An Application of Trimethylsilyl Derivatives with Temperature Programmed Gas Chromatography to the Paul B. Kelterl University of Wisconsin-Eau Claire, Eau Claire, WI 54701 J a m e s D. Carr University of Nebraska-Lincoln,
Lincoln, NB 68588
In the past several years automated temperature programming gas chromatographs (TPGC) have come into common use for the separation of species which vary widely in molecular weirht or uolaritv or both. The basic idea behind
H-N
/CH2C00H 'CH~COOH
coefficient, K, is related to temperature, T, by eqn. (1)
where AGO = standard free energy change for the gas-liquid partitioning equilibrium R
Figure
1. The reaction of IDA and BSTFA to give the derivatized product.
= gas constant
I t can he shown2that Va a KVs = t n therefore Equipment Necessary equipment is where VR = retention volume, Vs = stationary phase volume, t~ = retention time, and C = a constant. Eqn. (3) says that the hotter a column gets, the shorter will he the retention time of a given compound on that column. Many substances of interest undergo hydrogen-bonding which causes an unusually high boiling point. Bulkier nonpolar groups, such as a trimethylsilyl (TMS) group can he substituted thus decreasing hydrogen-bonding and lowering the boiling point of the compound. This results in greater volatility; therefore, separations may he made much more readily. Iminodiacetic acid (IDA) is an example of a compound which does not elute from a GC because of intermolecular hydrogen bonding. It and its TMS derivatized product which does elute are depicted in Figure 1. In our instrumental analysis programs we have devised an experimental procedure to teach students about TPGC and the importance of derivatizing many classes of substrates to be separated. The three classes that have worked wellin the laboratory are Fatty acids (laurie, myristic, stearie) Amino acids (mast work well) Substituted amino earhoxylstes (NTA3, IDA, MIDA, Gly)
The procedure described herein has taken students, working in pairs, about 3 hours (1 lab period) to complete for each class of compounds.
' Author to whom correspondence should be addressed.
Peters, D. G., Hayes, J. M., and Hieftje, G. M. "Chemical Separations and Measurements, Theory and Practice of Analytical Chemistry," Saunders Company, 1974, Ch. 17. Abbreviations used are NTA = nitrilotriacetic acid, IDA = iminodiacetic acid, MIDA = methyliminiodiacetic acid, and Gly = glycine. "Gehrke, C. W.. and Leimer, K., J. Chromatogr. 57, 219 (1971).
A temperature programmable gas chromatograph with aflame ionization detector. A 5-ft 1.5% OV-17 on Chromosorb W glass column (or comparable nonpolar column) 200 ml silicone oil in a 250-ml k k e r a hot plate a 10 syringe some 2 ml glass vials with caps three 0.5 ml pipets a bottle of BSTFA ($40-$100 for 100 g) 1.00 & 0.01 g substrate in 100 ml HzO 1.00 0.01 g phenanthrene in 100 ml CHzClz as an internal standard
.
+
Procedure for Making a TMS Derivative The procedure for the formation of TMS derivatives is a modified version of that described by Gehrke et al.* A 150°C oil bath consisting of 200 ml of silicone oil in a 250-ml beaker on a hot plate is made ready Care must be taken not to fill the beaker too full, because the ail will expand upon heatimg and may overflow. When hot, the oil level should be about 1cm from the top of the beaker. Into a '12 dram (2 ml) vial with a Teflon"-lined cap add the desired amount of substrate and phenanthrene (internal standard) solutions (0.50 ml of phenanthrene work very well). Note that because the phenanthrene is in CH&, it will be the top layer in the vial. The vial is immersed without the cap until it is about a half-inch into solution. A stream of dry nitrogen or air is then gently blown on to the solution until dried (about 2-3 min). The nitrogen stream is removed and two 0.5 ml aliquots of dry CH2Ch are added in succession (the solutions are taken to dryness after each addition of CH2Clz).This is done to remove traces ofwater which will hydrolyze the BSTFA. Approximately 0.5 ml of BSTFA is added to the vial while it is still in the oil bath and the cap is screwed on. If good technique has been used, the solution should boil very slightly or not a t all. A heavy boil indicates same traces of moisture in the vial. Heating is continued far -20 min. The vial is taken out of the oil bath andcouled a t room temperature for 5-10rnin. Aliquots are injected into the gas chromatograph. Some caveats for students when working with this procedure:
Volume 60
Number 5
May 1983
437
TEMPERATURE Figure 2. Gas chromatogram of fatty acids. detector and injector = 220°C initial temperature = 150°C temperature ramp = 10DC/min final temperature = 230°C
.
a b c d
= myristlc acid = phenanthrene = stearic acid
BSTFA is extremely toxic and has a disagreeable odor. Always do the derivatizatians in a hood! Never touch the silicone oil beaker while it is hot. Silicone ail burns are very nasty.
Applications Students are given known amounts of lauric, myristic, and stearic acids (1 gI100 ml of HzO) and are asked to derivatize each acid and develop an appropriate temperature program to monitor all three substrates. A good starting point might be detector and injector temperature: 220% initial temperature: 150°C. temoerature ramp: 10°C/min for 8 min, and final t e m p e r & e ~ 2 3 0 " ~ . A sample output with conditions is " eiven in Figure 2. The students are then given a n unknown mixture of the three substrates and are asked to determine how much of each of the three compounds they were given. The same protocol can be applied t o the separation of protein amino acids or arninocarhoxylates. Amino acids require a temperature program that spans a wider range (typically 70°C to 190°C), while aminocarboxylates require holding the initial temperature for 1or 2 min, and a higher temperature programming range (llO°C to 210°C). A typical aminocarhoxylate TPGC is given in Figure 3. The most serious problems that students encounter when doing this procedure are
..
No ~ e a k due s to water in the derivative Poor or no peaks due to moisture on the column. This can be remedied hv 10 ul .,iniwtine -~,~ -of a silvlatineaeent such as Silvl-8
~-~-
-
--
on the column and after'10 min reinjecting your sample No peaks because the flame in the detector went out. Example Calculation A student derivatized 1.00 ml each of three standard salutions which contained 1.00 g of compound in 100 ml HzO, and 0.50 rnl of phenanthrene internal standard solution at 1.00 gin 100ml CH~CIZ. The injection was done with a temperatureprogram of 150°C to 230°C with a 10 Co/min ramp. The following results were obtained: Compound
Temperature of Elmion
Peak Height
Relative Peak Height
Lauric Acid Myristic Acid Phenanthrene Stearic Acid
169'C 183'C 195'C 217'C
97 units 81 units 63 units 60 units
1.54 1.59 1.00 0.95
A flame ionization detector (FID) measures the conductivity (as evidenced by ion formation) in a flame between the cathode and anode. An increase in conductivity is proportional to an increase in the number of carbon-carbon or carbon-hydrogen bonds. As such, the response of an FID will vary for different organic compounds and
438
Journal of Chemical Education
TEMPERATURE
= laurlc acid
Figure 3. Gas chromatagram of aminmarboxylates. detector and injector = 220°C initial temperature = 80°C temperature ramp = 15°C/min final temperature = 220°C d = IDA e = phenanihrene f = NTA
a = solvent related peak b = Gly c = MIDA
generally increase with an increase in the number of C-C or C-H bonds in the compound. As a benchmark, however, the typical full scale resoonse is obtained usine about 1X 10V elm1 solution. (See ref. ( 1 ) for a more com~letediscussion.)
lowing results were ohtained: Peak number
Temperature of Elution
Peak Hebht
Relative Peak Heisht
1 2
170'6 182'C 195'C 216'C
80 unite 42 units 55 units 61 units
1.48 0.76 1.00 1.11
3 4
The unknown contains 5.8 mglml myristic acid in 2.00 ml or 2.9 mglml. Using the same procedure, the unknown contains 4.7 mg of lauric acid/ml and 5.8 rng of stearic acidlml. The careful student should be able to get a precision (measured as relative standard deviation (RSD)) of within about *3% and an accuracy of within about 5% of the true answers. These values are typical for gas chromatographic analyses of such low amounts of the substrates. Far an additional discussion on errors in gas chromatography, see reference (I).
Conclusion We have found that an experiment involving - t e m-~ e r a t u r e programming and a derivaiization can be done easily and safelv in a three-hour senior analvtical lahoratorv. There are man; compounds that lend themselves to this experiment, and students have expressed interest in these types of experiments which apply very up-to-date analytical techniques. Acknowledgment We wish to thank CarolModl for her help in preparing this manuscript. We also wish to thank William Smith for the use of his fatty acid chromatogram.
