Compound-Specific
Stable Isotope
Analysis


Analysis

Pricing

Sample Preparation

Tips

Sample Submission











































































UC Davis Plant Sciences


Amino Acid Sample Preparation

Acid Hydrolysis

Sample proteins are acid hydrolyzed with 6N HCl at 150°C for 70 min under N2.  Norleucine is added as an internal reference material (0.1 mg per mg sample).  After hydrolysis, the samples are dried at 50°C under a gentle stream of N2, and then re-dissolved in 200 µL 0.4 N HCl.  If necessary, the sample solution can be further purified by filtration and cation-exchange chromatography. Analysis of free amino acids may also possible, please contact us for details.

 

Derivatization using methyl chloroformate (MCF)

Hydrolyzed samples are directly derivatized by a mixture solution of methanol, pyridine and methyl chloroformate (MCF).  Amino acid-MCF derivatives, methoxycarbonyl amino acid methyl esters, are then separated from the reaction mixtures by liquid:liquid extraction using chloroform.  The amino acid-MCF derivatives are then suitable for GC-C-IRMS analysis. The resulting compounds contain very little derivative carbon, facilitating both δ13C and δ15N analysis of amino acids.  
Fig1

Fig. 1.Derivatization reaction of valine with methyl chloroformate in the presence of methanol and pyridine (simplified from Chen et al. 2010). 

 

 

 

Fig2

 Fig. 2. A typical chromatograph of δ15N analysis of amino acid-MCF derivatives. 

Compound list: 3=Alanine, 4=Valine, 5=Glycine, 6= Isoleucine, 7=Leucine, 8=Norleucine (I.S. #1), 9=Proline, 10=Aspartic acid, 11=Threonine, 12= Methionine (unoxidized), 13=Glutamic acid (as pyroGlu), 14=Phenylalanine, 15=Homophenyalanine (I.S. #2), 16=Lysine, 17=Tyrosine


Due to acid hydrolysis, some amino acids cannot be detected by GC-C-IRMS.  They include glutamine and asparagine, which are converted to their respective acid forms (i.e. Glu, Asp).  Tryptophan is completely destroyed during acid hydrolysis, while cysteine and methionine are partially degraded during and yields are not quantitative.  Note that all of these amino acids may be measured using MCF as free amino acids, just not from acid hydrosylates.  Serine and histidine recoveries are not sufficient for measurement and arginine cannot be derivatized using MCF, in any experimental context.  Table 1, below, lists all of the amino acids that can be measured from acid hydrolysates using MCF, with analytical precision for both δ13C and δ15N in those amino acids.

 

           

 

Retention time and standard deviation for both δ13C and δ15N in amino acid std (n=20)

Amino Acid

           

δ13C

δ15N

RT

s

s

Alanine

950

0.51

0.55

Valine

1087

0.34

0.36

Glycine

1140

0.75

0.84

Isoleucine

1268

0.40

0.94

Leucine

1291

0.44

0.82

Proline

1637

0.41

0.55

Aspartic Acid

2120

0.71

0.64

Threonine

2160

0.65

0.64

Glutamic Acid (as pyroGlu)

2740

0.59

1.22

Phenylalanine

2850

0.24

0.75

Lysine

3740

0.71

1.04

Tyrosine

3968

0.68

1.31

 

 

Sample requirements

The required sample weight is 6-9 mg for animal tissue, and 20-30 mg for algae and plant materials; smaller samples sizes may be suitable, but please contact us.  Samples must be dried and homogenized prior to sample submission to the SIF. 

 

 




 
e-mail: sif@ucdavis.edu | phone: 530-752-8100 | fax: 530-752-4361
UC Davis Stable Isotope Facility | Department of Plant Sciences
One Shields Avenue | Davis, California, 95616 | USA