chm 418, experiment 08 - design of experiments using a hewlett-packard 5890 series ii gas...

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Design of Experiments using a Hewlett-Packard 5890 Series II Gas

Chromatography Machine

Tyler Hacker, W. Daniel Smith & Nathaniel Wise

Bob Jones University, Division of Science, Department of Chemistry 1700 Wade Hampton Blvd. Greenville, SC 29614

Abstract

Chromatography is an important analysis tool for the chemist. Learning to properly utilize

instruments available will aid tremendously in the chemist’s career. To that end, this lab group

performed a design of experiments lab, using software to predict the optimal conditions for

adequate resolution of peaks and minimum retention time. Thirty samples were run with different

values for several variables. These were then used to give conditions for a final set of three runs, allof which exhibited good resolution and tolerable retention times.

Introduction

Gas chromatography is one of the more

widely used types of chromatography. It is

used for the separation and analysis of

compounds that can be vaporized. Many

times, gas chromatography is used to test thepurity of a substance, or to help in the

identification of the solution analyzed. In

separation chromatography, there are two

phases. The first is the mobile phase, in which

the sample is dissolved. The mobile phase is

then forced through the stationary phase,

which is fixed in place on a column. The

mobile phase in gas chromatography is a

carrier gas, which can be either an inert gas or

an unreactive gas. For this experiment,nitrogen, and unreactive gas, was used as the

carrier gas. The stationary phase for this

experiment was inside a fused-silica capillary

column. Different compounds being analyzed

interact with the stationary phase on the walls

of the column, and this causes the

compounds to pass through the column, or to

be eluted, at different times which are called

retention times. Many different types of

detectors are used with gas chromatography.

The most common of these are flame

ionization detectors (FID), which are more

sensitive to hydrocarbons. Effluent from the

column is passed through a hydrogen flame, which will produce ions and electrons at the

temperature of the flame. The current

produced by these charges are monitored as

the ions and electrons move toward the

collector electrode, located above the flame.

The resulting current is measured, and the

chromatogram is acquired.

In this experiment, four fatty acid methyl

esters were analyzed. This was done by

varying the initial injection temperature, initial

column oven temperature, final column oven

temperature, and the temperature ramping

rate. Using these data, a design of experiment

(DOE) was conducted. From the acquired

data from the first part of the experiment,

calibration curves were generated. With these



curves, the ideal separation conditions for the

fatty acid methyl esters could be predicted.

Also, unknown samples could be assessed to

determine their identity as well.

Another example of the use of calibrationcurves is found in an article found in the

Journal of Analytical Chemistry. The authors

in the reported experiment needed to find the

best coating possible for the materials used to

make stents. In order to do this, calibration

curves were established from data acquired

from confocal Raman microscopy

measurements. From the values obtained

from the calibration curve, a coating with the

right percentages of the chemicals sirolimus,PBMA, and PEVA along with others, were

found. (Balss, K., et al. Multivariate Analysis

Applied to the Study of Spatial Distributions Found

in Drug-Eluting Stent Coatings by Confocal Raman

Microscopy . J. Anal. Chem. 2008, 80, 4853–

4859.)

Also, and example of the use of gas

chromatography is found in an article in the

Journal of Agriculture and Food Chemistry.For this experiment, the authors used gas

chromatography along with chemometric data

analysis and solid-phase microextraction to

determine whether or not apple puree had

been added to samples of strawberry. When

comparing the chromatograms of adulterated

strawberry and unadulterated strawberry

samples, the differences were considered

obvious. However, the authors concluded that

only the sample adulterated to about seventy percent puree was easily distinguishable.

(Reid, L., et al. Potential of SPME-GC and

Chemometrics To Detect Adulteration of Soft Fruit

Purees . J. Agric. Food Chem. 2004, 52, 421-

427.)

Methodsand

Materials

This experiment used a Hewlett-Packard 5890

Series II gas chromatography machine. After

prepping the GC instrument and allowing it

to warm up, we programmed the auto sampler

to the conditions specified. The initial time

was set to 1.00 min; final time to 25 min;

dectector temperature to 250° C; and split

ratio to 75/1. The instrument automatically

ran through all the samples, and we processed

the data on the computer. It is presentedbelow. Once the computer gave us the

optimal values for the final runs, we

programmed those in and completed the

experiment.

Results&

Discussion

In designing our experiment, we operated on

four variables that affect the elution times and

the resolution of the gas chromatography

instrument. The first variable was the

injection temperature with a range of 200-

300°C. The second was the initial oven

temperature with a range of 100-200°C. The

third was the oven ramp rate with ranges of

5°C/min-50°C/min. The fourth variable we

set limits for was the final oven temperatureof 180-220°C.

By modifying these variables, we had four

outcomes that we looked at: the last peak

elution time which we wanted to be

minimized, and the three resolution values



between the four fatty acid methyl ester peaks

which we wanted to be maximized.

The DOE took our limiting variables and

gave us a list of specific variables to test.

These specific runs we limited to 30 and there

are 6 duplicates to test for instrumental erroras well as several outliers to make sure our

confining limits are reasonably set. The

remaining of the 30 runs were selected

through a randomization process that the

DOE will analyze later. The following chart

gives the list of variables the DOE had us test

and their corresponding results.



To determine the peak resolution, we used the output from the GC in this format.



By using the peaks width listed here and the retention time, we can determine the resolution by

this formula.

(Note: Within

our

calculations,

we

did

not

need

to

multiply

by

2

in

the

numerator

because

the

width values given in the GC readout were ½ height widths.)

The following pictures are the DOE analysis of the data and its prediction:



Estimate of error in the model.



Last peak retention time analysis:



Resolution between peaks 1-2 analysis:



Optimum conditions and prediction:



To verify these predictions we did 3 runs. The results follow.

RUN 1 RetTime(min Width(min)

1 0.762 7.55E-03time last peak eluted 7.391

2 2.645 1.44E-02 rs. b/t 1-2 19.81707

3 3.295 0.0184 rs. b/t 2-3 19.757714 4.192 0.027 rs. b/t 3-4 38.77576

5 7.391 0.0555





2 2.648 1.43E-02 rs. b/t 1-2 19.96933

3 3.299 0.0183 rs. b/t 2-3 20.08949

4 4.197 0.0264 rs. b/t 3-4 37.19258

5 7.403 0.0598





2 2.65 1.40E-02 rs. b/t 1-2 19.90826

3 3.301 0.0187 rs. b/t 2-3 19.97778

4 4.2 0.0263 rs. b/t 3-4 38.83777

5 7.408 0.0563

For each of these runs, the experiment

values of time of last peak and the resolution

values between the peaks were within 2

standard deviations of the predicted value of

the DOE.

The experimental data we obtained

was accurate and very usable; however, the

process can be sped up. During in-class

discussion we determined that a resolution

value over 1 is sufficient and that the process

can be done in much faster time with

acceptable results. After talking with Dr. Lee,

we determined that the reason the DOE did

not predict these results was because we did

not set up a full hierarchy in terms of

importance and value of results. This means

that we did not specify that a smaller last peak

elution time is much more important than a

large resolution value and that any value over

1 is quite sufficient. Because of this, the DOEsought to find the best medium between the

maximized resolution values and the

minimized last peak elution value.

chm 418, experiment 08 - design of experiments using a hewlett-packard 5890 series ii gas...

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