Investigating GCxGC separations using selective column chemistry and compound derivatization pairings for common metabolomics chemical compounds

Transcription

1 Investigating GCxGC separations using selective column chemistry and compound derivatization pairings for common metabolomics chemical compounds Julie Kowalski, Michelle Misselwitz and Jack Cochran Restek Corporation

2 The Power of GCxGC

3 Rxi-17Sil MS 1 m x 0.25mm x 0.25µm a lot of GC GC open space TMS Derivatives Amino acids rganic acids Carbohydrates Free fatty acids Trimethyl silyl derivatives most popular Rxi-5Sil MS 30m x 0.25mm x 0.25µm

4 Thinking.Can we do better?

5 general chemistry turtle Fortunately, we can use general chemistry principles to select columns and derivatives

6 Dimethyl Si Phenylmethyl Si column chemistry Common Stationary Phases Diphenyl Si Trifluoropropyl methyl CF 3 C 2 4 Si Cyanopropyl Phenyl C N (C 2 ) 3 Si Polyethylene glycol (PEG) C C C N (C 2 ) 3 Si (C 2 ) 3 C N Biscyanopropyl GC column have different chemical functionalities. We often think of these in terms of polarity but let s think about chemical interactions.

7 5 phase 5% diphenyl 95% dimethyl Si Si hydrophobic interactions Carbon rich moieties Polarity: Uses: Properties: slight to intermediate aromatics, flavors, environmental samples, and aromatic hydrocarbons min. temp. (-60 C), max. temp. (340 C)

8 17 phase 50% diphenyl 50% dimethyl pi-pi Interactions Double bonds and conjugated rings Si Si hydrophobic interactions Polarity: Uses: Properties: intermediate pesticides, herbicides, rosin acids, triglycerides, phthalate esters, and sterols. min. temp. (0 C), max. temp. (320 C)

9 200 phase Trifluoropropylmethyl polysiloxane polar interactions CF 3 C 2 4 Si lone pair electrons N,, Cl, F Polarity: Uses: Properties: polar, selective for lone pair electrons phenols, nitrosamines, organochlorine pesticides, chorinated hydrocarbons min. temp. (-20 C), max. temp. (340 C)

10 Biological groups free fatty acids hydrophobic 3 C Polar/lone e - C8 through C24 Several unsaturated: C16:1, C18:1, C18:2, C18:3, C22:1

11 Sucrose MW carbohydrates D-(+)-Maltose monohydrate MW D-( )-Fructose MW Polar/lone e - D-(+)-Glucose MW D-(+)-Mannose MW D-( )-Ribose MW

12 Acetic acid Adipic acid L-Ascorbic acid Polar/lone e - organic acids xalic acid Succinic acid pi-pi pi-pi Benzoic acid Butyric acid Citric acid Polar/lone e - Maleic acid Malonic acid Isobutyric acid Formic acid L-(+)-Lactic acid Propionic acid (-)Quinic acid Fumaric acid Shikimic acid D-( )-Tartaric acid pi-pi

13 amino acids L-Alanine Polar/lone e - Polar/lone e - L-Arginine L-Cystine L-Glutamic acid L-Aspartic acid Glycine L-Serine L-Isoleucine L-Leucine L-istidine L-Proline L-Threonine pi-pi L-Lysine L-Methionine L-Valine L-Phenylalanine pi-pi pi-pi L-Tyrosine

14 Split injection Precision split liner with wool (various ratios) 0.5 µl C Transfer line: 300 C FAME mix for reference GCxGC-TFMS Conditions Rxi-5Sil MS Rtx-200 Rxi-17Sil MS GC Conditions 30m x 0.25mm x 0.25µm 1 m x 0.25mm x 0.25µm 2 ml/min, constant flow GC oven program: 1 st :40 C (1 min), 5 C/min to 310 C old 5 min 2 nd :45 C (1 min), 5 C/min to 315 C hold 2 min LEC Pegasus 4D Source temperature: 225 C Electron ionization: 70 ev Modulation time: 2 sec Stored mass range: 45 to 550 u Acquisition rate: 200 spectra/sec

15 Moonlighting as an rganic chemist

16 4 column configurations Too many derivatization reactions 1 st dimension x 2 nd dimension Trimethyl silylation Methylation Ethylation Acylations Fluorination Sequential

17 3 C common TMS with 5 x 17 column set Polar/lone e - Polar/lone e - C 3 Polar/lone e - 3 C 3 C 3 C C C 3 3 Si Si hydrophobic 3 C Si 3C 3 C 3 C Si 3 C Si hydrophobic Polar/lone e - pi-pi Polar/lone e - pi-pi hydrophobic 3 C 3 C Si C 3 3 C Si 3 C 3 C N 2 hydrophobic Si N 3 C Si C 3 3 C Si Si 3 C hydrophobic Si

18 h no! Decreasing selectivity

19 methylation and ethylation 3 C 3 C N 2 N 2 3 C

20 Pentafluro- and acylation 3 C F F F N F F F F F 3 C F F F F F F F acylation ethylation/acylation 3 C N 2 3 C 3 C 3 C 3 C N

21 Rxi-17Sil MS 1 m x 0.25mm x 0.25µm Remember a lot of GC GC open space TMS Derivatives Amino acids rganic acids Carbohydrates Free fatty acids Rxi-5Sil MS 30m x 0.25mm x 0.25µm

22 Rxi-5Sil MS 1 m x 0.25mm x 0.25µm Derivatized compounds, especially fatty acids, are retained well in the 2 nd dimension with a 5 phase column that relies heavily on hydrophobic interactions. ethyl derivatives Amino acids rganic acids Carbohydrates Free fatty acids amino acids organic acids carbohydrates Derivatized amino acids, organic acids and carbohydrates do not use the full x-axis because the 17 retains compounds with double bonds and conjugated rings. Many of the derivatives do not contain these functionalities. Rxi-17Sil MS 30m x 0.25mm x 0.25µm

23 Rtx m x 0.25mm x 0.25µm organic acids derivatives amino acids derivatives Ethyl/acetyl derivatives Amino acids rganic acids Free fatty acids Addition of the ethyl group will provide retention along the x-axis using the 5 column. Longer retention of amino acids and organic acids in the y-axis results from both a smaller hydrophobic group (compared to TMS) and addition of an acetyl moiety. The acetyl group is selective for the 200 column because of the oxygen containing lone pair electrons. Rxi-5Sil MS 30m x 0.25mm x 0.25µm

24 Rxi-17Sil MS 1 m x 0.25mm x 0.25µm amino acids derivatives organic acids derivatives Ethyl/acetyl derivatives Amino acids rganic acids Free fatty acids Longer retention of amino acids and organic acids in the y-axis results from the addition of an acetyl moiety. The acetyl group is likely selective for the 17 column because of the addition of a carbon-oxygen double bond. Fatty acid derivatives amino acids derivatives organic acids derivatives Rxi-5Sil MS 30m x 0.25mm x 0.25µm

25 Rxi-17Sil MS 1 m x 0.25mm x 0.25µm These fluorinated derivatives do not add conjugation to the analytes so there is not a large increase in 2 nd dimension retention. amino acids derivatives Fluorinated Fluorinated derivatives derivatives Amino acids Amino acids rganic acids Carbohydrates Carbohydrates Free fatty acids Free fatty acids carbohydrate derivatives Rxi-5Sil MS Fatty acid derivatives 30m x 0.25mm x 0.25µm

26 Rxi-5Sil MS 1 m x 0.25mm x 0.25µm Fatty acid derivatives Fluorinated derivatives Amino acids rganic acids Carbohydrates Free fatty acids These fluorinated derivatives do not add conjugation to the analytes so there is not a large increase in 1 nd dimension retention. By reversing the column set from the previous slide, we can move the open space. carbohydrate derivatives Rxi-17Sil MS 30m x 0.25mm x 0.25µm

27 Rtx m x 0.25mm x 0.25µm amino acids derivatives carbohydrate derivatives organic acids derivatives The fluorinated groups provide many lone pairs of electrons to interact with the 200 column. Notice the long retention in the 2 nd dimension causing wrap around for the small polar compounds. Fluorinated derivatives Amino acids rganic acids Carbohydrates Free fatty acids amino acids derivatives Fatty acid derivatives Rxi-5Sil MS 30m x 0.25mm x 0.25µm

28 Rxi-5Sil MS 1 m x 0.25mm x 0.25µm amino acids derivatives wrapped one time These are the typical amino acid TMS derivatives but with a 200 x 5 column set. Shows better use of 2D space. TMS derivatives Amino acids Rtx m x 0.25mm x 0.25µm

29 Rxi-5Sil MS 1 m x 0.25mm x 0.25µm organic acids derivatives No wrap TMS derivatives rganic acids Shows better use of 2D space. Rtx m x 0.25mm x 0.25µm

30 Rxi-5Sil MS 1 m x 0.25mm x 0.25µm Fatty acid derivatives TMS derivatives Free fatty acids Shows better use of 2D space. Fatty acid derivatives Wrapped one time Rtx m x 0.25mm x 0.25µm

31 Rxi-5Sil MS 1 m x 0.25mm x 0.25µm carbohydrate derivatives wrapped TMS derivatives Carbohydrates Shows better use of 2D space. Rtx m x 0.25mm x 0.25µm

32 methyl derivatives Free fatty acids Comparing methyl and ethyl fatty acids shows longer primary column retention on a 5 phase column for ethyl derivatives. This is expected because the dominate interaction is hydrophobic for a 5 phase column and ethyl groups are more hydrophobic than methyl groups. Rxi-5Sil MS 30m x 0.25mm x 0.25µm Rtx-200 1m x 0.25mm x 0.25µm ethyl derivatives Free fatty acids

33 Ethyl derivatives Amino acids Met Phe Leu Pro Asp Glu Ethyl, acetyl derivatives Amino acids Derivatives resulting from sequential ethylation and acylation reactions produce compounds with ethyl and C0C3 groups. The 200 column can interact with the oxygen atom lone pairs of electrons causing longer retention in the 1 st dimension with respect to ethylated only analogs. Rtx m x 0.25mm x 0.25µm Rxi-5Sil MS 1m x 0.25mm x 0.25µm

34 Gly ILe Rxi-5Sil MS x Rtx-200 Ala Leu Pro Met is The fluorinated derivatives are highly selective for the 200 column due to many pairs of electrons on the fluorine atoms. Placing this column in the second dimension helps take advantage of the 2D chromatographic space. Pentafluoroderivatives Amino acids Phe Rtx-200 x Rxi-5Sil MS is Gly Leu Ala ILe Pro Phe Met

35 Rxi-5Sil MS x Rtx-200 Wrapped!! The fluorinated derivatives are highly selective for the 200 column due to many pairs of electrons on the fluorine atoms. Carbohydrates can have up to eight possible reaction sites that is 40 fluorine atoms! These derivatives are greatly retained and wrapped around many times. Pentafluoroderivatives carbohydrates Rtx-200 x Rxi-Sil MS

36 Selectivity Column chemistry Derivatization chemistry Design separations Use 2D chromatographic space

37 Acknowledgments Jack Cochran Snoop, lab dog Chris Rattray Michelle Misselwitz