There seem to be a disproportionate number of problems encountered in the concentration region near the limit of detection and lower limit of quantification with calibration curves used for liquid chromatography (LC) methods. Some of these problems relate to improperly selecting the model used for calibration. The choice of the limits of detection and quantification is an important decision that can be confusing. This month's installment of "LC Troubleshooting" is the first of a series about calibration curves. This month we will discuss some different calibration models, how to decide if a calibration curve goes through zero, and some problems that can occur if the wrong choices are made. In future columns, we will look at how to determine the lower limits of a method as well as standardization techniques: internal standardization, external standardization, and the method of standard additions. We also will look in more detail at normal errors as they relate to signal-to-noise ratio in trace analysis. 

Most LC methods are used for quantitative analysis — that is, the methods that answer the "how much is there?" question. Nearly all of these methods rely on comparison of the peak area (or less often, peak height) of a sample with that of a reference standard. To do this, we use a calibration curve (also referred to as a standard curve or sometimes "line"). The two most popular methods of calibration are external and internal standardization. In external standardization, the calibration is based upon comparing the response of the reference standard with its concentration. For internal standardization, a constant concentration of an internal standard is added to each sample and the ratio of the response of the analyte to that of the internal standard is compared with the concentration. The external-standard method is simpler and will be used for all the examples discussed here; the details of these two methods will be discussed in a future article. 

There are several different ways to use a calibration curve to quantify samples. Because UV detection is used for most LC methods, and the UV detector response vs. peak area is linear over five or more orders of magnitude, we can assume linear response is possible when using a method calibration that covers a wide range of concentrations. Some other detection methods, for example, mass spectrometry (MS) or evaporative light scattering detection (ELSD), have narrower linear ranges, so a linear calibration might not be appropriate. If a UV detector is used, we assume that the well-behaved calibration curve will be linear. Linearity is defined as  

-intercept. (Even though the relationship is linear, we still call it a curve.) When the curve goes through the origin (

 = 0, and the curve can be expressed as  

In its simplest form, equation 2 can be used for single-point calibration. In this case, one or more calibrators are injected at a single concentration and the concentration of an unknown sample is directly proportional to the response (or average response) of the calibrator or calibrators. For example, if the calibrator has a response of 100,000 area units for a 10-μL injection of a 1-μg/mL solution and the sample has a response of 95,342 area units, its concentration is (95,342/100,000) x 1 μg/mL x 0.95 μg/mL. Technically, single-point calibration should be valid whenever the standard curve behaves as in equation 2. However, it is best to inject calibration standards near the concentration of the analyte, so as to verify that the response continues to be as expected. For this reason, single-point calibration usually is reserved for samples that cluster tightly around a single concentration. A good example is a content-uniformity method to determine the concentration of a drug in a pharmaceutical product. All samples are expected to have the same concentration, so a single calibration standard is injected at the expected 100% concentration. From a practical standpoint, several calibrator injections are averaged to minimize error. Often two calibrator injections are followed by several sample injections (for example, 5–10), two more calibrators, and so forth. The four bracketing calibrators are averaged and used for calculating the concentration of the unknowns. 

Another popular application of equation 2 is to methods where the expected range of sample concentrations is narrow, for example less than one order of magnitude. In this case, a two-point calibration is used. Calibrators at two concentrations that bracket the expected sample concentrations are injected. For example, a group of samples that have a specification of ±5% from the target concentration might have calibrators formulated at 90% and 110% of the expected concentration. These would be injected and used to calculate 

 in equation 2. Each injection would have a concentration and response: (

) for the 90% and 110% concentrations, respectively. The slope would be determined as 

). (Because the range is narrow, and will have been shown to be linear in the validation, it doesn't matter if the curve passes through the origin or not.) Now unknown samples can be injected and the concentration can be determined, using the calculated value of 

The third common calibration technique is the multipoint calibration. As the name implies, several different points on the calibration curve are used to calculate the response vs. concentration relationship. Multipoint calibration is used for methods that cover a wide concentration range (for example, several orders of magnitude) and for methods where the calibration curve fits equation 1, but does not pass through the origin (

 μ 0). For example, multipoint calibration is used for methods to determine the concentration of a drug in plasma for a pharmacokinetic studies, where concentrations of <1% of the maximum concentration (

) need to be reported. Regulatory guidelines suggest using a minimum of five (1) or six (2) concentrations across the range to determine linearity of a method. Two different dilution schemes are used for selecting calibrator concentrations. For a method spanning 1–1000 ng/mL, a linear dilution might use concentrations of 1, 100, 200, 300, . . . 900, 1000 ng/mL, each differing by 100 ng/mL. A more popular scheme is an exponential dilution (sometimes improperly called "logarithmic"), where concentrations might be 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 ng/mL. For the remainder of this article, we will consider only multipoint calibration. 

Table I: Concentration versus response data 

When a multipoint calibration curve is used, you must decide whether to allow 

  0 (equation 1) or to force the curve through the origin (equation 2) when fitting the curve to the calibration data. Fortunately, this does not need to be an arbitrary decision, but can be based upon the regression statistics from the calibration data. As an example, we will use the data of Table I, where the response is listed for a 10-point, exponential-dilution calibration curve with a range of 1–1000 ng/mL. A linear regression of the data can be obtained from an Excel spreadsheet (Tools menu – Data Analysis – Regression). Most data system software also will perform linear regression calculations. The key regression statistics for the data of Table I are listed in Table II. The Excel nomenclature is used in Table II, with additional information added in parentheses. 

Table II: Regression statistics from Excel 

The decision to force zero or not on the calibration curve is based upon how close the calculated 

-intercept is to zero. This is difficult to do visually from a plot of the response vs. concentration, as in Figure 1, but is easy (and more reliable) to do statistically. If 

 is less than one standard deviation (SD) away from zero, it can be assumed that this is normal variation and 

 = 0 can be used in the standard curve. We can test this with the regression statistics using the standard error (SE), since we don't have enough data points to calculate the SD of 

 = 0. There are two values of SE included in the Excel data of Table II. The first is the SE for the entire curve, labeled simply "Standard Error" (1.4134). The second is the standard error of the 

-intercept (0.5244), also labeled "Standard Error," but included in the information about the y-intercept at the bottom of the table. The SE for the curve is based upon the variability of the curve throughout its range, including both high and low concentrations. The SE for the 

-intercept is based upon estimates of variability at the 

-intercept, not the entire curve, and will always be smaller. The SE of the 

) is the appropriate value to use to test if the curve passes through the origin: 

Examining the data of Table II, we can see that the 

 is 0.5244, so the relationship of equation 4 applies. The calibration curve is described properly as 

 + 0.8101; this is the calibration curve plotted in Figure 1. You can see the practical impact of choosing the proper origin by examining the last two columns of Table I. Here the %-error is shown for each point on the curve. This is calculated as (experimental value – calculated value)/calculated value, and expressed as a percentage. The calculated value is determined by the regression equation for the calibration curve (shown in the footnote of Table I). If the curve is forced through zero (third column), errors as large as 45% occur, whereas these are reduced by a factor of 2 or more for low concentrations if the proper curve fit is used (fourth column). Errors are larger at low concentrations, as expected; this will be discussed in more detail next month. 

There are three potential errors that can occur relative to the information discussed previously: the wrong choice of the calibration model, the wrong choice about forcing zero, and the wrong test of a zero intercept. The choice of the calibration model to use should be straightforward. Usually, there will be similar method in use in your laboratory, and the same calibration model will be used for all of them. The single-point model usually is limited to samples where the expected values are nominally identical. The two-point model will be used for methods with a narrow range, usually less than one order of magnitude. The multipoint model will be used when the method range spans several orders of magnitude and for cases when the curve cannot be forced through the origin. 

The decision about whether or not to force the curve through zero is an important one. As shown in the data of Table I, forcing 

 = 0 when it is not appropriate can generate larger errors, especially at low concentrations. The reciprocal also is true — not forcing zero when it should be forced — although the errors might or might not be significant. (With Excel, force zero by clicking the "constant is zero" checkbox in the regression dialog box.) 

-intercept also can have unintended consequences. In the example of Table II, the SE of the curve is 1.4134 and the SE of the 

-intercept is 0.5244. The y intercept is 0.8101 — midway between the two SE-values. It can be seen that using SE of the curve for the test would mean (wrongly) forcing 

 = 0, with the corresponding higher errors shown in Table I. 

Finally, linear regression of calibration data is a mathematical model meant to minimize the errors when making generalizations from the calibration curve. The more points on the calibration curve the better, and the more replicate injections at each concentration the better. This strongly suggests that the choice of the type of calibration curve and whether or not to force the curve through zero should be determined during method validation when data from many calibration curves are available to pool. Decisions based upon a single calibration curve are estimates of estimates, and clearly are not as good as results from larger data sets. 

"LC Troubleshooting" Editor John W. Dolan is Vice-President of LC Resources, Walnut Creek, California; and a member of LCGC's editorial advisory board. Direct correspondence about this column to "LC Troubleshooting," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

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

(1) Harmonized Tripartite Guideline, Validation of Analytical Procedures, Text and Methodology, Q2 (R1), International Conference on Harmonization, (November 2005), 

http://www.ich.org/LOB/media/MEDIA417.pdf

(2) Guidance for Industry, Bioanalytical Method Validation, USFDA-CDER (May 2001), 

http://www.fda.gov/cder/guidance/4252fnl.pdf

Articles

LC Troubleshooting

This month's "LC Troubleshooting" looks at some different calibration models, how to decide if a calibration curve goes through zero, and some problems that can occur if the wrong choices are made.

Calibration Curves, Part I: To b or Not to b?

Electrostatic Repulsion Hydrophilic Interaction Chromatography — A New Tool for Enrichment of Phospho- and Glycopeptides

Introduction of modifications into the protein backbone or its amino acid side chains following the translation of the mRNA code enables a single polypeptide chain to be transformed into diverse species with enhanced or modified biological functions. These posttranslational modifications (PTMs) can affect the cellular location of a protein, its biological lifetime, and its function or activity level. Of the huge variety of PTMs observed in the proteome, phosphorylation and glycosylation are among the most important. Reversible phosphorylation at serine, threonine, and tyrosine residues is implicated in control of cell growth and differentiation, cell death, gene expression, and signal transduction. Glycosylation at serine, threonine, and asparagine residues modulates the structure and function of membrane and secreted proteins and is important in proper protein folding and subcellular localization. Given the importance of these two posttranslational modifications in cell metabolism, the characterization of phosphoprotein and glycoprotein structure and function is vital to the identification of drug targets and development of novel therapeutics. 

Posttranslationally modified sites in proteins are identified generally by digestion of the protein and identification of its constituent peptides. The low abundance of a particular posttranslationally modified species is a major impediment to its characterization in a complex mixture such as a body fluid or cell lysate. To obtain sufficient material for study and to eliminate the vast majority of interfering proteins, selective enrichment techniques are required. Enrichment can be performed at the protein level, or, more commonly, at the peptide level after enzymatic cleavage of the protein. Enrichment techniques for glycoproteins include lectin chromatography (1–3), size exclusion chromatography (4), hydrazide-based covalent coupling (5), and (in the case of sialic acid–containing glycopeptides) cation-exchange chromatography (6). Enrichment techniques for phosphoproteins and phosphopeptides were reviewed in the June issue of "Directions in Discovery" (7). These include immunoprecipitation (8,9), chemical modification by β-elimination (10) or using phosphoramidate chemistry (11), immobilized metal affinity chromatography (IMAC) (12), and enrichment on metal oxides such as titanium dioxide (13) or zirconium dioxide (14). Currently, IMAC or metal oxides are the most popular methods for enrichment of phosphopeptides. 

The available enrichment techniques for phosphopeptides have two limitations. First, they tend to yield phosphopeptides as a class, with little or no discrimination among peptides differing in the level of phosphorylation. Second, no single enrichment technique delivers the entire phosphoproteome. A comparative study of phosphoramidate chemistry, IMAC, and TiO

 enrichment (15) demonstrated that overlap of the three techniques was poor, suggesting that each technique isolates a different subset of the phosphoproteome. The study of proteomics would benefit from a technique that would resolve phosphopeptides with different levels of phosphorylation and that would provide better (or complementary) representation of the phosphoproteome. Recently, electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) has emerged as a candidate technique. ERLIC is a variation of hydrophilic interaction chromatography (HILIC) that shows promise for separation and enrichment of phosphopeptides (16) and glycopeptides (17). 

HILIC describes a type of normal-phase chromatography that employs a polar stationary phase with aqueous–organic mobile phases (18). In contrast to reversed-phase chromatography, the aqueous component of the mobile phase (for example, water or buffer) serves as the strong solvent, and the organic component (for example, acetonitrile) is the weak solvent. Analytes are eluted in order of increasing hydrophilicity. It has been proposed that retention occurs as the analyte partitions between the bulk mobile phase and a water-enriched layer which hydrates the hydrophilic stationary phase. This is in contrast to retention in conventional normal-phase chromatography, which occurs by adsorption at the polar stationary phase surface. To a first approximation, the retention order of a series of analytes in HILIC is the opposite of that in reversed-phase chromatography. HILIC has several advantages. First, it provides a means of achieving retention and separation of very hydrophilic water-soluble analytes, which are intractable to reversed-phase and ion-exchange chromatography. Second, the acetonitrile typically used as the organic component of the mobile phase has low UV transparency (for better detection sensitivity) and low viscosity (for high chromatographic efficiency). Third, a mobile phase composed of a low fraction of water (strong solvent) and a large fraction of acetonitrile (weak solvent) provides an ideal composition for efficient desolvation in electrospray ionization mass spectrometry (MS). HILIC has been used successfully for a wide variety of small molecule pharmaceuticals, carbohydrates, and peptides (19). It has been less successful for proteins because of their generally poor solubility in high concentrations of organic solvents. An exception is the application of HILIC to the separation of histones and membrane proteins. 

Very basic and very hydrophilic species exhibit strong retention in HILIC, and, when present in a multicomponent mixture of less polar analytes, require gradient elution for separation within a reasonable time frame. The technique of ERLIC provides an alternative strategy for dealing with complex mixtures with components at the extreme of retention (poorly vs. highly retained). 

Electrostatic Repulsion–Hydrophilic Interaction Chromatography

Hydrophilic interaction can be superimposed as a mixed mode on an ion-exchange column in the presence of high concentrations of an organic solvent. The important feature of this combination is the independence of hydrophilic interaction and electrostatic effects, allowing the two to be manipulated independently. In this way, the behavior of charged solutes can be controlled to address extremes of retention. Three cases can be considered. The first case is the late elution of very acidic peptides containing aspartate or glutamate residues on an anion-exchange column. In this case, reduction of mobile phase pH below the p

 of the side chains reduces their retention. The second case is the strong retention of basic peptides on a HILIC column by hydrophilic interaction. In this case, use of an anion-exchange column in HILIC mode reduces retention by electrostatic repulsion of the basic groups from the stationary phase. The third case is the elution of basic peptides in the void volume of an anion-exchange column operated in anion-exchange mode. In this case, addition of sufficient organic solvent to the mobile phase introduces a HILIC contribution to the separation, and the retention of basic peptides is increased by hydrophilic interactions, which counteract the electrostatic repulsion of basic groups. In this case, the two superimposed modes antagonize each other's extremes of retention and can permit isocratic elution of complex peptide mixtures. This last case forms the basis for ERLIC of peptides (16). 

The issue addressed in the third case is illustrated in Figure 1, in which a weak-anion-exchange column was operated in ion-exchange mode. Vasopressin has a net positive charge, is repelled electrostatically, and is eluted before the void volume since it is excluded from the pore system. The comparative performance of HILIC and ERLIC to resolve a mixture of peptide standards is shown in Figure 2. In HILIC mode, a concentration of acetonitrile that promotes retention of the basic peptides 15 and 17 affords inadequate retention of acidic and neutral peptides. In the ERLIC mode, electrostatic repulsion of peptides 15 and 17 allows the acetonitrile concentration to be increased to a level where all peptides can be retained and resolved. 

Most tryptic peptides carry at least two positive charges, one on the N-terminal amino group and the other on the C-terminal lysine or arginine residue. In anion-exchange chromatography, these positive charges dominate the chromatography, and nonphosphorylated peptides (represented as peptide A in Figure 3) are eluted within the void volume. The presence of a single phosphate residue (which reduces the net positive charge of the peptide by one unit) is inadequate to promote retention of the peptide in the anion-exchange mode, which still is eluted near the void volume (represented by peptide B in the lower trace of Figure 3). Addition of acetonitrile to the mobile phase drives the hydrophilic interaction of the phosphate with the stationary phase, promoting retention of peptide B (ERLIC mode, upper trace of Figure 3). Peptides with two or more phosphates are well-retained in either mode. Similar behavior can be seen with mono- and tetraphosphorylated peptides in a tryptic digest of β-casein (Figure 4). 

The selectivity of ERLIC for phosphopeptides under conditions where unphosphorylated peptides are unretained suggests that the technique could be an effective means of separating and enriching phosphopeptides from complex mixtures in proteomics applications. This was investigated using a tryptic digest of HeLa cell proteins (20,21). For such a complex sample, which could potentially contain thousands of phosphopeptides with variable levels of phosphorylation, gradient elution was required to elute mono- and multi-phosphorylated peptides within a reasonable time frame. In the study summarized in Figures 5–7, the digest was separated using a weak-anion-exchange column and the same solvents used in Figures 3 and 4 for standard peptides. The initial solvent was 20 mM sodium methylphosphonate (pH 2.0) containing 70% acetonitrile. An initial 10-min isocratic elution with this solvent was used to elute the poorly retained unphosphorylated peptides. Then a gradient to 200 mM triethylamine phosphate (TEAP, pH 2.0) containing 60% acetonitrile was performed. Note that this is a gradient in both the HILIC mode (decreasing acetonitrile) and the ion-exchange mode (TEAP is a stronger eluent than sodium methylphosphonate for phosphate groups). The low mobile phase pH served to uncharged aspartate and glutamate residues (suppressing their ion-exchange interactions), and to fully charge amine groups (promoting electrostatic repulsion). Fractions were collected across the elution time and each fraction was analyzed by reversed-phase liquid chromatography (LC)–MS-MS to identify phosphopeptides. As expected, unphosphorylated peptides eluted mainly in the early fractions, while phosphorylated peptides were retained and eluted with increasing concentration of TEAP (Figures 5 and 6). Moreover, separation based upon phosphorylation states was possible, with monophosphorylated peptides eluted first and multiply phosphorylated peptides eluted later in the gradient (Figures 7 and 8a). The number of phosphopeptides identified in the HeLa cell lysate compared favorably with the best results reported using titania or IMAC for enrichment. 

A determination of the relative ratios of peptides phosphorylated on serine, threonine, or tyrosine residues (Figure 8b) revealed that 6% of the total was phosphorylated on tyrosine residues, which is significantly higher than previously reported values of ~2% (22). This might reflect the facts that ERLIC is more efficient in enriching multiphosphorylated peptides, and that 76% of the pY residues were found on such peptides. 

ERLIC Enrichment and Separation of Glycopeptides

Based upon their observation that glycopeptides, like phosphopeptides, are retained poorly in strong cation chromatography using salt gradient elution, Lewandroski and colleagues (17) concluded that ERLIC conditions might be applied successfully to glycopeptide enrichment. They applied the technique to enrichment of tryptic glycopeptides derived from human plasma membrane proteins that had been extracted from platelets by PEG-dextran two-phase partitioning. The ERLIC separation conditions were identical to those described previously for phosphopeptide separations (PolyWAX weak-anion-exchange column using gradients from high to low acetonitrile concentration and from methylphosphonic acid to TEAP at low pH). Fractions collected during ERLIC elution were digested with PNGase, which removed N-linked oligosaccharides and introduced a +1 Da mass shift by deamidation. Subsequent LC–MS-MS was used to distinguish glycopeptides from nonglycopeptides based upon the deamidation mass shift and to identify the glycoproteins from which the glycopeptides were derived. As anticipated, nonglycopeptides were eluted in the early fractions, while the deamidated glycopeptides were eluted in later fractions (Figure 9). Most of the detected glycopeptides were eluted as broad peaks, with elution times of 5–6 min or greater. The authors interpreted this as a reflection of microheterogeneity at the glycosylation sites resulting in the separation of peptide isoforms. They conclude that ERLIC provides a promising means of enriching glycopeptides from complex peptide mixtures with the potential for resolving glycopeptide isoforms. The separation appears to be based upon hydrophilic interactions of carbohydrate groups, with a contribution from electrostatic interaction of sialic acid groups (p

 ~2.6) with the anion exchange phase. Possible interference by coeluted phosphopeptides could be eliminated by phosphatase treatment of the sample before ERLIC separation. Nearly half the deamidated peptides identified did not fit the consensus sequence for a glycation site. These might be peptides containing isoaspartic acid residues (p

 ~3.1) resulting from nonenzymatic deamidation. 

Comprehensive analysis of phosphopeptides or glycopeptides is hampered by the failure of any single enrichment technique to completely represent the total population of posttranslationallymodified species. Moreover, enrichment techniques tend to yield PTMs as a bulk mixture, with no resolution based upon PTM structure or site occupancy. ERLIC, a new technique in which the selectivities of HILIC and anion exchange are overlaid in the same separation, has been applied to the enrichment of both phosphopeptides and sialylated glycopeptides. In addition to serving as an enrichment tool, ERLIC can provide resolution based upon the number of modified sites (phosphopeptides) or the structure of the modified site (glycopeptides). These characteristic make ERLIC a promising alternative or complementary approach to phospho- and glycopeptides analysis. 

The author would like to thank Andy Alpert for providing figures for this column, and for discussion in its preparation. 

"Directions in Discovery" editor Tim Wehr is staff scientist at Bio-Rad Laboratories, Hercules, California. Direct correspondence about this column to "Directions in Discovery," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

(1)  J. Ji, A. Chakraborty, M. Geng, X. Zhang, A. Amini, M. Bina, and F. Regnier, 

(3)  M. Geng, X. Zhang, M. Bina, and F.E. Regnier, 

(4)  G. Alvarez-Manilla, J. Atwood 3rd, Y. Guo, N.L. Warren, R. Orlando, and M. Pierce, 

(5) H. Zhang, X.J. Li, D.B. Martin, and R. Aebersold, 

(6)   U. Lewandrowski, R.P. Zahedi, J. Moebius, U. Walter, and A. Sickmann, 

(8)   S. Morandell, T. Stasyk, K Grosstessner-Hain, E. Roitinger, K. Mechtler, G.K. Bonn, and L.A. Huber, 

(11) H. Zhou, J.D. Watts, and R. Aebersold, 

(13) M.W. Pinkse, P.M. Uitto, M.J. Hilhorst, B. Ooms, and A.J. Heck, 

(15) B. Bodenmiller, L.N. Mueller, M. Mueller, B. Domon, and R. Aebersold, 

(17) U. Lewandrowski, K. Lohrig, R. P. lZahedi, D. Wolters, and A. Sickmann, Clin. Proteom. DOI 10.1007/s12014-008-9007-z. 

Directions in Discovery

ERLIC is a new technique that shows promise as an alternative or complementary technique for both enrichment and separation of these posttranslationally-modified peptides.

Electrostatic Repulsion Hydrophilic Interaction Chromatography

The accurate measurement of vitamins in conventional dietary supplement tablets and dietary supplement drinks that are consumed widely by the general public is of utmost importance. Vitamins are necessary for proper body function, and consuming too little or too much can be harmful to an individual's health. This makes it imperative that values reported for the amounts of vitamins in these supplements are measured accurately. Dwyer and colleagues (2) have shown that while analytically validated information on the vitamin content in food is relatively complete, the same information for dietary supplements is not. According to the authors, the differences between label values and actual content of vitamins in dietary supplements have not been studied extensively. Labels are required to reflect minimum content of nutrients according to U.S. regulatory requirements, so the actual nutrient content often is higher than stated. The amount of the overage is dependent upon several factors, including the cost of the vitamin and its stability.     

Many methods, including USP methods (3) currently used for the separation and quantitation of water-soluble vitamins use mobile phase additives including ion-pair reagents and EDTA. Ion-pair reagents are used as mobile phase additives to allow successful separation of polar substances on reversed-phase high performance liquid chromatography (HPLC) columns. They improve resolution and peak shape, particularly by reducing tailing. However, their use can result in irreversible changes in column performance due to adsorption of the ion-pair reagent onto the stationary phase (4), resulting in either a change in selectivity of the column or longer equilibration times. Through the elimination of ion-pair reagents, the proposed method avoids this damage to the chromatographic column. In addition, the simplified mobile phase lowers the cost of analysis and avoids interferences with the separation and problems with detection during gradient elution.  

: Analytical grade vitamin standards were purchased from Sigma-Aldrich (Milwaukee, Wisconsin). See Figure 1 for structures. Water (EMD Chemicals, Gibbstown, New Jersey) and methanol (Burdick and Jackson, Muskegan, Mchigan) were HPLC grade. Sodium dihydrogen phosphate, ultrapure bioreagent grade, was purchased from J.T.Baker, Phillipsburg, New Jersey, and phosphoric acid, HPLC grade, was purchased from Fisher Scientific, Pittsburgh, Pennsylvania.  

: An Agilent 1200 SL Rapid Resolution liquid chromatograph with a binary pump, autosampler, inline degasser, temperature-controlled column compartment, and an 80 Hz diode-array detector (Agilent Technologies, Inc., Santa Rosa, California) were used for the analysis. The detector flow cell selected for this study was a micro flow cell with a 2-µL volume. Sample peaks were identified positively by two methods: by matching retention times and by comparison of UV spectra from a spectral database created with this instrument. Spectra also were used to determine the peak purity. Chemstation for LC 3D Systems, Rev. B.03.01, was used for data collection and analysis.  

 Individual standards used for retention time determination and preparation of spectral database were prepared by weighing the appropriate amount of standard using a microbalance and then dissolving in 10 mL of water. Calibration standards were prepared by serial dilution of a stock solution that included all the vitamins being analyzed.  

 Aliquots of VitaminWater (Glaceau, Flushing, New York) were injected without dilution. A minimum of 30 vitamin tablets (Berkley & Jensen Children's Chewable Complete Multi Vitamins and Minerals Supplement, Natick, Massachusetts, and Equate Adult Multivitamin, Walmart, Bentonville, Arkansas) were ground and the equivalent of one tablet was weighed out and extracted in water in an ultrasonic bath for 5 min.  

All samples were filtered before injection with Agilent syringe Econofilter, regenerated cellulose, 25-mm diameter, 0.20-µm porosity. Due to decomposition issues all solutions were prepared daily (5,6).  

 All separation conditions are reported in Table I. The flow rate was kept constant for all three column dimensions, but the gradient was changed as shown in Table II to keep the 

* values equivalent. The injection volume also was scaled based upon the column dimensions.  

Figure 2 shows chromatograms of vitamin standards injected on three columns of different dimensions for comparison. Peaks are identified in Table III. Seven water-soluble vitamins were separated without the use of ion-pair reagents. The average tailing factors for the analytes on all the columns was < 1.2, well within the acceptable limits for tailing factors for these compounds as stated by the USP (3). The average % RSD for retention times on the 1.8-µm column was 0.4 % with none greater than 1.4 %, showing that the chromatographic method is precise for seven replicate injections.  

As shown in Figure 2, the separation was quite good, with all the peaks being well resolved. Based upon this observation, the method could have been optimized further by increasing the flow rate. This was done at a flow rate of 1.5 mL/min without negative effects on the separation of solution standards. When the vitamin supplements were analyzed using the faster flow rate, however, the identification of some of the vitamins of interest could not be performed due to overlapping unidentified peaks from other ingredients in the vitamins. Because of this, the flow rate was kept at 1.0 mL/min for analysis of the vitamin supplements.  

Figures 3 and 4 show chromatograms of the adult and children's chewable vitamin supplements, respectively. The solutions of these supplements were prepared as indicated in the experimental section, with no additional clean up before injection. In both vitamin supplements, six of seven vitamins of interest were identified. In the VitaminWater vitamin supplement drink, only five of the vitamins of interest were present, and four of these were positively identified. Sample peaks were identified positively by two methods: by matching retention times and by comparison with their reference spectra. Cyanocobalamin was not identified in any of the vitamin supplements or the vitamin drink, because it is needed in only very small amounts in the body, and only a few micrograms are present in the samples. The vitamin-containing water was not as complex a matrix to analyze and displayed only a few well resolved peaks. The chromatogram was not included. 

Quantitation was performed for pyridoxine and niacin in both the supplements and the vitamin drink. These analytes were selected because they were present in all three samples, and they did not require additional clean up procedures to eliminate interferences. The results for the quantitation of these analytes are shown in Table IV. The expected values are given to the number of significant figures listed on the label or provided by the manufacturer. As expected due to U.S. regulatory requirements, all except one of the measured values was greater than the label value for the two vitamins used for quantitation. 

The final goal was to investigate the effects of column dimension and particle size on resolution, separation time, pressure, and sensitivity. The three columns used in this study were manufactured using the same type of base silica. The variable column dimensions are given in Table I. The columns all have the same inner diameter, so the same flow rate can be used and the linear velocity will remain constant. The variables that differ for these columns are the length and the particle sizes.  

The dwell volume was the same for all the separations, so no adjustment needed to be made for this. A gradient was used in this separation, so it must be adjusted to account for the difference in column length. This involves calculating the number of column volumes in each segment of the original gradient and adjusting the gradient times used for each column to give the same number of column volumes between each segment on each column. Equation 1 was used to calculate the gradient times for the three columns, and the resulting values are given in Table II. In this equation, 

The injection volumes also needed to be adjusted for each column because the three columns had different dimensions and therefore different sample capacities. This is done to avoid phase overload, keeping the chromatograms consistent from column to column. Equation 2 was used to calculate the appropriate injection volumes for the three columns, and the resulting values are given in Table I. In this equation, the variables are the same as Equation 1, and 

As seen in Figure 2, the separation times decreased as the shorter columns were used; however, the separation remained consistent without loss of resolution. The column pressure did increase from 170 bar to 300 bar as the particle size was decreased from 5 µm to 1.8 µm. However, at these pressures, the separation still could be performed on a traditional HPLC instrument as long as care was taken in reducing the extra column volume.  

The increase in sensitivity as the column configurations were changed is probably the most striking difference in the comparison of the three columns. Table V shows the gain in sensitivity for cyanocobalamin. For this portion of the experiment, the standard vitamin mixture was injected into the three different columns. The injection volume was scaled proportionally to column length, so the peak heights are not compared directly. To compare the sensitivity, the amount injected is normalized, making the ratio of the peak areas to injected amounts the same. The data in Table V show that there is still a significant difference in the ratio of the heights and the injected amounts. This means there is a significant increase in the sensitivity of the 3.5-µm column compared with the 5-µm column, and another significant increase in the sensitivity of the 1.8-µm column. The values shown in Table V are for the gain in sensitivity in comparison to the 5-µm column. The gain in sensitivity when the column is changed from a 5-µm column to a 3.5-µm column is 34%, while the gain in sensitivity when the column is changed from a 5-µm column to a 1.8-µm column is 52%. This gain in sensitivity is due to the higher efficiency (that is, lower dilution factors) of the smaller particles, not simply because the analytes spend less time in the column (7). 

Separation, identification, and accurate quantitation of vitamins in commercially available supplements is very important because the health of consumers may be dependent upon the amount of vitamins consumed through their diet and the use of supplements. U.S. regulatory requirements state that labels must reflect the minimum amount of the vitamins present, which does not necessarily give consumers the information they need to make informed choices. A method has been presented that can be used for the determination of seven water-soluble vitamins in different commercially available supplements. This method does not require the use of ion-pair reagents or many additives to the mobile phase, as many previous methods have.  

By using three column dimensions, the data also can be used to illustrate the effect of decreasing particle size in the columns and changing the lengths of chromatographic columns on different separation attributes. Knowledge of these effects is of great importance as modern HPLC technologies are tending toward smaller particle sizes and faster separations. 

Different types of supplements contain different binders and other substances that can interfere with the quantitation of vitamins. Each type of sample requires a specific method of sample preparation and clean up before introduction into the HPLC column. Some of these samples require methods as simple as the use of syringe filters or centrifugation, while others require methods such as solid phase extraction. If adequate sample clean up is performed before HPLC analysis, the flow rate used for the analysis can be increased without loss of separation. 

The Authors would like to thank Ron Majors and Agilent Technologies for the loan of the instrument, donation of the columns used, and funding to complete this project. 

Karyn M. Usher, Anna Glinko, Michael J. Bozym, and Michelle L. Owens

				Department of Chemistry, West Chester University of Pennsylvania, West Chester, Pennsylvania			

The Vitamins: Fundamental Aspects in Nutrition and Health

(3)  U.S. Pharmacopeia National Formulary, USP 31 NF 26, May 1, 2008. 

(4)  L.R. Snyder, J.L. Glajch, and J.J. Kirkland, 

(7)  A. Glinko, M.J. Bozym, M.L. Owens, K.M. Usher, R. Majors, Agilent Technologies Pharmaceutical Food/Beverage application note, 5989-9313EN, (2008). 

The authors set out to perform a separation of seven water-soluble vitamins without the use of ion-pair reagents.

Separation, Identification, and Quantitation of Water-Soluble Vitamins by HPLC

Pittcon 2009, the 60th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, was held in the massive McCormick Place in Chicago, Illinois, on March 8–13, 2009, the sixth time the Conference has been held there. This year's event hosted nearly 1000 instrument manufacturers and laboratory suppliers in more than 2200 booths. In addition to attending the exposition, the conferees listened to 2500 technical presentations, checked numerous company seminar rooms, or attended one or more of 120 short courses. 

Undoubtedly, Pittcon remains the most important yearly international analytical exhibition, where companies introduce their latest instruments, instrument accessories, software, columns, sample preparation equipment, and other consumable products. Because many past attendees have purchased one or more new products within three months after attending the show, most exhibitors attempt to maximize their booth traffic to meet as many potential customers as possible. 

The purpose of this report is to provide information about many of the new separation consumables and accessory products that will be displayed at Pittcon 2009. In some cases, products that were introduced during 2008 but after Pittcon 2008 (1,2) might be included for reasons of completeness. The information is based upon manufacturers' responses to a questionnaire mailed in December 2008. Because of space limitations and the fact that some manufacturers did not respond to the questionnaire, this report cannot be considered an exhaustive listing of all new products that were introduced in Chicago. However, over the years, these Pittcon introduction summaries have provided a good source of information that would be difficult for one individual to gather during the four days of the exhibition. In addition, the products introduced have shown definite correlations to current research, development, and application activity in the separation sciences. 

As in previous years, columns and other products recommended by their manufacturers primarily for biomolecule separations or sample preparation are denoted in the tables with the designation BIO. Some of these products can be used for general high performance liquid chromatography (HPLC) separations as well, but their main emphasis is for biological samples.  

In this month's coverage, I will describe new introductions in the areas of HPLC: reversed-phase, normal- and bonded-phase, ion-exchange and ion, size-exclusion, and large- and preparative-scale chromatography; specialty chromatography columns; and thin-layer chromatography (TLC). Next month, I will look at gas chromatography (GC) columns, sample preparation products, and hardware, accessories and kits for chromatography, and sample preparation. 

 This year, I observed that there was a continued trend in high-throughput techniques, especially in the HPLC and sample preparation areas. More devices and instrumentation that support automation made their appearance, and autosamplers with sample preparation functionality have been reappearing. Many application-specific columns for HPLC and GC and various devices for solid-phase extraction (SPE) and sample preparation were introduced. Again at Pittcon, a considerable number of new accessories, hardware, software, and devices were introduced that could make chromatographic laboratories more productive. 

 At Pittcon 2009, reversed-phase HPLC maintained its dominance and over half of the introductions were the ever-popular C18 phase. Hydrophilic interaction chromatography (HILIC) columns also were quite evident. Polymer-based HPLC columns are ever-increasing and now dominate the ion-exchange and size-exclusion modes. Mixed-mode columns have now established themselves as an alternative to single-phase columns. Mixed mechanisms give rise to subtle (and sometimes substantial) selectivity changes compared to regular alkyl phases. From an applications standpoint, chiral and protein–peptide columns dominated the introductions.  

 Ultra-inert GC columns are the latest differentiating feature in the marketplace. New porous layer open tubular (PLOT) column introductions show that the separation of small molecules and gases is still of interest. A selection of application-specific columns, especially for biodiesel and bioethanol separations and regulated methods, show that interest in specialty GC columns is still prevalent. 

 A variety of formats in SPE were introduced: cartridges, pipette tips, and 96-well plates and some interesting new SPE application-specific phases for environmental and biological fluid samples. Combination products of protein precipitation and SPE were introduced to provide cleaner, phospholipid-free extracts of biofluids for LC with tandem mass spectrometry (LC–MS-MS) analysis. Additional QuEChERS kits made their appearance, for simple, low-cost sample preparation for the analysis of pesticides in fruits and vegetables. Some unique approaches to sample filtration are included. 

 With the current shortage of acetonitrile, solvent recyclers could become an important accessory. Products to make method development simpler in HPLC were introduced including software, column selector, and a low-pressure solvent mixer. Some convenient GC accessories such as a new hydrogen generator, leak detector, semiautomated vial cap crimper, and low gas alarm system were seen at the show. Some useful semiautomated sample preparation devices like evaporators, digesters, mixers, concentrators, and aspirators take some of the drudgery out of sample preparation.  

Autosamplers for GC and LC with sample preparation capabilities are making their appearance.  

Each year, manufacturers introduce entire new families of HPLC columns at Pittcon. For the sake of brevity, I have elected to cover those series here instead of listing each column under individual headings in the modes' tables. In some cases, the series are extensions of existing products but cover additional phases while in other cases, the families are entirely new to the market. 

Silicycle (Quebec, Quebec, Canada) is a company specializing in silica-based columns and catalysts. This year, the company introduced a line of SiliaChrom HPLC columns for analytical HPLC. The packings, based upon high-purity, spherical 3- and 5-μm diameter silica with 100–150 Å pores, are currently available for reversed-phase (C18, phase coverage 10–22% carbon), adsorption, and HILIC. Columns with 2.1- and 4.6-mm internal diameters and lengths from 30 to 200 mm are available. Varian (Palo Alto, California) introduced its SepTech bulk media that are optimized for preparative to process chromatography. Based upon 10-μm high-purity silica with low metal content, the media are available in two pore sizes, ST 60 Å and ST 150 Å. The media are suitable for small molecules and larger natural compounds and biomolecules, respectively. Their bonding chemistry with endcapping provides optimum ligand density (3.5 and 3.8 μmol/m

 for ST 60 and ST 150, respectively; corresponding carbon loadings are 25% and 15%) for high-capacity separations of hydrophobic and hydrophilic compounds. Bulk quantities from 100 g to 25 kg can be purchased as standard. 

Another silica gel supplier, Grace Davison Discovery Sciences (Deerfield, Illinois), introduced its VisionHT LC family of columns. A broad range of phases suitable for reversed-phase (C18), adsorption, and HILIC modes in particle sizes 1.5, 3.0, 5, and 10 μm were shown. Such a range of particle sizes allows seamless method transfer between various column configurations in analytical and preparative chromatography. The phases are based upon silica particles with 100- and 120-Å pores. Column dimensions are 10–250 mm x 1–10 mm. Zirchrom Separations (Anoka, Minnesota), best known for its zirconia-based columns, has introduced 2-μm versions of all of their phases. All products have a pore size of 300 Å. Column dimensions cover the capillary to analytical range; custom columns can be provided. Zirchrom also provides bulk material for those who wish to pack their own columns. 

Chiral Technologies (West Chester, Pennsylvania), a supplier of chiral columns, has extended its popular line of Chiralpak and Chiralcel columns with the addition of added phase chemistries. The columns are based upon 3- and 5-μm spherical silica gels; the pore size of over 1000 Å is required for the successful coating of the polysaccharide (cellulose or amylase) chiral stationary phases. These columns are all useful for separations in the reversed-phase mode. Various column dimensions are available including 50, 100, and 250 mm x 4.6 mm. Chiralcel OZ, with a 5-μm particle size, has a chiral selector based upon cellulose tris-(3-chloro-4-methylphenylcarbamate), while Chiralcel OD-3R has the same chiral selector but is based upon a 3-μm higher efficiency silica. ChiralPak AZ columns, packed with 5-μm particles, have a chiral selector amylose tris-(3-chloro-4-methylphenylcarbamate) while the ChiralPak AY columns, also packed with 5-μm particles, have the chiral selector amylase tris-(3-chloro-4-methylphenylcarbamate). Finally, ChiralPak AD-3R, packed with 3-μm particles, has the chiral selector amylase tris-(3,5-dimethylphenylcarbamate). Although the chiral selectors appear to be quite similar, such diverse functionalities can provide subtle differeneces in enantiomeric selectivity. To illustrate the separation power of chiral specific phases, Figure 1 shows the separation of the metolachlor (Novartis), a chloroacetamide herbicide which is the second most widely used herbicide in the world.  Its structure is also shown in Figure 1. The unusual functionalization pattern of this molecule renders the amine functionality extremely sterically hindered. As a consequence, metolachlor has two chiral elements: a chiral axis (atropisomerism) and a stereogenic center, giving rise to four stereoisomers, each of which undergoes enantioselective degradation in agricultural runoffs. So the ability to separate the isomers in order to follow this degradation is important. Note that the separation of these four stereoisomers is extremely difficult with most chiral stationary phases. The Chiralpak AY-H was able to provide nearly a baseline separation using an ethanol-hexane mobile phase. 

As usual, many new reversed-phase columns were introduced at Pittcon 2009 (see Table I). If one also adds all the reversed-phase columns covered in the new series and in specialty columns (Table II), one can see clearly the dominance of this operational mode. Over 53 new reversed-phase columns were added to the huge numbers that already exist from past introductions. Silica-based bonded phase columns dominated the introductions again (75%) but polymer-based reversed-phase columns (20%) were more visible this year. Polymer columns are more rugged with respect to pH range and solvent compatibility and can go to higher operating temperatures. For example, the ProSwift RP-4H column from Dionex can be rinsed with 1 M sodium hydroxide and the Shodex ET-RP1 column can withstand temperatures up to 150 °C for extended periods of time. 

Table I: Reversed-phase chromatography			

The C18 (octadecylsilane) functionality year proved again the most popular with over half of all reversed-phase columns being based upon this alkyl phase. However, this phase is not always the optimum for all separations and C4, C8, polar-embedded C8 and C18, phenyl, biphenyl, mixed phases, polymer-coated, silica-hydride, and cyanopropyl phases were made available by various suppliers to present selectivity alternatives. The HILIC columns that have some hydrophobic interaction possibilities are found in the Specialty column Table II. This year one silica-based monolith and one polymer-based monolith were introduced. 

Although C18 is the phase leader, mixed-mode reversed phases have come into popularity in recent years. Phenyl hexyl columns are one example; this year Advanced Chromatography Technologies (Aberdeen, UK) introduced a C18 combined with a phenyl. Possessing both hydrophobic and aromatic functionality, such columns can provide a different selectivity than either phase alone. Perhaps compounds that can be unresolved on a single stationary phase functionality could be better separated on such a mixed-phase column. Due to the addition of a polar moiety into an alkyl chain, polar-embedded stationary phases also give dramatically different selectivities than alkyl only phases; two new ones were introduced at Pittcon 2009. 

As far as particle sizes go, the workhorse columns are now packed with 3-μm particles. This trend has been evident over the last few years as shorter columns with smaller particles come into favor. In addition, the 2- and sub-2-μm particles are still being introduced because their use in fast, high-throughput columns is now well established. At least four companies introduced reversed-phase columns with these smaller particles. Some suppliers still feel that particles in the 2–3 μm diameters offer improved efficiency over 3–3.5 μm particles but lower pressure drops than the sub-2-μm columns. The superficially porous particles (SPP) of 2.7-μm diameter offer lower pressure with the same efficiency as the sub-2-μm columns. There seems to be growing interest by researchers to try out these SPP so it will be of interest to see what marketshare they can command. Although originally thought to have much lower surface area and therefore sample capacity, the SPP columns show only around 25% less capacity than a totally porous particle of the same diameter. 

Normal-Phase and Adsorption Chromatography

The term "normal-phase chromatography" implies that a nonpolar mobile phase and a polar stationary phase is used for the separation but columns with polar functional groups like cyano, amino, and diol have been used for other modes such as reversed phase, hydrophilic interaction, and ion exchange. This year, two companies introduced columns that were suited for bonded normal-phase chromatography: Imtakt (Kyoto, Japan) and Fortis (Neston, Cheshire, UK). 

Imtakt is represented in the U.S. by Silvertone Sciences (Philadelphia, Pennsylvania). Its Unison UK-Amino column is based upon a 3-μm silica with an aminopropyl bonded phase. When used in aqueous solution under acidic conditions under which the amino group is protonated, the column functions as a weak anion exchanger and when used with water–acetonitrile mobile phases for sugar separations, often with low UV detection, it functions in the HILIC mode. It also can function as a mixed-mode phase under the appropriate experimental conditions. The pH range for the Unison UK column is 2–9. The company's cartridge design columns are available from nanoLC to preparative sizes. Guard columns are also available. 

Fortis, a relatively new company in the columns business, established in 2005, has been expanding its portfolio and this year added a cyano column. Particles range from 2.1, 3.0, to 5 μm and can be packed into just about any analytical internal diameter (for example, 2.1, 3.0, and 4.6 mm). A prepacked guard column also is available. Due to the base silica's relatively high surface area (for example, 380 m

/g), the column shows rather high loadability. Separations of explosives, pesticides, and polar analytes by normal-phase chromatography have been demonstrated. 

In addition to the ion-exchange columns introduced as part of a family and the mixed-mode columns shown in Table III, two companies, OraChrom (Woburn, Massachusetts) and Tosoh Bioscience (Montgomeryville, Pennsylvania) introduced new ion-exchange columns. All of these columns were based upon polymeric packings; in general, silica gel-based packings do not seem to stand up to the rigorous mobile phase conditions often used in ion-exchange separations. 

The TSK-Gel STAT columns from Tosoh Bioscience are based upon polymethacrylate chemistry, which provides a hydrophilic polymer base material, making them more suitable for biomolecules in aqueous mobile phases. The polymer has been functionalized with quaternary ammonium (DNA-STAT), carboxymethyl (CM-STAT) and sulfopropyl (SP-STAT) to give strong anion, weak cation, and strong cation exchange character, respectively. Particle sizes of 5, 7, and 10 μm are available. Each of the STAT series columns is suited to a specific type of biomolecule separations including separation of DNA fragments, proteins including PEGylated proteins, peptides including digests, monoclonal antibodies, various protein aggregates, and nucleic acid constituents. The unique feature of the TSK-Gel STAT surface is its open network of multilayered ion-exchange groups. This feature, combined with a relatively large particle size, results in an improved loading capacity, low operating pressure, and good efficiency for both high and low molecular weight molecules. Static binding capacity is increased through novel bonding chemistry even though the surface area of the larger particles is decreased slightly.  

The OraChrom offerings are based upon its hard, gigaporous Styros PS-DVB methacrylate copolymers that the company terms a "simulated monolith." Columns packed with these materials show a low-pressure drop due to large flow-through pores, somewhat reminiscent of a polymeric monolith column. The chemistries are based upon covalent bonding of various functionalities with the polymer surface. This year the company has expanded its line with the addition of three new anion exchangers: Styros ANX based upon primary and secondary amine functionality (weak anion exchanger); Styros DEAE based upon diethylaminoethyl functionality (weak anion exchanger); and Styros SAX based upon a quaternary amine functionality (strong anion exchanger). The columns are well suited for the separation of various biomolecules by anion-exchange techniques. Columns of PEEK and stainless steel construction with 1–250 mm x 0.01–20 mm dimensions are available, as are guard columns. 

In the past few years, only a few new size-exclusion chromatography (SEC) columns have been introduced at Pittcon. That does not mean that SEC is not a highly practiced separation mode. It is a standard technique for the separation and characterization of organic polymers, both organic- and water-soluble. In addition, the mode is used widely for the separation of proteins and other biopolymers, sometimes known as gel filtration chromatography. Two companies introduced columns for SEC: Jordi Labs (Bellingham, Massachusetts) and Sepax Technologies (Newark, Delaware). 

Jordi, a company specializing in polymeric packings, introduced its xStream H2O column for the separation of common and unusual polar and cationic polymers, especially dextrans, polysaccharides, and vinyl ether–maleic acid copolymers in pure water eluents. The column can function in any range of mobile phases including 100% aqueous, 100% organic, or any mixture of solvents. Based upon polyamide chemistry, the column has been used with tetrahydrofuran, chloroform, DMSO, DMAC, and DMF and HFIP; the latter in cases in which sample-column interactions have been minimized, thereby providing excellent resolution of nylons and PET polymers. Other polymers separated include phenoxy resins, poly(n-butyl methacrylate), polycaprolactone, several styrenic polymers, PMMA, and other methacrylic polymers. In addition to a wide variety of packed guard, analytical, and preparative columns, Jordi also will provide bulk packing. 

Sepax introduced its silica-based column line called SRT Zenix, designed for aqueous mobile phase systems. The columns are recommended for the SEC separation of biological molecules, water-soluble polymers, nanoparticles, biological bacteria, and viruses. The stationary phase, on 3-μm silica, is hydrophilic and neutral with a coating of nanometer thickness. Two pore sizes, 150 and 300 Å, are available; high pore volume provides high capacity and high resolution; compatible with and stable in a wide range of salt concentrations (10 mM to 2.0 M). The SRT Zenix columns comes in standard dimensions of 300 mm x 2.1, 4.6, and 7.8 mm, but special sizes are available on request; bulk packing also can be purchased.  

TLC and HPTLC (high performance TLC) are simple techniques that many chromatographers overlook, even when they need only a cursory separation or purity check. Sorbent Technologies (Atlanta, Georgia) has made the technique easier by packaging a method development 12-pack of standard HPTLC plates. The company's Plate Spice Rack consists of two each of six unique silica-coated plates (10 cm x 10 cm) with the following phases: amino, cyano, C18, C2, diol, and silica gel. The "try before you buy" package saves money by cutting the cost of unusable full boxes. The plates are glass-backed and have an embedded fluorescent indicator activated by a 254-nm UV lamp for viewing separated spots.  

Specialty columns are HPLC columns that have been developed for specific separations that are difficult to achieve on a standard column. However, sometimes manufacturers will use a standard column but test it specifically for a certain class or compounds and provide a recommended set of chromatographic conditions. In some cases, the specialty column comes as part of a "total solution" kit with reagents, standards, and a method. Most specialty columns will be delivered with a test chromatogram from an analysis performed at the factory before shipment and some are guaranteed for a specific separation. Table II shows the 27 specialty columns that were presented at Pittcon 2009. Silica gel-based columns were again a distinct majority but polymer-based specialty columns were clearly in evidence. 

As depicted in Table II, the biggest specialty column application area was the separation of proteins and other large biomolecules. Columns of this type ranged from polymeric monoliths with various ionic and affinity functionalities to "simulated" monoliths with gigapores, to conventional wide pore silica and silica hydrides with 300-Å pores. Such large pores are needed to allow large biomolecules to diffuse inside of the particles to interact with specific ligands. Monolith columns provide lower pressure drops than particulate-based columns, and being polymeric, can withstand more aggressive solvent treatment. To illustrate the use of monolithic columns, Figure 2 shows the use of the BIA Separations (Ljubjana, Slovenia) two-column separation kit for plasmid DNA starting from a bacterial lysate obtained by an alkaline lysis procedure. After adjustment and clarification, the lysate is injected onto the first column (CIM DEAE, weak anion exchanger), where impurities were removed and the pDNA peak collected and then reinjected onto column 2 (CIM C4 HLD, hydrophobic interaction mechanism), where the supercoiled plamid DNA is separated from the open circular form, genomic DNA and remaining endotoxins. 

A most interesting protein column introduced by Invenca (Simpsonville, South Carolina) consisted of bundles of tightly packed nonporous fibers inside of a standard column format. This bundled fiber column is based upon the work of Kenneth Marcus and coworkers from Clemson University. Affinity chromatography is one of the more selective techniques for IgG and other monoclonal antibody purification. Protein A is one of the favored affinity ligands for this separation because it binds to the Fc portion of IgG. Three companies introduced products for monoclonal antibody separations using protein A. In addition, immobilized metal affinity chromatography (IMAC) is also a very selective approach for specific proteins such as His-tagged proteins. Natrix Separations' (Burlington, Ontario, Canada) Quantum media, which combines the binding capacity of resins with the ease of use of membranes, can perform IMAC separations at low pressures. 

Similar to last year (1), HILIC columns were prominent in the area of specialty columns. Some HILIC columns were featured in the new series section as part of a family but in Table II, additional HILIC columns based upon bare silica, silica hydride, and PS-DVB-methacrylate copolymers with polar functional groups were presented for the first time at Pittcon 2009. HILIC 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, polar bonded phase (for example, diol), or certain mixed-mode or zwitterionic phases. Operating conditions usually require a high percentage of nonpolar mobile phase, 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 can partition selectively. In addition, water-miscible organic solvents are used. Under HILIC, polar analytes are well retained and are eluted in order of increasing hydrophilicity. HILIC is especially favored by mass spectroscopists because ionization efficiency often is enhanced in organic solvents and the presence of lower concentrations of volatile buffer salt compared to reversed-phase chromatography. Most HILIC columns are silica-based, but Styros HILIC is a PS-DVB-methacrylate-based packing but with hydroxyl functionality. A simple separation illustrating the HILIC mode is shown in Figure 3. Typical of HILIC, the elution order is reversed from what one would expect of a reversed-phase column. Naphthalene is eluted before uracil and cytosine. The MS-compatible mobile phase is mostly organic at 90:10 acetonitrile–0.2 M aqueous ammonium formate. 

Chiral separations are still in the forefront, especially in the pharmaceutical industry. Separations using HPLC and supercritical fluid chromatography (SFC) can be achieved by a variety of chiral stationary phases (CSPs). A total of 10 new chiral columns were introduced at Pittcon 2009 (see Table II and new series). The most popular are those based upon modified polysaccharides (amylose- or cellulose-based) phases. Because many analytical separations are scaled up to obtain larger quantities of purified enantiomers, preparative particle sizes are frequently part of column introductions. 

Column Watch

Ron Majors brings readers his yearly review of all that was new and innovative at the annual Pittsburgh Conference.

New Chromatography Columns and Accessories at Pittcon 2009: Part I

 handed out its annual awards at Pittcon 2009 in Chicago, Illinois this year, at a press conference held on Monday, March 9 in the McCormick Place Convention Center. On hand were many members of the chromatography community, including presenters Robert Classon of Shimadzu and Ron Majors of Agilent Technologies. 

The awards themselves were the same as last year, with an Emerging Leader Award presented to the scientist making the greatest strides at an early stage of his or her career and a Lifetime Achievement Award presented to an industry veteran whose career has significantly influenced and advanced the field of separation science.  

The 2009 Emerging Leader Award went to Kevin Schug, an assistant professor at the University of Texas at Arlington, where he leads a group of nine researchers whose work focuses primarily on LC–MS research, specifically ESI-MS/HPLC.  

 Winners of the LCGC Emerging Leader Award 

This year's Lifetime Achievement Award went to a well known figure in the world of analytical chemistry, Harold McNair, professor emeritus of Virginia Tech's chemistry department. Dr. McNair has been active in the field for over 50 years, with a lengthy list of awards and accomplishments on his resume. Most recently, he won the  Horvath Medal in 2003 and the EAS Award in Analytical Chemistry in 2004. 

 Winners of the LCGC Lifetime Achievement Award 

 extends its warmest congratulations to this year's award winners and thanks everyone who participated whether by voting, submitting nominations, or contributing a  recommendation for a candidate. 

LCGC hands out a pair of awards at Pittcon 2009 in Chicago.

The 2009 LCGC Awards

In an ideal world, chromatographic peaks always would be perfectly shaped, symmetrical Gaussian entities, but in the real world of inlets, columns, and detectors, much can happen to distort peaks' shapes and lead to peak tailing — the appearance upon elution of a significant portion of a peak's mass away from the apex toward later times.  

The familiar Gaussian peak shape really is only an approximation. It arises from a simplified peak elution theory that assumes, among other things, that interactions between solutes and the chromatographic system are symmetrical and independent of solute concentration. In a real instrument, solutes encounter extra mobile phase volumes in inlets, connections, and detectors, and the solutes' chemical interactions with the system are composed of multiple, often nonideal, concentration dependent encounters with the stationary phase, the support, and other materials.  

 Solutes move through a chromatographic system while dissolved in the mobile phase — in the carrier gas when performing gas chromatography (GC). Solutes diffuse in all directions in the mobile phase as they are carried along. This is well and good as long as the flow path is free of extra void volumes or unswept areas. Figure 1 illustrates what can happen when void or unswept volumes are present. As a peak moves past an unswept area, some of the peak's mass can diffuse into it. After the peak moves on, solute molecules that diverted into the unswept area can diffuse back into the main mobile phase stream, but now they join the back of the peak and appear in the peak tail. The solute concentration in the unswept area declines at an approximately exponential decay rate as solute leaves the area and is replaced by carrier gas molecules. 

A number of problems can cause volumetric peak tailing in GC. Installing a fused-silica column with the tip of the column too low in a split–splitless inlet can create tailing peaks because the full carrier gas split flow cannot reach the column entrance and sweep residual sample away. A similar problem exists in some detectors if makeup gas is not used: solute molecules can become trapped in unswept detector areas. 

Volumetric peak tailing is a potential problem in splitless injection as well. Sample vapors expand rapidly and fill the inlet liner during the initial splitless injection period after sample has been deposited in the inlet liner. The split vent flow is disabled, so sample vapors flow through the liner and enter the column at the column flow rate: as much as 2 min can be required to transfer 80% or more of the vaporized sample into the column. Allowing this low flow condition to persist for much longer results in a severely tailing solvent peak that might obscure early eluted peaks. By establishing a high split flow after sample transfer into the column, the back of the solvent peak is cut off effectively. 

Lack of sufficient septum-purge flow also can cause peak tailing when some solute or solvent escapes from the inlet liner up into the septum area during injection and is trapped there. The volatile material will escape slowly, and if not swept away by septum purge flow, it will be free to diffuse back into the carrier gas split flow and on into the column. 

 As solutes move through a chromatographic system, they can undergo interactions other than normal adsorption–desorption onto or solution–dissolution into the stationary phase. One effect familiar to gas chromatographers is caused by active sites on surfaces exposed to the solutes — such as a poorly deactivated inlet liner or exposed areas of the column wall or support material. Such sites can engage in additional solute retardation mechanisms that hold solutes back from the main peak and cause tailing peak shapes. These interactions tend to manifest at lower solute concentrations, and as such, usually are present to some degree. Higher solute concentrations can provide enough additional material to saturate the active sites so that only a small fraction of the total solute present is affected.  

Poorly cut capillary column ends can expose active surfaces to the sample, as will the careless insertion of a column into a ferrule without making a clean cut afterwards. Accumulation of nonvolatile sample residues in the inlet or the column entrance also can lead to peak tailing, which will be cumulative as the residues build up from one run to the next. Sometimes overheating a column, or exposure to large amounts of the wrong solvent, will remove or strip the stationary phase and result in peak tailing from the exposed, more active surfaces. 

It is difficult to avoid some degree of peak tailing for polar or labile peaks that strongly interact with surface imperfections. Carboxylic acids as well as strongly basic compounds will tail strongly, although specialized stationary phases are available for many such problem samples. 

 Ideal stationary phase retention does not depend upon the solute concentration: the strength of retention, as expressed by the distribution coefficient 

, should be constant across an entire peak, from start to finish. In a sense, solute molecules should behave as if infinitely dilute in the stationary phase and should not be affected by the presence of other solute molecules.  

Peak tailing arises when solute interactions with the stationary phase change with concentration. As solute concentrations rise during the front half of a peak, the value of 

 might change if the chemical nature of the stationary phase is modified by the presence of larger solute concentrations, so that the solute molecules themselves become an active part of the stationary phase. As a result, those solute molecules present near the middle of the peak at sufficiently high concentrations will be shifted relative to those in low concentration areas near the start and end of the peak. Peak fronting results if concentration-related changes in retention across a peak cause the peak's center of mass to shift to later retention times, while tailing occurs if the opposite is true. Figure 2 illustrates these situations. 

Fortunately, concentrations sufficiently high to cause these effects are not encountered normally in elution gas chromatography, except in the case of a large solvent peak such as encountered in splitless injection. Figure 3 shows what a fronting splitless solvent peak looks like when viewed with the entire splitless solvent peak on scale, as compared with the more familiar square looking peak viewed at sensitivity high enough to reveal solute peaks at normal GC concentrations. Splitless injection uses this solvent peak fronting effect to advantage by engendering a rapidly declining solvent peak, which makes it easier to discern peaks that are eluted close after the solvent peak. This effect is in addition to the solvent peak cutoff that occurs when split flow is restored after a splitless injection. 

Several peak-tailing measurements are in use today. Most of these measurements do not correlate directly or scale with each other, so it is important to use one measurement method consistently. There is no standard symbol for chromatographic peak symmetry or asymmetry: in this article, the symbols 

, respectively, will be used. The subscripts will be followed by an indication of the type of measurement used for the calculation, for example, the term 

 refers to an asymmetry calculation made on the basis of peak measurements at 10% of the peak height. 

Peak symmetry and asymmetry are terms used in this context to describe either the lack or extent of peak tailing or fronting. Peak asymmetry is equal to one over the symmetry, as calculated from the same measured parts of a peak: 

Peak asymmetry is sometimes called the tailing factor; however, this is true only for tailing peaks. A value of 1.0 indicates the peak is symmetrical — its front and back sections are nominally the same width. Asymmetry measurements for the peak in Figure 4 are given in Table I for each of the following calculation methods. 

 The simplest type of peak symmetry measurements relate the width of the peak before and after its retention time. Figure 4 shows a tailing peak and various measurements of the times between the peak's rising side, its retention time, and the falling side of the peak. 

Table I: Asymmetry and other measurements of the tailing chromatographic peak in Figure 4			

One commonly used (1) asymmetry measurement takes the ratio of the front to back widths at 10% of the peak height: 

In this measurement, an asymmetry value of greater than one indicates a tailing peak, and an asymmetry less than 1 indicates a fronting peak profile. 

A related measurement uses the front and back widths at the baseline, where the lines tangent to the peak's inflection points intersect it: 

The United States Pharmacopeia (USP) definition of asymmetry (2) uses peak widths at 5% of the peak height to divide twice the front peak width into the total width: 

This definition assumes that, for moderately tailing peaks, the front width of the peak is close to the front width of a nontailing peak: the term 2

 is an approximation of the width at 5% that a nontailing peak would exhibit. Thus, equation 4 gives the ratio of the actual peak width at 5% to an approximation of the total peak width that might be observed if there were no tailing. The more strongly a peak tails, the larger the value of 

It is important to note that equation 2 (now calculated with peak widths measured at 5% of the peak height) and equation 4 do not produce the same values except when the front and back widths are the same and the asymmetry is equal to 1.0: 

If the peak has a fronting profile, the 2

 term in equation 4 no longer approximates a nontailing peak's width. Instead, an estimate of twice the peak's backwidth would be suitable in the fronting-peak case. Ettre (3) defines asymmetry in terms of the absolute difference Δ

 between the front and back portions of a peak: 

The asymmetry of a peak can then be written as

Asymmetry calculated according to equation 4 is equal to that calculated using equation 7 with measurements at 5% of peak height when the back part of a peak is larger than the front part, in other words, for a tailing peak. For a fronting peak, equation 7 gives the same asymmetry value as for an equivalent but reversed tailing peak. In this case, however, it is necessary to specify whether the peak in question has a tailing or fronting profile. 

 Two other measurements of peak tailing should be mentioned. First, some authors have used the ratio of the area of a peak after the apex, to the area before the apex: 

This metric is more difficult to calculate unless it is preprogrammed into a chromatography data-handling system. 

Another method uses the statistical moments or their estimates to compute asymmetry. The statistical moments themselves, as computed from the raw chromatographic data points, are prone to large fluctuations due to the statistical accumulation of noise present in the base chromatographic signal. Pseudomoments can be estimated from area slices across sections of a peak and then used to calculate asymmetry. Interested readers should refer online to reference 4 for a detailed description, which is too long to include here. The calculation of asymmetry from pseudomoments of the peak in Figure 4 was performed by adding the relevant computations described in reference 4 to a proprietary chromatography data-handling system, and not with the commercial system mentioned in the reference. Due to the nature of ongoing software development, subsequent revisions to the commercial software can produce different results than described in the reference. 

Finally, it is possible to fit a theoretical peak shape to the real chromatographic data, and then derive the asymmetry plus many other statistics from the fitted peak. Felinger's comprehensive book 

Data Analysis and Signal Processing in Chromatography

 (5) describes at least 11 variations on various empirical peak-shape models that can be applied. The exponentially modified Gaussian (EMG) method was used to generate the peak in Figure 4. Again, however, these methods must be programmed into a computer system for practical implementation. 

Most chromatographic peaks tail to some degree. Small amounts of peak tailing are tolerated and not usually observed except at very low concentrations. The presence of significant peak tailing indicates a potential problem with extra unswept dead volumes, low flow rates, active sites, or nonlinear retention effects.  

Peak asymmetry can be calculated by a number of different methods that are not directly related. Chromatographers should ensure that peak asymmetry and other statistical measurements are measured consistently with the same methods from well controlled chromatograms whenever such metrics are to be used for comparative, quality control (QC), regulatory, or system suitability requirements. 

The causes and cures for peak tailing are discussed, along with the various ways that chromatographers measure tailing peaks.

LCGC North America-03-01-2009

Calibration Curves, Part I: To b or Not to b?

Electrostatic Repulsion Hydrophilic Interaction Chromatography

Separation, Identification, and Quantitation of Water-Soluble Vitamins by HPLC

New Chromatography Columns and Accessories at Pittcon 2009: Part I

The 2009 LCGC Awards

Pinning Down Tailing Peaks

Calendar