The Theory of HPLC Band Broadenng Wherever you see ths symbol, t s mportant to access the on-lne course as there s nteractve materal that cannot be fully shown n ths reference manual.
Ams and Objectves Ams and Objectves Ams To llustrate and explan the prncple of Band Broadenng n HPLC To defne the Van Deemter equaton and explan the terms of the equaton To demonstrate the effects of Eddy Dffuson, Longtudnal Dffuson and Mass Transfer on the Effcency of Chromatographc Peaks Objectves At the end of ths Secton you should be able to: To use the Van Deemter coeffcents to llustrate how to optmse the Effcency (N) of chromatographc separatons and to reduce Band Broadenng
Content Band Broadenng 3 The Van Deemter Equaton 3 Eddy Dffuson 4 Longtudnal Dffuson 5 Mass Transfer 7 Optmsng Flow Rate 9 Optmsng Partcle Sze 10 Mnmsng System Volumen 11 Glossary 12 Crawford Scentfc www.chromacademy.com 2
Band Broadenng The Van Deemter Equaton Band broadenng s a phenomenon that reduces the effcency of the separaton beng carred out leadng to poor resoluton and chromatographc performance. Ths s problematcal n terms of both the qualty of the separaton obtaned and the accuracy wth whch sample components can be quantfed. The degree of band broadenng (loss of effcency) naturally ncreases wth the age of the chromatographc column beng used, but there are measures that can be taken to slow these processes and to optmse column and nstrument condtons to ensure maxmum effcency and mnmum band broadenng. In 1956 J.J. Van Deemter derved an equaton that ncluded the man factors contrbutng to column band broadenng. He descrbed the ndvdual terms (A,B,C & D) and also derved a composte curve whch related plate heght (HETP) to lnear velocty of the moble phase flowng through the column. Whlst t s not mportant to necessarly know and use the equaton on a daly bass t s mportant to understand the terms (or factors) that contrbute to band broadenng, so that we can optmse our separatons. For example, the nteractve dagram opposte shows the reducton n chromatographc performance sustaned when movng from a system extra column volume of 20μL to 80μL. The Van Deemter Equaton and graphcal representaton of the contrbutng terms Comparatve chromatograms from HPLC systems wth 20 and 80μL dead volume showng effects on effcency and resoluton. Crawford Scentfc www.chromacademy.com 3
Eddy Dffuson The frst of the factors relatng to band broadenng s that of Eddy Dffuson. Ths s a generc term, often used to descrbe varatons n moble phase flow or analyte flow path wthn the chromatographc column. Eddy dffuson tself relates to the fact that an analyte molecule, wthn a band of analytes, can take one of many paths through the column. These multple paths arse due to nhomogenetes n column packng and small varatons n the partcle sze of the packng materal. Ths multple path effect tends to make the band of analytes broader as t moves through the column. Large Partcles Small Partcles Band broadenng due to Eddy Dffuson (A Term) n columns wth large and small partcles effects on chromatographc peak shape (Effcency (N)). Mnmse Eddy Dffuson by: Selectng well packed columns Usng smaller statonary phase partcles Usng partcles wth a narrow sze dstrbuton Crawford Scentfc www.chromacademy.com 4
In fact, the Eddy Dffuson term n the Van Deemter equaton (the A term), s often called the packng term as t reflects the qualty of column packng. Lamnar flow occurs wth standard sze HPLC column packng materals at flow rates up to approxmately 4mL/mn. Non-lamnar flow occurs when usng very large partcle sze packng materal (>40μm) and at hgh flow rates (>5mL/mn.). Ths stuaton s also sometmes referred to as Turbulent Flow and does not occur under normal HPLC condtons customsed HPLC equpment and columns are requred for Turbulent Flow Chromatography. Band broadenng due to Eddy Dffuson (A Term) n columns wth n Lamnar and Non Lamnar moble phase flow profles. One other flow varaton n column chromatography comes from the lamnar flow profle adopted by lquds flowng under pressure through tubes. The flow profle s sad to be lamnar, where lnear velocty near the nner walls of the tubng s lower than n the centre of the column. Ths also tends to produce a broader band of analyte molecules. Longtudnal Dffuson A band of analyte molecules contaned n the njecton solvent wll tend to dsperse n every drecton due to the concentraton gradent at the outer edges of the band. Ths broadenng factor s called Longtudnal dffuson because nsde tubes, the greatest scope for broadenng s along the axs of flow. The band wll broaden n all system tubng, but the worst effects wll be encountered n the column tself. Longtudnal dffuson occurs whenever the HPLC system contans nternal volumes that are larger than necessary and some nstances of ths are: Tubng length too long Tubng that s too wde (nternal dameter) Tubng joned by unons Incorrectly connected Zero Dead Volume fttngs Usng the wrong column nuts and ferrules Usng a detector flow cell that has a large nternal volume Crawford Scentfc www.chromacademy.com 5
As can be seen, Longtudnal dffuson has a much larger effect at low moble phase velocty (flow). Therefore, usng hgh lnear velocty (hgh moble phase flow wth narrow columns), wll reduce the effects of ths broadenng factor. Band broadenng due to Longtudnal Dffuson (B Term) n columns wth low and hgh moble phase lnear velocty effects on chromatographc peak shape (Effcency (N)). Mnmse Longtudnal Dffuson by: Usng hgher moble phase flow rates Keep system tubng short and as a narrow as possble (careful wth back-pressure) (<0.12mm.d. s deal) Use correct nuts, ferrules and fttngs wherever possble Crawford Scentfc www.chromacademy.com 6
Mass Transfer Ths term n the Van Deemter equaton arses largely due to the fact that the statonary phase materal s porous and the moble phase wthn the pores s stagnant or statonary. The packng materal s porous to allow a very large surface area for separaton to occur. As the analyte molecules move through the stagnant moble phase to reach the surface of the packng materal, they do so by dffuson only. Analyte molecules enterng the pore, those that don t enter the pore and those that penetrate more deeply nto the pore, wll all be held up at that pont to dfferent extents causng a broadenng of the band. Ths s the C term. Mass Transfer (C Term) band broadenng processes n the pore structure of statonary phase partcles Further, the analyte resdence tme n (or on) the statonary phase s also varable agan causng a varaton n eluton tme and band broadenng. These effects may be mnmsed by reducng the sze (dameter) of the packng materal partcle sze to make the pores as shallow as possble. The effects of mass transfer are also lower at lower lnear velocty of the moble phase. Crawford Scentfc www.chromacademy.com 7
Large Statonary Phase Partcles Greater possble pore dstance for analyte dffuson Dffuson tme ncreased Dfferences n dffuson tmes out of the pore are amplfed Peak effcency decreases (peak broadens) Small Statonary Phase Partcles Reduced possble pore dstance for analyte dffuson Dffuson tme decreased Dfferences n dffuson tmes out of the pore are reduced Peak effcency ncreases (peak becomes narrower) Mnmse Mass Transfer effects by: Usng smaller (dameter) statonary phase partcles Usng lower moble phase flow rates Heatng the column (at hgher temperatures the dffuson processes are speeded up and the dfferences n eluton tme from the partcle pore are reduced) Effect of moble phase lnear velocty (top) and statonary phase partcle sze (bottom) on Mass Transfer effects and peak shape (Effcency (N)) Crawford Scentfc www.chromacademy.com 8
Optmsng Flow Rate Let s look at the composte Van Deemter curve and equaton once agan ths tme usng real world examples - to nvestgate optons for mnmsng band broadenng and optmsng peak effcency. Durng the prevous pages you have seen that the moble phase velocty and the partcle sze of packng materal used, have a fundamental effect on effcency (and therefore band broadenng). Van Deemter relatonshp between moble phase flow rate and peak effcency n HPLC The On-lne course contans an nteractve experment n whch the eluent flow rate can be changed to alter the HETP parameter Although the change n partcle sze does not appear to have a marked effect on the effcency and resoluton of ths separaton, you should study the results carefully Reducng partcle sze (even when usng tradtonal column nternal dameters), has a marked effect on plate heght, whch can be usefully used to mproved the resoluton of separatons, especally where selectvty optons have faled to produce baselne separated peaks The plate heghts obtaned wth very small partcles are extremely low. By combnng short columns wth tradtonal nternal dameter (0.46cm), and small dameter slca packng materal (1.8μm) very short analyss tmes may be acheved. When usng smaller dameter column packng materals, system backpressure should be carefully observed as ncreased backpressure results from the use of small slca partcles Startng wth an nvestgaton of moble phase flow (lnear velocty) you should see that the moble phase flow rate can have an effect on band broadenng, plate heght and hence resoluton. It s mportant to note that there s an optmum flow rate for each separaton (the mnmum pont on the curve correspondng to the lowest plate heght). Whlst ths effect s notceable, t s mnor n comparson wth other varables affectng resoluton and therefore flow rate would be used to fne tune a separaton only. Of course, f you have enough effcency (and resoluton) to run at hgher flow rates then ths wll make the analyss tme shorter however, care should be taken not to exceed the pressure lmt of your HPLC pump! Crawford Scentfc www.chromacademy.com 9
Optmsng Partcle Sze Another mportant factor to consder regardng band broadenng s the partcle sze of the statonary phase used. We have seen several nstances where the use of smaller dameter partcles s advantageous to both effcency and resoluton. Van Deemter relatonshp between statonary phase partcle sze and peak effcency n HPLC The On-lne course contans an nteractve experment n whch the effects of changng partcle dameter (d p ) on effcency (band broadenng) and resoluton are presented Although the change n partcle sze does not appear to have a marked effect on the effcency and resoluton of ths separaton, you should study the results carefully Reducng partcle sze (even when usng tradtonal column nternal dameters), has a marked effect on plate heght, whch can be usefully used to mproved the resoluton of separatons, especally where selectvty optons have faled to produce baselne separated peaks The plate heghts obtaned wth very small partcles are extremely low. By combnng short columns wth tradtonal nternal dameter (0.46cm), and small dameter slca packng materal (1.8μm) very short analyss tmes may be acheved. When usng smaller dameter column packng materals, system backpressure should be carefully observed as ncreased backpressure results from the use of small slca partcles It should be noted that whlst smaller dameter partcles are favourable there s a trade wth system backpressure. Small partcles may be used n conjuncton wth hgher moble phase flow rates to produce very fast analyses, but system backpressure needs to be carefully montored. The use of smaller dameter slca partcles (<2μm), wth tradtonal column nternal dameters (.e. 4.6mm), means extra-column dead volume s less mportant than when usng narrow bore columns. Ths can be an advantage when fast HPLC analyss s requred, wthout havng to change the analytcal system hardware to very low dead volume, or very hgh pressure versons. Crawford Scentfc www.chromacademy.com 10
Mnmsng System Volumen As prevously dscussed, systems contanng large extra-column volume (tubng, unons, flow cell etc.), show low effcency and hence broadened chromatographc peaks and loss of resoluton. You can use the slder bar to nvestgate the effects of extra column volume. You may want to consder how you could reduce these volumes on your system. Typcal ways mght nclude: reduce tubng length and nternal dameter / reduce the number of unons between tubng / ft column end fttngs approprate to the column type beng used / reduce the njecton loop volume / reduce the detector flow cell volume. Van Deemter relatonshp between extra column dead volume and peak effcency n HPLC Important: The system extra column volume can have a crtcal effect on effcency and therefore resoluton of a separaton You need to also consder the column dead volume, and f n any doubt about the qualty of the column packng, use a new column. Column vods from wthn columns over tme addng very large dead volumes to the system Extra column dead volumes below 20μL should be easly achevable on modern HPLC systems You should consder the volume of the detector flow cell as beng a major contrbutor to extra column volume, along wth any hgh pressure mxng chambers on the HPLC pump System dead volume becomes more crtcal as the nternal dameter, (and to a lesser extent partcle sze), of the column used are reduced It s straghtforward to measure the extra column volume of a system: Remove the column and jon the tubng wth a Zero Dead Volume unon Inject 10μL of 100% strong solvent (acetontrle works wth UV at 200nm) or a soluton of 1% acetone (montor at 265nm) The apex retenton tme (t R ) of the baselne perturbaton due to the passage of the solvent gves the extra column hold up tme of the system (expressed n mnutes) Multply ths tme by the flow rate (n mllltres per mnute) to obtan the extra column volume (n mllltres) Crawford Scentfc www.chromacademy.com 11
Apex solvent retenton tme used to determne extra column volume of an HPLC system. Glossary Lnear Velocty The lnear velocty of the moble phase across the average crosssecton of the chromatographc bed or column. It can be calculated from the column flowrate at column temperature (Fc ), the cross-sectonal area of the column (Ac ) and the nterpartcle porosty ( ε): = Fc /(εac) In practce the moble phase velocty s usually calculated by dvdng the column length (L) by the retenton tme of an unretaned compound (tm) = L/tM System Extra Column Volume the volume of the HPLC system from the pont of njecton to the pont at whch the sample passes out of the detector, MINUS the column dead volume. Ths typcally ncludes the njecton loop, any tubng and unons as well as the detector flow cell where approprate. Tubng Unons Fttngs whch allow the couplng of two peces of tubng n HPLC. May be made from Stanless Steel or and nert polymerc materal such as PEEK (polyether ether ketone). Zero Dead Volume refers to a fttng n whch the nut, ferrule and tubng exactly match the nternal volume of the fttng, leavng no dead space. Ths dead volume tends to cause band broadenng n HPLC by artfcally holdng up analyte molecules n the stagnant (not movng) phase that resdes n the dead volume wthn the fttng. Crawford Scentfc www.chromacademy.com 12
Dead Volume Wrong column nuts and ferrules - each column manufacturer uses partcular end fttng types. Specally desgn stanless nuts and ferrules must be used n order to obtan a satsfactory zero dead volume unon wth the column. Some workers use PEEK fttngs that deform to ft the nternal geometry columns from any manufacturer. However, these fttngs do loose ther plastcty and should be regularly replaced to avod band broadenng. Incorrect ferrule and tubng fttng Crawford Scentfc www.chromacademy.com 13