A new type of method development that uses modelling to find the "best" separation for high performance liquid chromatography (HPLC) was investigated. Principles of Quality by Design (QbD) were followed when planning the investigation. A simple Design of Experiments (DoE) with only three measured factors — gradient time, pH, and temperature — was used with different columns. The basic experiments were saved in an electronic file with the peak tracking results. After calculating a Design Space, the best prediction was compared with a confirmation run. The process delivered precise results and the method was able be transferred to a routine quality control (QC) laboratory.

For many years, a trial-and-error approach has been commonly practiced in high performance liquid chromatography (HPLC) method development — a "pick the winner" strategy. After the validation process had begun, several surprising observations were often made such as new peaks, the disappearance of other peaks, and changes in critical peak pairs. The typical response was to go back to the development process. To try to improve the separation, several test steps would be carried out, to interject quality into the method. This time-consuming process can be avoided by applying Quality by Design (QbD) principles, ensuring the quality of the method at the design stages (1). A well-established means of implementing QbD principles is to use modelling software. 

To apply QbD principles to the development of HPLC methods, focus must be placed on method understanding and solid science. We took two-dimensional modelling and extended it to include a third measured factor, establishing the "Cube" (2). In a 2011 paper we demonstrated that predictions made in this way are of high quality (3). In QbD-related method development processes, trial-and-error approaches should be avoided. The best method should be developed in a planned manner, based on solid science (4). There is a great number of different factor combinations that influence retention and resolution of the components in a mixture. The first step is column selection (5). In this study, we tested over 10 different columns and selected the Acquity BEH C18 column (Waters). To establish the number of peaks, the sample was first tested with a "scouting gradient." 

Which Design of Experiments (DoE) is the simplest? The most successful design is the t

 = temperature). The most successful design is simple because it requires only four runs: Two gradients, t

 (or the slope), as was shown by Snyder and Dolan (6). We extended this design with the addition of a third factor, either pH or ternary composition, t

 (2). The columns used had dimensions of 50 mm × 2.1 mm packed with 1.7-μm particles. We also used 4 and 12 min run times, with two different temperatures, 

, here 20 °C and 50 °C. The four runs were performed following a simple batch protocol. The main points are: Not to change the injection volume during the investigation; not to interrupt the process if new peaks appear; and to use a representative sample containing all possible, even unknown, components.

The next step was to organize the received chromatograms and make sure peak movements were realized and understood — a process called

 Every component is aligned in a horizontal line. This can be supported by the peak area of the sample, which is fairly constant in a t

 model. In the case of overlapping peaks, the areas are additive. 

When the peak table was finished, the software calculated mathematical functions for each component. It allowed the visual examination of the resulting chromatograms, including peak movements, peak overlaps, and changes in elution order.  

Compound retention behaviour is modelled so that the influence of all significant parameters (alone and in combination) is known, quantified, and visualized (2). In the HPLC method development process, relationships between parameters and their effect on the results are in this way well understood. This translates into a richer chromatographic understanding and facilitates a better scientific work.  

Rather than running random test arrangements — necessary when applying statistical analysis — to fulfill QbD conditions, HPLC method development with three-dimensional models is achieved by running three t

models with only 12 simple experiments. From these, a large number (over 10

) of virtual experiments could be derived and the best one found quickly. The obtained retention models were used for robustness testing, continuous quality control, and method transfer, and could serve as a fundament for Knowledge Management. In the following, we planned to compare an older method that took approximately 40 min with the new approach, based on a practical example from an industrial production process, using t

pH studies to enhance column selectivity choices in ultrahigh–pressure liquid chromatography (UHPLC).

The mobile phase was a mixture of acetonitrile and 5 mM ammonium dihydrogen phosphate buffer. Acetonitrile (gradient-grade), methanol, ammonium dihydrogen phosphate, phosphoric acid, and standard reference buffers (pH 2.00, 4.01, and 7.00) were obtained from Merck. For measurements, water was prepared fresh using ELGA Purelab UHQ water. The buffer was filtered before use on a regenerated cellulose filter membrane, 0.2-μm pore size (Sartorius). 

Samples contained 10–30 μg/mL amlodipine and its 

 impurities (A, B, D, E, F, G, H) and 1 mg/mL amlodipine spiked at 0.1% (European Directorate for the Quality of Medicines & HealthCare).  

An Acquity UHPLC system (Waters), Empower software, and Acquity BEH C18 50 mm × 2.1 mm, 1.7-μm column (Waters) were used. This system had a 5-μL injection loop and a 500-nL flow cell. The dwell volume of the system was measured to be 0.125 mL. The pH was measured with a MP 225 pH meter (Mettler Toledo).  

Method development and method modelling were performed using DryLab 2010 HPLC/GC optimization software, consisting of the DryLab Core-module v.3.9, PeakMatch v.3.6, and the 3D-Resolution Space feature called the "Cube" (Molnár-Institute).  

The problem of older HPLC methods, which may be available in pharmacopeias, is that the columns are often of an old type which are sometimes not available anymore. Furthermore, many old methods are very slow. This causes, in a modern pharmaceutical environment, great delays in the production of certain drugs. We need an easy replacement for older methods to be able to use more modern instrumentation and columns. We tried, in our case, to reduce the analysis time of the old method from 40 min to hopefully less than 10 min. 

There were several approaches to the selection of the proper design in the literature (7,8). As DoE we use the one shown in Figure 1 with 12 experiments (2). The advantage here is the fast evaluation of the sample using gradients. The creation of a cube was based on reversed-phase gradient elution (9) with the columns mentioned above, and was done in 3–4 h, representing more than 10

  virtual experiments. Column selectivity was evaluated quickly under consideration of both stationary and mobile phase influences.  

Figure 1: Design of experiments (DoE) for the simultaneous optimization of gradient time (tG), temperature (T ), and pH of the eluent (A); or ternary composition (tC) of the eluent (B). Circles represent the 12 experiments inputted for the 3D model.			

Experimental design for simultaneous optimization of gradient time, temperature, and pH and ternary eluent composition t

, required 12 experiments, as illustrated in Figure 2 (2). Two basic gradients with different rates (4 and 12 min gradient time, in a range of 30–90% B) were carried out at two different column temperatures (20 and 50 °C). The mobile phase "A" consisted of 5 mM ammonium dihydrogen phosphate buffer (pH = 2.0, 2.5, and 3.0), "B1" was acetonitrile, and "B2" was methanol. The flow-rate was at 0.5 mL/min. The injection volume was 1 μL.  

Figure 2: Experimental designs for the simultaneous optimization of gradient time, temperature, and pH for three organic solvent compositions. Circles represent 12 experiments input for the 3D model.			

We were able to create a four-dimensional model, as we prepared three cubes in identical pH ranges, but with three different ternary eluents, methanol, acetonitrile, and 50:50 (v/v) (methanol:acetonitrile). Then we selected a pH value, where our separation was robust in terms of pH with little risk of losing efficiency. From the three cubes we exported 3 × 4 chromatograms as the reference points of a new cube.  

 = 40 °C; pH = 2.3; and ternary composition (t

 sheets that belong to pH 2.3, as shown in Figure 3.  

Figure 3: Three-dimensional tG-T-pH models (Cubes) with three different organic solvents. Red colours mean regions above Rs (crit > 3.0), blue colours indicate co-elution (Rs,crit = 0) of the closest ("critical") peak pair.			

In Figure 4 we can see that the system is robust for pH changes, but not robust at two ternary eluents: With 100% acetonitrile (AN) the resolution between ImpG and ImpB is small. In the case of methanol (MeOH) the components ImpE and ImpG exhibit partial overlaps. Using 50:50 (v/v) methanol:acetonitrile as eluent B, we achieve a much better separation of the critical impurity components.  

Figure 4: Predicted chromatograms are showing that in all three eluent compositions robust separations are possible at pH 2.3 (tG = 8 min, 30â70% B, T = 40 Â°C).			

The next step was the selection of the "working point" at 40 °C and t

: 5 min (30–90% B) where B was 75:25 (v/v) acetonitrile:methanol.  

Figure 5: Four-dimensional tG-T-ternary model (fourth dimension was pH) and tG-T-sheet with "working point".			

Finally, the conformation of the prediction in Figure 6(a) revealed an excellent separation of all components, which could be well confirmed, as shown in practice with a mix of the standards in Figure 6(b) and with the drug product in Figure 6(c).  

Figure 6: (a) Predicted chromatogram, (b) experimental chromatogram, and (c) experimental spiked chromatogram at 0.1% level.			

The precision of the predicted vs. modelled experiments are showing only small deviation of a few hundredths of a min (11–13). Looking at the last two peaks, the retention times are 3.145 vs. 3.148 min, a difference of 0.003 min and 4.944 vs. 4.911, a difference of 0.033 min.  

Figure 7: Experimental designs for simultaneous optimization of gradient time, temperature, and ternary composition at three different pH values of the eluent A. Circles represent the 12 experiments input for the 3D model.			

After these steps we turned the process in the experimental design around and produced first three Cubes with a constant ternary composition region, but with three different pH values. For the creation of a new Cube we selected one ternary composition, and from the three Cubes we exported 12 runs. We received at the same working point the same separation as in the first part of the study (see Figures 7–10).  

Figure 8: Three-dimensional tG-T-ternary models (Cubes) with three different pH values (2.0, 2.5, and 3.0). Red colours mean regions above Rs (crit > 3.0); blue colours indicate coelution (Rs, crit = 0) of the closest ("critical") peak pair.			

In Figure 9, the "working point" was selected at t

 = 40 °C and pH 2.3. The predicted chromatogram has a run time of less than 5 min.  

Figure 9: Four-dimensional tG-T-pH model (fourth dimension was ternary composition) and tG-T-sheet with "working point".			

Using the software we were also able to increase the speed of the separation by modelling the flow rate.  

Figure 10: Predicted four-dimensional chromatogram.			

 = 2 min) and an increased flow rate of 0.8 mL/min, we were able to further reduce the analysis time to less than 1.8 min.  

 = 2 min; gradient range 30–90% B; flow rate: 0.8 mL/min; 

 = 75:25 (v/v) acetonitrile:methanol we receive the prediction shown in Figure 11(a) with the correspon ading confirmation run in Figure 11(b). In this way, the analysis time could be reduced to less than 1.8 min.  

Figure 11: (a) Predicted chromatogram with increased flow rate to 0.8 mL/min; (b) Experimental spiked chromatogram at 0.1% level. Conditions are as described in the text for Figure 11(a).			

 method is very long and this specifies only three impurities (14) while our method takes less than two minutes and seven impurities are specified (see Figure 12).  

Figure 12: Chromatogram of amlodipine and its impurities from the European Pharmacopeia 7.4, Amlodipine besilate 04/2012:1491 p.4275-4276. The Ph.Eur. method is very long and specifies only three impurities, while our method has a run time of less than 2 min and specifies seven impurities. 			

In this study, we were able to reduce the analysis time of a pharmaceutical preparation from 40 min to below 1.8 min using UHPLC instrumentation, modern short UHPLC columns, and modelling software. With the short analysis time the production of the drug is faster and more economical than in the past. The use of the modelling software allowed us to fulfill QbD criteria for the method, increasing flexibility in a routine operation. Retention and critical resolution phenomena became more transparent than they were before. Method transfer is also easier using the Knowledge Management protocol described in the paper. In case of renewing older methods we recommend the above technology because of its reliability and speed. 

, president of Molnár-Institute, is a former coworker of Prof. Csaba Horváth at Yale University, where he developed the fundamentals of reversed-phase chromatography and the well-known theory of "solvophobic interactions." In 1981 he founded the Molnár-Institute for Applied Chromatography in Berlin, Germany. He specializes in pharmaceutical research and analysis and conducts a series of HPLC courses on reversed-phase HPLC and the development of robust HPLC methods. Since 1984 he has worked with LC Resources Inc. on the development of the DryLab software, which is now widely used in industry and in the life science community as a modelling tool for complex separations, enabling the user to minimize the number of experiments required to develop an optimized and robust method. Since 2006 DryLab has been developed in Berlin further; the new version DryLab 4 is written in C#. Direct correspondence to: 

, vice president and product manager, has been working with the Molnár-Institute since 1999 as a chemist, specializing in software programming. In cooperation with Dr. Molnár, he is responsible for the development of new modules of DryLab for better peak tracking, visualizing the Design Space, and robustness calculations as well. He also teaches DryLab-courses on an international scale. E-mail: 

is a chemist with a Master of Science degree from the University of Debrecen in Hungary. He completed his education with an additional degree in chromatography and analytical chemistry from the Institute of Analytical Chemistry of the Technical University Budapest in the department of Prof. Dr. Jenö Fekete. Mr. Kormány specializes in UHPLC method developments for the separation of pharmaceutical compounds using computer simulation. E-mail: 

Articles

The experimental design that considers gradient time and temperature was expanded to include a third factor, either pH or ternary composition, and modelling software facilitated the analysis.

Application of Quality by Design Principles to a Pharmaceutical Sample Using UHPLC Method Development with Modelling Technologies

High performance liquid chromatography (HPLC) is a sophisticated technique. Chromatographers must not forget the basics of method development to reap the full benefits in practice. Obvious parameters to optimize include the choice of chromatographic column, mobile phase, injection volume, flow rate, and detector settings. However, it is also beneficial to know how to determine carryover, the properties of the needle wash solvent used, and the importance of monitoring instrument back pressure. This article will discuss issues related to carryover, instrument back pressure, and the choice of wavelength.  

Wavelength selection, back pressure, and injection-to-injection carryover were investigated to determine considerations during method development. The goal of the experiments was to control the overall precision of a technical assay measurement. We have investigated these parameters for two commercial agricultural products and report the results here. 

Carryover is a common problem in high performance liquid chromatography (HPLC). Carryover is detected by the unexpected presence of small peaks when a blank sample is injected following the injection of analyte samples. It can also be detected by unexpectedly poor injection-to-injection precision following multiple sample injections. If a calibration curve is constructed, deviations from linearity may signal the presence of carryover (1).  

The design of modern HPLC autosamplers has been greatly improved, decreasing the likelihood and magnitude of carryover from injection to injection. Manufacturers have changed the physical designs of instrument hardware to utilize features such as flow-through needles and active needle washes. In addition, instrument control software can offer different methods of washing the needle and the injector valves. 

To make the best choices regarding carryover, operators should become familiar with the options installed on the instrument. Even if familiar, they still have to make the right choices to take full advantage of them.  

The easiest way to determine if there is carryover in a method is to run blanks between sample injections. Blanks can be composed of the mobile phase organic modifier or sample diluent, among other options. During method development, it is important to run blanks regularly, whether to identify the amount of carryover, or to show that it is non-existent or negligible. It is insufficient to assume that a clear chromatogram for one blank injection following the first sample injection means that carryover is not an issue for the sample and method being used. Even after method development completion, it is good practice to continue to inject blanks interspersed with your samples and standards. This guarantees the validity of the original determinations and that the sample matrix does not change chromatographic behaviour. 

If carryover is identified, many steps can be taken to correct it. First, it is important to determine what type of carryover is present. The problem may be hardware — a small amount of the sample may be held up in the system and injected onto the column in subsequent injections. Some solutions to this are to check: Auto-sampler hardware; all compression fittings and tubing connections; and the wash solvent and station (2).  

The problem may also be an issue with the chosen washing mechanism; autosamplers have active needle washes to rinse the outside of the needle to prevent carryover (2,3). Users need to follow the manufacturer's recommendation for priming and flushing these needle wash systems. An alternative option is to wash the needle in a vial of solvent. If used, it is important to change the vial often, depending on the analyte of interest. This paper shows how calculated injection-to-injection reproducibility can be compromised by carryover, simply because the optimum wash solvent was replaced with one that did not work as well. 

Another consideration for HPLC users is instrument back pressure. When high pressures are discussed in relation to HPLC, it is typically with a negative connotation. However, high pressure pumps are what allow the mobile phase to be pushed through the chromatographic column's packed bed, driving separation. Newer generation, fully porous columns are being produced with smaller particle sizes and longer lengths, increasing efficiency. Therefore the pressure required to force the mobile phase through these columns is increasing. Many manufacturers now offer instrumentation that offers much higher maximum pressures, allowing the user to achieve more efficient separations with current column technology. This makes it increasingly important to monitor back pressure and to pay special attention to the maximum back pressure rating on columns, fittings, and instruments. 

For years, it seemed that the maximum pressure rating on instruments, fittings, parts, and columns was 400 bar. This was also the maximum pump pressure for the instrument before it would shut itself off, so it may not have seemed important to check the pressure limits carefully. However, with the introduction of ultrahigh-pressure liquid chromatography (UHPLC) instruments, technology, and columns, products are now commercially available that each have their own maximum pressure rating. Special attention should be paid to the stated maximum pressure ratings when replacing fittings, and when purchasing and using columns. If columns are used at pressures above their maximum rating, the packing can be fractured, resulting in fine particles that can move through the column toward the outlet frit, increasing the back pressure. Possible causes of high back pressure were discussed along with ways to deal with these issues in a previous 

Back pressure should be initially checked when the column is installed and equilibrated. It should then be continually monitored before, during, and after the analysis. As a safety consideration, it is also important to check that maximum pressure settings on the instrument do not exceed the maximum pressures allowed for the fittings and columns so that these pressures are not exceeded during analysis. This can be particularly important for method transfers to other instruments and will be discussed later in more detail. 

Lastly, this article will discuss the choice of wavelength for each analysis. The detector handbook for the two liquid chromatography instruments used gave all of the following information about optimizing detector wavelength (5,6):  					

Choose a longer wavelength than the cutoff wavelength of the mobile phase. Solvents used as HPLC mobile phases have some absorbance in the ultraviolet (UV) region. The cut-off wavelength is the wavelength below which the mobile phase has high absorbance and could interfere with the analysis. 

Choose a wavelength where the analytes have strong absorptivity to achieve the lowest possible detection limit.

Choose a wavelength with moderate absorptivity for high concentrations. 

Set the sample wavelength to coincide with a peak or valley in the spectrum to achieve the best linearity. 

Analytical chemistry textbooks typically suggest choosing wavelength at an absorbance maximum and bandwidth that gives the required signal-to-noise ratio (

). Models representing the correlation between diode array UV detector responses, the detector band pass, and the analyte's absorbance spectra were presented in a paper published by Dose and Guiochon (7). The work shows that when using a UV detector, there has to be a compromise between the linearity of the response and the accuracy of the absorbance measurement. For chromatographers who typically use the calibration curve to quantitate, linearity is of utmost importance. As a result, the wavelength is typically chosen at a spectral maximum (7). We have performed a study to determine whether the choice of wavelength, whether at an absorbance maxima, at an absorbance valley, or on a sloped portion of the curve, would have an effect on the precision of the results calculated from the experiment.  

Diuron and rimsulphuron standards were obtained from DuPont Crop Protection. Phenyl sulphone (Aldrich Chemicals) and 3–methyl-1,1-diphenylurea (Aldrich Chemicals) were the internal standards. HPLC-grade acetonitrile (EMD Millipore Corp.), and phosphoric acid (EM Science), and house deionized water were used for mobile phase preparation. Diuron sample solutions were used immediately or stored in the refrigerator. Rimsulphuron samples, because of solution issues, were used immediately. 

 Diuron standards and samples were prepared in acetonitrile. The standards ranged in concentration from 0.75 to 1.25 mg/mL and samples were prepared at a concentration of 1 mg/mL. Precision data were calculated based on eight individually prepared samples with duplicate injections of each sample. The internal standard was 3–methyl-1,1-diphenylurea. The final internal standard concentration was 1 mg/mL The chromatographic conditions are given in Table 1. The two HPLC systems used were an Agilent 1290 Infinity HPLC system and an Agilent 1100 HPLC system, both with diode array detection (Agilent Technologies Inc.).  

Table 1: Chromatographic conditions used in experiments.			

Rimsulphuron standards and samples were prepared in acetonitrile. The standards ranged in concentration from 0.25–1.5 mg/mL and the samples were prepared at a concentration of 0.75 mg/mL. Precision data were calculated based on eight individually prepared samples with duplicate injections of each sample. The internal standard was phenyl sulphone. The final internal standard concentration was 1.95 mg/mL The chromatographic conditions are given in Table 1. The HPLC system used was an Agilent 1100 HPLC system with diode array detection (Agilent Technologies Inc).  

If carryover is detected, the options are to wash the needle wash in a flush port, in a sample vial, or wash in multiple sample vials. An active needle wash is best used when the analytes in the sample have similar solubility. The solvent or solvent mixture and the length of wash time can be optimized for the sample. It is important, if using an active needle wash, that the solvent reservoir be checked regularly and the needle wash system primed or flushed with the solvent prior to analysis (in the same way that it is necessary to prime and flush LC pumps). It is also good practice to rinse the needle flush port with at least two times its volume before the analysis. If an active needle wash is not available, or if more than one solvent is needed to clean the needle, then it may be necessary to wash the needle in solvent placed into capped solvent vials. If this method is used, it is important to regularly change the vials of solvent used, which can increase method execution time. For samples where carryover is a significant problem, it may be necessary to wash the needle after every few injections in a new vial of solvent. Figure 1 shows the results of an experiment where the analyte was known to have issues with carryover. The graph shows the specifications for injection precision for the instrument used. It also shows the difference in the method precision (based on %RSD of peak areas) based on eight replicate injections, when two different needle wash solvents were used. The results show that for this particular analyte, 50% acetonitrile in water was not the right choice. When the solvent was changed to 100% acetonitrile, the %RSD calculated for the method were closer to those expected for the instrument used.  

Figure 1: Method precision for a diuron UHPLC method when different needle wash solvents are used.			

 When developing methods, back pressure needs to be a consideration for several reasons. First, it is often one of the early signs of either instrument or column failure. If a column, guard column, needle seat, or a piece of instrument tubing is becoming clogged, the pressure may slowly creep up. Knowledge of the typical back pressure for the instrument, without the column installed, can help to quickly determine if the column or a portion of the instrument is the cause of high back pressure. The user should also have an idea of what back pressures to expect when using a particular method.  

Monitoring back pressure can also be important for smooth method transfer and column longevity. When using a gradient method, the back pressure can vary greatly over the course of one run. This is especially true for methods involving methanol. The viscosity of binary methanol/water mixtures changes greatly as the percentage of each solvent in the mixture changes. If a method starts with low back pressure (within the range for the instrument and column), but during the method nears or exceeds the higher end of the range for the column, it can result in premature column failure and increased cost. When transferring a method to another instrument, this becomes even more important. If the second system (instrument and column) has a higher overall back pressure than the first, then the method may exceed the pressure maximum causing the method to fail, preventing successful method transfer. The method would then need to be modified and revalidated.  

Figure 2: Rimsulphuron and phenyl sulphone (IS) absorbance spectra overlayed with detector wavelengths indicated.			

Diuron and rimsulphuron were chosen for this portion of the study. Standard solutions of the analyte and an internal standard were prepared along with eight individually prepared samples. Data were simultaneously collected at several wavelengths. The wavelengths were chosen at a spectral maximum, at a spectral valley, and also on the sloped portion of the spectrum. Note that the wavelengths (shown in Figures 2 and 3) were picked by analysing the spectra for the analyte and internal standard. Calibration curves were prepared for both an internal standard calibration and an external standard calibration. The calibration curves were then used to determine the percent recovery for the samples. The correlation coefficients and standard deviation values for the calculated percent recoveries are shown in Tables 2 and 3. The results shown for the internal standard and external standard methods were calculated using data from the same experiments. Class A volumetric glassware was used for solution preparation so that data collected would give information for both methods. For the external standard method, only the data for the analyte were used and a calibration curve was prepared by plotting the peak area for the analyte against the concentration of the analyte in each standard solution. For the internal standard method, the calibration curve was prepared by calculating the ratio of the analyte peak area to the internal standard peak area and plotting this ratio against the ratio of the analyte amount to the internal standard amount. The resulting calibration curves were used to calculate the percent recoveries for each analyte. The standard deviations of the calculated percent recoveries are shown in Tables 2 and 3; the correlation coefficients for the calibration curves are also included.  

Figure 3: Diuron and 3-methyl-1,1-diphenylurea (IS) absorbance spectra overlay with detector wavelengths indicated.			

Analysis of correlation coefficients for the calibration curves produced for different wavelengths showed no striking differences. Analysis of the standard deviations was not so clear,  so we turned to statistics for a proper analysis.  

Table 2: Precision results (calculated from percent recovery results for eight standards injected in duplicate) and correlation coefficients for calibration curves for rimsulphuron. Data collected on an Agilent 1100 instrument.			

For rimsulphuron samples there were slight differences in the calculated standard deviations at different wavelengths for both the internal standard method and the external standard method. Using the F-test, it was determined that the calculated standard deviations were not significantly different, independent of what wavelength was used and the method of calibration.  

Table 3: Precision results (calculated from percent recovery results for eight standards injected in duplicate) and correlation coefficients for calibration curves for diuron. Data collected on an Agilent 1100 instrument.			

For diuron samples the F-test showed a significant difference in the standard deviations calculated using the internal standard method and the external standard method. However, the F-test did not show a significant difference between the results calculated at different wavelengths. 

The choice of wavelength is known to be important as it determines how strongly the analytes absorb. Therefore it determines how large the signal and 

ratio will be. However, this experiment suggests that as long as the 

 ratio meets requirements, and the peaks are large enough to be easily and accurately integrated, there is a lot of flexibility in wavelength choice. Therefore as long as the calibration curve is linear and the samples fall within the appropriate range of this calibration curve, the wavelength can be chosen to allow for other improvements in the experiment. For example: A wavelength where the analyte has lower absorbance may be chosen so that the amount of analyte being weighed for the experiment can be increased.  

Chromatographers have a wide variety of new options in instruments and columns that allow for better separation and quantitation of their samples. However, we must not forget to go "back to basics" when developing methods, or the full effects of all the recent improvements may not be realized. Some essential factors to consider during method development, and also later as the methods are being used routinely, are: Sample carryover, system back pressure, and wavelength choice. 

 is an associate professor of Chemistry at West Chester University, Pennsylvania, USA. She contributed to this work during a sabbatical as a visiting scientist at DuPont Crop Protection.  

 is a senior research associate at E.I. DuPont de Nemours and Company.  

 is a technical fellow at E.I. DuPont de Nemours and Company, and a member of the editorial advisory board of 

http://www.lcgceurope.com/lcgceurope/data/articlestandard//lcgceurope/042002/7657/article.pdf

(5) Agilent Technologies 1200 Series Variable Wavelength Detector Service Manual, 2nd edition (2006) G1314–90110. 

(6) Agilent Technologies 1200 Series Diode Array and Multiple Wavelength Detectors, 2nd edition, (2006) G1315B / G1365B. 

The parameters that should be considered in the optimization of HPLC methods are explained and then illustrated through the analysis of two commercial agricultural products.

Back to the Basics: Considering Carryover, Column Back Pressure, and Wavelength When Developing Chromatographic Methods

At the turn of the 21st century, supercritical fluid chromatography (SFC) experienced a re-birth prompted by a demand to separate polar analytes, initially in the pharmaceutical industry. Instrument design and performance improved in tandem with this demand. Preparative SFC has also improved significantly over the last decade and chiral stationary phases have been introduced for the SFC of racemic drug-related mixtures. The introduction of more polar stationary phases and unique mobile phase additives has enabled separation of water-soluble, ionic, and high molecular mass analytes. This article looks at the past, present, and future of SFC.

Supercritical fluid chromatography (SFC) was first proposed in 1958 at an international gas chromatography (GC) meeting by James Lovelock, one of the research pioneers in GC, while at Yale. Lovelock conceived the idea of using supercritical fluids as chromatographic mobile phases to increase solvating power, and to facilitate the elution of nonvolatile substances (1). In 1962, Ernst Klesper was the first person to use a supercritical fluid as a chromatographic mobile phase, utilizing it for the analysis of nonvolatile materials. Klesper used a 30-in. long column packed with 33% Carbowax 20M on Chromosorb W to separate a mixture of nickel porphyrins with supercritical dichlorodifluoromethane and monochlorodifluoromethane mobile phases. The technique was called "high pressure gas chromatography" (2). Work in the area of supercritical fluid technology continued with a number of applications, including the analysis of petroleum mixtures and silicone polymers (3). Between 1962 and 1981 nearly all of the 91 SFC-related papers published involved the use of packed columns (4), but SFC remained relatively underdeveloped as a viable separation alternative. In 1983, Hewlett Packard (HP) modified a limited number of their Model 1084 liquid chromatography (LC) instruments for packed column SFC (pcSFC). They were flow-controlled and able to accommodate the use of various modifiers. In the mid-1980s, a second commercial instrument was introduced that incorporated open tubular, wall coated fused silica column technology (5). This action afforded unprecedented litigation that prevented further packed column SFC development for several years. During this time, capillary SFC experienced explosive growth because it was seen as an extension of GC where the conventional sample base could be extended to less volatile, more reactive analytes. Nevertheless, many experimental issues associated with capillary SFC were not readily addressed, despite its wide acceptance at the time. 

Despite worldwide acceptance, in the late 1980s SFC using capillary columns was in bad shape. There were some prominent, unsolved experimental issues:  									

UV detection was feasible but not practical

Fragile fused silica restrictors were employed

Extension of the technology to small bore packed columns by Lee Scientific and Brownlee, Inc. afforded minimal advantage. Regardless, the technique was viewed as an extension of GC that was amenable to primarily high molecular mass, non-polar analytes. Separation scientists generally agreed that SFC seemed to be finding its niche as a technique that could handle larger molecules than GC, at higher efficiencies than high performance liquid chromatography (HPLC). However, although numerous SFC vendors offered GC instruments with basic modifications, there was no long-term vendor commitment to the technology. Columns tended to be operated well above the critical temperature of the supercritical fluid, while pressure (or density) programming replaced conventional temperature programming. Capillary SFC was most often practiced using (a) columns with inner diameters considerably smaller than GC columns (50 μm i.d.); and (b) highly cross-linked non-polar stationary phases to avoid significant CO

 promoted column bleeding. Capillary columns dominated the development of both SFC theory and instrumentation during this period (5). A syringe pump was used as a pressure source, with a fixed restrictor at the column outlet limiting flow through the small inner diameter open tubular column. Primary vendors in the development of capillary SFC during the 1980s were Dionex and Isco Inc. 

Packed column SFC was not viewed with much favour because it almost always used: (a) Silica-bonded stationary phases such as cyano– and amino–propyl designed specifically for HPLC; (b) binary or ternary CO

-based mobile phases; and (c) ultraviolet (UV) detection via a high pressure flow cell. Once modifiers were added to the CO

, mobile phase composition became more important than CO

 pressure or density in determining retention. By necessity, capillary SFC exclusively focused on non-polar analytes; whereas packed column SFC found limited application in the separation of polar analytes (6). Packed columns were usually operated near the critical temperature of the fluid, with less than precise flow control pumps and electronically controlled back pressure regulators. The combination of upstream flow control and downstream pressure control allowed, to a limited extent, both volumetric mixing of the main fluid and modifier. With these developments, packed column SFC was increasingly viewed as an extension or subset of HPLC. The most important idea that supercritical fluids brought to separation science during this period was probably the recognition of the unity in the separation methods, and that a continuum existed between gases and liquids.  

In 1992, Gilson, Inc. and Hewlett Packard, Inc. both introduced commercial analytical scale SFC systems that allowed computer control of all variables. The HP system was capable of both packed and capillary column operation; while the Gilson instrument delivered a higher flow rate. This prompted several unsuccessful attempts to create a small-scale semi-preparative instrument using 1-cm inner diameter columns. The HP product line was sold in 1995 to Berger Instrument where semi-preparative SFC was found to be successful for both achiral library purification and chiral-like stacked injections.  

Packed Column SFC Rediscovered: 1990–2012

SFC with packed columns underwent a renaissance at the end of the 1990s when (a) the limitations of capillary SFC became obvious to separation scientists; and (b) important progress in pressure gradient techniques of mixed mobile phases was achieved. Advancements in this area were halted by misconceptions about the role of modifiers in the SFC chromatographic system. For example, it was widely thought that modifiers only covered active sites on the support and did not dramatically increase solvent strength. Currently, the overall effect of adding a modifier to the mobile phase is considered to be a combination of mobile phase modification effects and stationary phase effects. Despite the early success enjoyed by SFC with modifiers and additives, at the time there was considerable resistance to the use of more complex and expensive binary pumping systems, and UV detectors with packed columns (7).  

Today, pcSFC is generally agreed to be an ideal replacement for normal phase LC. SFC (a) exhibits faster retention; (b) re-equilibrates extremely fast; (c) allows steep gradients; (d) tolerates significant amounts of water; (e) allows very long columns; (f) can incorporate very small particles with modest column pressure drops; and (g) is "green" technology. SFC outperforms normal-phase HPLC and even reversed-phase HPLC for some applications. Interest in packed column SFC is higher than ever, as it is capable of generating peak efficiencies similar to those observed in GC, and separations can be run at much higher flow rates than in HPLC. Furthermore, pcSFC is scaleable, detector-friendly, and relatively economical. Unlike reversed-phase HPLC, in which almost all work is performed on a variant of a single stationary phase (C18), SFC employs a wide range of polar stationary phases with significantly different selectivities (8). 

During this period, pcSFC was observed to offer many advantages for the rapid separation of enantiomeric products over HPLC because of its greater separation efficiency per unit time (9). Lower temperature was beneficial as chiral selectivity usually increased with decreasing temperature. Low temperature also reduced the risk of analyte racemization and thermal decomposition. To successfully perform chiral pcSFC, a polar modifier was added to the CO

 mobile phase and, in many cases, operation across a gradient became desirable during the separation. pcSFC was ideal for chiral screening that provided rapid identification of the most appropriate column and modifier. Finding a highly selective chiral stationary phase was a key step in method development and the column screening strategy proved to be highly successful. 

The use of SFC for preparative separations (PrepSFC) has received considerable attention in the past three years (10). The volume of solvent and waste generated is reduced, offering an economic bonus, and making PrepSFC especially attractive for the provision of purified materials on a kilogram scale. The product of SFC is recovered in a more concentrated form relative to HPLC, reducing the amount of solvent that must be evaporated. The higher pcSFC flow rates also contribute to higher productivity relative to HPLC methods. The faster SFC process makes the separation cycle time significantly shorter, so that it is practical to make purification runs by "stacking" small injections in short time windows, without compromising throughput. In this way, the utilization rate of expensive column material is much higher. 

Throughput in preparative chromatography is dependent on separation factors and the solubility of compounds in mobile phases. While chiral discrimination is required for purification, good racemate solubility (ideally greater than 20 mg/mL) is also necessary for a successful preparative resolution. A compound with poor solubility is more problematic in preparative purification than analytical chromatography because the typical ratio of sample to both mobile and stationary phases is much higher in preparative chromatography (11). Traditionally, mobile phase solubility measurement is overlooked when performing preparative SFC, because it is difficult and time-consuming. 

The use of PrepSFC for high-throughput purifications initially focused on UV-based fraction triggering systems. Productivity enhancements quickly higlighted the need to have mass-directed PrepSFC capabilities. The principal advantage of mass guided preparative chromatography is that the collection of fractions can be limited to only those compounds eluting within the target mass range. Mass-directed systems accomplish this by monitoring the extracted ion signal from the mass spectrometer to trigger fraction collection (12). The triggering mechanism is compound specific, in contrast to previous unselective UV triggering mode. 

In summary, pcSFC (as have most analytical techniques) has had a tortuous development history. However, it now appears that analytical- and preparative-scale SFC methods for purification are currently on the strongest foundation ever with vendors that are strongly committed to advancing the technology. New developments regarding achiral pcSFC and a broader spectrum of applications in the field are anticipated in the future. 

(a) Method Development for Analytical SFC:

 Even though academic interest in SFC has basically died and numerous publications by private industry have not been forthcoming, the future looks exceedingly bright. From an internet and scientific meeting perspective, pcSFC applications in the food, environmental, biomedical, natural products, and energy areas are expanding and complementing many of the well-publicized pharmaceutical areas. The most widely used approach for resolving complex mixtures has been to change selectivity of the chromatographic system (not increase plates or increase retention time). One can, therefore, expect the introduction of a new range of polar stationary phases for pcSFC in the future, such as the popular ethyl pyridine phase which was introduced several years ago by Princeton Chromatography, Inc. specifically for pcSFC. While these developments will focus on nitrogenous bases such as amide and imidazole functionalities, the future continues to look promising for highly pure, bare silica, and related hydrophillic interaction liquid chromatography (HILIC) phases for separation of highly polar analytes. Phases that reduce the need for mobile phase additives in the separation of ionizable analytes will also be of interest for PrepSFC. Porous silica particles that are smaller than 5 μm will prove beneficial along with porous surface layer particles of similar size as faster, more efficient separations are desired. Additional polar stationary phases and the introduction of mobile phase additives that are either acidic, basic, or neutral have led to promising separations of water-soluble, ionic, and high molecular mass analytes. Ion-pairing–SFC and HILIC–SFC are expected to be fertile areas for expansion into applications of achiral separation of biomarkers and metabolites for example (13). The maturity of SFC–UV technology has truly created a seamless interface with both gas chromatography–flame ionization detection (GC–FID) and HPLC–UV. The overall acceptance of pcSFC will depend on its future robustness and analytical sensitivity in comparison with conventional and equally popular analytical techniques (14).  

Figure 1: Suggested separation strategies for achiral SFC illustrating that SFC spans a wide range of polarity. Adapted with permission from Aurora SFC Systems.			

While method development in chiral pcSFC has evolved into an economically expensive, multi-column, trial and error approach, achiral pcSFC development will not be receptive to this type of strategy as analytes have greater variation. Forty years ago, pcSFC was thought to be most applicable for non-polar analytes, but today (and into the future) pcSFC will "fit-in" anywhere between nonpolars and highly charged analytes (see Figure 1). Four protocols can be envisioned with packed column technology based upon preliminary reported studies (13): 

 for elution of hydrocarbons, ethers, and esters. 

 + methanol (or acetonitrile) for elution of alcohols, amides, and aniline.  

 + methanol + trifluoroacetic acid (or isopropyl amine) for elution of ionizable acids and bases. 

 + methanol + ammonium acetate + water for elution of amphoterics, peptides, carbohydrates, water soluble vitamins, and inorganic ions. 

 There are a number of manufacturers, past and present, producing equipment for pcSFC. Most users appear to assume that all instruments produce the same, or similar, flow and composition performance with minor variations even though there are at least three different reported kinds of pumps used to perform pcSFC. There also appears to be no recognition that there are significant pumping differences in various designs. In other words, no consensus exists among the separation community as to what constitutes a standard approach (15). Furthermore, there appears to be no analysis of how design differences might affect validation of methods when transferring a method from one laboratory to another (that is, robustness). For the record, there is only one reference in the literature that proposes specifications for the validation of equipment for pcSFC in the pharmaceutical industry (16). Although more than 10 years old, this proposal has not been adopted. Adding to the difficulty, many suggested measurement techniques are not very accurate or reproducible. Specifications of control variables, such as pressure or flow rate, must allow a wider range of variability in the measurement result than the variability in the sensors used to monitor the process (15). Furthermore, flow values should be corrected to conditions at the pump rather than to condition at the meter. Steps toward validation and robustness in pcSFC must be considered by vendors in the future. 

High sample throughput is often required in applications with large numbers of samples such as library screening. It is often true that chromatograms can be generated in less than a minute, but the system cycle time is extended by an additional 1–2 min to prepare to inject, perform detector balance, store the method with the previous runs raw data, external needle wash, needle and loop wash steps, and so on. Throughput can be significantly increased by reducing the delay time between the end of one run and the injection of the next sample. Efficient user-friendly protocols should be perfected by all vendors in the future. 

SFC is almost never used for trace analysis. Poor sensitivity and inadequate dynamic range are two prevalent issues. These limitations have, in the past, discouraged the use of pcSFC in environments such as quality assurance (QA) and quality control (QC) and relegated it to major–minor component analysis. A significant improvement in sensitivity could expand the role of pcSFC out of pharmaceutical analysis into a much wider range of applications. A review of the literature only produces a limited number of papers attempting to quantify impurities or metabolites at the 0.1% level and below. In traditional pcSFC systems, the compression of carbon dioxide is thought to be the dominant cause of UV detector noise. By isolating compression and metering, the detection limits and dynamic range have been shown by Berger and colleagues (17) to be improved well over an order of magnitude. In pcSFC, the compressibility is as much as 20 times higher than in aqueous-based HPLC, thus greatly exaggerating compression problems. The investigators in this work believe the low noise is the result of separating compression from the metering of the fluid. In most pumping systems, the same pump both compresses and meters the flow. Direct quantitation, however, means both the main peak (100%) and the trace contaminants (>0.1% of the main peak) are in a linear region of the detector with adequate signal to noise. If the main peak is 2 AU tall and S/N>10:1 is required to quantitate the trace components, the noise (peak to peak) must be <0.2 mAU and preferably lower to provide margin. The improvements noted here appear to be related to a new approach to pumping carbon dioxide which should open the door for more widespread use of pcSFC in regulated environments that require an extended dynamic range and better sensitivity. The future for pcSFC will hopefully see more vendors embrace technology that enhances pcSFC–UV sensitivity. 

In summary, with faster separation, higher sample throughput, less organic solvent usage, and normal-phase mode purification, packed column-SFC has become a viable option for the separation scientist in diverse areas. Its applicability for achiral separations, its ability to separate water soluble analytes, and its feasibility for coupling columns of varied chemistries is only beginning to be appreciated. New areas of application can be envisioned such as flash SFC and its correlation with thin layer chromatography. The role of mobile phase components in changing both the polarity of the stationary phase and the mechanisms of retention are worthy of study (18). While the SFC area was given its first "push" as an extension of GC over 30 years ago, the field now seems more adequately described as an extension of HPLC. As a matter of fact, the area might be more readily advanced if it were thought to be "carbon dioxide-based HPLC". 

Informed discussions with Mehdi Ashraf-Khorassani (Virginia Tech, Blacksburg, Virginia, USA) regarding the preparation of the manuscript have been very helpful. 

 is emeritus professor of chemistry at Virginia Tech in Blacksburg, Virginia, USA. Please direct correspondence to: 

(1) Analytical Supercritical Fluid Chromatography and Extraction,

 (Chromatography Conferences, Inc., New York, 1990) 5–95. 

(2) J.C. Giddings, M.N. Myers, L. McLauren, and R.A. Keller, 

(4) N.M. Karayannis, A.H. Corwin, E.W. Baker, E. Klesper, and J.A. Walter, 

(5) M. Novotny, S.R. Springston, P.A. Peaden, J.C. Fjeldsted, and M.L. Lee, 

(8) T.A. Berger, J. Smith, K. Fogelman, and K. Kruluts, 

Supercritical fluid chromatography has become a viable option for the separation scientist in diverse areas, and the field now seems more adequately described as an extension of HPLC, or perhaps as "carbon dioxide–based HPLC".

Past, Current, and Future Directions in Supercritical Fluid Chromatography

Traditional high performance liquid chromatography (HPLC) method development simply required careful definition of column, mobile phase, and detector conditions. However, for ultrahigh-pressure liquid chromatography (UHPLC), which offers very high column efficiency and narrow sample elution bands, the UHPLC instrument components must be carefully matched in performance with columns to achieve the desired resolution. If UHPLC underperforms, it can be difficult to determine whether the instrument or column is the problem. This article will describe the design of modern UHPLC instruments and offer simple procedures to identify the causes of poor column and method performance. Modern textbooks do not yet adequately address the complex topic of UHPLC, therefore the reader is encouraged to collect all references to learn important, additional details.

For almost two decades, high performance liquid chromatography (HPLC) research has focused on smaller column particles. This coined the widespread use of the term 

 to describe the achievement of higher performance by using smaller particles and higher operating pressure. In 1987, Kirkland (1) published on the use of 2-μm porous, wide-pore silica for the separation of peptides and proteins, referring to the particles as 

. In 1994, Jorgenson began publishing on ultrahigh-pressure separations in capillary dimensions using 1.0-μm solid ODS silica (2) and has prepared a comprehensive review of his work (3).  

In 1998, Poppe (4) compared retention and loadability advantages of Kirkland's 1.8-μm porous ODS silica over 1.5-μm solid ODS silica. In 2000, Kirkland (5) used the term ultrafast HPLC in a review article when comparing the performance of small porous and superficially porous silica particles. Swartz (6) described ultra-performance liquid chromatography columns and instruments in 2005. While many articles have documented the benefits of reducing particle size to improve column efficiency 

, practical applications require that all small particles must have adequate surface area and stationary phase loading to fully utilize retention and selectivity terms in the resolution equation.  

The benefits of higher efficiency to achieve speed and resolution can be fully appreciated by examining the resolution and van Deemter equations. Figure 1 compares theoretical van Deemter plots for several particles using a simple form of the equation, and also shows the relationships between plates and pressure as particle size is reduced. Doubling the efficiency (by halving the particle size) creates a 40% increase in resolution for the same column length and flow rate, but requires about four times the amount of pressure. Particle size can be halved and still generate about the same efficiency and resolution with only half the column length, requiring about half the time at the same flow rate. The shorter column with smaller particles would require only slightly higher pressure. In addition to lower 

, the van Deemter plot shows that smaller particles have the advantage of much flatter 

 values at higher flow velocities. This exciting result with small particle columns predicts that speed can not only be doubled by halving particle diameter, but can also be doubled and doubled again by operating at higher flow velocities. No significant resolution will be lost as long as instruments can keep up with the flow rate and pressure requirements. Ultrahigh-pressure instruments were developed specifically to take full advantage of ultrahigh-performance columns operated at high linear velocities. Although the constraint of having to operate near the minimum in 

has been removed by modern column technology, higher flow and pressure causes frictional heating and uneven temperatures inside the column that may alter both efficiency and selectivity. Gritti and Guiochon (7) have recently published an excellent overview of present and future issues in UHPLC.  

Figure 1: Van Deemter plots for porous particles. Note that only sub-2-Î¼m porous particles fall below 5-Î¼m plate height, H. Since N = L/H, column efficiency exceeds 200,000 plates/m, which can be considered the minimum performance level that defines a UHPLC column. Core-type particles in the sub-3-Î¼m range and certain monoliths can also achieve this level of column performance when losses because of instrument dispersion are small or can be corrected.			

In HPLC or UHPLC, there are clear distinctions between columns, instruments, and systems that must be understood to appreciate what can happen in UHPLC experiments to create disappointing system performance. A modern column must be matched to the right instrument to create a system with good performance. 

Sample peaks spend time both outside and inside the column. The flow path outside the column promotes rapid band spreading and should be minimized relative to the flow path inside the column, which resists band spreading. Majors published a review in 2003 (8) of how uncontrolled HPLC instrument or extracolumn effects can reduce column performance. Majors used the following variance equation:  

where column peak variance increases with 

 while instrument variance remains fixed for a given flow rate:  

A certain amount of band dispersion, σ, from the different components always exists and can be estimated from equations or measured experimentally. The dispersion from each component must be added as the variance, σ

, as shown. For example, if each of the three components in equation 3 adds 5% extra bandspreading (σ) to the solute peak, the total peak dispersion added by the instrument would only be 8.6% (not 15%), as follows  

In 1960, during the infancy of column chromatography, Klinkenberg (9) reported that instrument (extracolumn) dispersion should be maintained below 5% for good system performance. Variance and dispersion are usually estimated or measured from bandwidth of sample peaks and reported in volume units such as microlitres (μL).  

Figure 2: UHPLC instrument specifications: Five different UHPLC instrument brands (AâE) are shown compared in pressure limit and flow rate range to a classic or traditional 400 bar instrument (F). 			

In about 2004, before the introduction of UHPLC instruments, columns with sub-2-μm particle performance could only be used effectively with research instruments that had optimized, low-volume components. Traditional instruments with 400 bar (6000 psi) pressure limits and 0–10 mL/min flow range were designed to barely meet the needs of columns with 3-μm particles and quickly exceeded pressure limits at high flow velocities with even short 3-μm columns. In addition, most traditional instruments used tubing with 0.010 in. (0.25 mm) or 0.007 in. (0.17 mm) i.d. When coupled with other components in the sample flow path, instrument band spreading became too large to be compatible with the narrow peak widths of early solutes emerging from modern columns. Although traditional instruments with factory components usually degrade efficiency of modern columns, they can be readily optimized by changing components. In 2005, Henry and Bell offered guidelines (10) to optimize liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography–ultraviolet (LC–UV) systems for ultrafast analysis.  

Table 1: Changes in UHPLC versus traditional HPLC instruments.			

In late 2004, commercial UHPLC instruments began to appear with higher pressure specifications over a reduced flow rate range, as shown in Figure 2 for five different vendors. A traditional HPLC instrument is included for comparison (labelled "vendor F"). As well as having components rated for higher operating pressure, UHPLC instruments feature much smaller volumes in the sample flow path and greatly reduced instrument bandwidth. Table 1 summarizes changes that have occurred in UHPLC instruments to meet the pressure and performance needs of modern columns with small particles operated at high flow velocities.  

Figure 3: Ultrahigh speed UHPLC separation: 2.7-Î¼m core-type C18 column, 50 mm Ã 3 mm i.d.; elution order: uracil, phenol, acetophenone, benzene, toluene, naphthalene; run (a) can be accomplished on a traditional HPLC instrument; run (b) requires a UHPLC instrument; both require very low instrument dispersion. 			

The performance of a modern UHPLC system (column plus instrument) is shown in Figure 3 for a 5-cm core-type column at two flow rates. Chromatogram A shows a 0.8 min separation with 11,000 plates (220,000 

/m) under optimum conditions at 0.6 mL/min while chromatogram B shows a 15 s separation with a very small loss of performance at 2.0 mL/min, which is made possible by the flat van Deemter plot in Figure 1. Note that peaks in run B are only about 1 s wide and elute in a volume of about 30 μL (measured by width at base). In this case, a normally fast detector response time of 0.1 s and data collection rate of 10 Hz may be too slow for accurate analysis. Users should investigate how detector and data system electronic settings affect efficiency, resolution, and sensitivity for ultrafast separations.  

Figure 4: Measurement of instrument bandwidth, IBW: Two important elements of dispersion can be learned by sample injection with no column: 1) the internal volume of sample flow path can be estimated from retention time and flow rate; before optimizing, sample retention was 0.70 min Ã 100 Î¼L/min = 70 Î¼L; after optimizing, retention was 0.22 min Ã 100 Î¼L/min = 22 Î¼L; instrument bandwidth (width at base, 4Ï) was estimated manually to be 44.7 Î¼L and 13.1 Î¼L, respectively. 			

Direct Measurement of Instrument Dispersion:

 Excessive instrument dispersion and injection of large sample volumes are major operational problems that can rob LC systems of performance. Gritti and Guiochon have published detailed articles about extracolumn band-broadening effects (11,12). Dong (13) proposed measurement of instrument bandwidth to characterize instruments and allow the problem to be identified and addressed. Figure 4 describes how to measure instrument bandwith (IBW) by replacing the column with a very low volume union. By injecting a very small sample (≤ 1 μL) and recording an instrument peak at fast detector response time, one can estimate peak width at base in microlitres at several operating conditions. A more accurate value for IBW can be obtained from a data system efficiency report and solving for σ in the equation, 

, where retention time is converted to elution volume in microlitres. Users can modify traditional instruments to approach UHPLC instruments in IBW, but not in pressure. Figure 5 shows that detector response setting can significantly impact measured instrument bandwidth.  

Figure 5: Peak broadening caused by slow detector electronics: IBW should be measured at several flow rates to match assays; measuring IBW at 1 mL/min is a tough test for a detector; at least 0.1 s and 20 Hz may be needed; users should confirm accuracy by changing settings as shown to determine whether results are affected; lowest IBW is the true instrument value.			

Scott has studied instrument dispersion extensively (14) and published a relationship derived from the Taylor-Aris equation:  

/s), which changes with solute structure and solvent viscosity. Figure 6 plots band spreading (σ) for different lengths of tubing at several flow rates and tube diameters. Volume has been converted from millilitres to microlitres for easier comparison to IBW. Just a few cm of 0.25 mm (0.010 in.) tubing can contribute more dispersion than the total amount present in a modern UHPLC instrument.  

Figure 6: Dispersion caused by different tubing diameters: The use of 0.010-in. (0.25 mm) i.d. tubing in modern HPLC is clearly unacceptable because of excessive dispersion; note that tubing dispersion increases dramatically with tube diameter (TD) and substantially with flow (F); short lengths of 0.004â0.005-in. tubing are reasonable compromises between low dispersion and high flow resistance to optimize instrument IBW for good system performance.			

Clearly, one should not try to optimize any other component in traditional instruments until the column connection tubing is reduced to 0.005-in. i.d. or smaller. Virtually all UHPLC instruments use smaller tubing than 0.005 in. Equation 6 is not highly accurate because there are unknown and uncontrolled variables (including temperature) in the high velocity sample flow path outside the column; however, the plots should be qualitatively correct and confirm that connection tube diameter is very critical to determining instrument dispersion. Connector length should also be reduced. Figure 7 illustrates that IBW increases significantly with flow rate, but flow rate is dictated by the column and method and cannot be optimized. IBW values should be recorded over a range of flows if instruments are to be evaluated and ranked for performance. IBW will improve at elevated temperatures because solute diffusion rate increases, which is a possible argument for conducting UHPLC at higher temperatures. Fountain and colleagues published a detailed paper about optimizing extracolumn effects in the design of a modern UHPLC instrument (15).  

Figure 7: Effect of flow rate on measured IBW: IBW is often measured at low flows to show lowest values, but IBW increases with flow rate (equation 6); IBW measurement is rapid and should be made at several flows; two instrument designs may show differences in IBW flow sensitivity.			

Although dispersion decreases rapidly as tubing diameter becomes smaller, one must exercise restraint because pressure drop across that component increases rapidly. The Hagen-Poiseuille equation of pressure with flow is:  

is tube radius (all in cgs units). The result is that UHPLC instruments achieve very low dispersion at the expense of high background pressure. Instrument back pressure makes the use of pressure to diagnose column blockage more complicated. Figure 8 shows the relative pressure that exists across a popular UHPLC instrument for several column and flow rate situations.  

Figure 8: Effect of flow rate on measured IBW: IBW is often measured at low flows to show lowest values, but IBW increases with flow rate (equation 6); IBW measurement is rapid and should be made at several flows; two instrument designs may show differences in IBW flow sensitivity.			

Detecting Excessive Instrument Dispersion: 

Sometimes the existence of excessive dispersion is obvious when high column efficiency measured with an optimized instrument is lower by 50% or more when the same column is installed on another, often older, instrument. Chester published (16) useful guidelines for optimizing a traditional HPLC instrument to allow use of a column with 2.7-μm, core-type particle. Solid-core particles can match the performance of smaller porous particles and operate at significantly lower pressure to permit use of optimized traditional instruments.  

Performance of a 10-cm column with solid-core particles had already been measured by Chester at 24,000 plates (240,000 

/m) using an UHPLC instrument. When the column was installed in a traditional HPLC instrument with factory tubing, Chester measured low system efficiency of 8500 plates and estimated that the instrument was contributing more (ca. 70%) to peak width than the column (ca. 30%). He reported that IBW came almost entirely from column inlet and outlet tubing plus an unnecessary column selector valve. After changing system column tubing from 0.25-mm (0.010 in.) i.d. to 0.12-mm (0.005 in.) i.d. and removing the selector valve, Chester reversed the ratio to 70% bandwidth from column and 30% from instrument, with only about 8% of total bandwidth contributed by the tubing. System efficiency rose to over 20,000 plates. Modifying other components such as detector flow cell and heat exchanger tubing were not as convenient, but this performance level was quite acceptable to make the traditional instrument usable with high performance 4.6-mm i.d. columns. Chester's optimization approach could also be used for sub-2-μm porous particles in shorter columns to stay within 400 bar pressure limits. Gritti and colleagues (17) have recently published an article about optimizing traditional HPLC instruments for use with modern columns.  

Figure 9: Detection of instrument dispersion using N vs. k plots: Peaks are clearly getting broader and system is losing efficiency as column i.d. becomes smaller; if columns are known to have about equal performance, this indicates too much instrument dispersion; after reduction of internal volume, plots should nearly overlay one another; traditional instruments can be optimized to perform very well with 4.6-mm and 3.0-mm i.d. columns, but UHPLC designs will be essential to maintain performance with 2.1 mm and smaller columns. 			

There are several ways to detect the existence of excessive instrument dispersion without comparing it to an optimized instrument. Good methods involve the injection of small volumes of a multicomponent mixture. In one case, a column is installed and sample is injected with several resolved components from 

reveals excessive extracolumn dispersion in peaks with lower 

values because they elute in smaller volumes and become a difficult test for the instrument. If the instrument has low variance compared to column peak variance (optimized traditional HPLC and UHPLC), all peaks should show nearly the same high efficiency. As instrument variance increases, early peaks will show reduced plates, but later peaks will still show close to the expected or true column efficiency for that particle. Figure 9 shows results with three columns. This instrument should be considered adequate for using short 4.6-mm i.d. columns, but it would be very marginal for using short 3-mm i.d. columns, and would not be suitable at all for using 2.1-mm i.d. columns. UHPLC instruments and those with very low IBW should plot with a nearly straight line for all three column internal diameters; even low dispersion instruments will likely show reduced efficiency with smaller i.d. columns at low 

 values. For a fair test of IBW, injection volumes should be reduced in proportion to void volume as column internal diameter is reduced.  

Figure 10: Detection of instrument dispersion using van Deemter plots with a multicomponent sample: (a) shows a plot for 50 mm Ã 4.6 mm column with 3.0-Î¼m Zirconia-PBD column using a traditional HPLC instrument, where all five solute data plots overlay; this instrument with factory configuration has low enough dispersion to have negligible impact on efficiency, which is just noticeable for the shortest k (benzene) peak (lowest peak volume); (b) shows the same plot for a 50 Ã 4.6 mm column with 1.9-Î¼m Zirconia-PBD column, where plots no longer overlay; this same factory instrument is therefore not suitable for this column because instrument dispersion is too large for the narrower peaks; (c) shows improvement created by adding a micro flow cell and replacing 0.007 in. tubing with 0.005 in. tubing to reduce dispersion to an acceptable level; (d) shows the effect of adding a short length of heat-exchange tubing to allow separation at elevated temperature; the instrument is now adequately optimized to perform an ultrahigh speed drug assay.			

A van Deemter plot reveals valuable information about the measuring instrument when multiple component samples are studied. Figure 10(a) shows a fast separation of five components on a 5 cm × 4.6-mm column with 3-μm Zr-PBD (Zirchrom) particles at increasing flow rates. A 400 bar instrument with factory tubing and flow cell configuration was used. Note that all five solutes show the same high performance as indicated by curves that overlay at an approximate plate height of 

 = 6 μm (the expected value for this particle size). Only the earliest eluting benzene peak shows slightly higher 

The authors explain modern UHPLC instrument design and outline simple procedures to identify causes of poor column and method performance.

Special Issues-10-02-2013 | LCGC Supplements

Application of Quality by Design Principles to a Pharmaceutical Sample Using UHPLC Method Development with Modelling Technologies

Back to the Basics: Considering Carryover, Column Back Pressure, and Wavelength When Developing Chromatographic Methods

Past, Current, and Future Directions in Supercritical Fluid Chromatography

Practical Tips for Operating UHPLC Instruments and Columns

Introduction: Recent Developments in HPLC/UHPLC

Vol 26 No s10 LCGC Europe October 2013 Recent Developments in HPLC and UHPLC Supplement PDF