Guidelines for the use of UHPLC Instruments

Transcription

1 Guielines for the use of UHPLC Instruments Requirements for UHPLC instruments, metho evelopment in UHPLC an metho transfer from regular HPLC to UHPLC Authors: Dr. Davy Guillarme, Prof. Jean-Luc Veuthey Laboratory of Analytical Pharmaceutical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevar Yvoy 0, Geneva 4, Switzerlan - Phone: Fax: jean-luc.veuthey@unige.ch. INTRODUCTION Over the past few years, there has been tremenous interest in approaches to spee up an/or increase the resolving power of the analytical separation process, particularly with the evelopment of columns packe with porous sub-µm particles use in very high pressure conitions (namely UHPLC, for ultra high pressure liqui chromatography). Many laboratories want to transition some of their conventional HPLC methos to fast UHPLC methos, but their lack of experience sometimes acts as a eterrent to trying. In this white paper, we share our experience in HPLC-to-UHPLC metho transfer in the form of a tutorial introuctory guie to help those who want to try this new an exciting UHPLC methoology to improve their prouctivity. Figure : Columns packe with sub-µm particles: concept an interest It is well known in liqui chromatography that the use of small particle size results in higher plate numbers, as well as faster separations. These effects are ue to the fact that i) the chromatographic efficiency, N, is irectly proportional to the ratio of column length an particle iameter, L/ p an ii) the mobile phase linear velocity, u, is inversely proportional to the particle iameter, p. As illustrate in figure, for high throughput separations it is inee possible to maintain an equivalent efficiency between a 50mm column packe with 5µm particles an a 50mm column packe with sub-µm particles, while the analysis time is ivie by 9- fol. It is also theoretically possible to maintain the analysis time equivalent between a conventional HPLC columns an a 450mm column packe with sub-µm, but with an efficiency enhancement by 9-fol with the latter. - - Guielines for the use of UHPLC instruments

2 However, the particle size reuction also generates a high backpressure (> 400 bar) not compatible with conventional instrumentation. Therefore, to benefit from the full potential of columns packe with small particles, it is recommene to work with a chromatographic system that withstans pressures up to 600 an even 000 bar. By comparing the intrinsic performance of such packing size with other existing techniques, such as monoliths, fuse-core technology or high temperature liqui chromatography with conventional particle size, it is shown here that UHPLC, with a maximal pressure of 000 bar is a very attractive strategy (i.e. approach that generates the lowest analysis time for a given efficiency) in the range,000 to 80,000 plates. Only the monolithic approach performs better than UHPLC for efficiencies higher than 80,000 plates (such efficiency is however often beyon the nees of a conventional LC analysis).. Requirements for UHPLC Experiments.. UHPLC instrumentation To work with columns packe with small particles, a specialize chromatographic system is neee. First, this instrument shoul be able to withstan the very high pressures generate by small particles an this is generally achieve by extening the pressure capabilities of the pumping system an by improving the sample introuction evice to make it compatible with ultra-high pressures. Secon, the chromatographic system shoul also be aapte to operate in fast an ultra-fast moe with reuce column volumes. Inee, small iameter columns ( an. mm I.D.) limit the frictional heating (generate uner high pressure rops an high flow rates) an reuce the organic solvent consumption but require small extra-column volumes ue to etection, tubing, an injection volume (figure ). The following criteria have to be fulfille to perform efficient separations: The tubing volume shoul be reuce as much as possible. For this reason, the tubing length shoul be as short as possible an its iameter selecte as a compromise between an acceptable generate pressure an a low volume. For this reason, a system plumbe with I.D. stainless steel tubing an zeroea volume fittings is generally preferre for UHPLC experiments. tot tot column Injector Connection tubing σ = σ + σ inj col σ col Figure σ total et A Detector ext σ = σ + σ + σ B tubing The injection volume shoul be selecte in agreement with column geometry. A rule of thumb is to maintain the injecte volume between an 5% of the column ea volume. As most of the experiments carrie out in UHPLC are performe with a 50x. mm column (V 0 = 0 µl), the injecte volume shoul be inclue between an 5 µl, to limit ban broaening. In aition, a fast injection cycle time is manatory for analysis times lower than one or two minutes. Last but not least, the etector cell volume, time constant an acquisition rate shoul be carefully selecte. The etector use in UHPLC shoul ieally possess a low cell volume ( µl or lower) while the sensitivity shouln t be lowere compare to that of a conventional HPLC instrument. Detector time constant has to be fast enough (τ 00 ms) because peak withs are very small in UHPLC (only a few secons). Finally, the etector sampling rate must be sufficiently high to acquire a suitable amount of ata points across each peak (> 0 Hz). It is noteworthy to note that to reuce the importance of extra-column volumes an avoi an unacceptable loss in efficiency, chromatographic conitions leaing to high retention factors (k at least equal to 3) are often recommene in UHPLC (i.e. high σ² col values inucing lower influence of σ² ext ). However, ue to the very small column ea time in UHPLC (e.g. t 0 = 0.0 min for conventional UHPLC conitions with a 50x. mm,.9 µm columns operating at 600 µl/min), a high retention factor woul not be etrimental for the analysis time. As example, a retention factor of 5 in conventional HPLC leas to an analysis time of 0 minutes while it correspons to only minute in UHPLC. - - Guielines for the use of UHPLC instruments

3 The last critical parameter to perform ultra-fast separations in the graient moe is the well volume of the system which correspons to the time necessary for the graient to reach the column inlet. To work with ultra-fast separations, a small graient elay volume is require. With a large well volume, fast separations are compromise because an isocratic hol is generate at the beginning of the graient, inucing potential changes in selectivity an longer analysis times. To check the compatibility of an UHPLC instrumentation with a given column geometry, it is recommene to characterize the chromatographic system by etermining the extra-column an well volumes. For optimal compatibility with ultra-fast separations, the former shoul be lower than 0µL while the latter shoul be no more than a few hunre µl... Quality of mobile phase an buffers It is important to consier that the size of the frits an particles containe within UHPLC columns are much smaller than on regular HPLC packings. For example, the inlet frit pore size of an HPLC column is often equal to µm while it coul be equal to only 0. µm in UHPLC (this value strongly epens on the column provier). Therefore, small particles, potentially present within the mobile phase an which o not affect HPLC materials, can become critical in the UHPLC configuration. Therefore, it is important to check the absence of insoluble particles in the solvents an, for this purpose, several key rules have to be followe in UHPLC for the mobile phase preparation: Use only high grae organic solvents (ieally filtere through a 0. µm membrane) it is even possible to fin acetonitrile of UHPLC grae from several suppliers (Biosolve, JT Baker, Fisher Scientific, etc ) Use high quality salts to prepare buffere mobile phases The water shoul be ultra pure an filtere through a 0. µm membrane (Milli-Q system or similar high quality water is recommene) Be careful with the microbiological growth (particularly when using phosphate buffer): always use freshly prepare mobile phases Be vigilant with the cleaning of glassware an o not top-off the bottles. To limit the microbiological growth, the chromatographic system an columns shoul ieally be store with pure organic solvents (methanol or acetonitrile)..3. UHPLC columns One of the main criticisms mae by early UHPLC users has been the reuce lifetime of columns packe with sub-µm, compare to conventional columns. It is true that UHPLC columns are always expose to very high pressures, but the packing pressure has been increase proportionally. In our laboratory, we have observe that lifetimes of UHPLC an regular HPLC columns are comparable. However, column lifetime also epens on the number of injections, number of column volumes or perio of time use. With the latest generation of columns packe with sub-µm, commercialize by ifferent suppliers, it is possible to perform between 500 an,000 injections or even more on a single column. Such values correspon to about 5,000-0,000 column volumes, which are fully comparable with those obtaine on stanar HPLC columns. However, when consiering the corresponing perio of time, it is significantly reuce compare to conventional HPLC because of the higher throughput in UHPLC. For example, in a routine laboratory which performs a UHPLC analysis in to 5 minutes, one thousan injections can be performe in a very short perio of time. This time is significantly reuce compare to that using conventional instrumentation (0-fol longer) while a similar amount of work has been carrie out. In aition, the re-equilibrating time neee uring a UHPLC graient shoul be rastically reuce compare to the regular HPLC, to limit the number of column volumes percolate. Nevertheless, a real issue of UHPLC packing is linke to the very low volume of the column in conjunction with the high mobile phase linear velocity employe. Inee, it is not recommene to let the system Guielines for the use of UHPLC instruments

4 continue pumping without performing any analysis because the column lifetime can be rapily egrae (50 column volumes percolate through the column in only 0 minutes). 3. Metho Development in UHPLC The rules for eveloping a new metho in UHPLC are slightly ifferent from those of conventional HPLC because it is necessary to account for the backpressure constraint generate by the use of small particles. 3.. Choice of column imensions Depening on the supplier, it is possible to fin some columns eicate to UHPLC with internal iameters of,. an 4.6 mm. As iscusse previously, the 4.6 mm I.D. column is not of great interest because of the significant frictional heating generate by high mobile phase flow rates, generating a lack of repeatability of retention times an some potential ifficulties in transferring methos from conventional HPLC. In aition, the consumption of organic solvent is critical as the flow rate shoul be in the range 3-5 ml/min. Concerning the mm I.D column, the effect of frictional heating shoul be neglecte even at 000 bar but the compatibility between this column geometry an any UHPLC instrument is extremely critical (particularly for tubing geometry). Due to these statements, the. mm I.D. column shoul be consiere as optimal for UHPLC operation. Regaring the column length, it shoul be selecte accoring to the require efficiency (in isocratic moe) or peak capacity (in graient moe). It has to be note that there is no real limitation in UHPLC column length. Numerous suppliers propose some 50mm columns which can be couple in series using low volume tubing if an experiment has to be performe with very long columns. In the isocratic moe, it is well known from the basic equations of chromatography that efficiency is irectly proportional to the column length. Therefore, a 50x. mm,.9 µm column shoul generate aroun 0,000 plates, while the efficiency is increase to 0,000 an 30,000 plates with a 00 an 50x. mm,.9 µm column respectively. Therefore, the column length shoul be chosen accoring to the requirement of the separation an the longest column always provies the highest efficiency. However, with 50 mm or longer columns, the mobile phase flow rate shoul be aapte to avoi over-pressurizing the analytical system. In the graient moe, the relationship between column length an chromatographic performance (expresse as peak capacity in graient moe) is not trivial. In fact, the peak capacity epens both on efficiency an column ea time, but each to a ifferent extent. Therefore, the column length shoul be aapte accoring to the graient time an the longest column oesn t necessarily provie the highest peak capacity. It can be emonstrate that a 50x. mm,.9 µm column has to be selecte for graient times lower than 5 minutes. The 00x. mm,.9 µm column gives optimal performance for graient times between 5 an 0 minutes an finally, the 50x. mm,.9 µm column shoul only be use with graients longer than 0 minutes. 3.. Choice of mobile phase conitions In UHPLC, the mobile phase flow rate has to be selecte accoring to the Van Deemter curve (similarly to conventional HPLC) but also, accoring to the backpressure generate. For compouns with molecular weights in the range g.mol -, the optimal flow rate in isocratic moe for a. mm I.D. column packe with.9 µm particles is aroun µl/min. Due to the low mass transfer resistance generate by small particles (because of the reuce iffusion path), it is even possible to work up to 000 µl/min, with a limite loss in efficiency (aroun 0%). When ealing with larger molecules, the mobile phase flow rate shoul be reuce to µl/min ue to a reuction of iffusion coefficients. The rules in graient moe are ifferent, where the highest flow rate always provies the best peak capacity because it is epenent on the column ea time an, to a lesser extent, on efficiency. Therefore, the flow rate for graient UHPLC experiments shoul be elevate but at the maximum equal to 80-90% of the maximum pressure withstoo by the instrument. This solution is recommene to attain a sufficient level of robustness an to hanle unexpecte changes in column backpressure after hunres of injections Guielines for the use of UHPLC instruments

5 Regaring the mobile phase temperature, it coul be valuable to work in UHPLC at a mobile phase temperature of C instea of room temperature. With this strategy, the mobile phase viscosity is reuce an the backpressure iminishes by about 30% (at 50 C for an acetonitrile-water mobile phase) without affecting chromatographic performance. Finally, it is well known that the viscosity of acetonitrile-water hyro-organic mixtures is on average.5 to -fol lower than that of methanol-water. Therefore, in the case of metho evelopment, the initial choice for mobile phase is acetonitrile as it generates significantly lower backpressure an more possibilities in UHPLC compare to methanol (particularly for the choice of column length) Decision tree for metho evelopment On the basis of the above iscussion, it is possible to establish some generic conitions for the UHPLC metho evelopment: the column shoul be initially a C8 with geometry of 50x. mm,.9 µm operating at a temperature of C. The mobile phase shoul c onsist in a mixture of acetonitrile an buffer. It is generally better to begin the experiments in the graient moe at a mobile phase flow rate close to the maximal pressure accepte by the UHPLC instrument. Regaring the choice of graient time, it is extremely ifferent in UHPLC compare to regular HPLC. Therefore, table summarizes the optimal graient time for various sets of UHPLC conitions. Table : Calculate optimal graient time accoring to column geometry. Column geometry Flow rate Graient profile Optimal graient time 50x4.6 mm, 5 µm ml/min 5-95% 30 min 50x. mm,.9 µm 600 µl/min 5-95% 3.5 min 50x. mm,.9 µm ml/min 5-95%.0 min 50x. mm,.9 µm 600 µl/min 0-60%.0 min 00x. mm,.9 µm 300 µl/min 5-95% 4 min 00x. mm,.9 µm 300 µl/min 0-60% 8 min 50x. mm,.9 µm 00 µl/min 5-95% 30 min 50x. mm,.9 µm 00 µl/min 0-60% 7 min If the selectivity with the generic conitions previously escribe is not acceptable, it is possible to aapt various parameters (e.g. mobile phase ph, nature of the stationary phase an organic moifier). In UHPLC, the first parameter to change is the mobile phase ph, then the column chemistry an finally the organic moifier nature (because of the constraint in pressure with UHPLC). 4. Metho Transfer in UHPLC In various fiels of application (i.e. pharmaceutical, environment, foo, ), it is important to be able to to transfer existing methos (performe in conventional HPLC conitions) to faster separations involving the use of columns packe with sub-µm particles. As most of the proviers now offer equivalent stationary phases packe with 5, 3 an sub-µm particles, a geometrical transfer can be performe if the stationary phase chemistry remains ientical between the original an final sets of conitions. For this purpose, some rules have to be applie in both isocratic an graient moes Guielines for the use of UHPLC instruments

6 4.. Case of isocratic moe In isocratic moe, two important parameters have to be moifie, namely the injection volume an the mobile phase flow-rate. To avoi a etrimental extra-column ban broaening an maintain equivalent sensitivity, it is necessary to aapt the injection volume in line with the change of column imensions. In liqui chromatography, the injecte volume shoul represent only -5% of the column volume. The latter can be calculate simply from the column internal iameter ( c ) an length (L). Therefore, the injection volume is inepenent of the particle size an only proportional to the column volume. The new injecte volume (V inj ) can be etermine simply by maintaining the ratio of column ea volume an injecte volume constant between regular HPLC an UHPLC. Thus, the injecte volume in UHPLC (V inj ) can be calculate accoring to the following equation: c L Vinj = Vinj () L c In this equation, subscripts an are relate to HPLC an UHPLC column imensions, respectively. For example, from a conventional 50x4.6 mm, 5 µm column to an UHPLC 50x. mm,.9 µm column, the injecte volume shoul be ecrease by 4-fol. It can be note that larger injection volumes than preicte can be use to maximize sensitivity. However, the sample shoul be issolve in a solvent of weaker eluent strength than the initial mobile phase composition. This approach escribe as sample focusing (peak compression) allows the enrichment of the analytes on the top of the column. Regaring mobile phase flow rate, it shoul be aapte to be close to the optimal value of the Van Deemter curve representation (H=f(u)). In liqui chromatography, it is well-known that the mobile phase linear velocity (u) is irectly proportional to the square of column iameter an also epens on the particle size of the support. It is however, completely inepenent of the column length. For a successful metho transfer, it is manatory to maintain the prouct u* p constant, to take into account simultaneous changes in column iameter an particle size of the support. Therefore, for a geometrical transfer, the UHPLC flow rate (F ) can be calculate with the following equation: F = F () c c ² ² p p As an example, from a regular 50x4.6 mm, 5 µm column to a UHPLC 50x. mm,.9 µm column, the mobile phase flow rate shoul be ecrease by.8-fol. The expecte analysis time of the transferre metho (t ana ) is irectly proportional to the change in column ea time an can be estimate accoring to: t ana F c L = tana (3) F c L The expecte backpressure ( P ) can be calculate from the Darcy s law which shows that P is inversely proportional to p 3 an strictly relate to the column length: 3 L p P = P 3 (4) L p Guielines for the use of UHPLC instruments

7 Finally, the expecte solvent consumption of the transferre metho (V ) can be calculate by taking into account the change in column internal iameter, particle size an analysis time. V c p ana = V (5) c ² p t ana ² t Therefore, from a regular 50x4.6mm, 5µm column to an UHPLC 50x. mm,.9 µm column, the analysis time is reuce by 8-fol. It has also to be note that for the above-mentione transfer, the efficiency woul be ientical, while the backpressure shoul be 6-fol higher an the solvent consumption reuce by 4- fol. This shows the benefits of the UHPLC strategy but also emonstrates the nee to work with an instrument that withstans pressure higher than 400 bar. Table : Optimal conitions to work with 6 column geometries that provie a similar theoretical efficiency of 0,000 plates. Changes in analysis time, backpressure an solvent consumption were also inicate. Column geometry Injecte volume Mobile phase flow rate Change in analysis time Change in backpressure Change in solvent consumption 50x4.6 mm, 5 µm 0µL.00 ml/min x4.6 mm, 3.5 µm 3.3 µl.43 ml/min. x x4.6 mm,.9 µm 6.7 µl.63 ml/min 7.9 x x. mm, 5 µm 4. µl 0. ml/min x. mm, 3.5 µm.8 µl 0.30 ml/min. x x. mm,.9 µm.4 µl 0.55 ml/min 7.9 x Table summarizes the injecte volumes, mobile phase flow rates, analysis times, column backpressures an solvent consumption calculate with equations to 5 for various typical column geometries. It is possible to fin in the literature a quantity of applications showing the possibility to transfer isocratic HPLC methos to columns packe with sub-µm particles. For sake of clarity, only one example is reporte but the approach can be applie to a wie variety of compouns an matrices. Figure 3 presents a metho transfer from a conventional 50x4.6 mm, 5 µm column to a 50x. mm,.9 µm column. Both columns provie an equivalent efficiency of aroun 0,000 plates (similar L/p ratio). As shown on the chromatograms, efficiency, selectivity an resolution remain equivalent for the separation of seven common anxiolitic agents. After ajustment of mobile phase flow rate, the analysis time coul be ecrease by a factor of 7 ( vs. 3. min), as expecte from theory for a transfer from 5 to.9 µm particles Guielines for the use of UHPLC instruments

8 REGULAR HPLC METHOD Anxiolytiques 4.6 x 50 mm, 5 µm F = 000 µl/min V inj = 0 µl F = 550 µl/min V inj =.4 µl mv x 50 mm,.9 µm 0.08 AU Minutes min Equivalent efficiency UHPLC METHOD Equivalent selectivity Equivalent resolution Analysis time: gain 7x min Minutes. bromazepam,. nitrazepam, 3. oxazepam, 4. clonazepam, 5. alprazolam, 6. flunitrazepam, 7. iazepam Figure 3: Isocratic metho transfer from regular HPLC to UHPLC 4.. Case of graient moe The rules for graient metho transfer are much more complex than isocratic ones but also base on basic principles of chromatography. First, the injection volume an mobile phase flow rate shoul be aapte in a similar way as the isocratic moe (see equations an ). In linear or multi-linear graient elution, the graient profile can be ecompose as the combination of isocratic an graient segments. The rules for efficient graient transfer originally establishe by Snyer an Dolan () an recently upate by Carr et al. () shoul be strictly followe. For both parts, it is important to scale the graient volume in proportion to the number of column volumes, to yiel ientical elution patterns, while the initial an final composition shoul be maintaine constant. In fact, the number of column volumes percolate uring the graient in the regular HPLC system shoul be equivalent to that of the UHPLC setup. For any isocratic step within the graient (i.e. initial isocratic step, isocratic step uring a multi-linear graient an also re-equilibrating time), the ratio between isocratic step time (t iso ) an column ea time (which epens on the mobile phase flow rate, column I.D. an length) shoul be maintaine equivalent between conventional HPLC an UHPLC conitions. Therefore, the UHPLC isocratic step (t iso ) can be etermine with: t iso F c L = tiso (6) F c L As an example, from a regular 50x4.6 mm, 5 µm column to a UHPLC 50x. mm,.9 µm column, the isocratic steps which occurre uring the graient process (incluing re-equilibrating time) shoul be reuce by 8-fol Guielines for the use of UHPLC instruments

9 For slope segments, it is manatory to keep the initial an final graient composition (%B) constant. The new graient time (t gra ) can be expresse as: t gra (%Bfinal %Binitial ) = (7) slope The graient slope (slope ) shoul be calculate to maintain the prouct of graient slope an column ea time constant. The new graient slope (slope ) can be expresse as: slope c L F = slope (8) c L F As an example, from a regular 50x4.6 mm, 5 µm column to a UHPLC 50x. mm,.9 µm column, the graient slope uring the graient process shoul be increase by 8-fol. When transferring a graient metho from regular HPLC to UHPLC, some changes in selectivity coul occur uring the graient run because of ifferences in well volume between the original an the UHPLC configuration. The system well volume (V ) is also known as graient elay volume. It refers to the volume between the mixing point of solvents an the hea of the analytical column, as shown in figure 4. Lowpressure mixing systems possess generally larger well volumes than high-pressure mixing systems. After starting the graient, it will take time until the selecte proportion of solvent reaches the column. It means that the sample is subjecte to an aitional isocratic migration in the initial mobile phase conition. Since the graient well volume may iffer from one system to another, this extra isocratic step woul be ifferent an coul result in retention time variations affecting resolution for early eluting peaks when transferring a metho. To overcome this problem, the ratio of system well time (t ) an column ea time (t 0 ) must be hel constant while changing column imensions, particle size or mobile phase flow rate. Figure 4: Importance of well volume in graient metho transfer As the column ea time is reuce by aroun 8-fol between a regular 50x4.6 mm, 5 µm column an a 50x. mm,.9 µm column, the system well time shoul be reuce by a similar factor. Therefore, it is manatory to work in UHPLC with a system possessing a very low well volume (no more than a few hunre µl) to limit the influence of V. When the ifference between t /t 0 ratios remains too large, it is also possible to a an isocratic hol at the beginning of the UHPLC graient Guielines for the use of UHPLC instruments

10 Again, the applicability of the approach iscusse can be foun in the literature for metho transfer between conventional an sub-µm packings. One example has been selecte an is presente in figure 5. The separation of pharmaceutical compouns was originally achieve using a mm, 5 µm, C 8 column an further transferre to UHPLC with a 50. mm,.7 µm C 8 column possessing strictly similar chemistry. The original separation was performe in approximately 7 minutes an efficiently transferre to UHPLC in less than 3 min (reuction by a factor of 9). In aition, both separations were equivalent in terms of sensitivity, peak capacities an resolution, mainly because of an aequate reuction of system well volume (from.3 ml to 30 µl for HPLC an UHPLC, respectively). Another relevant avantage of UHPLC is the re-equilibrating time reuction. In HPLC (50x4.6 mm column at ml/min), re-equilibration took about 0 minutes, while using a short column packe with sub- µm particles (50x. mm column at 600 µl/min), the re-equilibrating time ecreases to only minutes. 4.6 x 50 mm, 5 µm F = 000 µl/min V inj = 0 µl P = 80 bars AU REGULAR HPLC METHOD min Minutes UHPLC METHOD Equivalent Peak capacity Equivalent Selectivity Equivalent Resolution Equivalent Sensitivity Analysis time gain 9. x 50 mm,.7 µm F = 600 µl/min V inj =.5 µl P = 50 bars AU min Minutes Figure 5: Graient metho transfer from regular HPLC to UHPLC A last recommenation when working in UHPLC is to be careful when increasing mobile phase flow rate in the graient moe. For an aequate transfer, it is necessary to aapt the graient profile when increasing the flow rate, as illustrate by equations 6 an 8. Therefore, when changing mobile phase flow rate, the graient slope an time of any isocratic step shoul be aapte in orer to maintain equivalent selectivities. Equations to 8 can be use for etermining the new parameters of a transferre isocratic or graient metho. However, at the University of Geneva, we evelope a free program calle HPLC calculator poste on our website ( which gives optimal conitions for metho transfer in isocratic an graient moes. In this calculator, the system well volume can also be consiere for calculation in graient moe. 5. Conclusion The UHPLC technology (efine as columns packe with sub-µm particles use in very high pressure conitions) has prove to be a powerful approach to improving chromatographic analysis in terms of throughput an resolving power. By comparing its intrinsic performance with other existing techniques, such as monoliths or high temperature liqui chromatography, it is a very attractive strategy for improving chromatographic efficiencies in the range,000 80,000 plates Guielines for the use of UHPLC instruments

11 An important factor in obtaining suitable performance with columns packe with sub-µm particles is the choice of instrumentation. Inee, it is manatory to work with a chromatographic system that withstans pressure higher than 400 bar. The system shoul also possess very low extra-column volume, limite well volume an a sufficient etection acquisition rate. The rules for metho evelopment in UHPLC are slightly ifferent from that of regular HPLC because the high backpressure generate by the columns packe with very small particles has to be taken into account. For example, acetonitrile is the first choice for mobile phase organic moifier because of the lower viscosity compare to methanol. In aition, it is always beneficial to work at a temperature slightly higher than ambient (40-50 C). One of the main avantages of UHPLC remains the possibility to transfer easily the existing HPLC methos to columns packe with sub-µm particles, using basic equations of chromatography. In the isocratic moe, only the injecte volume an mobile phase flow rate have to be ajuste. In the case of graient elution, injecte volume, flow rate, isocratic step uration an graient slope must be aapte an well volume shoul be carefully consiere. 6. References. J.W. Dolan, L.R. Snyer, Maintaining fixe ban spacing when changing column imensions in graient elution, J. Chromatogr. A 799 (998) -34. A.P. Schellinger, P.W. Carr, A practical approach to transferring linear graient elution methos, J. Chromatogr. A 077 (005) List of Abbreviations Symbols: c : column internal iameter p : particle size of the support F: mobile phase flow rate k: retention factor of the compoun L: column length N: chromatographic efficiency t 0 : column ea time Greek symbols: P: column backpressure σ² ext : extra-column ispersion σ² col : column ispersion τ : time constant of the etector %B initial : initial percentage of organic moifier %Bfinal: final percentage of organic moifier t ana : total analysis time t : system well time t iso : initial isocratic hol t gra : graient time u: mobile phase linear velocity V: Volume of solvent consume V 0 : column ea volume V inj : injection volume - - Guielines for the use of UHPLC instruments