Lecture 4. Detectors for GC.

DETECTORS FOR GC.

General requirements: sensitivity; low noise; wide linear range; stable. The most important GC detectors are:

Thermal conductivity detector (TCD). The TCD is not as sensitive as other detectors but it is non-specific and non-destructive. Flame-ionization detector (FID). The !D is e"tremely sensitive with a large dynamic range# its only disadvantage is that it destroys the sample. Electron-ca ture detector (ECD). The $CD is as sensitive as the !D but has a limited dynamic range and finds its greatest application in analysis organic molecules that contain electronegative functional groups# such as halogens# phosphorous# and nitro groups.

Other ty e! o" detector!# %tomic-emission detector &%$D' (itrogen-phosphorus detector &()D' lame-photometric detector & )D' )hotoioni*ation detector &)!D'

Lecture 4. Detectors for GC.

Thermal conductivity detector (TCD). % TCD detector consists of an electrically-heated wire or thermistor &tungstenrhenium wire'. The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes in thermal conductivity# such as when organic molecules displace some of the carrier gas# cause a temperature rise in the element which is sensed as a change in resistance. Response is universal and proportional to concentration. ,est gases for TCD: -. or -e# because of highest thermal conductivity &/.+0/ and /.+1+ 23&45m5s'# respectively# for (. /./.1'. or helium and hydrogen the temperature conductivity lowers when solute is eluted. The sensitivity is inversely proportional to flow rate: The detector is more sensitive at lower flow rates. Detection limit: 1// pg3ml. 6inear response range: 7 +/8. Gas flow out

Gas flow in

ilament To increase sensitivity the temperature of the bloc9 should be maintained at lowest temperature that allows all the solutes remain gaseous.

Lecture 4. Detectors for GC.

Flame-Ionization Detector! (FID). The eluate is burned in a mi"ture of hydrogen and air. Carbon atoms &e"cept -C:; and -C;;-' produce C- radical# which are thought to produce C-;< ions in the flame. The ions are collected by cathode. %mount of ions is proportional to the number of carbon atoms &=+ in +////'# and therefore# the response is proportional to sample mass. ,est gases for !D: -e# (.# %r. Detection limit: . pg3s. !t is reduced 8/> when nitrogen is used as a carrier gas instead of helium; nitrogen is often used as a makeup gas before the eluate enters the detector. 6inear response range: 7+/0 (ot detectable: inorganics &including water'# C;# C;.

Lecture 4. Detectors for GC.

Electron-Ca ture Detector (ECD).

The $CD uses a radioactive ,eta emitter &electrons' to ioni*e some of the carrier gas and produce a current between a biased pair of electrodes. @hen organic molecules that contain electronegative functional groups (Hal, conjugated -C=O, -CN, -NO , organometall!cs" pass by the detector# they capture some of the electrons and reduce the current measured between the electrodes. The detector responds by varying the frequency of voltage pulses between the anode and cathode to maintain a constant current. 6ow sensitivity for: hydrocarbons# -;-# 9etones. Detection limit: %s low as 8 fg3s 6inear response range: +/1

Lecture 4. Detectors for GC.

$tomic emi!!ion detector ($ED).

The detector is able to simultaneously determine the atomic emissions of many of the elements in analytes that elute from a GC capillary column &called eluants or solutes in some boo9s'. %s eluants come off the capillary column they are fed into a microwave powered plasma &or discharge' cavity where the compounds are destroyed and their atoms are e"cited by the energy of the plasma. The light that is emitted by the e"cited particles is separated into individual lines via a photodiode array. The associated computer then sorts out the individual emission lines and can produce chromatograms made up of pea9s from eluants that contain only a specific element.

)erforms quantitative analysis using nearly constant response factors. Aou can calibrate with any readily available compound containing the element. %naly*es higher-boiling compounds with operation up to 18/ BC. Conducts trace-level analysis. ive times more sensitive than GC- !D for carbon.

Very expensive!!! 08/// CDD

Lecture 4. Detectors for GC.

%itro&en- ho! horu! detector.

The (itrogen )hosphorus Detector & ()D ' is similar in design to the !D# e"cept that the hydrogen flow rate is reduced to about ? ml3min and an electrically heated thermionic bead & al9ali bead# containing Eb.D;1 ' is positioned near the Fet orifice. (itrogen or phosphorus containing molecules e"iting the column collide with the hot bead and undergo a catalytic surface chemistry reaction. The ions created in this reaction are attracted to a collector electrode# amplified# and output to the data system. The ()D is commonly used to detect pesticides# herbicides# and drugs of abuse because it responds to (-) compounds about +//#/// stronger than normal hydrocarbons# ma9ing it very selective. Detection limit: +// fg3s. 6inear response range: +/8

Lecture 4. Detectors for GC.

Flame- hotometric detector (F'D).

The )D uses one of two available band pass filters over a photomultiplier tube &)HT' to selectively detect compounds containing sulfur or phosphorus as they combust in the hydrogen flame. @hen compounds are burned in the )D flame# they emit photons of distinct wavelengths &D. ?I1 nm# -); 8+/-8.G nm doublet'. ;nly those photons that are within the frequency range of the filter specifications can pass through the filter to the )HT. The )HT converts the photons it JseesK through the bandpass filter to an analog signal# which is acquired by the )ea9 Dimple data system.

Detection limit: + L +/ pg3s &) L D'. 6inear response range: +/1 L +/?

Lecture 4. Detectors for GC.

'hotoionization detector ('ID). The photoioni*ation detector &)!D' utili*es ultraviolet light to ioni*e gas molecules# and is commonly employed in the detection of volatile organic compounds &M;Cs'. Duitable for aromatics and unsaturated compounds. 6ow sensitivity to halocarbons and hydrocarbons.

Detection limit: /.+ppm &for isobutylene'.

Lecture 4. Detectors for GC.

($SS S'ECTRO(ETR). GC-(S and *C-(S.

HD-detector can be combined with GC or 6C chromatograph. This is called #$p#enated tec#n!%ue. or technical reasons GC-HD is easier to build up. The mass spectrometer is an instrument designed to separate gas phase ions according to their m3* &mass to charge ratio' value.

( + e- , (+ + -e-

The mass spectrometer includes: O % vacuum system O Tools to introduce the sample &6C# GC P' O Tools to produce the gas phase ions from the sample molecules O Tools to fragment the ions# in order to obtain structural information# or to get more selective detection O % detection system O Doftware and computing &#e anal$'er uses electrical or magnetic fields# or combination of both# to move the ions from the region where they are produced# to a detector# where they produce a signal which is amplified. &#e anal$'er is operated under high vacuum# so that the ions can travel to the detector with a sufficient yield. &#e mot!on and separat!on of !ons !s (ased on electr!cal or magnet!c f!elds. )t !s t#e mass to c#arge rat!o, and not onl$ t#e mass, *#!c# !s of !mportance.
I

Lecture 4. Detectors for GC.

GC-(S inter"acin&. The pressure inside HD is =+/-8 torr. %n interface should eliminate the solvent# generate gas phase ions# and ensure pressure reduction from atmospheric to high vacuum. +et separator (t#e eluent flo* !s spl!t",

D!rect coupl!ng (eluent flo* !s - ml.m!n",

+/

Lecture 4. Detectors for GC.

*C-(S inter"ace!.

separates the sample from the solvent; allows the introduction of the sample in the form of dry particles into the high vacuum region.

Host common approaches nowadays are:

Atmospheric pressure ionisation (API) technique: solvent elimination and ionisation steps are combined in the source and ta9e place at atmospheric pressure; %)! is soft ioni*ation method with almost no fragmentation# and only molecular ions are observed in the spectra. %)! includes: ESI L electrospray ioni*ation; $'CI L atmospheric pressure chemical ioni*ation. Electron impact ionisation (EI): the solvent elimination and ionisation steps are separate; usually coupled with GC; due to high energy electron impact &0/ M'# e"tensive fragmentation of the molecule occurs# thus providing information about structure. $lectron impact is of interest: for molecules which do not ioni*e with %)! technique; when an electron impact spectrum is necessary# since it provides spectral information independent of the sample introduction technique &GC or 6C# or direct introduction' and instrument supplier; $! is the hardest method of ioni*ation# and provides high fragmentation.
++

Lecture 4. Detectors for GC.

(a!! de"inition. re!olution. accuracy.

$vera&e ma!!# --/.0123 )t !s (ased on t#e a/erage atom!c masses. %ominal ma!!# --/ )t !s calculated on t#e nom!nal mass of most a(undant !sotopes. E4act (monoi!oto ic) ma!!# --/.5-26 )t !s calculated on t#e e0act mass of most a(undant !sotopes. The mass spectrometer measures the e"act mass. The value is slightly different from the e"pected ..../.0N and .+I./.0N because these spectra were obtained with a %uadrupole !nstrument# which does not provide sufficient mass resolution and mass accuracy for obtaining the e"act mass. The ne"t smaller pea9s correspond to the C+? and Cl?0 isotopes. $"ample of high resolution and high accuracy:

% high resolution instrument &t!me of fl!g#t, sector, 1&23P'# properly used with a reference compound provides the mass information with an accuracy better than 8ppm# which is enough to unambiguously determine the elemental composition.

+.

Lecture 4. Detectors for GC.

($SS $%$*)7ERS.
the most widely used analy*er due to its ease of use# mass range covered# good linearity for quantitative wor9# resolution and quality of mass spectra. %ll this for a relatively accessible price. 9or:in& ma!! ran&e# +/ to 1/// %.H.C. Re!olution# usually operated at a resolution : +///# but resolution can be reasonably pushed up to 1/// &resolution E : m 3 @-H' (a!! accuracy# /.+ to /.. %.H.C. Scan ! eed# up to 8/// %.H.C per second. The life time of an ion from its formation to detection is 8/ - +// microsecond.

The 8uadru ole analyzer#

The quadrupole is composed of two pairs of metallic rods. ;ne set of rod is at a positive electrical potential# and the other one at a negative potential. % combination of ! and R" &radio frequency' voltages is applied on each set .

4 t =4 dc 4 rf cos t

4 t =4 dc 4 rf cos t

+?

Lecture 4. Detectors for GC.

;o< it <or:!. E voltage applied on the quadrupole acts li9e a hi#h pass $ilter: The low m.' ions have a greater acceleration rate so the wave for these ions has a greater amplitude. !f this amplitude is great enough the ions will collide with the electrodes and can not reach the detector. The low m.' value cutoff of the quadrupole is changed by adFusting the E potential or the E frequency. %ny ions with a m.' greater than this cutoff are transmitted by the quadrupole. DC voltage combined with the E potential acts li9e a lo% pass $ilter: -igh m.' ions do not refocus as quic9ly during the E cycle. -igh m.' ions slowly drift away from the center of the quadrupole under DC potential. %t the end of the analy*er# high m.' ions are too far off-a"is to stri9e the detector. 6ow m.' ions are not affected# because their traFectories are stabili*ed by E . or a given amplitude of the DC and E voltages# only the ions of a given m.' &mass to charge' ratio will resonate# have a stable traFectory to pass the quadrupole and be detected. ;ther ions will be destabili*ed and hit the rods. The performance &i.e. ability to separate two adFacent masses across the applicable range' depends on the quad geometry# on the electronics# on the voltage settings and on the quality of the manufacturing. !ncreasing the resolution means that fewer ions will reach the detector# and consequently impacts the sensitivity. The quadrupole is scanned with DC3E : constant; the resolution depends on the slope of the scan line. The Hathieu stability diagram.

!f the continuous voltage DC is switched off# the scan line is the Q a"is: @e have now a transfer only device.
+1

Lecture 4. Detectors for GC.

SC$% and SI( mode!. D!H - single ion monitoring mode &or D!E - single ion recording': the amplitudes of the DC and E voltages are set to observe only a specific mass# or a selection of specific masses; D!H mode provides the highest sensitivity for specific ions or fragments# since more time can be spent on each mass; that time can be adFusted; it is called the d*ell t!me; the mass window for observing an ion in D!H mode can be adFusted# in order to compensate small mass calibration shift. This is the span factor. DC%( mode: the E 3DC ratio is constant; the amplitudes of the DC and E voltages are ramped to obtain the mass spectrum over the required mass range; the sensitivity is a function of the scanned mass range# scan speed# and resolution. @ith most 6C3HD instrument# it is possible to do positive3negative switching# in order to analy*e in the same run molecules that will ioni*e in positive and negative modes.

+8

Lecture 4. Detectors for GC.

To &et !tructure in"ormation# (S=(S. Tri le 8uadru ole!. The analy*er of a Jtriple quadK instrument consists in two quadrupoles# separated by a collision cell. Duch a configuration is often referred as a Rtandem in spaceR instrument.

Collision induced dissociation: ions enter a collision3reaction cell. % collision3reaction gas such as argon or helium is then bled into the cell. The E -only field has the effect of focusing the ions# which then collide and react with molecules of the collision3 reaction gas to produce fragmented product ions. 2odes to use tr!ple %uadrupoles,

irst two e"periments are Jsingle quadrupole modeK. Decond two e"periments can be used for quantitation# because two subsequent analy*ers improve selectivity and signal-to-noise ratio. &#e l!m!t!ng factor for %uant!tat!on *!t# an$ 23 !s 5!on suppress!on6, and !t s#ould (e al*a$s accounted.

+G

Lecture 4. Detectors for GC.

Ion tra analyzer.

The quadrupole ion trap &Q!T' mass analy*er consists of three hyperbolic electrodes: the ring electrode# the entrance endcap electrode and the e"it endcap electrode. These electrodes form a cavity in which it is possible to trap and analy*e ions. ,oth endcap electrodes have a small hole in their centres through which the ions can travel. The ring electrode is located halfway between the two endcap electrodes.

,y ramping the E voltage# or by applying supplementary voltages on the end cap electrodes# or by combination of both# it is possible to: destabili*e the ions# and eFect them progressively from the trap; 9eep only one ion of a given m3* value in the trap# and then eFect it to observe it specifically; 9eep only one ion in the trap# fragment it by inducing vibrations# and observe the fragments; repeat the last operation a few times to progressively fragment the ions. % helium gas of about + mTorr within the trapping volume contracts the ion traFectories to the center of the trap and reduces the 9inetic energy of the ions. The ion pac9et is eFected more quic9ly and efficiently than a diffuse cloud of ions may be eFected# thus improving resolution.

+0

Lecture 4. Detectors for GC.

;o< it <or:!. Dtability domain of the ions in the trap &Hathieu graph':

1 e81 . mr / . N eDC 9= . . mr / *#ere e = c#arge , m = mass r / = rad!us (et*een caps , = 81 fre%uenc$ , 81 = rad!o fre%uenc$ /oltage , DC = d!rect current /oltage 7=
The ions are stable in the trap if the Q value lies between /.? and /.I This parameter has important consequences when doing fragmentation &in other words HD3HD' in a trap: the fragments which are smaller than about one third of the precursor ion will have a Q value 7 /.I and will be lost. ,y increasing the E voltage it is possible to e"tract an ion &m3* is fi"ed' from the trap. !t is also possible to play with the voltage on the end cap electrodes# which affects the % value. ,y combining both actions# it is possible to eliminate from the trap the high masses and low masses# and 9eep in the trap only the desired ions. 8esolut!on and performance in an ion trap are dependent upon the charge density of ions in the trap: !f too many ions are present at the same time in the trap# the electrical fields are distorted# and also collisions between the ions may occur# leading to une"pected dissociation or chemical reactions. % short pre-scan is achieved automatically to determine the optimum ion sampling time so that enough# but not too many ions will be introduced. The optimum is to have between ?// and +/// ions present in the trap. Eesolution of Q!T is similar to this of Q%# but can be greatly improved by scanning narrow window for longer time. Scan ! eed# few hundred Daltons in a fraction of a second. Re!olution# up to 8///.

5.1 > ? > 5.3

+N

Lecture 4. Detectors for GC.

The Time o" Fli&ht $nalyzer. (a!! ran&e# more than 8// 9Da. !t (ecomes !ncreas!ngl$ d!ff!cult to d!scr!m!nate (et*een t!mes of arr!/al at t#e detector as t#e m.' /alue (ecomes large: /er$ large molecules are d!ff!cult to !on!'e: ;s!ng an !on!sat!on tec#n!%ue *#!c# produces mult!pl$ c#arged !ons, l!ke electrospra$ !on!sat!on, e0tends t#e *ork!ng range of t#e &O1 anal$'er. Re!olution# up to +//// @-H with a T; instrument. (a!! accuracy# better than 8 ppm; that allows unambiguous formula determination of small organic molecules !ons formed in an ion source are e"tracted and accelerated to a high velocity by an electric field into an analy*er consisting of a long straight Sdrift tubeT. The ions pass along the tube until they reach a detector. %fter the initial acceleration phase# the velocity reached by an ion is inversely proportional to the square root of its m.' value.

!n order to increase the resolution# the ion traFectory is bent by an electronic mirror# t#e reflectron. @hen going through the reflectron# the dispersion of ions of the same m.' value is minimi*ed# leading to a great improvement of resolution. or HD3HD# the T; is associated with Quadrupole &QT; '# or to another T; &T; -T; ' or to !on Trap &Q!T3T; '.

+I

Lecture 4. Detectors for GC.

Ionization method!. Electro! ray ionization (ESI).

The compound of interest must be ioni*ed in solution &it is possible to use post column addition to get appropriate conditions'.

-)6C line is connected to the electrospray probe# which consists of a metallic capillary surrounded with a nitrogen flow. !n the electrical field# at the tip of the capillary# the surface of the droplets containing the ioni*ed compound will get charged. Due to the solvent evaporation# the si*e of the droplet reduces# and the density of charges at the droplet surface increases. The repulsion forces between the charges increase until there is an e"plosion of the droplet. This process repeats until analyte ions evaporate from the droplet.

Hultiply charged ions can be obtained depending on the chemical structure of the analyte. This is why $D! is the technique of choice for analy*ing proteins and other biopolymers on quadrupole or ion trap analy*ers. Ty ical ion! roduced @y electro! ray ioni!ation# <os!t!/e mode, UH<-V< protonated molecule UH<(aV <# UH<4V < P adducts UH<C-?C(<-V < protonated# < solvent adducts Negat!/e mode, UH--V - deprotonated molecule UH<-C;; -V -# P adducts
./

Lecture 4. Detectors for GC.

$tmo! heric re!!ure chemical ionization ($'CI).

The -)6C line is is connected to the %)C! probe which consists normally of a glass capillary surrounded with a nitrogen flow used for mobili*ation. % metallic needle at potential of a few 9ilovolts is located close to the probe &Rcorona discharge electrodeR'. The eluent vapours are ioni*ed by the corona effect# and react chemically with the analyte molecules in the gas phase. !f the eluent is not suitable for ioni*ation# a small amount of modifier &reagent gas' can be added &e.g. methane# which forms e"cellent proton donor C-8<'. %pplicability conditions: the analyte must be volatile and thermally stable; the mobile phase must be suitable for gas phase acid-base reactions: - for wor9ing in positive mode# the proton affinity of the analyte must be higher than the proton affinity of the eluent &in other words# the analyte can catch a proton from the protonated solvent'

S;+ + ( , S + (;+

- for wor9ing in negative mode# the gas phase acidity of the analyte must be lower than the gas phase acidity of the eluent &in other words# the analyte can give a proton to the deprotonated solvent'

AS B ;C- + ( , S + A( B ;CB

.+

Lecture 4. Detectors for GC.

$'I !ource de!i&n. Curta!n gas des!gn, a flow of heated gas is protects the orifice plate and helps in the desolvatation.

The sample is introduced through a series of differentially pumped stages. This maintains the large pressure difference between the ion source and the mass spectrometer without using e"tremely large vacuum pumps. !n addition a drying gas is used to brea9 up the clusters that form as the solvent evaporates. ,ecause the analyte molecules have more momentum than the solvent and air molecules# they travel through the pumping stages to the mass analy*er. J)epper potK design: the ions have to travel through the channels of the metallic counter electrode# which is heated. The counter electrode protects the sampling cone orifice and helps in ion desolvatation.

..

Lecture 4. Detectors for GC.

Ionization method! com ari!on#

Ionization method Ty ical $nalyte! Eelatively small volatile Eelatively small volatile )eptides )roteins nonvolatile Sam le Introduction GC or liquid3solid probe GC or liquid3solid probe 6iquid Chromatography or syringe (a!! Ran&e to +#/// Daltons to +#/// Daltons to .//#/// Daltons (ethod ;i&hli&ht! -ard method versatile provides structure info Doft method molecular ion pea9 UH<-V< Doft method ions often multiply charged

$lectron !mpact &$!'

Chemical !oni*ation &C!'

$lectrospray &$D!'

ast %tom ,ombardment & %,' Hatri" %ssisted 6aser Desorption &H%6D!'

Carbohydrates ;rganometallics Dample mi"ed in viscous )eptides matri" nonvolatile )eptides )roteins (ucleotides Dample mi"ed in solid matri"

Doft method to but harder G#/// than $D! or Daltons H%6D! to Doft method 8//#/// very high Daltons mass

%: negative %)C!; ,: negative %)C! with in source fragmentation; C: electron impact ioni*ation.
.?

Lecture 4. Detectors for GC.

$lcohol %n alcoholWs molecular ion is small or non-e"istent. Cleavage of the C-C bond ne"t to the o"ygen usually occurs. % loss of -.; may occur as in the spectra below. 1-'entanol CD;/-O (9 E 66./D

$ldehyde Cleavage of bonds ne"t to the carbo"yl group results in the loss of hydrogen &molecular ion less +' or the loss of C-; &molecular ion less .I'. 1-'henyl--- ro enal C3;6O (9 E /1-./0

$l:ane Holecular ion pea9s are present# possibly with low intensity. The fragmentation pattern contains clusters of pea9s +1 mass units apart &which represent loss of &C-.'nC-?'. ;e4ane C0;/F (9 E 60./6

.1

Lecture 4. Detectors for GC.

$mide )rimary amides show a base pea9 due to the Hc6afferty rearrangement. 1-(ethyl@utyramide CD;//%O (9 E /5/./D

$mine Holecular ion pea9 is an odd number. %lpha-cleavage dominates aliphatic amines. n-Gutylamine CF;//% (9 E 21./1

%nother e"ample is a secondary amine shown below. %gain# the molecular ion pea9 is an odd number. The base pea9 is from the C-C cleavage adFacent to the C-( bond. n-(ethyl@enzylamine C6;//% (9 E /-/./6

.8

Lecture 4. Detectors for GC.

$romatic Holecular ion pea9s are strong due to the stable structure. %a hthalene C/5;6 (9 E /-6./2

Car@o4ylic $cid !n short chain acids# pea9s due to the loss of ;- &molecular ion less +0' and C;;&molecular ion less 18' are prominent due to cleavage of bonds ne"t to C:;. --Gutenoic acid CF;0O(9 E 60.53

E!ter ragments appear due to bond cleavage ne"t to C:; &al9o"y group loss# -;E' and hydrogen rearrangements. Ethyl acetate CF;6O(9 E 66.//

.G

Lecture 4. Detectors for GC.

Ether ragmentation tends to occur alpha to the o"ygen atom &C-C bond ne"t to the o"ygen'. Ethyl methyl ether C1;6O (9 E 05./5

;alide The presence of chlorine or bromine atoms is usually recogni*able from isotopic pea9s. /-Gromo ro ane C1;2Gr (9 E /-1.55

Hetone HaFor fragmentation pea9s result from cleavage of the C-C bonds adFacent to the carbonyl. F-;e tanone C2;/FO (9 E //F./3

&#e e0amples are from #ttp,..***.c#em.ar!'ona.edu.massspec.e0ample=#tml.e0amples.#tml

.0

Lecture 4. Detectors for GC.

?I$*IT$TIJE $%D ?I$%TIT$TIJE $%$*)SIS. ?ualitative analy!i!.

@e typically thin9 of GC and 6C as quantitative tools. !n general# chromatography is a JblindK method. !t indicates the presence of a substance# but not what it is. Qualitative data can also be obtained even with non-discriminating detectors. Eetention data L can be used for qualitative wor9. The retention time is characteristic of substance# compared to a standard. Eeproducibility of absolute retention data depends on several e"perimental conditions. %dFusted retention time or volume can be used for identification of un9nown compound. or a homologous series MWE can be accurately determined by:

ln 4 > N = a (N

therefore# for an un9nown carbon number:

ln 4 ? ln 4 N@ 0 = N + N . N + ln 4 N ln 4 N@ N . 0 N +

This can be used for straight chain compounds only. or non-paraffins the inde" value can be calculated li9e it was a paraffin. %bsolute retention inde":

ln 4 ? ln 4 N@ ) < = N + N . N + ln 4 N ln 4 N@ N . 0 N +

and (+ and (. are reference paraffins.

.N

Lecture 4. Detectors for GC.

4ovatWs retention inde" is a modification of the absolute inde":

) A =+// ) < = ln 4 ? ln 4 N@ =+// N + +// N . N + ln 4 N ln 4 N@ N . 0 N +

This inde" is available from reference tables for large number of compounds at different temperatures. Eelative retention can be used to identify your own compound:

;nly single standard is required.

t > 8 u 4 > 8 u k u r ! , std = = = t > 8 std 4 > 8 std k std

Eelative retention should be measured with internal standard L a standard is a part of the sample or added to it before separation. Dtandard should elute somewhere in the middle of chromatogram# and sample si*e should be small. Malues may vary from column to column# but remains quite constant with the same column. Dimple retention time data is suitable for simple assays li9e process quality control# when it is already 9nown# what the sample is# and how many components are present. @hen new un9nown compound is observed# the retention time does not give much of positive information. Eetention plot for homologous. Mery often semi log plot of retention time vs carbon number give a linear relationship. This can be used to pic9 out potential series members. !n 6C# the eluent can be varied# and elution orders and times will change. @ith additional standards this provides more information about analyte structure. The detectors provide some information about analyte structure: T!E# CM3Mis# $mission# HD.

.I

Lecture 4. Detectors for GC.

?uantitative analy!i!. %ny detector produce a signal. The signal intensity is proportional to sample amount or sample concentration. The pea9 area is the quantitative characteristic of the sample. The response is substance dependent and# therefore# standards must always be used. or quantitative measurements the pea9s of interest should be well resolved# with distinct pea9Ws beginning# end and ma"imum. %ppro"imation for very sharp and symmetrical pea9s# which are usually produced by capillary columns: peak #e!g#t is proportional to concentration. <eak area is more reliable source of information. )ea9 area can be measured automatically by detector3interface program# or can be calculated by operator. )ea9 shape and tailing should be ta9en into account to get accurate values.

Conversion of detector response &pea9 area' to sample concentration can be done by following methods: $DTD L e"ternal standard method; !DTD L internal standard method.

?/

Lecture 4. Detectors for GC.

ESTD. E4ternal !tandard cali@ration method. Hethod of e"ternal standard assumes that response is linearly proportional to concentration. The area of linear response should be determined with standards of different concentrations. !f the same inFection volume was used for both un9nown and standard# then:

9rea unkno*n conc unkno*n = conc kno*n 9rea kno*n

Eequirements for proper use: standard solution contains all analytes; standard analytes are of similar concentration as un9nowns; standard and sample matri" are as similar as possible; analysis conditions are identical. $X%H)6$. Compound: DH%) Dtandard concentration: /.. mg3ml !nFection volume &both standard and un9nown': 8 Yl. %rea DH%)std : +N// units %rea DH%)un9 : 1+// units ind concentration of DH%) in un9nown sample. Dolution: Cun9: /.. Z1+//3+N// : /.18G mg3ml $DTD calibration plot. )ea9 area is assigned to certain concentration of the sample.
Dtandard + . ? 1 Conc.# ng3ml 8/ +// .// 1// %rea +?8// .1/// 8I/// IG///

ESTD calibration
120000 100000 80000 peak area 60000 40000 20000 0 0 100 200 300 400 500 conc., ng/ml

?+

Lecture 4. Detectors for GC.

ISTD. Internal !tandard cali@ration method. !DTD is the most reliable method. The 9nown substance at a constant concentration is added to all standards and samples. Eequirements for an internal standard: standardWs concentration in all samples is constant; standard is stable and measurable under analysis conditions; standard does not overlap with sample components. !nternal standard can be introduced as: weighted portion of the standard; 9nown volume of stoc9 solution. !DTD calibration plot. Eelation %reaC(43%rea!DT is assigned to certain concentration of the sample.
Dtd + . ? 1 conc.# ng3ml %reaC(4 8/ +?8// +// .1/// .// 8I/// 1// IG/// %rea!DT ??.// ?+.// ?81// .I8// %reaC(43%rea!DT /#1/0 /#0GI +#GG0 ?#.81

To create !DTD calibration plot# the !DT concentration is maintained constant while changing the concentration of analyte.
ISTD calibration

3,5
AreaUNK/AreaIST

3 2,5 2 1,5 1 0,5 0

0 100 200 300 400 500 conc., ng/ml

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