Gas chromatography (GC) uses a wide range of gases including helium, hydrogen, and nitrogen as the carrier gas. In the U.S., the most common carrier gas is helium, as it provides good separations, is inert, is readily available, and it is safe to use. In many other countries, helium is less available or is very expensive, and hydrogen is commonly employed. In recent years, the availability of helium has decreased and its cost has increased significantly, so many chromatographers have considered switching to the use of hydrogen (1). In this article, we compare the use of helium and hydrogen in GC and discuss the advantages of hydrogen. In addition, we will discuss how a chromatographer can obtain hydrogen in a convenient, safe, and economical manner to meet the needs of the laboratory.  

Helium is a minor component of natural gas formed by the natural breakdown of uranium. Fractional distillation of natural gas allows for the purification of helium from the natural gas. The largest helium concentrations found in natural gas are in Texas, Oklahoma, and Kansas. Sites outside the U.S. with high concentrations of helium are in Algeria and Qatar.  

Helium has many uses and applications. These include (by order of size) cryogenic cooling (28%), tank purging (26%), welding cover gas (20%), as a controlled atmosphere (13%), leak detection (4%), and for breathing mixtures (2%) (2). The use of the gas as a carrier gas for GC falls under a miscellaneous category and is a relatively small fraction of the available supply. Many larger users of helium are seeing considerable growth in their use of the gas and will need even larger quantities in the future.  

Helium is a critical and national resource for the U.S. During World War II, huge caverns were sealed and used to store the helium, creating the USA Helium National Reserves. In 1995, the government decided to allow the sale of 600 million ft

  of helium between January 1, 2005 and January 1, 2015 (The Helium Privatization Act of 1996- Public Law 104-273) (3). Sales are ongoing and continue in the U.S. and to foreign countries, leading to reduced supplies and much higher prices for helium. In addition, breakdowns in manufacturing facilities have also reduced available supplies (4). The end result of the increase in demand and the decrease in supply has required the withdrawal of the gas from the national reserves and there are significant concerns about the ability to obtain helium on a reliable basis. Many users worry more about the ability to obtain helium when they need it than the increased price. 

Hydrogen and helium are both very efficient gases to use for GC and chromatographers can switch from one to the other with little to no difficulty. While the nature of the separation is the same with the two gases, the differences in the properties of the two gases will lead to differences in the efficiency of the separation. A plot of the efficiency of a column is a van Deemter (4) (packed column) or Golay (5) (capillary column) plot that depicts the efficiency of a column versus different operating velocities of various carrier gases. The van Deemter equation (equation 1) is used to calculate the height equivalent to a theoretical plate (HETP). It describes the column efficiency or a measure of its ability to separate peaks. The desired HETP value would be the smallest value obtainable.   

 µ = linear velocity of carrier gas (mobile phase) 

 = A constant that accounts for the effects of "eddy" diffusion in the column. (The 

 term is not used with capillary columns because there is only one flow path and no packing material in a capillary column) 

 = A constant that accounts for the effect of molecular diffusion of the vapor in the direction of the column axis 

 = A constant proportional to the resistance of the column to mass transfer of solute through it. 

Figure 1: Plot of HETP versus linear velocity (5).  

Figure 1 shows a plot of HETP versus linear velocity and depicts the areas of the plot that represent the 

 = Retention time in seconds of a nonretained compound (typically methane).  

 term in the van Deemter expression defines the slope of the curve after the minimum (or bottom) or lowest HETP value. It is this slope and the minimum that displays the difference between the use of hydrogen and helium.  

From an operational perspective, the HETP values are determined experimentally at various carrier gas velocities. A plot of HETP versus the linear velocity is generated using equation 3 and equation 4.  

 = Retention time of the peak being measured 

Measure the peak width for packed columns at the base line and for capillary columns at the half height of the peak.  

The chromatographer would desire conditions that provide the smallest value for HETP, as this would provide the accumulation of the most plates or optimize the separation power within a column. When looking at the typical plot of HETP versus the linear gas rate (LGR), there is not much difference between the minimum value for the HETP for helium and hydrogen, but there is a large difference for the average linear velocity or linear gas rate values obtained for the HETP minimum for each gas. When using helium with typical capillary columns, the usable range of LGR is 20–30 cm/s, while for hydrogen, we see a much wider range of LGR of 25–65 cm/s. The slope of the HETP versus LGR curve for hydrogen (C term) is smaller than that for helium and shows only a 25% reduction in the optimum HETP value over the 25–65 cm/s range. 

Figure 2: Typical plot for various carrier gases with capillary columns (6).  

When you switch from helium to hydrogen, there are two options:  

Choose to duplicate your analysis using hydrogen with little to no loss of efficiency.

Choose to take advantage of the higher LGR available with hydrogen to speed up your analysis as the higher linear velocity used for hydrogen leads to shorter retention times and a shorter analysis. 

Comparing the Use of Hydrogen and Helium at the Same LGR:

 The chromatograms in Figure 3 compare the separation of a complex sample of bacterial acid methyl esters on an Equity-1 column with a linear velocity of 25 cm/s for both helium and hydrogen. Notice that at the same LGRs that even with this complex sample the retention times for these components are about the same.  

Figure 3: Separation of bacterial acid methyl esters (6).  

The chromatograms shown in Figure 3 shows the separation of bacterial acid methyl esters for helium and hydrogen with the helium being at the optimum LGR for helium and the run for hydrogen also being at 25 cm/s but not at its optimum (hydrogen is the top chromatogram and helium is the bottom chromatogram.) The run shown for hydrogen shows a total run time of 19.5 min. What would this run look like at the optimum of 50 cm/s with hydrogen?  

The chromatogram in Figure 4 is the same column run at 50 cm/s using hydrogen at the same program rate as used in Figure 3. The total run time is at 16 min. Though there is an improvement in the reduction of the run time, you do not see a great reduction. This is because this separation is very temperature sensitive and needs a higher temperature program rate to speed it up. To obtain a faster separation you would increase your temperature program rate by close to twice that used for the run in Figure 3. (See later in this article for temperature program concerns.)  

Figure 4: Separation of bacterial acid methyl esters using hydrogen (50 cm/s) (6).  

 The use of hydrogen provides the chromatographer with a number of benefits:  

Increased speed: Increasing the linear flow rate allows for shorter run times, thereby increasing the throughput of a laboratory.

Achieve lower temperature separations: At the faster elution times, it might not be necessary to increase the column temperature run rate. You might be able to lower the maximum temperature needed for the analysis or remain at those temperatures for shorter periods. 

Longer column life: Lower temperatures lead to less column bleed and can provide longer column life. In addition, hydrogen is a reducing gas and can remove potential acidic sites inside of the column. The removal of these sites leads to less sample absorption and less phase breakdown (column bleed). The result is a longer usable life for the column. 

Environmental concerns: Hydrogen is available readily via the electrolysis of water and is not a critical national resource. In contrast, helium is a by-product of natural gas or petroleum production and there are environmental concerns with the production and purification of the gas. Hydrogen is a green gas in that its production does not contribute to environmental pollution. 

Articles

Article

The authors explain the operational considerations for switching from helium to hydrogen in the separations lab, as well as discussing options.

Considerations on Switching from Helium to Hydrogen

Gas chromatography (GC) had its beginnings in the early 1950s with the famous Nobel Prize winner A.J.P. Martin and his colleague A.T. James demonstrating the feasilibility of GC (1). They provided the guidelines for separations in the gas phase but GC had its development via very smart scientists like Desty (2), Golay (3), Ettre (4), Giddings (5), and many others, who had a remarkable power of combining technologies with creativity to foresee unique solutions for separation challenges. Initially, the work started with chromatographic separations using packed-bed columns, but the level of separation power moved to another magnitude by using the capillary columns. In the early days of capillary GC, most of the work was done using metal columns, which gave many issues in efficiency and inertness, but when glass capillaries were used (Figure 1), it was possible to make highly inert and efficient columns. The internal glass surfaces could be modified relatively easily to coat nonpolar as well as polar phases using surface roughening and silane-deactivation techniques.

Despite the excellent chromatographic characteristics of glass, the practical issues of column installation were always a big problem. Because both of the column ends had to be inherently straight for 10–12 cm to fit into injector and detector, this was a challenge for every chromatographer who had to install a glass capillary column. This was a problem not only for the user, but also for the manufacturer. I remember in the early days of our column manufacturing operation at Chrompak, our biggest problem was to find a suitable way to package the glass capillary columns to ship them all over the world while keeping the straightened ends in shape for future installation. Cutting off a piece of the column, as well as straightening the ends, was an art. However, some users found a way around that problem. For certain brands of GC columns, there were already cassette systems in which the glass capillary columns were mounted in a fixed frame. At the time, this was a very practical way to solve the shipping problem and even today, such a system would be highly appreciated. 

To prevent having to straighten the ends, many columns were supplied with platinum-iridium tubing. This metal tubing is relatively inert and could be melted onto the ends of the glass capillary columns, creating flexible ends on both sides. However, there was also a challenge to straighten the platinum-iridium capillary tubing because it always tended to bend. Another challenge was to find the correct ferrule and get a good gas-tight seal. The outer diameter of the Pt/Ir tubing was 0.4–0.5 mm, considered very small in those days. Graphite ferrules had to be deformed heavily with all the risks of cold flow and resultant leaks.  

Another approach to packaging was the coating of a paraffin-film around the column. The glass column was doped in liquified "candle wax" before shipping. Of course, an application sheet was sent with the column instructing the user to first put the column into hot water to remove the candle wax. As might be expected, we received some interesting phone calls from customers who exclaimed that their GC instrument was "smoking." Indeed, they had forgotten to take off the candle wax before placing the column in the hot oven! 

The big change came in 1979 with the invention of fused silica (6). In just a few years, the glass capillary was completely replaced by the fused-silica columns, which solved a lot of the problems very simply because it was easy to use. The fused-silica column was flexible, so there was little risk for breakage. It was very easy to install as the ends were self-straightening. When column was contaminated, a piece could be cut off. Columns also could be coupled, and the surface was high-purity silica, so few impurities were present to interact with analytes. This application of fused silica was the start of a revolution in capillary GC, and the technique grew exponentially. 

A great deal of work has been done to explore the scientific applications and limitations of capillary GC. I had the opportunity to work with the former research group at the University of Eindhoven, The Netherlands (7), who did a tremendous job in laying out new concepts and foundations for high-speed capillary GC applications.  

In addition to the fused-silica capillary advancements the stationary phase chemistry developed very fast, initially with the coating techniques, but also with the development in the bonding and cross-linking of different types of phases. Most of these phases were polymeric and were based on siloxane chemistry. Even today, this chemistry is still playing an important role in the development of new phases.  

In my research work, I was personally interested in separation of volatile compounds. To separate volatile compounds using polysiloxane liquid phases, we always ended up making very thick films, which lose their efficiency rapidly because of the slow stationary phase mass transfer (that is, the resistance in the liquid phase when the component is dissolved and tries to get out). Due to their higher capacity, adsorption on solid sorbents coated onto the capillary tubing walls can separate volatile compounds without having to use thick layers. 

My interest in adsorbents started with packed-column applications. From the beginning of GC, volatile components were analyzed mainly using particulate adsorbents such as molecular sieves, porous polymers, carbon, silica gel, and alumina as stationary phases. On occasion, a combination of solids and liquids was used by coating active adsorbent surfaces with thin films of liquid phases. The adsorbents were packed into glass or metal tubes with outer diameters ranging from 1/16 to 1/4 in. (1–5.3 mm i.d.) for analytical work. Adsorption on these solid materials resulted in strong analyte interactions but packed columns were much slower and less efficient compared to the liquid coated open tubular capillary columns. 

To mimic the packed columns, these solid adsorbents had to be coated onto the internal surface of the fused-silica capillary tubing. The coating processes for adsorbents are significantly different from the polysiloxane type phases used in gas–liquid chromatography. These new adsorbent-coated capillary columns were (and still are) referred to as porous layer open tubular (PLOT) capillary columns. Physically, the PLOT columns look different as the coating of a white or black "powder" will give the column a unique appearance, as can be seen in Figure 2. The chromatography using these adsorbent solids is also remarkably different from gas–liquid phases. For instance, column overloading in gas–liquid chromatography results in a leading peak, while overloading in adsorption chromatography results in a more tailing peak as can be noted in Figure 3. Compared to liquid-coated phases, adsorbents are known to be very stable over time and they are difficult to "destroy." Liquid phase–coated columns can develop activity by hydrolysis or oxidation. In the case of adsorbents, the driving separation mechanism is already an active surface. Any additional activity created by hydrolysis or oxidation will generally have minor impact.  

Figure 2: PLOT column, with typical "yellow" appearance due to white solid coating. 

However, adsorbents have high retention for organic substances and any contamination in injector or carrier gas will accumulate on an adsorption column. For example, if PLOT columns are left overnight in flowing carrier gas steams at room temperature, in the morning they will need extensive conditioning as septum bleed and gas impurities are all trapped on the adsorbent surface.  

Figure 3: Peak shape of overloaded component in gasâliquid and gasâsolid chromatography. 

In looking at the challenge for light hydrocarbon separations, early on, alumina showed very interesting selectivity. However, the coating of alumina "particles" instead of soluble polymers was a new challenge for making GC capillary columns. Deposition of solid layers of adsorbents for PLOT column production required very stable suspensions. A very frequent problem for making such columns was always the plugging of PLOT columns after they were coated. After the adsorbent layer was deposited, it had to be dried and activated while the particles remained in place. 

The first alumina PLOT columns made showed very high retention and far too much activity. To reduce the activity, the sorbent had to be wetted with water. One way to accomplish this wetting was to have the carrier gas deliver the water in a controlled manner. A nice solution to this problem was found by using copper sulfate crystals placed in a filter cartridge. By keeping this cartridge under constant pressure and temperature, the carrier gas was "humidified" in a constant and controllable way. This gave beautiful separations for hydrocarbons, but the setup could only be used under isothermal conditions, which limited the upper hydrocarbon range. Also, the slightest variation of laboratory temperature resulted in a change of water level and thus a change of retention. 

The solution to this problem was not all that difficult: change the water for a more permanent deactivating molecule, a procedure that was accomplished based upon the work of Schneider, Frohne, and Brudereck (8), who did the work on glass capillary columns. Our group had to transfer this process from glass to fused silica. Initially, the dynamic coating process was very challenging to obtain sufficient layer thickness. After optimizing the thixotropic behavior of suspensions, this coating also was successful on fused-silica columns. 

Instead of water, the columns were permanently deactivated with potassium chloride. As a result, the alumina PLOT column could be used in temperature-programmed analysis up to 200 °C, thus allowing hydrocarbons to be eluted up to decane. We also found that column polarity could be changed by using different deactivation salts. Sodium sulfate as well as sodium molybdate also could be used, which created more polar alumina surfaces. Later, this technology also was applied in smaller internal diameter columns. At Restek (Bellefonte, Pennsylvania), we went down to 0.18 mm diameter with Rt-Alumina BOND/KCl (Figure 4). However, with smaller internal diameter columns, the chromatography became very challenging due to minimal loadability.  

Figure 4: Light hydrocarbons separated on Rt AluminaBOND/KCl. Column dimensions: 30 m Ã 0.18 mm; oven temperature: 100 Â°C. Peaks: methane, ethane, ethylene, propane, cyclopropane, propylene, acetylene, propadiene, isobutane, butane, trans-3-butene,1-butene, isobutene, cis-2-butene, isopentane, propyne, pentane, 1,3-butadiene. 

Regretfully, until now, the alumina PLOT columns have been applied mainly to hydrocarbon separations. If water, CO

 or any other polar compounds are present in the sample or carrier gas, they will be adsorbed by the alumina and the retention times for hydrocarbons will change. After commercialization of the first fused-silica alumina column, workers in the hydrocarbon field appreciated the selectivity the alumina for hydrocarbons and this type of column became available from different commercial suppliers. Investigations were started in "other" adsorption-type columns. Alumina also was investigated for use in other application fields, like the chlorofluorocarbon (CFC) ozone precursors. Alumina separates these components very well, but the activity of the column does not allow measurement of every CFC component. Some compounds showed in-column decomposition, which is not unexpected considering the high activity and the high temperatures applied. By more intense deactivation, the reactivity can be reduced, but not completely eliminated (9). 

The next challenge in PLOT capillary column technology was the application of molecular sieves targeting the separation of permanent gases. We initially looked at the zeolite chemistries and were surprised by their immense possibilities. The growing of zeolites in situ under autoclave conditions was possible, and we investigated this technology. The experiment failed as the column exploded, leaving a GC oven full of dust along with a terrible smell caused by burning the polyimide outside coating on the fused-silica capillary column! 

For the zeolite column, there was a lot of early work done by adsorption specialist Prof. Dr. Manfred Mohnke, who was working at the University of Leipzig, East Germany. At that time, the Cold War was still on and it was not easy to get in contact with scientists in the DDR. Dr. Mohnke was the developer of many adsorption columns in the early days of GC (10). However, a successful collaboration between our two labs was initiated with the goal of investigating different coating techniques for making molecular sieve (molsieve) columns. The challenge with molsieve compared with alumina is that the layers required are 10× thicker to get sufficient retention for permanent gases at temperatures above ambient (for example, 50 μm in 0.53-mm i.d. capillaries). 

Molsieve columns were developed successfully, but we ran into testing challenges. To detect permanent gases with capillary columns, we also needed detection systems that could be used. The only systems available were the thermal conductivity detection (TCD) systems that were used for packed columns but those systems had huge cell volumes and minimum flow was 10–20 mL/min. Because the molsieve PLOT columns used only 2–3 mL/min, we needed to add make up gas, but lost sensitivity as a result. GowMac (Madison, New Jersey) was one of first instrument companies who made micro TCD systems and we successfully used one for our work. Later, Hewlett-Packard (now Agilent Technologies, Santa Clara, California) developed their flipflop TCD system for capillary columns. Still, compared with flame ionization detection (FID), TCD is 50–100× less sensitive, which meant that the molsieve column needed to possess a higher loadability. For this reason, the use of smaller bore molsieve columns for laboratory applications had been limited. Things changed when the chip TCD systems were developed. Those systems have detection limits approaching 1 ppm and they can work with flow rates produced by 0.15–0.25 mm columns. 

Working with molsieves also opened a new separation mechanism in GC that is based upon size exclusion. Permanent gases are separated because they have access to the enormous surface inside the zeolite pores. Molecules that cannot enter the pores will be retained only by the outside surface, which has a much smaller surface area. Components that cannot enter the pore will be eluted faster. For example, isobutane is eluted before methane, having a retention factor of approx 3 at 30 °C. While 

-butane, because it is linear, can enter the pore and shows huge retention with a retention factor  >> 100. 

Molsieves are used widely, but have very limited application range. Similar to alumina, water and CO

 are adsorbed and need very high temperature to elute them from the zeolite PLOT columns. It would be nice to see the development of some hydrophobic molsieve materials. 

The porous polymers were widely known under the names Porapak (Waters, Milford, Massachusetts) and Hayesep (Hayes Separations, Bandera, Texas) in the packed-column era. The base material is a porous structure of polydivinylbenzene. With this polymeric material, different functional groups can be incorporated to create various selectivities. Most widely known is the "Q" type polymer. The porous polymers have interesting characteristics as they have high retention, are highly inert, and elute both polar and nonpolar compounds. Also, the porous polymers can be coated onto the fused-silica capillary columns, but the coating process becomes more challenging. Porous polymers have the habit of developing an electrical potential, making the particles quite static, especially when particle-size has to be reduced. Once the particles are in suspension, this is not an issue but after the solvent is removed, when particles are isolated where they can move, one can expect trouble. If such effects occur inside the PLOT capillary column, the coatings can become unstable and the particles can be eluted and plug the columns. When columns are transported, used in valve switching or placed a vibrating environment, the polymer particles can be released. 

To prevent particulates from reaching the detector, where they can produce spikes, or to switching valves that can be damaged, a simple solution was developed that was called a "particle trap." Such particle traps are 1–2 m long pieces of normal polydimethylsiloxane (PDMS)-coated fused-silica capillary column coupled between the column and the valve or between the column and the detector. The PDMS phase in the particle trap will act as "glue," trapping any particles that are released. The extra retention generated by the PDMS capillary column can be neglected because the retention of the adsorbent is several magnitudes higher than the liquid phase. This solution works quite well if the particle release is moderate. If particles are released by the thousands, the particle trap will also start to plug. 

There has been ongoing research in stabilization of porous polymers using stabilizers, in situ preparation of porous layers (11,12) and in situ gluing but the stability still has not been at a comparable level to bonded liquid phases. When column switching is to be used, it is recommended to use the flow (so-called Deans) switching approach, in which pressure shocks are not as big as seen with valves. Because the porous polymer PLOT columns always can have some free-particle formation during transport, it is recommended to precondition fresh columns at a flow rate 50% higher than what will be used for analysis. To build in long-term security, one can always add particle traps or other screens between the column and switching device. 

Chemically, the porous polymer PLOT columns are highly inert and they separate polar volatiles like alcohols and aldehydes as well as hydrocarbons — Figure 5 shows the use of an Rt-U-BOND column (Restek Corp.) to separate polar volatiles. Porous polymers can be modified by including vinyl-pyridine or acrylate groups to create higher polarity. As with the liquid phases, the maximum operation temperatures of porous polymers drops when the polarity of the phase is increased.  

Figure 5: Polar volatiles on porous polymer PLOT column. Column: 30 m Ã 0.53 mm fused-silica Rt-U-BOND, df = 20Î¼m; oven temperature: 100 Â°C; carrier gas: H2; injection: split; detection: TCD. Peaks: 1 = N2 + O2 + CO, 2 = CO2, 3 = formaldehyde,  4 = water, 5 = methanol. 

Silica gel is not used widely in GC, as only few applications have been developed on this material. It remains an interesting material because it has high retention for hydrocarbons and it also can be used to separate a range of semipolar compounds. Silica gel is the perfect precolumn if low parts-per-million oxygen has to be measured in hydrocarbon stream. Normally, porous polymers could be used, but trace oxygen gets removed by the porous polymer, not by the silica gel. Commercialization of silica-coated columns like Gaspro (Supelco, Bellefonte, Pennsylvania) and SilicaPLOT (Agilent Technologies) (13), revealed that selectivity wise, the silica did very well in the C

 isomers tend to be eluted closer as also can been seen with porous polymers. An important characteristic of silica is that this material separates sulfur compounds at parts-per-billion levels. The sulfur compounds are well resolved from hydrocarbons, which made the parts-per-billion analysis of H

S and COS in propylene–propane possible, as can be seen in Figure 6. Silica-coated columns are also much less sensitive to moisture, a big advantage over alumina. However, the poor selectivity for C

 hydrocarbon isomers limits its use for light hydrocarbons. Aside from the sulfur compounds, chloro-fluoro compounds and ethers can be separated on silica PLOT columns. One of the big challenges using silica is, similar to high performance liquid chromatography (HPLC) packings, that the manufacture of reproducible silica surfaces is difficult. One of the outcomes of this challenge is the apparent variation of trace sulfur response that seems to be difficult to control among columns as well as among suppliers.  

Figure 6: Separation of trace sulfur compounds in propylene using a silica PLOT column. Column: 30 m Ã 0.32 mm CP-SilicaPLOT; oven temperature: 50 Â°C, 1 min to 120 Â°C, 10Â°C/min; detection: Antek SCD. Peaks: 1 = H2S, 34 ppb; 2 = COS, 108 ppb. (Varian application note 1293 at www.varianinc.com/cgi-bin/scanweb/scanview.) 

Active carbon is also of interest because of its low polarity and huge surface area. Like the porous polymers, it is very nonpolar, but with much higher retention and significantly lower retention compared to zeolite molecular sieves. With carbon, CO

 is eluted as a sharp peak and if layers can be built to 10 μm or thicker, CO can be resolved from air. However, thick layers of carbon are not easy to make. Also, working with these powder suspensions is very challenging because any contact will immediately give black spots (fingers as well as labware). 

There are different ways to make carbon-type PLOT columns, suspensions, pyrrolysis, or carbon nanotubes. It's all about getting enough surface. A unique application of carbon PLOT columns is the separation of acetylene from the main ethylene matrix. If impurity analysis is the goal, it is usually preferable to have the impurity eluted before the main component. The carbon PLOT column shows ideal selectivity because carbon materials are very nonpolar as can be seen in Figure 7, showing the analysis of 3-ppm acetylene in ethylene using a CarboBOND (Agilent Technologies) column.  

Figure 7: Separation of impurities in ethylene. Column: 25 m Ã 0.53 mm CP-CarboBOND, df = 10 Î¼m; oven temperature: 35 Â°C, 1 min to 180 Â°C, 10Â°C/min; sample: 3-ppm acetylene in ethylene. Peaks: 1 = methane, 2 = acetylene, 3 = ethylene, 4 = ethane, 5 = propane. (Varian application note 1433 at www.varianinc.com/cgi-bin/scanweb/scanview.) 

 values, the use of packed columns remains an interesting option. Recently, packed columns have been miniaturized, by filling 0.53-mm deactivated stainless steel tubing with adsorbents like carbon, molecular sieves, and porous polymers (14). Such columns show promise for simple fast separations that can be performed in any instrument. Figure 8 shows an example on a 1 m × 0.53 mm MXT Shincarbon (Restek Corp) micropacked column. Such columns can be coiled in small diameters and operate at flow rates of 2–4 mL/min.  

Figure 8: Separation of CO and CO2 using carbon 0.53-mm i.d. micropacked column. Column: 1 m Ã 0.53 mm MXT, Shincarbon 80/100 mesh (Restek); oven temperature: 100 Â°C, carrier gas: He, 165 kPa; split: 1:30; detection: micro-TCD. Peaks: 1 = hydrogen, 2 = oxygen + nitrogen, 3 = CO, 4 = methane, 5 = CO2. 

Special Selective PLOT Columns for Oxygenates

Several highly selective PLOT columns have been developed (CP-Lowox, OxyPLOT, Agilent Technologies) with a unique selectivity toward oxygenated compounds (13). Though the technology is proprietary, these columns have a polarity that is tremendously high. Methanol is eluted after C

, allowing oxygenated compounds to be separated nicely from a paraffin matrix (see Figure 9). As oxygenated compounds are known as "catalyst-killers" in hydrocarbon processing, the determination of trace oxygenates is a very important analysis. Using oxygen-selective columns allows the accurate determination of parts-per-billion levels of oxygenates. When used with a nonpolar precolumn in which the higher hydrocarbons are separated from the volatile fraction, one can use this setup for measuring parts-per-billion levels of oxygenates in naphtha streams.  

Figure 9: Highly selective PLOT column for oxygenated compounds. Column: 10 m Ã 0.53 mm CP-Lowox; oven temperature: 175 Â°C, 2 min to 175 Â°C, 10 Â°C/min; carrier gas: He, 70 kPa. Peaks: 1 = C14, 2 = methanol, 3 = C15, 4 = C16, 5 = C18, 6 = C20, 7 = C22, 8 = C24. (Varian application note 1363 at www.varianinc.com/cgi-bin/scanweb/scanview.) 

Column Watch

GC expert Jaap de Zeeuw explores the historical development of PLOT columns, covers details of the most popular stationary phases, and provides practical hints.

The Development and Applications of PLOT Columns in Gas–Solid Chromatography

I recently received an inquiry from a reader complaining about the difficulty he was having meeting system suitability for a liquid chromatography (LC) method that had been transferred into his laboratory. The system suitability test called for a column plate number 

 of ≤1.5. The column was a 150 mm × 4.6 mm reversed-phase column packed with 5-µm diameter particles. The mobile phase was acetonitrile–buffer, with a buffer of triethylamine adjusted to pH 5.3 with dilute phosphoric acid. He could meet system suitability with a new column, but after fewer than 50 injections, the method would no longer pass. His inquiry had to do with what he could do to improve things without changing the method so that it needed to be revalidated. 

As I look at the system suitability requirements, I'm not surprised that the method fails. Neither of the required performance criteria seem to be too demanding on the surface, but let's pull back and examine the potential for problems with this method. To do this, we have to look at what is reasonable and ideal performance for a column in terms of plate number and then how the tailing factor plays into this. The bottom line is that I think the only way out of his predicament is to live with short column life or rework the method. 

Most of us are familiar with the column plate number, or column efficiency, which we abbreviate as 

 is by drawing tangents to the sides of the peak and measuring the peak width at the baseline between where the tangents intersect, as indicated in Figure 1. Then 

 is the width at the half-height. The half-height method is more convenient, because tangents don't need to be drawn. The value of 

 obtained at the baseline and half-height should be about the same, because the tangents usually coincide with the curve at the half height. 

 which also can be referred to as the height-equivalent of a theoretical plate, HETP. This is done by dividing 

 can be useful when comparing the performance of columns of different lengths, it does not help much when comparing columns of different particle size. One technique that is used to compare column performance for columns of different particle sizes and lengths is to use the reduced plate height, 

. This is calculated by dividing the plate height by the particle diameter, 

The reduced plate height also gives us an idea of how well a column is performing relative to the ideal or theoretical performance. A very well-packed column operated under ideal conditions with well-behaved analytes, such as toluene or naphthalene, will give 

 ≈ 2. More realistically, real LC methods with real compounds will generate reduced plate heights of 3 or more. 

Figure 1: Measurement of column plate number at the peak baseline and half-height (equations 1 and 2). 

So how well would the 150 mm × 4.6 mm, 5-µm column do under ideal and realistic conditions? After rearranging equation 4, we can see that 

 = 0.015 mm. We can rearrange equation 3 to calculate that 

 = 3 case. (Be sure to keep careful track of the units when you make these calculations: micrometers, millimeters, and centimeters seem to get mixed up if you aren't careful!) Similarly, we can calculate that the value of 

 = 8000 for the system suitability test corresponds to 

 = 3.75, which should not be too hard to obtain with most compounds if we are careful. So far so good. 

In the pharmaceutical industry, peak asymmetry is calculated as the tailing factor, 

 (also called the USP or EP tailing factor based on instructions in the United States Pharmacopoeia or European Pharmacopoeia) as shown in Figure 2, where the half-widths of the peak are measured at 5% of the peak height: 

 is the back half-width. If we rearrange equation 5, we can calculate that 

 in the limiting case, where the system suitability limit is 

 = 1.5. That is, the back half-width is twice as wide as the front-half-width with a tailing factor of 1.5.  

Figure 2: Calculation of peak tailing factor at 5% of the peak height (equation 5). 

For the rest of the discussion, we'll assume that 

 is unchanged from the perfectly symmetrical peak and all the tailing comes as a result of an increase in 

. This is unlikely to be completely true, but it simplifies the discussion. 

If we want to see how the tailing factor influences the plate number, things get a little more complicated. Although we could deal with this based upon some involved calculations, perhaps the simplest way to see what is happening is to look at some typical examples. For a symmetrical peak, we assume that the shape is a Gaussian curve, but this rarely occurs in real chromatography — the peaks almost always tail a little. Peak tailing can originate from several different causes, and different causes can cause different looking tails. However, the most common way to model a tailing peak is to use an exponentially modified Gaussian curve. There is a good description of this in reference 1, and a bonus is that this book comes with a CD containing an Excel spreadsheet designed to calculate LC separations with different amounts of tailing and overlap. 

In Figure 3, I've shown three examples of tailing peaks using the method of reference 1, where 

 = 1.24 (Figure 3a), 1.42 (Figure 3b), and 1.58 (Figure 3c). I have also included an overlay of all three peaks (Figure 3d), because visual comparisons sometimes are difficult for stand-alone peaks. In Figures 3a–3c, I've drawn tangents to the peak to illustrate the intersection of the tangents with the chromatogram at the half-height.Thus, the plate number calculated from equation 1 or 2 should be equivalent; I'll use the half-height method here, because it is more convenient. You'll also notice that the tailing dramatically increases near the baseline, as is observed in real chromatograms.  

Figure 3: Peak tailing modeled with exponentially modified Gaussian peaks. Tailing factor (TF): (a) 1.24, (b) 1.42, and (c) 1.58. (d) overlay of a, b, and c. 

To compare the influence of the plate number, we need to calculate 

 with and without the presence of tailing. If we assume that the tailing is all in the back half of the peak, we can double the front-half-width and calculate 

 based upon this for the non-tailing case (

) and compare it to the full peak-width method (

). In Table I, I've summarized the results of my calculations based on measurements of the peaks in Figure 3. The plate number based on the nontailing peak is the same in each case, 

Table I: Half-height plate numbers calculated from Figure 3 

 = 1.24 peak of Figure 3a, I can't measure any difference in the front and back half-widths measured at the half-height, although this is likely a limitation of my measurement technique. In any event, it looks like a tailing factor of less than approximately 1.25 will have little influence on the plate number. But as the peak tailing increases to 1.42 or larger, we can no longer pass the plate number requirement.  

The examples of Figure 3 illustrate the interaction of the plate number and tailing factor, but are by no means absolute limits. They do, however, show how you can get into trouble if the system suitability requirements for a method are not well thought out. 

There is another, perhaps more serious problem, with the method that also deserves comment, and that is the pH of the mobile phase. The buffer comprises triethylamine and phosphoric acid. Recall from your first year chemistry class that buffers are effective ±1 pH unit from their p

 values: 2.1, 7.2, and 12.3. Note that the pH of the mobile phase (5.3) is well away from the buffering range of either of the buffer components. In other words, the "buffer" really isn't acting as a buffer. If buffering is needed to control ionization of the analyte and as a result, likely needed to help to control peak tailing, I would expect to have problems with the current mobile phase conditions.  

Another red flag for me in the method is the presence of triethylamine at all. Triethylamine was a common additive to reduce peak tailing with the older, low-purity, type-A silica columns, but is rarely used with today's higher-purity, type-B silica columns. The column mentioned by the reader was a type-B column, so I suspect that the triethylamine is not needed. This also suggests that either the triethylamine as added for good measure or because "we've always done it that way." Or perhaps the method has migrated over the years from an older column to a newer one. Although the triethylamine is not likely to cause problems, it also is not likely to be needed. And I'm a strong adherent to the KISS (Keep It Simple, Stupid) principle when it comes to mobile phases — the more stuff you put in a mobile phase, the more likely you'll have problems. 

So you can see that there are some potential problems with the mobile phase. I would be interested to see how the method performed with a real buffer in the system. One option would be to reduce the pH to the 2 < pH < 3 region, where phosphate buffers. Another option would be to use acetate (p

 = 4.8) as the buffer, because it would provide true buffering at pH = 5.3. 

While the preceding discussion might be more than you want to know about the relationship between peak tailing and plate number, I hope the illustration gives you some ideas about how to examine data to see if the requirements of the method are realistic or not. In this case, a system suitability requirement of 

 ≤ 1.5 each seemed reasonable, but in combination, the requirements may be a bit too stringent for a routine method with this column. 

Similarly, it is important to critically examine the method conditions to see if they make sense from a chemical and chromatographic point of view. In the present case, the pH of the mobile phase is outside of the buffering region of either of the buffer components, so there is little if any buffering going on. Because peak tailing is a significant problem with this method, adequate buffering is likely to be an important component of the method. 

Finally, there is a bit of a conundrum about how to proceed with this method. It appears that the method works with a new column, but it fails system suitability much earlier than one would expect with a robust method. Any changes to the method would have to be validated, creating more work than would be required to transfer a previously validated method. So one has to balance the cost of method modification and revalidation against the cost of method transfer, early failure of the column, and possibly required sample analysis repeats because of failure of system suitability during a run. If the method has a long projected lifetime, it is likely to be best to fix the problems and revalidate. 

"LC Troubleshooting" Editor John Dolan has been writing "LC Troubleshooting" for 

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

's editorial advisory board. Direct correspondence about this column to "LC Troubleshooting," 

, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

For an ongoing discussion of LC troubleshooting with John Dolan and other chromatographers, visit the Chromatography Forum discussion group at 

H.-J. Kuss and S. Kromidas, Eds. (Wiley-VCH, 2009), Ch. 4. 

LC Troubleshooting

John Dolan discusses an inquiry he received from a reader complaining about the difficulty he was having meeting system suitability for an LC method that had been transferred into his lab.

Are the Method Requirements Reasonable?

Part I of this series (1) reviewed the calculation of the resolution of two adjacent peaks, repeated here in equation 1. A version of the resolution equation commonly used in practice is given in equation 2.  

 is the peak width-at-base as measured at 13.4% of the peak height, and 

 is the peak width at it's half-height. The subscript numbers indicate which peak. Measurements of the peak retention times and widths for the purposes of determining resolution are shown in Figure 1 for a pair of peaks with 

These relationships are cast in terms of a pair of identical symmetrical peaks, but the definition of resolution does not limit the relative sizes of the peaks or their shapes. Resolution calculations are performed routinely on pairs of peaks that can be quite different in size and shape, yet the effects of disparities in peak shapes on quantitative measurements are rarely assessed. Part I of this series (1) showed that increasing the size of the first peak up to 10 times the size of the second did not materially affect quantitative measurements, as long as the peak resolution was at the "baseline" level of 1.5 or greater and a simple vertical peak boundary at the valley point — I'll call this method valley integration — delineated the first peak from the second. With a less-than-baseline resolution of 1.0, however, where the difference in the retention times of symmetrical peaks is equal to the average of their widths at base, significant measurement errors of up to 10% for the smaller peak's area were incurred for valley integration. Even greater errors were caused by either tangential or exponential skimming methods of peak integration. Thus for this limited set of examples, a requirement that symmetrical peak pairs have resolution of 1.5 or greater is well justified. 

Figure 1: Peak measurements for calculation of resolution. Peak 1 and peak 2 are identical nontailing Gaussian peaks with Ï = 4.0 s and Ï = 0.0 s. The peaks are spaced 8Ï apart (32 s), and Rs = 2.0. 			

In reality, chromatographic peaks are not truly Gaussian in nature. This symmetrical peak shape arises from purely theoretical and idealized treatments of mechanisms of retention and transport through a chromatographic system. As soon as significant dead volumes, adsorption, nonlinear mass transfer, thermal gradients, or other real-world effects occur, peak shapes depart from the ideal. Non-Gaussian peak profiles, tailing or fronting peaks, and other distortions are the rule rather than the exception. Of these, peak tailing is perhaps the most commonly encountered effect. Tailing can be simulated by the convolution of a Gaussian peak profile with an exponential decay function. Granted, this technique takes idealized symmetrical peaks and adds idealized peak tailing, and so does not represent real-world peaks any better than the purely Gaussian shapes, but it comes close and it certainly provides a useful model for discussion. 

Figure 2: Exponentially modified Gaussian (EMG) peak model. Three peaks with increasing tailing and identical areas and retention times (peak apex): (a) symmetrical Gaussian peak with Ï = 4.0 and Ï = 0.0, the same as in Figure 1; (b) same as peak 1 with Ï = 4.0; (c) same as peak 1 with Ï = 8.0. 			

Figure 2 illustrates the effect on a Gaussian peak of imposing increasingly stronger peak tailing. The degree of peak tailing is determined by an exponential decay factor τ, sometimes referred to as the exponential time constant, using an exponentially modified Gaussian peak mathematical model (2). Figure 2a shows a purely Gaussian peak, Figure 2b shows the effect of an exponential decay factor τ equal to the peak's standard deviation σ, and Figure 2c shows an exponential decay factor of twice the standard deviation. From experience, most gas chromatographers would agree that peaks which lie between the purely symmetrical shape of Figure 2a, where the asymmetry factor 

 = 1.0, and the slightly tailing shape of Figure 2b, where 

 = 1.4, would be acceptable, while the more strongly tailing peak of Figure 2c, where 

 = 2.1, might be considered as exhibiting excessive tailing. Table I lists measurements of the widths and asymmetry factors for these three peaks. In particular, it is significant that the ratio of the peaks widths at base to the widths at half-height are roughly constant. This implies that we can apply the alternative version of the resolution equation given in equation 2 for determining the resolution of slightly to moderately tailing peaks, with asymmetry factors ranging from 1.0 to 2.0. The measurement of single peak asymmetry and the effects of asymmetry on peak area determination were discussed in two previous "GC Connections" columns (3,4). Now let's consider the effects of peak tailing on the resolution of a pair of peaks with the characteristics of the peak shown in Figure 2b. 

Figure 3: Resolution and overlap of tailing peaks. Peak shapes as in Figure 2b. Resolution: (a) 1.0, (b) 1.5, (c) 2.0. 			

The resolution of a pair of peaks is defined in terms of their separation and widths. Peak tailing increases the widths of affected peaks, so to maintain a proscribed resolution level, the separation between a pair of peaks must increase in proportion to the amount of peak tailing. In the previous "GC Connections" installment the characteristics of a pair of symmetrical peaks were measured at 

 = 1.0, 1.5, and 2.0. The symmetrical peak retention time data is given here in the first part of Table II and the peak overlap data is given in the first part of Table III. These symmetrical peaks have the peak shape from Figure 2a. 

Table I: Peak measurements from Figure 2. Measurement of As is at 10% of peak height (2). 			

For comparison, the second parts of Tables II and III show how far apart the asymmetrical peaks must stand to achieve the stated resolution, and to what degree the peaks overlap when a moderate degree of tailing, with 

 = 1.4, is applied to both peaks by using the peak shape from Figure 2b. Looking first at Table II for the retention data, an increase in the second peak's retention time of 4–7 s is required, depending upon the selected resolution level, to maintain the same resolution as peak tailing increases. Thus, considering only the resolution measurement for the moment, it is evident that when designing a robust chromatographic method that anticipates a certain degree of peak tailing, the degree of separation of the peaks must be significantly greater than would be required by accommodating only nontailing peaks. 

Table II: Retention times to attain specified resolution for equal-size peaks. The symmetrical peaks are those shown in Figure 2a, and the tailing peaks are those shown in Figure 2b. The data on symmetrical peaks are from the first part of this series (1). (a), (b), and (c) refer to the parts of Figure 3. 			

But now consider the meaning of peak resolution. It is tied directly to the degree of overlap of a pair of peaks. The goal is to provide an acceptable tradeoff between area measurement inaccuracies and the degree of separation. The expectations set by the overlap of symmetrical peaks are listed in the first part of Table III. Here we can see that the overlap of a pair of symmetrical peaks with resolution of 1.5 is about 0.14%. This low overlap is the desired result of separating peaks at baseline resolution. It is the basis for the familiar guideline that baseline resolution allows for adequately accurate area measurements.   

Table III: Peak area measurements from Figure 3. Overlap is the percentage of the area of one peak that extends into the other. The data on symmetrical peaks are from the first part of this series (1). The peaks all have the same area. (a), (b), and (c) refer to the parts of Figure 3. 

When peaks start to tail, however, this assumption breaks down. The first peak in the pair of tailing peaks under consideration, when separated sufficiently to produce a measured resolution of 1.5, extends into the second peak with an area that is 7.6× greater than is obtained for the pair of symmetrical peaks at the same resolution. At a resolution of 2.0, the overlap area of the first asymmetrical peak into the second is about equal to the degree of overlap for the pair of symmetrical peaks at a resolution of 1.5. Thus, the degree of peak tailing has a direct effect on the meaning of baseline resolution. Higher resolution is required for tailing peaks to achieve the same degree of area overlap as nontailing peaks. 

For the development of robust methods, a good guideline is to require the initial resolution of critical peak pairs to be at least 2.0, and preferably higher. Then, as peaks start to tail while  columns age or inlets become contaminated with use, there will still be sufficient resolution available to achieve an acceptable separation. Providing a somewhat higher resolution also grants some leeway for drifting retention times due to aging, and it facilitates the acceptability of new columns over the lifetime of a method as column manufacturers' normal variabilities come into play. 

This installment of "GC Connections" characterized the behavior of symmetrical and nonsymmetrical peaks in terms of the effects of peak tailing on measured resolution and area overlap between peak pairs. For the moderately tailing peaks examined, greater differences in retention time are required to achieve a given resolution compared to symmetrical peaks, as might be expected. But the degree of overlap from the first peak into the second peak is greater for tailing peaks than for symmetrical peaks at the same resolution level, so additional resolution is required to achieve baseline separation of tailing peaks. The next installment will examine the effects that peak tailing has on measured areas and will take a look at some metrics other than resolution for characterization of the quality or degree of separations. 

 "GC Connections" editor John V. Hinshaw is senior Research Scientist at Serveron Corp., Hillsboro, Oregon, and a member of LCGC's editorial advisory board. Direct correspondence about this column to LCGC, Advanstar Communications, 485 Rt. 1 S, Bldg F, 1st Floor, Iselin, NJ 08830, or contact the author via e-mail:  

Data Analysis and Signal Processing in Chromatography

 (Elsevier, Amsterdam, The Netherlands, 1998). 

GC Connections

In Part II of this series, John Hinshaw covers the effect on tailing peak measurements when the peaks start to merge, following on from Part I, which discussed nontailing peaks.

When Peaks Collide, Part II

In the early days of high performance liquid chromatography (HPLC), detection for qualitative or quantitative analyses often was carried out by collecting fractions and analyzing them off-line using gravimetric or wet chemical techniques. It wasn't until the 1940s and 1950s that the first online detectors for liquid chromatography, the refractive index (RI), and conductivity detectors appeared on the scene (3, 4), and while they were certainly an improvement over off-line approaches, neither detector was particularly sensitive. The search for more sensitive universal detectors for HPLC (much like flame ionization detection [FID] for gas chromatography [GC]) over the years led researchers to adapt GC detectors for use in HPLC (5–8), but the removal of the HPLC mobile phase through evaporation originally limited any real applicability. However, in the 1960s, the first ultraviolet (UV) detector for HPLC was introduced (9), and subsequent improvements in design led to better sensitivity (10) and improvements such as variable wavelength and diode array UV detectors. While a truly universal HPLC detector with the kind of sensitivity achieved in GC–FID is still elusive, many different types of detectors have been developed since the early UV, RI, and conductivity detectors that have been very successful for a wide variety of general or specific HPLC applications. 

			Detectors for HPLC are designed to take advantage of some physical or chemical attribute of either the solute or mobile phase in the chromatographic process in one of four ways (2):									

A bulk property or differential measurement

Bulk property detectors are the most universal detectors for HPLC as they measure properties common to all analytes by measuring differences in the mobile phase with and without the sample. One of the most common bulk property detectors is the RI detector. Given the universal nature of bulk property detectors, they respond to all analytes, placing more emphasis on the selectivity of the chromatographic column. They are, however, inherently somewhat limited in sensitivity because they are the chromatographic equivalent of determining the weight of a sailor by weighing the battleship before and after the sailor departs for shore leave. 

Analyte-specific property detectors respond to a characteristic that is unique to an analyte. The UV detector is the most common example of an analyte-specific property detector, responding to analytes that absorb UV light at a particular wavelength. UV detectors are usually thought of as somewhat specific, responding only to compounds with chromophores, but at low UV wavelengths (<210 nm), where just about every organic compounds absorb, UV detectors are actually somewhat universal. Other analyte specific detectors include fluorescence, conductivity, and electrochemical. 

Mobile phase modification detectors change the mobile phase postcolumn to induce a change in the properties of the analyte — for example, by creating particles suspended in a gas phase. Evaporative light scattering and corona discharge detectors fit into this category. Pre- or postcolumn derivatization of the analyte also is considered to fit into this category.  

Table I: Desired detectorcharacteristics 

Hyphenated techniques refer to the coupling of a separate independent analytical technology to an HPLC system. The most common is mass spectrometry (LC–MS), and technologies such as infrared spectrometry (LC–IR) and nuclear magnetic resonance (LC–NMR) also have been used. While it can be argued that MS is seeing more and more use as a routine HPLC detector, good reviews of its use as well as other hyphenated techniques are available and can be consulted for more information (11–13). 

Table II: Example detector selection criteria 			

There are many characteristics to consider when choosing a detector, and Table I lists some of them. For additional details on many of the HPLC detectors addressed here, the reader is encouraged to consult the references in each individual section along with additional general reviews (14–16).  

Table III: Common HPLC detector properties 			

Because no one detector has all of these characteristics, over time, a multitude of detectors have been designed, produced, and sold to answer one particular challenge or another. Ease of use, predictability, and reproducibility are all very important characteristics, however, recently, there has been an increased emphasis on the flow-cell contribution to band broadening and faster detector responses. This emphasis is due to new, low dispersion ultrahigh-pressure liquid chromatography (UHPLC) systems designed to take full advantage of sub-2-µm particle size column packings (17,18). A common rule of thumb says that for good peak integration, a minimum of 20 points should be collected across the peak. With the chromatographic efficiency of sub-2-µm particle size column packings often resulting in peaks that are less than 1-s wide, faster data rates are required to maintain good integration, sensitivity, and resolution. Narrower peaks also will require smaller volume detector flow cells to maintain peak concentration, and this lower dispersion shouldn't come at a loss of signal. Many detectors, including UV, photodiode-array (PDA), fluorescence, evaporative light scattering, and charged aerosol detectors are available commercially in both HPLC and UHPLC version depending upon the application. Some additional considerations to be made when selecting a detector are summarized in Table II. Most of the considerations in Table II are relatively straightforward; however, the use of complementary or orthogonal detectors used in combinations (series or parallel, depending upon back pressure limitations and whether or not they are destructive) should be noted as it is gaining in popularity, particularly in drug discovery or other screening-type applications. Table III lists some properties of the common HPLC detectors in use today, and a summary of some of their key attributes is presented in Table IV. 

Table IV: Common HPLC detector attributes 			

The UV–vis absorbance detector is the most common HPLC detector in use today because many compounds of interest absorb in the UV (or visible) region (from 190 to 600 nm). Sample concentration, output as absorbance, is determined by the fraction of light transmitted through the detector cell by Beer's law:  

 is the intensity of the transmitted light, ε is the molar extinction coefficient of the sample, 

 is the pathlength of the cell in centimeters, and 

 is the molar sample concentration. UV absorbance occurs as a result of the transition of electrons from π→π*, n→π*, or n→σ* molecular orbitals; most aromatic compounds absorb strongly at or below 260 nm, compounds with one or more double bonds (for example. carbonyls and olefins) at ~215 nm, and aliphatic compounds at ~205 nm (19,20). Mobile phase solvent and buffer selection is also important for optimum UV sensitivity and linearity; UV cutoffs (the wavelength at which the solvent absorbs) become particularly significant at low wavelengths. 

There are three different types of UV detectors: fixed-wavelength detectors that rely upon distinct wavelengths, and variable-wavelength and photodiode-array detectors that rely upon one or more wavelengths generated from a broad spectrum lamp. Fixed-wavelength detectors, the backbone of early HPLC systems, are cheap and simple, but are in limited use today. The most common fixed wavelength detectors use the 254-nm output from a low-pressure mercury lamp, the reason many variable-wavelength and photodiode-array applications today still use this wavelength out of sheer habit.  

Variable wavelength detectors can be tuned to operate at the absorbance maximum of an analyte or at a wavelength that provides more selectivity. They can also be programmed to change wavelengths during a chromatographic run to compensate for response of different analytes. In a variable-wavelength detector, light from a broad-spectrum (for UV deuterium is common, tungsten for visible) lamp is directed through a slit to a diffraction grating that spreads the light out into its constituent wavelengths. The grating is then rotated to direct a single wavelength of light through a slit, through the detector cell, to a photodiode.  

Photodiode-array detectors have an optical path similar to variable-wavelength detectors except the light passes through the flow cell before hitting the grating, allowing it to spread the spectrum across an array of photodiodes. Z-path or tapered detector cells designs are commonly used in most UV detectors for HPLC. Using a 100 mm × 4.6 mm column (3-µm particle size) detector cell volumes of 8–10 µL are required (for example, 1-mm diameter, 10-mm pathlength). An 8-µL flow-cell volume can lead to unacceptable extracolumn band broadening using smaller column diameters and particle sizes, however, reducing the path length to decrease extracolumn volume will decrease the signal. In addition, early PDA detectors were not as sensitive as single-wavelength UV, but the gap has been closing in recent years thanks to new detector cell technology of the type in use in some detectors today, as illustrated in Figure 1. 

Figure 1: Schematic representation of a light guided total internal reflectance detector flow cell. (Figure courtesy of Agilent Corporation.) 			

This new detector cell type consists of a light-guided flow cell equivalent to an optical fiber. Light is transferred efficiently down the flow cell through a noncoated fused-silica fiber in an internal reflectance mode that maintains a long flow-cell pathlength with a low volume; extending the pathlength while maintaining low dispersion. Light-guided flow cells are particularly useful in maintaining low dispersion in UHPLC systems while providing increased sensitivity, as shown in Figure 2. 

Figure 2: Detector cell sensitivity comparison. An increase in signal-to-noise ratio of over 11 was obtained using UHPLC and the total internal reflectance detector cell versus the standard HPLC cell. Column: 150 mm Ã 4.6 mm, 5-Âµm Zorbax SB C18 (Agilent Corporation); mobile phase: 20:80 waterâacetonitrile; flow rate: 1.5 mL/min; sample: 5 ÂµL injection of anthracene (835 pg/ÂµL); detection: PDA (251/4 nm, Ref = 450/80 nm, 2.5Hz, slit width 4 nm). (Figure courtesy of Agilent Corporation.) 			

PDA detectors extend the utility of UV detection by providing spectra of eluted peaks that can be used to aid in peak identification, and to monitor for coelutions, helpful during method development. They can also serve as a multiwavelength UV–vis detector. The spectra collected at the chromatographic peak apex can be used to create a library that can in turn be used to compare subsequent spectra for identification purposes, and spectra collected across the peak at each data point can be compared to evaluate peak homogeneity or purity.  

Fluorescence detectors measure the optical emission of light by solute molecules after they have been excited at a higher energy wavelength, and they can be very sensitive for compounds that have native fluorescence or that can be made to fluoresce through derivatization (13). Schematically, they resemble UV detectors, except that the grating is replaced by a filter or monochromator at a right angle to the incident light to simplify the optics and reduce background noise. The light source is usually a broad spectrum deuterium or xenon flash lamp. The excitation wavelength (often close to the UV λ

) is selected by a filter or monochromator between the lamp and the flow cell, always at a higher energy (lower wavelength) than the emission wavelength. Laser-induced fluorescence (LIF) detectors using lasers as the excitation source are sometimes used in micro- or capillary-LC systems in which the higher energy of the laser provides better sensitivity in the small diameter flow cells necessary to limit dispersion. 

Figure 3: Example of fluorescence detection for the UHPLC analysis of a series of polycyclic aromatics.  Column: 50 mm Ã 4.6 mm, 3-Âµm SUPELCOSIL LC-PAH; temperature: 40 Â°C; mobile phase A: water, B: acetonitrile; gradient: 50% B (0â0.5 min), 50â88% B (0.5â3.0 min), 88% B (3.0â4.2 min), 100% B (4.21â4.5 min), 50% B (4.51â5.0 min); flow rate: 3.0 mL/min; excitation/emission wavelengths switched in each interval as follows: A, 270-nm excitation wavelength, 330-nm emission wavelength; B, 250-nm excitation wavelength, 370-nm emission wavelength; C, 330-nm excitation wavelength, 430-nm emission wavelength; D, 270-nm excitation wavelength, 390-nm emission wavelength; E, 290-nm excitation wavelength, 430-nm emission wavelength; F, 370-nm excitation wavelength, 460-nm emission wavelength; G, 270-nm excitation wavelength, 330-nm emission wavelength. (Figure courtesy of Shimadzu Corporation.) 			

The principles and attributes of today's HPLC detectors are discussed, and the advantages and disadvantages of the various detectors are compared and contrasted.

LCGC North America-10-01-2010

Considerations on Switching from Helium to Hydrogen

The Development and Applications of PLOT Columns in Gas–Solid Chromatography

Are the Method Requirements Reasonable?

When Peaks Collide, Part II

Seeing is Believing: Detectors for HPLC

The LCGC Awards: Opening Doors

Vol 28 No 10 LCGC North America October 2010 Regular Issue PDF