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GC Connections
In this third part of a series, John Hinshaw examines some techniques that simulate gas chromatography results under varying conditions. He provides a route to improved separations that is easier and less time-consuming than performing repetitive brute-force elution experiments.

Strategies for GC Optimization, Part III Software

John V. Hinshaw
GC Connections Editor

n part I of this series (1), I discussed general strategies and considerations for achieving improved results in gas chromatography (GC). Part II (2) gave examples of ways to optimize column stationary phase and dimensions to improve resolution or speed of analysis. In this third part, Ill look at some techniques that compute GC results under varying conditions and provide a route to improved separations. The techniques Ill describe are easier and less time-consuming than performing repetitive elution experiments. The manipulation of capillary column parameters stationary phase, inner diameter, length, and lm thickness gives chromatographers control over column efficiency, resolution, and the speed of analysis. The limits of inlet pressure, sampling systems, and detectors imposed by available instrumentation and methodology establish practical boundaries for the column variables and resulting chromatograms. The choice of stationary phase determines, rst and foremost, for a given sample how much column efficiency a separation will require to meet minimum standards of resolution. How rapidly that separation is generated is a function of column dimension and elution condition choices. When presented with a large number of closely related peaks or with an unknown separation, chromatographers should err on the side of high resolution to ensure that peaks are not coeluted. For a wellcharacterized separation, however, optimization for speed or resolution may be attractive. For situations in which peaks are well separated with excess resolution, chromatographers can increase the speed of analysis by using shorter columns, higher carrier-gas linear velocities, or columns with larger inner diameters. When one or more peak pairs are marginally resolved, longer or narrower columns will deliver higher plate numbers and increased resolution, but they will sacrice analysis speed to do so. Alternatively, small changes in temperature and pressure

conditions can provide a needed resolution boost. An alternative solution may lie in choosing a different stationary phase that better separates the peaks of interest. The interplay of all the chromatographic variables presents a complex picture, even to experienced chromatographers. A number of commercially available computer programs can alleviate this complexity and facilitate a visualization of the effects that result from changing the variables. In addition, the programs perform various types of optimization calculations directed toward achieving a set of dened goals. The optimization programs discussed in this column manipulate elution conditions such as temperature program rate, carriergas ow, and column dimensions to realize improved resolution, higher speed of analysis, or the use of less expensive columns. I searched the Internet as well as manufacturers information to locate the products listed in Table I. The descriptions in the table come from the suppliers web site, catalog, or from the software descriptions contained in its help les. The programs are suitable for specic types of optimization problems; each targets aspects of GC optimization and approaches its goals in different ways. Some, such as OnLine GC Lab, address specic tasks; in its case, on-line isothermal GC. Others, such as DryLab or Pro ezGC, are more general in nature. I chose these programs as examples of chromatographic optimization software, and their mention in this GC Connections column does not constitute an endorsement of any sort, nor do I intend this article to review or rate the programs in any way. Interested readers should investigate the programs themselves by visiting the suppliers web sites for detailed information and by trying the available demonstration versions.
Computer Optimization

Optimization by computer program is based on two fundamental components: a

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Table I: Software for GC optimization Product Name DryLab chromatography modeling software GC Capillary Column Method Translation software Manufacturer* LC Resources Description Models changes in separation conditions using a PC to generate chromatograms that correspond to selected separation conditions based on two or more calibration runs. Ports an existing GC method to another while ensuring that relative retention order is maintained. Works for changes in carrier-gas type, column dimensions, phase ratio or lm thickness, column outlet pressure, and carriergas ow rate. Simulations determine the effects of varying temperature, pressure, and column size conditions on separations. Column modeling and optimization program for modeling and predicting the isothermal performance of fused-silica open tubular columns with bonded phases. The model is valid for single-phase column or dual-phase series coupled (end-to-end) systems. A computer algorithm calculates thermodynamic retention indices for sample components, and the software predicts the best combinations of column dimensions, temperature program, and ow conditions.

Agilent Technologies

GC-SOS gas chromatography simulation and optimization software OnLine GC Lab

ChemSW

OnLine Analytics

Pro ezGC method development software

Restek

* Agilent Technologies (Palo Alto, California); ChemSW, Inc. (North Faireld, California); LC Resources Inc. (Walnut Creek, California); OnLine Analytics, Inc. (Duxbury, Massachusetts); Restek Corp. (Bellefonte, Pennsylvania).

system model and an optimization engine. The system model provides information about how a system chromatographic peak elution in this case behaves as independent variables change. The optimization engine uses the system model to compute sets of variables that most nearly achieve a desired goal. For GC optimization, the independent variables are the familiar list of column dimensions, stationary-phase chemistry, carrier-gas pressure, and column temperature. Other inuences that are external to the column, such as injection bandwidth or detector response times, may come into play in simulations that also render peak shapes. Chromatographers operating the software sometimes play the role of the optimization engine by examining the results of GC simulations throughout a range of variables and then picking out the most suitable conditions. Computer-based optimization engines range from a minimum resolution threshold calculation to more complex simplex optimization schemes. They select sets of conditions that satisfy chromatographers optimization criteria. All the optimization results rely on the accurate input of one or more sets of real chromatographic retention times and conditions. If the input data were inaccurate, predicted results might fail to mimic realworld chromatograms closely enough to be useful. Secondary to the input data, the system model must encompass sufficient degrees of freedom and response functionality to ensure accurate results prediction.

System models for GC: The system models that GC optimization programs use vary somewhat in their implementation, but they all are founded on thermodynamics models of GC separations; namely, the dependence of peak retention on temperature, pressure, and column variables. Hand-calculator or spreadsheet calculations suffice for isothermal models that can predict retention times, separation factors, and resolution with very good accuracy for a single column. Matters become more complex, however, when it comes to making predictions of temperature- or pressureprogrammed runs or to forecasting the effects of changing column dimensions. It is possible to use a spreadsheet to compute programmed-elution predictions, but the process is like using a penknife to carve an intricate wood sculpture its slow, laborious, and difficult to repeat. Chromatographers who are skilled in computer programming or who have access to programmers might choose to author a program for these purposes, but most will be better off purchasing a commercial product and taking advantage of the program creators experience. Isothermal elution: For isothermal, isobaric (constant-pressure) elution, the measurement of peak retention parameters at several temperatures reveals a linear relationship between the reciprocal absolute column temperature (Tc, measured in kelvins) and the log of the peaks retention factors (k)

log k 5

A 1B Tc

[1]

where A and B are arbitrary constants that are specic to individual compounds on a particular column. Most solutes retention times decrease by one-half for every 1520 C increase in the column temperature. This relationship is independent of a capillary columns pressure drop, because the retention factor does not depend upon it. Thus, equation 1 predicts the retention behavior of peaks whose retention factors are known for at least two temperatures, and variations in pressure drop or ow with temperature are accommodated by the use of the retention factor. Figure 1 plots experimental data and curves tted by linear regression to equation 1 for several solutes in a test mixture across a range of temperatures. This example appeared in a GC Troubleshooting column, in which I discussed several schemes for manually applying these types of data and plots to isothermal GC optimization (3). It is a fairly simple matter to compute coefficients A and B for each peak of interest by linear regression and then nd the peaks predicted retention factors at specic temperatures. However, the peaks predicted retention times are needed for chromatogram simulation and resolution calculations. The scenario gets a little more complex when I begin to pursue peak retention times, but bear with me for a few more

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equations. Have another cup of coffee if youd like. The retention times (tR) are a function of their individual retention factors and the unretained peak time (tM): tR tM(k 1) [2]

P pi/po

[5]

Therefore, in addition to knowing the retention factors from equation 1, chromatographers need to predict the unretained peak time at the desired temperatures. Fortunately, if not simply, the unretained peak time is a function of the column dimensions, pressure drop, carriergas type, and column temperature; it can be calculated for simulation purposes as follows. The pressure drop across an opentubular column is related to the column dimensions and the carrier-gas viscosity according to these relationships, which are derived from the HagenPoiseuille equation (4):
Dp j 9 5 32L h u d2 c P1 1
2 2

In equations 35, p is the pressure drop across the column, j is the reduced pressure correction factor, L is the column is length, is the carrier gas viscosity, u the carrier-gas average linear velocity, dc is the column inner diameter, P is the relative pressure, and pi and po are the absolute inlet and outlet pressures, respectively. The unretained peak time can be related to the pressure drop, column dimensions, and carrier-gas viscosity by combining the familiar relationship between column length, unretained peak time, and average carrier-gas linear velocity tM L/u with equations 35 to yield
L dc
2

the same unretained peak time as a 15 m 100 m column at the same temperature and pressure drop, for example. The carrier-gas viscosity () increases with rising temperatures according to the following relationship:
T 0(T/273.15)x

[7]

t M 5 32

h Dp j 9

[6]

[3]

j9 5 0.75

[4]

P 1 P1 1

Interestingly, equation 6 predicts that at constant temperature and pressure drop, the unretained peak time will be unchanged if the ratio of the column length to the diameter remains constant. A 30 m 200 m column should have

In this relationship, T is the viscosity at temperature T, 0 is the viscosity at 273.15 K, and x is an exponent specic to the type of carrier gas (5). As a bonus, by incorporating the column and carrier-gas variables with the unretained peak time calculation, the simulation can be extended to accommodate dimensional and carrier-gas changes. To do so, however, I will have to make some changes to equation 1 rst. Equation 1 is written in terms of the retention factor, which is independent of the carrier gas and pressure drop but dependent on the column dimensions. The peaks thermodynamic distribution coefficients (K ) remain constant across column dimensional changes, including changes to the stationary-phase lm thickness. Rewriting equation 1 with K and changing it to the natural log yields equation 8:
ln K 5 a 1b Tc

[8]

1.60 1.40 1.20 1.00

n-Dodecane Naphthalene 2, 4-DMA 2, 6-DMP 1-Octanol

25 20

10

This equation is essentially the same as equation 1, except that the new constants a and b are directly related to the entropic and enthalpic thermodynamic coefficients H and S, respectively,
a5 2 DH R DS R

5 0.80 0.60 0.40 0.20 130 120 110 100 90 80 Column temperature (C) 2

tR (min)

[9]

log k

b5

[10]

Figure 1: Plot of log k and tR versus 1/Tc for ve peaks at various temperatures. The individual points represent experimentally measured retention values; the lines represent best-fit linear regression of the experimental points for each component. Column: 30 m 530 m, coated with 3-m 5% diphenyl95% dimethylpolysiloxane at a constant 12 mL/min helium ow rate. Sample: 0.1 L direct injections of a 2-g/mL 1-propanol solution. The temperature scale indicates degrees Celsius for clarity of presentation; the data are plotted against reciprocal kelvins (3).

where R is the universal gas constant. This relationship derives from equilibrium partition theory (6), and it is the close correlation of the theory with experimental results that makes accurate GC simulations possible. To use equation 8, I will need to relate the retention factor to the distribution coefficient. Chromatographers can measure retention factors directly from the chromatogram if the unretained peak time is known. The distribution coefficient in equation 8 is not directly evident; however, it can be computed from the measured retention factor, if the column lm thick-

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ness (df ) and inner diameter (dc ) are known. These two column measurements determine the phase ratio (), which is the ratio of the gas to stationary-phase volumes in the column:
b dc 2 4df 4df

[11]

The retention factor and the distribution coefficient are related by the column phase ratio, as shown in the following equation: K k [12]

So if I know the column phase ratio and the peaks retention factors at various temperatures, I can compute the distribution coefficients. With that information I can nd the thermodynamic parameters that characterize the peaks on a specic stationary phase. Optimization programs use at least two and often three sets of known retention data at different temperatures. By making some assumptions about the value of H, one set of calibration data can be used, but the results will be approximate. Alternatively, some optimization programs provide predetermined libraries of compounds already calibrated on several popular stationary phases. If a peak of interest is in this type of library, then no additional calibration may be necessary as long as the calibration column and the experimental column dimensions are known with good accuracy, the calibration GC oven temperature and experimental oven temperatures are standardized, and the pressure drops and ambient pressures for the calibration and experimental sys-

tems are known accurately. If these conditions are not true, then the simulations will be less accurate. Small errors in these areas will not distort simulated results so much that peak elution order and relative retention will be meaningless. Even when simulations are not exact to the second, they provide a wealth of useful information about peak retention behavior under a range of test conditions. Now, I am in a position to compute peak retention times in terms of their measured thermodynamic coefficients, the column dimensions, the carrier-gas type, the pressure drop, and the column temperature. By combining equations 6 and 2, I obtain the following relationship:
t R,T 5 32 1 1 kT L dc
2

hT Dp j 9

[13]

Figure 2: Example of isothermal GC chromatogram simulation. The Analysis tab (shown) displays the elution conditions plus the retention times, peak widths, effective theoretical plate numbers and resolution for the two peaks anking the cursor. Program: GC-SOS v 5.1.

In this equation, the subscripted Ts refer to the parameters values at the desired simulated column temperature. The retention factor at column temperature (kT ) is computed from equations 8, 11, and 12. Known retention and unretained peak times from calibration runs at two or more temperatures provide the thermodynamic coefficients a and b for this purpose. The viscosity at column temperature comes from equation 7. By using equation 13, algebraically competent chromatographers can predict the effects of changing column dimensions, linear velocity, phase ratio, carrier gas, pressure drop, and temperature on simulated isothermal retention times for peaks whose retention times are known at two or more temperatures on a single stationary phase. They also can achieve these manipulations using a hand calculator and a lot of effort or more easily using a computer spreadsheet that can also graph the results. Naturally, the commercial programs listed in Table I excel at these calculations, although they dont all use formulations exactly the same as the ones I have provided here. Figure 2 shows an example screen of an isothermal GC simulation. In this computer program, the Temperature, Pressure, and Column tabs in the display permit users to set elution conditions, including multiramp temperature and pressure programming. I happened to choose this software for an isothermal example, but it isnt limited to this case. The Auto-Optimize tab performs a minimum-resolutionoriented optimization calculation, which

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determines a set of conditions that lie within specied limits and meet the minimum resolution criterion. Programmed elution: When it comes to programmed-temperature or -pressure elution, GC simulations divide the programmed elution run into a large number of discrete steps or into small time increments. Instead of computing the retention time, equation 13 is modied to calculate the distance a peak moves along the column in the z direction during each interval. The motion of each peak is summed with each step to simulate transport, separation, and elution. The transformation of equation 13 from the temperature domain to time incorporates an arbitrary temperature and pressure program, including static plateaus and negative ramps. The local carrier-gas linear velocity at the peaks position in the column (u z ) determines the distance the peak moves during one step. When the peaks position equals the column length (L), the peak is eluted from the end of the column, and that time is equal to the retention time. If I consider the increments to be innitely small, then I can write an integral to express the process of peak elution:
tR 0

uzdt 5 L

[14]

This deceptively simple expression incorporates all the variables mentioned previously and includes the temperature and pressure programs as discrete timebased functions. The solution to the integral is complex, even for simple cases. It

becomes quite unwieldy for discontinuous temperature or pressure programs, so a numerical solution is the most common approach. This kind of computationintensive elucidation lends itself to a computer program, and it or similar processes form the basis of the simulation programs that incorporate programmed-temperature and -pressure solutions. Figure 3 is an example screen of a programmed-temperature simulation. In this example, one chromatogram from a set of chromatograms that satisfy minimum resolution criteria is displayed in the top window, and a synopsis of several of the solution sets appears below it. The program varies the column length, the temperature program, and the pressure drop across dened ranges to arrive at a limited set of acceptable solutions. Solutions to chromatogram simulation exist for isothermal, isobaric elution, as well as more complex and more common programmed-temperature and -pressure separations. The discussion above presents the basics of a theoretical treatment that can be applied to these computations. I havent addressed a number of second-order effects that also come into play, including additional temperature dependencies; compensation for small errors in actual column dimensions, pressures, and temperatures; and masstransport effects that stem from the compressible nature of the carrier gas. Some of these factors may be included in the simu-

Figure 3: Example of temperatureprogrammed GC chromatogram simulation. The upper window displays the simulated chromatogram and notation. The lower window lists a series of solution sets that meet the users criteria for minimum resolution of the closesteluted peaks across a range of elution conditions. Program: Pro ezGC for Windows.

Figure 4: Example of window diagram presentation of optimization results. The upper window graphs the resolution of the leastresolved peak pair as a function of temperature program rate. The lower window simulates the chromatogram corresponding to the program rate indicated by the arrow and dotted line in the upper window. The areas to the left describe the column and give information about the peak selected by the arrow under the chromatogram in the lower window. Program: DryLab 2000.

lation programs listed here, because they constitute implementation details that improve the accuracy of simulation. Optimization engines: With an accurate simulation model in place, an optimization program is in a position to select conditions that satisfy criteria for improved resolution, faster speed of analysis, and different column dimensions. Programs that do so include an optimization routine that computes simulation results within a dened range of independent variables, displays the simulation results as a function of one or more of the variables, and indicates the optima. Window diagrams: An elegant and highly useful approach to optimization, window diagrams display aggregate peak resolution as a function of one or two independent variables such as temperature or pressure. A window diagram plots resolution curves or surfaces against simulated changing elution conditions. Chromatographers can see quickly which conditions produce the best tradeoffs between, for example, analysis speed and resolution. Figure 4 illustrates a window diagram and chromatogram display. In this gure, the window diagram appears in the top half of the display. Resolution is plotted against temperature program rate. The ascending and descending curve represents the predicted resolution of the closesteluting peak pair at each program rate. The curve is discontinuous wherever two pairs of peaks exhibit the same minimum resolution, for example at approximately 6 C/min. One pair has the least resolution of all peaks at lower rates, and a different pair has the least resolution at higher rates, as high as approximately 10.5 C/min. At this program rate, coelution occurs and the minimum resolution drops to zero. As the rate increases further, the two coeluted peaks begin to separate with higher resolution until at approximately 19 C/min another peak pair displays a lower resolution. The vertical red lines at 1 and 20 C/min represent the lower and upper bounds of the optimization experiment chosen by the operator. The vertical dotted line above the arrow at the bottom of the window diagram indicates the program rate for the simulated chromatogram displayed in the lower window on the screen. The effect of program rate on speed and resolution is apparent in Figure 5. In this gure, the selected program rate of 19.5 C/min causes the last simulated peak to be eluted in approximately 8 min, com-

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pared with 33 min at 1.5 C/min in Figure 4. At the lower optimum program rate, the minimum resolution is 1.64, which is just greater than the baseline resolution of 1.5. At the higher program rate, it drops off to 1.39, which is slightly less than baseline. Remember that this is a simulation. If analysis speed were a concern in this case, it would be worthwhile to make a couple of real chromatographic runs at those two program rates and determine the real system performance. The actual resolution could be somewhat greater or less than the predicted amount. This example makes the advantage of chromatogram simulation clear. Two real runs were acquired for the system calibration at different initial and nal temperatures than were used for the illustrated simulations. Two more runs at the predicted optimum rates of 1.5 and 19.5 C/min would be enough to conrm the simulated results. Without chromatogram simulation, operators would have to acquire many more runs and guess at the best set of conditions. The programs described in this column possess various additional simulation capabilities, such as modeling the effects of pressure programming, column dimensional changes, serially coupled columns, and peak broadening. As I stated in the introduction, chromatographers who are interested in these programs should determine for themselves which is best suited to their needs. The Capillary Column Method Translation software from Agilent Technologies (Palo Alto, California) is a very useful tool for translating a method across column dimensional changes while maintaining peak elution order. It does not simulate chromatograms, and it does not compute optima. It helps to achieve increased speed of analysis by computing a new method at

reduced column diameter and length that affects the chromatogram only by compressing it into a shorter time. Analysts also can use this software to compute a method that is compensated for the effects of changing from a ame ionization detector that holds the column exit at room pressure to a mass spectrometry detector with the column end at vacuum.
Conclusion

Gas chromatographers occasionally need to adjust elution conditions and column dimensions to optimize a separation. They may want to increase the analysis speed, improve the resolution of a critical pair of peaks, or translate a method to a less expensive column or different detection system. The GC elution process can be modeled thermodynamically by calibrating a theoretical representation of a GC system with known peak elution data. Selected conditions can be modied and the results simulated in a manner directed toward achieving a stated goal. A number of commercially available software programs perform this kind of optimization process to assist chromatographers with the otherwise time-consuming and laborious task of obtaining real chromatograms throughout a sufficiently wide range of variables.
References
(1) (2) (3) (4) J.V. Hinshaw, LCGC 18(3), 296304 (2000). J.V. Hinshaw, LCGC 18(6), 610616 (2000). J.V. Hinshaw, LCGC 9(2), 9498 (1991). L.S. Ettre and J.V. Hinshaw, Basic Relationships of Gas Chromatography (Advanstar Communications, Cleveland, Ohio, 1993), 3643. (5) J.V. Hinshaw and L.S. Ettre, J. High Resolut. Chromatogr. 20(9), 471581 (1997). (6) B.L. Karger, L.R. Snyder, and Cs. Horvth, An Introduction to Separation Science (John Wiley & Sons, New York, 1973).

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John V. Hinshaw
GC Connections editor John V. Hinshaw is president and principal scientist of ChromSource, Franklin, Tennessee, and a member of LCGCs editorial advisory board. Direct correspondence about this column to GC Connections, LCGC, 859 Willamette Street, Eugene, OR 97401, e-mail lcgcedit@lcgcmag.com. For an ongoing discussion of GC issues with John Hinshaw and other chromatographers, visit the Chromatography Forum discussion group at www.chromforum.com.

Figure 5: Window diagram display with a simulated chromatogram at an elevated temperature program rate. Circle 28