Encyclopedia of Chromatography 2004 Update Supplement

1679 Pages·2004·68.016 MB·English

by  Jack Cazes

bb-Agonist Residues in Food, Analysis by LC
Nikolaos A. Botsoglou
Aristotle University, Thessaloniki, Greece
INTRODUCTION animals for growth-promoting purposes has been pro-
hibited by regulatory agencies in Europe, Asia, and the
b-Agonistsaresyntheticallyproducedcompoundsthat,in Americas. Clenbuterol, in particular, has been banned by
addition to their regular therapeutic role in veterinary the FDA for any animal application in the United States,
medicine as bronchodilatory and tocolytic agents, can whereas it is highly likely to be banned even for
promoteliveweightgaininfood-producinganimals.They therapeutic use in the United States in the near future.
are also referred to as repartitioning agents because their However, veterinary use of some b-agonists, such as
effectoncarcasscompositionistoincreasethedeposition clenbuterol,cimaterol,andractopamine,isstilllicensedin
of protein while reducing fat accumulation. For use in several parts of the world for therapeutic purposes.
lean-meat production, doses of 5 to 15 times greater than
the recommended therapeutic dose would be required,
together with a more prolonged period of in-feed
MONITORING
administration, which is often quite near to slaughter to
obviatetheeliminationproblem.Suchusewouldresultin
Monitoring programs have shown that b-agonists have
significant residue levels in edible tissues of treated
been used illegally in parts of Europe and United States
animals, which might in turn exert adverse effects in the
by some livestock producers.[1] In addition, newly
cardiovascular and central nervous systems of the
developed analogues, often with modified structural
consumers.[1]
properties, are continuously introduced in the illegal
There are a number of well-documented cases where
practice of application of growth-promoting b-agonists
consumption of liver and meat from animals that have
in cattle raising. As a result, specific knowledge of
been illegally treated with these compounds, particularly
the target residues appropriate to surveillance is very
clenbuterol, has resulted in massive human intoxifica-
limited for many of the b-agonists that have potential
tion.[1] In Spain, a foodborne clenbuterol poisoning
black market use.[2] Hence, continuous improvement
outbreak occurred in 1989–1990, affecting 135 persons.
of detection methods is necessary to keep pace with
Consumption of liver containing clenbuterol in the range
the rapid development of these new, heretofore unknown
160–291ppbwasidentifiedasthecommonpointinthe43
b-agonists. Both gas and liquid chromatographic meth-
families affected, while symptoms were observed in 97%
ods can be used for the determination of b-agonist
of all family members who consumed liver. In 1992,
residuesinbiologicalsamples.However,LCmethodsare
another outbreak occurred in Spain, affecting this time
receiving wider acceptance because gas chromatographic
232persons.Clinicalsignsofpoisoninginmorethanhalf
methods are generally complicated by the necessity of
of the patients included muscle tremors and tachycardia,
derivatization ofthepolarhydroxylandaminofunctional
frequently accompanied by nervousness, headaches, and
groups of b-agonists. In this article, an overview of the
myalgia. Clenbuterol levels in the urine of the patients
analyticalmethodologyforthedeterminationofb-agonist
were found to range from 11 to 486 ppb. In addition, an
in food is provided.
incident of food poisoning by residues of clenbuterol in
veal liver occurred in the fall of 1990 in the cities of
Roanne and Clermont-Ferrand, France. Twenty-two
persons from eight families were affected. Apart from ANALYSIS OF bb-AGONISTS BY LC
the mentioned cases, two farmers in Ireland were also
reported to have died while preparing clenbuterol for Included in this group of drugs are certain synthetical-
feeding to livestock. ly produced phenethanolamines such as bambuterol,
Although, without exception, these incidents have all bromobuterol, carbuterol, cimaterol, clenbuterol, dobut-
been caused by the toxicity of clenbuterol, the entire amine, fenoterol, isoproterenol, mabuterol, mapenterol,
group of b-agonists are now treated with great suspicion metaproterenol, pirbuterol, ractopamine, reproterol, rimi-
byregulatoryauthorities,anduseofallb-agonistsinfarm terol, ritodrine, salbutamol, salmeterol, terbutaline, and
EncyclopediaofChromatography 1
DOI:10.1081/E-ECHR120028860
CopyrightD2004byMarcelDekker,Inc.Allrightsreserved.
ORDER REPRINTS
2 b-AgonistResiduesinFood,AnalysisbyLC
tulobuterol. These drugs fall into two major categories, ether/n-butanol as extraction solvents.[5,7,8] The organic
i.e., substituted anilines, including clenbuterol, and extracts are then either concentrated to dryness, or repar-
substituted phenols, including salbutamol. This distinc- titionedwithdiluteacidtofacilitatebackextractionofthe
tion is important because most methods for drugs in the analytesintotheacidicsolution.Aliteraturesurveyshows
former category depend on pH adjustment to partition that liquid–liquid partitioning cleanup resulted in good
the analytes between organic and aqueous phases. The recoveries of substituted anilines such as clenbuterol,[7,8]
pH dependence is not valid, however, for drugs within but it was less effective for more polar compounds such
the latter category, because phenolic compounds are as salbutamol.[5] Diphasic dialysis can also be used for
charged under all practical pH conditions. purificationoftheprimarysampleextract.Thisprocedure
was only applied in the determination of clenbuterol re-
sidues in liver using tert-butylmethyl ether as the ex-
traction solvent.[6]
EXTRACTION PROCEDURES
b-Agonists are relatively polar compounds that are
soluble in methanol and ethanol, slightly soluble in SOLID-PHASE EXTRACTION
chloroform, and almost insoluble in benzene. When
analyzing liquid samples for residues of b-agonists, Solid-phase extraction is, generally, better suited to the
deconjugation of bound residues, using 2-glucuronidase/ multiresidue analysis of b-agonists. This procedure has
sulfatase enzyme hydrolysis prior to sample extraction, become the method of choice for the determination of
is often recommended.[3,4] Semisolid samples, such as b-agonists in biological matrices because it is not labor
liver and muscle, require usually more intensive sample and material intensive. It is particularly advantageous
pretreatment for tissue breakup. The most popular ap- because it allows better extraction of the more hy-
proach is sample homogenization in dilute acids such drophilic b-agonists, including salbutamol. b-Agonists
as hydrochloric or perchloric acid or aqueous buffer.[3–6] are better suited to reversed-phase solid-phase extraction
In general, dilute acids allow high extraction yields due,inpart,totheirrelativelynon-polaraliphaticmoiety,
for all categories of b-agonists, because the aromatic which can interact with the hydrophobic octadecyl- and
moiety of these analytes is uncharged under acidic con- octyl-based sorbents of the cartridge.[9–11] By adjusting
ditions, whereas their aliphatic amino group is positively the pH of the sample extracts at values greater than 10,
ionized. Following centrifugation of the extract, the optimum retention of the analytes can be achieved.
supernatant may be further treated with b-glucuronidase/ Adsorption solid-phase extraction, using a neutral
sulfatase or subtilisin A to allow hydrolysis of the con- alumina sorbent, has also been recommended for
jugated residues. improved cleanup of liver homogenates.[5] Ion-exchange
solid-phase extraction is another cleanup procedure that
hasbeensuccessfullyusedinthepurificationofliverand
tissue homogenates.[12] Because multiresidue solid-phase
CLEANUP PROCEDURES extraction procedures covering b-agonists of different
types generally present analytical problems, mixed-phase
The primary sample extract is subsequently subjected to solid-phase extraction sorbents, which contained a
cleanup using several different approaches, including mixture of reversed-phase and ion-exchange material,
conventional liquid–liquid partitioning, diphasic dialysis, werealsousedtoimprovetheretentionofthemorepolar
solid-phase extraction, and immunoaffinity chromatogra- compounds. Toward this goal, several different sorbents
phy cleanup. In some instances, more than one of these were designed, and procedures that utilized both in-
procedures is applied in combination to achieve better teraction mechanisms have been described.[5,9,13]
extract purification.
IMMUNOAFFINITY CHROMATOGRAPHY
LIQUID–LIQUID PARTITION
Owing to its high specificity and sample cleanup
Liquid–liquidpartitioningcleanupisgenerallyperformed efficiency, immunoaffinity chromatography has also
at alkaline conditions using ethyl acetate, ethyl acetate/ received widespread acceptance for the determination of
tert-butanol mixture, diethyl ether, or tert-butylmethyl b-agonists in biological matrices.[3,4,12,14] The potential
ORDER REPRINTS
b-AgonistResiduesinFood,AnalysisbyLC 3
of online immunoaffinity extraction for the multiresidue CONCLUSION
determination of b-agonists in bovine urine was recently
demonstrated, using an automated column switching This literature overview shows that a wide range of
system.[14] efficient extraction, cleanup, separation, and detection
proceduresisavailableforthedeterminationofb-agonists
in food. However, continuous improvement of detection
methods is necessary to keep pace with the ongoing
SEPARATION PROCEDURES introduction ofnew unknown b-agonists that have poten-
tial black market use, in the illegal practice.
Following extraction and cleanup, b-agonist residues are
analyzedbyliquidchromatography.Gaschromatographic
separation of b-agonists is generally complicated by the
REFERENCES
necessity of derivatization of their polar hydroxyl and
amino functional groups. LC reversed-phase columns are
1. Botsoglou, N.A.; Fletouris, D.J. Drug Residues in Food.
commonlyusedfortheseparationofthevariousb-agonist
Pharmacology,FoodSafety,andAnalysis;MarcelDekker:
residuesduetotheirhydrophobicinteractionwiththeC
18 New York, 2001.
sorbent. Efficient reversed-phase ion-pair separation of
2. Kuiper, H.A.; Noordam, M.Y.; Van Dooren-Flipsen,
b-agonists has also been reported, using sodium dodecyl M.M.H.; Schilt, R.; Roos, A.H. Illegal use of beta-
sulfate as the pairing counterion.[15] adrenergic agonists—European Community.J. Anim.Sci.
1998, 76, 195–207.
3. Van Ginkel, L.A.; Stephany, R.W.; Van Rossum, H.J.
Development and validation of a multiresidue method for
DETECTION PROCEDURES beta-agonists in biological samples and animal feed.
J.AOAC Int. 1992, 75,554–560.
4. Visser, T.; Vredenbregt, M.J.; De Jong, A.P.J.M.; Van
Following LC separation, detection is often performed in
Ginkel, L.A.; Van Rossum, H.J.; Stephany, R.W. Cryo-
the ultraviolet region at wavelengths of 245 or 260 nm.
trapping gas-chromatography Fourier-transform infrared
However, poor sensitivity and interference from coex-
spectrometry—Anewtechniquetoconfirmthepresenceof
tractives may appear at these low detection wavelengths
beta-agonists in animal material. Anal. Chim. Acta 1993,
unless sample extracts are extensively cleaned up and 275, 205–214.
concentrated. This problem may be overcome by post- 5. Leyssens, L.; Driessen, C.; Jacobs, A.; Czech, J.; Raus, J.
column derivatization of the aromatic amino group of Determination of beta-2-receptor agonists in bovine urine
the b-agonist molecules to the corresponding diazo dyes and liver by gas-chromatography tandem mass-spectrom-
through a Bratton-Marshall reaction, and subsequent de- etry. J.Chromatogr.1991, 564,515–527.
tection at 494 nm.[15] Although spectrophotometric de- 6. Gonzalez, P.; Fente, C.A.; Franco, C.; Vazquez, B.;
Quinto, E.; Cepeda, A. Determination of residues of the
tection is generally acceptable, electrochemical detection
beta-agonistclenbuterolinliverofmedicatedfarm-animals
appears more appropriate for the analysis of b-agonists
by gas-chromatography mass-spectrometry using diphasic
duetothepresenceonthearomaticpartoftheirmolecule
dialysis as an extraction procedure. J. Chromatogr. 1997,
of oxidizable hydroxyl and amino groups. This method
693,321–326.
of detection has been applied in the determination of
7. Wilson, R.T.; Groneck, J.M.; Holland, K.P.; Henry, A.C.
clenbuterolresiduesinbovineretinaltissuewithsufficient Determination of clenbuterol in cattle, sheep, and swine
sensitivity for this tissue.[8] tissues by electron ionization gas-chromatography mass-
spectrometry. J.AOAC Int. 1994, 77,917–924.
8. Lin, L.A.; Tomlinson, J.A.; Satzger, R.D. Detection of
clenbuterol in bovine retinal tissue by high performance
CONFIRMATION PROCEDURES liquid-chromatography with electrochemical detection.
J.Chromatogr. 1997,762,275–280.
9. Elliott, C.T.; Thompson, C.S.; Arts, C.J.M.; Crooks,
Confirmatoryanalysisofsuspectedliquidchromatograph-
S.R.H.; Van Baak, M.J.; Verheij, E.R.; Baxter, G.A.
ic peaks can be accomplished by coupling liquid chro-
Screening and confirmatory determination of ractopamine
matography with mass spectrometry. Ion spray LC-MS-
residuesincalvestreatedwithgrowth-promoting dosesof
MS has been used to monitor five b-agonists in bovine
the beta-agonist. Analyst1998, 123,1103–1107.
urine,[14] whereas atmospheric-pressure chemical ioniza- 10. Van Rhijn, J.A.; Heskamp, H.H.; Essers, M.L.; Van de
tion LC-MS-MS has been used for the identification of Wetering,H.J.;Kleijnen,H.C.H.;Roos,A.H.Possibilities
ractopamine residues in bovine urine.[9] for confirmatory analysis of some beta-agonists using 2
ORDER REPRINTS
4 b-AgonistResiduesinFood,AnalysisbyLC
differentderivativessimultaneously.J.Chromatogr.1995, ylboronicandbutylboronicderivatizationforconfirmatory
665, 395–398. analysis by gas-chromatography mass-spectrometry. J.
11. Gaillard, Y.; Balland, A.; Doucet, F.;Pepin, G. Detection Chromatogr.1998, 716, 366–370.
of illegal clenbuterol use in calves using hair analysis. 14. Cai,J.;Henion,J.Quantitativemulti-residuedetermination
J.Chromatogr.1997,703, 85–95. of beta-agonists in bovine urine using online immunoaf-
12. Lawrence, J.F.; Menard, C. Determination of clenbuterol finity extraction coupled-column packed capillary liquid-
in beef-liver and muscle-tissue using immunoaffinity chromatography tandem mass-spectrometry. J. Chroma-
chromatographic cleanup and liquid-chromatography with togr.1997,691, 357–370.
ultraviolet absorbency detection. J. Chromatogr. 1997, 15. Courtheyn,D.;Desaever,C.;Verhe,R.High-performance
696, 291–297. liquid-chromatographic determination of clenbuterol and
13. Ramos, F.; Santos, C.; Silva, A.; Da Silveira, M.I.N. cimaterolusingpostcolumnderivatization.J.Chromatogr.
Beta(2)-adrenergic agonist residues—Simultaneous meth- 1991, 564,537–549.
Absorbance Detection in Capillary Electrophoresis
Robert Weinberger
CE Technologies, Inc., Chappaqua, New York, U.S.A.
Introduction The CLOD can be calculated using Beer’s Law:
A 5(cid:2)10(cid:3)5
Most forms of detection in High-Performance Capil- CLOD(cid:4) (cid:4) (cid:4)2(cid:2)10(cid:3)6M (1)
lary Electrophoresis (HPCE)employon-capillaryde- ab 15000215(cid:2)102(cid:3)3
tection. Exceptions are techniques that use a sheath where Ais the absorbance (AU), ais the molar absorptiv-
flow such as laser-induced fluorescence [1] and elec- ity (AU/cm/M), bis the capillary diameter or optical path
trospray ionization mass spectrometry [2]. length (cm), and CLOD is the concentration (M). The
In high-performance liquid chromatography noise of a good detector is typically 5(cid:2)10(cid:3)5 AU.A mod-
(HPLC), postcolumn detection is generally used. This est chromophore has a molar absorptivity of 5000. Then
means that all solutes are traveling at the same velocity in a 50-(cid:2)m-inner diameter (i.d.) capillary, a CLOD of 2 (cid:2)
when they pass through the detector flow cell. In HPCE 10(cid:3)6 Mis obtained at a signal-to-noise ratio of 1, assum-
with on-capillary detection, the velocity of the solute de- ing no other sources of band broadening.
termines the residence time in the flow cell. This means
that slowly migrating solutes spend more time in the op-
Detector Linear Dynamic Range
tical path and thus accumulate more area counts [3].
Because peak areas are used for quantitative deter-
The noise level of the best detectors is about 5 (cid:2)10(cid:3)5
minations, the areas must be normalized when quanti-
AU. Using a 50-(cid:2)m-i.d. capillary, the maximum signal
tating without standards. Quantitation without stan-
that can be obtained while yielding reasonable peak
dards is often used when determining impurity profiles
shape is 5 (cid:2) 10(cid:3)1 AU. This provides a linear dynamic
in pharmaceuticals,chiralimpurities,andcertainDNA
range of about 104. This can be improved somewhat
applications. The correction is made by normalizing
through the use of an extended path-length flow cell.
(dividing) the raw peak area by the migration time.
In any event, if the background absorbance of the elec-
When a matching standard is used, it is unnecessary to
trolyte is high, the noise of the system will increase re-
perform this correction. Ifthemigrationtimesarenot
gardless of the flow cell utilized.
reproducible,thecorrection may help, but it is better
to correct the situation causing this problem.
Classes of Absorbance Detectors
Limits of Detection
Ultraviolet/visible absorption detection is the most
common technique found in HPCE. Several types of
The limit of detection (LOD) of a system can be
absorption detectors are available on commercial in-
defined in two ways: the concentration limit of detec-
strumentation, including the following:
tion (CLOD) and the mass limit of detection
(MLOD). The CLOD of a typical peptide is about 1. Fixed-wavelength detector using mercury, zinc,
1 (cid:2)g/mL using absorbance detection at 200 nm. If or cadmium lamps with wavelength selection
10 nL are injected, this translates to an MLOD of by filters
10pg at three times the baseline noise. The MLOD il- 2. Variable-wavelength detector using a deu-
lustrates the measuring capability of the instrument. terium or tungsten lamp with wavelength selec-
The more important parameter is the CLOD, which tion by a monochromator
relates to the sample itself. The CLOD for HPCE is 3. Filter photometer using a deuterium lamp with
relatively poor, whereas the MLOD is quite good, es- wavelength selection by filters
pecially when compared to HPLC. In HPLC, the in- 4. Scanning ultraviolet (UV) detector
jection size can be 1000 times greater compared to 5. Photodiode array detector
HPCE.
Encyclopedia of Chromatography 1
DOI: 10.1081/E-Echr 120004560
Copyright © 2002 by Marcel Dekker, Inc. All rights reserved.
2 Absorbance Detection in Capillary Electrophoresis
Each of these absorption detectors have certain at- Indirect Absorbance Detection
tributesthatareusefulinHPCE.Multiwavelengthde-
tectors such as the photodiode array or scanning UV To determine ions that do not absorb in the UV, indi-
detector are valuable because spectral as well as elec- rect detection is often utilized [6]. In this technique, a
trophoretic information can be displayed. The filter UV-absorbing reagent of the same charge (a co-ion) as
photometer is invaluable for low-UV detection. The the solutes is added to the BGE. The reagent elevates
use of the 185-nm mercury line becomes practical in the baseline, and when nonabsorbing solute ions are
HPCE with phosphate buffers because the short opti- present, they displace the additive. As the separated
cal path length minimizes the background absorption. ions migrate past the detector window, they are meas-
Photoacoustic, thermo-optical, or photothermal de- ured as negative peaks relative to the high baseline.
tectors have been reported in the literature [4]. These For anions, additives such as trimellitic acid, phthalic
detectors measure the nonradiative return of the ex- acid, or chromate ions are used at 2–10 mM concen-
cited molecule to the ground state. Although these can trations. For cations, creatinine, imidazole, or cop-
be quite sensitive, it is unlikely that they will be used in per(II) are often used. Other buffer materials are ei-
commercial instrumentation. ther not used or added in only small amounts to avoid
interfering with the detection process.
Optimization of Detector Wavelength It is best to match the mobility of the reagent to the
average mobilities of the solutes to minimize
Because of the short optical path length defined by the electrodispersion, which causes band broadening [7].
capillary, the optimal detection wavelength is fre- When anions are determined, a cationic surfactant is
quently much lower into the UV compared to HPLC. added to the BGE toslow or even reverse the electro-
In HPCE with a variable-wavelength absorption de- osmotic flow (EOF). When the EOF is reversed, both
tector, the optimal signal-to-noise (S/N) ratio for pep- electrophoresis and electro-osmosis move in the same
tides is found at 200 nm. To optimize the detector direction. Anion separations are performed using re-
wavelength, it is best to plot the S/N ratio at various versed polarity.
wavelengths. The optimal S/N is then easily selected. Indirect detection is used to determine simple ions
such as chloride, sulfate, sodium, and potassium. The
technique is also applicable to aliphatic amines,
Extended Path-Length Capillaries
aliphatic carboxylic acids, and simple sugars [8].
Increasing the optical path length of the capillary win-
dow should increase S/N simply as a result of Beer’s References
Law. This has been achieved using a z cell (LC Pack-
ings, San FranciscoCA)[5],bubblecell(AgilentTech- 1. Y. F. Cheng and N. J. Dovichi, SPIE, 910: 111 (1988).
nologies, Wilmington, DE), or a high-sensitivity cell 2. E. C. Huang, T. Wachs, J. J. Conboy, and J. D. Henion,
(AgilentTechnologies). Both the zcell and bubble cell Anal. Chem. 62: 713 (1990).
are integral to the capillary. The high-sensitivity cell 3. X. Huang, W. F. Coleman, and R. N. Zare, J. Chro-
comesinthreeparts:aninletcapillary, an outlet capil- matogr. 480: 95 (1989).
4. J. M. Saz and J. C. Diez-Masa, J. Liq. Chromatogr. 17:
lary, and the cell body. Careful assembly permits the
499 (1994).
use of this cell without current leakage. The bubble
5. J. P. Chervet, R. E. J. van Soest, and M. Ursem, J.
cell provides approximately a threefold improvement
Chromatogr. 543: 439 (1991).
in sensitivity using a 50-(cid:2)m capillary, whereas the zcell
6. P. Jandik, W. R. Jones, A. Weston, and P. R. Brown, LC–
or high-sensitivity cell improves things by an order of
GC 9: 634 (1991).
magnitude. This holds true only when the background 7. R. Weinberger, Am. Lab. 28: 24 (1996).
electrolyte (BGE) has low absorbance at the monitor- 8. X. Xu, W. T. Kok, and H. Poppe, J. Chromatogr. A 716:
ing wavelength. 231 (1995).
Acoustic Field-Flow Fractionation for Particle Separation
Niem Tri
Ronald Beckett
Monash University, Melbourne, Australia
Introduction ing sound wave will be pushed in the direction of sound
propagation. Therefore, sized-based separations may
Field-flow fractionation (FFF) is a suite of elution be possible if this force is applied to generate selective
methods suitable for the separation and sizing of transport of different components in a mixture. In a
macromolecules and particles [1]. It relies on the com- FFF channel, it is likely that the receiving wall will
bined effects of an applied force interacting with sam- reflect at least some of the emitted wave. If the channel
ple components and the parabolic velocity profile of thickness corresponds exactly to one-half wavelength,
carrier fluid in the channel. For this to be effective, the then a single standing wave will be created (see Fig.1).
channel is unpacked and the flow must be under lami- For a single standing wave, it is interesting to note that
nar conditions. Field or gradients that are commonly three pressure (force) nodes are generated, one at each
used in generating the applied force are gravity, cen- wall and one in the center of the channel.
trifugation, fluid flow, temperature gradient, and elec- Yasuda and Kamakura [3] and Mandralis and co-
trical and magnetic fields. Each field or gradient pro- workers [4] have demonstrated that it is possible to
duces a different subtechnique of FFF, which separates generate standing-wave fields between a transducer
samples on the basis of a particular property of the and a reflecting wall, although of much larger dimen-
molecules or particles. sions (1–20 cm) than across a FFF channel. Sound
travels at a velocity of 1500 m/s through water, which
translates to a wave of frequency of approximately 6
Research and Developments
MHz for a 120-µm thick FFF channel.
The force experienced by a particle in a stationary
The potential for using acoustic radiation forces gen-
acoustic wave was reported by Yosioka and Kawasima
erated by ultrasonic waves to extend the versatility of
[5] to be
FFF seems very promising. Although only very pre-
liminary experiments have been performed so far, the
F (cid:2) 4pr3kE A sin 2kx (2)
possibility of using such a gentle force would appear to ac ac 1 2
have huge potential in biology, medicine, and environ-
mental studies. where ris the particle radius, kis the wave number, Eac
is the time-averaged acoustic energy density, and A is
Acoustic radiation or ultrasonic waves are currently
the acoustic contrast factor given by
being exploited as a noncontact particle micromanipu-
lation technique [2]. The main drive to develop such
techniques comes from the desire to manipulate bio- A(cid:2) 1 5rp(cid:3)2rl (cid:3) gp (3)
logical cells and blood constituents in biotechnology 3a r (cid:4)2r g b
l p l
and fine powders in material engineering.
In a propagating wave, the acoustic force, Fac,acting where r and g are the particle density and compress-
p p
on a particle is a function of size given by [1] ibility, respectively, and r and g are the liquid density
l l
and compressibility, respectively. Thus, in a propagat-
F (cid:2)pr2EY (1) ing wave, the force on a particle has a second-order de-
ac p
pendence, andinastandingwave,theforceisthirdor-
where r is the particle radius, E is the sound energy der. This should give rise to increased selectivity for
density, and Ypis a complicated function depending on separations being carried out in a standing wave [6].
the characteristics of the particle which approaches Due to the nature of the acoustic fields, the distri-
unity if the wavelength used is much smaller than the bution of the particles will depend on the particle size
particle. Particles in a solution subjected to a propagat- and the compressibility and density of the particle rel-
Encyclopedia of Chromatography 1
DOI: 10.1081/E-Echr 120004561
Copyright © 2002 by Marcel Dekker, Inc. All rights reserved.
2 Acoustic FFF for Particle Separation
(a)
(b)
Fig. 1 Acoustic FFF channels suitable for particles with (a) A , 0 and (b) A . 0, utilizing a divided acoustic FFF channel.
ative to the fluid medium. Closer examination of the fected the retention time of a sphere of 3.8 µm diame-
acoustic contrast factor shows that is may be negative ter when subjected to varying acoustic fields. However,
(usually applicable to biological cells which are more the high resolution inherent in FFF has not yet been
compressible and less dense relative to the surround- exploited.
ing medium) or positive (as is in many inorganic and Naturally, with some design modifications to the
polymer colloids). Therefore, acoustic FFF (AcFFF) FFF channel, SPLITT cells could be used for sample
has tremendous potential in very clean separations of concentration or fluid clarification.
cells from other particles. One important application
may be for the separation of bacterial and algal cells in
References
soils and sediments.
If the acoustic contrast factor A,0, then a conven-
1. J. C. Giddings, J. Chem. Phys. 49: 81 (1968).
tional FFF channel will enable normal and steric mode
2. T. Kozuka, T. Tuziuti, H. Mitome, and T. Fukuda, Proc.
FFF separations to be carried out (Fig.1a). IEEE435 (1996).
However, if A.0, then the particles will migrate to- 3. K. Yasuda and T. Kamakura, Appl. Phys. Lett. 71: 1771
ward the center of the channel. In this case, a divided (1997).
FFF cell could be used as shown in Fig. 1b. This en- 4. Z. Mandralis, W. Bolek, W. Burger, E. Benes, and D. L.
sures that particles are driven to an accumulation wall Feke, Ultrasonics 32: 113 (1994).
rather than the center of the channel where the veloc- 5. K. Yosioka and Y. Kawasima, Acustica 5: 167 (1955).
6. A. Berthod and D. W. Armstrong, Anal. Chem. 59: 2410
ity profile is quite flat and selectivity would be minimal.
(1987).
Johnson and Feke [7] effectively demonstrated that
7. D. A. Johnson and D. L. Feke, Separ. Technol. 5: 251
latex spheres migrate to the nodes (center of the cell)
(1995).
and Hawkes and co-workers [8] showed that yeast cells
8. J. J. Hawkes, D. Barrow, and W. T. Coakley, Ultrasonics
migrate to the antinodes (walls of the cell). These au-
36: 925 (1998).
thors used a method similar to SPLITT, which is an- 9. J. C. Giddings, Anal. Chem. 57: 945 (1985).
other technique closely related to FFF, also originally 10. S. N. Semyonov and K. I. Maslow, J. Chromatogr. 446:
developed by Giddings [9]. Semyonov and Maslow [10] 151 (1998).
demonstrated that acoustic fields in a FFF channel af-
Additives in Biopolymers, Analysis by
Chromatographic Techniques A
Roxana A. Ruseckaite
University of Mardel Plata,Mar delPlata, Argentina
Alfonso Jime´nez
University of Alicante,Alicante, Spain
INTRODUCTION and polydroxyalcanoates (PHAs).[4] Many of these mate-
rials are commercially available, e.g., Ecostar (polyethyl-
Biopolymers are naturally occurring polymers that are ene/starch/unsaturated fatty acids), Mater Bi Z (polycap-
formed in nature during the growth cycles of all orga- rolactone/starch/natural additives) and Mater Bi Y
nisms; they are also referred to as natural polymers.[1] (polyvinylalchol-co-ethylene/starch/natural additives).
Their synthesis generally involves enzyme-catalyzed, Natural additives are mainly polyols.
chain growth polymerization reactions, typically per- The proteins, which have found many applications,
formed within cells by metabolic processes. are, for the most part, neither soluble nor fusible without
Biodegradable polymers can be processed into useful degradation.Therefore,theyareusedintheforminwhich
plastic materials and used to supplement blends of the theyarefoundinnature.[1]Gelatin,ananimalprotein,isa
synthetic and microbial polymer.[2] Among the polysac- water-soluble and biodegradable polymer that is exten-
charides, cellulose and starch have been the most sively used in industrial, pharmaceutical, and biomedical
extensively used. Cellulose represents an appreciable applications.[2]Amethodtodevelopflexiblegelatinfilms
fraction of the waste products. The main source of cel- is by adding polyglycerols. Quite recently, gelatin was
lulose is wood, but it can also be obtained from agri- blended with poly(vinyl alcohol) and sugar cane bagasse
cultural resources. Cellulose is used worldwide in the inordertoobtainfilmsthatcanundergobiodegradationin
paper industry, and as a raw material to prepare a large soil. The results demonstrated the potential use of such
variety of cellulose derivatives. Among all the cellulose films as self-fertilizing mulches.[5]
derivatives, esters and ethers are the most important, Other kinds of natural polymers, which are produced
mainly cellulose acetate, which is the most abundantly by a wide variety of bacteria as intracellular reserve ma-
producedcelluloseester.Theyareusuallyappliedasfilms terial, are receiving increasing scientific and industrial
(packaging), fibers (textile fibers, cigarette filters), and attention, for possible applications as melt processable
plastic molding compounds. Citric esters (triethyl and polymers. The members of this family of thermoplastic
acetyltriethylacetate) wererecentlyintroducedasbiode- biopolymers are the polyhydroxyalcanoates (PHAs).
gradable plasticizers in order to improve the rheological Poly-(3-hydroxy)butyrate(PHB),andpoly(3-hydroxy)bu-
response of cellulose acetate.[2] tyrate-hydroxyvalerate (PHBV) copolymers, which are
Starch is an enormous source of biomass and most microbial polyesters exhibiting useful mechanical prop-
applications are based on this natural polymer. It has a erties,presenttheadvantagesofbiodegradabilityandbio-
semicrystalline structure in which their native granules compatibilityoverotherthermoplastics.Poly(3-hydroxy)-
are either destroyed or reorganized. Water and, recently, butyrate has been blended with a variety of low- and
low-molecular-weight polyols,[2] are frequently used to high-costpolymersinordertoapplyPHB-basedblendsin
produce thermoplastic starches. Starch can be directly packaging materials or agricultural foils. Blends with
used as a biodegradable plastic for film production be- nonbiodegradablepolymers,includingpoly(vinylacetate)
cause of the increasing prices and decreasing availability (PVAc), poly(vinyl chloride) (PVC), and poly(methyl-
of conventional film-forming materials. Starch can be methacrylate) (PMMA), are reported in the literature.[4]
incorporated into plastics as thermoplastic starch or in its Poly(3-hydroxy)butyrate has been also blended with syn-
granular form. Recently, starch has been used in various thetic biodegradable polyesters, such as poly(lactic acid)
formulations based on biodegradable synthetic polymers (PLA), poly(caprolactone), and natural polymers includ-
in order to obtain totally biodegradable materials. Ther- ing cellulose and starch.[2] Plasticizers are also included
moplastic and granular starch was blended with polycap- into the formulations in order to prevent degradation
rolactone(PCL),[3]polyvinylalcoholanditscopolymers, of the polymer during processing. Polyethylene glycol,
EncyclopediaofChromatography 1
DOI:10.1081/E-ECHR120018660
CopyrightD2003byMarcelDekker,Inc.Allrightsreserved.