The Advantages of a Separate Calibration

File in Galaxie™ Chromatography Software

By Gary Burce

Abstract
Galaxie uses a separate file for a calibration curve. This allows a curve to be used with several different methods
customized to different sample matrixes or different sample concentrations that require different data handling.
An example of this is found in the analysis of anions and cations in boiler water.

Introduction...............................................................................................................................2

Background...............................................................................................................................2

The Galaxie solution.................................................................................................................2

Cation sample analysis ............................................................................................................2

Anion sample analysis .............................................................................................................6

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Introduction
With the Varian, Inc. Galaxie™ Chromatography Software, the same calibration file can be used
with different methods, saving time and simplifying operation. Samples and standards, which
require different peak processing or different peak identification, can be run without the operator
modifying the method either manually or during automation and without running separate
standards for each method.

Background
Generally, once the instrumental parameters of a chromatographic method have been
developed, the method can be utilized for samples of significantly different concentrations and
for samples in significantly different matrices. If the method is robust, changes in concentration
and sample matrix still produce separated peaks.
Although a single set of instrumental parameters will produce an acceptable separation, the
peak processing may need to be changed for different types of samples. For example, widely
diverse concentrations may produce differences in retention times and peak shapes that need
different peak processing or peak identification parameters. Also, significant differences in
sample matrices and interfering compounds may require different peak processing parameters.
These variations demand either different data handling methods or manually modified integration
for many of the chromatograms.
If different methods are used, most chromatography software require separate calibrations for
each method because the calibration curve is permanently stored as part of the method.
Therefore, the chromatographer must either modify the method peak processing parameters for
each standard or sample type or run many different methods each with its own peak
identification and peak processing parameters. This can add significant complexity and
overhead to the operation of a routine lab.

The Galaxie solution

Galaxie avoids this problem completely by having a separate calibration file. Whenever a
method, which uses this calibration file, is run, standards can be added to the calibration for
determining the calibration curve. In addition, samples can be analyzed with this curve, even if
they have completely different peak processing or peak identification parameters in the method.
The Galaxie separate calibration file can be used by any number of methods both for calibration
and analysis.

Cation sample analysis

Anions and cations in boiler water can be analyzed on an ion chromatograph. Typical cations
that are analyzed include lithium, sodium, ammonium, phosphate, magnesium and calcium.
Typical anions include fluoride, chloride, nitrate, phosphate and sulphate.
A typical separation of cations at high and low concentrations is shown in Figure 1.

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 Figure 1. Cation standards at high, medium and low concentrations.

The retention times for the later eluting peaks, magnesium and calcium, are significantly different
between the concentration standards. Therefore, the peak identification table requires
significantly different retention time targets as shown in Table 1.
Table 1. Expected retention times for the high, medium, and low concentration cation standards.

Compound Retention time (min) Retention time (min) Medium Retention time (min)
High concentration concentration Low concentration
Lithium 4.15 4.20 4.20
Sodium 5.15 5.15 5.15
Ammonium 6.05 6.10 6.20
Potassium 8.00 8.00 8.25
Calcium 12.50 14.00 14.00
Magnesium 16.00 17.90 18.10
Note that the changes in retention times are not proportional across the entire chromatogram.
Therefore, relative retention time identification using reference peaks cannot properly identify the
peaks. Also, even though there is a significant difference in the peak shape, Galaxie™ easily
handles these changes with a single set of peak integration parameters.

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Calibration is done by running three different methods, Cation Hi, Cation Mid and Cation Low,
with three different standards but referencing the same calibration file in the method. In Galaxie,
the calibration file is referenced in the method as shown in Figure 2.

Figure 2. Galaxie calibration file referenced in each method

Galaxie adds peaks to the calibration curve by name, not by retention time. Therefore, peaks
with the same name will be part of the same curve, even if they have different retention times in
different methods. As shown on the chromatogram and in the table above, the peaks changed
retention times significantly. Even so, calibration resulted in excellent calibration curves for the
peaks, as seen in Figures 3 and 4.

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Figure 3. Calibration curve for magnesium

Figure 4. Calibration curve for sodium

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 These calibration curves are then used to calculate the results from two different types of
samples, a “raw” water sample and a “boiler feed” water sample.

Figure 5. Chromatograms for the low standard, raw water sample and boiler feed water sample.

The boiler feed water has a longer retention time for calcium than the low standard. Therefore,
another method is created to identify these peaks. Now, routine analysis can be performed
based on the method optimized for the type of sample and a single calibration file.

Anion sample analysis

The analysis of anions presents a different problem. The retention times are consistent between
the standards at different concentrations. (As with cations, the high standards have slightly
shorter retention times than the low standards.)The samples also have slightly different retention
times as shown in Figure 6.

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Figure 6. Anion standards and boiler feed sample.
Figure 6 also shows that the chromatographic baseline is significantly different for the early
eluting peaks. The higher concentration standard has a flat baseline. The lower concentration
standard and the boiler feed sample have significant baseline upsets near the fluoride and
chloride peaks. Different timed peak processing events are needed to optimally process these
peaks. Table 2 lists the different peak processing events for each method.

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 Table 2. Galaxie integration events.
Time Low Standards High Standards Boiler Feed Water
0.00 Width = 0.1 Width = 0.5 Width = 0.1
0.00 Threshold = 100 Threshold = 100 Threshold = 100
0.00 Turn Integration Off Turn Integration Off Turn Integration Off
0.00 Min Area = 5 Min Area = 100 Min Area = 5
0.80 -------------- Turn Integration On -----------------
0.88 Turn Integration On --------------------- -------------------
0.9 Baseline Now
1.05 Turn Integration On
1.30 Baseline Now --------------------
2.00 Width = 0.5 -------------------- Width = 0.5
2.00 Threshold = 100 -------------------- Threshold = 100
Raw water, which has concentrations similar to the high concentration standards, uses slightly
different peak processing events to obtain optimal results, as noted in the Figure 7 annotations.

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Figure 7. Comparison of timed events for the raw water method and the high standards method.

The entire process of calibration and analysis can be done automatically. A Galaxie™
sequence, as shown in Figure 8, can be used for calibration and analysis in a single run.

Figure 8. The Galaxie sequence for anion analysis.

Six methods are run in sequence. The first two lines are used for calibration at two levels using
two different methods. The remainder of the lines are used with methods optimized for both raw
water and water from different boilers. By having a different method for each boiler, the data
files can be easily given distinctive names and stored in distinctive folders, making the data self
organizing.

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Benefits of a separate calibration file:
Accurate and precise results can be obtained for different types of samples by using the
different peak processing time events to set the baseline correctly for each type of sample.
Operations are simplified when methods are optimized to each type of sample so that plant
operators can easily use them to analyze samples without any manual peak processing.
The data is virtually self-organizing as a result of methods being dedicated to different sample
sources, allowing different file names to be preset for each sample source.
Galaxie saves the time for running multiple redundant calibration standards by enabling the
same calibration curve to be used for different methods.
Galaxie™ makes this possible because the calibration file is separate from the peak processing
and peak identification parameters in a method. See Figure 9.

Figure 9. Calibration file for anions entered into the calibration section of a Galaxie method

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