Chem 219 GCMS summary

3/6/2018

Chem 219 Lab Summary Report Guide: GCMS of Amino Acids

Alyssa Hogan

Lab Partner: Shuai Yang

TA: Kevin Fischer

Date Preformed: 2/20/2018

Date Report Submitted: 3/6/2018

Alyssa Hogan Chem 219 Page: Lab: GCMS of Amino Acids 3/6/2018

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ABSTRACT

The purpose of this experiment was to use gas chromatography and mass spectrometry for

different amino acids samples. Because some amino acids are polar, they need to be derived

before they could be separated by gas chromatography. Doing the derivations by organic

reactions in screw cap vials, the amino acids became more volatile and less reactive. An

experimental element that was key to this experiment was replacing active hydrogens on OH,

NH2 and SH groups with nonpolar moieties. The major results were that certain amino acids

separated well by GC, while others did not separate well. Some of the compounds separated

faster than others. Some of the amino acids did not appear via spectra. The major problem with

some of these compounds were that even after the derivation, the volatility can actually decrease

more depending on the compound of interest.

INTRODUCTION

The initial challenge of this experiment was overcoming the fact that amino acids do not

separate well by GC. This is the case because for amino acids because depending on the specific

functional group attached, this adds polarity to the compound and sometimes a positive or

negative charge as well. While polarity helps their solubility in water, it does not help in the

separation of the compounds for identification purposes by GC because their vapor pressure is

low.

The work of this experiment was important for several reasons. First of all, GCMS

provides a lot of information about the compounds of study. The mass spectrometry identifies

the molecular weight of the substances which is a powerful identification tool. The gas


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chromatography also aids in identification purposes because it detects elution time of the

compounds present. GC is also frequently used because the detection requires a very small

quantity of the analyte. This identification tool is very useful for substances that are sufficiently

thermally stable and reasonably volatile. This experiment was important because it highlights

that certain characteristics are needed to run a successful separation such as vapor pressure,

polarity, column temperature, carrier gas flow rate, column length and the amount of material

injected (1).

The experimental approach used for this experiment to overcome the low volatility and

polar character of the compounds was to use derivation chemistry. This included cleavage of C-

C bonds next to the oxygen in an alcohol functional group. Also, the cleavage of bonds next to

the carbonyl group in an aldehyde results in loss of the hydrogen or the CHO group. In ether

groups, fragmentation tends to occur alpha to the oxygen atom (2). All of the are derivatives are

different examples of how the functional groups could be fragmented in order to respond better

to GC. These examples of the changes made were crucial to the experiment because polar

compounds have long retention times on polar stationary phases and shorter retention times on

non-polar columns using the same temperature (1).

EXPERIMENTAL

Theory. As discussed, the polar nature of amino acids requires derivation in order for

the analysis to be effective. The goal of the derivation was to make the analyte more volatile and

less reactive. “In the case of amino acids, the derivatization that occurred replaced active

hydrogens on OH, NH2 and SH polar groups with a nonpolar moiety. A common technique is

through silylation. In this experiment, the silylation reagent N-tert-butyldimethylsilyl-N-


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methyltrifluoroacetamide (MTBSTFA) was used because it formed tert-butyl dimethylsilyl

(TBDMS) derivatives when reacted with polar functional groups containing an active hydrogen”

(4).

Instrumentation. 1) Varian Saturn 2100T ion trap gas chromatograph-mass spectrometer

with a Varian 3900 GC and CP-8400 autosampler. This separated compounds in the gas phase

for identification. 2) Micro-derivatization equipment. This measured out microliters worth of

material.

Procedures. Two vials of 50 µL of 4-AA (Ala, Leu, Lys and Phe) were obtained by a

hand-held disposable tip pipetter. Then, two vials of 58 µL of 2H-AA were obtained. Next, the

ion exchange column was prepared. First, 1-mL of cation exchange resin was added to each of

the columns. This was rinsed with about 4 mL of distilled water. Then, 2-mL of 1-N acetic acid

and vortex was added. The hydrolysate, 150 µL, was then poured onto the columns. After all of

the fluid passed through, the columns were rinsed three times with about 2 mL of distilled water.

The vials were then placed under the columns. Next, 2-mL of 3-N ammonium hydroxide was

added to each column and the eluate was collected. Two vials of BSA and 1 mL of BSA + 2H-

AA were collected. All of the vials were ran under nitrogen gas. Then, 100 µL of 1:1 mix of

MTBSTFA + acetonitrile was added to each vial. They were placed on a heating block at 110° C

for 30 minutes. They were allowed to cool in the fridge. The following week, the GCMS analysis

was taken of one of each of the samples.

Data Analysis. The main tool for determining which amino acids were in each mixture

was through the amino acid fragmentation table. The molecular weights of all of the amino acids

were given in accordance to which functional group was or was not present on each specific

amino acid.


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RESULTS

Table 1: 4-AA Sample

Species [M]+ [M-tB]+ [M-COOtBDMS]+ [M-COtB]+

Ala 260.0 158.2 234.1

Leu 302.2 200.2 274.2

Phe 394.3 336.2 234.3 308.2

Lys 488.3 431.3

Ala

Leu

Phe

Lys


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At 5.7 minutes (Ala)

At 7.7 minutes (Leu)


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At 12.4 minutes (Phe)

At 15.6 minutes (Lys)


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Table 2: BSA Sample


Ala (5.7 min) 260.2 158.2 233.3

Leu (7.67 min) 302.2 200.2 274.2

Val (7.2 min) 288.2 186.2 261.2

Val (8.546 min) 286.2 184.2 258.2

Phe (12.44 min) 394.2 336.2 234.3 308.2

Orn (13.2 min) 418.3 316.5

Lys (14.44 min) 432.5 330.4 404.5

Ile (15.6 min) 301.2 198.3 272.3

Thr (18.08 min) 302.3

Pro (II) (22.8) 258.2


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Table 3: 2D-AA Sample


Ala (+4 diff.) (5.663 min)

264.2 162.2 236.3

Leu (+10 diff.) (7.612 min)

312.3 210.3 284.4

Phe (+8) (12.416 min)

344.2 242.3 316.3

Lys (+6) (14.423 min)

437.3


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Table 4: BSA + 2D-AA Sample


Ala (5.7 min) 264.2 162.2 236.3

Leu (7.60 min) 312.3 210.3 284.4

Val (7.1 min) 284.4

Phe (12.40 min) 344.2 242.3 316.3

Orn (13.2 min) 418.3 316.5

Lys (14.45 min) 432.5 330.4

Ile (15.62 min) 308 206 280

Thr (18.09 min) 308.3

Pro (II) (22.8) 258.4


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DISCUSSION

A few observations were made throughout this experiment. If the initial temperature of

the programming was changed, the amino acids would have separated faster if the temperature

was raised and if the temperature was lowered, it would have taken longer for the elution of the

amino acids. The first amino acid to elute was Ala at around 5.7 minutes for each of the four

samples. Pro (II) and Thr eluted late at about 22.8 and 18.1 minutes each. This was true for the

rates because Ala was non-polar to begin with so in general it is readily able to be separated by

GC. On the contrary, Thr is polar and is therefore difficult to separate. Because of the structure

of Pro (2), it is very reactive making it also difficult to elute. Asp and Glu are examples of amino

acids that did not give a peak or spectrum during this experiment. This is most likely so because

these two compounds are polar with a negative charge. In order to get a spectrum of these amino

acids, the negative charge would need to be neutralized. The molecular (M+) ions were recorded

for the amino acids: Phe and Lys. For the EI TBDMS amino acids, common characteristic

fragmentations included that there was no (M+) ions present except for Phe and [M-

COOtBDMS]+ was extremely common for most of the amino acids identified. The spectra for

2H-amino acids was helpful in identifying the remaining amino acids in the other samples

because there were many overlaps between the Ala, Leu, Phe and Lys acids throughout the rest

of the samples.

The most common derivatization reaction is through alkylation. “This represents the

replacement of active hydrogen by an aliphatic or aliphatic-aromatic group in a process referred

to as esterification. This converts organic acids into esters, especially methyl esters” (3). Again,

some peaks might have not occurred because the derivatization could not occur for certain amino

acids. The elution of amino acids relies on their ability to become volatile, low reactive species.


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LITERATURE CITED

1. http://www.chem.ucla.edu/~bacher/General/30BL/gc/theory.html (accessed Mar 5, 2018).

2. Libretexts. Mass Spectroscopy: Fragmentation Patterns

https://chem.libretexts.org/Core/Analytical_Chemistry/Instrumental_Analysis/Mass_Spec

trometry/Mass_Spec/Mass_Spectroscopy%3A_Fragmentation_Patterns (accessed Mar 5,

2018).

3. Orata, F. Derivatization Reactions and Reagents for Gas Chromatography

Analysis. Advanced Gas Chromatography - Progress in Agricultural, Biomedical and

Industrial Applications 2012 DOI: 10.5772/33098.

4. T. G. Sobolevsky, A. I. Revelsky; Barbara Miller, Vincent Oriedo, E. S Chernetsova, I.A

Revelsky, J. Sep. Sci. 2003, 26, 1474-1478.


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APPENDIX

BSA Sample:

At 5.7 min (Ala)

At 7.2 min (Val)


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At 7.671 min (Leu)

At 8.546 min (Val)


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At 12.44 min (Phe)

At 13.169 min (Orn)


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At 14.44 min (Lys)

At 15.6 min (Ile)


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At 18.079 (Thr)

At 22.768 min (Pro 2)

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Chem 219 GCMS summary