selective elution of ionically bound analytes may be attained by manipulating the

charge of either the analyte [when bound to strong ion exchangers] or of the sorbent [for analytes

bound to weak ion exchangers].

SPE Method Development Summary

The following table summarizes the foregoing discussion of the modes of SPE:

Summary of Utility and Practice of Principal LC Modes for Solid-Phase Extraction [SPE]

Reversed Phase Normal Phase Ion Exchange

Analyte Moderate to low

polarity

Low to high

polarity/neutral

Charged or Ionizable

Separation

Mechanism

Separation based

on hydrophobicity

Separation based on

polarity

Separation based on charge

Sample Matrix Aqueous Non-polar organic

solvent

Aqueous/ Low ionic strength

Condition/

Equilibrate SPE

Sorbent

1. Solvate with

polar organic

2. Water

Non-polar organic Low ionic strength buffer

Preliminary Wash

Step

Aqueous/buffer Non-polar organic Low ionic strength buffer

Elution Steps Increase polar

organic content

Increase eluotropic

strength of organic

solvent mixture

Stronger buffers - ionic strength or

pH to neutralize the charge

AX

[Anion

Exchange]

CX

[Cation

Exchange]

Sorbent

Functionality

C18, tC18, C8, tC2, CN,

NH2, HLB, RDX, Rxn

RP

Silica, Alumina,

Florisil, Diol, CN, NH2

Accell Plus QMA,

NH2, SAX, MAX,

WAX

Accell Plus CM,

SCX, MCX, WCX,

Rxn CX

Sorbent Surface

Polarity

Low to Medium High to Medium High High

Typical Solvent

Polarity Range

High to Medium Low to Medium High High

Typical Sample Water, low Hexane, chloroform, Water, low Water, low

Loading Solvent strength buffer methylene chloride strength buffer strength buffer

Typical Elution

Solvent

CH3OH/water,

CH3CN/water

Ethyl acetate,

acetone, CH3CN

Buffers, salts

with high ionic

strength,

increase pH

Buffers, salts with

high ionic

strength,

decrease pH

Sample Elution

Order

Most polar sample

components first

Least polar sample

components first

Most weakly

ionized sample

component first

Most weakly

ionized sample

component first

Mobile Phase

Solvent Change

Required to Elute

Compounds

Decrease solvent

polarity

Increase solvent

polarity

Increase ionic

strength or

increase pH

Increase ionic

strength, or lower

pH

This has been a brief introduction to sample enrichment and purification using solid-phase

extraction [SPE]. The best way to start using SPE is to first learn what others have done with

analytes and/or matrices similar to those of interest to you. You will find > 7,700 references to the

use of SPE in the Resource Library on waters.com. Fill in the blank with a partial compound or

matrix name in the following search phrase:

“Sep-Pak” OR “Oasis” AND ______*

NOTE: Rather than risk a spelling error, use an asterisk [*] with a root name for best results. Using

this same search string, even more references [> 60,000] may be found on GOOGLE Scholar.

Further reading:

J.C. Arsenault and P.D. McDonald, Beginners Guide to Liquid Chromatography, Waters [2007]; Order P/N

715001531 on waters.com

P.D. McDonald and E.S.P. Bouvier, A Sample Preparation Primer and Guide to Solid-Phase Extraction Methods

Development, Waters [2001] Search for WA20300 on waters.com

Waters, Purity by SPE [2008]; Search for 720001692en on waters.com

U.D. Neue, P.D. McDonald, Topics in Solid-Phase Extraction. Part 1. Ion Suppression in LC/MS Analysis: A Review.

Strategies for its elimination by well-designed, multidimensional solid-phase extraction [SPE] protocols and methods for its

quantitative assessment [2005]; Search for 720001273en on waters.com

Beginner's Guide to SPE [Solid-Phase Extraction]

A Powerful Tool for Improved Sample Preparation

As an analytical scientist, you are faced with many challenges when determining what tools you

can best use to achieve the desired result. Determining which sample preparation tools and

approaches are important considerations that can significantly impact your success.

Ideally, you would be happy if you did not have to do any sample preparation. In reality, however,

sample preparation is often necessary. You may need to optimize a method for an existing sample

to improve throughput or lower the cost per analysis. Or, you may be asked to analyze a wide

variety of different types of samples to report on new compounds of interest. Each new sample

type can present different analytical challenges. In addition, scientists today are faced with the

significant challenge of reporting values at lower concentration levels than ever before, without

compromising accuracy and precision.

This book is designed to help you explore and understand a very powerful tool in sample

preparation technology: solid-phase extraction [SPE]. You will see how this technology, which uses

devices with chromatographic packing material, can help meet your analytical challenges.

Definiton of Solid-Phase Extraction

SPE is a sample preparation technology that uses solid particle, chromatographic packing material,

usually contained in a cartridge type device, to chemically separate the different components of a

sample. Samples are nearly always in the liquid state [although specialty applications may be run

with some samples in the gas phase]. Figure 1 shows a sample, which appears black, being

processed on a SPE device so that the individual dye compounds, which make up the sample, are

chromatographically separated.

Figure 1: Examples of an SPE Method

The chromatographic bed can be used to separate the different compounds in a sample, to make

subsequent analytical testing more successful. For example, SPE is often used for the selective

removal of interferences.

The technically correct name for this technology is “Liquid-Solid Phase Extraction,” since the

chromatographic particles are solid and the sample is in the liquid state. The same basic

chromatographic principles of liquid chromatography that are used in HPLC are also used here, but

in a different format and for a different reason. Here, chromatography is used to better prepare a

sample before it is submitted for analytical testing.

In sample preparation, samples can come from a wide range of sources. They can be biological

fluids such as plasma, saliva, or urine; environmental samples such as water, air, or soil; food

products such as grains, meat, and seafood; pharmaceuticals; nutraceuticals; beverages; or

industrial products. Even mosquito heads can be the sample! When a scientist needed to analyze

neuropeptides extracted from the brains of mosquitoes, SPE was the sample preparation method of

choice [Waters Applications Database, 1983].

Four Major Benefits of SPE

There are many benefits to using SPE, but four major benefits deserve special attention.

1. Simplification of Complex Sample Matrix along with Compound Purification

One of the most difficult problems for an analytical chemist is when compounds of interest are

contained in a complex sample matrix, such as mycotoxins in grains, antibiotic residues in shrimp,

or drug metabolites in plasma, serum, or urine. T he large number of interfering constituents or

substances in the sample matrix along with the compounds of interest makes analysis extremely

difficult.

The first problem to solve is the resulting complexity of the analysis itself due to the presence of so

many entities which must be separated in order to identify and quantitate the compound[s] of

interest. See Figure 2.

Figure 2: Example of a Complex Sample

The robustness of the assay may not be adequate, because any slight change could impact the

resolution of the separation of a critical pair of analytes.

Another consideration is that the presence of all the interferences in the original sample matrix can

result in instrument downtime due to a buildup of contamination with each injection. If the

interferences were removed as part of the sample preparation, then the compounds of interest

could be analyzed with a simpler, more robust method. This can be seen in Figure 3, which

compares the original sample on top to the new SPE-prepared sample on the bottom.

Figure 3: Comparison of Sample Matrix Complexities

An additional benefit of simplifying the sample matrix is improved quantitation accuracy. T he top

blue trace for compound 1 in Figure 4, initially appears to be acceptable. However, it really has

some contamination from the sample matrix when compared to the blank sample matrix trace in

red shown just beneath it. With a proper SPE protocol, the lower traces show the same compounds

with no problems with interference, making quantitation much more accurate.

Figure 4: Improved Quantitation with Better Sample Preparation

Another example is shown in Figure 5. T he upper trace shows significant interference from the

sample matrix on both compounds 1 and 2. T he lower trace shows much improved results [clean

base line] due to proper sample preparation with SPE. Notice a much cleaner baseline improves the

accuracy of the analytical results. Also, a much purer extract can be obtained if the sample

requires isolation and purification of that compound.

Figure 5: Significant Improvement in Baseline Using SPE Technology

2. Reduce Ion Suppression or Enhancement in MS Applications

The second problem with complex sample matrices can be seen when we look at mass

spectrometer output [LC/MS or LC/MS/MS]. For proper MS signal response [sensitivity], the

compound ion must be allowed to form properly. In cases where the formation of the compound ion

is suppressed by interferences in the sample matrix, the signal strength is greatly diminished.

We can see this effect in Figure 6. The upper output is the signal for our compounds of interest

when injected in a saline solution. The lower trace shows significant reduction in response [> 90%

suppression] of these same compounds when they were analyzed in human plasma. For the lower

trace, only a common protein precipitation step was performed. This technique does not clean up

the matrix interferences that cause the ion suppression, resulting in poor signal response.

Figure 6: Example of Ion Suppression Due to Sample Matrix

Another good example of this suppression effect can be seen in Figure 7. In the upper trace of the

MS output, where the plasma sample was prepared with just a protein precipitation step, we can

see that the terfenadine peak is suppressed by 80%. In the lower trace, where the same sample

was prepared with a SPE method, we can see minimal ion suppression. Because the interferences

from the sample matrix were removed, this allowed the compound ion to form properly, creating a

better signal.

Figure 7: Reduced Ion Suppression with Proper SPE

In some instances, interferences from the sample matrix can artificially increase the signal

reported for a compound. This is called ion enhancement, resulting in an inaccurately high reported

value. A proper SPE method will minimize this effect by cleaning away the interferences from the

compound, resulting in a more accurate reported value.

3. Capability to Fractionate Sample Matrix to Analyze Compounds by Class

An analyst may be faced with a sample that contains many compounds, with a need to separate

them by class so that further analysis can be carried out much more efficiently. For example, a soft

drink beverage contains a wide range of compounds in its formulation. An SPE method could be

developed to separate the different classes of compounds, for example by their polarity. The polar

compounds could be collected, as a separated fraction, from the more non-polar compounds.

These two fractions could then be separately analyzed in a much more efficient way because their

compounds would be more similar.

An example of the power of fractionation by SPE is shown in Figure 8. Here, a complex sample of a

dry powder [purple grape drink mix] is easily separated into four fractions: a fraction of just the

polar compounds, a purified red compound, a purified blue compound, and a fraction containing all

the remaining very non-polar compounds. You will see elsewhere in this book how very powerful

this capability can be.

Figure 8: Sample Preparation by SPE

For a more detailed discussion of sample fraction by SPE see page 125 in the Method Development

section.

4. Trace Concentration [Enrichment] of Very Low Level Compounds

Analysts today often need to report on compounds at far lower concentration levels than ever

before, as little as parts per trillion [ppt] and even lower. Typically these levels are lower in the

neat sample than the sensitivity capability of the analytical instruments.

A good example of this is the analysis for trace contaminants in environmental samples or

metabolite development over time in biological fluids. The upper trace in Figure 9 shows the poor

response of the original neat sample for the compound of interest. Using the same analytical

conditions but with the sample prepared with SPE used in a trace concentration strategy, the lower

trace shows a dramatic increase in signal strength for this compound. With this result, an accurate

calculation of the original compound concentration in the neat sample can be made.

Figure 9: Example of Trace Concentration

Without the retention capability of chromatographic packing materials in SPE, the ability to trace

concentrate a specific compound[s] would be very difficult, if not impossible, with other sample

preparation approaches.

Summary

As we’ve seen, an SPE device with a chromatographic bed can perform four critical functions to

make the analysis of the sample more successful. See Figure 10.

Figure 10: The Power of SPE

In this book, we have endeavored to provide all of the SPE fundamentals and success techniques

derived from scientists from all over the world who have counted on this technology in the past

thirty years. Today, scientists are finding SPE more useful than ever in solving difficult sample

preparation and analytical problems.

We hope this book will enable you to understand and master the capabilities of SPE, so that you

too can put the power of this technology to use in your laboratory.

SPE - Sample Enrichment and Purification using Solid-Phase

Extraction

Three Reasons To Use Solid-Phase Extraction (SPE)1. You need to remove specific interferences from your sample so that they do not cause problems

for the detection and quantitation of analytes of interest. In the example shown here, an

inadequate sample preparation protocol failed to remove interferences, as seen by the residual

yellow color of the extract and many peaks overlapping the analytes of interest in the

chromatogram.

2. You need to increase the concentration of the analyte of interest in the original sample so that it

can be more readily detected and more accurately quantitated by your analytical technique. A

large sample volume may be loaded onto an SPE column if the analyte of interest is strongly

retained. Then the analyte may be eluted in a very small volume, thereby increasing its

concentration in the sample aliquot presented to your chosen analytical tool.

3. You need to remove interferences in your sample that, though invisible, suppress the signal for

the analyte of interest as detected by mass spectrometry. In the example shown here, protein

precipitation failed to remove the phospholipids from a plasma extract, causing severe ion

suppression. An optimized mixed-mode SPE protocol provides the cleanest extract and minimizes

ion suppression.

Goals and Benefits of SPE

What is Solid-Phase Extraction (SPE)?

Don't be confused by the term solid-phase extraction [SPE]. A typical SPE device has 50 times

more separation power than a simple, single liquid-liquid extraction. SPE is actually column liquid-

solid chromatography. Since SPE is liquid chromatography [LC], its practice is governed by LC

principles. A sample is introduced into a column or a cartridge device containing a bed of

appropriate particles, or other form, of a chromatographic packing material [stationary phase].

Solvent [mobile phase] flows through the bed. By choosing an appropriate combination of mobile

and stationary phases, sample components may pass directly through the column bed, or they

may be selectively retained.

Individual compounds in the sample each typically appear to travel at different speeds through the

device. Using a weaker solvent causes them to move slowly and/or be strongly retained. A stronger

solvent speeds up their passage through the bed and elutes the analyte(s) in a more concentrated

volume. Elution from an SPE device is usually done by increasing the strength of the mobile phase

in a series of discrete, rather than continuous, steps during which selected analytes or

interferences are either fully retained or rapidly eluted-this variation of gradient elution called a

step gradient.

Most commonly, SPE is practiced using miniature column or cartridge devices. An example is

shown here. A mixture of three dyes is loaded onto the cartridge in a weak solvent, causing strong

sample retention in a narrow band that appears black at the column inlet. Subsequent gradient

steps, each with a successively stronger solvent, are used to elute the dyes individually [yellow,

red, then blue].

Typical SPE cartridges are low-pressure devices-constructed of solvent-resistant plastic or glass-

filled with particles ≥30 µm in diameter. Suitable flow rates may be achieved by gravity or with the

assistance of vacuum or low positive pressure. [The latter requires putting a cap on the open inlet

of a column or using a sealed device with inlet and outlet fittings.]

Importance of Sample Preparation

In the last two decades, dramatic advances in analytical instrumentation and laboratory

information management systems shifted the analyst's predominant tasks from assay

measurements to sample preparation and data processing. As the stringency of requirements for

higher sensitivity, selectivity, accuracy, precision, and number of samples to be processed has

escalated, the corresponding increases in speed and sophistication of analysis and data collection

have outpaced improvements in the many traditional techniques of sample collection and

preparation. By some estimates, 75 to 80% of the work activity and operating cost in a

contemporary analytical lab is spent processing and preparing samples for introduction or injection

into an analytical separation and/or measurement device. Clearly, efforts directed and products

designed to streamline sample preparation protocols are essential to future progress in analytical

science.

Goals of Sample Preparation

Successful sample preparation for most analytical techniques [HPLC, GC, spectrophotometry, RIA,

etc.] has a threefold objective: namely, to provide the sample component of interest

in solution

free from interfering matrix elements

at a concentration appropriate for detection or measurement.

To accomplish these goals, a sample, or a representative portion thereof [not always easy to

obtain], is prepared via traditional methods of dissolution, homogenization, extraction [liquid- or

solid-phase], filtration, concentration, evaporation, separation, chemical derivatization,

standardization [internal or external], etc.

Usually such methods are used in combinations of multiple steps, which form a sample prep

protocol. The fewer steps and methods used in any given protocol, the simpler, more convenient,

cost effective, and less time consuming it is. Simpler protocols lend themselves more readily to

automation and also lead to increased accuracy, reliability, reproducibility, and safety.

Innovation in Sample Preparation Methods

There are many ways to combine standard tools and techniques to accomplish the goals of sample

prep. However, it is best to seek innovative means to streamline sample prep protocols:

to combine the functions of several steps, if possible, into one operation;

to eliminate needless sample transfers and manipulations;

to reduce the scale as much as practicable [gaining economies of time, labor, and cost];

to use new tools in creative ways.

Benefits of Solid-Phase Extraction [SPE] Cartridges

When compared to other sample preparation processes, solid-phase extraction using SPE

cartridges offers:

Lower Cost • lower solvent consumption

• lower reagent consumption

• less apparatus

Greater Recoveries • minimal sample transfer

Faster Protocol • fewer steps

Greater Safety • less exposure to toxic agents

Greater Accuracy • no cross contamination

No Emulsion Problems • less sample handling

• fewer steps

No Transporting of Samples to Lab • direct field sampling

Reduced Harm to Labile Samples • minimal evaporation

Minimal Glass Breakage • less glassware used, less to wash

Achieving Sample Preparation Objectives with Solid-Phase Extraction [SPE]

To remove sample constituents that elute after the analytes of interest or are strongly adsorbed:

use solid-phase extraction with sorbent surface chemistry that is the same as that in the analytical

HPLC column.

tailor the gradient steps to elute analytes selectively.

To remove sample constituents that coelute with an analyte of interest:

use solid-phase extraction with sorbent surface chemistry and/or separation mode different from that

in the analytical column.

tailor the gradient steps to elute analytes selectively.

To enrich sample components present in low concentration:

tailor the gradient steps to elute analytes selectively.

use "large" sample volumes in adsorption-promoting solvent.

use "small" collection volume in desorption-promoting solvent.

use sorbent chemistry tailored to the analyte, independent of that in analytical column.

carefully choose chemistry of solid-phase extraction column so further sample prep will be

unnecessary.

To desalt samples:

first, adsorb analytes on reversed-phase sorbent while salt breaks through unretained.

then, after using water to wash away residual salt, desorb analytes using water-miscible organic

solvent.

To exchange solvents:

adsorb the sample completely onto a strongly retentive sorbent and flush away the original solvent

with a weaker eluent.

elute the analyte with the desired solvent.

To fractionate classes of compounds:

use a step-gradient sequence to divide a sample-on the basis of hydrophobicity, polarity, or charge-

into fractions containing groups of analytes that share common properties.

To derivatize analytes using solid-phase reagents:

adsorb a derivatization reagent on the surface of the sorbent; then, collect the sample (usually a gas)

under conditions that favor complete adsorption of the analyte; wait for the reaction to occur and then

selectively elute the derivative.