![CHRONATOG-
302
a)Bulk property detectors, which compare an over all changes in a
physical nrne
operty of the m
phase with and without an eluting solute. Examples of such detectors are
refractive index and
cond
tivity detectors.
(b) Solute property detectors, which respond to a
physical property of the solt
hibited by the pure mobile phase. Such type of detectors are about
a detectahle signal for a few nanograms of sample. Ultraviolet, visible
electrochemical detectors have achieved popularity in this category of detectors
Ihe response time of the detector is critical and should be atleast 10 times smaller
wdth in time units. Ulimate detection limits in any detection system depend
pure solvent for mobile phases.
In general, detectors are also classified as.
(a Those detectors which respond to changes of the solute concentraion in the eluent
e whuch s
1000times more
ensitive. gE
le adsorption, fluorescenc
an
thanthe
nd upon the
availability of
tration sensitive detectors.
eecr
b) Thase detectors which respond to changes of the solute mass flow rate into the dete
detector, ie, signal is
proportion
These detertors are sensitive to the mass flow rate of solute to the
is
the praduct of solute concentration by mobile phase flow rate. If the tlow rate is zero, the signal
hatever the concentration.
The signal in concentration sensitive detector (group a) is proportional to the concentration s
solute in the eluent and is independent of the solute mass flow rate, ie. of the flow velocity cithe
in the detector cell. These detectors are generally non-destructive like the differential refractomet
the
spectrophotometers. When the flow of solvent is stopped, the signal remains constant, but
(a) The baseline and the response factors sometimes depend on the flow rate.
b) The concentration ofsolute in the detector cells changes slowly due to moleculardifusion.
Within these limits, the peak area is inversely proportional to the flow rate and the peak heightrem
almost constant if the flow rate is close to optimum (minimum plate height)
Detectors of group (b) are usually destructive. When the eluent flow is stopped. the signal decres
1o zero very rapidly, with a time constant equal to the detector response time. The peak area is indepeni=
of the flow rate. The peak height increases constantly with increasing flow rate. Electrochemical detas
and the mass
spectrometer belong to this group.
Most LC detectors, however, belong to the group (a). The peak area measured with all
detectars
the integral of the signal vs time, while the sample size is , in the case of concentration sensiuve deterions
the integral of the concenuration vs volume of effluent flowing through the detector cell. Hence. the
pea
area is proportional to the sample size, the response factor and the reverse of the flow rate.
AccordinE
it is
important to stabilise the flow rate.
An ultraviolet photometer operating at fixed wavelengths of 254 or 280 nm is one of
the m
widely used detectors for HPLC, because of its relatively low cost, high sensitivity achieved t07
ma
compounds of chemical and biological interest that absorb UV light, and its insensitivity to
chang*
temperature, flow rate, and mobile phase composition. Compounds absorbing UV light includ? s
stances having one or more double bonds and substances having unshared nonbonded electrons.
example. olefins, aromatics, and compounds containing the carbonyl, thiocarbonyl. nitroso and
azo
CHARACTERISTICs OF DETECTOR
Sensituvity, linear response and the type of response are the various important characterisu
detector to be ued in HPLC. Sensitivity, expressed as the noise equivalent concenration he
of
concentration [C,], which produces a
signal equal to the detector noise level. The lower the
va
for a solute, the more sensitive is the detector for the solute. The linear range of a
detector
regarded as the concentration range over which its response is proportional to the
conce
solute. Whether a detector is universal or sensitive depends upon the type of
respons electv
detector. A universal detector is expected to sense all the constituents of the
sample, itiin1
detector will respond only to certain components of the solute. Selectivity relates the se
ration](https://image.slidesharecdn.com/adobescan01-sep-2022-220913142232-807c2ab7/85/Adobe-Scan-01-Sep-2022-pdf-2-320.jpg)
Adobe Scan 01-Sep-2022.pdf
- 1. DETECTORS The function of the detector in HPLC is to monitor the mobile phase as it emerges from the column. The detector for HPLC consists of a photometric detector fitted with a low volume flow cell (about 10l, suitable). The detector response is usually presented as a record trace displaying the components Deaks on a time scale. A number of detectors used in LC are also suitable in case of HPLC and in act detectors are usually of two types:
- 2. CHRONATOG- 302 a)Bulk property detectors, which compare an over all changes in a physical nrne operty of the m phase with and without an eluting solute. Examples of such detectors are refractive index and cond tivity detectors. (b) Solute property detectors, which respond to a physical property of the solt hibited by the pure mobile phase. Such type of detectors are about a detectahle signal for a few nanograms of sample. Ultraviolet, visible electrochemical detectors have achieved popularity in this category of detectors Ihe response time of the detector is critical and should be atleast 10 times smaller wdth in time units. Ulimate detection limits in any detection system depend pure solvent for mobile phases. In general, detectors are also classified as. (a Those detectors which respond to changes of the solute concentraion in the eluent e whuch s 1000times more ensitive. gE le adsorption, fluorescenc an thanthe nd upon the availability of tration sensitive detectors. eecr b) Thase detectors which respond to changes of the solute mass flow rate into the dete detector, ie, signal is proportion These detertors are sensitive to the mass flow rate of solute to the is the praduct of solute concentration by mobile phase flow rate. If the tlow rate is zero, the signal hatever the concentration. The signal in concentration sensitive detector (group a) is proportional to the concentration s solute in the eluent and is independent of the solute mass flow rate, ie. of the flow velocity cithe in the detector cell. These detectors are generally non-destructive like the differential refractomet the spectrophotometers. When the flow of solvent is stopped, the signal remains constant, but (a) The baseline and the response factors sometimes depend on the flow rate. b) The concentration ofsolute in the detector cells changes slowly due to moleculardifusion. Within these limits, the peak area is inversely proportional to the flow rate and the peak heightrem almost constant if the flow rate is close to optimum (minimum plate height) Detectors of group (b) are usually destructive. When the eluent flow is stopped. the signal decres 1o zero very rapidly, with a time constant equal to the detector response time. The peak area is indepeni= of the flow rate. The peak height increases constantly with increasing flow rate. Electrochemical detas and the mass spectrometer belong to this group. Most LC detectors, however, belong to the group (a). The peak area measured with all detectars the integral of the signal vs time, while the sample size is , in the case of concentration sensiuve deterions the integral of the concenuration vs volume of effluent flowing through the detector cell. Hence. the pea area is proportional to the sample size, the response factor and the reverse of the flow rate. AccordinE it is important to stabilise the flow rate. An ultraviolet photometer operating at fixed wavelengths of 254 or 280 nm is one of the m widely used detectors for HPLC, because of its relatively low cost, high sensitivity achieved t07 ma compounds of chemical and biological interest that absorb UV light, and its insensitivity to chang* temperature, flow rate, and mobile phase composition. Compounds absorbing UV light includ? s stances having one or more double bonds and substances having unshared nonbonded electrons. example. olefins, aromatics, and compounds containing the carbonyl, thiocarbonyl. nitroso and azo CHARACTERISTICs OF DETECTOR Sensituvity, linear response and the type of response are the various important characterisu detector to be ued in HPLC. Sensitivity, expressed as the noise equivalent concenration he of concentration [C,], which produces a signal equal to the detector noise level. The lower the va for a solute, the more sensitive is the detector for the solute. The linear range of a detector regarded as the concentration range over which its response is proportional to the conce solute. Whether a detector is universal or sensitive depends upon the type of respons electv detector. A universal detector is expected to sense all the constituents of the sample, itiin1 detector will respond only to certain components of the solute. Selectivity relates the se ration
- 3. HROMATOGRAPHY jcal n a t u r e ofthe. anse factor and the sample. Ideally dctection limits should he determined direct cach property of a detec ector, and specially the range of lnearty SOME ETECTORS USED IN HPLC from the column w.r.l pure mobile phase. of the principal types of detectors used in HPLC is given helow / a c c o u n t he bulk property detectors are based pefractive index detectors detectors function unction by measuring the change in refractive index in t sample cell. Inorder to avoid drift at high sensitivity themenetting of the ra:tive inder ows th ofthecluent fron on the change in refraectve ugh the samplo n e a n d r e t C r e n c e 4 l t h u g h They lack in high sensitivin. efcrence cels to better than 0.001'C is required l u t e the eluent widcly becn used in HPLC, they suffer from several disadvantage detectors have They are not much suitable for gradient elution. rict temperature control to be operated at their higher sensitivity They need strict Theelfectof eluent is compared these limitations limitations are reduced to some extent by using differential systems in which the with areference flow of pure mobile phase. tector measure the variation of the refractive index of the column effluent refraction detec measurement with d i f i e r e n t i a l ent with respect to pure eluent permits a greater stability by using a diffe uhich minimises the effect of temperature fluctuations. It is easier to achieve a tem- erical design which clUedifi uctuations ofthe af less than 1x l0 "C between the reference and measurement cells than to reduce absolute temperature ller that 2 to 5x10° index unit with a pure solvent and 1x 10 with a of these cells 1x 10C. Nevertheless, it is very difficult to line noise smalle evea base. Enl mixture. Jthough there are some excep- 5egCS). the refractive index Tganic compounds lies between and 1.50. The difference be- eentthe refractive index of two pounds can be as large as 0.17 nene in methanol) or as low as 5diethyl ether in acetonitrile) Lsually be of the order of 0.10. 5. therefore, always necessary to 7rate the RI detector. Moreover, agood RI detector, the mini- n detectable signal being about , the detector sensitivity wi KOeed ppm and the detection S USually in tens of ppm. Column mobile phase -Light beam Mirror -Reflected beam 3 90 Reference solvent ne reiractive index does not inearly with the concentration Solute. The RI detector is not above 2-5% which is not very Fig. 7. Refractive index detector AONS a limitation in liqu lquid chromatography but it is not possible to use the difterence in refraction and solute to derive response factors esofthe solvent the detector is sen indly lOT cell has to be eKt of the solvent OIs sensitive to flow rate fluctuations orrather to pressure pulsations because the dintained under slight back pressure inorder to avoid bubble formation and permit am through the outlet tubing. Because the capacity of the LC system is small pulsations only slightly. oumn dam mpens the pump
- 4. C304 CHROMATOGRAP- At its best, RI detection is about three orders ofmagnitude less sensitive than UV detection and of about 1 part in 10° (comesponding to about lug injected) provide the detection limit of RI deta These detectors, no doubt, have the advantage that many UV non-absorbing compounds can be d Usef leve measured without derivatisation and in cases where sensitivity 1s not very important, this is clearly nce In fact, RI detectors can be used in gradient elution systems, because the base line continuousl as the changing solvent mixture causes refractive index variations. In order to avoid this, a step pra system may e used where the mobile phase is changed abruptly and the baseline settles before the clute. Alernatively, flow programming can be used with RI detectors. The impertant types of refractive index detectors are given below: eam ) Deflection refractometer- In this type of detector, the detlection of a monochromatic lightr ai glass divide. No deflection in the light beam takes place, if both reterence and sample celi contain solvea the same composition. If, however, the composition of the column mobile phase is changed due he The is measured by a double prismn in which reference and sample cells are separated by a diagonal presence of a solute, then refractive index changes and this also causes the beam to be deflected ase. magnitude ofthis deflection in the beam depends upon the solute concentration in the mobile phase (i) Fresnel refractometer This type of detector is based on the measurement of change in the fractio of reflected and transmitted light at a glass-iiquid interface as a result of change in the refractive inder of the liquid. In this detecior, the column mobile phase as well as reference flow of solvent are passe through small cells on the back surface of a prism. When the two liquids are identical there is no difference between the two beams reaching the photocell. There is, however, a change in the amount of light trang mitted to he photocell, if mobile phase containing solute passes through the cell, and as a result a signal is produced. (2) Ultraviolet detectors- As seen above, UV absorption detectors have widely been used in HPLC because they are based on the principle of absorption of UV visible light as the effluent from the colurmn is passed through a small flow cell held in the radiation beam. Movable Hg lamp Quartz lens calibrated filter SOurce Sample photocel Reference 1 photocell Dual Compound channel cell UV filter Fig. 8. Block diagram of a double beam UV detector. The UV detector measures the change in the UV absorption as the solute passes through a flow Cel (usually 1OuL volume) in a UV transparent solvent. The principle of UV detector is very simple. A gnt source delivers a monochromatic parallel light beam which passes through a cell swept by the colun effluent, and falls on a photocell. A signal proportional to the amount of light received is measured recorded. The source oflight of the simple UV photometers is generally a mercurylamp providing at 254 nm (eg 280 nm or 365 nm using proper interference filters to isolate the desiredmercury The need for lower wavelength lines has led to the development of miniatured zinc (214nm)andcadm (229 nm) gas discharge lamps. A very large fraction of organic compounds absorbs at 214 nm. so UV photometer with a zinc lamp is almost a universal detector. In order to increase the sensitivity ofdetection, variable wavelength detectors have been used. incorporate a deuterium lamp and sometimes oftungsten lamp to extend the wavelength range to
- 5. 3NMANE) LI DCNROMATOARAPay 205 azNNIts the seloton the desired wavelength In recent cell deugns nN oty Inae n the par1 of the ligha heam whch stikes the cet h N al, we eOcPS Wnh a cel volume as small as 0 Rdic More eoent diovdes have gond response, down to he kow Ta Nn snIVe deetOS and have the advantage that they do not destroy eAYS deoeviar are: hange an mperaneT* and Jiw rae, because u is a solnie property l e ir ahemt edai eAS Mamy Shens used in HPL do not absorb to any ddouble beam UV detectars anre commereialty available. fig 8 ilustrates a richanne) detertar is anoher UN detoctor, in which polychromatic light is passed T h emergng radhn s htfrad by a rating and then allowed to fall on to an diae raavng a ditferent namow wavelength band. A micro pracessor s many tnes a sond ai the sptrum so odtained may de displaycd on the SCT ise he fitnd emmission lin afa mercury ane lamp (254 nm) inorder uo allow e of a faorescent eminer with suiazble slters farther detecion af a range of other izaiwueiengohs (eS 2 is also pessible. r arder z provide e veriable wnrlength detector, the conrinuozs emission of the deuteriurm lamp he used in cnjaganion with a nochromator. This variable waelength technique is remei zsefal way efgaining increasrd sensihin in diicult anahses. because solutes can be mumiared ar their wavelengt ofmzrimumahsorpio Thesaubrelin of recen: detectors is such thar cancentration in the eluent of about 1 part in 10 of TieCALeswit high maolareuincies coeticiens oanbe detected ubie beum UV detectors are available which are oapable ef econding the UV spectrum of the | R Tiow isstoppedwhile thesolute passesthnugh thecell. k ection. Important examples include bile cids, lipids, sugas and most amino acids. BE ibilities is to form UV absorbing derivatives of these nmolecules. For example, bile acds i ied by their end absorption at short wavelengths (190-210 nnm) and this technique could hs techeique e molecuks possess a sufficiently strong UV absorption or stron UV chromophoe tor che ecuon of bromo-organics, sugars and steroids with isolated double bonds. The success l cpends on the purity of the solvent. However, with some anomatics an increase in scence detectors When molecules absorb high frequency electromagnetic radiation (UV ined by monitoring at short wavelength. V visible)) they Camental sale in o r several steps, one of which being the emission of one or several photons, ys lower than that of the absorbed photon. When the emission follows very shortly are excited to higher electronic states from which they return to the nomal or OCess is cailed fluorescence and the îluorescenoe spoctrum is a function of the h deteIhe fluorescence detectors are based on the principle that a UV beam of light is the ethe nh Cell, swept by the column eluent. Light emitted in perpendicular direction is ency always lo m wavelengt ei i the dett t o combine such a fluorescence detector to a UV photometer. eas Argecnts stueam tocell and its inte intensity is measured as a function of concentration of elute in the tam. It is tescen solutes p present in the nobile phase are detected by these detectors by passing the column
- 6. RMANCE) LIQUD CHROMATOGRAP th LC eftucnts though the plasma tacks aaiey tx Is noM sunable tor the analysas of metlac elemes C This detect. Aut provdes ecellent sensitivity and selectrvty for metals s euge pery sefgl the plane of olarisation of a plana polarsed bea - 7 1 0 degree. This allows -The rotatron of have onc or several asymmetncal carbom aoms have a sma even a large optical activity. especialy ckose to he man V rgi of can detection of optical 1somers srong ogec a n p o u n d s but sone have may be sutable in o lange changes detection of biochemücal compounds Ths has he advaage in eluent composition (gradiens chun fuorescing molecules whach abscrd a ph s t i c d e t e c t o r N o n a d subsequently return to ground o Dormal staie folowng a deaxtirvating cosaom a pihoecm ased ea temperaure, whach causes a pressure jmp ha can be deec ed laser, which can afford a high energy gh soare w strong local increase in temperature 0ma staie f 0cn ae The use of pulsed ulation. allows a sensitive detection down to i0-100 ppb. depending on the comgounds e m T o p h o n e . cLerimental conditions. thermal lens-A I. beam passing through a solutioa is shghtly bsorbed The hea s nire of the beam. Since the refractive inder of bqmds depen lager in the centi the radial temperature gradient resuits in the solution beheving s a lense The e n e r a tected and recorded by measuing the overfiow of the lxe ben eyond er beam can be deted detector. is however. less sensitive. EDlectrochemical detectory nce have widely been ing HPerture. This ors Although the optical detectors sach a hoe tased on ahsorbnz HPLC the electrochemical (EC) detectors are siowiy emrgng for trace analysis. The populanty of he electrochemical deectors is probably dr erTul tool sensiüvity and selectivi of etection. Without the ue of seocndary reacions he ne d ectrolysis al a pre-selected potential is limited Mareover. EC detccas ith microbore colunmns, which make them very atractive imvic f he which can undergo elec suitabie for operation with towards miniaturisation ot LC equipment The overall detection sensitvity of EC detecsors the mass transfer and electron ransfer proes, which are complex funcions of he soluic on gradient in the cell, solvent iiow rate and ViscosTy. tenperature, cell gemery and eaton namics and kinetics. BC detector is a transducer which permits a direct conversion of chemical infomaton ino ciecc TheEC reaction takes place at the electrode surface which makes it possibie to rodace te el 10 2 thin layer of minimum thickness. In this way cell cohumns of less than a ulL ak easy aRn recent years, EC detector with a cell volume of less than InL has beea developod aai d CuOn with capillary columns. Problems with electrochemical detectors invohe ovntaminanon o 2erodes by adsorbed impurities. edurs which measure the current : ance with their geometries, the flow throush EC cells are classtied as Channel type. (b) Tubular, and (c) Walljet tnpe Dúing on the electrolysis. EC detectors can be subdivided into amperoaetric and coukometr associated with the oxidation or reduction of the sodute ln Ctor, only approximately 5% of the total number of eleciroactive molkuks will Tetric EC detector, only go the reaction, while in tors, hoe Ccoulometric EC detectors, the conversion efficiency is 100. Ihe oulo mpanied by a concurTen fford lower detection sensitivity because an increase n ekcradk area s neN ommodation of electrodes and lower flow rates can degrade the perfmance ent increase in background current and noise. In additioa larger vell se of iectrochemical reducion interfere dt so that electro by reduction of oxygen in the mobile phase. Complete removal ot ovgen 1on as a means of detection in HPLC is, however, vey dafficul ectrochemical detect is usually based on oxidation of the solute. Suk deiev tors
- 7. CHROMATPA to umpounds whiuh nls, uromatie wi the vse all compounds do not undergn eler t r o rhemic ul o r i d a t i o n C wetenay deteted bv electrochemical nrtidation ane aldehydes, ketones, phenat aR heenNwth' aitrogen compounds. Electo hemual d e t e c t i o n requires ie use of conduCtin. ne e . cvntaining inorganic salts or m i x t u r e s of water with water v l u b l e ganic soe hn S aPe however, not suitahle for ter hnigues other than r e v e r s e d phuse 9olvents. Sw se and in exchanp hemic ofhigh sensitivity, the a m p e r o m e t i r d e t e r 1 o r tf ne mosi Widelyused electroe."nr the electractive deneae Threeelectrodet uted in a m p r r o m e l t i r d e t e c t o r are aWorkingelectrode. speces s monitered b)Referenceelectr mtemuby Because made of glassy carton, is the e l e c t r o d e at which reproducible vohtap c) Auriliary electrode, made of stainless stecl, is the current carrying electrode The posituoning of the reference and auxihiary e l e c t r o d e s with respect to the working clectrrd mportat because it may affect the noise and response times. Carbon paste and glassy carton h R l widely used working ekctmde materials for the anaiysis ofeasily o X I d i s a b l e and reducible uhe Carheon pasie gives a lorwer backzround current, but it can not be Used with mobile phases cr more than 21 Uh of organic modifier. Glassy cartn electrodes are more rugged and compatihle rgaRic solvents. Mercury andanalgaTnatedgold are generally used for reductions Mayrity ofEC detectors ofesale on a 3-clectrode potentiostatic system which ensures the como of the working potential sy iust potential drift and ohnic drop. In additii9 to the single working electr arangements, multiple ciectrodes designs have also been used for further increasing the specificity of t derectun and toi 1Case its use. When the two working electrodes are connected in parallel and maintaine a different ptcntials, the currents generated can be recorded simultaneously and their ratios used solute chera ierisation. Wth the serial arrangement, however, the upstrearn elecirode acts as a post colu reactn for the successive reaction at the second eiectrode. The upstrean electrode can also be replac i a nknnetric cell if complete elimination of the interferences is required Inorder n enhunte the selectivity as well as sensitivity, several derivative techniques, such as difle eralpulse,reverse pulse und square wave voltummetry have been proposed, in addition to the controlld pnential methods Due to high sensstivity of EC detecton, its operation with HPLC, particularly in the reversed phae chatmatarngaphy 1s tecoming an extremely useful technique of the analysis of trace compounds in complax matwes of muiogical, environnental and pharmaceutical fields. Non electroactive compounds can e analyed usng "te comept of post codumn chemikal reductions. A ppnenially veful and selective range of detection systems can be envisaged involving reactions hstels) after eluiwn from the clurnn. Such a system must employ a very fast chemical reaction otherwie fhe whute will bezsme unaceptably diluted by diffusion. Moreover, tubing and mixing chambers rnust ricly limied in vohume. Several such useful post column reaction detectors include the well knoin nntrydrin resctr tor arinne acids, the2,4dinitrophenyl hydrazine (24-DNP) reactor for carbonyl con onunds and the cerun deecton fon carbohydrate analysis. lectrode, usually a Ag AgC1 clectrode) which gives a stable rep omtaining e wi Berasie theproblemsassniated with the designofa microdrop mercury electrode and the limited nde potential ranje in ttntinuuu: npertim (which precludes the use in the electrooridation mode) te pruluranruphit denctorg have heen tompietely replaced by amperometric detectors. A large nasnter of othes detectons have aism been used in recent years for specific applications. So them inelude, transprrt deteciors, spray impact detectors, radioactivity detectors, electrica ductivity detetturs, gas ehromatography detectors, polymer specific detectors, etc. The principle f transprt detertor consisus in eliminating the volatíle solvent and carry M-vthatile ute in a sepatate atea where it can te detecied and quantized. This detectr has ai ten imaoved bry usng a moving wire on a gold chain but it has several disadvantages which n ack oh ensitivity, linearty and reprducibility. Some of these defects are due to mechanica The eluent strearn coats a wite tn chain which passes through it, but unfortunately can take oniy a frae ion, deerding tm the surface tensiun of he solution, Th film i include
- 8. h g h h em'1alure nnivation e t e v e n w h e r e whee it i pyrolyodd nidined into (0, he pytodyssa prru ts are wep well an the methane olhtained by the red tiem of (0, also been poposrd in which the effluent is nehuliserl in a warm ge strearm e t s hrough a drifn tube, wlhere the nlvent is vr sed, and then thrrgh olets olid patelesof non volatile suutesr atter ight and the seattered h a n e h e soution dooplets thingh a ddri e n t p r n o i p l e hi h e a m The dre e c t e d a n d u n e d aN usedana m e a s t U r e of act detee orming a Npray. In this procesn electrie eharges are separated by a still a T C s h e Nolute concentratio the eluent stream is orced through a narrow tuhe t form a jet impinging aallkr I n p r a d o n a e l e c i r o d e dows through the electronde, the spray droplets being positively charged sed The curent intennity is strongly dependent on the concentratinn f Obile phase. With pure wHler, detection limits between 2 ppm and I pph in a c u r e n t lows throug p o x e s s , a n d d e negatively c h a r g e d . The Jw nds in the m o b i l e phase. Wit e d e t e c t o r have been found. the ppb range for polar, ionised species like nitro phenol features of spray impact dctector arc With water/organic solvent mixtures the detection limsts yanhnYoundy S o m e of the m p o r t a n i al Small d e a d v o l u m e hile they a r e still b Fast r e s p o n s e , a n d entially very little o r low the beginning of tor their clution using diffe nise between the requiremer ive countings, which The radioactive de detection limits for ionised compounds. a of column chromatography, biochemists using radio labelled compounds have ferent kinds of counters. The main problem is the design of a ments of HPLC, ie, low dcad volume, fast response etc., and are just the opposite, to achieve good counting efficiency and low detectors are necessary for the analysis of radio labclled compounds. ve a LC using wider bore columns than usual inorder to allow the hough thcir use would require large sample size. S n c e m p i t c d t o m o n i a b l e c o m p r o r t o f r a d i o a c t i hey shoui be designed to have volume counting cells, altho conductivity detectors, he measurement of the resistance of an ionic solution, using I n e l e c t r i c a l s l t e m a t i v e voltage to eliminate spurious effects inciple for ionic species. One of the most important advantages of such a detector is the very >cts because of clectrode polarisation can be used as a i dolume. When used with buffer solutions detection limit is very small, being in the low ppm With non-ionic mobile phases of low electrical conductivity the detection limit can be decreased sveral orders of magnitude. I should be noted that combination of the elactrical conductivity detector with ion exchange chro- avgaphic columns has led to the technique, known as ion chromatography. In spite of the excellent ecivity of ion exchange resins, the analysis of non- UV absorbing ions has been hampered essentially ztothe lack of suitable universal detector. Inorder to suppress the overwhelming conductivity of the goUnd eluent electrolyte, an ion exchange suppressor column with opposite charge groups (ie,an wCAChange suppressor column for cation separation and a cation exchange suppressor column,tor raon has been introduced between the ion exchange separation column and the conductivity a y , the eluent is neutralised and the conductivity of the elute ions can be convenientuy WI chromatography ondetecto detectors are sensitive as well as reliable. Some of them such as the flame ind The suppressor column increases the analysis time and the band broadening and may give some ired effects such as ion exclusion, reaction with some ions etc. aion detector ionisati are non- selective. Others like electron capture detectors, the flame pholomeiricdetector Others like electron capture detectors, the flame photometric detector etector are selective for s o m e groups ofcompounds. These detectors are described e cha mal ionisation detector napter of gas chromatography. ution (M distrib uaton curve ojective ofthe analysis of poiy of known detcctor a MWnstruciono tion ( M u S s p o l y m e r s , especially those ofsynthetic origin, is to supply a molecular obtained with a concentra re obrai D) of the sample of known chemical s t r u c t u r e . This can be done by converting ion curve relating the elution c e n t r a t i o n sensitive detector to a MWD c u r v e with the help of a me to the m o l e c u l a r weight. As the c o n s t r u c t i o n of such cali on relatino c o n c e n t r a t i o n sensitive detector to a MWD curve with the helpota
- 9. CHROMATOGRAP C-310 bration curves is sometimes difficult or impossible, it has been found useful to combine i. sensitive detector with molecular weight sensitive detector. Various classical static techni. characterisation have been found to be amenable to the flow through of these are viscometric detector and light scattering detector. The vIscometric dectector is bascd on pressure drop measurement in a small capillary . . sca:tering detector, the usc of the laser technology allows measurements of the scattered ioh n ligh o Combine the conccntrat iques of polyrme nts of mdern IC. 'TWo light to be made at low angle (< 5'). This simplifies the relationship betwcen the scattered light and the solutio detector) the product of the mass conccntration by the molccular weight of the polymer solution a Caureof Jv, IR or Sluorescence de significantly. The measurement of the cxcess Raylcigh factor basically provides a continu propere wing through the cell. The combination with a concentration detcctor (refractíve index, UV, IR allows casy extraction of concentration and molecular weight, which provides the reacquired MWD Cu does In general, the solute property detectors arc more sensitive particularly if the mobile phase contribute to the property being measured. ndex detectors The two detectors which have widely bee used are the absorption and refractive indo Diagrams for these two instruments are illustrated in the figures 9 and 10. Mirror Sample C B Light Mask A Source Lens Recorcer Amplifier and Power Supply Detectobr Optical Reference Lero Zero Adjust Fig. 9. Ultraviolet-Visible detector. Mirror -I:j Reference Cell F Lens Lens Amplifier Outet and Phototubes Recorder Sample Cell Monochromator - Beam Lens Lens Source Splitter Inlet Fig. 10. Refractive index detector. index In general, the effluent, as it emerges from the column. passes into the absorption or relrace cell. If a solute is present, the absorption or refractive index changes and is plotted on a rco 0 provide the chromatogram. The cells should be small in volume (< 0.5 mL) inorder to Pr corder Hen the eluting agent minus the sample is passed through a reference cell and the absorptionor ed. ve inorder to preventdií of the band into a larger volume. In the better detection system, a double beam princip refract Henct index of the unknown cell (column effluent) is measured in comparison to the relerence
- 10. FORMANCE) LIQUID CHROMATOGRAPu ESSURE(OR PERFC substances have a refractive index and hence a refractive index deteetor ean ,311 PERFORMANCE he efficiency is define in terms of the number of theoretical plater per metre (N by the sed for C o h u m . N .54.V, L.W2 there,VR Distance along the base line ween the point of injection and a perpendicular droppe h m i h e L The length oft = The the maximum of peak of interest. column in metres. idth of the peak of interest at half peak height, measured in the sam olotionfactor ( dby the expression. uoion factor (R) between the measured es peaks on the chromatogram shall be geater than 10 and is same units as V,. The whereVR» nd VRs are the the maximum of two adjacent peaks. Whh and Wh that values of Whs, Whb VRs and VRb must be expressed in the same units of R= (RVR cularsdropped from red at half peak height. Whs+ Whb the distances along the baseline between the point at injection and perpen hs are the respective peak wdis Here it The symmetry factor of a peak is Symmetry factor n Z S u r e m e n t . calculated from the expression, 2A where Wx is the width of the peak at one-twentieth of the peak height and A is the distance berw perpendicular dropped from the he peak maximum and the leading edge of the peak a one-wenieh peak The capacity factor (k) is defined by the expression k Vo where V= The distance along the baseline between the point of injection and a perpendicular droppei n maximum of the peak of interest. V, The distance along the baseline between the point of injection and a perpendicular dropped iam te maximum of an unretained peak. VR and Vo are expressed in the same unit of measuremenc The linear capacity of an adsorbent column is the amount of solute which may be appued u te TIabefore a 10% loss in efficiency is observed. This generally occurs typically for 10- 10" g i gram of support. lt has been shown that using very efficient columas (reduced plane deghs af ) he ficiency (plate height) pends linearly on sample load down to very low sampie loats (1 4g a measure of adsorbed capacity the term relative capacity has been proposed, whuch is the sampie al columns, the minimum amount of solute dissolved in less than 5 ul of solvent sdouli give anple per g. of support). per gm of adsorbent that increases the HETP of the column by 0.1 mm from the minimum value. OWeye Lunear capaciy has also been measured by the amount of solute required to cause a10% decrease , it has been observed that a substantial decrease in efficiency on the very efficient cplunn Howeve been accompanie ional sanied by any corresponding decrease in K value. Column capacity has been found nal to the surface area relativeuace area of the adsorbent and depends on the mobile phase and vaue with relative capacity apacity values being inverselyproportionaltolog K.