John Dolan has been writing "LC Troubleshooting" for 

 for over 25 years. One of the industry's most respected professionals, John is currently a principal instructor for LC Resources, Walnut Creek, California He is also a member of 

How suitable is the column plate number for system suitability testing?

	I was recently contacted by a colleague inquiring about whether or not the column plate number, N, is a good measurement to include in system suitability testing (SST) for liquid chromatography (LC) methods. He had noticed that monographs in the United States Pharmacopoeia (USP) often had minimum plate number requirements as part of SST for those methods, but that often it was specified that N > 2000 was sufficient. He wondered about the origin of a N > 2000 requirement and if it really added any value to SST, especially in light of the performance of modern LC columns. This is a topic of wide enough interest that I feel it is worth a discussion here.

	The column plate number usually is part of the specification for new columns. Traditionally, N is calculated based on manual measurements of the retention time, tR, and the baseline width, wb, measured between tangents to a chromatographic peak, as shown in Figure 1 using the formula

	N = 16(tR/wb)2                [1]

	More commonly today, N is measured by data systems based on the more easily measured width at half the peak height, w0.5, using

	The method of equations 1 or 2 only applies to isocratic separations, where the mobile-phase composition is constant, and should not be used for gradient separations.

	A well-packed, reversed-phase column containing totally porous particles tested under ideal conditions should generate plate numbers of

	where L is the length of the column (in millimetres) and dp is the particle diameter (in micrometres). However, this requires an LC system with very low extracolumn volume, a small injection, a well-behaved, small molecular weight test solute, an optimum flow rate, and other conditions that do not reflect typical application of the column in the routine laboratory. Also, if equation 3 were used for acceptance criteria of a new column, column manufacturers would have to discard many otherwise satisfactory columns. As a result, equation 3 may be adjusted to use 2.5 dp (or some other value) rather than 2 dp as the denominator for a quality control specification in the column manufacturing process.

	When an LC column is used for a real LC method, usually the conditions are much less ideal than those used by the manufacturer for testing the column. The typical LC system will have more extracolumn volume than the test system. The sample solutes will be less wellâbehaved, and other chromatographic conditions (temperature, flow rate, and so forth) may also degrade the column performance. It is nice to have a rule of thumb for column performance under typical, “real” conditions to make sure the column is performing adequately. For totally porous particle columns I like to use the estimate

	N ≈ (300L)/dp               [4]

	In Table 1, I have compared the calculated plate numbers for two of the most popular column configurations today: a 150-mm, 5-µm dp column and a 100-mm, 3-µm column.

	Let’s consider some of the ways the knowledge of the plate number for a particular column can be useful. You can see from Table 1 that the plate number for the 150 mm, 5 µm column is approximately the same as the 100 mm, 3 µm one. This means that these columns should give the same separation if the same column chemistry (brand and product line) and operating conditions are used. This can be helpful in evaluating tradeoffs when changing column dimensions - if the 150-mm column had a 15-min run time, it would only take 10 min with the 100âmm column and would reduce solvent consumption by a third.

	You can also monitor the column performance over time for help in deciding when to replace the column. I usually consider that the column should be replaced when you see ~30% loss in N relative to the value measured when the column is new. So, if you measured N = 9000 for a new column and your method, when it dropped to N ≈ 6000, it probably would be time to replace it. The replacement decision should also be based on other factors, such as changes in peak shape and back pressure.

	The column plate number can be helpful in evaluating overall performance of the LC system. If you suspect that the cause of a drop in plate number is external to the column, you can replace the column with a new one and compare the plate number (and other performance parameters) to historic values when the system was known to be operating well. If N is still low with a new column, look for problems external to the column. Extracolumn band broadening can be a critical factor in such cases, especially with the increased popularity of ultrahigh performance LC (UHPLC), where 50 mm × 2.1 mm columns packed with ≤2-µm diameter particles are popular. A fitting that has slipped a bit because of system over-pressure or poor assembly is not visible to the naked eye, but often will cause noticeable deterioration of chromatographic performance, including N.

	Resolution, Rs, is another parameter that is used to evaluate the suitability of a method for analysis. It is useful to compare the utility of N and Rs and their roles in system suitability, so let’s take a slight detour and review how we determine resolution.

	Resolution can be calculated using the retention times of the two peaks (tR1 and tR2) and the peak widths at baseline (wb1 and wb2):

	Rs = (tR2 – tR1)/(0.5(wb1 + wb2))                        [5]

	Alternatively, the widths at half-height (w0.5,1 and w0.5,2) can be used:

	Rs = (tR2 – tR1)/(1.7 × 0.5(w0.5,1 + w0.5,2))      [6]

	Rs = (1.18 ((tR2 – tR1))/(w0.5,1 + w0.5,2)           [7]

	As with the calculations of N at half the peak height, equation 6 (or 7) is a simpler measurement for the data system than peak widths at the baseline. A further advantage of the half-height technique is that if Rs < 1.5, baseline peak widths will be difficult or impossible to determine even for well-shaped peaks, so the method of equation 5 cannot be used. As you can see from equations 5, 6, and 7, resolution is directly related to the peak width.

	Another way to determine resolution is with the fundamental resolution equation:

	Rs = 0.25N0.5(α – 1)(k/(1 + k))                           [8]

	where k is the retention factor and α is the selectivity, calculated as the ratio of retention factors for two adjacent peaks. Values of k, and thus α, are controlled by the chemistry of the mobile and stationary phases as well as the temperature. So, if chemistry and temperature are constant, as is normally the case, resolution is a function of the square root of the plate number.

	Often the column plate number is included as part of system suitability testing, such as the N > 2000 requirement mentioned in the introduction. If, as is the case most of the time, one goal of an LC method is to separate two compounds, the resolution (Rs) of the two peaks will be included as part of SST. If resolution measurement is required, does measurement of N add any useful information to SST?

	To help compare and contrast Rs and N, I’ve prepared Table 2 and Figure 2. Table 2 tabulates peak and chromatogram characteristics for different values of N. The various rows show data corresponding to columns with plate numbers ranging from 10,000 down to 2000 in 20% increments (data in columns 1 and 2). This progression might occur when we start with a new, 150-mm, 5-µm dp column (N ≈ 10,000 per Table 1) and watch the plate number drop over the lifetime of the column. If the chemistry of the system stays the same, the retention will stay constant, so decreasing N will increase the peak width as a square root function (see equations 1 or 2); the peak width is shown in the third column of Table 2. For constant sample size and detector response, the peak height will drop in proportion to the increase in peak width (column 4). As we saw above in equation 8, resolution will be reduced by the square root of the drop in N; resolution relative to the starting condition is shown in column 5. Notice that it takes a fourfold drop in N to halve Rs. Finally, columns 6 and 7 translate this relative loss of resolution into numeric values for separations starting with Rs = 2.0 or 1.7.

	Figure 2 comprises simulated partial chromatograms based on the data in Table 2. In each chromatogram, a wellâresolved peak precedes a peak pair whose resolution is tracked as follows. The top row of chromatograms corresponds to column 6 of Table 2 where Rs = 2.0 initially (left) and drops progressing to the right as the plate number drops to N = 2000 and Rs = 0.89. The bottom row of Figure 2 shows the corresponding chromatograms starting with Rs = 1.7.

	One thing that is quickly apparent when studying the chromatograms in Figure 2 is that a change in N may be less likely to be noticed than a change in Rs. For example, the loss of ~25% in peak resolution is obvious when comparing the chromatograms for N = 10,000 and N = 6000, whereas the loss of N, often noticed by increased peak width, is much less obvious without careful study. In Figure 2, the loss of peak height is obvious as N is decreased, but here all the chromatograms are on the same scale; if the peak heights were normalized to the tallest peak, a 25% change in peak height might be overlooked in a visual evaluation of the chromatogram.

	Another problem with the plate number as a measure of quality is that the absolute value of the plate number does not necessarily reflect the quality of the separation. For example, compare the initial Rs = 2.0 separation after the column degrades to N = 6000 to the original Rs = 1.7 separation with the N = 8000 column. Both have the same resolution (Rs = 1.5), but N differs by 25%. Thus, setting some arbitrary minimum acceptable value of N, as was proposed in the introduction for N > 2000, makes no sense as a general requirement. Instead, the minimum acceptable plate number can be determined only in light of the minimum acceptable value of resolution for each method. On the other hand, a specified value of resolution will always translate into the same visual appearance of the chromatogram regardless of the method or the column plate number.

	Does resolution always win out over the plate number as an effective SST parameter? Not necessarily. A value for resolution is of little practical use once two peaks are well separated at the baseline (for example, Rs > 3). This is apparent for the resolution between the first and second peaks of each partial chromatogram in Figure 2; in no case is a numeric value of resolution very useful in alerting us to potential problems with the separation. The plate number, on the other hand, does give us an idea that something is not going well when the value drops precipitously.

	The plate number also gives us information regarding how well the column performs relative to an ideal case (manufacturer’s test), a generic rule of thumb for “good” performance (equation 4), or the same column when it was new. Tracking the plate number can help us determine when the column is nearing the end of its useful life so we can order a replacement. Following changes in the column plate number can also help to anticipate problems with detection limits. As the plate number drops, the width increases and the peak height decreases (Table 2) so that peak area remains constant. Whereas most of us use peak area for quantification, peak height is the critical measurement when it comes to detection limits, which depends on the signal-to-noise ratio. Shorter peaks will have smaller signal-to-noise ratios, and thus poorer detection limits. When methods are used for trace analysis, the plate number may serve as a very useful tool to help determine when to retire a column so as to maintain suitable lower quantification limits.

	A final consideration for the plate number is reflected in the speed of separation. Larger plate numbers (for the same column length) always translate into narrower peaks. Narrower peaks allow peaks to be more closely eluted for any given resolution than for broader peaks. Therefore, larger plate numbers can be translated into faster separations. So the use of columns that generate substandard values of N will require longer run times and thus increase analysis costs.

	We have looked at the column plate number as a system suitability parameter to include in LC system suitability testing. We have seen that resolution is much more useful than N if the peaks are close together (for example, Rs < 3), but its usefulness drops for very well separated peaks. Therefore, the plate number can be a useful way to predict method performance when the separation is not challenging. It can also help to determine the overall health of the column and its performance compared to some specified reference condition. Because both N and Rs can be calculated easily and automatically by chromatographic data systems, I think it is a good idea to keep track of both measurements. Just be sure to use the data appropriately to evaluate current or potential problems with an LC method. And an arbitrary lower limit of N >  2000? I don’t think this is a good idea.

	So where did that N > 2000 requirement come from, anyway? As far as I can tell, it originated in a United States Food and Drug Administration (FDA) document (1). This document is designed to help FDA inspectors evaluate chromatographic methods when they conduct laboratory audits. In the guidance, the recommendation is, “The theoretical plate number depends on the elution time but in general should be >2000”. The document is dated 1994, and, although 5 µm dp columns were widely used by that time, older columns packed with 10-µm irregularly shaped particles were still common. There may have been some justification for N > 2000 20 years ago, but not today, and certainly not as a generic requirement.

		U.S. Food and Drug Administration, Reviewer Guidance. Validation of Chromatographic Methods (USFDA-CDER, Rockville, Maryland, USA, November 1994).

 has been writing “LC Troubleshooting” for 

 for more than 30 years. One of the industry’s most respected professionals, John is currently the Vice President of and a principal instructor for LC Resources in Lafayette, California. He is also a member of 

Columns

Column Plate Number and System Suitability

John V. Hinshaw is senior staff engineer at Serveron Corp., Hillsboro, Oregon, and a member of LCGC's editorial advisory board. Direct correspondence about this column to "GC Connections," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

. For an ongoing discussion of GC issues with John Hinshaw and other chromatographers, visit the Chromatography Forum discussion group at 

Thermal desorption sampling often provides a means for bringing otherwise intractable samples to a gas chromatography (GC) column for separation and detection. In this instalment, John Hinshaw describes the principles of thermal desorption sampling in relationship to other analysis techniques for volatile solutes.

	When asked about common sampling techniques, gas chromatographers first envision a liquid inlet system such as what is found on most gas chromatography (GC) systems. Injection of a relatively clean liquid sample into a vaporizing inlet system often suffices to transfer analyte components from their physical state outside the GC column into a state suitable for GC analysis. A vaporizing inlet system heats the sample to vaporize the components of interest, mixes the sample vapour with carrier gas, and then transfers the sample into the GC column. Such injection techniques include direct, split, splitless, on-column, and programmedâtemperature methods.

	Many other samples present a different problem: They contain nonvolatile materials that can impair a vaporizing inlet’s function when significant quantities of residue are deposited in the inlet or in the beginning of the column. The builtâup residue interferes with subsequent analyte transport through the inlet system by causing peak-shape distortion, irreversible adsorption, and thermolytic decomposition. Other samples such as resins, powders, fibres, plastics, and biological fluids present a matrix that is simply incompatible with direct injection techniques.

	In such cases, gas chromatographers can call upon a number of related techniques that extract relatively volatile analytes from a less volatile sample matrix. Analyte concentration and focusing steps before transfer onto the GC column often accompany such procedures. Purge-and-trap, headspace, and thermal desorption sampling, plus solid-phase microextraction (SPME) add a number of sampling-stage steps that make them suitable for solid and liquid samples in difficult matrices.

	Thermal desorption sampling refers to a number of related techniques that include one or more steps in which sample heating releases volatile analytes into the carrier-gas stream from an adsorbent or other involatile material. One or more adsorption–desorption steps serve to concentrate analytes as well as reduce their bandwidths to meet column injection requirements. Volatiles enter the desorption tube from a variety of sources, including directly from the atmosphere, from the headspace over a solid or liquid sample, by sparging a liquid sample with inert gas, or as the effluent from a chemical process or engine exhaust. A related technique, thermal extraction, heats a solid or semisolid sample directly in the carrier-gas stream using essentially the same equipment as thermal desorption. To distinguish it from pyrolysis injectors, thermal desorption operates at lower temperatures and does not intentionally cause sample thermolysis. For example, chromatographers can use thermal extraction to sample polymers, waxes, powders, pharmaceutical formulations, solid foods, and cosmetics for their volatile constituents.

	The bandwidth or volume that analytes occupy as they approach an openâtubular column is often large enough to prevent the column from delivering all of its peakâresolving potential, especially for early-eluted peaks. Ancillary cold trapping or secondary focusing techniques help reduce analyte bandwidths appropriately. Other problems found in conventional liquid inlet systems from excessive amounts of solvent or nonvolatile residue deposition in the inlet are absent in volatiles extraction techniques such as thermal desorption. Water vapour from atmospheric or aqueous samples, however, may interfere in some cases, and may require additional sample management steps to prevent waterâinduced side effects.

	In its simplest form, thermal desorption involves heating a sample tube with carrier gas flowing through it. The emerging gas is conducted directly to the GC column. Figure 1 illustrates a simple thermal desorption setup. A tube containing an adsorbent or sample material (or both) is positioned in the carrier gas stream before the column inlet. After a short delay to flush out residual air, the tube is heated and analytes are desorbed or thermally extracted directly from the sample. Carrier flow conducts the released volatile compounds into the column. In practice, an existing inlet can provide a convenient base for a top-mounted desorption system, or it can act as the terminus of a heated transfer line for benchtop systems. After desorption has finished, the tube cools down and normal carrier-gas flow is restored.

	This simple scheme suffers from a number of potential difficulties and generally is not used in practice without enhancements. Researchers have developed a variety of solutions, some of which are generally applicable to thermal desorption, while others are more useful for specific sample types. Thermal desorption problem areas include controlling the amount of sample amount entering the column, flow rates through the column and the sample tube, solute breakthrough volumes, and peak bandwidths. These areas are discussed in more detail below.

 Capillary GC columns tolerate a limited analyte mass beyond which column overloading occurs, characterized by peak distortion and broadening plus retention time shifts. Column sample capacity ranges from about 100 ng on a 0.25-mm i.d., 0.25-mm film column up to about 5 mg on a 0.53-mm i.d. column with a 5-mm stationary phase film. Analyte amounts released from thermal desorption or thermal extraction often exceed these levels, and serious performance losses could occur if all the analyte mass contained in a sample tube were permitted to enter the column.

	Thermal desorption systems can use in-line sample splitting to control the analyte amounts that traverse the sampling system. A modified capillary column inlet splitter works well for top-mounted samplers, while benchtop systems often use self-contained splitters. Figure 2(a) shows an outlet splitter inserted after the sample desorption tube. A needle valve or electronic device (not shown) controls the outlet split flow and thus the split ratio from the desorption tube to the column. Split control with electronic pneumatics is possible when the in-line capillary GC inlet system supports it. For trace-level samples, direct transfer conducts all of the material onto the column. Sample splitting also helps to balance flow rates and reduce peak bandwidths by permitting faster tube flow rates and concomitantly faster desorption times.

Thermal desorption tubes generally have inner diameters of 3–4 mm. With lengths of about 100 mm, tube internal volumes range from 2.8–5.0 cm3. With a typical packing factor of about 0.4, tube void volumes are 1.1–2.0 cm3. Desorption tubes are not packed tightly with adsorbent from end to end, however, to allow space for glass wool or other materials that restrain the packing inside the tube within the heated zone. For this reason, typical void volumes are on the high side of this range. During desorption, when the tube is heated and analytes are no longer retained on the tube packing, a gas volume of at least 4–8 times the void volume should pass through the tube to transfer analytes to the column quantitatively. A desorption tube with the above dimensions needs at least 30 s to heat its contents to the desorption temperature. With these assumptions, a desorption volume of at least 10 cm3 at flow rates of 20 cm3/min or greater is suitable in most cases. If the tube packing slightly retains high-boiling analytes at elevated desorption temperatures, however, the gas volume and the time required for complete desorption increases significantly. In practice, longer desorption times help ensure quantitative transfer from the desorption tube.

	For packed columns, desorption flow matches column flow well. However, a disparity exists between the desired desorption flow rate and optimum capillary column flows. At an average helium carrier-gas linear velocity of 40 cm/s and a temperature of 50 °C, the corrected outlet flow rate of a 25-m long, 0.25-mm i.d. capillary column is 1.75 cm3/min, about five times less than the desired desorption flow. For a 25-m long, 0.53âmm i.d. column, the corresponding corrected outlet flow rate is 5.4 cm3/min. This value is closer to the desorption tube flow rate, but still lower by a factor of 2. Without modification, direct connection to a capillary column would require excessively long desorption times to pass even a minimum amount of carrier gas through the tube: about 2–4 min for a wide-bore column and 6–12 min for a narrow-bore column.

	A simple solution to this problem is to increase the column flow rate during desorption. This remedy is feasible for wide-bore columns, where the increase is limited to two or three times the optimum flow, and in many cases a wide-bore column can deliver good results with direct sample transfer. For narrow-bore columns, however, the needed increase in flow rate of greater than six times will affect analyte trapping efficiency and focusing at the head of the column, and may require more column inlet pressure than is available.

	In-line stream splitting as shown in Figure 2(a) provides a better solution for nontrace samples with capillary columns. Flow from the desorption tube splits between the capillary column and the vent. Minimum desorption times remain in the 30-s range, and optimum flow persists in the capillary column. However, stream splitting alone may not suffice with traceâlevel samples for which a high split ratio would reduce analyte amounts below a minimum quantifiable limit. Alternative approaches such as secondary adsorbent trapping can be advantageous, as discussed below.

For peaks that are significantly retained at the initial column temperature, the time it takes for them to be desorbed from a sample tube and pass onto the column is not of too much concern. Such peaks will be focused at the head of the column by trapping in the stationary phase if their retention factors (k) are greater than about 10 at the initial temperature. Analytes that the column does not retain significantly at the initial temperature, however, hit the column and keep going. The column will not deliver the highest resolution possible for these peaks. Since the minimum desorption time is on the order of 30 s, thermal desorption sampling almost always requires some form of band focusing. Several solutions can help establish narrower initial peak widths for these early eluting analytes.

	A cryogenic cooling device at the beginning of the column can effectively trap the more volatile analytes in a sample, along with those less volatile ones that would be trapped anyway, without such a device. Figure 2(b) shows a cryofocusing trap in a thermal desorption system, positioned just inside the column oven, after a stream splitter. Liquid nitrogen or carbon dioxide cools the cryofocusing zone to below 0 °C during sample tube desorption, and analytes from the sample tube, including the more volatile ones, are trapped there. After desorption is completed, cryogenic cooling is turned off and the cooled area is heated quickly to release the analytes trapped there.

	A potential drawback of on-column cryofocusing is the accumulation of water at the head of the GC column when moist materials - for example, foodstuffs - are sampled by thermal extraction. Ice crystals can block the column, and ice on the column surface can affect trapping efficiency. Large amounts of water that are eluted from the column can cause detector problems, too.

An important characteristic of thermal desorption sampling is the GC column–like behaviour of sample tubes packed with active adsorbents. As sample gas bearing analytes passes through an adsorbent bed, the analytes migrate along the length of the tube, just as they would in a GC column, and it is possible for them to traverse the length of the tube and emerge from its exit. The gas volume required for an analyte to do so is termed the breakthrough volume. It is expressed in units of gas volume per gram of adsorbent at a specific temperature, and is equivalent to the specific retention volume, Vgθ, on a packed column if the analyte were to be injected onto the adsorbent trap in a narrow plug. Breakthrough volumes help characterize adsorbents by determining not only the maximum gas volume when accumulating a sample, but also by indicating appropriate desorption temperatures.

	If the breakthrough volume is too low at the sampling temperature, which is usually ambient temperature, then the trap will not adsorb the analyte quantitatively from a large sample gas volume. For a safety margin, the gas sample amount should not exceed half the breakthrough volume. If sample splitting is used, then the minimum quantifiable concentration increases accordingly.

	After the sample compounds are adsorbed onto a sample tube, all of them must desorb at temperatures less than the maximum limit for the adsorbent. When an analyst is selecting a desorption temperature under the maximum, the highestâboiling or most strongly adsorbed compound determines the desired desorption temperature. The breakthrough volume decreases logarithmically with increasing temperature.

	It is imperative to consider all the compounds of interest in a sample in terms of their breakthrough volumes. If lighter, less strongly adsorbed materials are present, and sample collection cannot occur at subambient temperatures, then a second, stronger adsorbent material can augment the sample trapping scheme and extend the overall volatility range of thermal desorption sampling. Figure 3 illustrates a dualâadsorbent sampling tube. Sample gas passes through the weaker adsorbent first, and the higher-boiling, more strongly adsorbed components are trapped there. The lower-boiling, weakly adsorbed components break through the weak adsorbent into the stronger adsorbent where they are trapped. Care must be taken not to allow the higherâboiling components to break through the weak adsorbent; they will be permanently trapped on the strong adsorbent.

	During thermal desorption the flow through a two-adsorbent trap must reverse and backflush the trap. If thermal desorption were to take place in the forward flow direction, then high-boiling materials from the weaker adsorbent would irreversibly encounter the strong adsorbent, just as if they had broken through to it during sampling. During backflush desorption, material from the strong adsorbent passes through the weak adsorbent without effect on its way out of the sample tube.

	Multiple adsorbents extend the range of thermal desorption sampling to include compounds that quantitatively adsorb and desorb from either of two adsorbents in the same sample tube. The two adsorbents should be complementary in the range of suitable compounds. The maximum desorption temperature for a multiple-adsorbent sample tube is the lowest of the maximum temperatures for all included adsorbents. Good sources of information about adsorbents and trapping of specific compounds are available from the manufacturers of thermal desorption hardware and adsorbent tubes.

	With single or multiple adsorbents, forward- or back-flushed, the desorption time, volume, and flow rate from a conventional sample tube are not well matched to capillary column requirements. If the sample tube were much smaller, its desorption and flow characteristics would be a better match because it could be heated more rapidly, but its breakthrough volumes and sampling characteristics would not be suitable for collection of larger sample volumes. Nor would such a tube be well suited to direct thermal extraction. However, a small additional adsorbent trap positioned in-line between the sample tube and the column can serve both as a focusing device for desorbed analytes as well as a means to better balance the flow rates of the column and the sample tube. Figure 4 shows a small secondary adsorbent trap positioned between the sample tube and the column. Additional valving (not shown) creates the flow paths shown in Figures 4(a) and 4(b). In Figure 4(a), carrier gas flows directly into the column and isolates the two traps from the column. During sample desorption or thermal extraction, the sample tube heats up and desorption gas flow conducts released analytes past the first split point into the secondary adsorption trap, which is cooled to ambient temperature or below.

	After desorption is completed, flow switches to the arrangement shown in Figure 4(b). At that point, desorption flow continues to purge the sample tube, while carrier gas flows through the secondary trap in the backflushed direction. The secondary trap is heated rapidly to desorb the analytes quickly. They pass through an optional second split point and then into the column.

	The secondary trap contains only a small amount of an adsorbent, 10–20 mg, which can be the same single or multiple adsorbent material as used in the main sample tube. Adsorption breakthrough volumes on the secondary trap are concomitantly lower, but they are greater than the desorption volumes from the primary sample tube. Operation of the secondary trap at subambient temperatures also helps to increase the breakthrough volumes there.

	The smaller inner diameter and shorter length of the secondary trap bring its desorption time, flow, and volume down to capillary column levels. A typical heating rate for the packing inside the secondary trap is 40 °C/s, so that it can reach a 200 °C desorption temperature in around 5 s. At elevated temperatures, the minimum desorption volume in such a trap is 1–2 mL, which is about 10 times less than for a primary sample tube. This lower volume opens the possibility for splitless transfer into a capillary column with trace-level samples, although some form of on-column focusing is still required at narrow-bore capillary column flow rates.

	A secondary trap also helps with direct thermal extraction. With suitable hydrophobic adsorbents and trap temperatures, water from moist samples will not be adsorbed on the secondary trap, but will pass through the trap to vent. Water that does condense or freeze in the secondary trap is better tolerated there than at the head of the GC column; ice crystals or water droplets will not block gas flow. Subsequent stream splitting during secondary trap desorption reduces the amount of water actually entering the column.

	The secondary trapping scheme is not without its drawbacks. It is considerably more complex than other systems and requires a more in-depth knowledge of thermal desorption principles for effective use. The mechanical implementation includes extra valves and tubing, all of which must be inert towards the sample materials and free of cold spots. But the additional complexity may be justified by the ability to handle difficult samples and the flexibility that comes along with secondary adsorbent trapping.

	GC sampling techniques for volatiles are highly developed. Analysts can call upon a wide variety of methods for sampling volatiles from difficult matrices, including headspace, thermal desorption, solid-phase microextraction, and purgeâandâtrap sampling. Thermal desorption techniques present good methods for the concentration of gaseous samples from a variety of sources, including the headspace over solids and liquids, atmospheric samples, and process effluents. Direct thermal extraction of otherwise intractable samples is also possible with thermal desorption equipment.

	Researchers have developed a variety of thermal desorption techniques. The need to accommodate capillary columns for large gas samples that span a wide volatility range has spurred development of ancillary methods for thermal desorption samplers, including on-column cryotrapping, sample stream splitting, multipleâadsorbent sample tubes, and secondary adsorbent traps. To be successful with thermal desorption sampling, chromatographers must pay careful attention to the relationships between the adsorption–desorption characteristics of trapping materials, the range of sample components and sample matrices to be analyzed, and the kind of equipment that will perform the sampling and analysis.

 is a Senior Scientist at Serveron Corporation in Beaverton, Oregon, USA, and a member of 

 editorial advisory board. Direct correspondence about this column should be addressed to “GC Connections”, 

, Hinderton Point, Lloyd Drive, Ellesmere Port, Cheshire, CH65 9HQ, UK, or e-mail the editor-inâchief, Alasdair Matheson, at amatheson@advanstar.com

Thermal Desorption Sampling

Szabolcs Fekete, Jean-Luc Veuthey, and Davy Guillarme

, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.

The recent trends in column technology for reversed-phase liquid chromatography (LC), sizeâexclusion chromatography (SEC), ion-exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) for analysis of biopharmaceuticals at the protein level is critically discussed.

	Therapeutic proteins are large and heterogeneous molecules subjected to a variety of enzymatic and chemical modifications during expression, purification, and longâterm storage. These changes include several possible modifications, such as oxidation, deamidation, glycosylation, aggregation, misfolding, or adsorption, leading to a potential loss of therapeutic efficacy or unwanted immune reactions. Regulatory bodies require a detailed characterization (for example, verifying primary structure and appropriate postâtranslational modifications, secondary and tertiary structure), lotâtoâlot and batch-toâbatch comparisons, stability studies, impurity profiling, glycoprofiling, determination of related proteins and excipients as well as determination of protein aggregates. For this purpose, a single analytical technique is generally not sufficient, and a variety of orthogonal methods are required to fully describe such a complex sample.

	Today, one of the most widely used analytical techniques for therapeutic protein characterization is liquid chromatography (LC). This is probably a direct result of the remarkable developments of the past few years, which have enabled a new level of chromatographic performance. These developments include ultrahigh-pressure LC (UHPLC), columns packed with wideâpore superficially porous particles (SPPs), and organic monolith columns, which have allowed a dramatic increase in separation efficiency, even with large intact biomolecules.

	This article will review the possibilities and trends of current state-of-the-art LC column technology applied for different modes of chromatography for the characterization of therapeutic proteins.

	Hydrophobic interaction chromatography (HIC) has been historically used for protein purification; more recently, the two main application fields have been in the determination of the drug-toâantibody ratio (DAR) of antibody-drug conjugates (ADCs) and in monitoring post-translational modifications of monoclonal antibodies (mAbs).

	In HIC, proteins are retained and separated on the basis of their hydrophobicity as a result of the van der Waals forces between the hydrophobic ligands of the stationary phase and the non-polar regions of proteins (1). The binding of proteins to a hydrophobic surface is affected by a number of factors including the type of ligand, the ligand density on the solid support, the backbone material of the stationary phase, the hydrophobic nature of the protein, and the type of salt added to the mobile phase. During the separation, a negative salt gradient (typically from 2–3 M to 0 M) is applied under aqueous mobile phase at around pH 6.8–7.0. The structural damage to the biomolecules is therefore minimal and its biological activity is maintained (2).

	Analytical-scale HIC columns are based either on silica or polymer particles. Both porous and nonâporous particles are available. Highly cross-linked nonâporous poly(styrene–divinylbenzene) (PS/DVB) and polymethacrylate-based particles are frequently used in protein separations as a result of their advantageous mass transfer properties (the main contribution to the band broadening of large biomolecules, namely transâparticle mass transfer resistance is negligible). Table 1 summarizes the most widely used and the latest HIC columns applied for mAb and ADC separations.

	These materials can now withstand pressure drops of up to 100–400 bar. Columns are typically packed with 10-, 7-, 5-, 3-, and even 2.5-µm particles. Column diameters between 2 mm and 8 mm are available but 4.6-mm i.d. columns are the most widely used in current HIC applications. It is worth mentioning that there is a need for 150 × 2.1 mm column formats, which are often applied for the analysis of proteins in modern chromatographic practice.

	HIC allows both the characterization of the distribution of drug-linked species and the determination of average DAR of ADCs (3). Conjugation of the drug-linker to the antibody increases the hydrophobicity; therefore HIC appears as a suitable tool to separate the different DAR species. A good example of the HIC profile of a native IgG1 ADC is shown in Figure 1 (4).

	Recently an off-line mass spectrometric (MS) detection was applied for the characterization of Brentuximab-vedotin. Each individual HIC peak was collected, buffer exchanged, and analyzed by native MS (5). HIC was also successfully applied for monitoring various postâtranslational modifications, including proteolytic fragments, domain misfolding, tryptophan oxidation, and aspartic acid isomerization in therapeutic mAbs (6).

	Ion-exchange chromatography (IEX) is widely used for the characterization of therapeutic proteins and can be considered as a reference marker and powerful technique for the qualitative and quantitative evaluation of charge heterogeneity. Among the different IEX modes, cationâexchange chromatography (CEX) is the most widely used for protein characterization (7).

	Two modes of elution are often applied for protein characterization, namely the (i) salt-gradient and the (ii) pH-gradient. In salt-gradient mode, solutes are eluted in order of increasing binding charge, which correlates more or less with the isoelectric point (pI) and equilibrium constant. In this case, the mobile phase pH is kept constant, while the ionic strength is continuously increased. In pH-gradient mode, the ionic strength is kept constant and the pH is varied during the gradient programme. This mode of elution is often referred to as chromatofocusing.

	Regarding the stationary phase, there are two main aspects that need to be considered for successful IEX separation: (i) the strength of interaction and associated retention (strong or weak ion exchanger) and (ii) the achievable peak widths (efficiency) (8). Both cation and anion exchangers can be classified as either weak or strong exchangers. Weak cation exchangers are comprised of a weak acid that gradually loses its charge as the pH decreases (for example, carboxymethyl groups), while strong cation exchangers are comprised of a strong acid that is able to sustain its charge over a wide pH range (for example, sulphopropyl groups). On the other hand, strong anion exchangers contain quaternary amine functional groups, while weak anion exchanger possesses diethylaminoethane (DEAE) groups. As a rule of thumb, it is preferable to begin the method development with a strong exchanger, to ensure that a broad pH range can be worked on. Strong exchangers are also useful if the maximum resolution occurs at an extreme pH. However, silica-based ion exchangers can only be operated in a restricted pH range. In contrast, polymeric ion exchangers can be used over a wide pH range.

	Commercially available IEX columns are based on silica or polymer particles but organicâpolymeric monoliths are also available. Both porous and nonâporous particles are available but for large molecules, which possess low diffusivity, non-porous materials are clearly preferred. Highly cross-linked nonâporous PS/DVB materials are most frequently used in protein separations because of their high pH stability (2 ≤ pH ≤ 12). These materials can now withstand pressure drop of up to 500–600 bar in some cases. Columns packed with 10-, 5-, or 3-µm non-porous particles are often used, but sub-2-µm materials are also available to perform UHPLC separations (see Table 1). Suitable peak capacity can be attained with large biomolecules on those columns within a reasonable analysis time (for example, 15–20 min). However, some limitations can be expected in terms of loading capacity and retention when applying non-porous materials.

	A recent study systematically compared the latest state-of-the-art cation exchanger columns applied for the characterization of therapeutic mAbs in pH- and saltâgradient modes (8).

	Figure 2 shows an example of the separation of four intact antibody charge variants using a 100 × 4.6 mm, 5-µm strong cation polymeric exchanger column packed with nonâporous particles and a 20-min long gradient (9)

	Size-exclusion chromatography (SEC) is a powerful technique for the qualitative and quantitative evaluation of protein aggregates. The main advantage of SEC is the mild mobile phase conditions that permit the characterization of proteins with minimal impact on the conformational structure and local environment.

	SEC separates biomolecules according to their hydrodynamic radius. The stationary phase consists of spherical porous particles with a carefully controlled pore size, through which the biomolecules diffuse based on their molecular size difference using an aqueous buffer as the mobile phase. Basically, SEC is an entropically controlled separation process in which molecules are separated on the basis of molecular size differences (filtering) rather than by their chemical properties (10). Therefore, retention factor (thermodynamic) in SEC is different from other chromatographic modes. Here, the thermodynamic retention factor is the fraction of the intraparticle pore volume that is accessible to the analyte (11).

	Since no retention occurs in SEC, large pore volumes (high porosity) are required to ensure appropriate resolution. Generally, this large pore volume is provided by long-and wide-bore columns. In routine SEC applications, a 30-cm column length with internal diameters (i.d.) of 7.8-, 8.0-, or 10-mm is generally employed. These SEC columns are referred to as standard bore columns. Now, several vendors offer narrow bore columns with 4.6âmm i.d. and 15-cm length that are packed with very efficient, small particles of ~3 µm. Similar separation power can be attained using these columns as with 5-µm particles in 30-cm standard bore columns, but the analysis time can be reduced by a factor of 3 to 4 (12).

	There are mainly two types of SEC packing materials: (i) silica, with or without surface modification, and (ii) cross-linked polymeric packings, which possess nonâpolar (hydrophobic), hydrophilic, or ionic character (10). The most common silica packing consists of chemically bonded 1,2-propanediol functional groups that provide a hydrophilic surface. This stationary phase blocks or reacts with many of the acidic silanol groups allowing the surface to be neutralized. Bare silica is also a suitable packing material for non-aqueous polar or non-polar organic mobile phases; however, it is not recommended with aqueous mobile phases because of the presence of active silanol sites. The latest type of silica-related packing is an ethyleneâbridged hybrid inorganic-organic (BEH) material that is currently available at particle sizes of 1.7 µm - the first sub-2-µm SEC packing - and 2.5 µm (13). Compared to regular silica packings, BEH particles have improved chemical stability as well as reduced silanol activity. The 1.7âµm BEH material can be operated at up to 600 bar.

	There have been a number of different hydrophilic cross-linked packings developed for the SEC of biopolymers. Most of these packings are proprietary hydroxylated derivatives of cross-linked polymethacrylates (10). Unusual polymeric packings for aqueous SEC include sulphonated cross-linked polystyrene, polydivinylbenzene derivatized with glucose or anion exchange groups, a polyamide polymer, and high-performance, crossed-linked agarose (10).

	Today, columns for aqueous and non-aqueous SEC applications with pore sizes of 125 to 900 Å are commercially available (14). Very fast separations of peptides, myoglobin, and insulin aggregates have been demonstrated with 1.7-µm SEC columns (15). These columns were also applied for the characterization of recombinant mAbs (13).

	Applying 1.7- and 2.5-µm particles in SEC has opened up a new level of separation performance, but it should be kept in mind that on very fine particles, the separation quality is improved at the cost of pressure (and frictional heating temperature gradients). Therefore, there is a risk of creating on-column aggregates when analyzing sensitive proteins under high pressure (> 200 bar) conditions (13).

	In reversed-phase liquid chromatography (LC), the solute retention is predominantly mediated through hydrophobic interactions between the non-polar amino acid residues of the proteins and the bonded n-alkyl ligands of the stationary phase. Compared to the HIC mode, the reversed-phase LC mobile phase typically consists of water, acetonitrile or methanol, and 0.1–0.2% trifluoroacetic acid or formic acid. The separation mechanism is based on a combination of solvophobic and electrostatic interactions, the latter being governed by the interaction of TFA with basic side chains of a few amino acids (that is, arginine, lysine, and histidine) and the N-terminus as well as ionic interactions between the positive charges at the surface of the protein and the negatively charged residual silanols (16). The efficiency of reversed-phase LC is always superior to other chromatographic modes and its superior robustness makes it well suited for use in a routine environment (17).

	Current reversed-phase LC stationary phases used for proteins analysis can be classified as silicaâbased particulate materials and organic monoliths. The pore size of particulate phases is an important factor that must be considered. For the analysis of peptides and small proteins, a pore size between 100–200 Å may be acceptable. However, porous materials with pore sizes of more than 200 Å are mandatory for the separation of larger proteins or mAbs fragments because the solute molecular diameter must be approximately one-tenth the size of the pore diameter to avoid the restricted diffusion of the solute and to allow the total surface area of the sorbent material to be accessible. An average pore size between 250–300 Å is often mentioned as the reference value for protein separations, but recently it was shown that 400 Å particles completely eliminated restricted diffusion effects for molecules up to about 500 kDa.

	The two main trends today in reversed-phase LC analysis of therapeutic proteins are the use of (i) fully porous small particles (FPPs) (sub-2-µm) and (ii) superficially porous particles (SPPs), which possess particle sizes between 3â and 4-µm.

	Columns packed with FPPs have constraints in separation speed and efficiency because of limitations in the stationary phase mass transfer, which results from the relatively long diffusion times required for proteins to cross the porous structure. Therefore, Horváth first applied the concept of SPPs in the late 1960s (18,19). They were initially intended for the analysis of macromolecules such as peptides and proteins. SPPs are made of a solid, non-porous silica core surrounded by a porous shell layer. They have similar properties to the fully porous materials conventionally used in HPLC. The rationale behind this concept was to improve column efficiency by shortening the diffusion path that molecules must travel, in addition to improving their mass transfer kinetics.

	It was recently shown that columns packed with wideâpore 3.6-µm and 3.4-µm SPPs showed significant gain in analysis time and peak capacity compared to FPPs for intact protein analysis (20,21). These wide-pore SPPs are now available with C4, C8, and C18 chemistries and can be operated up to 600 bar. Figure 3 shows an example of fast separation of heavy-chain (Hc) and lightâchain (Lc) variants of an IgG1 mAb performed on a wide-pore C4 SPP column.

	In another study, efficiency and analysis times of 1.7âµm SPPs and FPPs were compared for peptides and moderate size intact proteins (22). This study suggests a twoâfold increase in terms of achievable peak capacity and analysis time for large proteins when using SPPs compared to FPPs of the same size. For the separation of peptides and moderate size proteins, a 160 Å SP packing was also introduced (23,24). Recently 1.3- and 1.6-µm SPPs were also applied for peptide mapping of mAb samples (25,26). By combining long columns
	(200–300 mm) with extended analysis time, peak capacity around 1000 can be reached with 1.3âµm SPPs for 0.5–2 kDa peptides.

	An alternative, carbonânanoâdiamond based C18 superficially porous material was recently introduced (27). The core of this material is a carbonized poly(divinylbenzene) particle with a diameter of approximately 3.4âμm. Poly(allylamine)-nanodiamond heteroâlayers are deposited onto the surface of the carbonized core by a modified layer-by-layer method. The resulting core-shell is synthesized to a shell thickness of ca. 0.1 μm and a finished particle size of 3.6 μm. This superficially porous carbon-based material was successfully applied for real life protein separations.

	Another interesting alternative to SPPs was proposed by Hayes et al. (28). The so-called sphereâonâsphere (SOS) approach provides a simple and fast one-pot synthesis in which the thickness, porosity, and chemical substituents of the shell can be controlled by using the appropriate reagents and conditions (29). SOS particles have been shown to be microporous with a pore diameter of less than 2 nm. However, while the surface of the material might not exhibit significant porosity, when packed into a HPLC column the spaces between surface nanospheres provide superficial macroporosity. It has been proposed that for large molecules, larger pores as well as reduction of the shell thickness can be advantageous, because of the shorter diffusion distance and greater access to the surface area of the material (30). SOS particles were demonstrated to have similar chromatographic performance compared to commercial SPP materials (28). Figure 4 shows the separation of reduced ADC (Brentuximab-Vedotin) fragments on a column packed with SOS particles.

	As an alternative to particle-based stationary phase formats for the LC separations of proteins, organic polymer-based monoliths offer some advantages, including high permeability and rapid mass transfer (31). Polymeric monolithic stationary phases have shown great potential for the reversed-phase LC separations of large biomolecules, including intact proteins, oligonucleotides, and peptides. With this material, the mass transfer is mainly driven by convection, rather than diffusion, because of the absence of mesopores (32). The fact that the solvent is forced to pass through the macropores of the polymer because of pressure leads to faster convective mass transfer compared to the slow diffusion process into the stagnant pore liquid that is present in porous beads packed columns. As a result of their open channel structure, monoliths generally possess a high permeability, allowing the application of elevated flow rates at moderate back pressure. It was previously demonstrated that polymeric stationary phases led to superior performance over silicaâbased materials in the reversed phase analysis of very large proteins (MW > 50 kDa) (33).

	There is always a need to use several chromatographic methods to draw reliable conclusions regarding the quality of biopharmaceuticals. IEX, SEC, and HIC are historical techniques and are still used in any laboratory dealing with the analytical characterization of mAbs or ADCs. These techniques were known to offer poor resolving power, which is why the stationary phases employed in IEX, SEC, and HIC have strongly evolved over the last few years, in terms of chemistries, dimensions, and chemical stability.

	The most important improvements for protein analysis were brought to reversed-phase LC materials. In the past, this technique has rarely been used for biopharmaceutical characterization. However, because this is the only chromatographic approach directly compatible with MS, providers have improved and developed their existing materials. The performance that can be achieved today with columns packed with wide pore sub-2-µm fully porous or sub-4-µm superficially porous are highly competitive, and even if the selectivity of reversed-phase LC is still limited for separating charge or size variants of proteins, this is (at least partially) compensated by the high kinetic performance generated by modern reversed-phase LC columns.

	The authors acknowledge Alain Beck (Pierre Fabre, Saint-Julien Genevois, France) for providing mAb and ADC samples, and Stephanie Schuster (Advanced Materials Technology) and Tony Edge and Richard Hayes (Thermo Fisher Scientific) for providing stationary phases.

	Davy Guillarme wishes to thank the Swiss National Science Foundation for support through a fellowship to Szabolcs Fekete (31003A_159494).

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 holds a PhD degree in analytical chemistry from the Technical University of Budapest, Hungary. He worked at the Chemical Works of Gedeon Richter Plc at the analytical R&D department for 10 years. Since 2011, he has worked at the University of Geneva in Switzerland. He has contributed 70 journal articles and authored book chapters. His main interests include liquid chromatography, column technology, pharmaceutical, and protein analysis.

 is professor at the School of Pharmaceutical Sciences, University of Geneva, Switzerland. He has also acted as President of the School of Pharmaceutical Sciences, Vice-Dean of the Faculty of Sciences, and finally Vice-Rector of the University of Geneva. His research domains include development of separation techniques in pharmaceutical sciences, and, more precisely, the study of the impact of sample preparation procedures in the analytical process; fundamental studies in liquid and supercritical chromatography; separation techniques coupled with mass spectrometry; and analysis of drugs and drugs of abuse in different matrices. He has published more than 300 articles in peer-reviewed journals.

 holds a PhD degree in analytical chemistry from the University of Lyon, France. He is senior lecturer at the University of Geneva in Switzerland. He has authored 140 journal articles related to pharmaceutical analysis. His expertise includes HPLC, UHPLC, HILIC, LC–MS, SFC, and analysis of proteins and mAbs. He is an editorial advisory board member of several journals including Journal of Chromatography A, Journal of Separation Science, and LCGC North America.

Cover Story

The recent trends in column technology for reversed-phase LC, SEC, IEX, and HIC for analysis of biopharmaceuticals at the protein level is critically discussed.

Modern Column Technologies for the Analytical Characterization of Biopharmaceuticals in Various Liquid Chromatographic Modes

Issue PDF

Click the title above to open the LCGC Asia Pacific March 2016 regular issue, Vol 19, No 1, in an interactive PDF format. 

LCGC Asia Pacific-03-08-2016