Can you qualify or calibrate your chromatograph using a system suitability test? No. However, for the doubters amongst you I'll spend the rest of this column and the next one explaining why!

In this two-part column I'll be discussing the role of system suitability tests (SSTs) in the context of analytical instrument qualification (AIQ) and the generation of quality analytical results. I'll look at this first from the perspective of regulatory requirements based upon the 

  However, as much of this discussion is also good analytical science I will also look at this from the perspective of ISO 17025,

  the interpretation of the ISO 9000 quality management system for testing and calibration laboratories. In this column I'll use the ISO 17025 term of calibration to be equivalent to AIQ and discuss the reason why I have taken this approach.  

Specifically, I want to discuss the question why system suitability tests (SSTs) are not a substitute for AIQ for the initial qualification or re-qualification of a chromatograph (i.e., a periodic check or operational qualification) under GMP or calibration under ISO 17025. The reason for this is that there is a common misconception in some laboratories that SSTs can be used to qualify a chromatograph. This is wrong. Furthermore, using SSTs as the sole instrument qualification approach will leave any laboratory exposed to regulatory action or non-conformance as the chromatographs cannot be demonstrated as being fit for their intended purposes. Therefore, we will explore the reasons for this from both the regulatory and quality perspectives. 

An SST is simply a check to see if the chromatographic system (instrument modules, column and mobile phase) is capable of undertaking the analysis you require. It is performed immediately before the samples you have prepared in the laboratory are committed for analysis. At it's simplest, an SST is the single injection of a standard solution of reference compound(s) to see if the separation is 'OK'. The only criterion used for assessing if the system is acceptable is a visual check by the chromatographer to assess if the separation is within expected boundaries. However, there are no measurements made: just a simple visual assessment that things are right.  

At the other end of the scale are the formal SSTs described in the 

 used in pharmaceutical QC laboratories. Here five replicate injections of a standard are injected and the calculated peak areas and other chromatographic criteria (retention time window, peak shape, peak tailing, resolution between peaks etc.) are objectively compared with predefined specifications defined by the 

 (peak area repeatability) and the laboratory (all other chromatographic criteria). If the SST injections meet all the predefined criteria then the chromatographic system is deemed acceptable and the samples committed for analysis.  

Good manufacturing practice (GMP) regulations from either the US or Europe state simply that equipment used in the manufacture of drug products should be fit for their intended use as can be seen in Table 1, which presents the US regulations.

  Also, shown in Table 1 are the requirements for ensuring that scientifically sound specifications for the qualification work are written and that qualification and calibration of instruments is documented correctly. Note also that if an instrument fails to meet its specification it must be taken out of service until the problem is rectified and retested to show that it meets its specification. 

Table 1: Selected US good manufacturing practice (GMP) regulations for instruments and equipment.			

However, for chromatographic equipment these GMP regulations are expanded by either the 

  which have both specified requirements for SST for chromatographic analysis to demonstrate that a chromatograph is fit for the analysis it will undertake on the day of analysis. System suitability tests must be run each time before an analysis is undertaken and each SST is specific for an individual method with pre-defined acceptance criteria, for example, precision, peak shape and resolution from other analytes. If an SST fails, then the samples cannot be assayed.  

The principle of a point of use check, such as an SST, is applicable to any analytical instrument or system and this is performed just before an analysis to demonstrate correct performance, for example, a check to see if the chromatographic system or an analytical balance works before committing samples. This point of use check is used in other types of instrumental analysis, for example, balance and pH meter checks so it is not an unusual approach and is good analytical science. 

 <1058> on analytical instrument qualification (AIQ) in an earlier 

  where we looked at the impact of this on the chromatography laboratory. This informational general chapter consists of six major sections:  					

Analytical instrument qualification process.

 <1058> that concern our debate of AIQ versus SST are the components of data quality and the analytical instrument qualification process. The quotations from 

 <1058> come from these two sections. I will focus my comments on the first section of 

 <1058> that discusses the components of analytical data quality. In my earlier column

  I skipped over this section but I would like to go into further detail, as the content is good analytical science and is applicable to all laboratories. 

OK so what is ISO 170252 and what is accreditation? First question first, ISO 17025 is an international standard that both testing and calibration laboratories can work against; for the purpose of this column I will focus on testing laboratories mainly. A major difference between ISO 17025 and GMP is that compliance with the requirements of the ISO standard is voluntary rather than imposed by government agencies. However, the advantage of working to ISO 17025 is that a laboratory is working against an internationally recognized standard. To answer the second question, accreditation is the process of ensuring that the laboratory has implemented and complies with the requirements of the ISO 17025 standard and continues to do so. This is achieved by an independent audit by technical officers from the local accreditation body coupled with external experts in the specific areas offered by a laboratory. Each country in the European Union has an accreditation body (UKAS for the UK, COFRAC for France etc.) that is responsible for the accreditation of laboratories in their respective country. However, ISO 17025 goes further than GMP in that the test methods used by a laboratory and the performance of each one are also covered within the scope of a laboratory's accreditation and are included in the assessments made by accreditation bodies. 

  we have to remember that this standard is a specific interpretation of ISO 9000 for testing and calibration laboratories. In this column I will focus mainly on the requirements for testing laboratories that use chromatographic equipment to perform their work. The applicable sections of ISO 17025 are presented in Table 2 and will now be discussed and interpreted in more detail below. Please note that key portions of the standard, as were the US GMP regulations, are highlighted for the purposes of this column and the full sections should be interpreted in practice.   

Table 2: Selection of the main ISO 17025 sections covering instrument qualification.			

The first requirement for equipment is in section 5.5.2 that notes that all laboratories are required to implement and operate a programme for the maintenance and calibration of equipment used in the laboratory. However, these calibration checks are only required where the equipment setting can significantly affect the test or analytical result (e.g., oven temperature, gas or liquid flow, detector wavelength accuracy and photometric linearity for GC or LC modules). Proper equipment use combined with periodic servicing and calibration does not necessarily ensure that an instrument, in this case a chromatograph, is performing adequately, there also has to be point of use checks of overall system performance that can be built into test methods (e.g., chromatographic system suitability tests). These checks should be documented and should be satisfactorily completed before the equipment is used or before results are accepted.  

Reconciling GMP Regulations and ISO 17025 Requirements

How can we have a common understanding of both the GMP and ISO 17025 requirements so that we can have a debate that is independent of applicable regulations or requirements? This all comes down to definitions and what we mean by specific words. So here are the key terms used in either regulations or standards and let's see how we can reconcile them to help us in the debate:  					

 In GMP the term "equipment" is used to cover anything used in the manufacture of pharmaceutical products that includes the laboratory. Similarly, ISO 17025 uses the term equipment to cover the wide range of equipment, instruments and systems used in the testing laboratory. This is in contrast to 

 <1058> that uses the term instrument. However, in practice the two terms are synonymous as we can refer to either chromatographic instruments or chromatographic equipment. I prefer the term instrument and will use that in the remainder of this column.

 Both GMP and ISO 17025 refer to calibration in contrast to qualification used by 

 <1058>. Are the two terms equivalent? Yes in my view. Calibration is a simpler term to understand and has been defined by EU GMP as the set of operations which establish, under specified conditions, the relationship between values indicated by a measuring instrument or measuring system, or values represented by a material measure and the corresponding known values of a reference standard.

  I would argue that the work undertaken in qualification and calibration of an instrument is essentially the same: does it work for your purpose and can you demonstrate that by using calibrated test instruments and traceable reference standards?

 All regulations and guidance refer to these terms so there is full agreement and thus no need for interpretation  	

An example of how a laboratory can fall foul of a regulator is found in the US Food and Drug Administration (FDA) warning letters publicly available on the Agency website (

). Shaanxi Hanjiang Pharmaceutical Group, a Chinese company, received a warning letter in January 2008 following an inspection of its facilities in September 2007.8 Quoting from the warning letter we can see that: 

3. Calibration of measuring devices was not traceable to the national standards. 

Several balances and thermometers were not traceable to national standards. We acknowledge your firm's submission of a certificate of metrology <redacted> which expires on 16th April 2008. However, this certificate is deficient in that it does not include identification of the instrument for which it is meant for and does not include a statement of traceability. 

So we can see the requirement for traceability outlined in ISO 17025 can be mirrored in GMP. The only problem is that there is no explicitly written requirement for this as you can see in Table 1. However, as you can see in 211.160(b) in Table 1 there is a need for the laboratory controls to be 'scientifically sound', as traceability of calibration is good science. Therefore, if you have not calibrated or qualified your instruments with appropriate and traceable reference standards then the data you produce from those instruments will be poor.  

In this column, I've looked at the regulations and quality standards for qualification and calibration of chromatographic instruments. In part 2 we'll look at how the data quality triangle can be applied to this from a practical perspective. 

 is principal at McDowall Consulting, Bromley, Kent, UK. He is also a member of 

 Editorial Advisory Board. Direct correspondence about this column to "Questions of Quality", 

, Poplar House, Park West, Sealand Road, Chester CH1 4RN, UK, or e-mail Alasdair Matheson, the editor, at 

1. United States Pharmacopoeia general chapter <1058> Analytical Instrument Qualification. 

2.  ISO 17025 General requirements for the competence of testing and calibration laboratories, International Standards Organisation, 2005. 

3. Good Manufacturing practice for finished pharmaceuticals (21 CFR 211), 2008. 

4.  United States Pharmacopoeia general chapter <621> Chromatography. 

5.  EP Chromatography chapter 2.2.46 Chromatography. 

6.  R.D. McDowall, Questions of Quality, 

7.  EU Good Manufacturing Practice Glossary. 

Articles

Questions of Quality

Can you qualify or calibrate your chromatograph using a system suitability test? No. However, for the doubters out there this column will explain why.

Why System Suitability Tests Are Not a Substitute for Analytical Instrument Qualification or Calibration (Part 1)

Two previous "GC Connections" instalments addressed the reasons for, and the effects of, tailing peaks on detection and quantification.

  This instalment addreses the effects on peak resolution and quantification of two overlapping peaks.

The goal of quantitative chromatography is to separate two or more substances sufficiently so that they can be measured with a desired degree of accuracy and precision. The resolution of a peak pair provides a standardized measurement of the extent of a separation but strictly speaking, resolution is defined only for two Gaussian peaks of equal size. This month's "GC Connections" discusses resolution and the effects that relative peak size have on area measurements. 

The resolution of a pair of adjacent peaks is defined in the chromatographic literature and in standard nomenclatures such as IUPAC

For a Gaussian shaped peak the width at half-height is related to the width at base by a factor of 1.699: 

It is usually more convenient to calculate resolution from the width at half-height of the second peak using this related formula: 

The width at half-height is more easily measured than the width at base, and it is measured and used by many chromatography data-handling systems in, for example, system suitability calculations. The width of the second peak is used because it will generally be either equal to or slightly greater than the first peak's width and thus, produces a more conservative result while not requiring measurement of the widths of both peaks.  

Most chromatographers are familiar with the appearance of partially resolved, baseline resolved and fully resolved peak pairs. Figure 1 shows an ideal pair of generated Gaussian peaks of equal sizes and widths with partial resolution at 

 = 1.0 [Figure 1(a)], baseline resolution at 

 = 1.5 [Figure 1(b)], and full resolution at 

 = 2.0 [Figure 1(c)]. In this illustration, resolution increases as the separation between the peaks increases, while the same peak shape is maintained. Keeping the same separation while decreasing the peak widths would also increase their resolution proportionately. Figure 1(c) illustrates the relationship between the peak width at half-height and base, and it also shows how the width at base is determined from the baseline intersection points of lines drawn tangent to the peak's upward and downward slopes. A common misconception is that the width of a peak at base is the time between when the peak starts to rise above the baseline until it subsides back into the baseline — the peak integration start and stop times — but in fact, the base width of a Gaussian peak is measured at 13.4% of the peak height above the baseline, at the times corresponding to the tangent–baseline intersection points. 

Table 1 lists the retention times, peak width measurements and resolution for the three cases shown in Figure 1. At a resolution of 1.0 the valley point between the peaks corresponds with the end of the first peak's base width and the start of the second peak's base width. At baseline resolution, where 

 = 1.5, the ending and starting base widths of the first and second peaks, respectively, are separated by 0.5 times the base width. At resolution 2.0, the ending and starting base widths of the first and second peak are each spaced at intervals equal to the base width. 

Table 1: Resolution measurements from Figure 1. (a), (b) and (c) refer to the parts of Figure 1. 

The identification of specific peaks by their retention times becomes more certain as resolution increases. It is possible to identify peaks on the basis of a partial separation, but the certainty of identification improves with increasing resolution. The detector signal comes closer to baseline between the peaks and the presence of additional components becomes more apparent. A mass-selective detector helps to identify merged peaks on the basis of their mass spectra, but closely eluted peaks often are isomeric or otherwise chemically similar. They may have similar spectra that preclude unambiguous identification by spectral library matching.  

Each component in a group of two or more merged peaks should be identified with separate analyses of the pure components and the influence of matrix components should be determined through blank injections. Bear in mind, too, that the elution order of close peaks will be influenced by changes in the column temperature conditions: a change in temperature programme rate of as little as 2 °C/min can reverse elution order. When faced with the partial resolution of a critical peak pair, or with the presence of a new matrix compound that interferes, make sure that the current separation conditions are optimal and then consider upgrading the column resolving power by installing a longer or narrower-bore column. If all else fails, then try changing to a different stationary phase that better separates the peaks of interest. 

Measurement of a partially resolved peak pair can be difficult to achieve with acceptable levels of accuracy. The simple but unrealistic case of two equal-sized peaks is a good starting point for understanding the effects that colliding peaks have on quantitative analysis. Both the influence of the portion of one peak that extends into another and the effects that merged peak shapes have on peak integration play strongly into the accuracy of quantitative measurement of merged peaks. 

Returning to Figure 1, the degree of area overlap of the coinciding portions of the peaks decreases as resolution increases. At 

 = 1.0 in Figure 1, the contribution of either peak to the other's total area is about 2.3%, as measured from where a vertical dividing line for peak area integration is dropped from the valley point between the two peaks to the baseline. I measured the overlap areas with a measurement tool in my data system for the first generated peak alone by starting at the time of the valley point, which corresponds with the end of the peak's segment, and extending to the end of the peak. In this case, the peaks are symmetrical and identical so the overlap and total areas are the same for each. 

The overlapping areas are highlighted in colour in Figure 1(a) and in the inset in Figure 1(b). As the resolution increases to 1.5 — baseline resolution — the overlap between the two peaks drops toward 0.1% and at a resolution of 2.0 no measurable overlap remains. Table 2 gives the area measurements from Figure 1. The values for each peak are very close to the known areas as set when the peaks were generated. 

Table 2: Peak area measurements from Figure 1. Overlap is the amount of area or percentage of one peak into another. (a), (b) and (c) refer to the parts of Figure 1. 

In this idealized situation, there is no significant error between the measured and known peak areas, even down at a resolution of 1.0. However, real peaks are not equal in area and width because their amounts, detector response factors and shapes vary. Also, real-world peaks incorporate uncertainty in their measurement due to injection variability, detector noise and slight shifts in retention time from run to run. It is instructive to consider what happens when one peak is considerably larger than the other and what the effects imply about the influence of peak resolution on quantitative analysis. 

Increasing the size of the first peak in our example affects both the peak separation and area measurements. The disparity in peak sizes changes the degree of peak overlap, but more significantly, it has a profound effect on quantitative measurements. Figure 2 shows the same three cases as in Figure 1, but the first peak is now 10 times larger. A question arises of whether the classical resolution measurement, which is based upon two equal-sized peaks, is applicable when the peak sizes are significantly different. 

We can use the degree of area overlap between the peak pair as an indicator of the degree of peak resolution and compare the case of equal-sized peaks to that of unequal peaks. Looking first at Figure 2(a) (the case of partial resolution of 1.0 of peaks with a size ratio of 10:1), the first peak's percentage of overlap into the second, as listed in Table 3, is similar to that of a pair of equal-sized peaks when there is a simple vertical division between the two peaks at the valley point. In the case of baseline resolution, in Figure 2(b), Table 3 lists the overlap of the larger first peak into the smaller second peak at 0.31% of the second peak's area, while the second peak's overlap into the first is only 0.06%. The overlap for the unequal peaks is small and similar to the degree of overlap of 0.14% for a pair of equal-sized peaks, however, so the definition of the resolution of equal-sized peaks seems to extend to at least at a peak area ratio of 10:1. In the case of resolution equal to 2.0, in Figure 2(c), there is no measurable influence of either peak on the other: they are indeed fully resolved and independent. 

Table 3: Peak areas and overlap of unequal peaks. Overlap is the amount of area or percentage of one peak into the other. (a), (b) and (c) refer to the parts of Figure 2. 

Quantitative area measurements in the case of baseline or better resolution of unequal peaks are fairly accurate. The measured and known areas in Table 3 for cases of baseline resolution [Figure 2(b)] and full resolution [Figure 2(c)] compare very well. Going from a 1:1 area ratio to a 10:1 area ratio with a pair of peaks at baseline resolution or better should yield a nearly linear trend between peak size and measured area. At some point, as the area ratio increases above 10:1, however, the degree of influence of the larger peak on the smaller one's measured area will become significant and the linear relationship between peak size and measured area will break down. Greater resolution between the peaks will raise this ceiling on linearity.  

 = 1.0 the story is quite different. Now, there is a much larger overall error in measured area counts with a peak size ratio of 10:1, which was not hinted at when only the similar degree of area overlap for equal and nonequal peak sizes was considered. Not surprisingly the largest errors occur for the smaller peak. 

The error level depends strongly upon the type of baseline allocation selected for the data system. Figure 3 illustrates three common choices. First is a simple vertical separator dropped from the valley point to the baseline, which we've used throughout this discussion so far, shown [Figure 3(a)]. This choice produces an error of about +1% for the larger peak, while the smaller peak comes back with an area that's about 10% smaller than the known amount, as listed in the first line of Table 4. This effect is seen graphically in the inset of Figure 3(a), where the relative amount of the second peak that's overlapped into the first is a significant fraction of the second peak's size. 

Table 4: Influence of integration type on areas of partially resolved unequal peaks Rs = 1.0. (a), (b) and (c) refer to parts of Figure 3. 

A tangent skim baseline choice puts a straight line at the valley point and tangential to its intersection with the declining side of the smaller peak. When a tangent skim is selected, the error in the second peak's area becomes unacceptably huge at –63%. This effect is visualized easily by examination of the inset in Figure 3(b), where a very large portion of the second peak is lost into the area of the first. This shift of area also biases the first peak's area upward by 4.5%.  

Finally, using an exponential skim or curve fit off of the first peak reduces the error relative to the tangent skim, but for the second peak the loss of area is still very large at –31%. Looking at the inset in Figure 3(c), the curve-fit of the exponential skim to the first peak is fairly good and overestimates the peak size by only 1%. The loss of a significant portion of the second peak is obvious.  

The errors associated with these three different baseline allocation choices depend on the relative sizes of the peaks and their resolution. What I've shown here is just a snapshot of one artificial situation. Chromatography data-handling systems include a conditional code that allows users to select boundaries for cases in which different baseline allocations are to be applied, based upon relative peak heights, valley points and other parameters. This means that the choices for baseline allocation must be made carefully and validated across the entire targeted range of analyte concentrations.  

The resolution of a pair of Gaussian chromatographic peaks provides a useful metric for gaugeing the ability to achieve reliable qualitative peak identification as well as predicting the accuracy of area measurement. As long as peak resolution is at baseline or better levels, quantitative errors can be very low, even for adjacent peaks at size ratios up to ten to one. When the resolution falls below the baseline level, the measurement of peak areas becomes difficult. Careful choices for area integration in data-handling systems can minimize the errors but must be checked across the entire operating range of a method. Selecting conditions and columns that better resolve critical peak pairs can be a better choice than relying on the data system's rules of area integration to slice away partially resolved peaks from each other. 

 is a senior research scientist at Serveron Corp., Hillsboro, Oregon, and a member of 

Direct correspondence about this column should be sent to Alasdair Matheson, editor, 

, Advanstar Communications, Poplar House, Park West, Sealand Road, Chester CH1 4RN, UK or e-mail: 

 IUPAC Compendium of Chemical Terminology

 (International Union of Pure and Applied Chemistry, Research Triangle Park, North Carolina, 27709-3757, USA, 1997). 

Standard Practice for Gas Chromatography Terms and Relationships," Annual Book of Standards

, Vol. 03.06 (ASTM International, Conshohocken, Pennsylvania). 

GC Connections

This instalment addreses the effects on peak resolution and quantification of two overlapping peaks.

When Peaks Collide

Continuing professional development for chromatographers

CHROMacademy is a chromatography e-learning programme that provides continuing professional development for chromatographers to make them more effective in the work place. There are over 3000 pages of e-learning on HPLC, GC, MS and Sample Preparation techniques to improve practical knowledge for the absolute novice (

) to the expert who needs a refresher in a particular topic (

Advanced Topics in LC-MS Spectral Interpretation

, for example). The site can be also used for induction training or programmed learning activities and there are tutorials and assessments built into all learning modules to check progress. The first official webinar: 

Essential Guide 1: Split /Splitless Injection for Capillary GC

 attracted 568 visitors. The recorded session is available via the CHROMmunity at 

 (under the WEBCASTS Tab). Other Webcasts include 

Essential Guide 2: Mobile Phase Optimization Strategies for Reversed Phase HPLC; Essential Guide 3: Understanding and Optimizing Detectors for Capillary GC

Essential Guide 4: Mobile Phase Optimization Strategies for Reversed Phase HPLC

ChromaTOF 4.30 is now available for all LECO instrument systems.New features are reported to include variable modulation times within each GC×GC chromatographic run for a more specialized analysis, which maximizes resolution in both the first and second dimensions. Also available is the LECO/Fiehn metabolomics library with over 1100 spectra of 700 unique metabolomics for metabolite identification of the most complex samples. Fully integrated within the system, the library works seamlessly to search for analyte matches without the need for importing or exporting data. 

DataApex has announced the release of Clarity version 3.0. This version is reported to bring more than 100 enhancements that increase users' comfort and add new functions, including new calibration and sequence options (such as calibration bracketing, standard addition calibration), new export into Excel format, or enhancement to GLP requirements. New control modules have been introduced so that the software can now control more than 270 different instruments from one environment. Users can download the latest version from DataApex webpages. The detailed list of changes in the new version is available from the company's webpages for easy navigation in the new version.  

Bio-Rad has released a fully integrated software and database package to provide multiple tools, including database building and management, search, analysis, structure drawing and reporting within a single user interface. According to the company, the KnowItAll Informatics System 8.2 includes a series of new report templates for more efficient output and improved design and a function to preview chromatograms and spectra to be used for searching, interpretation or database creation before the file is actually opened. The informatics system is reported to offer a fully integrated environment with flexible and expandable software and database solutions for chromatography, MS, cheminformatics; chemometrics, metabolomics and more. 

Departments

Digital Update

SFC 2010 — the 4th International Conference on Packed Column SFC 

— will take place in Stockholm, Sweden at the Radisson Blu Royal Viking Hotel from 

. The organizers, The Green Chemistry Group, aim to attract leading practitioners in SFC from research institutes, industry and academia across the US and Europe. Shalini Andersson of AstraZeneca in Molndal, Sweden and Eric Francotte of Novartis in Basel, Switzerland will co-chair this year's event.  

The conference was launched because of the lack of SFC presentations and posters at the standard industry conferences. Most meetings downplayed the significance of SFC as powerful new technology that could improve not only analytical and preparative chromatography, but aid the development process in the pharmaceutical, cosmetic, petrochemical and food industries.  

After speaking with other scientists in the supercritical fluid community, Todd Palcic, then vice president of Thar Instruments, realized that another broad, all-inclusive supercritical symposium would continue to disappoint those scientists and their employers who desired an industry-led event. To meet this need, he contacted potential sponsors and found them receptive to the idea of a meeting where the customers present their chemistry problems and SFC solutions.   

Palcic founded The Green Chemistry Group as a platform for new technology meetings and promotion of environmentally sustainable chemical research and development throughout the world. Board members include J. David Pinkston, Larry Taylor, Larry Miller, Francis Mannerino, Frank Riley and Nicholas Mitchell. The group is located in Pittsburgh, Pennsylvania, USA and can be found on the web: 

 will feature a scientific programme of oral and poster presentations and an exhibition. Retired Professor Larry Taylor and Amgen scientist Larry Miller head the Scientific Committee. Topics include natural product analysis, hydrocarbon analysis, enantiomeric separations and mass-directed purification of discovery compound libraries. They currently seek abstracts from presenters working on new research or applications in the field of supercritical fluid chromatography. The deadline for oral presentations is 

Vendors can take advantage of the opportunity to showcase the latest SFC technology to a diverse audience by exhibiting at a booth, sponsoring student travel or giving workshops. Current sponsors include Agilent, Amgen, 

, Pfizer, PIC Solution, Regis Technologies and Waters. 

 13th Symposium on Preparative and Industrial Chromatography and Allied Techniques,

 which is also held in Stockholm this year from 

As supercritical fluid chromatography grows in stature as a cutting-edge technology, this conference provides a forum for new developments and applications. To ensure the global nature of the SFC series of meetings, the next conference will likely be held in Asia for the first time in 2011. 

16th International Symposium on Separation Science

Conference Halls, National Research Council Headquarters, Rome, Italy 

28th International Symposium on Chromatography

21st Annual International Light Scattering Colloqium

Four Seasons Biltmore Resort, Santa Barbara, USA 

wyatt.com/events/ilsc/21st-annual-international-light-scattering-colloqium.html

XII Chemometrics in Analytical Chemistry Conference (CAC 2010)

Recent Advances in Sample Preparation Techniques

Event News

The realization that supercritical fluid chromatography (SFC) is now viewed as modern normal phase (NP) liquid chromatography (LC) has prompted a re-examination of bare silica as a viable stationary phase for packed column SFC. Published documents that appeared before and after 2000 involving bare silica and CO

/methanol mobile phases are reviewed. A current study which experimentally compares five bonded phases with bare silica for the SFC separation of a 19-component mixture of polar analytes is reported.

The evolution of stationary phases in supercritical fluid chromatography (SFC) continues to take place, even though the technique is now over 50 years old. Initially packed columns were used for SFC in the 1960s, but in 1981 attention switched to cross-linked polymer wall coated open tubular columns (WCOT). The most popular stationary phases (SP) were siloxane-based aliphatic and/or aromatic hydrocarbons that were analogous to (at the time) popular gas chromatographic phases. SFC open tubular columns, however, had smaller inner diameters than conventional GC columns. Packed columns (PCs) were regarded as being too retentive due to the active nature of the packing, although Berger insisted that the retentiveness was solely due to differences in the "phase ratio" of packed versus open tubular columns (OTCs).

  The controversy quickly died as it became clear that the two techniques (i.e., OTC versus PC) tended to best address different problems.  

Nowadays, the future of SFC is considered to rest unmistakably with packed, bonded silica-based columns and carbon dioxide-based mobile phases (MPs). The early goal of using only pure carbon dioxide was, however, realized only in part with nonpolar and weakly polar analytes. The limited success was attributed to many unsuccessful attempts to deactivate silica-based packing materials.

  The obvious strategy of conventional endcapping for each HPLC phases was not effective in that all silanol sites were not reacted and at temperatures above 100 °C the endcapped reagent was not stable.  

For a short while a more satisfactory solution to this dilemma involved the use of hydrosiloxane polymers that were coated and chemically bonded to the porous silica particle. A surface coating technique similar to one previously developed for a capillary GC column was used. The bonded phase would ideally cap or shield any remaining active sites from involvement in the separation mechanism. Results from this approach or its modification were surprisingly successful; resulting in the elution of moderately polar aliphatic amines with 100% CO

  Nevertheless, it became apparent to workers in the field that binary phases of CO

 and an organic polar modifier such as methanol would be required for elution of highly polar analytes that would be of interest to the pharmaceutical industry.  

SFC is now a separation technique most similar to normal phase HPLC. It almost always uses carbon dioxide as the main component in a binary or ternary mobile phase. It is usually performed as gradient elution where the composition of the mobile phase is changed versus time. Pressure programming has become uncommon. Small changes in temperature are used to change the selectivity of difficult analyte pairs. While SFC is quite amenable to the separation of volatile, non-polar analytes, it usually does not compete very favourably with gas chromatography (GC). The future of SFC is, therefore, the separation of highly polar analytes, which was the case many years ago with normal phase liquid chromatography (NPLC). SFC is now more applicable, more efficient, faster, greener and scalable for preparative chromatography than conventional NPLC.

  Considered by most workers in the field to be modern NP chromatography, SFC generally requires polar stationary phases to separate most polar solutes. In this manuscript, no differentiation will be made here between what is sometimes called subcritical fluid chromatography, enhanced fluidity chromatography and SFC. At sufficiently high pressure, transitions between these pseudo states are undetectable chromatographically and the instrumentation used is identical. 

HPLC studies have shown that a monolayer of the strongest solvent component of the mobile phase commonly exists on the surface of the stationary phase. Comparable studies have shown that somewhat similar effects are observed for SFC with CO

 as the mobile phase. Adsorption isotherms for supercritical CO

 displayed maximum adsorption at pressures close to the critical pressure with silica, octadecyl-, cyano- and diol-adsorbents.

  Multilayer adsorption occurred in each system and the type of bonded phase had little or no effect on the adsorption. In other words, under conditions normally used for packed column SFC, significant amounts of not only CO

 but also modifier are adsorbed on the solid adsorbent and function as components of the stationary phase. 

Parcher has thus suggested that polar modifiers can possibly affect the performance of an SFC system in a variety of ways.

  For example, (a) the modifier may alter the density and solvent power of the MP, (b) the modifier can block active adsorption sites on the SP inhibiting adsorption of solutes, (c) sorbed modifier may act as a component of the SP, (d) sorbed modifier may increase the volume of the SP while decreasing the volume of the MP thereby changing the phase ratio for the column and (e) the modifier may also solvate around a polar solute to form clusters in the MP. The combination of all these factors could result in either enhanced or diminished retention of solutes with variations in modifier concentration. All five effects may operate in any given SFC procedure that uses subcritical, polar modifiers. It is safe to assume, nevertheless, that an important aspect in the use of modifiers, besides changing the density and solvating power of the MP, is the influence of the modifiers and supercritical fluids on the physical and chemical properties of the SP. The effects of temperature, pressure and composition on the adsorption of both methanol and CO

 have been determined over a range of conditions with supercritical fluid MP. Under the conditions normally used for packed-column SFC, significant amounts of CO

 and modifier were both adsorbed on the solid adsorbent and functioned as components of the SP.  

Many acidic, basic and amphoteric compounds do not elute or elute with poor peak shapes even when the mobile phase consists of a binary mixture of CO

 and a modifier such as methanol and acetonitrile. The addition of a very polar secondary modifier (i.e., additive) appears to allow elution of symmetrical peaks.

  Just as with primary modifiers, additives may (a) suppress solute ionization, (b) adsorb on active sites of the SP and (c) form neutral ion pairs with ionic solutes. Additives adsorb onto many, but not all stationary phases. Surface coverage between 0.4 and 21% was measured at millimolar or lower mobile phase concentrations. The amount adsorbed increased with increasing stationary phase polarity. Changing concentration of the modifier appeared to affect the amount of additive adsorbed. The higher the concentration of modifier, the smaller the amount of additive adsorbed.

In light of the observation that all components of the polar modified MP in supercritical fluid chromatography are sorbed to different degrees on the SP, one might question the nature of the SP that was purchased versus the nature of the SP that is actually being used for separation. In other words, would a bare silica column suffice just as well as a bonded silica phase for SFC because mobile phase components are modifying the nature of the polar bonded phase silica? With the advent of purer and better defined silica that contains less metal ion contamination by major vendors, the question seems worth asking. Because silica columns appear to be more economical today than bonded phases, the question takes on added significance, especially for preparative chromatography. A recent review of the published literature has suggested that bare silica continues to find increased use in packed column SFC. 

Silica serves as the solid support for many stationary phases, but historically bare silica has been most often used as the stationary phase for absorption liquid chromatography (i.e., normal phase). In their pioneering SFC work, Klesper et al. mentioned having tried silica gel in different publications. Soon after this early work spherical porous silica particles became available that provided more uniform shape, higher surface area and greater sample capacity that packed more efficiently. Particle sizes varied from 3–10 μm in diameter with pore sizes ranging from 60 to 300 angstroms corresponding to a surface area of approximately 100 to 300 m

. The surface activity of silica packing materials proved to be a serious limitation when CO

 was used to elute moderately polar analytes. Chromatographic results, however, became markedly different when binary and ternary mobile phases were used with silica. 

In 1986 the influence of the physical state of CO

 concerning solute retention on bare silica was studied.

 was found to be very similar to that observed in HPLC with an apolar solvent such as hexane. SFC retention decreased when pressure (i.e., density) increased at constant temperature. However, when temperature was increased at constant pressure with phenanthrene and naphthalene as analytes, two situations were encountered. Below 200 bar, retention increased as solubility decreased, which was to be expected. Above 200 bar, solubility increased with temperature but retention surprisingly increased also. This increase in solute–stationary phase interaction could only be explained by a change in the apparent nature of silica caused by the presence of water which could affect SP activity. Water solubility in SF CO

 with temperature at 200 bar. For example, at 25 °C, solubility is 1500 ppm; while at 75 °C, solubility is 4500 ppm. Thus, when temperature increases, some water molecules are transferred from the SP to the MP and the number of free silanol groups covered by water molecules decreases. The adsorption energy of the solutes on the free silanol sites should be higher; thus there can be a simultaneous increase in retention and solubility. 

After this study, non-bonded silica was observed to give a higher resolution than bonded aminopropylsilica for the analysis of narcotine, thebaine, codeine, cryptopine and morphine.

/methanol/methylamine/water mixture (83.37:16.25:0.15:0.23). The procedure was later applied to a poppy straw extract and to the determination of three alkaloids.  

In another early study, capacity ratios and selectivity were determined for basic compounds, fat-soluble vitamins and carboxylic acids.

  Retention behaviour of the model compounds was compared for SP of unmodified and chemically modified silica particles. Unmodified silica gave higher plate numbers for more of the test solutes than the bonded stationary phases. Therefore, pure silica was recommended when the substances of interest could be eluted with reasonable capacity ratios and adequate selectivity. 

The separation and identification of sesquiterpene hydrocarbon mixtures on bare silica as a stationary phase with Fourier transform infrared detection and 100% CO

 was also reported approximately 20 years ago.

  The separation of these nonpolar but extremely thermosensitive organic compounds required low temperature rather than low density. These chromatographic conditions preserved structural information about the sesquiterpene hydrocarbons such as the differentiation of methyl and methylene groups and the geminal or single nature of methyl groups. In a related investigation, bare silica stationary phase with a high specific surface area was considered to give higher selectivity than a partitioning SP because of the similar chemical structures of the analytes.

  PCSFC with 0.5% methanol was found to be useful for the separation (10 min) of thermosensitive sesquiterpene alcohols such as nerolidol, alpha-bisbolol, farnesol and geranyllinalol. The elution order on silica was the same as observed in adsorption HPLC with 2,3,4-trimethylethane-ether mixtures. 

The application of SFC with 10% methanol-modified CO

 for the separation of polymethoxylated flavones (PMF) has been reported.

  Bare silica with UV detection (313 nm) was used. Selectivity was found to be different with SFC than with HPLC. Sinensetin, nobiletin, taneretin, heptamethoxylflavone and tetramethylscutellarein could be quantitatively determined using an internal standard (IS). The method was applied to the determination of PMFs in several 

 oils. SFC has also been more recently reported as a method of analysis for the determination of 4-hydroxybenzylglucosinolate degradation products.

The use of bare silica has been reported for the separation of ionic analytes such as sodium aryl sulphonates with ionic additive and polar modifier in the MP.

  The effect of additive concentration and additive functionality on analyte retention was investigated. The additives were lithium acetate, ammonium acetate, tetramethylammonium acetate and ammonium chloride dissolved in methanol. While the positive effect of ammonium acetate was expected from previous reports, results for the other additives salts were not expected and suggested a general phenomenon that seemed to not be cation or anion specific. A similar effect was also observed on the two cyanopropyl bonded phases that were investigated in this work. The fact that bare silica yielded analogous results to the two bonded phases suggested that some alteration of the SP by the additive was strategically involved. Interestingly, the aryl sulphonates were found to be retained more strongly on the silica column when the concentration of ammonium acetate in methanol was increased, which was inverse of the trend with the bonded phases. This observation suggested that different elution mechanisms may be operating with bare silica and the bonded phase silica. The elution mechanism envisioned with bare silica was suggested to be ion exchange; whereas an ion-pairing mechanism was postulated for the bonded phases. The elution of the totally ionic analytes with a CO

-based MP from a highly active, non-bonded silica packed column SP was not predicted and was deemed noteworthy. 

Bare silica along with zirconia and titania have been compared for their ability to separate diesel samples into saturates, mono-, di-, tri- and polyaromatics by SFC using pure CO

  Conventional Type A bare silica column HGS, Lichrospher Si 60, Lichrosorb Si 60, as well as a low metals Type B silica Kromasil Si column were studied to determine how well bare silica columns performed group-type separations of diesel samples. None of the silica columns adequately separated all of the model compounds according to group type, with the separation of the di- and tri-aromatics proving to be the most difficult. Yet, all of the columns studied met the ASTM D 5186-03 method requirements. A titania column coupled in series to a silica column was found to provide the highest overall group-type resolution based on 20 model compounds with resolutions as high as 14.7 for saturates versus monoaromatics and 11.8 for monoaromatics versus diaromatics. An oil sands-derived Synfuel light diesel, a commercial Ontario diesel and a heavy Shell Canada Ltd diesel blending feedstock were studied on both the bare silica column and the coupled columns. The Synfuel results were similar, but the heavier commercial diesel and diesel blending feedstock samples were different. 

The separation of cytarabine (ara-C) from the endogenous compounds in mouse plasma by packed column SFC with an isocratic MP composed of CO

/MeOH and ammonium acetate as an additive has recently been reported.

  The packed column was integrated with a tandem APCI-MS–MS to enhance sensitivity, selectivity and speed of analysis. No change in retention for ara-C was observed with an increase of water content in the modifier from 0.01 to 1.0%. The mouse plasma levels of ara-C (ng/mL) were found to be consistent with those determined by various reversed phase, HPLC methods using either a porous graphite carbon column, a mixed-mode column, or a C18 column in conjunction with an ion pairing agent coupled to a tandem mass spectrometer. 

Given the relatively few references discussed in this treatise, we feel that this review of packed column SFC with non-bonded silica is nearly exhaustive. The use of bare silica for packed column SFC has been known as long as SFC has been known. Its use, however, has not been embraced by many workers in the area and reported publications have been rather spotty. No doubt silica impurity, traces of uncontrollable mobile phase moisture and a host of newly developed, competing bonded polar phases have led to a lack of interest in silica for SFC purposes. In those instances where it has been used the results appear favourable compared with both HPLC and with SFC that embraces packed bonded silica phases. As the quality of bare silica has improved, no doubt its properties as a stationary phase have become more attractive, especially because water has been demonstrated to be an excellent neutral additive in conjunction with silica and various polar bonded phases. 			

We have experimentally compared five bonded phases with bare silica for the separation of a 19-component mixture of polar analytes (Table 1) with CO

, methanol modifier, and 3% water/10 mM ammonium acetate (AA) as a binary additive (Figure 1). All separations were performed using a SFC instrument (Thar, Pittsburgh, Pennsylvania, USA) equipped with a fluid delivery module, column oven, autosampler, automated back pressure regulator and a 2998 photodiode array detector (Waters, Milford, Massachusetts, USA ). All chemicals were obtained from Sigma-Aldrich Inc. (Saint. Louis, Missouri, USA); while all solvents were HPLC grade from Thermo Fisher Scientific (Pittsburgh, Pennsylvania, USA). Bare silica, 2-ethylpyridine, diol and cyanopropyl packed columns (250 × 4.6 mm, 

 = 5 μm, 60 Å) were obtained from Princeton Chromatography (Princeton, New Jersey, USA). Octadecyl and phenyl-hexyl columns of similar dimension were obtained from Phenomenex (Torrence, California, USA). All chromatographic separations were performed at 3.5 mL/min with the following step gradient of mobile phase components: Time zero: 90/10 CO

/modifier; time 1 min: 60/40, time 10 min: 60/40, time 3 min: 90/10, time 5 min: 90/10. Additives were incorporated into the methanol modifier (v/v) before mixing with CO

 pressure and temperature were 200 atm and 60 °C. The sample solvent was methanol and the injection volume was 5 μL. Two injections separated by approximately 11 min (during which time the column was equilibrated with the mobile phase) were made for each run. The results of the second injection were accepted and are shown here. Water and ammonium acetate proved to be ideal additives for the separation.

  Overall peak shape and selectivity with silica are as good as or better than with any of the remaining five phases using identical chromatographic conditions. The superiority of polar phases for the separation of polar analytes is clearly demonstrated in the set of six chromatograms. Selected, clearly resolved components of the analyte mixture are identified for the silica column in Table 1. 

Table 1: Components of chromatographic mixture with selected silica retention times in minutes noted.			

In summary, the way to succesfully improve SFC chromatographic peak shapes of polar solutes is by using polar modifiers and even more polar additives with polar stationary phases. The dominant retention mechanism for solutes in SFC then becomes the interaction between the solutes and the most polar entity on the chromatographic support. Berger and Deye suggested that to be successful in SFC the stationary phase should be more polar than the solutes and probably more polar than a silanol site.

  Thus, it is now assumed that a film of modifier and CO

 is a significant, if not dominant part of the true packed column stationary phase because the film tends to further decrease the intensity of the solute—silanol interaction. Under supercritical fluid conditions, surface molar excesses of mobile phase additives may also act to change the chemical nature of the stationary phase surface thereby making it more polar than the mobile phase. The excellent separation of polar analytes on bare silica clearly supports these hypotheses. 

The greatest success for improving SFC chromatographic peak shapes of polar solutes is achieved using polar modifiers and even more polar additives with polar stationary phases. The dominant retention mechanism for solutes in SFC then becomes the interaction between the solutes and the most polar entity on the chromatographic support. Berger and Deye suggested many years ago that to be successful in SFC, the stationary phase should be more polar than the solutes and probably more polar than a silanol site.

 is a significant, if not dominant part of the true packed column stationary phase because the film tends to further decrease the intensity of the solute—silanol interaction. Under supercritical fluid conditions, surface molar excesses of mobile phase additives may also act to change the chemical nature of the stationary phase surface, thereby making it more polar than the mobile phase. The excellent separation of polar analytes on bare silica clearly supports these hypotheses. 

The loan of a Waters SFC (Milford, Massachusetts, USA) instrument is greatly appreciated. Financial support of this work was provided in part by Applied Analytical Inc. Discussions with Frank Riley and Jian Wang at Pfizer (Groton, Connecticut, USA) and Rui Chen (Waters) were extremely helpful. 

 received his Ph.d in Analytical Chemistry from Virginia Tech in 1998. From early 1992 till mid-1994 he joined Trabriat Modaress University (Tehran, Iran) as an assistant professor). In 1994 he joined Virginia Tech as Senior Research Scientist in the chermistry department. 

 is Emeritus Professor of Chemistry, Virginia Tech. He has served as the Chair of the Scientiific Committee for SFC 2008 (Zurich), SFC 2009 (Philadelphia), and SFC 2010 (Stockholm). He is the author or co-author of approximately 400 peer-reviewed publications and 12 patents. 

1. T.A. Berger, "Packed Column SFC", RSC Chromatography Monograph Series, Royal Society of Chemistry, Cambridge, Chapter 4 (1995). 

SFC is now regarded as a modern normal phase LC technique prompting a re-examination of the role of bare silica as a useful stationary phase.

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