Introduction to Modern Liquid Chromatography, Third Edition part 81 doc

758 SAMPLE PREPARATION16.10 SAMPLE PREPARATION FOR BIOCHROMATOGRAPHY, 797 16.11 SAMPLE PREPARATION FOR LC-MS, 800 16.12 DERIVATIZATION IN HPLC, 802 Sample preparation is an essential par

CHAPTER SIXTEEN

SAMPLE PREPARATION

with Ronald Majors

16.1 INTRODUCTION, 758

16.2 TYPES OF SAMPLES, 759

16.3 PRELIMINARY PROCESSING OF SOLID AND SEMI-SOLID

SAMPLES, 760

16.3.1 Sample Particle-Size Reduction, 760

16.3.2 Sample Drying, 762

16.3.3 Filtration, 763

16.4 SAMPLE PREPARATION FOR LIQUID SAMPLES, 764

16.5 LIQUID–LIQUID EXTRACTION, 764

16.5.1 Theory, 766

16.5.2 Practice, 766

16.5.3 Problems, 768

16.5.4 Special Approaches to Liquid– Liquid Extraction, 770

16.6 SOLID-PHASE EXTRACTION, 771

16.6.1 SPE and HPLC Compared, 772

16.6.2 Uses of SPE, 772

16.6.3 SPE Devices, 774

16.6.4 SPE Apparatus, 777

16.6.5 SPE Method Development, 778

16.6.6 Example of SPE Method Development: Isolation of Albuterol from Human Plasma, 784

16.6.7 Special Topics in SPE, 785

16.7 MEMBRANE TECHNIQUES IN SAMPLE PREPARATION, 790 16.8 SAMPLE PREPARATION METHODS FOR SOLID SAMPLES, 791

16.8.1 Traditional Extraction Methods, 792

16.8.2 Modern Methods for Extracting Solids, 793

16.9 COLUMN-SWITCHING, 796

Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R Snyder,

Joseph J Kirkland, and John W Dolan

Copyright © 2010 John Wiley & Sons, Inc.

757

758 SAMPLE PREPARATION

16.10 SAMPLE PREPARATION FOR BIOCHROMATOGRAPHY, 797 16.11 SAMPLE PREPARATION FOR LC-MS, 800

16.12 DERIVATIZATION IN HPLC, 802

Sample preparation is an essential part of HPLC analysis, intended to provide a rep-resentative, reproducible, and homogenous solution that is suitable for injection into the column The aim of sample preparation is to provide a sample aliquot that (1) is relatively free of interferences, (2) will not damage the column, and (3) is compatible with the intended HPLC separation and detection methods The sample solvent should dissolve in the mobile phase without affecting sample retention or resolution, and without interfering with detection It may also be necessary to concentrate the analytes and/or derivatize them for improved detection or better separation Sample preparation begins at the point of collection, extends to sample injection onto the HPLC column, and encompasses the various operations summarized in Table 16.1 Options 1 to 4 of Table 16.1—which include sample collection, transport, storage, preliminary processing, laboratory sampling, and subsequent weighing/dilution— all form an important part of sample preparation Although these four steps in the HPLC assay can have a critical effect on the accuracy, precision, and convenience of the final method, only option 3 (preliminary sample processing) will be (briefly) discussed here See [1–4] for a discussion of options 1, 2, and 4 This chapter will be devoted mainly to options 5 to 8 of Table 16.1, which encompass what

is usually meant by sample pretreatment or sample preparation (‘‘sample prep’’).

Whereas HPLC is predominantly an automated procedure, sample pretreat-ment often is performed manually As a result sample pretreatpretreat-ment can require 60%

or more of the total time devoted to routine analysis Sample pretreatment includes a large number of methodologies, as well as multiple operational steps, and can there-fore be a challenging part of HPLC method development Finally, method precision and accuracy are often largely determined by the sample-pretreatment procedure [5–6], including operations such as weighing and dilution For all these reasons the development of a sample-pretreatment procedure deserves careful planning

A sample-pretreatment procedure should provide quantitative recovery of analytes, involve a minimum number of steps, and (if possible) be easily automated Quantitative (99+%) recovery of each analyte enhances sensitivity and assay precision, although this does not mean that all of the analyte present in the original sample must be included in the final injected sample For example, in a given method that includes a series of sample-pretreatment steps, aliquots of intermediate fractions may be used for further sample preparation or for injection If analyte recovery is significantly less than 100%, it must be reproducible A smaller number

Table 16.1

Sample Preparation Options Option Number Option Comment

1 Sample collection Obtain representative sample using statistically valid

procedures.

preservation

Use appropriate inert, tightly sealed containers and storage conditions; stabilize volatile, unstable, or reactive samples, if necessary; biological samples may require freezing.

processing

Disperse or divide sample (drying, sieving, grinding, etc.) for more representative sample and to improve dissolution or extraction.

dilution

Take necessary precautions for reactive, unstable, or biological materials; for dilution, use calibrated volumetric glassware.

processing methods

Consider, among these, solvent exchange, desalting, evaporation, or freeze drying.

6 Removal of particulates Use filtration, centrifugation, solid-phase extraction.

7 Sample extraction For methods for liquid samples, see Table 16.2; for

solid samples, Tables 16.8 and 16.9.

separation; extra steps may add time, complexity and potential loss of sample (Section 16.12).

of sample-pretreatment steps—plus automation— reduces the overall time and effort required, improves assay precision, and decreases the opportunity for errors

by the analyst

Many sample-preparation techniques have been automated, and appropriate instrumentation is commercially available Approaches to automation vary from using a robot to perform manual tasks, to dedicated instruments that perform a specific sample-preparation procedure Although automation can be expensive and elaborate, it is often desirable when large numbers of samples must be analyzed, and the time or labor (per sample) required for manual sample preparation would

be excessive The decision to automate a sample-preparation procedure is often based on a cost justification or, in some cases, operator safety (e.g., to minimize exposure to toxic substances or other possible health hazards) A full coverage of sample-preparation automation is beyond the scope of this chapter; the reader is referred to recent textbooks on the subject [7–8]

Sample matrices can be broadly classified as organic (including biological) or inor-ganic, and may be further subdivided into solids, semi-solids (including creams, gels, suspensions, colloids), liquids, and gases For nearly every matrix some form of sam-ple pretreatment will be required prior to HPLC analysis, even if only simsam-ple dilution

760 SAMPLE PREPARATION

Gaseous samples usually are analyzed by gas chromatography rather than

by HPLC Techniques such as canister collection, direct sampling via sample loops, headspace sampling, and purge-and-trap are used to collect and inject gases However, volatile analytes that are labile, thermally unstable, or prone to adsorb onto metal surfaces in the vapor state are sometimes better handled by HPLC Trapping is required to analyze gaseous samples by HPLC The gas sample is either (1) passed through a solid support and subsequently eluted with a solubilizing liquid or (2) bubbled through a liquid that traps the analyte(s) An example of the HPLC analysis of a gaseous sample is the American Society for Testing Materials (ASTM) Method D5197-03 and United States Environmental Protection Agency (EPA) Method TO-11 for volatile aldehydes and ketones [9] An air sample is passed through an adsorbent trap coated with 2,4-dinitrophenylhydrazine, which quantitatively converts aldehydes and ketones into 2,4-dinitrophenylhydrazones The hydrazones are then eluted from the trap with acetonitrile and separated by reversed-phase HPLC (RPC)

Table 16.2 provides an overview of typical sample preparation procedures used for liquids and suspensions The remainder of this chapter will be devoted to the pretreatment of samples of most concern: semi-volatile and non-volatile analytes

in various liquid and solid matrices

Sample preparation for solid samples can be more demanding than for liquid samples In some cases, the sample is easily dissolved and is then ready for injection or further pretreatment In other cases, the sample matrix may be insoluble in common solvents, and the analytes must be extracted from the solid matrix There are also cases where the analytes are not easily removable from an insoluble matrix—because

of inclusion or adsorption Here more rigorous techniques such as Soxhlet extraction, pressurized fluid extraction (PFE), ultrasonication, or solid–liquid extraction may

be necessary (Section 16.8.2) Table 16.8 lists some traditional methods for the recovery of analytes from solid samples, while Table 16.9 describes some more recent procedures Once analytes have been quantitatively extracted from a solid sample, the resulting liquid fraction can either be injected directly into the HPLC instrument or subjected to further pretreatment

Compared to gases or solids, liquid samples are much easier to prepare for HPLC Many HPLC analyses are based on a ‘‘dilute and shoot’’ procedure, whereby the solubilized analyte concentration is reduced by dilution so as to not overload the column or saturate the detector, or to make the injection solvent more compatible with the mobile phase

SEMI-SOLID SAMPLES

16.3.1 Sample Particle-Size Reduction

Solid samples should be reduced in particle size because finely divided samples (1) are more homogeneous, allowing more representative sampling with greater precision and accuracy, and (2) dissolve faster and are easier to extract because of their greater surface area Methods for reducing the particle size of solid samples are outlined in Table 16.3

Table 16.2

Typical Sample-Preparation Methods for Liquids and Suspensions Sample Principles of Technique Comments

Preparation

Method

Solid phase

extraction

(SPE)

Similar process to HPLC Sample is

applied to, and liquid is passed through, a column packed with a solid phase that selectively removes analytes (or interferences) (Section 16.6).

Wide variety of stationary phases is available for the selective removal of desired inorganic, organic, and biological analytes.

Liquid– liquid

extraction

(LLE)

Sample is partitioned between two

immiscible phases Interference-free analytes are then recovered from one

of the two phases (Section 16.5).

Beware of formation of emulsions.

Values of K Dcan be optimized by the use of different solvents or additives; continuous extraction or large volumes can be used for low

K D-values.

Dilution Sample is diluted with a solvent that is

compatible with the HPLC mobile phase.

To avoid excess peak broadening or distortion, dilution solvent should be miscible with, and preferably weaker than, the HPLC mobile phase Evaporation Liquid is removed by gentle heating

with flowing air or inert gas.

Do not evaporate too quickly; avoid sample loss on wall of container; don’t overheat to dryness; best with inert gas like N2.

Distillation Sample is heated to boiling point of

solvent and volatile analytes in the vapor phase are condensed and collected.

Mainly for samples that can be easily volatilized; some samples may decompose if heated too strongly Vacuum distillation for high boilers Microdialysis A semi-permeable membrane is placed

between two aqueous liquid phases, and analytes transfer from one liquid

to the other, based on their differential concentration.

Enrichment techniques such as SPE are required to concentrate dialysates; dialysis with molecular-weight-cutoff membranes can be used on-line to deproteinate samples prior to HPLC; ultrafiltration and reverse osmosis can also be used in a similar manner Lyophilization Aqueous sample is frozen, and water is

removed by sublimation under vacuum.

Good for nonvolatile organics; large sample-volume can be handled; possible loss of volatile analytes; good for recovery of thermally unstable analytes—especially biologicals Filtration Liquid is passed through a paper or

membrane filter or a SPE cartridge/disk to remove suspended particulates.

Highly recommended to prevent back-pressure problems and to preserve column life.

Centrifugation Sample is placed in a tapered centrifuge

tube and spun at high force (several

times gravity, G); supernatant liquid is

decanted.

Ultracentrifugation is not normally used for simple particulate removal.

Sedimentation Sample is allowed to settle when left

undisturbed; settling rate is dependent

on Stoke’s radius.

Extremely slow process; manual recovery of different size particulates

at different levels, depending on settling rate.

762 SAMPLE PREPARATION

Table 16.3

Methods for Reducing Sample Particle-Size

Particle-Size Reduction Method Description of Procedure

Blending Mechanical blender is used to chop a semi-soft substance into

smaller parts or blend a nonhomogeneous sample into a more consistent form.

Chopping Process of mechanically cutting a sample into smaller parts Crushing Tungsten-carbide variable-jaw crushers can reduce the size of

large, hard samples.

Cutting Cutting mills can reduce soft-to-medium hard materials

(<100-mm diameter).

Grinding Manual or automated mortar-and-pestle are the most popular

choice; both wet and dry grinding are used; particle sizes of

≈10 μm can be achieved.

Homogenizing Any process used to make sample more uniform in texture

and consistency by breaking down into smaller parts and blending.

Macerating Process of breaking down a soft material into smaller parts by

tearing, chopping, cutting, etc.

Milling Various disk, rotor-speed, or ball mills can reduce

soft-to-medium hard and fibrous materials to 80–100- μm size.

Mincing Process of breaking down a meat or vegetable product into

smaller parts by tearing, chopping, cutting, dicing, etc Pressing Generally, the process of squeezing liquid from a semi-solid

material (e.g., plants, fruit, meat).

Pulverizing Electromechanically driven rod or vibrating base used to

reduce particle size for either wet or dry samples; a freezer mill can be used with liquid N2to treat malleable samples Sieving Process of passing a sample through a metal or plastic mesh of

a uniform cross-sectional area (square openings of 3–123 μm) in order to separate particles into uniform sizes; both wet and dry sieving can be used.

16.3.2 Sample Drying

Solid samples are often received for analysis in a damp or wet state Removal of water or drying the sample to constant weight is usually necessary for reliable assay Inorganic samples, such as soil, should be heated to 100–110◦C to ensure the removal of moisture Hydrophobic organic samples seldom require heating, since water absorption is minimal However, organic vapors can be adsorbed

by solid organic samples, and a heating step can remove these contaminants For hydroscopic or reactive samples (e.g., acid anhydrides) drying in a vacuum dessicator

is recommended Samples that can oxidize when heated in the presence of air should

be dried under vacuum or nitrogen Biological samples generally should not be heated to>100◦C, and temperatures above ambient should be avoided to prevent

sample decomposition Sensitive biological compounds (e.g., enzymes) often are prepared in a cold room at<4◦C to minimize decomposition Such samples should

be maintained at these low temperatures until the HPLC analysis step Freeze-drying (lyophilization) often is used to preserve the integrity of heat-sensitive samples (especially biologicals) Lyophilization is performed by quick-freezing the sample, followed by removal of frozen water by sublimation under vacuum

16.3.3 Filtration

Particulates should be removed from liquid samples prior to injection, because

of their adverse effect on column lifetime as well as possible damage to tubing, injection valves, and frits The most common methods for removing particulates from the sample are filtration, centrifugation, and sedimentation Several approaches

to filtration are described in Table 16.4 The lower the porosity of the filter medium, the cleaner is the filtrate, but the longer is the filtering time Vacuum filtration speeds the process Membrane filters in a disk format can be purchased for use with commercial filter holders/housings However, most users prefer disposable filters

Table 16.4

Filtration in HPLC Filtration Typical Products Recommended Use Comments Media

particles (<40 μm) Beware of filter-paperfibers getting into

sample; ensure solvent compatibility of filter paper.

Membrane

filters

Nylon, PTFE,

polypropylene, polyester, polyethersulfone, polycarbonate, polyvinylpyrolidone

Removal of small particles (>10 μm) Prefilter may be neededfor dirty samples prior

to filtration; avoid solvent incompatibility.

Functionalized

membranes

Ion-exchange membranes,

affinity membranes

Can remove both particulates and matrix interferences

Prefilter may be needed for dirty samples prior

to filtration; avoid solvent incompatibility SPE cartridges Silica- and polymer-based Can remove both

particulates and matrix interferences

Particles of silica-bonded phase can pass into filtrate; beware of plugging.

SPE disks PTFE- and

fiberglass-based

Can remove both particulates and matrix interferences

PTFE membranes are delicate, so handle with care; can pass a large volume at high flow rate; beware of plugging.

764 SAMPLE PREPARATION

equipped with Luer fittings The sample is placed in a syringe and filtered through the membrane using gentle pressure

A variety of membrane materials, nominal porosities, and dimensions are available for filtration, and the manufacturers’ literature provides specifications The large cross-sectional areas of the membrane-disk-type filter allows for good flow characteristics and minimizes plugging For most samples encountered in HPLC, filters in the range of 0.25- to 2-μm nominal porosity are recommended The porosity values are approximate, and the type of membrane can have some influence

on the filtration characteristics The most popular sizes for sample filtration are 0.25- and 0.45-μm porosities Membranes with 0.25-μm pores remove the tiniest particles (and large macromolecules) If the sample contains colloidal material or

a large amount of fines, considerable pressure may be required to force the liquid sample through the filter Sometimes a prefilter or depth filter (a thick filter with a large capacity for trapping larger particulates) is placed on top of the membrane to prevent plugging with samples containing these types of particulates

An important consideration in filter selection is solvent compatibility with the membrane If an inappropriate solvent is used, the filter may dissolve (or soften) and contaminate the filtrate Manufacturers of membrane filters usually provide detailed information on the solvent compatibility of their products More expensive functionalized membranes and SPE disks and cartridges are used not only for chemical interference removal but also to remove particulates

Table 16.2 provides an introduction to sample preparation methods for liquid sam-ples Most laboratories need only a few of these procedures For example, distillation

is limited to volatile compounds, although vacuum distillation for high-boiling com-pounds in environmental samples can extend the application of this technique [10] Lyophilization is usually restricted to the purification and handling of biological samples In the present discussion we will emphasize two methods used most often

in most HPLC laboratories: liquid–liquid extraction (Section 16.5) and liquid–solid

or solid–phase extraction (Section 16.6)

Liquid– liquid extraction (LLE) is useful for separating analytes from interferences

by partitioning the sample between two immiscible liquids or phases One phase in

LLE will usually be aqueous, and the second phase will be an organic solvent More hydrophilic compounds prefer the polar aqueous phase, whereas more hydrophobic compounds will be found mainly in the organic solvent Analytes extracted into the organic phase are easily recovered by evaporation of the solvent; analytes extracted into the aqueous phase often can be injected directly onto a RPC column The following discussion assumes that an analyte is preferentially concentrated into the organic phase; similar approaches can be used when the analyte is extracted into the aqueous phase

Liquid Sample

Dissolve in suitable solvent

Chose solvent suitable for later extraction step

Make chemical

adjustment (e.g., pH)

Place sample

in sep funnel

Add immiscible solvent and shake vigorously

Allow phases

to separate

Measure solute in each phase

Draw off each phase

Break emulsion

Evaporate to appropriate concentration

Injection or further sample pretreatment

Are the two liquids clear?

Are solutes extracted quantitatively?

Is sample in solution?

Yes

Yes

Yes

No

No

No (repeat extraction)

Figure16.1 Summary of the steps involved in liquid-liquid extraction (LLE)

Figure 16.1 summarizes the steps involved in a LLE separation Since extraction

is an equilibrium process with limited efficiency (only one ‘‘theoretical plate’’),

significant amounts of the analyte can remain in both phases—even when K D

(or 1/K D) 1 Chemical equilibria involving changes in pH, ion-pairing, and

complexation, for example, can be used to enhance values of K D and improve analyte recovery and/or eliminate interferences

The following characteristics are desirable for a LLE organic solvent:

• low solubility in water (<10%)

• volatility for easy removal and concentration after extraction (Table I.3)

• compatibility with the HPLC detector (e.g., low UV absorbance) (Table I.3)

• polarity and hydrogen-bonding properties that enhance recovery of the analytes in the organic phase (Section 2.3.2.1) (Table I.4)

• high purity to minimize sample contamination