7 Gas Chromatography in Food Analysis - vscht.cz

7 Gas Chromatographyin Food Analysis

� 2008 by Taylor & Fra

Jana Hajslova and Tomas Cajka

CONTENTS

7.1 Introduction .......................................................................................................................... 1197.2 Sample Introduction............................................................................................................. 120

7.2.1 Split=Splitless Injection............................................................................................ 1207.2.2 Cold On-Column Injection ...................................................................................... 1227.2.3 Programmable Temperature Vaporization Injection................................................ 1237.2.4 Direct Sample Introduction=Difficult Matrix Introduction ...................................... 1257.2.5 Solid-Phase Microextraction.................................................................................... 125

7.3 Sample Separation................................................................................................................ 1267.3.1 Capillary Columns for GC....................................................................................... 1267.3.2 Fast Gas Chromatography ....................................................................................... 1267.3.3 Comprehensive Two-Dimensional Gas Chromatography....................................... 130

7.3.3.1 GC�GC Setup ........................................................................................ 1317.3.3.2 Optimization of Operation Conditions and Instrumental

Requirements in GC�GC ....................................................................... 1317.3.3.3 Advantages of GC�GC .......................................................................... 133

7.4 Sample Detection ................................................................................................................. 1347.4.1 Flame Ionization Detector ....................................................................................... 1357.4.2 Thermal Conductivity Detector ............................................................................... 1367.4.3 Electron Capture Detector ....................................................................................... 1367.4.4 Nitrogen–Phosphorus Detector ................................................................................ 1367.4.5 Flame Photometric Detector and Pulsed Flame Photometric Detector ................... 1367.4.6 Photo-Ionization Detector ........................................................................................ 1367.4.7 Electrolytic Conductivity Detector .......................................................................... 1367.4.8 Atomic-Emission Detector....................................................................................... 1367.4.9 Mass Spectrometric Detector................................................................................... 137

7.5 Matrix Effects ...................................................................................................................... 1377.6 Food Analysis Applications................................................................................................. 1407.7 Conclusion and Future Trends............................................................................................. 142Acknowledgments......................................................................................................................... 142References ..................................................................................................................................... 142

7.1 INTRODUCTION

In food analysis, gas chromatography (GC) represents one of the key separation techniques formany groups of (semi)volatile compounds. The high separation power of GC in a combination witha wide range of the detectors makes GC an important tool in the determination of variouscomponents that may occur in such complex matrices as food crops and products.

ncis Group, LLC.

Samplepreparation

Sampleintroduction

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1D-G

C2D

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Separation Detection

Data analysis

Con

vent

iona

lM

ass

spec

.

Split

Classical splitless

Pulsed splitless

Cold on-column

Programmable temperaturevaporiser

Direct sample introduction/Difficult matrix introductionSolid-phase microextraction

Conventional GC

Fast GC

Very fast GC

Flame ionisation

Thermal conductivity

Electron capture

Nitrogen–phosphorus

(Pulsed) flame photometricPhoto-ionisation

Electrolytic conductivity

Atomic-emission

Quadrupole

Quadrupole ion trap

Magnetic sector

Time-of-flightHybrid instruments

Ultra-fast GC

Heart-cut GC

Comprehensive two-dimensional GC

FIGURE 7.1 Basic steps typically involved in the determinative step of gas chromatographic analysis oforganic food compounds.

In practice, a GC-ba sed method consi sts typicall y of the follow ing steps : (1) isol ation ofanalyt es from a representat ive sample (extr action); (2) separa tion of co-extract ed mat rix componen ts(clea nup); (3) ident i fication and quanti fica tion of targe t analyt es (dete rminativ e step) , and if theneed is imp ortan t enough, this is foll owed by (4) con firmation of results by an addit ional analys is(Figur e 7.1). In any case, the sample prepar atio n pract ice plays a cruci al role for obtaining requiredparam eters of a parti cular analytical method. Under some cond itions, especi ally when polar analytesare to be analyze d, derivatiz ation is ca rried out prior to the GC step to avoid hydroge n bondin g,hence incre asing the a nalyte volatil ity and reducing interacti on wi th acti ve sit es in the syst em.

In Figure 7.2, the interrel ationshi p between solut e amoun ts and inst rumental options (inlet,colum n, and detector) is illust rated. GC users shoul d examine the relations hip of analyz ed samp lesto the operat ing range of the instrum ent syst em. If the analyt e concent ration lies outside this range, adiff erent injectio n technique, column dim ension, or detector may be appropr iate.

7.2 SAMPLE INTRODUCTION

The re are a numbe r of options avail able for GC inlet syst ems; the most common (charac terizedbelow) being spli t=splitless, progra mmed temperat ure vap orizer, and cold on-column (COC)inje ctor. The choice of an optimum samp le introduct ion strategy depend s mainly on the c oncentra-tion range of targe t analyt es, thei r p hysico-chem ical proper ties, and the amount and natur e of mat rixco-ext racts presen t in the samp le.

7.2.1 S PLIT=SPLITLESS INJECTION

Split=splitl ess inje ction remains the main samp le introduct ion techniq ue in the analysis ofGC-am enable food compo nents mai nly due to its easy operat ions.

� 2008 by Taylor & Francis Group, LLC.

Femtogramsparts per trillion

Mas

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1 10 100 1 10 100 1 10 100 1 10 100 1000

Picogramsparts per billion

Nanogramsparts per million

Microgramsparts per thousand

Percentage:

Solute mass (g)

Split 100:1

Splitless

530 µm 0.1 µm 5.0 µm

5.0 µm

1.0 µm

0.5 µm

0.5 µm

Thermal conductivity

Flame ionization

Nitrogen–phosphorus

Electron–capture

MS (scan)MS (single-ion monitoring)

Solute mass (g)

0.1 µm

0.1 µm

0.1 µm

0.1 µm

320 µm

250 µm

180 µm

100 µm

dc df

10–15 10–14 10–13 10–12 10–11 10–10 10–9 10–8 10–7 10–6 10–5 10–4 10–3

10–15 10–14 10–13 10–12 10–11 10–10 10–9 10–8 10–7 10–6 10–5 10–4 10–3

10–15 10–14 10–13 10–12 10–11 10–10 10–9 10–8 10–7 10–6 10–5 10–4 10–3

0.1% 1% 10% 100%

FIGURE 7.2 GC dynamic range nomogram. Concentrations expressed in grams per microliter (g=mL).(Reproduced from Hinshaw, J.V., LC GC Eur., 20, 138, 2007. With permission.)

In a split inje ction mode, typically small volum e of samp le extra ct (0.1 –2 m L) is rapid lydelivered into a heated glass line r foll owed by its splitting into tw o streams: the large r part isvented , while the smal ler part is trans ferred onto the column. Conside ring that the most of injectedsamp le is lost, this techniqu e is obviou sly not suitable for trace analys is, where very low detectionlimits are requi red. Anot her problem associated with split injectio n is a potent ial discrim ination dueto the heati ng of the syri nge during its introduct ion into a hot inje ctor resul ting in a change ofrelative abund ances of samp le compo nents when a mixture of analytes large ly diff ering in boilingpoints is analyz ed. Another advers e phenomen on relat ed to this technique is n onlinear splitting dueto adsorp tion of samp le compo nents on liner surfa ces or deposi ted mat rix ‘‘dirt. ’’

Nowaday s, hot splitl ess injectio n repres ents the most comm only used injection techniq ue intrace quanti tative ana lysis since e ntire inje cted samp le is intr oduced onto the GC capillary . Themajor limitati on of this inlet is that it suffe rs from the potential therm al degrada tion and adsorptionof susceptible analytes that may result either in matrix-induced response enhancement or itsdiminishment. In addition, the volume of injected sample=solvent is typically limited to 1 mL (forsome solvents even less) due to the expansi on volume of solve nt used (T able 7.1), since the totalliner volume is in a range of 150–1000 mL and the safety limit is typically 75% in maximum of thetotal liner volume.


TABLE 7.1Expan sion Vol ume of So lvents Used in GC

Expansion Volume (mL)

Solvent1 mL at 2508C

and 69 kPa (10 psig)1 mL at 2508C

and 345 kPa (50 psig)5 mL at 2508C

and 345 kPa (50 psig)

Water 1414 540 2700Methanol 631 241 1205

Acetonitrile 487 186 929Acetone 347 133 663Ethyl acetate 261 100 498

Toluene 241 92 460Hexane 195 75 373Isooctane 155 59 295

Source: From Hewlett-Packard FlowCalc 2.0 software. Available at http:==www.chem.agilent.com=cag=servsup=usersoft=files=GCFC.htm via the Internet. Accessed July 1, 2007.

Note: Calculated using Hewlett-Packard FlowCalc 2.0 software.

To overcome, or at least partly compensate for these problems, pulsed splitless injection can beapplied. Increased column head pressure for a short period during the sample injection (usually 1–2min)leads to a higher carrier gas flow rate through the injector (8–9 versus 0.5–1 mL=min during classicalsplitless injection), thus faster transport of sample vapors onto the GC column. In this way, the residencetime of analytes and, consequently, their interaction with active sites in the GC inlet is fairly reduced [3].The detection limits of troublesome compounds obtained with pulsed splitless injection are thus lowerand their further improvement can be obtained by injection of higher sample volumes (for most liners upto 5 mL) without the risk of backflash (Table 7.1) [4]. It should be noted that for injections > 1–2 mL, aretention gap prior to the analytical column is generally required to avoid excessive contamination ofseparation column and peak distortion (Figure 7.3).

7.2.2 C OLD ON-COLUMN INJECTION

In COC injectio n, a sample aliq uot is directly introduced by a speci al syri nge onto the analyt icalcolum n or a reten tion gap at tem peratures lower (608 C –80 8C) than those typicall y used in hot

(A) (B)

Pulsed splitless Pulsed splitless

3 µL

5 µL

4 µL

3 µL

2 µL

1 µL

2 µL

1 µL

Splitless 1 µL

FIGURE 7.3 Peak shapes obtained by pulsed splitless injections of different volumes of standard solutiononto the GC column (A) without a retention gap; (B) with an installed retention gap. (Reproduced from Godula,M., Hajslova, J., and Alterova, K., J. High Resolut. Chromatogr., 22, 395, 1999. With permission.)


http://www.chem.agilent.com


split=splitless mode (2008C–3008C). COC is therefore expected to cause less thermal stresson analytes during the injection process. This low-temperature injection eliminates both syringeneedle and inlet discrimination and is suitable namely for high-boiling analytes. On the other hand,the introduction of the entire sample (both analytes and matrix components co-isolated from foodmatrix) into the GC system is associated with increased demands for cleaning and maintenancewhen such complex samples as food is analyzed [5].

There are two alternative approaches available to perform on-column injection.

1. Small volume on-column injection: In this approach, a small volume of the sample (up to1–2 mL) is injected onto the separation column, or preferably, in the case of dirty samples,onto a retention gap [6].

2. Large volume on-column injection: In this mode, a large volume of the sample (up to1000 mL) can be introduced into the GC system [7]. The bulk of solvent is usuallyeliminated via a special solvent vapor exit. Once the venting is finished, the solventvapor exit is closed and analytes, together with remaining traces of solvent, are transferredonto the analytical column. However, a modification of the GC system is required in thiscase to include a large diameter retention gap (10–15 m� 0.53 mm), connected to aretaining precolumn (3–5 m� 0.32 mm) assisting in retention of volatile analytes. Inaddition, injection speed has to be slowed down to prevent flooding of the system duringlarge volume injections (LVI) with injection speeds in LVI-COC of 20–300 mL=min ascompared to LVI-PTV at 50–1500 mL=min [8].

7.2.3 PROGRAMMABLE TEMPERATURE VAPORIZATION INJECTION

A programmable temperature vaporization (PTV) injector represents the most versatile GC inletoffering significant reduction of most problems typically present when using hot vaporizing devices(splitless and=or cool on-column inlets) in trace analysis. The most important fact is that a PTVinjector chamber is cool at the moment of injection. A rapid temperature increase, followingwithdrawal of the syringe from the inlet, allows efficient transfer of the volatile analytes onto theGC column while leaving behind nonvolatiles in the injection liner. With regard to these operationalfeatures, PTV is ideally suited for thermally labile analytes and samples with a wide boiling range(when needed, PTV operating temperature can be programmed even higher than the usual columntemperature allowing injection of analytes that would not be vaporized through a classic split=split-less inlet). In addition eliminating a discrimination phenomenon and diminishing adverse affects ofnonvolatile matrix deposits on the recovery of injected analytes, PTV enables to introduce largesample volumes (up to hundreds of microliters) into the GC system. No retention gaps or pre-columns are needed for this purpose; instead of that, the liner size is increased. This feature makesPTV particularly suitable for trace analysis and also enables its online coupling with variousenrichment and cleanup techniques such as automated solid-phase extraction (SPE) approaches.From practical point of view, PTV is compatible with any capillary GC column diameter includingmicrobore columns. However, to attain optimal PTV performance in particular application, manyparameters have to be optimized (e.g., initial and final injector temperature, inlet heating rate,venting time, flow and pressure, transfer time, injection volume, type of liner). Due to the inherentcomplexity of this inlet, method development might become on some occasions a rather demandingtask. Despite that, the use of PTV in food analysis is rapidly growing. The paragraphs belowdescribe two most commonly used PTV operation modes.

1. PTV splitless injection. The sample is introduced at a temperature below or close to theboiling point of the solvent. A split exit is closed during the sample evaporation andsolvent vapors are vented via a GC column. PTV splitless injection can be employed forboth LVI of up to 20 mL of sample and for conventional small volume injections [9]. The


advantage of this technique is that no losses of volatile analytes occur. Operating para-meters have to be carefully optimized to avoid inlet overflow by sample vapors (losses ofvolatile compounds) as well as column flooding by excessive solvent (poor peak shapes ofmore volatile analytes). It has been reported that for some analytes the PTV splitlessinjection may produce better stability of responses and less matrix influence [10].

2. PTV solvent vent injection. When employing this technique, a sample is injected at temper-atures well below the boiling point of the solvent, holding the temperature of the injectionport at low a value, thus enabling elimination of solvent vapors via a split exit. After theventing step, the inlet is rapidly heated and analytes are transferred onto the front part of aGC column. In this way, sample volumes of up to hundreds of microliters can be injected[11,12]. For injection of large volumes, injector liner is often packed with various sorbents(e.g., Tennax, polyimide, Chromosorb, glass wool, glass beads, PTFE, Dexsil) in orderto protect solvent from reaching bottom of injector what may lead to column floodingwith liquid sample [13]. However, some labile compounds can be prone to degradation=rearrangement due to the catalytic effects of the sorbent; alternatively, strong binding ontothe packing material causes poor desorption (Figure 7.4). If selection of a suitable inactivesorbent fails to prevent these adverse effects, then the only viable solution is the use of anempty or open liner. Under these conditions, rather smaller volumes (in maximum about50 mL) of sample can be injected, typically employing a concept of multiple injections toget a larger injection volume. Obtaining good performance of the PTV injector in solventvent mode requires thorough experimental optimization of all relevant parameters asdescribed above.

pA140012001000

800600400200

0

(A)

(B)

10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 min

pA

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13.9

7514

.194

15.7

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17.7

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19.1

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10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 min

12.2

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FIGURE 7.4 Chromatograms obtained by programmable temperature vaporization (PTV) injection into(A) empty liner and (B) liner packed with glass wool plug. The differences in responses of sensitive analyteswhen injections were carried out into empty multibaffle liner and into single-baffle liner packed with glasswool. (Reproduced from Godula, M. et al., J. Sep. Sci., 24, 355, 2001. With permission.)


7.2.4 DIRECT SAMPLE INTRODUCTION=DIFFICULT MATRIX INTRODUCTION

Direct sample introduction (DSI) or its fully automated version, difficult matrix introduction (DMI),represents a novel LVI technique. The DSI approach involves adding up to 30 mL of the extract to amicrovial that is placed in the adapted GC liner. The solvent is evaporated and vented at a relativelylow temperature. After that, the injector is ballistically heated to volatilize the GC-amenablecompounds, which are then focused at the front of a relatively cold GC column. The column thenundergoes normal temperature programming to separate the analytes and cool to initial conditions,at which time the microvial is removed and discarded along with the nonvolatile matrix compo-nents that it contains. Only those compounds with the volatility range of the analytes enter thecolumn [14]. In the commercial DMI approach, the entire liner along with the microvial is replacedafter each injection [15].

In this way, time-consuming and expensive purification steps can be omitted=significantlyreduced for some matrices [15,16]. Since the bulk (semi)volatile matrix components introducedfrom the sample into the injector may influence the quantitative aspects of the injection process andinterfere in analytes detection, instruments with MS analyzers (single or tandem) providing moreaccurate results should be preferably used [17]. In Figure 7.5 the distinct improvement obtained bysample cleanup is illustrated. Regardless sample preparation strategy, reduced demands for the GCsystem maintenance represents a positive feature of this technique.

7.2.5 SOLID-PHASE MICROEXTRACTION

Solid-phase microextraction (SPME) represents a solvent-free sampling technique employing afused-silica fibre that is coated on the outside with an appropriate stationary phase. Volatile analytesemitted from the analyzed sample are isolated from the headspace or by direct immersion into theliquid sample and concentrated in fibre coating. After the extraction, thermal desorption in the hotGC injection port follows [18]. The main features of SPME include unattended operation viarobotics (if a fully automated option is available) and the elimination of maintenance of the linerand column. However, this sample introduction technique is associated with strong matrix effects,

1 2

Metalcap (septum inside)

O-rings

Needle guide

Microvial

Liner

3

FIGURE 7.5 Difficult matrix introduction (DMI) liner after injection of (1) purified baby-food extract,(2) crude extract, and (3) detail of microvials used for introduction of crude extracts. (Reproduced fromCajka, T. et al., J. Sep. Sci., 28, 1048, 2005. With permission.)


thus compl ications in quanti fi cation. In addit ion, varia bility of limit of de tections for differentanalyt es depends on the equil ibrium b etween the coati ng mat erial and the matrix.

7.3 SAMPLE SEPARATION

To be amena ble for the GC analys is, an analyt e shoul d posses s not only appreci able volatil ity attem peratures below 350 8C –400 8 C, but also must be able to wi thstand relative high tem peratureswithout degrada tion and reaction with other compo unds presen t in the GC syste m.

Wit h regard to a typicall y co mplex mixture of matrix components occurring in food extrac ts(oft en even after its puri fication ), the optimizati on of GC separa tion requires careful attentio n to anumbe r of importan t varia bles and their interaction. Both physical (column length, internal dia-met er, and stationar y phase incl uding its film thickness) , and param etric (temperat ure an d flowveloci ty) column varia bles affect the separa tion proces s.

7.3.1 C APILLARY COLUMNS FOR GC

To illustrate a wide range of combinations to be considered when selecting capillary GC column, theoverview of commonly available internal diameters is shown in Table 7.2.

Tab le 7. 3 shows relative polariti es of comm ercially available stationar y phases . The range ofstationary phases including also those dedicated for specific applications (e.g., volatiles, fatty acids,dioxins) is growing and also their quality characterized by reduced bleed and increased uppertemperature limit is improving.

In addition, the limits of inlet pressure, sampling system, and mass spectrometric detector(MSD) parameters have to be involved into consideration.

7.3.2 FAST GAS CHROMATOGRAPHY

A higher sample throughput together with the need to reduce laboratory operating costs has broughtattention of many laboratories to the implementation of high-speed GC (HSGC) systems. Althoughthe basic principles and theory of HSGC were formulated as early as in the 1960s, its practicaldevelopment occurred at the end of last century from introduction of novel technologies such as newmethods of fast and reproducible column heating, inlet devices allowing large sample volumeintroduction, and MS detectors with fast acquisition rates. It should be noted that full exploitationof the potential of this technique in routine practice is conditioned by reduction of sample

TABLE 7.2Classification of Capillary Column

CategoryColumn Diameter

Range (mm)Standard Commercial

Column Diameters (mm)Max Flow Rate

(mL=min)a

Megabore �0.5 0.53 �660

Wide bore �0.3 to<0.5 0.32, 0.45 �85 to<660Narrow bore �0.2 to<0.3 0.20, 0.25, 0.28 �17 to<86Microbore �0.1 to<0.2 0.10, 0.15, 0.18 �1 to<17Sub-microbore <0.1 Various <1

Source: Reproduced from Mastovska, K. and Lehotay, S.J., J. Chromatogr. A, 1000, 153, 2003.With permission.

a Flow rate calculated using helium carrier gas at 690 kPa, 2008C oven, vacuum outlet conditions,and 10 m column length.


TABLE 7.3Characterization of Stationary Phases Used in GC Analysis

Polarity Phase Composition Commercial Description

Nonpolar 100% Dimethylpolysiloxane DB-1, DB-1 ms, HP-1, HP-1 ms, Ultra 1, DB-1ht,Equity-1, SPB-1, AT-1, AT-1MS, Optima 1, Optima-1 ms, BP-1, VF-1MS, CP Sil 5 CB, CP Sil 5 CB MS,

ZB-1, 007-1, Elite-1, Rxi-1 ms, Rtx-1, Rtx-1MS5% Diphenyl–95% dimethylpolysiloxane DB-5, HP-5, DB-5 ms, HP-5 ms, Ultra 2, DB-5ht,

Equity-5, SPB-5, AT-5, AT-5MS, Optima 5, Optima-5

ms, BP-5, BPX-5, VF-5MS, CP Sil 8 CB, CP Sil 8 CBMS, ZB-5, 007-2, PE-2, Rxi-5 ms, Rtx-5, Rtx-5 ms,Rtx-5Sil MS

20% Diphenyl–80% dimethylpolysiloxane Rtx-20, SPB-20, At-20, 007-7

6% Cyanopropyl-phenyl–94%dimethylpolysiloxane

DB-1301, HP-1301, Rtx-1301, SPB-1301, AT-624,Optima 1301, 007-1301

35% Diphenyl–65% dimethylpolysiloxane DB-35, HP-35, DB-35 ms, Rtx-35, Rtx-35MS, SPB-35,

AT-35, AT-35MS, BPX-35, VF-35MS, ZB-35, 007-11,PE-11

Moderately polar 50% Diphenyl–50% dimethylpolysiloxane DB-17, DB-17 ms, HP-50þ , DB-17ht, Rtx-17,

VF-17MS, SPB-50, AT-50, AT-50MS, Optima 17,BPX-50, CP Sil 24 CB, ZB-50, 007-17, PE-17

14% Cyanopropyl-phenyl–86%

dimethylpolysiloxane

DB-1701, HP-1701, SPB-1701, AT-1701, Optima 1701,

BP-10, CP Sil 19 CB, ZB-1701, 007-1701, PE-170150% Cyanopropyl-phenyl–50%dimethylpolysiloxane

DB-23, DB-225, DB-225 ms, Rtx-225, AT-225,Optima 225, BP-225, CP Sil 43 CB, 007-225, PE-225

Polar Polyethylene glycol DB-WAX, HP-INNOWax, Rtx-WAX, Stabilwax,

Supelcowax-10, AT-Wax, Optima WAX, BP-20, CPWax 52 CB, ZB-WAX, 007-CW, PE-CW

Highly polar 70% Cyanopropyl-phenyl–30%


BPX-70

100% Cyanopropylsiloxane SP-2340

preparation time and other operations limiting laboratory throughput. Using approximate terms, theclassification of GC analyses on the basis of their speed is summarized in Table 7.4.

Alike in conventional GC, the separation time is defined as the retention time (tR) for the lasttarget component peak eluting from the column:

TABLE 7.4Classification of GC Analyses Based on Speed of SampleSeparation

Type ofGC Analysis

Typical SeparationTime (min)

Full Width atHalf-Maximum

Conventional >10 >1 s

Fast 1–10 200–1000 msVery fast 0.1–1 30–200 msUltra-fast <0.1 5–30 ms


tR ¼ L

u ( k þ 1) (7:1)

wher ek is the solut e c apacity ratio (capacity facto r, reten tion facto r) for the last compo undL is the column lengthu� is the average linear carrier gas velocity

On the b asis of this equation, the faster GC separa tion can be achiev ed by follow ing ways[19,20] :

. Red uced c olumn length ( # L)

. Decr eased retention factor ( # k): (1) increased isotherm al temperat ure, (2) faster tempera-ture programm ing, (3) alte red stat ionary phase to imp rove selectivity, (4) thinner film ofthe stationar y phase, (5) large r diameter c apillary colum n (for fixed length)

. Increas ed carrier gas veloci ty ( " u�): (1) higher than opti mum carrier gas velocity,(2) increased optimum carrier gas veloci ty (hydro gen as a carrier gas and vac uum outletoperat ion)

The increase in separa tion speed generally requi res a compromi se in terms of reduced resoluti on ( R)and=or samp le capacity ( Qc ). The accept ability of these losses has to be considered for eachparticu lar case separa tely . Availab ility of compa tible samp le introduct ion technique and detectionparam eters play an imp ortant role in selection of the analyt ical strategy.

Red uction of column length repres ents a very sim ple approac h to decreas e time of GC analys is.In practice, almo st all fast GC analys es are perfor med wi th short colum ns (usually �10 m) in acombi nation with other approac hes (Figur e 7.6). On this accou nt, reduct ion of the length of a givencolum n results in reduced resol ution ( R ~

p L), which can be compe nsated to some extent by

suitable MS detector (spectral resolutio n).Use of a colum n with a small internal diam eter is anothe r attracti ve way tow ards faster GC

analys is. How ever, the inst rumental requireme nts especiall y the dif fi culties with the samp le intro-ducti on of large r sample volumes and also the lower samp le capaci ty limit their applicati on in manyreal- world analyses.

Use of a column with a thin fi lm of stationar y phase results in the decreas e of the capacity(ret ention) factor and thus in the faster GC analysis. In addit ion, due to the decreas ed contribut ion ofmass trans fer in the stationar y phase, separa tion ef ficiency is incre ased. On the other hand, reducedruggedn ess and samp le capacity are the fees for analys is speed.

Fa st temperat ure progra mming is the most popula r approac h in applicati on of fast GC in foodanalys is. Ei ther convect ion heating facil itated by a convent ional GC ov en or resistive heati ng can beempl oyed. If ‘‘fast ’’ separa tion in terms of class ifi cation show n in Tab le 7.4 is required, a convent ionalGC oven can be used. At fast er progra mming rates , heat losses from the oven to the surrounding maycause poor oven tempe rature pro file, hence lower reprod ucibili ty of analyt e elution.

Oper ation of colum n outlet at low p ressure (low-pr essure GC) is anothe r fast GC alte rnative thatmay find a wide use in routine labor atories concern ed wi th food analysis. Because o f o perating amegab ore separa tion colum n (typicall y 10 m length � 0.53 mm inte rnal diameter � 0.25 –1 mmphase) at low pressure, optimum carrier gas linear velocity is attained at higher value because ofincreased diffusivity of the solute in the gas phase. Consequently, faster GC separations can beachieved with a disproportionately smaller loss of separation power [22]. The main attractivefeatu res of LP- GC –MS invol ve (1) reduced peak tailing and width (F igure 7.7) thus their improveddetection limits, (2) increased sample capacity of megabore column allowing injection of highersample volume resulting in lower detection limits for compounds not limited by matrix interfer-ences, and (3) reduced thermal degradation of thermally labile analytes [23,24].


Inte

nsity

Inte

nsity

50000

40000

30000

20000

10000

80000

60000

40000

20000

0 10 20 30 40 50 min

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31

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6

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910

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14

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192122

23

2425

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28

(B)

(A)

2930

31

20

1.0 1.5 2.0 min

FIGURE 7.6 GC–FID chromatograms of fatty acid methyl esters obtained under conditions of (A) con-ventional (column: Rtx-WAX, 30 m� 0.25 mm� 0.25 mm; injection: split 1:100; oven temperatureprogram: 508C, 38C=min to 2808C; acquisition rate: 12.5 Hz) and (B) fast GC (column: Supelcowax,10 m� 0.10 mm� 0.10 mm; injection: split 1:200; oven temperature program: 508C, 808C=min to 1508C,708C=min to 2508C, 508C=min to 2808C (1 min); acquisition rate: 50 Hz). (Reproduced from Mondello, L.et al., J. Chromatogr A., 1035, 237, 2004. With permission.)

Hydrogen can be used as a carrier gas because with the highest diffusion coefficient it isobviously the best carrier gas for fast GC. Its low viscosity also results in lower inlet pressurerequirements. In practice, however, helium is usually preferred as a carrier gas flow for safety andinertness reasons.


(A) Conventional GC–MS (B) LP-GC–MS

Abundance Abundance

AbundanceAbundance

3000m /z 201 m /z 201

m /z 283 m /z 283

4000

3000

2000

1000

4000

3000

2000

1000

2000

1000

0 0

0

3000

2000

1000

0

Time−> 9.50

Time−> 9.50 Time−>3.00

Time−> 3.00 4.00 min

4.00 min10.50 min

10.50 min

FIGURE 7.7 Comparison of peak shapes of thiabendazole (m=z 201) and procymidone (m=z 283) obtained by(A) conventional GC–MS (column: Rtx-5MS, 30 m� 0.25 mm� 0.25 mm; injection: splitless, 1 mL; oventemperature program: 908C (0.5 min), 208C=min to 2208C, 58C=min to 2408C, 208C=min to 2908C (6.5 min))and (B) LP-GC–MS (column: Rtx-5Sil MS, 10 m� 0.53 mm� 1.0 mm coupled to 3 m� 0.15 mm restrictioncolumn; injection: splitless, 1 mL; oven temperature program: 908C (0.5 min), 608C=min to 2908C (3.0 min)).(Reproduced from Mastovska, K., Lehotay, S.J., Hajslova, J., J. Chromatogr. A, 926, 291, 2001. Withpermission.)

7.3.3 COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY

In the analysis of complex mixtures, such as food extracts, by one-dimensional chromatography(1D-GC), overlap of some sample components unavoidably occurs. To achieve a considerableincrease in peak capacity, two independent separation processes with peak capacities n1 and n2 canbe employed in the sample analysis. Supposing that separations are based on two differentmechanisms (orthogonality criterion), the maximum peak capacity calculated as n1� n2 is typicallyenhanced by at least one order of magnitude.

Most of the successful applications reported in food analysis since 1960 up to 1990 employedso-called heart-cut mode, in which only narrow fraction(s) containing analytes of interest is (are)transported for further separation onto the second column.

However, this approach has limitations. Increasing the width of the first column fraction orisolating too many parts of 1D-GC analysis to subject them to two-dimensional gas chromato-graphy (2D-GC) separation becomes troublesome. Also, time-demanding reconstruction ofgenerated chromatograms may become a serious problem. The introduction of systems thatallow the entire sample from the first column to be analyzed on the second column has enabledand improved both target and nontarget screening of food components in a wide range of matrices.This approach, called comprehensive two-dimensional (GC�GC), is introduced in the followingsections in a greater detail.


7.3.3. 1 GC � GC Setup

The hea rt of the GC �GC syst em is a modul ator that connect s the fi rst-dimensi on convent ional-si zecolumn with a short microbore column in the second dimensi on (Figure 7.8).

There are three fundamental functions of this interface: (1) trapping of small adjacent fractions(typically 2–10 s) of the effluent from the first separation column, (2) refocusing these fractions (either intime or in space), and (3) injection of the refocused fractions as narrow pulses into the second-dimensioncolumn. The separation on the latter column is extremely fast and takes only 2–10 s versus 20–120 minfor the first dimension, and is therefore performed under essentially isothermal conditions.

A large seri es of high-speed chromatogr ams as the outcome of the transfer of chromatogr aphicband from the first to the second dim ension are generated durin g the GC � GC run. As shown inFigure 7.9, these adjace nt pulse s are usual ly stack ed side- by-side by a special soft ware to form a 2Dchromatogram with one dimension representing the retention time on the first column (tR1) and theother, the retention time on the second column (tR2). The most convenient way to visualize GC�GCdata is as contour plots representing the bird’s eye view, where peaks are displayed as spots on aplane using colors and shading to indicate the signal intensity (Figure 7.9).

7.3.3.2 Optimization of Operation Conditions and InstrumentalRequirements in GC�GC

Compared to conventional 1D-GC, the optimization of GC�GC analysis requires a more complexapproach. The changes in operational parameters such as oven temperature or carrier gas flow ratehave different impacts on the performance of separation columns since these differ both in theirgeometry and separation mechanism. Furthermore, new parameters such as modulation frequencyand modulator temperature have to be optimized [25].

Conventional columns, typically 15–30 m length� 0.25–0.32 mm internal diameter� 0.1–1 mmfilm thickness, are used in the first dimension. This allows application of virtually all sampleintroduction techniques (split=splitless, on column, LVI-PTV, DMI=DSI, and=or SPME). Stationaryphases commonly used in first-dimension columns are typically 100% dimethylpolysiloxane or (5%phenylene)-dimethylpolysiloxane. The separation on these nonpolar columns is governed mainly byanalyte volatility. The size of columns for second dimension is commonly in a range of 0.5–2 mlength� 0.1 mm internal diameter� 0.1 mm film thickness. More polar stationary phases suchas 35%–50% phenylene–65%–50% dimethylpolysiloxane, polyethylene glycol, carborane, and=orcyanopropyl–phenyl–dimethylpolysiloxane are employed. Analytes interact with these medium-polar=polar phases via various mechanisms such as p–p interactions, hydrogen bonding, etc.,hence the requirement for different, independent separation principle is met. In most applications,orthogonality is achieved using nonpolar� polar separation mechanisms.

To obtain acceptable separation in both dimensions, a compromise has to be made with regardto both columns. The linear velocity of the carrier gas in the (narrow bore) first-dimension column isusually rather lower than optimal (about 30 cm=s) while, at the same time, the linear velocity in the(microbore) second-dimension capillary is relatively high, typically exceeding 100 cm=s. Also whensetting the temperature programming rate, the requirement for obtaining at least four modulations

Injector Modulator

Detector

First column Second column

FIGURE 7.8 GC�GC instrument configuration.


1D chromatogram

Modulation

Transformation

Raw 2D chromatogram

(second column outlet)

Second dimensionalchromatograms

Second dim

ension

First dimension

Visualization

2D plot 3D plot

(first column outlet)

First dimension

Sec

ond

dim

ensi

on

Sec

ond

dim

ensi

on

First dimension

FIGURE 7.9 Generation and visualization of a GC�GC chromatogram. (Reproduced from Zrostlikova, J.,Hajslova, J., and Cajka, T., J. Chromatogr. A, 1019, 173, 2003. With permission.)

over each first-dimension peak (so-called modulation criterion) has to be taken into account. In mostanalyses, this is achieved by using programming rates as low as 0.58C–58C=min, which is less thanin conventional 1D-GC [26]. It should be noted, however, that even steeper programming rates (thusfaster GC separation) can be employed (108C–208C=min) in GC�GC, which typically results in twomodulations over each first-dimension peak. Under these conditions, the separation accomplished inthe first column might be lost. However, because of different activity coefficients on the secondcolumn, the analytes can be completely separated (with higher chromatographic resolution than inthe case of 1D-GC) in the second dimension [27,28]. For better tuning of the GC�GC setup,systems with a programmable second oven are preferred.

Effective and robust modulation is a key process in the GC�GC analysis. Thermal modulation ina capillary GC can be performed by both heating and cooling. While heated modulators use a thick-film modulation capillary to trap subsequent sample fractions eluting from the first column by means


of stationar y phase focusing (com pounds are released by the temperat ure incre ase), the cryogen icallycooled modul ators do not use a modulati on capil lary. Inste ad, they trap and focus the sample fractionseluting from the first column at the front part of the seconda ry colum n itself. Initially, movingheated=cooled modulato rs were used but they exhibi ted relativel y low robustness (frag ile capillarycan be easily broken) . The se shortcomi ngs of movi ng modulato rs have bee n overcom e by two-st agejet modul ators that use a stream of nitrogen or carbon dioxide for cooling a short secti on of the secondcolumn for trapp ing=focusing of the analytes elut ing from the fi rst colum n [29,30] .

In practice, fixed modul ation freque ncy, typically in a range of 0.1 –10 Hz is employ ed durin gthe analys is. Under ideal experi ment al condition s, the reten tion time of the most retaine d compo undin the second d imensio n is shorter than a modul ation time. If this is not the case, i.e., analyt es do notelute in their modul ation cycle, so-called wrap-arou nd, which may cause co-elutio ns, occurs.Avoidin g this phenom enon can be achiev ed, e.g., by an incre ase of the second- dimensi on colum ntemperat ure (if a n indepe ndent ove n is avail able). In any case, optimal funct ion of modulato r isessential for the q uality of separa tion and detection proces s.

The fast separa tion on a short and mic robore second- dime nsion column results in very n arrowpeaks with widths of 50 –1000 ms at the ba seline. Althoug h fast analogue detectors such as a flameionizati on d etector o r electron captur e detect or (E CD) are full y compa tible with fast chromatog-raphy and provi de reliable peak recogni tion, they do not provi de stru ctural informat ion. Coupli ngGC � GC separation with MS detect or resul ts into the three-dim ensional syst em that may contribut eto the identi fication of 2D separa ted peaks and brings a dee per unders tanding of stru cturedchrom atograms [26]. However , convent ional scanning MS detectors are typi cally too slow and donot provi de reliable spectra an d peak reprod ucti on. At presen t, only time-of - flight mass spect ro-meters (see Cha pter 10) can acquir e the 50 or more mass spectra per second, whi ch are requi red forthe proper recons truction of the chromatogr am and for quanti ficati on in GC � GC [35].

7.3.3. 3 Adva ntages of GC � GC

A nu mber of charact eristics of GC � GC have been reported that documen ts superi ority of thistechniqu e over convent ional 1D-GC [26].

High peak capaci ty. The peak c apacity, characteri zed as a maximal numbe r of chromatogr aphicpeaks that can be placed side by side into the available separa tion space (chromato gram ), issigni fica ntly en hanced. Unde r the real-world condition s, the total peak capaci ty in GC � GC is rathe rlower than the calcul ated value due to the imperfect ions in the sample transfer betw een the twocolumns; however, it still great ly exc eeds the limits of convent ional GC. As an examp le, the meri t inpesticide residue analys is resultin g from the separa tion powe r is shown in Figure 7.10.

Enh anced sensi tivity . Compar ed to 1D- GC separa tion, pronoun ced imp rovement of detectionlimits in GC � GC syst em is o btained; thanks to compr essing the peak in the modulation capillaryand front part of the second colum n (following fast chromatogr aphy avoids band broaden ing offocuse d peak s). Furthe rmore, thanks to imp roved separa tion of analytes and mat rix interfere nces(chem ical noise ) in the GC � GC system, the signa l to noise ratio is also improved. An e xample isgiven in Figure 7 .11 that ill ustrates diff erences in 1D-GC versus GC � GC a nalysis of limonene.

Struc tured chrom atogra ms. Thanks to compleme ntary separa tion mecha nisms occurr ing in bothcolumns, the chromatogr ams resultin g from particula r GC �GC setup are ordered, i.e., moleculeshave thei r de finite locations in the reten tion space based on their stru cture. In the recons tructed 2Dcontou r plots, characteri stic patt erns are obtained, in which the members of homologi cal seri esdiffering in thei r volatility are ordere d along the first-dim ension axis (nonpol ar capillary is typicall yemploye d in first dim ension), whereas the compounds differing by polarity are spread along thesecond-dimension axis. The formation of clusters of the various subgroups of compounds in aGC�GC contour plot may be useful for the group type analysis.

Improved identification of unknowns. Nontarget screening allows obtaining of overview of thesample constituents. This approach consists from: (1) peak finding and deconvolution (algorithm for


(A)

(B)

20000

18000

16000

14000

12000

10000

8000

1

2

6000

4000

2000

Time (seconds)spectrum #

Time (seconds)spectrum #

1st Time (seconds)2nd Time (seconds)

spectrum #

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640697

6620.6

6620.8

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47

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109

128 145185

220

60

79

93

109

145 185 220

1000

500

1000

500

80 100 120 140 160 180 200 220 240

60

Peak true–sample

Library Hit - similarity 727, “Phosphoric acid,2,2-dichlorovinyldimethylester”

80 100 120 140 160 180 200 220 240

6621.8

662

32520 32560 32600

79 109 185

32640 32680 32720 32760 328002

650 660747 797

79 109 185

670847

680897

690947

650747

660797

670847

680897

690947

79 109 186

700997

FIGURE 7.10 Separation of dichlorvos (1) in apple extract at 0.01 mg=kg from matrix co-extract5-(hydroxymethyl)-5-furancarboxaldehyde (2). Plotted are three most abundant ions in the mass spectrum ofdichlorvos (79, 109, and 185). Chromatogram of (A) 1D-GC analysis of zoomed section shows the peakof dichlorvos (m=z 185) and matrix interference (m=z 79 and 109); and (B) GC�GC analysis (DB-XLB�DB-17columns); matrix interference resolved on medium polar DB-17 column. Data acquired by TOFMS atacquisition rates 5 spectra=s and 250 spectra=s, respectively.

recogni zing of partly co-el uting peaks in the GC –MS chrom atogram and obtai ning thei r ‘‘ pure ’’mass spect ra), (2) library searching, and (3) furt her post- processing. Since a large amoun t of datahave to be proces sed, automated data proces sing is employed.

7.4 SAMPLE DETECTION

Depe nding upon the type of food compo unds being measured severa l different detectors areavail able for this purpose (T able 7 .5), e ach with its own advantages and drawbacks. The followingsections briefly introduce various GC detectors most commonly in use today [32,33].


(B)(A)

4�104

3�104

2�104

1�104

570 590Retention time (s)

Abu

ndan

ce

Abu

ndan

ce

610

S/N = 639 S/N = 19570

3�104

2�104

1�104

590.80.22

593.80.22

1tR (s)2tR (s)

596.80.22

FIGURE 7.11 Improvement of detectability of limonene (m=z 93) isolated from honey headspace by SPMEunder the conditions of (A) 1D-GC and (B) GC�GC (DB-5 ms�Supelcowax-10 columns). Data acquired byTOFMS at acquisition rates of 10 spectra=s and 300 spectra=s, respectively. (Cajka, T. et al., J. Sep. Sci., 30,534, 2007.)

7.4.1 FLAME IONIZATION DETECTOR

Flame ionization detector (FID) represents one of the most widely used detectors. The effluent froman analytical column is mixed with hydrogen and air, and is directed into a flame, which breaksdown organic molecules and produces ions. A voltage potential is applied across the gap betweenthe burner tip and an electrode located just above the flame. The resulting current is then measuredand is proportional to the concentration of the components present.

TABLE 7.5Overview of GC Detectors Applicable for the Determination of Food Components

Detector Selectivity Detectability Linearity

Flame ionization detector (FID) No 2 pg C=s 107

Thermal conductivity detector (TCD) No �300 pg=mL 104–6

Electron capture detector (ECD) Halogens fg=s 104

Nitrogen–phosphorus detector (NPD) N, P fg–pg N, P=s 104–7

Halogen-specific detector (XSD) Halogens pg Cl=s 104

Thermionic ionization detector (TID) N, P �100 fg N=s, N: 105, P: 104

�100 fg P=s

Photoionization detector (PID) Aromatics pg 106

Flame photometric detector (FPD) S, P pga S: 103, P: 105

Pulsed flame photometric detector (PFPD) Tuneable for 28 elements pg S=s, S, P: 103

100 pg P=sa

Atomic-emission detector (AED) Tuneable for any element pg=sa 103–4

Electrolytic conductivity detector (ELCD)

or Hall electrolytic conductivity detector

S, N, halogens pg 106

Mass spectrometric detector (MSD) Yes fg–pg 104–7

Fourier transform infrared (FTIR) Yes pg 102

a The detectability considerably varies among particular elements.

pg, picogram; fg, femtogram.


7.4.2 THERMAL CONDUCTIVITY DETECTOR

Thermal conductivity detector (TCD) consists of an electrically heated wire or thermistor. Thetemperature of the sensing element depends on the thermal conductivity of the gas flowing around it.Changes in thermal conductivity cause a temperature rise in the element, which is sensed as achange in resistance.

7.4.3 ELECTRON CAPTURE DETECTOR

In ECD, the sample is introduced into the detector through an analytical column and passes over a63Ni radioactive source emitting b particles, which causes ionization of the carrier gas and thesubsequent release of electrons. When organic molecules containing electronegative functionalatoms or groups pass by the detector, they capture some of the electrons and reduce the currentmeasured between the electrodes.

7.4.4 NITROGEN–PHOSPHORUS DETECTOR

In nitrogen–phosphorus detector (NPD), a glass bead containing an alkali metal is electricallyheated until electrons are emitted. These electrons are then captured by stable intermediates toform a hydrogen plasma, which ionizes compounds from the column effluent. A polarizing fielddirects these ions to a collector anode creating a current.

7.4.5 FLAME PHOTOMETRIC DETECTOR AND PULSED FLAME PHOTOMETRIC DETECTOR

In flame photometric detector (FPD), a sample is burned in a hydrogen=air flame to produce molecularproducts that emit light by means of chemiluminescent chemical reactions. The emitted light is thenisolated from background emissions by narrow bandpass wavelength-selective filters and is detectedby a photomultiplier and then amplified. Unfortunately, the detectability of the FPD is limited by lightemissions of the continuous flame burning products. This disadvantage is eliminated by pulsed flamephotometric detector (PFPD), where a hydrogen=air mixture flows into the FPD so low that acontinuous flame could not be sustained. By inserting a constant ignition source into the gas flow,the hydrogen=air mixture would ignite, propagate back through a quartz combustor tube to aconstriction in the flow path, extinguish, then refill the detector, ignite, and repeat the cycle.

7.4.6 PHOTO-IONIZATION DETECTOR

In photo-ionization detector (PID), the column effluent is ionized by ultraviolet light and the current(proportional to the concentrations of the ionized material) produced by the ion flow is measured.

7.4.7 ELECTROLYTIC CONDUCTIVITY DETECTOR

In electrolytic conductivity detector (ELCD), compounds eluting from an analytical column areswept into a nickel reaction tube at the temperature up to 9008C. The components are stripped offtheir halogenated atoms and these atoms are carried into a conductivity cell. As the concentrations ofthe halogens change in this cell, the measured conductivity of a solution in the cell changesproportionally.

7.4.8 ATOMIC-EMISSION DETECTOR

In atomic-emission detector (AED), eluted compounds from an analytical column are transportedinto a microwave powered plasma (or discharge) cavity where those compounds are destroyed andtheir atoms are excited by the energy of the plasma. The emitted light by the excited particles isseparated into individual lines via a photodiode array. The individual emission lines are then sorted


and produce chromatogr ams co nsisting of peaks from eluant s that co ntain only a speci fic element. Inthis way, elem ental compo sition can be estimat ed. It shoul d be noted that the inte nsity of signa llarge ly varies among the elements and it is relat ively low for oxygen, nitrogen, chlorine, brom inewhile higher sensi tivity is obtai ned for carbon, phospho rus, and sulph ur.

7.4.9 MASS S PECTROMETRIC DETECTOR

The mass spect rome ter (MS) is by far the most powerful an d fle xible of the detectors used in theanalys is of GC-am enable food compone nts today. The advantage over all GC detectors descri bedabove is a possibil ity to ob tain, in addit ion to selec tive de tection of analyt e elut ed at certa in retentiontime, also stru ctural infor mation, enabli ng either con firmation of targe t compo und or identi ficati onof unknow n speci es. The charact er of data obtained largely depends on the type of mass analyz eremploye d. The princ iple s of this type of detection are thoro ughly discussed in Cha pter 10.

7.5 MATRIX EFFECTS

Unde r the real- world condition s, some residues of mat rix co-ext ractives u navoidably remain in thepuri fied samp le prepar ed for examinati on by GC analysis. Inaccur ate quanti fication, decreas edmethod ruggedn ess, low analyte detectabil ity, and ev en report ing of false positive o r negati veresults are the most serious matrix- associated probl ems, whic h c an b e e ncountered [3]. The extentof these phenomena depend on a wide range facto rs incl uding samp le compo sition and injectio ntechniqu e empl oyed.

Matr ix-induced chromato graphi c respon se enhancem ent, first descri bed b y Erney et al., ispresuma bly the most discu ssed matrix effect adversely imp acting quanti ficati on accuracy of c ertain,particula rly more polar analytes [34]. Its principle is as follow s: During inje ction of particula rcompo unds in neat solve nt, adsorp tion and=or therm o-degr adation of susceptibl e analytes on theactive sites (mainly free sil anol groups ) presen t in the inje ction port and in chromatogr aphic colum nmay occur. On this account, the number of analyte molecules reaching GC detector is reduced. Thisis, however, not the case when a real-world sample is analyzed. Co-injected matrix components tendto block the active sites in GC system thus reducing the analyte losses and, consequently, enhancingtheir signa ls as co mpared to the injectio n in neat solve nt (Figur es 7.12 and 7.13). If these facts areignored and calibration standards in solvent only are used for calculation of target analytes con-centration, recoveries as high as even several hundred percent might be obtained [3]. It is worthnoticing that hydrophobic, nonpolar substances, such as persistent organochlorine contaminants(with some exceptions such as DDT that may thermally degrade in a dirty hot injector), are notprone to these hot injection-related problems.

Repeated injections of nonvolatile matrix components, which are gradually deposited in the GCinlet and=or front part of the GC column, can give rise to successive formation of new active sites,which might be responsible for the effect, sometimes called matrix-induced diminishment [36].Gradual decrease in analyte responses associated with this phenomenon together with distorted peakshapes (broadening, tailing) and shifting the retention times towards higher values negatively impactruggedness, i.e., long-term repeatability of analyte peak intensities, shapes, and retention times,performance characteristic of high importance in routine trace analysis [24].

Three basic approaches and their combination should be considered as way to improved qualityassurance [3]: (1) elimination of primary causes, (2) optimization of calibration strategy enablingcompensation, and (3) optimization of injection and separation parameters.

Unfortunately, the first concept of the GC system free of active sites is in principle hardlyviable—not only because of commercial unavailability of virtually inert materials stable even underlong-term exposure to high temperatures that typically occur in a GC inlet port, but also due toimpossibility to control formation of new active sites from deposited nonvolatile matrix. In thiscontent, a more conceivable alternative might be based on avoiding sample matrix to be introduced


Standard

C C

Sample

Injection

Liner

Transfer ontothe GC column

C – X C – Y

X Y

FIGURE 7.12 Illustration of the cause of matrix-induced chromatographic enhancement effect; (C) numberof injected analyte molecules; (X, Y) number of free active sites for their adsorption in injector; (*) moleculesof analyte in injected sample; (.) portion of analyte molecules adsorbed in GC injector; (~)molecules of matrix components in injected sample; (~) portion of matrix compounds adsorbed in GC liner;(C�X)< (C� Y). (Reproduced from Hajslova, J. and Zrostlikova, J., J. Chromatogr. A, 1000, 181, 2003.With permission.)

260240220200

180

160140120100

8060

12.20 12.30

(P)

(S)

12.40 12.50Time−>

FIGURE 7.13 Matrix-induced enhancement response effect: 1 pg of 2-nitronaphthalene (m=z 173) injected inpure solvent (S) and in purified sample of pumpkin seed oil (P). (Reproduced from Dusek, B., Hajslova, J., andKocourek, V., J. Chromatogr. A, 982, 127, 2002. With permission.)


into the GC syst em. Unfortu nately, again, none of the comm on isolati on and=or cleanu p techni quesare selec tive eno ugh (mainly in the case of broad scope methods) to avoid the presen ce of residualsamp le compo nents in the analyt ical sample.

Since an effect ive elimin ation of the source s of the matrix effects is not like ly to occur inpract ice, their compen sation by using alte rnative cali bration methods is obviously the most feasibleoption. Se veral stra tegies are concei vable for this purpos e: (1) addition of isotopic ally labeledinternal standards, (2) the use of stand ards addition method, (3) the use of mat rix-mat chedstand ards, an d (4) the use of analyt e protectant s (introduce d only recent ly). The main disad van-tages=requi rements of these methods are summ arized in Tab le 7.6 [37].

As regards analyt e protec tants , these compound s are capabl e to strongly inte ract wi th acti vesites in the GC syst em, thus decreas ing de gradation an d adsorp tion of targe t analyt es [37]. The sameamoun t of the analyte protectant s is added to both samp le extra cts and mat rix-free standards, whichresults in maximi zation and equ alization of the matrix- induced respon se enh ancement effect andavoids overes timati on of results, which can occur with stand ards in neat solve nt [38]. A wide rangeof compo unds containing mul tiple polar=ionizable groups such as vario us po lyols and their deriv a-tives, carboxy lic a cids, amino acids , and deriv atives of basic nitrogen contai ning h eterocycles havebeen exp erimenta lly evalua ted as analyte prote ctant s. In a study con cerned with the analys isof mul tiple pesticide resi dues using hot splitless injection , a mixture of 3-ethoxyp ropane-1,2-di ol,L-gulo nic acid g-lact one, and D-gluc itol (in aceton itrile extra cts) was found to most effect ively covera wide volatility range of GC-amenable analytes [38]. This analyte protectant mixture worked alsovery well in the multi residue GC analysis of pesticides using DMI [15], which has more active glasssurfa ces that need effective deacti vation d uring each injectio n. Figure 7.14 show s chrom atograms

TABLE 7.6Quantification Strategies and Their Critical Assessment

Method Comments

Standard additions Extra labor effort required for preparation

Inaccuracies may occur because the matrix effect is concentration dependentIsotopically labeled standards Only a limited number of certified isotopically labeled standards is currently

commercially available; not available in wide scope methods

Restriction in the use of detection techniques other than mass spectrometryAdditional labor=time burden of developing analytical conditions for so manymore compounds

Matrix-matched standards Need for enough blank matrix (ideally identical as the samples) and its long-term storageExtra time, labor, and expense for preparing the blank extracts for calibrationstandards needed

Greater amount of matrix material injected onto the column in a sequence,which leads to greater requirements for GC maintenanceGreater potential for analyte degradation in the matrix solution

Analyte protectants Following criteria have to be met for analyte protectants:Unreactiveness with analytes in solution and the GC systemSufficient stability under the GC conditions (no thermodegradation,

re-arrangement, etc.)No deterioration of the GC column or detector performance (e.g., due toaccumulation)No interference with the detection process of analytes (i.e., low intensity, low

mass ions in its MS spectra)Good availability, low cost, no toxicityGood solubility in the solvent of interest


A) Without analyte protectants

(A) Lindane

(B) Phosalone

(C) o-Phenylphenol

CI

OH

10.6x

2.16x

3.98x

1.12x

Injection in:MatrixSolvent

OO

O

O

N S

S

P

CI

CI

CI

CI

CI

CI

B) With analyte protectants

FIGURE 7.14 Comparison of peak shapes and intensities of 100 ng=mL lindane (m=z 219), phosalone (m=z182), and o-phenylphenol (m=z 170) obtained by injection in matrix (mixed fruit extract) and solvent (MeCN)solutions (A) without and (B) with the addition of analyte protectants (3-ethoxypropane-1,2-diol, L-gulonic acidg-lactone, and D-glucitol at 10, 1, and 1 mg=mL in the injected sample, respectively). (Reproduced fromMastovska, K., Lehotay, S.J., and Anastassiades M., Anal. Chem., 77, 8129, 2005. With permission.)

for three pesticide s lindane, phosal one, and o-phenyl phenol obtained by hot splitl ess inje ction insolve nt a nd matrix-mat ched stand ards wi thout and with the above mixture of analyte prote ctants,demon strating dram atic improvem ent in peak shapes an d intensit ies wi th the use of analyt eprote ctants [38].

Bes ides the above compe nsation approac hes, also careful optimizati on of injectio n and separa tionparam eters (includin g the choice of suitable inje ction technique, temperat ure, and volume; liner sizeand its desig n; solvent expansi on volum e; column fl ow rate; colum n dimensi ons) can reduce to someextent the numbe r of active sites avail able for interacti on (lower surface area) and its durat ion [37].

7.6 FOOD ANALYSIS APPLICATIONS

Since a large range of food co mpounds are (semi)vo latile co mpounds, the GC is widely used for theirdeter minati on. The ch oice of an optimal GC setup depends on the requireme nts for the performanc echaract eristics of methods used, cost, speed, and several other factors. In Tab le 7.7, the curren t GCmethods for several groups of food constituents are summarized with special attention paid toapplicability of recent advances in the field of this technique for their analysis [39–43].


TABLE 7.7Overview of Typical Conditions of Most Common Applications Employing GCfor Separation

Injection Typical GC Column Phase Detection

Naturalsubstances

Lipids (fatty acids,mostly derivatized)

Split Polyethylene glycol70% Cyanopropyl-phenyl–30%


FID, MS

Aroma and flavorcompounds

Split, splitless, SPME 5% Diphenyl–95%dimethylpolysiloxane

MS, FID

Polyethylene glycolFoodcontaminants

Modern pesticides Splitless, PTV,DSI=DMI, SPME

5% Diphenyl–95%dimethylpolysiloxane

MS, ECD,NPD, FPD,

PFPD, AED,PID, ELCD

50% Diphenyl–50%dimethylpolysiloxane6% Cyanopropyl-phenyl–94%

dimethylpolysiloxane35% Diphenyl–65%dimethylpolysiloxane

Polychlorinated

biphenyls

Splitless, PTV 5% Diphenyl–95%


ECD, MS

50% Diphenyl–50%dimethylpolysiloxane

Polychlorinateddibenzo-p-dioxinsand dibenzofurans

Splitless, PTV 5% Diphenyl–95%dimethylpolysiloxane

MS

50% Cyanopropyl-phenyl–50%

dimethylpolysiloxaneother special phases

Brominated flameretardants

Splitless, PTV 100% Dimethylpolysiloxane MS, ECD5% Diphenyl–95%

dimethylpolysiloxane14% Cyanopropyl-phenyl–86%dimethylpolysiloxane

Polycyclic aromatichydrocarbons


MS, PID

50% Diphenyl–50%

dimethylpolysiloxaneVeterinary drugs(derivatized)

Splitless 100% Dimethylpolysiloxane MS5% Diphenyl–95%

dimethylpolysiloxaneMycotoxins(derivatized)

Splitless 5% Diphenyl–95%dimethylpolysiloxane

MS, ECD

Acrylamide Splitless, PTV, DSI 5% Diphenyl–95%

dimethylpolysiloxane(derivatized form)

MS (both

forms), ECD(derivatizedform)Polyethylene glycol

(nonderivatized form)Chloropropanols(derivatized)


MS

Heterocyclic amines(derivatized)

Splitless 5% Diphenyl–95%dimethylpolysiloxane50% diphenyl–50%dimethylpolysiloxane

MS

(continued )


TABLE 7.7 (continued)Overview of Typical Conditions of Most Common Applications Employing GCfor Separation

Injection Typical GC Column Phase Detection

Foodcontaminants

Phthalate andadipate esters

Splitless, SPME 5% Diphenyl–95%dimethylpolysiloxane

MS, ECD

Epoxy-compounds(derivatized)

Splitless 5% Diphenyl–95%dimethylpolysiloxane

MS

Note: AED, atomic-emission detector; DMI, difficult matrix introduction; DSI, Direct sample introduction; ECD, electron

capture detector; ELCD, electrolytic conductivity detector; FID, flame ionization detector; NPD, nitrogen–phosphorus detector; PFPD, pulsed flame photometric detector; PID, photo-ionization detector; PTV,programmable temperature vaporization; MS, mass spectrometry; SPME, solid-phase microextraction.

7.7 CONCLUSION AND FUTURE TRENDS

After severa l decades of GC on the mark et, the techno logy and its appli cations have improvedsigni fi cantly. Despit e that they have no t reached an end to the p ossibilities, which are con ceivable.The re are alwa ys new challenges for further imp rovements of performanc e and extend ing the scopeof applicati ons. Con sidering future uses o f GC in food analys is, the mai n trend fores een issucces sive replacemen t of conventional detection approac hes by MSD s employin g vario us typesof mass analyz ers. Fast GC –MS can be introdu ced in many applicati ons; thanks to the spectralresol ution of co-el uting compo unds that can compe nsate for low er GC resolution obtai ned in h igh-speed separa tions.

ACKNOWLEDGMENTS

This chapte r was financia lly suppor ted by the Mini stry of Edu cation, Youth and Sp orts of the CzechRep ublic (project MSM 604 6137305) .

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