Trends in HPLC Column Usage

December 28, 2009

LCGC North America

In this installment of "Column Watch," Ron Majors looks at the results of a recent HPLC column survey conducted by LCGC North America.

Occasionally, LCGC North America surveys its readers to obtain a current profile of users of high performance liquid chromatography (HPLC). The last survey on HPLC columns was conducted in 2007 (1). In the past, I have used results from these surveys to chart trends in column technology and in the practice of HPLC. In mid-2009, a web-based survey was conducted by LCGC North America. The survey was sent to subscribers whose primary chromatography technique was HPLC. The total number of subscribers who were sent the survey totaled 7428 and of those, 322 readers responded for a 4.3% response rate. This number of respondents was statistically sufficient to allow comparisons to previous survey data. The maximum statistical error at the 95% confidence level was 5.3%.

To ensure that the results of the current survey were compatible with those of previous surveys, I used the same methodology to report the results. Because many of the questions allowed respondents to give more than one answer, in some cases, I normalized response totals. Normalizing the results to a base of 100% makes it easier to compare the results of previous surveys with those of the present survey and to identify trends in the use of HPLC columns, modes, and packings. Questions pertaining to mode usage, column life, particle size usage, purchasing considerations, and possible future needs were explored to understand selection criteria.

Instrumentation Used

To understand the current usage rate of instruments, a question was asked pertaining to the number and type of instruments used per respondent. Table I shows that the average response for each category of instrument. The numbers reported should not be construed that every respondent uses every type of instrument but serves to provide an idea of the relative number of the various types of systems in use. Later, I will look at the numbers of columns used by these instruments.

Table I: Types of HPLC instruments used by survey respondents

According to the survey results, a user of conventional HPLC instruments is responsible for an average of 3.6 units. This number was derived from looking at the total number of instruments of the type identified by all respondents and then dividing this number of the total number of respondents. For example, some respondents reported that they have only one conventional HPLC system while others reported that they have five instruments for which they are responsible. Based upon the relative numbers of Table I, for every one of these respondents who uses a conventional LC system, only 1 in 9 would possess a capillary or nano LC system, 1 in 4 might have an instrument capable of working with microbore columns, and 1 in 5 could have a preparative instrument in his or her laboratory. These numbers are up from the 2007 survey (1), which could mean that through mergers and layoffs, there are more LC units available for those remaining in the laboratory or that laboratory managers responsible for multiple instruments and chemists who completed the survey skewed the results. According to the demographics, about 1/6 of the respondents reported that their job title was laboratory manager.

Mode and Phase Usage

Most liquid chromatographers use more than one chromatographic mode to solve specific separation problems. The total number of techniques used by respondents relative to the number of respondents revealed that each used 3.4 modes on the average, more than the results from the last survey (1). The normalized data in Table II compares the relative mode usage in 1997, 2007, and 2009. No surprise that the results again show that reversed-phase chromatography was the most popular mode; according to the raw data, over 94% of respondents use this mode, with normal bonded phase usage a distant second (42%).

Table II: Analytical HPLC mode and stationary phase usage

Although the data in Table II shows that reversed-phase chromatography has dropped on a relative basis, this drop is not due to its decrease in popularity, which remains extremely high, but rather from the increased use of other modes such as adsorption and hydrophilic interaction chromatography (HILIC). HILIC, where respondents indicated an 80% increase in usage since 2007, is a separation technique for highly polar analytes that gets around some of the problems associated with reversed-phase chromatography such as low retention or phase collapse (dewetting). HILIC uses a polar stationary phase such as bare silica gel or polar bonded phase (for example, diol) and requires a high percentage of a nonpolar mobile phase, usually acetonitrile, similar to the requirements for normal-phase chromatography. However, unlike normal-phase chromatography, which uses nonpolar solvents like hexane and methylene chloride and tries to exclude water from the mobile phase, HILIC requires some water in the mobile phase to maintain a stagnant, enriched water layer on the packing surface into which analytes may selectively partition. In addition, water-miscible organic solvents are used. Under HILIC, polar analytes are well retained and are eluted in order of increasing hydrophilicity. The unusually high increase in the use of adsorption chromatography from the historical usage level of 1.2–1.7% (1,2) of the respondents to 6.4% in the present survey undoubtedly reflects the increased use of bare silica columns for HILIC, even further strengthening the growth of this mode since the last survey. Bare silica is still the most widely used HILIC phase but the current survey did not explore its use for HILIC applications.

With regulatory bodies requiring evidence of enantiomeric purity, it is no surprise that use of chiral chromatography has increased by 50% in the last decade. Newer chiral columns appear to be more universal and some can be used in the reversed-phase and polar organic modes. Supercritical fluid chromatography (SFC), which employs columns similar to those used in HPLC, also has seen renewed interest, especially for preparative chiral separations. The use of chiral columns in SFC was not explored in the present survey.

The use of normal bonded phases continues to have a good level of application. This mode is favored over adsorption chromatography for the separation of geometric and positional isomers, cis–trans compounds, and other separations using organic mobile phases. Reversed-phase chromatography does not do a good job on certain isomeric compounds. The phases used in this mode such as diol, amino, and especially cyano are sometimes used with aqueous solvents also. Except for cyano phases, the survey did not solicit information about the uses of these stationary phases in aqueous versus organic mobile phases.

The use of ion-exchange chromatography showed a slight downward swing in usage and is back to its historical levels of the mid-to-late 1980s (3). Included in this usage is ion chromatography, which is sometimes treated as a separate separation technique but in reality is ion-exchange chromatography because it employs the same separation principles and uses columns with ionic or ionizable functionalities. Ion-exchange chromatography is frequently used in proteomics, where large numbers of tryptic peptides are fractionated by 2D LC first with ion exchange followed by reversed-phase LC–MS for separation and detection. Although ion-pair chromatography also separates ionic and ionizable compounds, it is generally lumped with reversed-phase chromatography because it employs the same packing materials. Many chromatographers familiar with reversed-phase chromatography prefer to use ion-pair chromatography for ionic and ionizable compounds because the columns are more familiar to them and the principles of reversed-phase chromatography generally can be applied in method development.

The normalized data in Table III breaks the reversed phases into individual organic moieties and clearly shows that C18 (octadecylsilane) was most popular (the raw data showed that 95% of all users use this phase at some time) followed by C8 (octylsilane). The use of phenyl bonded phase has shown continual increase as users exploit its unique selectivity compared to alkyl phases. For the first time, the survey explored the use of cyano phases in reversed-phase chromatography applications. I had suspected for a long time that many of the users of this phase treated it as a short chain alkyl phase with some polar interactions via the cyano functionality and this survey proved so. In fact, the use of cyano in aqueous solution actually was found to be 2.5 times higher than its use with organic mobile phases in the normal-phase mode. If one removes this added cyano phase from Table III, the percentages in parentheses show that reversed-phase usage has stayed constant in the last two years. In addition, I have corrected Table II to reflect the cyano columns used in reversed-phase chromatography while decreasing that percentage from the normal bonded phase category. Some newer biphenyl and diphenyl phases have appeared but, for the current survey, usage of these phases wasn't investigated.

Table III: Stationary phases used in reversed-phase chromatography

In the 2009 survey, the use of silica-based and polymer-based monolithic columns was queried. Both types of monoliths have been available commercially for a decade.

When the question of "Which of the following phases do you typically use?" was asked, 6.0% of the respondents indicated that they have used silica monoliths and 4.4% of respondents indicated that they use polymeric monoliths. Silica monoliths have been used commercially for small molecule separations while polymeric monoliths generally have been employed for larger biomolecules, although recently at the HPLC 2009 Symposium, several papers discussed the optimization of polymeric monoliths like poly(styrene–divinylbenzene) (PS-DVB) and methacrylates for the separation of small molecules (4). In a separate question pertaining to future use (in the next two years), 14% of the respondents said that they would consider trying a silica monolith while 15% indicated that they would consider polymeric monoliths. Other phases considered for future purchase by respondents not currently using them included alkyl phases with chain lengths greater than C18 (22%), HILIC columns (16%), cyano columns for reversed-phase use (12%), and phenyl reversed-phase columns (12%).

When survey respondents who indicated that they have used monoliths were asked why they use monoliths, their top three responses were lower pressure drop (69%), their ruggedness (48%), and better efficiency than their current columns (31%). Commercial silica monoliths have the efficiency of a 3–4 µm porous particle and their relative pressure drops are about 50–60% lower than an equivalent efficiency porous particulate column of the same dimensions. Evidently, these attributes are not powerful enough to convert conventional column users to monoliths now and in the future based on the results of this survey.

Particle Sizes in Analytical and Preparative LC

Since the beginning of HPLC in the late 1970s, the trend has been to use columns packed with smaller spherical particles. Throughout most of the 1970s, 10-µm irregular particles were the norm because they were available commercially and with slurry packing, column efficiency was quite good. In the late 1970s, 5-µm spherical particles gave better performance due to improved mass transfer and better packing ability. Through occasional LCGC column surveys, I started tracking particle size usage in the mid-1980s. Table IV provides data of the use of various particle sizes from 1985 until 2009 (current survey). As one can see from the first survey, columns packed with 10-µm particles already had given way to 5-µm particles. These particles have maintained their dominance through the 1990s and are still in widespread use today. There are many validated methods that have been developed on 5-µm particles and users don't want to take the time to revalidate their method unless there is a strong driving force to do so. Nevertheless, as can be seen in Table IV, use of even smaller particles (3–4 µm) began in earnest in the mid-1990s and now these particles have nearly caught up to the 5-µm particles. With the reduction in particle size, column efficiency improved further, allowing a reduction in column length. Shorter columns are now in vogue (Figure 1). Not only does a shorter column provide faster separations but also, solvent use is reduced. Recently, the use of sub-2-µm particles has become fashionable in even shorter column lengths. In just two years, the use of the sub-2-µm columns has doubled. Columns of 30–50 mm in length packed with these particles now provide plate counts formerly obtained on 15–25 cm columns packed with larger particles.

Table IV: Analytical HPLC particle-size trends 1985–2009*

In the present survey, we did not query the use of 2–3 µm particles, which have attracted interest by some users. These columns are intermediate between the popular 3.0–3.5 µm columns and the newer sub-2-µm columns. They show lower pressure drops and efficiency than the sub-2-µm particles but better efficiency and higher pressure drop than the 3.0–3.5 µm particles. In the next survey, it will be interesting to see if the trend on the increased use of small particles continues.

In comparing the use of preparative chromatography to that of analytical HPLC, 55% of those performing analytical separations do not use preparative chromatography. Not surprisingly, those who perform preparative separations tend to prefer larger particles, as depicted in Table V. Reasons for the preference of larger particles are cost, lower pressure drop, and loadability. For the latter consideration, the higher efficiency of smaller particles may be lost when overloaded conditions are employed. Even so, in the present survey, there has been an increase in respondents using smaller particles for preparative work (Table V). For example, 21% of respondents use 5-µm particles for their preparative work up from 9.7% in 2007.

Table V: Particle sizes used in preparative chromatography

Column Lengths in Analytical HPLC

In this year's survey, we included a question about column lengths used in analytical HPLC. For many years, when 5-µm particles were dominating analytical work, 25-cm columns were the standard (especially with an internal diameter of 4.6 mm). We did not query the column internal diameters being used, but Figure 1 shows that 25-cm columns have given way to 15-cm columns, which are now favored. The 15-cm columns are the standard with 3.0–3.5 µm particles and certainly the increase in the use of the smaller particles is partially responsible for the shift. For the sub-2-µm particles, 50-mm lengths are the most often used and this is reflected in the percentage of respondents using these shorter columns.

Figure 1

Column Purchase Patterns

Several questions in the survey were related to the purchase and cost of analytical HPLC columns on a per-instrument basis. Most of the respondents purchase prepacked columns and very few purchase empty columns and bulk packings, presumably to pack their own. Still, nearly 90% of respondents have no budget for empty or bulk packings. After prepacked columns, the largest expenditure is for guard columns. About a third of respondents have no budget for guard columns but for those that do, the average guard column expenditures are about half of those of the average analytical column budget.

The average annual purchase budget for prepacked analytical HPLC columns per instrument was approximately $3043, about 3% higher than the last survey results (1). Based upon the number of analytical columns used per instrument per year of 5.8 (Table VI), this would amount to an average cost of $525 per column, which is reasonable. For the 2/3 of respondents who do purchase guard columns, the average budget was about $1500 per year per instrument. For the average cost of a guard column of $60, this would amount to approximately 25 guard columns per instrument per year or about five guard columns per analytical column, again not an unreasonable number. Guard columns extend the life of analytical columns; therefore, without these expenditures, users would have to allocate a higher percentage of their budget for prepacked columns.

Table VI: Types of analytical columns used by respondents

Another question broke down the purchases of different dimensions of LC columns. Users were asked about their yearly purchases of conventional analytical columns (3.0–4.6 mm i.d.), microbore columns (2.0–2.1 mm i.d.), narrowbore columns (1.0-mm i.d.), and capillary columns (less than 1-mm i.d.). Table VI shows the column units used based upon a weighted average. For conventional HPLC column users (3.0–4.6 mm i.d.), an average of 5.8 columns per instrument per year are used. Microbore columns, which also can be used on conventional analytical instruments, were about half of the usage of conventional columns. Ultrahigh-pressure liquid chromatography (UHPLC) favors the use of microbore columns due to frictional heating effects displayed by large internal diameters and pump flow rate limitations at very high pressures (1000 bar) for some UHPLC systems. One would expect narrow bore and capillary columns to have lower usage based upon the smaller number of available instruments that accommodate these smaller internal diameter columns (see Table I).

A question asked if the average price of an HPLC column had changed in the past three years, and 60% of the respondents felt that column prices had stayed the same, while 35% felt that prices had increased. Only 5% felt that column prices had decreased in the last three years. The average perceived price increase for the latter group was 22% compared to three years ago or about 7% a year increase. In reality, judging from the column budgets discussed earlier, the increase in column prices was only 1% per year.

One of the questions asked chromatographers from which source they prefer to purchase prepacked HPLC columns. According to the survey, Table VII shows that the chromatography supply houses maintained their lead but their market share dropped since 1997 and 2007. The biggest drop off in the last couple of years was for the general laboratory suppliers; This year, 24% of the users surveyed purchase their columns from this source, a drop of 20% from 2007 (1). General laboratory distributors usually provide less technical assistance than the dedicated chromatography suppliers or instrument manufacturers but they promote themselves as one-stop shopping where users can get solvents, chemicals, laboratory glassware, as well as HPLC columns. The general laboratory distributors sometimes provide additional services such as electronic ordering, local stocking, volume discounts, and on-site services. The percentage of users loyal to a single instrument manufacturer has slipped a bit in favor of consideration of purchasing columns from any instrument manufacturer, which has picked up to drop off (Table VII). According to the present survey, the biggest increase in column sources was the independent column manufacturers who had seen a substantial drop in the share of the HPLC columns' market in the last survey. Surprisingly, a few people are packing their own, perhaps driven by the faltering economy.

Table VII: Sources for HPLC column procurement

Table VIII lists, on a normalized basis, the factors that respondents considered important when purchasing an HPLC column. Respondents were asked to name their top factors that influenced their buying decision. The most important factor — ranked the same in earlier surveys — was column-to-column reproducibility. Nobody wants to develop a method only to find out that the column that they purchase later for the same application does not perform in the same manner. However, column reproducibility has definitely improved over the years, driven by competitive pressure, quality initiatives such as ISO and 6-Sigma, and a better understanding of the manufacturing processes. The next factor was column lifetime, an issue that will be explored in more depth in the next session. In this survey, taken during one of the worst economic downturns in U.S. history, price was rated the third most important factor in considering a column supplier, while the company reputation dropped to fourth place. A big jump resulted in the consideration of column hardware design. With the rising popularity of UHPLC, column design has been found to be an important consideration not only in high-pressure operation but in providing the highest efficiency and the least plugging with the new sub-2-µm particles.

Table VIII: Factors considered when selecting an HPLC column supplier

Column Lifetime

As mentioned earlier, column lifetime is an important selection and operating parameter in HPLC. This survey specifically explored column lifetime from two standpoints — lifetime in months and by the number of injections. Although some of the modern liquid chromatographs do track the number of injections, many chromatographers do not. Usually, one will install an HPLC column and keep it in the instrument performing one or more methods until the column shows a loss of resolution or shows significant tailing that may affect quantitation. They may track the time of usage in a logbook.

As can be observed in Table IX, since the earliest days, column lifetime has increased continually through the years. When I first began asking about lifetime in column surveys in 1985, 21% of the respondents had columns that lasted less than three months. Now only 14% of respondents have lifetimes of less than three months. If one looks carefully at the data of Table IX, nearly 19% of the respondents now experience lifetimes of at least a year and 29% have columns that last over a year, higher than data reported in the 1990s. About 13% of the users don't track column lifetime by month at all.

Table IX: Average analytical column lifetime for those who track change

When the question was asked about column lifetime based upon the number of injections, a larger number (25%) of the respondents reported that they do not track their injections. Table X shows the normalized data of those who do monitor column lifetime based upon the number of injections. There has been a definite trend in columns surviving more injections over the last few years, especially comparing the data from 1997 to 2009. Some of this increase in lifetime is due to chromatographers becoming more aware of the limitations of silica-based HPLC columns, using specialty high- and low-pH columns, and exploring polymeric and other column types that are more rugged.

Table X: Normalized percentage of respondents who reported on column lifetimes by the number of sample injections for analytical columns

Although the data are not shown, a large percentage of preparative column users do not track monthly column lifetime (58%) nor the number of total samples injected (70%) before discarding a column. Intuitively, one would think that preparative columns would have shorter lifetimes because they tend to be used for dirty samples, at high flow rates, and higher sample loads than analytical columns. Surprisingly, the small number of respondents using preparative columns that do track lifetime reported that nearly 50% of them last longer than a year, similar to the results of previous surveys (1,2). This greater lifetime perhaps indicates that preparative columns are used less frequently than analytical columns and therefore tend to be stored and used at a later date when the need for preparative applications arises. Another possibility for longer lifetimes would be that preparative columns are substantially more expensive than analytical columns and users may take better care of them. A third possibility is that the larger particles used in preparative separations do not generate as high a back pressure as obtained with the smaller particle analytical columns. Higher back pressure tends to limit the column lifetime because more stress is placed on the column materials.

Future Column Interests

Future column interests were explored in the current survey. When asked about newer column technologies that respondents might be trying in the next two years, Figure 2 provides a normalized graphical representation of their responses. A number of choices were provided and respondents were permitted to select multiple responses. The actual percentage total showed that an average response was for 1.7 types of columns, which implies that they are interested in trying more than one column format. In actuality, the ultrahigh-pressure and sub-2-µm columns should probably be combined as they generally go hand in hand. Thus, the newer high-pressure, sub-2-µm columns would be the columns of most interest (38% of respondents) for future use, while hybrids are in second place (23%). Hybrid materials are packings consisting of both silica and polymeric backbones. They have a wider pH range than silica and are suitable for ultrahigh-pressure applications. Such packings are available in sub-2-µm and conventional particle sizes but the relative usage was not explored in the present survey. The third highest packing type of future interest was the superficially porous particles. These particles consist of a solid core surrounded by a thin porous layer of silica to which various phases are bonded. Columns packed with these particles show efficiencies and speeds rivaling the sub-2-µm particles but with the pressure drop of a 2.7-µm spherical particle. Often, they allow older HPLC systems with less than 400-bar pressure capability to provide the efficiency of a sub-2-µm column without requiring ultrahigh-pressure capability. Of course, with the narrow peak resulting from such columns, to maintain the high efficiency, extracolumn dead volumes must be minimized and detector time constants must be able to handle the fast eluted peaks.

Figure 2


This survey has shown some changes over the time period selected. As expected, reversed-phase chromatography maintains its dominance, with 94% of respondents using this technique and C18 and C8 being the most popular phases, but phenyl continues it growth. For the first time, we asked about the frequency of use of cyano columns in the reversed-phase mode and not surprisingly, the use of this phase in reversed-phase chromatography is much stronger than in normal-phase applications. By far, the biggest growth has been in the HILIC area, which has more than doubled during the two-year period. Although not queried, the greatly increased use of bare silica columns further indicates that HILIC is becoming a well-accepted alternative to reversed-phase chromatography for polar analytes.

The use of smaller particles is evident with 3.0–3.5 µm particles gaining in popularity, almost equaling the use of the always-popular 5-µm porous particles. The use of sub-2-µm columns has doubled since the last survey, coinciding with the acceptance of UHPLC. These smaller particles usually are packed in shorter columns and run at higher flow rates, but longer columns dictate the higher pressures of UHPLC.

Column purchasing questions revealed that column prices have increased only 3% in the last two years while usage for analytical columns has grown slightly to an average of 5.8 per conventional analytical instrument per year. The general laboratory distributors have lost a portion of the market taken up by the independent HPLC column producers. Column-to-column reproducibility is still the top factor in the buying decision, but price has risen as a bigger factor in the present economic climate.

Column lifetimes have remained strong and continue to increase; the increased use of guard columns and more stable phases has helped. Future column interests include the hybrid (organic–inorganic polymers), superficially porous particles, sub-2-µm, and high-pressure packings to go with the newer UHPLC system coming onto the market. Silica and polymeric monoliths continue to get minimal attention.

Ronald E. Majors

Ronald E. Majors

"Column Watch" Editor Ronald E. Majors is business development manager, Consumables and Accessories Business Unit, Agilent Technologies, Wilmington, Delaware, and is a member of LCGC's editorial advisory board. Direct correspondence about this column to "Column Watch," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail


(1) R.E. Majors, LCGC North America 25(6), 448–454 (2007).

(2) R.E. Majors, LCGC North America 15(11), 1008–1015 (1997).

(3) R.E. Majors, LCGC North America 7(6), 468–475 (1989).

(4) R.E. Majors, LCGC North America 27(9), 796–815 (2009).

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