CHROMATOGRAPHIC TERMS

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Symbol and Name Recommended Other Symbols and

by the IUPAC* Names in Use

K_c Distribution Constant (for GLC) K_p Partition Coefficient

Distribution Coefficient

k Retention Factor k Capacity Factor

N Plate Number n Theoretical Plate Number; No. of Theoretical Plates

H Plate Height HETP Height Equivalent to One Theoretical Plate

R Retardation Factor (in columns) R_R Retention Ratio

R_S Peak Resolution R Peak Resolution

a Separation Factor a Selectivity; Solvent Efficiency

t_R Retention Time

V_R Retention Volume

V_M Hold-Up Volume V_M Volume of the Mobile Phase

V_G Volume of the Gas Phase

V_O Void Volume, Dead Volume

Chromatography Common Abbreviations

a Selectivity; separation factor

AED Atomic Emission Detector

b Phase ratio

C4, C8, C18 Alkyl chain length of an LC reversed bonded phase

CE, CZE Capillary Electrophoresis, Capillary Zone Electrophoresis

CI Chemical Ionization

DAD, PDA (Photo) Diode Array Detector

dp Particle diameter

df Film thickness

ECD Electron Capture Detector

EI Electron Impact

ELCD Electrolytic Conductivity Detector

EPA Environmental Protection Agency

FAMEs Fatty Acid Methyl Esters

FID Flame Ionization Detector

FPD Flame Photometric Detector

FS Fused Silica

FT-IR Fourier Transform Infrared

GLC Gas Liquid Chromatography

GPC Gel Permeation Chromatography

GSC Gas Solid Chromatography

HETP or H Height Equivalent to one Theoretical Plate

HPLC High Performance Liquid Chromatography

HRGC High Resolution Gas Chromatography

I.D. Internal Diameter

IPC Ion Pair Chromatography

IRD Infrared Detector

IS Internal Standard

k (or k') Capacity factor (syn capacity ratio, partition ratio)

K Partition coeficcient

l Wave length

LSC Liquid Solid Chromatography

LIF Laser Induced Fluorescence (detector)

MAOT Maximum Allowable Operating Temperature

MDQ / MDL Minimum Detectable Quantity / Minimum Detectable Level

MIP Microwave Induced Plasma

MS Mass Spectrometry

MSD Mass Selective Detector

N Theoretical plate (number of)

NP Normal Phase

NPD Nitrogen Phosphorus Detector

O.D. Outer Diameter

ODS Octadecyl silane

PCBs Polychlorobiphenyls

PID Photo Ionization Detector

PLOT Porous-Layer Open Tubular (column)

PMT Photo-Multiplier Tube

PNAs Polynuclear Aromatics

ppb Part per billion

ppm Part per million

ppt Part per trillion

PTFE Polytetrafluoroethylene (Teflon®)

PTGC Programmed Temperature Gas Chromatography

RS Resolution

RP Reversed Phase

RSD Relative Standard Deviation

SCOT Surface Coated Open Tubular (column)

SEC Size (or Steric) Exclusion Chromatography

SFC Supercritical Fluid Chromatography

SFE Supercritical Fluid Extraction

SIM Single Ion Monitoring

S/N Signal to noise

TCD Thermal Conductivity Detector

TID Thermionic Detector (usually known as NPD)

TLC Thin Layer Chromatography

THMs Trihalomethanes

TMS Trimethylsilyl (derivative)

tR Retention time

UV Ultra-Violet (detector)

VR Retention volume

VOC Volatile Organic Compound

W Peak width

WCOT Wall Coated Open Tubular (column)

LIQUID CHROMATOGRAPHY AND GAS CHROMATOGRAPHY

GLOSSARY

absolute calibration - syn. direct calibration or external standard; method relating detector response to sample concentration in order to perform quantitative analysis. Standard solutions of a sample to be quantitated are prepared and equal volumes chromatographed. Peak heights or peak areas are plotted versus concentrations to produce a calibration curve.

absolute retention time - syn. retention time; the time elapsed between the introduction of the sample and the appearance of the peak maximum.

adsorbent - packing used in adsorption chromatography. Silica gel and alumina are the most frequently used adsorbents in HPLC.

adsorption - process which occurs at the surface of a liquid or solid as a result of the attractive forces between the adsorbate and solute; the basis for separation in GSC and LSC.

adsorption chromatography - one of the basic LC modes. elies on the adsorption process to effect the separation. Silica gel and alumina are the most frequently used supports; molecules are retained by the interaction of their polar functional groups with the surface functional group (e.g. silanols of silica).

adsorption isotherm - in adsorption, a plot of ilibrium concentration of sample in the mobile phase per unit volume us. the concentration in the stationary phase per unit weight. The shape of the adsorption isotherm can determine the chromatographic behavior of the solute: tailing, fronting, overload, etc.

affinity chromatrography - a technique in which a biospecific adsorbent is prepared by coupling a specific ligand (such as an enzyme, antigen, or hormone) for the macromolecule of interest to a solid support (or carrier). This immobilized ligand wil interact only with molecules that can selectively bind to it. Molecules that will not bind elute unretained. The retained compound can later be released in a purified state. Affinity chromatography is not a chromatographic technique as such, but is actually selective filtration.

alumina - an adsorbent sometimes used in adsorption chromatography. Aluminum oxide (Al2O3) is a porous adsorbent that is available with a sligtly basic surface. For this reason, it can have advantages over silica, which is considered to have an acidic surface.

analyte - the chemical species being investigated by an analytical method. Usually the identification and/or the amount is to be determined by a separation and estimation technique.

anion-exchange chromatography - the ion-exchange procedure used for the separation of anions. Both resins and bonded phases are available for this mode. The tetralkylammonium group is a typical strong anion-exchange functional group. An amino group on a bonded or coated stationary phase would be the example of a weak anion exchanger.

asymmetry - factor describing the shape of a chromatographic peak. Theory assumes a Gaussian shape and that peaks are symetrical. The peak asymetry factor is the ratio (at 10% of the peak height) of the distance between the peak apex and the back side of the chromatographic curve to the distance between the peak apex and the front side of the chromatographic curve. A value > 1 is a tailing peak, while a value <1 is a fronting peak.

band - mobile phase zone which contains a sample component; band is usually in the column; peak is the band signal on the chromatogram.

baseline - constant signal produced by the background level of the instrument; usually represented by a flat line on the recorder.

biocompatible - term to indicate that the column or instrument component will not irreversibly or strongly adsorb or deactivate biomolecules such as proteins.

bonded columns - silica is traditionally the common "normal" chromatography packing material. When another material is chemically bonded to the surface of the silica, a new column packing with unique separation properties can result. When an alkyl group is chemically joined to a silica surface, a hydrophobic paraffinic packing is obtained. Similarly, amine, sulfonic acid, or nitrile groups could be bonded to silica.

bonded-phase chromatography - (BPC) the most popular LC mode. A stationary phase chemically bonded to a support is used for the separation. The most popular support is microparticulate silica gel, and the most popular type of bonded phase is the organosilane, such as octadecyl (for reversed-phase chromatography). Approximately 70% of all HPLC is carried out on chemically bonded phases.

bleeding - column bleed; loss of the stationary phase due to its own volatility. Every phase has a maximum operating temperature.

C4, C8, C18, etc. - refers to the alkyl chain length of a reversed bonded phase.

capillary tubing - tubing for connecting various parts of the chromatograph. Most capillary tubing used in HPLC is <0.020 in. i.d. The smallest useful i.d. is about 0.004 in.

cartridge column - a type of column that has no endfittings and is held in a cartridge holder. The column is a tube; the packing is contained by frits in each end of the tube. Cartridges are easy to change and are less expensive and more convenient than conventional columns with endfittings.

cation-exchange chromatography - the form of ion-exchange chromatography that uses resins or packing with functional groups that can separate cations. A sulfonic acid would be an example of a strong cation-exchange group; a carboxylic acid would be a weak cation-exchange group.

chain length - the length of carbon chain in the hydrocarbon portion of a reversed-phase packing. It is expressed as the number of carbon atoms (e.g. C8, C18).

channeling - occurs when voids created in the packing material of a column may cause mobile phase and accompanying solutes to move more rapidly that the average flow velocity, resulting in band broadening. The voids are created by poor packing or by erosion of the packed bed.

chemisorption - sorption caused by a chemical reaction with the packing. Most such interactions are irreversible; usually occur on packings with reactive functional groups such as silanol or bonded amino phases.

chiral stationary phases (CSP) - stationary phases that are designed to separate enantiomeric compounds. They can be bonded to solid supports or created in situ on the surface of the solid support, or they can be surface cavities that allow specific interactions with one enantiomeric form.

column chromatography - any form of chromatography that uses a column or tube to hold the stationary phase. Open-column chromatography, HPLC, and open-tubular capillary chromatography are all examples.

corrected retention time (T) - the retention time corrected for the pressure drop developed along the chromatographic column; column inlet and outlet pressures need to be measured.

correction factor - syn. response factor; calculation coefficient which corrects the different response of detectors to different compounds; used to convert peak areas to numbers proportional to weight of sample.

counterion - in an ion-exchange process, the ion in solution used to displace the ion of interest from the ionic site. In ion pairing, it is the ion of opposite charge added to the mobile phase to form a neutral ion pair in solution.

coupled columns - a form of column switching. Uses a primary column connected to two secondary columns via a selector valve. Fractions from the first column can be selectively transferred to the other two columns for additional separation. Term also used to describe two or more columns connected in series to provide increased plate number.

coverage - refers to the amount of bonded phase on a silica support in bonded-phase chromatography. Coverage is usually described in mmol/m2 or in terms of %C.

cross-linking - during the process of copolymerization of resins to form a three-dimensional matrix, a disfunctional monomer is added to form cross-linkages between adjacent polymer chains. The degree of cross-linking is determined by the amount of this monomer added to the reaction. For example, divinylbenzene is a typical cross-linking agent for polystyrene ion-exchange resins. The swelling and diffusion characteristics of a resin are governed by its degree of cross-linking.

cyano phase - a stationary phase that usually consists of cyanopropylsilyl groups; used in both normal and reversed-phase chromatography.

deactivated support - support which has been chemically treated to reduce its surface activity; the most common treatments include acid washing (AW) and silanizing with dimethyl dichlorosilane (DMCS).

dead band - range within which the signal can change without causing a response in a potentiometric recorder.

dead volume (Vo) dead time (To) - syn. gas hold-up; volume of carrier gas required to transport a compound not retained by the stationary phase throughout the column; usually measured by injection of air or methane.

degassing - the process of removing dissolved gas from the mobile phase before or during use. Dissolved gas may come out of solution in the detector cell and cause baseline spikes and noise. Dissolved air can affect electrochemical detectors (by reaction) or fluorescence detectors (by quenching). Degassing is carried out by heating the solvent or by vacuum (in a vacuum flask), or on-line using evacuation of a tube made from a gas-permeable substance such as PTFE, or by helium sparging.

derivative(s) - compound(s) obtained from original sample, usually through chemical reactions which are more easily chromatographed; active protons such as those found in acids, amines, alcohols, and phenols are reacted to form more inert esters, ether, or silyl derivatives.

detection limit - syn. minimum detectable quantity; amount of sample which produces a signal twice the noise level.

detector - part of the gas chromatograph which constantly monitors the composition of the column effluent by measuring some physical property of the carrier gas and eluted compounds.

diaphragm pump - a reciprocating piston pump which acts not directly on the mobile phase, but on nydraulic fluid in a closed system. The pumped hydraulic fluid compresses a flexible steel diaphragm, which in turn pumps mobile phase with a reaction in flow stream pressure pulsation.

dimethyl dichlorosilane (DMCS) - reagent employed to block the silanol groups of the diatomaceous supports through chemical reaction.

displacement - a form of chromatography (like elution and frontal) where stronger solvents are used in series to displace (desorb) sample components; used occasionally in liquid chromatography.

distribution coefficient (K) - syn. partition coefficient; ratio of the concentration of solute or sample in the stationary phase to the concentration of the same in the gas phase.

dual piston pump - see "reciprocating piston pump".

eddy diffusion - phenomena occurring in packed columns due to lack of homogeneity of packing; expressed quantitatively by the first term of the van Deemter equation; produces peak broadening.

effective plate number - number of theoretical plates calculated by using the adjusted retention time instead of the absolute retention time; is considered to be a better measure of the efficiency of capillary columns.

efficiency of a column - column characteristic expressed quantitatively by the number of theoretical plates; efficient columns have many theoretical plates and show only limited band broadening.

effluent splitter - device which divides the column effluent in two or more streams; useful when two detectors measure the same sample simultaneously, or when part of the effluent is collected or passed to an auxiliary instrument.

electrochemical detector - an HPLC detector based upon an electroreductive or electro-oxidative process at a micro electrode in a low volume detector flowcell. The process is similar to polarographic techniques, and such electrochemical methods can be adapted to HPLC.

electronic integrator - instrument which converts the chromatographic signal into a frequency count proportional to peak area.

eluant - syn. carrier gas; general designation of mobile phase in chromatography.

eluate - that which is eluted from the column; designation of the sample when separated and dissolved in the mobile phase.

elution analysis - syn. elution technique; elution chromatography; the most commonly used technique in chromatography; the sample components are transported by the carrier gas and separated according to their partition coefficients.

elutropic series - a relative ranking of HPLC solvents ranging from non-polar to very polar properties. Very useful in choosing solvents for separation of analytes by column-solvent partitioning phenomena when developing an HPLC method. Polarity effects are due, in part, to dielectric constant, dipole moment and hydrophobic-hydrophillic properties.

endcapping - a column is said to be endcapped when a small silylating agent (e.g. trimethylchlorosilane) is used to bond residual silanol groups on a packing surface. Most often used with reversed-phase packings. May cut down on undesirable adsorption of basic or ionic compounds.

endfitting - the fitting at the end of the column that connects it to the injector or detector. Most HPLC endfittings contain a frit to hold the packing and have a low dead volume for minimum band spreading. Usually made of stainless steel.

exclusion limit - in SEC, the upper limit of molecular weight (or size), beyond which molecules will elute at the same retention volume, called the exclusion volume. Many SEC packings are referred to by their exclusion limit. For example, a 105 column of porous silica gel will exclude any compounds with a molecular weight higher than 100,000 based on a polystyrene calibration standard.

exclusion volume (Vc) - the retention volume of a molecule on an AEC packing; all molecules larger than the size of the largest pore are totally excluded and elute at the interstitial volume of the column.

external standard - syn. absolute calibration; quantitative analysis method in chromatography where dilutions of pure standard are compared to unknown samples; the standards are the components of interest.

extracolumn effects - the band-broadening effects of parts of the chromatographic system outside of the column itself. Extracolumn effects must be minimized in order to maintain the efficiency of the column. Areas of band broadening can include the injector, connecting tubing, endfittings, frits, detector cell volume, and internal detector tubing. The variances of all of these contributions are additive.

ferrule - part of fitting (see fittings).

fittings - pieces of plumbing used to connect the column to the instrument by high pressure, high temperature seal.

flow rate - volume of mobile phase per unit time passing through the column, usually reported as milliliters per minute.

flow splitters - (see effluent splitter).

fluorescence detector - a very sensitive and selective HPLC detector, equipped with two monochrometers at right angles to one another. The flow cell is illuminated at one face, and compounds which are excited by that light (can fluoresce) emit light at a different wavelength. This emitted light is measured via the second monochrometer using a photomultiplier tube.

frit - the porous element at either end of a column that serves to contain the column packing. It is placed at the very ends of the column tubs or, more commonly, in the endfitting. Frits are made from stainless steel or other inert metal or plastic, such as porous PTFE or polypropylene.

frontal analysis - a form of chromatography where pure sample flows through the column; each component breaks through at a different time depending on its affinity for the column; not commonly used today.

fronting - peak shape in which the front part of a peak (before the apex) in a chromatogram tapers in advance of the remainder of the peak. There is an asymmetric distribution with a leading edge. The asymmetry factor for a fronting peak has a value <1. The opposite effect is tailing. Fronting is related to the shape of the sorption isotherm.

gas chromatography (GC, GLC) - a form of chromatography where the mobile phase is gas.

Gaussian curve - a standard error curve, based on a mathematical function, that is a symmetrical, bell-shaped band or peak. Most chromatographic theory assumes a Gaussian peak.

gel filtration chromatography (GFC) - size-exclusion chromatography carried out with aqueous mobile phases. Generally refers to separations carried out on soft gels such as polydextrans. Most gel filtration separations involve biopolymers.

gel permeation chromatography (GPC) - SEC carried out with organic mobile phases. Used for the separation and characterization of polymers. SEC with aqueous mobile phases is referred to as a aqueous GPC, or GFC.

ghost-peak - spurious signal due to sample carry over in a syringe or injection valve.

gradient elution - technique for decreasing separation time by increasing mobile phase strength over time during the chromatographic separation. Also known as solvent programming. Gradients can be continuous or stepwise. Binary, ternary, and quaternary solvent gradients have been used routinely in HPLC.

guard column - a small column placed between the injector and the analytical column. Protects the analytical column against contamination by sample particulates and, perhaps, by strongly retained species. The guard column is usually packed with the same material as the analytical column and is often of the same i.d. It is much shorter, costs less, and is usually discarded when it becomes contaminated.

H - Same as HETP.

head pressure - the pressure above gravity at the head of the column. Expressed in psig, bar, atm, or MPa.

headspace analysis - analysis of the vapors above a liquid sample; commonly used for analysis of foods, flavors, fragrances, etc.

heart cutting - in preparative LC, refers to collection of the center of the peak, where purity should be maximum. Also used in column switching.

height equivalent to a theoretical plate (HETP) - value obtained by dividing the column length by the number of theoretical plates; taken as an indication of column quality.

HETP - height equivalent to a theoretical plate. A carryover from distillation theory; a measure of a column's efficiency. For a typical HPLC column well-packed with 5-mm particles, HETP (or H) values are usually between 0.01 and 0.03 mm. HETP = L/N, where L is column length, and N is the number of theoretical plates.

hexamethyl disilazane (HMDS) - sililating reagent employed to deactivate solid supports by blocking of silanol groups.

hydrophilic - "water-loving"; refers both to stationary phases that are compatible with water and to water-soluble molecules in general. Most columns used to separate proteins are hydrophilic in nature and should not sorb or denature protein in the aqueous environment.

hydrophobic - "water-hating"; refers both to stationary phases that are not compatible with water and to molecules in general that have little affinity for water. Hydrophobic molecules have few polar functional groups; most are hydrocarbons or have high hydrocarbon content.

hydrophobic interaction chromatography - a technique in which reversed-phase packings are used to separate molecules by virtue of the interactions between their hydrophobic moieties and the hydrophobic sites on the surface. High salt concentrations are used in the mobile phase; separations are effected by changing the salt concentration. The technique is analogous to "salting out" molecules from solution. Gradients are run by decreasing the salt concentration over time.

internal standards (IS) - substance used as reference in quantitative analysis; the internal standard is first mixed with standard solutions; later it is added to the unknown, and the ratio of peak heights (or areas) of internal standard and analyte is used for quantitative analysis.

interstitial volume - (Vo) - the total value of mobile phase within the length of the column. It is made up of the intraparticle volume (inside the packing itself) and interparticle volume (between the packing particles). Same as void volume. Also abbreviated Vi or Vm.

ion chromatography (IC) - An ion-exchange technique in which low concentrations of anions or cations are determined using low-capacity ion exchangers with weak buffers. Conductivity detectors are often used. Ion chromatography is practiced in two forms. In suppressed IC, a second column is used to remove the buffer ions so that sample ions can be more easily detected; membrane separator is sometimes used. In nonsuppressed IC, weakly conducting buffers at low concentration are carefully selected, and the entire effluent is passed through the detector; ions are detected above the background signal.

ion-exchange chromatography (IEC) - a mode of chromatography in which ionic substances are separated on cationic or anionic sites of the packing. The sample ion (and usually a counterion) will exchange with ions already on the ionogenic group of the packing. Retention is based on the affinity of different ions for the site and on a number of other solution parameters (pH, ionic strength, counterion type, etc.).

ion-exchange capacity - the number of ionic sites on the packing that can take part in the exchange process. Exchange capacity is expressed in mequiv/g; typical strong anion-exchange resin may have 3-5 mequiv/g capacity.

ion-pair chromatography - form of chromatogrpahy in which ions in solution can be "paired" or neutralized and separated as an ion pair on a reversed-phase column. Ion-pairing agents are usually ionic compounds that contain a hydrocarbon chain that imparts a certain hydrophobicity so that the ion pair can be retained on a reversed-phase column. Ion-pairing can also occur in normal-phase chromatography when one part of the pair is loaded onto a sorbent, but this technique is not as popular as the RPC technique.

irregular packing - refers to the shape of a silica gel based packing. Irregular silicas are available in microparticulate sizes. The packings are made by grinding silica gel into small particles and then sizing them into narrow fractions using classification machinery. Spherical packings are now used more often than irregular packings in HPLC, but less-expensive irregular packings are still widely used in prep LC.

irreversible adsoprtion - when a compound that has a very strong affinity for the adsorbent is injected onto a column, it can be adsorbed so strongly that it cannot be eluted from the column. A chemical reaction between the sample and the surface of the adsorbent is an example of irreversible adsorption.

isocratic - use of a constant-composition mobile phase in liquid chromatography.

Kovats index (Kovats retention index) - characterization system of the chromatographic behavior of substances in gas chromatography; normal alkanes are used as reference compounds to establish a scale of retention; widely used as a qualitative tool in Europe.

linear range - extension of the calibration plot (usually expressed in decades of concentration) within which the detector response is clearly linear.

linear velocity (u) - the velocity of the mobile phase moving through the column. Expressed in cm/s. Related to flow rate by the cross-sectional area of the column. Sometimes expressed as v.

linearity - proportionally between detector response and amount of sample; a calibration plot with a slope of 1.0 is the ideal case.

liquid-liquid chromatography (LLC) - same as partition chromatography. The earliest form of HPLC, it gave way to chemically bonded phases in the early 1970s.

liquid-solid chromatography (LSC) - same as adsorption chromatography.

loading - the amount of stationary phase coated or bonded onto a solid support. In liquid-liquid chromatography, the milligram amount of liquid phase per gram of packing. In BPC, the loading may be expressed in mmol/m2 - or in %C. See coverage.

longitudinal diffusion - same as molecular diffusion term/ B-term in van Deemter equation. See van Deemter equation.

mass transfer - band-broadening effect due to the lack of equilibrium between the mobile and stationary phase when partitioning the sample; this effect is expressed by the third term of the van Deemter equation.

mean pore diameter - the average pore diameter of the pore in a porous packing. The pore diameter is important in that it must allow free diffusion of solute molecules into and out of the pore so that the solute can interact with the stationary phase. In SEC, the packings have different pore diameters, and therefore molecules of different sizes can be separated. For a typical adsorbent such as silica gel, 60-Å and 100-Å pore diameters are most popular. For packings used for the separation of biomolecules, pore diameters ≥300 Å are used.

mesh - usual way to characterize particle size; the mesh number indicates the number of wires per inch of the sieve through which the particles are passed.

micellar chromatography - the addition of micelles to the mobile phase to effect separations. The micelles act as displacing or partitioning agents and provide another parameter that can be used to change selectivity.

micro LC - refers collectively to techniques in which a column of smaller-than-usual internal diameter (i.d.) is used for separation. In micor HPLC, columns of <0.5 mm i.d. are used.

minimum plate height - the minimum of the curve that results from a plot of H vs u. This value represents the most theoretical plates that can be obtained for a certain column and mobile phase system. Usually occurs at very low flow rates.

mobile phase - in LC the mobile phase is a solvent mixture such as methanol and water for reversed phase LC, or hexane for adsorption chromatography that flows continuously through the column.

modifier - additive that changes the character of the mobile phase. For example, in reversed phase, water is the weak solvent; methanol, the strong solvent, is sometimes called the modifier.

molecular diffusion - effect that produces band-broadening expressed by the second term of the van Deemter equation; the effect is due to the sample diffusion in the mobile phase.

molecular weight distribution - the distribution of molecular weight of molecules in a polymer sample. Distribution can be defined as weight average and number average.

monomeric phase - refers to a bonded phase in which single molecules are bonded to a support. For silica gel, monomeric phases are prepared by the reaction of an alkyl or aryl monochlorosilane. Polymeric phases are generally prepared from a di- or tri-chlorosilane reactant.

net retention time (Tog) - the adjusted retention time per gram of liquid phase corrected for the pressure drop along the chromatographic column.

noise - random fluctuation of the chromatographic signal; short-term noise (less than 1 sec) is often electrical in nature; long-term noise can be due to flow rate changes, temperature changes, or column "bleed".

normalization - quantitative method commonly employed when the entire sample is eluted from the column; the area percent is taken as weight percent composition; of limited usefulness since most detectors give different responses to different samples.

normal-phase chromatography - a mode of chromatography carried out with a polar stationary phase and a nonpolar mobile phase. Adsorption on silica gel using hexane as a mobile phase would be a typical normal-phase system. Also refers to the use of polar bonded phases, such as CN or NH2 . Sometimes referred to a straight-phase chromatography.

octadecylsilane (ODS) - the most popular reversed phase in HPLC. Octadecylsilane phases are bonded to silica or polymeric packings. Both monomeric and polymeric phases are available.

open tubular column - syn. capillary column, Golay column, WCOT column, SCOT column; a column with a hole down the middle, frequently called a capillary column.

overload - in preparative chromatography, the overload condition is defined as the mass of sample injection onto the column at which efficiency and resolution begin to be affected if the sample size is further increased. See sample capacity.

packing - material contained inside the column; responsible for the separation.

particle size (dp) - the average particle size of the packing in an LC column. A 5-mm column would be packed with particles having definite particle size distribution; packings are never monodisperse. See particle-size distribution.

particle-size distribution - a measure of the distribution of the particles used to pack the LC column. In HPLC, a narrow particle-size distribution is desirable. A particle-size distribution of dp ± 10% would mean that 90% of the particles fall between 9 and 11 mm for an average 10-mm dp packing.

partition - distribution phenomena of the sample between the mobile phase and the stationary phase.

partition chromatography - separation process in which one of the liquid phases is held stationary on a solid support while the other is allowed to flow freely down the column. Solutes partition themselves between the two phases based on their individual partition coefficients. Liquid-liquid chromatography is an example.

partition coefficient (K) - quantitative expression of the partition equilibrium; usually expressed as the ratio of concentration of the sample in the stationary phase and the mobile phase.

partition ratio (k) - syn. capacity ratio, capacity factor; column characteristic expressed as the ratio of the retention volumes to the dead volumes; k' = t'R/to.

phase ratio(b) - column characteristic defined as the ratio of mobile phase to stationary phase.

K = kb

phenyl phase - a nonpolar bonded phase prepared by the reaction of dimethylphenylchlorosilane with silica gel. Claimed to have affinity for aromatic compounds.

photoconductometric detector - an HPLC detector which works on a two fold principle. Firstly, high energy photoexcitation of a neutral molecule raises its energy level to generate a charged, ionized species. Secondly, this new charged species is detected by a conductometric measuring cell. Chloro-aromatics are one of the analytes measured at very low levels by this technique.

pirkle columns - chiral "brush-type" stationary phases, based on 3.5-dinitrobenzoylphenylglycine silica, used in the separation of a wide variety of enantiomers. Named after the developer, Dr. William Pirkle, University of Illinois.

plate height - syn. height equivalent to a theoretical plate, HETP; column length corresponding to a theoretical plate, normally found by dividing the column length by the number of theoretical plates.

plot columns - syn. porous layer open tubular columns; see open tubular column.

polarity - a measure of the separation of charges in a molecule; hydrocarbons are non-polar; alcohols are polar; more polar stationary phases interact more strongly with polar samples and usually provide a better separation.

polystyrene-divinylbenzene resin (PS-DVB) - the most common polymer base for ion-exchange chromatography. Ionic groups are incorporated by various chemical reactions. Neutral PS-DVB beads are used in reversed-phase chromatography. Porosity and mechanical stability can be altered by varying the cross-linking through the variation of the DVB content.

porosity - for a porous adsorbent, the ratio of the volume of the interstices to the volume of the solid particles. The pore volume is also used as a measure of porosity.

preparative chromatography - chromatographic technique in which the purpose is the separation of sizable amounts of pure materials.

programmed temperature gas chromatography (PTGC) - technique employed to speed up the elution of long-retained compounds by gradual heating of the column as the separation occurs.

pulsating flow - flow originating from a reciprocating pump. Normally, the pulses are dampened out by a pulse damper, by an electronic pressure feedback circuit, or by an active damper pump head. Some detectors (e.g. electrochemical) are affected by flow pulsations.

reciprocating piston pump - the most common HPLC pump design which is available in single or multiple-piston arrangements forces mobile phase through the column on a forward piston stroke, and refills the cylinder with mobile phase on the recycle stroke.

recorder - electromechanical instrument which transforms the chromatographic signal into a graphical record.

reduced plate height (h) - Used to measure efficiencies of columns. An HPLC column with an h value ≤2 is considered to be well-packed, h = H/dp.

reduced velocity (v) - along with the reduced plate height, is used to compare different chromatographic columns. It relates the solute diffusion coefficient (Dm) in the mobile phase to the particle size of the column packing (dp). v = dp/Dm.

relative retention time - syn. solvent efficiency; ratio between the net retention time of a substance and that of a standard compound.

residual silanols - the silanol (-Si-OH) groups that remain on the surface of a packing after a phase in chemically bonded onto its surface. These silanol groups may not be accessible to the reacting bulky organosilane (e.g. octadecyldimethylchlorosilane) but may be accessible to small polar compounds. Often they are removed by endcapping with a small organosilane such as trimethylchlorosilane. See endcapping.

resolution - a quantitative measure of the separation of two peaks; it accounts for narrowness of peaks and the separation of peak maxima.

response - detector characteristic which defines the detectable types of compounds by a particular detector.

retention index - see Kovats index.

retention time (adjusted) (t'R = tR- to) - the retention value obtained by subtracting the dead time from the uncorrected retention time.

retention time (uncorrected) (tR) - time elapsed between sample introduction and maximum of response.

retention volume (adjusted) (V) retention volume corrected by subtracting the dead volume.

retention volume (corrected) - retention volume corrected for pressure drop across the column.

retention volume (uncorrected) - volume of mobile phase required to elute the peak maximum of a compound; calculated by multiplying the retention time by the flow rate.

reversed-phase chromatography (RPC) - the most common HPLC mode. Uses hydrophobic packings such as octadecyl- or octylsilane phases bonded to silica or neutral polymeric beads. Mobile phase is usually water and a water-miscible organic solvent such as methanol or acetonitrile. There are many variations of RPC in which various mobile phase additives are used to impart a different selectivity. For example, for the RPC of anions, the addition of a buffer and tetraalkylammonium salt would allow ion pairing to occur and effect separations that rival ion-exchange chromatography.

sample capacity - refers to the amount of sample that can be injected onto a LC column without overload. Often expressed as grams of sample per gram of packing. Overload is defined as the sample mass injected at which the column efficiency falls to 90% of its normal value.

sample loop - a loop of calibrated volume used in a sample injection valve; normal volumes range from 5 to 100 microliters.

sample valve - syn. injection valve; a device used to inject fixed volumes of liquid sample; may be operated manually or automatically; provides very reproducible injection volumes.

SAX - strong anion exchanger. A typical strong anion exchange functional group would be tetraalkylammonium.

SCX - strong cation exchanger. A typical strong cation exchange functional group would be a sulfonic acid.

selectivity (a = (t'R(2)/t'R(1)) - measures the selective solubility of a stationary phase for 2 compounds; high a values mean high selectivity and easy separation.

sensitivity - term which quantitatively describes the signal obtained per amount of sample introduced.

separation factor - ratio of retention times of two peaks; has been replaced by selectivity a which is the ratio of adjusted retention times.

silanol - the Si-OH group found on the surface of silica gel. There are different strengths of silanols, depending on their location and relationship to each other. The strongest silanols are acidic and often lead to undesirable interactions with basic compounds during chromatography.

silica gel - the most commonly used packing in liquid chromatography. It has an amorphous structure, is porous, and consists of siloxane and silanol groups. It is sued as a bare packing for adsorption, as the support in liquid-liquid chromatography or for chemically bonded phases, and, with various pore sizes, as packing in size-exclusion chromatography. Micro-particulate silicas of 5- and 10-mm average particle diameter are used in HPLC.

siloxane - the Si-O-Si bond. A principal bond found in silica gel or for attachment of a silylated compound or bonded phase. Stable except at high pH values.

silylation - a chemical reaction involving a silane reagent such as trimethyl chlorosilane (TMS); used to silanize or deactivate active sites found in samples on solid support surfaces and even on glass tubing.

single-piston pump - see "reciprocating piston pump".

size-exclusion chromatography (SEC) - same as steric exclusion chromatography.

slurry packing - the technique most often used to pack HPLC columns with microparticles. The packing is suspended in a slurry (10% wt/vol) and is rapidly pumped into the empty column. Special high-pressure pumps are used.

solid phase extraction (SPE) - A sample-preparation technique that uses a solid-phase packing contained in a small plastic cartridge. The solid stationary phases are the same as HPLC packings; however, the principle is different from HPLC. Sometimes referred to as digital chromatography. The process as most often practiced requires four steps: conditioning the sorbent, adding the sample, washing away the impurities, and eluting the sample in as small a volume as possible with a strong solvent.

solid support - 5 to 20 mm particles contained in the column whose surface is coated with stationary phase in liquid-liquid chromatography, or derivatized with a bonded phase in BPC.

solute - syn. sample or analyte.

solvent strength - refers to the ability of a solvent to elute a particular solute or compound from a column. Described by Lloyd Snyder for LEAC (LSC) adsorption chromatography on alumina; solvents were quantitatively rated in an elutropic series. No elutropic series exists for other modes.

sorbent - refers to an adsorption packing used in liquid chromatography. A common sorbent is silica gel.

spherical packing - refers to spherical solid packing materials. Spherical packings are generally preferred over irregular particles.

standard addition - a method of quantitative analysis where standard amounts of sample are added to the unknown; from the increase in peak height the concentration in the unknown can be estimated.

stationary phase - syn. liquid phase; liquid covering the surface of the solid support in the column in LLC, or the derivatized particles in BPC.

steric exclusion chromatography (SEC) - a major LC mode in which samples are separated by virtue of their size in solution. Also known as size-exclusion, gel permeation, gel filtration, or gel chromatography.

supercritical fluid chromatography (SFC) - a technique that uses a supercritical fluid as the mobile phase. The technique has been applied to the separation of substances that cannot be handled effectively by liquid chromatography (because of detection problems) or gas chromatography (because of the lack of volatility). Examples are separations of triblycerides, hydrocarbons, and fatty acids. GC detectors and HPLC pumps have been used together in SFC.

support - refers to solid particles. Support can be naked or coated or can have a chemically bonded phase in HPLC.

suppressor column - in ion chromatography, refers to the column placed after the ion-exchange column. Its purpose is to remove or suppress the ionization of buffer ions so that sample ions can be observed in a weakly conducting background with a conductivity detector.

surface area - in an adsorbent, refers to the total area of the solid surface as determined by an accepted measurement technique such as the BET method using nitrogen adsorption. The surface area of a typical porous adsorbent such as silica gel can vary from 100 to 600 m2/g.

surface coverage - usually refers to the mass of stationary phase per unit area of an LC support. Often expressed in mmol/m2 of surface. Sometimes the %C is given as an indicator of surface coverage.

swelling - process in which resins and gels increase their volume because of their solvent environment. Solvent enters ion-exchange resin to dilute ions;; in gels, solvent penetrates pores. If swelling occurs in packed columns, blockage or increased back pressure can occur. In addition, column efficiency can be affected.

syringe pump - a popular and useful pump design for Micro-LC and SFC, consists of a single, large volume cylinder and piston assembly (typically 50 mL in volume), such that uninterrupted mobile phase flow is available without pulsation.

tailing - the phenomenon in which the normal Gaussian peak has an asymmetry factor >1. The peak will have skew in trailing edge. Tailing is caused by sites on the packing that have a stronger-than-normal retention for the solute. A typical example of a tailing phenomenon is the strong adsorption of amines on the residual silanol groups of a low-coverage reversed-phase packing.

theoretical plate - one equilibrium between the mobile and stationary phase; a measure of column efficiency; the more plates, the better is the column efficiency.

tortuosity - a property of a packed column that indicates the degree of unevenness of the path followed by the solute molecule as it passes down the column. Covered in the A-term of the van Deemter equation.

total permeation volume (Vp) - the retention volume on an SEC packing in which all molecules smaller than the smallest pore will elute. In other words, at Vp, all molecules totally permeate all of the pores and elute together.

trimethyl chlorosilane (TMS) - common reagent employed in the formation of derivatives, and in deactivation of packing materials.

uncorrected retention time - see retention time; time from injection to peak maximum.

UV detector - the most popular detector for HPLC, a UV spectrophotometer (for variable-wavelength detection) or photometer (for single-wavelength detection) equipped with a low-volume flow through "curvette", commonly referred to as a flow cell. Detects analytes which readily absorb light at the selected wavelength.

van Deemter equation - theoretical relationship which describes sample band broadening as a function of three phenomena; eddy diffusion, molecular diffusion, and mass transfer.

void - the formation of a space, usually at the head of the column, caused by a settling or dissolution of the packing. A void in the column leads to decreased efficiency and loss of resolution. Even a small void can be disastrous for small microparticlate columns. The void can sometimes be removed by filing it with glass beads or porous packing.

void volume (Vi) - the total volume of mobile phase in the column; the remainder of the column is taken up by packing material. Can be determined by injecting an unretained substance that measures void volume plus extracolumn volume. Also referred to as interstitial volume. Vo or Vm are sometimes used as a symbols.

wall effect - the consequence of the looser packing density near the walls of the rigid HPLC column. Mobile phase as a tendency to flow slightly faster near the wall because of the decreased permeability. The solute molecules that happen to be near the wall are carried along faster than the average of the solute band and, consequently, band spreading results.

WAX - weak anion exchanger. Ionizable groups such as primary, secondary, or tertiary amino groups on a packing are considered to be weak.

WCX - weak cation exchanger. Carboxylic groups on a packing are typical of a weak cation exchanger.

well coated open tubular column - syn. WCOT; capillary column, Golay column.

zwitterions - compounds that carry both positive and negative charges in solution.

Chromatography Common Abbreviations

a Selectivity; separation factor

AED Atomic Emission Detector

b Phase ratio

C4, C8, C18 Alkyl chain length of an LC reversed bonded phase

CE, CZE Capillary Electrophoresis, Capillary Zone Electrophoresis

CI Chemical Ionization

DAD, PDA (Photo) Diode Array Detector

dp Particle diameter

df Film thickness

ECD Electron Capture Detector

EI Electron Impact

ELCD Electrolytic Conductivity Detector

EPA Environmental Protection Agency

FAMEs Fatty Acid Methyl Esters

FID Flame Ionization Detector

FPD Flame Photometric Detector

FS Fused Silica

FT-IR Fourier Transform Infrared

GLC Gas Liquid Chromatography

GPC Gel Permeation Chromatography

GSC Gas Solid Chromatography

HETP or H Height Equivalent to one Theoretical Plate

HPLC High Performance Liquid Chromatography

HRGC High Resolution Gas Chromatography

I.D. Internal Diameter

IPC Ion Pair Chromatography

IRD Infrared Detector

IS Internal Standard

k (or k') Capacity factor (syn capacity ratio, partition ratio)

K Partition coeficcient

l Wave length

LSC Liquid Solid Chromatography

LIF Laser Induced Fluorescence (detector)

MAOT Maximum Allowable Operating Temperature

MDQ / MDL Minimum Detectable Quantity / Minimum Detectable Level

MIP Microwave Induced Plasma

MS Mass Spectrometry

MSD Mass Selective Detector

N Theoretical plate (number of)

NP Normal Phase

NPD Nitrogen Phosphorus Detector

O.D. Outer Diameter

ODS Octadecyl silane

PCBs Polychlorobiphenyls

PID Photo Ionization Detector

PLOT Porous-Layer Open Tubular (column)

PMT Photo-Multiplier Tube

PNAs Polynuclear Aromatics

ppb Part per billion

ppm Part per million

ppt Part per trillion

PTFE Polytetrafluoroethylene (Teflon®)

PTGC Programmed Temperature Gas Chromatography

RS Resolution

RP Reversed Phase

RSD Relative Standard Deviation

SCOT Surface Coated Open Tubular (column)

SEC Size (or Steric) Exclusion Chromatography

SFC Supercritical Fluid Chromatography

SFE Supercritical Fluid Extraction

SIM Single Ion Monitoring

S/N Signal to noise

TCD Thermal Conductivity Detector

TID Thermionic Detector (usually known as NPD)

TLC Thin Layer Chromatography

THMs Trihalomethanes

TMS Trimethylsilyl (derivative)

tR Retention time

UV Ultra-Violet (detector)

VR Retention volume

VOC Volatile Organic Compound

W Peak width

WCOT Wall Coated Open Tubular (column)

Structure-related sample treatment

(Bio-)Analytical Chemistry: From B(egin) to E(nd)

Hubertus Irth, Henk Lingeman

BioMolecular Separations Group, Faculty of Sciences, VU University Amsterdam, Amsterdam, the Netherlands

The start
Introduction

Sample treatment (ST) is the bottleneck in most chromatographic/electrophoretic separations. As a result the primary goal of the present module on “Structure related sample treatment” techniques and approaches that can be used for this purpose.

The main ST objectives discussed in this module are:

- Learn technologies and techniques:

o How do they work,

o What are the applications,

o What are the advantages and limitations,

- Possibilities to improve laboratory operations:

o Cost (labour intensive),

o Errors (accuracy, precision),

o Time,

- Productivity issues:

o Automation,

o Parallel versus serial processing,

o On-line versus off-line,

- Help to make the transition from SOP’s to (new) techniques a successful one:

o Criteria for choosing sample treatment techniques.

This means that the problems are addressed that are related to: time, labour, automation, enrichment, selectivity, organic solvents, inorganic salts, contamination, etc.

The resulting ST goals are:

1. Removal of interferences (high and low molecular weight compounds, organic and inorganic compounds);
2. Enrichment of the analyte(s);
3. Phase transfer of the solutes(s).

The main focus will be on the development of total (bio-)analytical procedures an in particular on the ST steps needed to determine these compounds with either gas chromatography (GC), liquid chromatography (LC), ion chromatography (IC), affinity chromatography (AC) or capillary electrophoresis (CE), with the emphasis on automated/hyphenated systems including high-throughput and bio-specific assays. This includes a discussion on the theoretical backgrounds, advantages and disadvantages of pharmaceutical, bio-analytical, food, clinical environmental and process analytical methods and procedures.

Figure 1.1: Relative speed of different steps in (bio-)analytical sample treatment.

The fact that the polarity of the compounds of interest is still increasing and the fact that sampling and sample preparation (SP) are the most time consuming parts of the total analytical scheme, explains the focus on the numerous combinations of sampling and SP procdures in combination with GC / LC and CE separation-detection approaches (Figure 1.1)

The most important options for ST, sampling plus sample preparation, are:

- Sample collection: Taking a representative sample;

- Sample storage and stabilization: Using proper containers and freezing of unstable samples;

- Initial (primary) sample preparation: Reducing the sample size;

- Weighing or volumetric dilution: Taking precautions for unstable and reactive samples;

- Alternative sample processing methods: Introducing solvent replacement, desalting, evaporation procedures etc.;

- Removal of particulates: Applying filtration and centrifugation steps;

- Selective (secondary) sample preparation: Introduction of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) approaches;

- Derivatization: Enhancing detection and improving separation.

In this module various definitions will be used:

- Sample treatment: Total process of analyzing a sample including sampling, initial (primary) and selective (secondary) sample preparation, analysis, detection, quantitation and validation.

- Sampling: Process of taking a reliable and representative sample.

- Initial sample preparation: Process of storing, stabilizing and preparing a sample for selective clean-up and analysis.

- Selective sample preparation: Process of sample concentration, selective sample clean-up and/or phase transfer of the analyte(s).

- Analysis: Process of selective (qualitative or quantitative) determination of the analyte(s).

- Detection: Process of identifying or detection of the analyte(s).

- Quantitation: Process of data acquisition, data reduction and data interpretation.

- Validation: Process of guaranteeing the repeatability / reproducibility, robustness / ruggedness of the overall sample treatment process.

These steps are summarized in the overall analytical procedure as given in Figure 1.2.

Figure 1.2: Overall analytical procedure.

Another reason that sampling and SP are so important is that looking at the time spend on the analysis, about 60% is related to sample processing. In addition to this about 30% of the errors during SP/ST is due to sample processing and another 20% to the operator. This means that automation (Figure 1.3) of SP/ST techniques certainly will improve the overall quality of the analytical procedure.

Figure 1.3: Lab-automation in combinatorial chemistry (www.pa.bosch.com).

Analytical chemistry plays an important role in separating, isolating and quantifying various chemical compounds. Virtually every item of commerce has been subjected to analytical testing (Figure 1.4) at one or more stages in its manufacturing process.

Figure 1.4: Analytical testing can be performed using numerous techniques and procedures (www.pennpharm.co.uk).

Apart from the classical methods, such as titrimetric and gravimetric techniques, many instrumental techniques have been developed, for the determination of not only the active ingredient, but also the quantification of related compounds or impurities associated with it (Figure 1.5).

Figure 1.5: Traditional techniques like titrimetric and gravimetric analysis are, nowadays, replaced by modern separation techniques.

The recently developed analytical methods have the advantage of not only using small amounts of sample, reagents and less time, but also produce accurate results. These analytical techniques may be:

- physico-chemical methods,

- electro-analytical methods

- separation-based methods.

The physico-chemical methods like spectroscopy include colorimetry and spectrophotometry covering ultra-violet and visible region or fluorimetry, nephelometry (Figure 1.6) or turbidimetry, and nuclear-magnetic resonance and mass spectrometry. The electro-analytical methods cover potentiometry, amperometry, voltammetry, electrophoresis and polarography.

Figure 1.6: Nephelometry is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (www.lib.mcg.edu).

The separation-based methods involve (high-performance) liquid chromatography (LC), (high-performance) thin-layer chromatography (HPTLC / TLC), capillary electrochromatography (CEC), supercritical-fluid chromatography (SFC) and gas chromatography (GC). The combination of GC and LC with mass spectrometry (MS), nowadays, are the most powerful tools employed for the quantification and identification of analytes in pure as well as in associated forms. In addition to chromatographic separation methods, electrophoretic techniques, like capillary electrophoresis (CE), isotachophoresis (ITP) (Figure 1.7) gel electrophoresis (GE) and isoelectric focusing (IEF), are relatively popular for the separation and quantitation of (charged) organic compounds.

Figure 1.7: Isotachophoresis is one of the electrophoretic techniques used for the separation of charged molecules (www.bd.com).

The still increasing interest on the determination of drugs has forced the analytical chemists to develop methods for their trace analysis in the presence of biological matrices. Since every year a large number of drug candidates are synthesized it means that also a large number of methods/procedures must be developed that are applicable for the routine analysis of these drug candidates. These methods should be rapid, precise, accurate and cost effective. Although costly sophisticated instruments like LC, HPTLC, GC, GC-MS and LC-MS are available, the spectrophotometer is being preferred by ordinary laboratories for its simple, economical and easy handling techniques (Table 1.1).

Table 0.1: Bio-analytical techniques.

In summary: The philosophy and concepts of sampling, sample preparation in analytical chemistry will be presented in a practical way to provide the analytical chemist with the necessary tools to determine low- and high-molecular-weights compounds in a variety of matrices using chromatographic / electrophoretic, spectroscopic or immunological methods.

In addition, the philosophy and concepts for the method development procedures will be based on the physico-chemical properties of the analyte(s) and the matrices in which these compounds are present (Figure 1.8).

Figure 1.7: Physico-chemical properties.
Physico-chemical properties

(& Structure-activity relationships of drugs)

In order to develop analytical procedures for low-molecular weight (LMW) compounds in pharmaceutical formulations and biological samples the starting point always must the physico-chemical properties of both the compounds of interest and the sample matrix. Therefore, the focus will be on these physico-chemical properties and the structure-activity relationships between the solutes and matrix components such as the interaction between drugs and biological matrix components (e.g., proteins, enzymes, receptors) or pesticides and humic substances.

Pharmaceutical chemistry Bio-analysis Pharmacochemistry

The intention is not to discus all the basic principles of analytical chemistry, physical chemistry, organic chemistry, thermodynamics and kinetics in detail, but just to provide the information necessary to develop new or to modify existing analytical methods that can be used in pharmaceutical chemistry, bio-analysis and pharmacochemistry.

For example, during the development of new drug entities (NCE) it is of utmost importance to optimize the structure of a new compound (drug candidate) for a certain target. This can be done by changing the 3-dimensional (3-D) structure, or in other words the way the functional groups are arranged around the basic molecule. In addition the physico-chemical and bio-pharmaceutical properties of a compound are determined by its structure (Figure 1.8).

Figure 1.8: Fit of NCE in receptor pocket (www.tripos.com).

In addition the physico-chemical and bio-pharmaceutical properties of a compound are determined by its structure. These properties, on its turn, are related to processes like solubility, absorption, protein binding, partitioning, elimination and metabolism (Figure 1.9).

Figure 1.9: Summary of metabolism process (www.elmhurst.edu).

These properties, on its turn, are related to processes like solubility, absorption, protein binding, partitioning, elimination and metabolism. In (bio)-analytical chemistry (BAC) these properties determine which analytical techniques can be used and what the actual conditions must be. It will be obvious that there is a strong correlation between the various physico-chemical properties (Figure 1.10).

Figure 1.10: Relation between physico-chemical properties.

In the case of drugs it can be stated that when the molecular weight is over 500, the number of H-bridge donors is over 5, the number of H-bridge acceptors is over 10 and the calculated partitioning coefficient is over 5, the compound will badly orally adsorbed (Figure 1.11). This gain shows the importance of studying the physico-chemical properties of organic compounds.

Figure 1.11: Solubility of chloramphenicol is determined by the presence of hydrophilic and lipophilic groups (www.auburn.edu).

From the example mentioned above it will be clear that the emphasis will be on the relation between the chemical structure, or in other words the physico-chemical properties of a compound, and the determination of these compounds in a (complex) sample matrix. The final objective is that the information provided can be used to develop simple qualitative and quantitative methods for organic compounds in a variety of matrices. In order to obtain this goal the following parameters will be discussed:

- Physico-chemical properties of organic molecules like polarity, acid-base properties, solubility, stability, absorption/adsorption, etc;

- Composition of sample matrices and their effect on the analytical results (e.g., analyte-matrix binding, stability);

- ST/SP procedures (e.g., filtration, centrifugation, extraction).

There are two magic words that will be discussed throughout the whole monograph: pH and pKa (Figure 1.12).

Figure 1.12: Questions to be answered!

After studying the module on “Structure-related sample treatment” it should be possible to answer the following starting questions (Figure 1.12) and the final question (Figure 1.13):

Figure 1.13: Final analytical question to be answered!

With respect to environmental, pharmaceutical and bio-analysis this means that the following topics features are of importance:

- Insight in structures of chemical compounds and physico-chemical properties like pK_a, polarity, stability, solubility, chromophores, etc;

- Composition of biological materials and, to lesser extent, pharmaceutical formulations and environmental samples, and their influence on the analytical results (e.g. stability, matrix binding);

- Initial (primary) sample treatment procedures (e.g., filtration, precipitation, extraction);

- Selective (secondary) sample preparation procedures (e.g. solid-phase extraction, immunological approaches, hyphenated and automated system

In order to do deal with these features a number of sub-questions should be answered. These sub-questions are related to the following physico-chemical properties of the analyte(s) or the matrix molecules:

- Name, molecular weight, structure;

- Appearance, colour, smell;

- Acid-base properties;

- Functional groups;

- Polarity (partitioning coefficients);

- Solubility;

- (Chemical) stability;

- Spectral properties;

- Bio-pharmaceutical properties;

- Sample treatment / sample preparation;

- Separation / detection;

- Quantitative aspects (validation).

Except for the relatively straightforward analytical applications in most cases an overall sample analysis scheme should be constructed (Figure 1.14). This because direct sample injection into a chromatographic/electrophoretic system, normally is not possible because unwanted matrix components can disturb the separation and/or detection or can clog the analytical device. In other cases the concentration of the analyte(s) or the degradation products or metabolites may be too low to allow a direct injection procedure. The result is that ST/SP is an important aspect of the total analytical procedure and that in order to provide an adequate ST/SP procedure accurate knowledge about the properties of the compounds to be separated and the sample matrix is a prerequisite.

Figure 1.14: Sample analysis scheme.

Bio-analytical chemistry

Bio-analytical chemistry (BAC) involves a number of different disciplines such as therapeutic drug monitoring (TDM), toxicology (clinical, forensic, post-mortem), drugs-of abuse, pharmaceutical analysis, food analysis and environmental analysis.

One of the most important disciplines is therapeutic drug monitoring (TDM) (Figure 1.15). TDM can be defined as:

‘The measurement and the clinical use of blood (serum/plasma) drug levels (concentrations) to adjust each patient’s individual drug dosage and schedule to each patient’s individual therapeutic requirement’.

Figure 1.15: Therapeutic drug monitoring in epilepsy (www.e-epilepsy.org.uk).

In order to perform TDM, quantitative methods for the determination of these drugs, their metabolites and their degradation products should be available.

A number of variables complicate bio-analytical procedures. For example, after a drug has been administered to an individual, it is metabolized into products that can be excreted more easily. On its way through the body multiple metabolites are formed meaning that depending on the bioactivity and relative concentrations, the parent drug and/or the metabolites should be determined.

Biomonitoring outshines the indirect assessment of exposure in determining which pollutants enter the body, and whether they cause disease or disability (Figure 1.16). Non-persistent toxicants move quickly out of the blood as they are metabolized to water-soluble compounds that can be extracted in urine. Some chemicals or metabolites may bind to proteins or to DNA and persist in the body for a longer period of time.

Figure 1.16: Biomonitoring of pollutants (www.nature.com).

Postmortem toxicology, for example, is used to determine if alcohol, drugs or other poisons have contributed to the death of a person (Figure 1.17). Fundamentally it is different from TDM and clinical toxicology. The reason is that it is far more difficult to interpret post-mortem results. Clinical toxicology is dealing with patients with suspected poisoning and the primary objective is to assist in the treatment of such a patient.

Figure 1.17: Postmortem toxicology (www.answers.com).

Drugs-of-Abuse can be defined as: ‘Any substance that, because of some desirable effect, is used for some purpose other than that intended’ (Figure 1:18).

Figure 1.18: Mechanism of action of high concentration of amphetamine (www.cnsforum.com).

The questions in pharmaceutical analysis are again quite different. Questions that are relevant are dealing with the identity, concentration and stability of the drug in the formulation (Figure 1.19). In addition information on the impurities, shelf-life and release-rate of the drug from the formulation are essential. The connection between pharmaceutical and bio-analysis is that after administration of the drug the concentration in tissue or a biological fluid is a key factor. While during drug development parameters like acid/base properties, polarity (log P values), solubility and stability are important features.

Figure 1.19: Pharmaceutical analysis (www.chem.agilent.com).

In food analysis the properties of foods and their constituents are characterized. Analytical procedures are used in food analysis to provide information about a wide variety of characteristics such as composition (e.g., lipids, proteins, water, carbohydrates, and minerals), structure, physico-chemical properties and sensory attributes. The data obtained are used to produce on an economical, safe and nutritious way.

Environmental system analysis is an important tool to analyze sustainability, e.g. environmental impact, optimal resource management and how outside factors affect farm management (Figure 1.20).

Figure 1.20: Environmental system analysis (www-mat21.slu.se).

Trace environmental quantitative analysis (TEQA) is not only dealing with the determination of organic and inorganic compounds in e.g., air, ground, water, plant effluent, leachate, soil, sediment), by also with the determination of these compounds in body fluids and tissues because the intake of environmental pollutants by humans, and the potential of biohazards is a key issue in environmental chemistry. This means that bioterrorism and biomonitoring are important issues in this respect.

Environmental monitoring is completely different from TDM (Figure 1.21).

Figure 1.21: Environmental monitoring (B.J. Alloway, D.C. Ayres, Chemical Principles of Environmental Pollution, Blackie Academic & Professional, London 1997).

Environmental analysis can be:

- Analytes entering the environment, amounts involved, original sources and spatial distribution;

- Effects of analytes on humans, crops, livestock and eco-systems;

- Trends in concentration of analytes over time and the reason for this;

- Extent inputs, concentrations, effects and trends can be modified;

- Establish baseline concentrations for comparison;

- Assess need for legislative controls;

- Activate emergency procedures;

- Determine suitability of resources for proposed uses.

In addition to in TDM BAC is important in Pharmacochemistry. The strategy in pharmcochemistry is to find and to document structure-activity relationships (SAR), with the goal to develop new drug candidates (Figure 1.22).

Figure 1.22: Structure-activity relationships of taxol (www.ch.ic.ac.uk).

Using the term activity means for example:

- Dynamic interactions with biological targets like receptors and enzymes;

- Kinetic parameters like membrane penetration and metabolic conversion;

- Toxicological properties like mutagenesis, cell and organ toxicity.

Quantitative-structure activity relationships (QSAR) play an important role in drug development (Figure 1.23).

Figure 1.23: Quantitative SAR plays an important role during drug development (www.goldenhelix.com).

` Pharmacochemistry can be divided a number of (sub) disciplines like:

- (Bio)organic chemistry (synthesis and combinatorial chemistry) (Figure 1.23);

- Physical chemistry and structural chemistry (SAR, computational chemistry);

- (Bio-)analytical chemistry;

- Molecular and structural biology (including bio-informatics);

- Pharmacology (including bio-transformation and partitioning);

- Molecular toxicology.

Figure 1.23: Electron density in the amino acid cystein calculated using a quantum-chemistry computer program (Computational Chemistry). The picture shows the surface where the electron density is 0.002 electrons/Å3 (meaning that nearly all electrons are inside the surface). The grey scale shows the electrostatic potential at this surface, darker portions representing negative potential (http://nobelprize.org).

The popularity of LC can be explained by the fact that in bio-analytical research analyte stability, metabolism (biotransformation), distribution as well as excretion are important and that the strength of LC is that qualitative as well as accurate quantitative information can be obtained of the parent compounds as well as of polar and extremely polar metabolites. LC also has some disadvantages: relatively time-consuming sample preparation techniques are normally needed and relatively large sample volumes (0.5 -1.0 mL) are normally required.

In contrast, immunological techniques require no sample preparation and only 50 -100-mL volumes are needed, but these techniques lack the selectivity of LC methods (Figure 1.24).

Figure 1.24: Immunoassay formats (www.piercenet.com).

For a considerable number of analytes radio-immunoassays (RIA) are more frequently used than LC methods, but although this is the case for, for example, cyclosporins, the RIA procedures overestimate the cyclosporin concentration because the polyclonal antibodies used react with cyclosporin as well as its metabolites (Figure 1.25).

Figure 1.25: Format of radio-immunoassay (http://users.rcn.com).

However, although cross-reaction is one of the major limitations of immunoassays (IA), the production of highly specific monoclonal antibodies can overcome this problem which means that especially enzyme-linked immunosorbent assay (ELISA) (Figure 1.26) will be an important bio-analytical tool next to LC.

Figure 1.26: Principle of enzyme-linked immunosorbent assay (www.biosystemdevelopment.com).

Since both techniques, LC and IA, have their own advantages and limitations, the combination of these two techniques, the so-called bio-specific detection, seems to be rather promising.

There are many reasons why an analytical procedure should be performed. There are an increasing number of substances that, for legal reasons must be monitored to ensure the safety of the individual as their presence may be detrimental to both humans and animals. There is an increased consumer awareness concerning the quality and safety of manufactured products and this makes manufacturer’s test their products as quality ensures that financial losses from law suits are reduced. The increased or decreased concentrations of the natural constituents of the body can be used to diagnose diseases and to monitor its treatment.

An analytical procedure is a means to an end; it provides information which can concern the composition of a sample or the status of a chemical process. This analytical information is then used as a basis for a decision: is a sample within specifications, is this water sample safe to drink or is the process under control?

The analytical laboratory is often on the critical path in many organizations as the assay results are needed before a decision can be taken. Therefore, the analytical laboratory is under increasing pressure to improve its efficiency and improve turnaround times while maintaining the quality and consistency of the data reported. Analytical data and information must not be confined within the laboratory, it is a resource that should be used and disseminated throughout the laboratory’s organization.

Contents

Structure-related sample treatment

The start

Introduction

Physico-chemical properties

Bio-analytical chemistry

Contents

Introduction

Summary

Introduction

Rationale sample treatment

Introduction

Choice sample treatment

Categorization techniques

Sensitivity & selectivity requirements

Quality & validation

Introduction

Quality & validation

Validation

Method validation (Why, how, when)

Metabolism & stability

Introduction

Metabolism

Degradation

Stability in solution

Stability in matrix

Method development

Introduction

Separation-detection systems

Liquid chromatography

Separation modes

Detection

Gas chromatography

Introduction

Stationary phase

Detection

Gas chromatography – mass spectrometry

Capillary electrophoresis

Introduction

System theoretical background

Conclusions

Sample treatment/preparation

Sample treatment

Sample preparation

Conclusions

Problems & questions

Molecular Properties (Non-covalent interactions)

Organic structures

Hetero atoms

Stereochemistry

Reaction kinetics

Reaction rate

Reaction constants

Non-covalent interactions

Electrostatic interactions

Dipole interactions

Hydrophobic interactions

Solubility parameters

Drug-protein interactions

Protein binding

Disruption of protein binding

Receptor-ligand binding

Chromatographic partitioning

Partitioning isotherms

Secondary chemical equilibria

Molecular Properties (Molecular effects)

Introduction

Acid, bases, buffers

Dissociation & pH

Ionization & Activity

Electronic effects

Electronegativity

Inductive & resonance effects

Hammett equation

Taft equation

Ionization constants

Microscopic constants

Macroscopic constants

Molecular Properties (Partition Related Processes)

Calculations of log P

Hansch method

Substituent additivity

Limitation of substituent additivity

Application of p-values

Rekker method

Relation p- and ¦-values

Collander equation

Correlation log P and protein binding

Solubility and dissolvation

Determination of solubility

Calculation of solubility

Solubility and log p

Solubility of liquids

Solubility of solids

Distribution coefficient

Partition coefficients & reversed-phase chromatography

Use of liquid chromatography

Relation log P/log D and pH

Partition coefficient and transport

Equilibrium models

Relation rate constants and log P

Bilinear model

Molecular Properties (Structure-Stability Aspects)

General stability features

Stability in solution

Matrix stability

Storage and stability

Degradation and stabilization

Hydrolysis

Minimizing hydrolysis

Racemization & epimerization

Oxidation

Minimizing oxidation

Photochemical reactions

Light absorption by the skin

Processes on molecular level

Photobiochemistry

Therapeutic applications

pH profiles

Calculation pH minimum

Calculations rate constants

Infuence ionic strength

Influence ionization

Bio-pharmaceutical determination of drugs

(Bio-)analytical strategy

How to start?

Appearance of solute(s)

Acid/base properties

Functional groups

Polarity

Solubility

Chemical stability

Spectral properties

Bio-pharmaceutical aspects

Sample preparation – chromatography

Sampling: Techniques & precautions

Sampling statistics

Sample size & number of samples

Sampling efficiency

Modes of sampling / analysis

Off-line sampling

On-line sampling

In-line sampling

Contact-less sampling

Sampling methods

Sampling of gases & liquids

Sampling of solids

Biological samples

Volatile compounds

Direct analysis of macromo-lecule-containing samples

Direct-sample introduction

Column-switching systems

Micellar systems

Wide-pore columns

Restricted-access materials

Protein-coated columns

Sample preparation: Introduction

Introduction

Division sample treatment

Summary procedures

Physico-chemical properties

Physico-chemical properties analytes

Physico-chemical properties matrix

Choice of sample

Choice of environmental sample

Choice of biological sample

Sample preparation: Basic techniques

Handling of liquid samples

Chemicals, reagents & vials

Equipment & procedures

Sampling: Techniques and precautions

Requirements for sampling

Preparation of biological samples

Initial sample preparation: Digestion / solubilization

Solubilization of samples

Release of analyte from biological matrix

Removal of endogenous compounds

Initial sample preparation: Techniques

Microwave irradiation

Principles

Applications

Filtration

Off-line methods

Materials

Selective materials

Influence materials on protein binding

Automation

Bio-analytical applications

Precipitation

Acid precipitation

Organic solvent precipitation

Inorganic salt precipitation

Precipitation efficiency

Precipitation of pigments / bile salts

Lyophilization

Equipment and procedures

Dissolution after lyophilization

Applications

Ultrafiltration

Comparison ultrafiltration – dialysis

Principles

Applications

Various techniques

Freezing & thawing

Homogenization

Saponification

Dilution

Dehydration

Ultrasonic techniques

In-vivo microdialysis

Sample preparation: Off-line procedures (Liquid extractions)

Introduction

Rationale off-line procedures

Extraction techniques

Liquid-liquid extractions

Soxhlet extractions

Accelerated extractions

Miniaturized extractions

Solid-liquid extractions

Supercritical-fluid extractions

Headspace extractions

Chemical modification procedures

Miscellaneous off-line techniques

Sample prepararion: Off-line procedures (Solid-phase extraction)

Principles

Non-selective systems

Sorbents

Applications

Selective systems

Sorbents

Applications

Membrane-based extractions

Miniaturized extractions

Special extraction modes

Solid-phase microextraction

Stir bar sorption extraction

Matrix solid phase dispersion

Molecular imprinted polymers

Restricted access materials

Sample preparation: Automated methods (Continuous-flow)

Rationale for automation

Continuous-flow systems

Introduction

Principles

Dispersion

Detection\

Applications

Membrane-based systems

Dialysis

Electrodialysis

Microdialysis

Liquid membranes

Ultrafiltration

Solid-phase extraction techniques

Non-selective systems

Selective systems

Sample preparation: Automated methods (Discrete systems)

Sample processor (dedicated automation)

Robotics (flexible automation)

Coupled column systems

Introduction

Principle columns switching

Column-switching systems

Coupling LC and GC

Coupling LC and CE

Coupling LC and TLC

Monitoring systems

Comparison of automated systems

Separation: Principles & applications

Liquid chromatography

Objectives

Developing new methods

Modes of chromatography

Chromatographic parameters

Principles

LC instrumentation

Method validation

Treatment of chromatographic data

Gas chromatography

Capillary electrophoresis

Various separation techniques

Detection: Principles & applications

Detection in LC

General aspects

Refractive-index detection

Absorbance detection

Amperometric detection

Fluorescence detection

Chemiluminescence detection

Phosphorescence detection

Mass-spectrometric detection

Radiochemical detection

Detectors in GC

Detection in GC

Detection in electrophoresis

Various detection techniques

Reaction-detection systems

Introduction

Rationale derivatizations

Types of derivatizations

Properties of derivatization reagents

Optimization derivatizations

Pre-column techniques and applications

Derivatization of amines

Derivatization of thiols

Derivatization of carboxylic acids

Derivatization of alcohols & phenols

Derivatization of carbonyls

Derivatization of other functionalities

Post-column techniques and applications

Principles

Reactors

Special applications

On-column techniques & applications

Solid-state derivatizations

Immobilized enzyme reactors

Immobilized enzyme reactors

Electrochemical detection

Optical detection

Molecular spectroscopy

General principles

Absorbance

Luminescence

Vibrational

Nuclear-magnetic resonance

Mass spectrometry

Hyphenated techniques

Mass spectrometry-based techniques

Sample treatment

Matrix effects

Traditional sample preparation

Recent developments

Protein labelling techniques

Isotope coded affinity tag in proteomics

Coupling of ICAT to MS

ICAT in practice

Other labelling techniques

Ion mobility mass spectrometry

IMS-MS for peptides and proteins

Theory

Experimental

Immunological procedures

Introduction

Principles

Standard curve

Modes

Evaluation

Radiolabelled immunoassays

Labelled antigen assays

Labelled antibody assays

Enzyme immunoassays

Homogeneous assays

Heterogeneous assays

Avidin-biotin systems

Fluorescence immunoassays

Homogeneous assays

Heterogeneous assays

Chemiluminescence immunoassays

Direct assays

Indirect assays

Electrochemical immunoassays

Liposome immunoassays

Homogeneous assays

Heterogeneous assays

Protein-based analytical procedures

(On-line ) digestion of proteins

Principles of proteolytic digestion

Digestion methods

Multidimensional systems

Applications

Validation analytical methods

Key issues in validation

Equipment qualification

Transfer of analytical methods

Regulatory validation issues

Computerized analytical systems

Evaluation (Bio-analytical strategies)

Integration of sample treatment

Comparison of sample treatment

Decision trees

Future trends

Structure-related sample treatment

(Bio-)Analytical Chemistry: From B(egin) to E(nd)

Hubertus Irth, Henk Lingeman

BioMolecular Separations Group, Faculty of Sciences, VU University Amsterdam, Amsterdam, the Netherlands

The start
Introduction

Sample treatment (ST) is the bottleneck in most chromatographic/electrophoretic separations. As a result the primary goal of the present module on “Structure related sample treatment” techniques and approaches that can be used for this purpose.

The main ST objectives discussed in this module are:

- Learn technologies and techniques:

o How do they work,

o What are the applications,

o What are the advantages and limitations,

- Possibilities to improve laboratory operations:

o Cost (labour intensive),

o Errors (accuracy, precision),

o Time,

- Productivity issues:

o Automation,

o Parallel versus serial processing,

o On-line versus off-line,

- Help to make the transition from SOP’s to (new) techniques a successful one:

o Criteria for choosing sample treatment techniques.

This means that the problems are addressed that are related to: time, labour, automation, enrichment, selectivity, organic solvents, inorganic salts, contamination, etc.

The resulting ST goals are:

1. Removal of interferences (high and low molecular weight compounds, organic and inorganic compounds);
2. Enrichment of the analyte(s);
3. Phase transfer of the solutes(s).

The main focus will be on the development of total (bio-)analytical procedures an in particular on the ST steps needed to determine these compounds with either gas chromatography (GC), liquid chromatography (LC), ion chromatography (IC), affinity chromatography (AC) or capillary electrophoresis (CE), with the emphasis on automated/hyphenated systems including high-throughput and bio-specific assays. This includes a discussion on the theoretical backgrounds, advantages and disadvantages of pharmaceutical, bio-analytical, food, clinical environmental and process analytical methods and procedures.

Figure 1.1: Relative speed of different steps in (bio-)analytical sample treatment.

The fact that the polarity of the compounds of interest is still increasing and the fact that sampling and sample preparation (SP) are the most time consuming parts of the total analytical scheme, explains the focus on the numerous combinations of sampling and SP procdures in combination with GC / LC and CE separation-detection approaches (Figure 1.1)

The most important options for ST, sampling plus sample preparation, are:

- Sample collection: Taking a representative sample;

- Sample storage and stabilization: Using proper containers and freezing of unstable samples;

- Initial (primary) sample preparation: Reducing the sample size;

- Weighing or volumetric dilution: Taking precautions for unstable and reactive samples;

- Alternative sample processing methods: Introducing solvent replacement, desalting, evaporation procedures etc.;

- Removal of particulates: Applying filtration and centrifugation steps;

- Selective (secondary) sample preparation: Introduction of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) approaches;

- Derivatization: Enhancing detection and improving separation.

In this module various definitions will be used:

- Sample treatment: Total process of analyzing a sample including sampling, initial (primary) and selective (secondary) sample preparation, analysis, detection, quantitation and validation.

- Sampling: Process of taking a reliable and representative sample.

- Initial sample preparation: Process of storing, stabilizing and preparing a sample for selective clean-up and analysis.

- Selective sample preparation: Process of sample concentration, selective sample clean-up and/or phase transfer of the analyte(s).

- Analysis: Process of selective (qualitative or quantitative) determination of the analyte(s).

- Detection: Process of identifying or detection of the analyte(s).

- Quantitation: Process of data acquisition, data reduction and data interpretation.

- Validation: Process of guaranteeing the repeatability / reproducibility, robustness / ruggedness of the overall sample treatment process.

These steps are summarized in the overall analytical procedure as given in Figure 1.2.

Figure 1.2: Overall analytical procedure.

Another reason that sampling and SP are so important is that looking at the time spend on the analysis, about 60% is related to sample processing. In addition to this about 30% of the errors during SP/ST is due to sample processing and another 20% to the operator. This means that automation (Figure 1.3) of SP/ST techniques certainly will improve the overall quality of the analytical procedure.

Figure 1.3: Lab-automation in combinatorial chemistry (www.pa.bosch.com).

Analytical chemistry plays an important role in separating, isolating and quantifying various chemical compounds. Virtually every item of commerce has been subjected to analytical testing (Figure 1.4) at one or more stages in its manufacturing process.

Figure 1.4: Analytical testing can be performed using numerous techniques and procedures (www.pennpharm.co.uk).

Apart from the classical methods, such as titrimetric and gravimetric techniques, many instrumental techniques have been developed, for the determination of not only the active ingredient, but also the quantification of related compounds or impurities associated with it (Figure 1.5).

Figure 1.5: Traditional techniques like titrimetric and gravimetric analysis are, nowadays, replaced by modern separation techniques.

The recently developed analytical methods have the advantage of not only using small amounts of sample, reagents and less time, but also produce accurate results. These analytical techniques may be:

- physico-chemical methods,

- electro-analytical methods

- separation-based methods.

The physico-chemical methods like spectroscopy include colorimetry and spectrophotometry covering ultra-violet and visible region or fluorimetry, nephelometry (Figure 1.6) or turbidimetry, and nuclear-magnetic resonance and mass spectrometry. The electro-analytical methods cover potentiometry, amperometry, voltammetry, electrophoresis and polarography.

Figure 1.6: Nephelometry is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (www.lib.mcg.edu).

The separation-based methods involve (high-performance) liquid chromatography (LC), (high-performance) thin-layer chromatography (HPTLC / TLC), capillary electrochromatography (CEC), supercritical-fluid chromatography (SFC) and gas chromatography (GC). The combination of GC and LC with mass spectrometry (MS), nowadays, are the most powerful tools employed for the quantification and identification of analytes in pure as well as in associated forms. In addition to chromatographic separation methods, electrophoretic techniques, like capillary electrophoresis (CE), isotachophoresis (ITP) (Figure 1.7) gel electrophoresis (GE) and isoelectric focusing (IEF), are relatively popular for the separation and quantitation of (charged) organic compounds.

Figure 1.7: Isotachophoresis is one of the electrophoretic techniques used for the separation of charged molecules (www.bd.com).

The still increasing interest on the determination of drugs has forced the analytical chemists to develop methods for their trace analysis in the presence of biological matrices. Since every year a large number of drug candidates are synthesized it means that also a large number of methods/procedures must be developed that are applicable for the routine analysis of these drug candidates. These methods should be rapid, precise, accurate and cost effective. Although costly sophisticated instruments like LC, HPTLC, GC, GC-MS and LC-MS are available, the spectrophotometer is being preferred by ordinary laboratories for its simple, economical and easy handling techniques (Table 1.1).

Table 0.1: Bio-analytical techniques.

In summary: The philosophy and concepts of sampling, sample preparation in analytical chemistry will be presented in a practical way to provide the analytical chemist with the necessary tools to determine low- and high-molecular-weights compounds in a variety of matrices using chromatographic / electrophoretic, spectroscopic or immunological methods.

In addition, the philosophy and concepts for the method development procedures will be based on the physico-chemical properties of the analyte(s) and the matrices in which these compounds are present (Figure 1.8).

Figure 1.7: Physico-chemical properties.
Physico-chemical properties

(& Structure-activity relationships of drugs)

In order to develop analytical procedures for low-molecular weight (LMW) compounds in pharmaceutical formulations and biological samples the starting point always must the physico-chemical properties of both the compounds of interest and the sample matrix. Therefore, the focus will be on these physico-chemical properties and the structure-activity relationships between the solutes and matrix components such as the interaction between drugs and biological matrix components (e.g., proteins, enzymes, receptors) or pesticides and humic substances.

Pharmaceutical chemistry Bio-analysis Pharmacochemistry

The intention is not to discus all the basic principles of analytical chemistry, physical chemistry, organic chemistry, thermodynamics and kinetics in detail, but just to provide the information necessary to develop new or to modify existing analytical methods that can be used in pharmaceutical chemistry, bio-analysis and pharmacochemistry.

For example, during the development of new drug entities (NCE) it is of utmost importance to optimize the structure of a new compound (drug candidate) for a certain target. This can be done by changing the 3-dimensional (3-D) structure, or in other words the way the functional groups are arranged around the basic molecule. In addition the physico-chemical and bio-pharmaceutical properties of a compound are determined by its structure (Figure 1.8).

Figure 1.8: Fit of NCE in receptor pocket (www.tripos.com).

In addition the physico-chemical and bio-pharmaceutical properties of a compound are determined by its structure. These properties, on its turn, are related to processes like solubility, absorption, protein binding, partitioning, elimination and metabolism (Figure 1.9).

Figure 1.9: Summary of metabolism process (www.elmhurst.edu).

These properties, on its turn, are related to processes like solubility, absorption, protein binding, partitioning, elimination and metabolism. In (bio)-analytical chemistry (BAC) these properties determine which analytical techniques can be used and what the actual conditions must be. It will be obvious that there is a strong correlation between the various physico-chemical properties (Figure 1.10).

Figure 1.10: Relation between physico-chemical properties.

In the case of drugs it can be stated that when the molecular weight is over 500, the number of H-bridge donors is over 5, the number of H-bridge acceptors is over 10 and the calculated partitioning coefficient is over 5, the compound will badly orally adsorbed (Figure 1.11). This gain shows the importance of studying the physico-chemical properties of organic compounds.

Figure 1.11: Solubility of chloramphenicol is determined by the presence of hydrophilic and lipophilic groups (www.auburn.edu).

From the example mentioned above it will be clear that the emphasis will be on the relation between the chemical structure, or in other words the physico-chemical properties of a compound, and the determination of these compounds in a (complex) sample matrix. The final objective is that the information provided can be used to develop simple qualitative and quantitative methods for organic compounds in a variety of matrices. In order to obtain this goal the following parameters will be discussed:

- Physico-chemical properties of organic molecules like polarity, acid-base properties, solubility, stability, absorption/adsorption, etc;

- Composition of sample matrices and their effect on the analytical results (e.g., analyte-matrix binding, stability);

- ST/SP procedures (e.g., filtration, centrifugation, extraction).

There are two magic words that will be discussed throughout the whole monograph: pH and pKa (Figure 1.12).

Figure 1.12: Questions to be answered!

After studying the module on “Structure-related sample treatment” it should be possible to answer the following starting questions (Figure 1.12) and the final question (Figure 1.13):

Figure 1.13: Final analytical question to be answered!

With respect to environmental, pharmaceutical and bio-analysis this means that the following topics features are of importance:

- Insight in structures of chemical compounds and physico-chemical properties like pK_a, polarity, stability, solubility, chromophores, etc;

- Composition of biological materials and, to lesser extent, pharmaceutical formulations and environmental samples, and their influence on the analytical results (e.g. stability, matrix binding);

- Initial (primary) sample treatment procedures (e.g., filtration, precipitation, extraction);

- Selective (secondary) sample preparation procedures (e.g. solid-phase extraction, immunological approaches, hyphenated and automated system

In order to do deal with these features a number of sub-questions should be answered. These sub-questions are related to the following physico-chemical properties of the analyte(s) or the matrix molecules:

- Name, molecular weight, structure;

- Appearance, colour, smell;

- Acid-base properties;

- Functional groups;

- Polarity (partitioning coefficients);

- Solubility;

- (Chemical) stability;

- Spectral properties;

- Bio-pharmaceutical properties;

- Sample treatment / sample preparation;

- Separation / detection;

- Quantitative aspects (validation).

Except for the relatively straightforward analytical applications in most cases an overall sample analysis scheme should be constructed (Figure 1.14). This because direct sample injection into a chromatographic/electrophoretic system, normally is not possible because unwanted matrix components can disturb the separation and/or detection or can clog the analytical device. In other cases the concentration of the analyte(s) or the degradation products or metabolites may be too low to allow a direct injection procedure. The result is that ST/SP is an important aspect of the total analytical procedure and that in order to provide an adequate ST/SP procedure accurate knowledge about the properties of the compounds to be separated and the sample matrix is a prerequisite.

Figure 1.14: Sample analysis scheme.

Bio-analytical chemistry

Bio-analytical chemistry (BAC) involves a number of different disciplines such as therapeutic drug monitoring (TDM), toxicology (clinical, forensic, post-mortem), drugs-of abuse, pharmaceutical analysis, food analysis and environmental analysis.

One of the most important disciplines is therapeutic drug monitoring (TDM) (Figure 1.15). TDM can be defined as:

‘The measurement and the clinical use of blood (serum/plasma) drug levels (concentrations) to adjust each patient’s individual drug dosage and schedule to each patient’s individual therapeutic requirement’.

Figure 1.15: Therapeutic drug monitoring in epilepsy (www.e-epilepsy.org.uk).

In order to perform TDM, quantitative methods for the determination of these drugs, their metabolites and their degradation products should be available.

A number of variables complicate bio-analytical procedures. For example, after a drug has been administered to an individual, it is metabolized into products that can be excreted more easily. On its way through the body multiple metabolites are formed meaning that depending on the bioactivity and relative concentrations, the parent drug and/or the metabolites should be determined.

Biomonitoring outshines the indirect assessment of exposure in determining which pollutants enter the body, and whether they cause disease or disability (Figure 1.16). Non-persistent toxicants move quickly out of the blood as they are metabolized to water-soluble compounds that can be extracted in urine. Some chemicals or metabolites may bind to proteins or to DNA and persist in the body for a longer period of time.

Figure 1.16: Biomonitoring of pollutants (www.nature.com).

Postmortem toxicology, for example, is used to determine if alcohol, drugs or other poisons have contributed to the death of a person (Figure 1.17). Fundamentally it is different from TDM and clinical toxicology. The reason is that it is far more difficult to interpret post-mortem results. Clinical toxicology is dealing with patients with suspected poisoning and the primary objective is to assist in the treatment of such a patient.

Figure 1.17: Postmortem toxicology (www.answers.com).

Drugs-of-Abuse can be defined as: ‘Any substance that, because of some desirable effect, is used for some purpose other than that intended’ (Figure 1:18).

Figure 1.18: Mechanism of action of high concentration of amphetamine (www.cnsforum.com).

The questions in pharmaceutical analysis are again quite different. Questions that are relevant are dealing with the identity, concentration and stability of the drug in the formulation (Figure 1.19). In addition information on the impurities, shelf-life and release-rate of the drug from the formulation are essential. The connection between pharmaceutical and bio-analysis is that after administration of the drug the concentration in tissue or a biological fluid is a key factor. While during drug development parameters like acid/base properties, polarity (log P values), solubility and stability are important features.

Figure 1.19: Pharmaceutical analysis (www.chem.agilent.com).

In food analysis the properties of foods and their constituents are characterized. Analytical procedures are used in food analysis to provide information about a wide variety of characteristics such as composition (e.g., lipids, proteins, water, carbohydrates, and minerals), structure, physico-chemical properties and sensory attributes. The data obtained are used to produce on an economical, safe and nutritious way.

Environmental system analysis is an important tool to analyze sustainability, e.g. environmental impact, optimal resource management and how outside factors affect farm management (Figure 1.20).

Figure 1.20: Environmental system analysis (www-mat21.slu.se).

Trace environmental quantitative analysis (TEQA) is not only dealing with the determination of organic and inorganic compounds in e.g., air, ground, water, plant effluent, leachate, soil, sediment), by also with the determination of these compounds in body fluids and tissues because the intake of environmental pollutants by humans, and the potential of biohazards is a key issue in environmental chemistry. This means that bioterrorism and biomonitoring are important issues in this respect.

Environmental monitoring is completely different from TDM (Figure 1.21).

Figure 1.21: Environmental monitoring (B.J. Alloway, D.C. Ayres, Chemical Principles of Environmental Pollution, Blackie Academic & Professional, London 1997).

Environmental analysis can be:

- Analytes entering the environment, amounts involved, original sources and spatial distribution;

- Effects of analytes on humans, crops, livestock and eco-systems;

- Trends in concentration of analytes over time and the reason for this;

- Extent inputs, concentrations, effects and trends can be modified;

- Establish baseline concentrations for comparison;

- Assess need for legislative controls;

- Activate emergency procedures;

- Determine suitability of resources for proposed uses.

In addition to in TDM BAC is important in Pharmacochemistry. The strategy in pharmcochemistry is to find and to document structure-activity relationships (SAR), with the goal to develop new drug candidates (Figure 1.22).

Figure 1.22: Structure-activity relationships of taxol (www.ch.ic.ac.uk).

Using the term activity means for example:

- Dynamic interactions with biological targets like receptors and enzymes;

- Kinetic parameters like membrane penetration and metabolic conversion;

- Toxicological properties like mutagenesis, cell and organ toxicity.

Quantitative-structure activity relationships (QSAR) play an important role in drug development (Figure 1.23).

Figure 1.23: Quantitative SAR plays an important role during drug development (www.goldenhelix.com).

` Pharmacochemistry can be divided a number of (sub) disciplines like:

- (Bio)organic chemistry (synthesis and combinatorial chemistry) (Figure 1.23);

- Physical chemistry and structural chemistry (SAR, computational chemistry);

- (Bio-)analytical chemistry;

- Molecular and structural biology (including bio-informatics);

- Pharmacology (including bio-transformation and partitioning);

- Molecular toxicology.

Figure 1.23: Electron density in the amino acid cystein calculated using a quantum-chemistry computer program (Computational Chemistry). The picture shows the surface where the electron density is 0.002 electrons/Å3 (meaning that nearly all electrons are inside the surface). The grey scale shows the electrostatic potential at this surface, darker portions representing negative potential (http://nobelprize.org).

The popularity of LC can be explained by the fact that in bio-analytical research analyte stability, metabolism (biotransformation), distribution as well as excretion are important and that the strength of LC is that qualitative as well as accurate quantitative information can be obtained of the parent compounds as well as of polar and extremely polar metabolites. LC also has some disadvantages: relatively time-consuming sample preparation techniques are normally needed and relatively large sample volumes (0.5 -1.0 mL) are normally required.

In contrast, immunological techniques require no sample preparation and only 50 -100-mL volumes are needed, but these techniques lack the selectivity of LC methods (Figure 1.24).

Figure 1.24: Immunoassay formats (www.piercenet.com).

For a considerable number of analytes radio-immunoassays (RIA) are more frequently used than LC methods, but although this is the case for, for example, cyclosporins, the RIA procedures overestimate the cyclosporin concentration because the polyclonal antibodies used react with cyclosporin as well as its metabolites (Figure 1.25).

Figure 1.25: Format of radio-immunoassay (http://users.rcn.com).

However, although cross-reaction is one of the major limitations of immunoassays (IA), the production of highly specific monoclonal antibodies can overcome this problem which means that especially enzyme-linked immunosorbent assay (ELISA) (Figure 1.26) will be an important bio-analytical tool next to LC.

Figure 1.26: Principle of enzyme-linked immunosorbent assay (www.biosystemdevelopment.com).

Since both techniques, LC and IA, have their own advantages and limitations, the combination of these two techniques, the so-called bio-specific detection, seems to be rather promising.

There are many reasons why an analytical procedure should be performed. There are an increasing number of substances that, for legal reasons must be monitored to ensure the safety of the individual as their presence may be detrimental to both humans and animals. There is an increased consumer awareness concerning the quality and safety of manufactured products and this makes manufacturer’s test their products as quality ensures that financial losses from law suits are reduced. The increased or decreased concentrations of the natural constituents of the body can be used to diagnose diseases and to monitor its treatment.

An analytical procedure is a means to an end; it provides information which can concern the composition of a sample or the status of a chemical process. This analytical information is then used as a basis for a decision: is a sample within specifications, is this water sample safe to drink or is the process under control?

The analytical laboratory is often on the critical path in many organizations as the assay results are needed before a decision can be taken. Therefore, the analytical laboratory is under increasing pressure to improve its efficiency and improve turnaround times while maintaining the quality and consistency of the data reported. Analytical data and information must not be confined within the laboratory, it is a resource that should be used and disseminated throughout the laboratory’s organization.

Contents

Structure-related sample treatment

The start

Introduction

Physico-chemical properties

Bio-analytical chemistry

Contents

Introduction

Summary

Introduction

Rationale sample treatment

Introduction

Choice sample treatment

Categorization techniques

Sensitivity & selectivity requirements

Quality & validation

Introduction

Quality & validation

Validation

Method validation (Why, how, when)

Metabolism & stability

Introduction

Metabolism

Degradation

Stability in solution

Stability in matrix

Method development

Introduction

Separation-detection systems

Liquid chromatography

Separation modes

Detection

Gas chromatography

Introduction

Stationary phase

Detection

Gas chromatography – mass spectrometry

Capillary electrophoresis

Introduction

System theoretical background

Conclusions

Sample treatment/preparation

Sample treatment

Sample preparation

Conclusions

Problems & questions

Molecular Properties (Non-covalent interactions)

Organic structures

Hetero atoms

Stereochemistry

Reaction kinetics

Reaction rate

Reaction constants

Non-covalent interactions

Electrostatic interactions

Dipole interactions

Hydrophobic interactions

Solubility parameters

Drug-protein interactions

Protein binding

Disruption of protein binding

Receptor-ligand binding

Chromatographic partitioning

Partitioning isotherms

Secondary chemical equilibria

Molecular Properties (Molecular effects)

Introduction

Acid, bases, buffers

Dissociation & pH

Ionization & Activity

Electronic effects

Electronegativity

Inductive & resonance effects

Hammett equation

Taft equation

Ionization constants

Microscopic constants

Macroscopic constants

Molecular Properties (Partition Related Processes)

Calculations of log P

Hansch method

Substituent additivity

Limitation of substituent additivity

Application of p-values

Rekker method

Relation p- and ¦-values

Collander equation

Correlation log P and protein binding

Solubility and dissolvation

Determination of solubility

Calculation of solubility

Solubility and log p

Solubility of liquids

Solubility of solids

Distribution coefficient

Partition coefficients & reversed-phase chromatography

Use of liquid chromatography

Relation log P/log D and pH

Partition coefficient and transport

Equilibrium models

Relation rate constants and log P

Bilinear model

Molecular Properties (Structure-Stability Aspects)

General stability features

Stability in solution

Matrix stability

Storage and stability

Degradation and stabilization

Hydrolysis

Minimizing hydrolysis

Racemization & epimerization

Oxidation

Minimizing oxidation

Photochemical reactions

Light absorption by the skin

Processes on molecular level

Photobiochemistry

Therapeutic applications

pH profiles

Calculation pH minimum

Calculations rate constants

Infuence ionic strength

Influence ionization

Bio-pharmaceutical determination of drugs

(Bio-)analytical strategy

How to start?

Appearance of solute(s)

Acid/base properties

Functional groups

Polarity

Solubility

Chemical stability

Spectral properties

Bio-pharmaceutical aspects

Sample preparation – chromatography

Sampling: Techniques & precautions

Sampling statistics

Sample size & number of samples

Sampling efficiency

Modes of sampling / analysis

Off-line sampling

On-line sampling

In-line sampling

Contact-less sampling

Sampling methods

Sampling of gases & liquids

Sampling of solids

Biological samples

Volatile compounds

Direct analysis of macromo-lecule-containing samples

Direct-sample introduction

Column-switching systems

Micellar systems

Wide-pore columns

Restricted-access materials

Protein-coated columns

Sample preparation: Introduction

Introduction

Division sample treatment

Summary procedures

Physico-chemical properties

Physico-chemical properties analytes

Physico-chemical properties matrix

Choice of sample

Choice of environmental sample

Choice of biological sample

Sample preparation: Basic techniques

Handling of liquid samples

Chemicals, reagents & vials

Equipment & procedures

Sampling: Techniques and precautions

Requirements for sampling

Preparation of biological samples

Initial sample preparation: Digestion / solubilization

Solubilization of samples

Release of analyte from biological matrix

Removal of endogenous compounds

Initial sample preparation: Techniques

Microwave irradiation

Principles

Applications

Filtration

Off-line methods

Materials

Selective materials

Influence materials on protein binding

Automation

Bio-analytical applications

Precipitation

Acid precipitation

Organic solvent precipitation

Inorganic salt precipitation

Precipitation efficiency

Precipitation of pigments / bile salts

Lyophilization

Equipment and procedures

Dissolution after lyophilization

Applications

Ultrafiltration

Comparison ultrafiltration – dialysis

Principles

Applications

Various techniques

Freezing & thawing

Homogenization

Saponification

Dilution

Dehydration

Ultrasonic techniques

In-vivo microdialysis

Sample preparation: Off-line procedures (Liquid extractions)

Introduction

Rationale off-line procedures

Extraction techniques

Liquid-liquid extractions

Soxhlet extractions

Accelerated extractions

Miniaturized extractions

Solid-liquid extractions

Supercritical-fluid extractions

Headspace extractions

Chemical modification procedures

Miscellaneous off-line techniques

Sample prepararion: Off-line procedures (Solid-phase extraction)

Principles

Non-selective systems

Sorbents

Applications

Selective systems

Sorbents

Applications

Membrane-based extractions

Miniaturized extractions

Special extraction modes

Solid-phase microextraction

Stir bar sorption extraction

Matrix solid phase dispersion

Molecular imprinted polymers

Restricted access materials

Sample preparation: Automated methods (Continuous-flow)

Rationale for automation

Continuous-flow systems

Introduction

Principles

Dispersion

Detection\

Applications

Membrane-based systems

Dialysis

Electrodialysis

Microdialysis

Liquid membranes

Ultrafiltration

Solid-phase extraction techniques

Non-selective systems

Selective systems

Sample preparation: Automated methods (Discrete systems)

Sample processor (dedicated automation)

Robotics (flexible automation)

Coupled column systems

Introduction

Principle columns switching

Column-switching systems

Coupling LC and GC

Coupling LC and CE

Coupling LC and TLC

Monitoring systems

Comparison of automated systems

Separation: Principles & applications

Liquid chromatography

Objectives

Developing new methods

Modes of chromatography

Chromatographic parameters

Principles

LC instrumentation

Method validation

Treatment of chromatographic data

Gas chromatography

Capillary electrophoresis

Various separation techniques

Detection: Principles & applications

Detection in LC

General aspects

Refractive-index detection

Absorbance detection

Amperometric detection

Fluorescence detection

Chemiluminescence detection

Phosphorescence detection

Mass-spectrometric detection

Radiochemical detection

Detectors in GC

Detection in GC

Detection in electrophoresis

Various detection techniques

Reaction-detection systems

Introduction

Rationale derivatizations

Types of derivatizations

Properties of derivatization reagents

Optimization derivatizations

Pre-column techniques and applications

Derivatization of amines

Derivatization of thiols

Derivatization of carboxylic acids

Derivatization of alcohols & phenols

Derivatization of carbonyls

Derivatization of other functionalities

Post-column techniques and applications

Principles

Reactors

Special applications

On-column techniques & applications

Solid-state derivatizations

Immobilized enzyme reactors

Immobilized enzyme reactors

Electrochemical detection

Optical detection

Molecular spectroscopy

General principles

Absorbance

Luminescence

Vibrational

Nuclear-magnetic resonance

Mass spectrometry

Hyphenated techniques

Mass spectrometry-based techniques

Sample treatment

Matrix effects

Traditional sample preparation

Recent developments

Protein labelling techniques

Isotope coded affinity tag in proteomics

Coupling of ICAT to MS

ICAT in practice

Other labelling techniques

Ion mobility mass spectrometry

IMS-MS for peptides and proteins

Theory

Experimental

Immunological procedures

Introduction

Principles

Standard curve

Modes

Evaluation

Radiolabelled immunoassays

Labelled antigen assays

Labelled antibody assays

Enzyme immunoassays

Homogeneous assays

Heterogeneous assays

Avidin-biotin systems

Fluorescence immunoassays

Homogeneous assays

Heterogeneous assays

Chemiluminescence immunoassays

Direct assays

Indirect assays

Electrochemical immunoassays

Liposome immunoassays

Homogeneous assays

Heterogeneous assays

Protein-based analytical procedures

(On-line ) digestion of proteins

Principles of proteolytic digestion

Digestion methods

Multidimensional systems

Applications

Validation analytical methods

Key issues in validation

Equipment qualification

Transfer of analytical methods

Regulatory validation issues

Computerized analytical systems

Evaluation (Bio-analytical strategies)

Integration of sample treatment

Comparison of sample treatment

Decision trees

Future trends

Introduction
Summary

The main objective of this monograph is to describe the overall analytical procedure. This means starting with the ‘Object’ and ending with the ‘Information’. These two parameters are connected with each other via the ‘Analytical Method’.

Figure 2.1: Analytical method based on liquid chromatography (www.toxics.usgs.gov).

In summary it can be stated that a (bio-)analytical procedure is used to obtain qualitative and/or quantitative information on a sample (Figure 2.2). In this “Introduction” section an overview will be given on the different stages of such a procedure emphasizing on sampling, sample preparation (SP) and separation/detection of the analytes.

Figure 2.2: Example of a flow diagram for a processing line of canned tuna fish in brine (www.fao.org).

The rationale for sample handling including the choice of the various sample preparation techniques with respect to their sensitivity and selectivity will be discussed. The influence of drug metabolism on the optimum sample treatment techniques will be overviewed. The objectives that will be highlighted are:

- New techniques and methods (i.e. principles, advantages/disadvantages, applications);

- Possibilities to improve laboratory operations (i.e. cost, accuracy, time);

- Throughput aspects (i.e. automation, parallel versus serial processing, off-line versus on-line);

- Strategies in choosing most successful method.

Finally, a short introduction will be given in chromatographic and electrophoretic techniques that are normally used in (bio-) analytical systems (Figure 2.3).

Figure 2.3: Proteomics chart (swehsc.pharmacy.arizona.edu/mass_spec.html).
Introduction

Normally a sample cannot be injected directly into a separation system. In the sample unwanted matrix components can be present that disturb the separation and/or detection of the analyte(s) or even damage the analytical system (Figure 2.4). In trace analysis, the concentration of the analyte frequently is so low that pre-concentration (trace-enrichment) should be performed to improve the detectability of the analyte(s). The result is that a number of sample handling – the combination of sample treatment / sample preparation (ST/SP) - steps should be performed to allow injection of the analyte(s) in the separation system.

Figure 2.4: Composition of milk (www.delaval-us.com).

Figure 2.5: Stage of the analytical process.

In this Figure 2.6 it can be seen that the ST/SP steps can be divided into three parts:

- Stabilizing of the sample (e.g., avoiding degradation);

- Initial (non-selective) sample preparation (e.g., removal of interfering matrix components, dissolution);

- (Selective) sample treatment (e.g., concentration, clean-up).

Figure 2.6: Division of sample treatment technique

The initial SP step is also called sample preservation or sample stabilization (Figure 2.7). This is rather important because there normally is a delay between sample collection and analysis. This means that sample preservation is needed to be sure that both the chemical and physical properties are changing during the analytical process.

Figure 2.7: Sample stability during storage at different temperatures (www.ipj.quintessenz.de).

The importance of proper ST/SP steps can be illustrated by comparing the selectivity, the degrees of freedom and the costs of the various steps of the analytical process (Table 2.1).

Table 2.1: Role of sample preparation / sample treatment in separation process.

From this Table it can be seen that especially optimizing the SP/ST steps can give an extra dimension to the analytical process.

In summary the following statements can be made:

- The isolation and determination of low concentrations of (organic) compounds is a real challenge;

- The lower the concentration of the analytes, the longer method development will take;

- The ST/SP procedure(s) always should be adapted to the final goal of the analytical procedure;

- Always the most simple SP/ST procedure should be chosen, which is in agreement with the goal of the procedure;

- Always the best compromise should be found between the selectivity of the sample ST/SP step and of the separation / detection procedure.

The first stage is