高等食品分析(Advanced Food Analysis) VII. GAS …web.nchu.edu.tw/pweb/users/mushroom/lesson/6402.pdf · 高等食品分析(Advanced Food Analysis) 90 VII. GAS CHROMATOGRAPHY

高等食品分析(Advanced Food Analysis)

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VII. GAS CHROMATOGRAPHY

*Based on the stationary phase: Gas-solid chromatography (GSC), GLC, Bonded phase GC (bonded or cross-linked phases).

Sample is injected and vaporized onto the chromatographic column and eluted by the flow of an inert gaseous mobile phase.

The mobile phase does not interact with molecules of analyte and its function is to transport the analyte through the column.

Solve the conventional distillation problem: Benzene (b.p. 80.1) and cyclohexane (80.8°C). => Easily separated by GC.

*Gas-solid chromatography (GSC): Based on adsorption of gaseous substances on solid surfaces.

Useful for separation of permanent gases and low boiling materials, up to a molecular weight of about 150.

Examples: Air, hydrogen sulfide, carbon disulfide, nitrogen oxides, carbon monoxide, carbon dioxide and the rare gases.

Performed with both packed and open tubular columns. Porous layer open tubular (PLOT) columns: A thin layer of

adsorbent is affixed to the inner walls of the capillary. Adsorbents: Alumina, silica gel, carbon, molecular sieves and

porous polymers. Quite limited in applicability: Due to mainly the tailing caused by

non-linear adsorption isotherms and partially excessive retention of reactive gases and surface catalysis.

* Adsorbents (solid phases):

1) Alumina: Modification needed due to its high polarity and highly catalytic activity.

Alumina coated with 10% Na2SO4, Na2MoO4, NaCl or Al2 (SO4)3 were found to be useful for separation of cis-trans isomers, chlorobenzene and various dichlorobenzenes.

Alumina/KCl porous layer open tubular (PLOT) columns are excellent for separating C1-C10 hydrocarbons.


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2) Silica gel: Modification needed due to its high polarity and highly catalytic activity.

Porasil is a porous spherical bead form of silica. Zipax is a related material in which the beads have impervious

silica cores surrounded by a porous layer. Silica surfaces were modified by salt, by chemical bonding of

materials such as silanes and by hydrothermal treatment. Hydrothermal treatment involves contact with steam at 850°C for

about 24 hr to enlarge the pores. Chemical modification: i) Silanization of the surface hydroxyl groups with chloro-

trimethyl- or dichlorodimethylsilane renders the surface non-polar and non-specific.

ii) Esterification: MeOH and EtOH were bonded to the surface. Example: Modified Porasil => Durapak, stable up to 150°C,

similar as the liquid stationary phase Carbowax 400. XAD amberlite resins are agglomerated microspheres, beads of

nearly continuous solid phase and pore phase, and widely used for sampling and trapping organics.

3) Carbon: Active carbons are the most difficult solids to prepare in reproducible form due to its non-polar surface as well as microporosity that leads to tailing.

Graphitization overcomes both polarity and microporosity problems and gives C with a high homogeneity of surface.

The surface of carbon can be modified with phthalcyanins to reduce the elution time.

Packed capillary of graphitized C is non-specific, has high permeability, small pressure drop across the column and high selectivity for certain systems, e.g. geometrical and structural isomers such as o-, m- and p-cresols and xylenes and polar compounds such as alcohols, and isotopic systems e.g. deuteroacetone and acetone.


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4) Molecular sieves: Aluminum silicate ion exchanger, whose pore size depends on the kind of cation present.

Particle sizes available from 40-60 to 100-120 mesh. Pore size of commercial molecular sieves: 4, 5, 10 and 13Å. Example: A 6-ft, 5Å packing at room temperature will separate a

mixture of helium, oxygen, nitrogen, methane and carbon monoxide in the order given.

5) Porous polymers: Beads of uniform size manufactured from styrene cross-linked with divinylbenzene.

Useful for separation of gaseous polar species such as hydrogen sulfide, oxides of nitrogen, water, carbon dioxide, methane and vinyl chloride.

Tenax-GC is a new porous polymer packing material based on 2,6-diphenyl-p-phenylene oxide and suitable for separation of high boiling polar compounds such as alcohols, polyethylene glycols, diols, phenols, amines, ethanolamines, amides, aldehydes and ketones.

*Gas chromatograph:


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*Carrier gas supply and controls: Viscosity and thermal conductivity of common carrier gas Gas Mol. wt Viscosity Thermal conductivity P, × 106 cal/sec•cm [(°C/cm) × 106] CO2 44.01 189 49 Ar 39.95 269 50 O2 32.00 256 77 N2 28.01 219 73 He 4.00 228 388 H2 2.02 108 490 Carrier gas: Must be chemically inert, including He, Ar, N2, CO2

and H2 and supplied from gas cylinder. The gases with smallest diffusion coefficients will give the best

column performance. High molecular weight gases, N2, Ar, CO2, give lower flow rates than hydrogen and helium.

For fast analysis, the ratio of viscosity to diffusion coefficient should be minimal, and therefore H2 and He are ideal, while to reduce diffusion effects that cause peak broadening, higher flow rates are used.

Hydrogen: Fire- and explosion-hazard, and chemical reactivity with reducible and unsaturated samples.

In practice, N2 for flame ionization detector (FID) and nitrogen- phosphorus detector (NPD); Ar for electron capture detector (ECD); He or H2 for katherometer detector or thermal conductivity detector (TCD).

Additional gases may be required for detectors, e.g. air or O2 and H2 for FID.

Associated with gas supply are pressure regulators, gauges and flow meters. The carrier gas system contains a molecular sieve to remove water or other impurity.

Flow rate is measured using a soap-bubble flow meter or linear velocity is measured by injecting the butane from the lighter.


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*Sample introduction/injector system:

Use of syringe to inject a liquid or gaseous sample through septum into an injection port or the use of valve to introduce sample.

Sample sizes: Few tenths of l to 20 l for packed, or ~10-2 l for capillary columns without split.

For packed columns: On-column and glass insert system. For capillary column: Split/splitless, direct injection (DI) and

on-column (OC) systems. The temperature of the injection port is normally higher (30-50°C)

than the final programmed temperature of the oven (column) to ensure the vaporization of the sample prior to getting onto the column.

On-column (OC) injection: For heat sensitive samples. Sample is directly injected onto the column without the heated

injection port. Direct injection: Sample is vaporized inside the injection port but

with no split. Split or splitless: Sample amount < 0.2 l without splitting. If flow-splitter is used, about 30-90% of sample is vented, while

the remainder goes onto the column. The vaporized volume of 1.0 l liquid with a mol. wt of 85 is

about 388 l at 250°C injection port and head pressure 10 psi. If the head pressure is 5 psi, then the vaporized volume will double.

Sample and solvent expansion volume = nRT/P (1) Where n: # moles of solvent and sample. T: Absolute temperature of injector. P: Column head pressure (atm) + 1 atm. R: Gas constant = 82.06 cc atm/mole degree.

Column head pressure:increased => Lower the retention time; decreased => May be better the resolution.


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*Derivatization of samples: For non- or low volatile compounds. Reasons for derivatization: i) To increase volatility of the sample. ii) To reduce thermal degradation of the sample by increasing

thermal stability. iii) To increase detector response by incorporating into the

derivative functional groups which produce higher detector signals such as CF3 for ECD.

iv) To improve separation and reduce tailing.


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*Derivatization of samples:

Most derivatives are thermal stable, although trimethylsilyl derivatives may be decomposed on the stainless steel of an injector port at > 210°C.

Pyridine is the commonly used solvent for derivatization, and act as an acid scavenger and basic catalyst if required. DMF, toluene and methanol are also used.

a) Silylation: Involving the replacement of labile acidic hydrogen in -OH, -COOH, -SH and -NH2 groups with an alkylsilyl group, e.g. SiMe3.

The derivatives are generally less polar, more volatile and more thermally stable.

1) Using trimethylchlorosilane (TMCS):

R-OH + Cl-SiMe3 => R-O-SiMe3 + HCl

2) Using hexamethyldisilazane (HMDS):

2 R-OH + Me3Si-N=N-SiMe3 => 2 (R-O-SiMe3) + N2 Silylation reactions generally proceed very rapidly (within 5 min)

with pyridine as the most frequently used solvent.

Other silylating agents: Substituted acetamides, e.g.

i) BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide),

ii) BSA (the non-fluorinated analog). Both react rapidly and quantitatively under mild conditions using

pyridine or DMF as solvent, forming esters, ethers or N-TMS derivatives.

The main advantage of BSTFA over BSA is that the by- products are more volatile and often elute with the solvent front.

R-OH + BSTFA => R-O-SiMe3 + CF3-CO-NH-SiMe3 or CF3-CO-NH2


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b) Acylation:

1) Forming perfluoroacyl derivatives of alcohols, phenols or amines, mainly for enhanced detector performance using an ECD. An added benefit is the increased volatility.

Using trifluoroacetyl from trifluoroacetic anhydride, TFAA:

R-OH + O-(COCF3)2 —> R-O-CF3

2) N-Fluoroacyl-imidazoles react smoothly to acylate hydroxyl groups and secondary or tertiary amines.

No acids are produced which could hydrolyze the products. The imidazole produced as by-product is relatively inert.

Using N-trifluoroacetylimidazole:

R-OH + CF3-CO- NN

=> R-O-COCF3 + imidazole

c) Alkylation: Addition of alkyl group to an active group.

Esterification to form methyl ester (methylation) is the most useful reaction since they are more volatile.

A number of reagents are available such as diazomethane, BF3- MeOH, H2SO4-MeOH, HCl-MeOH and sodium methoxide (CH3ONa), but boron trifluoride in MeOH is commonly used.

RCOOH + BF3/MeOH => RCOOMe

Other reagents commonly used include pentafluorobenzyl bromide developed for the analysis of acids, amides and phenols using an electron capture detector for enhanced sensitivity.

Dialkylacetals of DMF react instantaneously and quantitatively with acids, amines and amides, barbiturates and on-column derivatization is possible.

Methyl and butyl alkyls are available. The reaction mixture is injected immediately onto the GC without

washing, extraction and also contain no water.


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VII. GAS CHROMATOGRAPHY Flash alkylation: High temp of the injection port is used to form

derivatives on the injection of sample together with an appropriate reagent.

i) Quaternary alkylammonium hydroxides, e.g. tetrabutyl ammonium hydroxide (TBAH) (as 0.2 M solution in MeOH): Mainly for low mol. wt acids to increase retention times.

ii) General purpose reagent trimethylanilinium hydroxide (TMAH): Used when normal methylation might cause confusion with naturally occurring methyl derivatives in biological systems.

*Chromatographic column and oven: Separation process occurring in the column involves equilibrium

established by solutes between stationary and mobile phases. Relative retention of two components will decrease as the

stationary phase (column) temperature increases. Packed columns with larger amounts of stationary phase on the

support material will require higher temperature to obtain elution times equivalent to lower stationary phase loadings.

Decreasing the amount of stationary phase and reducing the column temperature results in the peaks eluted first having poorer separation.

A balance of stationary phase loadings: 10-15% w/w loading for small molecules up to C8 and up to 3-5% w/w for C9-C20.

Once the column was chosen, temperature and carrier gas flow rate are two variables that can be modified to optimize the separation of components.

Electronic pressure control (EPC) for HP 5890 Series II GC can control the flow rate for automatic optimization.

Control of temperature is important in order to obtain reproducible chromatograms.

Oven is operated from ca. 10°C above ambient temp up to 450°C. Temperature programming: Isothermal at programmed, e.g.

50°C/10 min, 5°C /min, 220°C /30 min.


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*Packed columns: Consist of a glass or metal (stainless steel) tube packed with adsorbent particles (GSC) or stationary coated particles (GLC), typically 60-80 or 80-100 mesh size range.

*Supports for packed columns: To provide a uniform, inert support for the stationary phase, and include diatomaceous materials and polytrifluoroethylene (PTFE).

Diatomaceous materials:

1) Pink firebrick-derived materials, e.g. Chromosorb P or Gas Chrom R, suitable for non-polar hydrocarbon compounds.

2) White diatomaceous materials derived from filter aids, e.g. Chromosorb W, Gas Chrom Q.

These support materials contain mineral impurities that promote catalytic reaction, and silanol (Si-OH) groups that are reactive, polar, forming H-bonds with suitable components.

The supports are then treated with HCl to remove minerals and silanized using dimethyldichlorosilane (DMDCS) or hexamethyldisilylazane (HMDS) to block the Si-OH groups with methylated siloxane bonds (Si-O-Si).

PTFE supports, e.g. Chromosorb T, are extremely inert and used for corrosive materials often with fluorocarbon oil (e.g. Kel-F, Fluoropak-80), polyethylene glycol and squalane stationary phase.

*Stationary phases for packed columns: Liquid at the operating temperature of GC columns.

The stationary phase should be thermally stable, unreactive, have negligible volatility, and have a reasonable column life (generally 2 years) over the operating temperature range.

The life of a stationary phase can be extended if use at the temp. of 20-50°C below the recommended maximum.


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*Stationary phases for packed columns: Polar stationary phases: -CN, -CO and -OH. Hydrocarbon-type stationary phases and dialkyl siloxanes are

nonpolar, while polyester phases are highly polar. Polar analytes include alcohols, acids and amines; species of

medium polarity include ethers, ketones and aldehydes. When the polarity of stationary phase matches that of the sample

components, the order of elution is determined by the boiling point of the eluants.

Bonded stationary phase: Stationary phase is bonded to the support through silyl ether linkages, prepared by reaction of the support silanol groups (Si-OH) with chlorosilanes.

*Capillary or open tubular columns: Long narrow tubing coated on the inner surface with about 1 m of stationary phase.

1) WCOT (wall-coated open tubular) columns: The stationary phase is coated directly on tubing surface. 2) PLOT (porous layer open tubular) columns: For GSC. Adsorbent such as Porapak, molecular sieve, aluminum oxide. 3) Micropacked columns: Packed column < 1 mm ID. 4) SCOT (support coated open tubular) columns: Stationary phase is coated on solid support to increase loadings. 5) FSOT (fused-silica open tubular) or new WCOT columns: Fused silica column is preferred due to its low catalytic activity.


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*Capillary columns: The efficiency of SCOT is less than that of WCOT but

significantly greater than that of packed columns. The length of capillaries can be 15, 30, 60 and 120 m. 180, 250 and 320 m capillaries: A sample splitter must be used to

reduce injected sample size. 530 and 750 m capillaries, megabore or wide bore columns:

Tolerate sample sizes that are similar to those for packed columns.

The performance characteristics of megabore OT columns are not as good as those of smaller diameter capillaries but are significantly better than those of packed columns.

*Column conditioning: Condition when a column is first installed or after solvent rinsing. Condition periodically to remove high b.p. components. Overnight conditioning at the maximum operating temp. or 20°C

below provides most stable baseline.


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*Capillary vs. packed columns:

Advantages:

a) Greater separating power: Narrow peaks and faster RT. b) Versatility: One capillary replaces many packed columns. c) Higher reproducibility: More controlled and individually

tested. d) Greater inertness: No support effects. e) Bonded stationary phases: minimal bleed, better longevity,

rinseable. f) Improved accuracy: Symmetrical peaks easier to integrate. g) Lower detectability: Narrower peaks = more response/time. h) Less leakage: Operates at lower pressure.

Disadvantages:

a) Lower sample capacity: ng vs. g. b) Smaller injections required: Lower operating pressure. c) Less tolerance to dirty samples: Required packed inlets/guard

columns. d) Require more knowledge: Such as inlets, pneumatics,

make-up, seals. e) Make-up gas required and high dead volume.


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Packed Capillary Length, m 1 - 6 5 - 105 I.D., mm 2 - 4 0.2 - 0.75 Flow, ml/min 10 - 60 0.5 - 15 Pressure drop, psi 10 - 40 3 - 40 Total effective plates 5,000 (2 m) 180,000 (60 m) Effective plates/m 2,500 (2 mm ID) 3,000 (0.25 mm ID) Capacity 10 g 50 ng (0.25 mm ID) Film thickness, m 1 - 10 0.1 - 7.0

*Guard columns (untreated or deactivated columns): To increase separation efficiency, decrease maintenance

requirements and minimize peak splitting. To protect columns from high mol. wt compounds, inorganic

residues, pyrolyzates, particulates, other contaminants, needle damage and water. => Increase the life of column.

*Detectors: A detector is used to monitor the GC column effluent; it does not identify the components except in the case of specific detectors.

When a component is eluted and detected, the signal produced is proportional to the concentration or mass of that component.

The temp of the detector port is normally higher (30-50°C) than the final programmed temp of the oven (column) to ensure that column effluent will get into the detector.

*Characteristics of the ideal detector: 1) Adequate sensitivity. Sensitivity of current detectors lies in

the range of 10-8 to 10-15 g analyte. 2) Good stability and reproducibility. 3) A linear response to analyte extending over several orders of

magnitude. 4) A temperature range from room temp. to at least 400°C. 5) A short response time independent of flow rate. 6) High reliability and ease of use.


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VII. GAS CHROMATOGRAPHY 7) Similarity in response to all analytes or to one or more classes

of analytes. 8) Nondestructive of sample.

*Compatible unretained compounds for various detectors: 1) TCD/FID—CH4 (methane)

2) ECD—CH2Cl2 (methylene chloride) vapor 3) NPD—Acetonitrile vapor 4) ELCD—Dichlorodifluoromethane vapor 5) MS—O2 or N2 (air) 6) PID—Ethylene or acetylene

Comparison of GC detectors

IRD

MSD

AED

TCD

FID

ECD

NPD(N)

NPD(P)

FPD(S)

FPD(P)

ELCD (C)

10-15

10-12

10-9

10-6

10-3

fg pg ng 痢 mg1 ppt 1 ppb 1 ppm 0.1% 100%

Sensitivity

PID


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*Minimum detection level (MDL): S/N ≥ 2 The level of sample measured by the detector at the peak

minimum, i.e. minimal concentration when the detector signal (S) is at least twice the mean noise signal level (N).

The MDL of the common detectors is as follows: FID: 10-12 -11g/ml NPD: 10-14 -13 g/ml FPD: 10-11 TCD: 10-9 -8 ECD: 10-14 -13

*Type of GC detectors:

a) Thermal conductivity detector (TCD) or katherometer.

A heated thin-wire filament or thermistor (semi-conductor of fused metal oxides) is positioned in the path of the effluent gas from the column; the other is positioned only in the path of the carrier gas.

Both filaments need the carrier gas to flow in, otherwise ≠ ! 2 or 4 resistive elements form the arms of Wheatstone bridge. When a sample component is eluted from the column, the thermal

conductivity of the gas in the detector cell changes, which causes a change in the temperature and resistance of the detecting element, and hence a change in the electrical current flowing in the bridge circuit.

The heated element may be a fine platinum, gold or tungsten wire, or alternatively a thermistor of higher sensitivity.

The advantages of TCD: Its simplicity; large linear dynamic range (~105); reproducible response to all types of organic and inorganic compounds including those not detector by FID; and non-destructive character, which allows collection of solutes after detection.

H2 and He carrier gases are preferred with TCD due to their high thermal conductivity.

However, the sensitivity of TCD is low => 1 ng. 10 ng


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b) Flame ionization detector (FID). Most widely used and generally applicable GC detectors,

particularly with capillary columns due to its great sensitivity. The effluent from the column is mixed with hydrogen and air and

then ignited electrically.

High sensitivity to virtually all organic compounds in proportional

to the number of carbons. Little or no response to water, CO2, the common carrier gas

impurities, giving a zero signal when no sample is present. Gives a stable baseline as it is not significantly affected by

fluctuations in temp or carrier gas flow rate and pressure. Has good linearity over a wide sample conc’n range (about 107). Functional groups such as carbonyl, alcohol, halogen and amine

yield fewer ions or none at all in a flame. FID is 1000-fold more sensitive than TCD. (1 pg vs. 1 ng). Disadvantage of FID: Destructive and only for organic comp’ds. Detection limits is lowered using Ar or He as the carrier gas, using

O2 instead of air for combustion, or providing pure carrier gas (make-up gas) directly into the detector (scavenging).


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c) Nitrogen-phosphorus detector (NPD), alkali flame ionization detector (AFID), flame thermionic detector (FTD), or thermionic detector (TID).

Commonly used in the detection of pesticide residues. The ionization processes in the flame were modified by the

presence of an alkali metal salt which brings an enrichment of the detector response towards P (500 folds) and N (50 folds).

Its response to a P atom is 10 x greater than to a N atom and 104~106 × larger than to a C atom.

To improve detector stability, modified design of NPD uses a glass bead as the alkali source which contains an essentially non-volatile, stable rubidium silicate.

d) Flame photometric detector (FPD).

Applicable in the detection of pesticides containing P (40 pg) and S (200 pg).

The detector consists of a hydrogen-air burner with a photo-multiplier detector optically coupled to it.

P and S in the flame produce emissions at 394 (HPO*) and 526 nm (S2*), respectively. Interchangeable optical filters permit selection of one or the other of the two elements.

e) Electron capture detector (ECD).

Non-destructive detector that utilizes the ability of compounds to capture free electrons.

Radiation source: Titanium foil containing tritium (T or 3H) or 63Ni are used as -ray sources, due to easy shielding against radiation hazard.

ECD is highly sensitive to electrophilic molecules such as halogens, peroxides, quinones and nitro groups, insensitive to amines, alcohols and hydrocarbons.

An important application of ECD is for the detection of chlorinated pesticides, such as DDT and lindane.


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f) Atomic emission detector (AED).

The eluent is introduced into a microwave-energized helium plasma that is coupled to a diode-array optical emission spectrometer, which is capable of detecting emitted radiation from ca. 170 to 780 nm.

The plasma atomizes all the elements in a sample and excites their characteristic atomic emission spectra.

Used to detect elements including C: 495.7 nm, H: 486.1 nm, O: 777.2 nm and Cl: 479.5 nm, and to establish the chemical formula of compounds.

Sensitive to water, S and organo-metals.

g) Electrolytic conductivity detector (ELCD) or Coulson conductivity detector (CCD).

Based on measurement of the electrolytic conductivity of water.

Organic substances in the column effluent are combusted in a stream of O2 and air over a platinum catalyst; the combustion products, such as SO2, SO3 and HCl, are dissolved in water; and the conductivity of the water is measured.

Specific for X-, S- and N-containing compounds.

CO2 is not quickly absorbed by water.

h) Photo-ionization detector (PID).

The column eluent is irradiated with an intense beam of UV radiation varying in energy from 8.3 to 11.7 eV (149 to 106 nm), which causes ionization of molecules.

Application of a potential across a cell containing the ions leads to an ion current, which is amplified and recorded.

For the detection of impurity (organic and inorganic gases) in high purity gases.


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i) Electron ionization detector (EID). GCD system provided by Hewlett Packard is based on HP 5890

GC equipped with a single, built-in electron ionization detector (EID) that is a compact HP 5970 MSD (mass selective detector).

GCD provides information of retention times, area for each chromatographic peak and (mainly EI) mass spectrum.

j) Mass spectrometric detector (MSD) in GC/MS.

k) Infrared Detector (IRD) in GC/FTIR.

Documents

高等食品分析(Advanced Food Analysis) VII. GAS …web.nchu.edu.tw/pweb/users/mushroom/lesson/6402.pdf · 高等食品分析(Advanced Food Analysis) 90 VII. GAS CHROMATOGRAPHY