Analysis of nitrogen isotope ratios in amino acids by GC/C/IRMS 27 March 2013 Yoshito Chikaraishi The initial version was released by YC in May 2009. The present version reflects feedbacks from Svenja Kruse (Univ. of British Columbia) and Shawn A. Steffan (Univ. of Wisconsin-Madison), Elizabeth Bosak (Univ. of Wisconsin-Madison) in March 2012.
Instrument Instruments in my lab: GC Agilent 6890N Injector Gerstel PTV Column Agilent HP Ultra-2 (50m, 0.32mm, 0.52µm) Flow rate (He) 1.4ml/min Oven program 40ºC (2.5 min) -15ºC/min - 110 (0 min) - 3ºC/min - 150 (0 min) - 6ºC/min - 220ºC (17.3min) Interface GC combustion III (oxidation at 1000ºC, reduction at 550ºC) IRMS Delta plus XP Standard mix of authentic amino acids: after HCl hydrolysis and derivatization, derivatives are stored at 20ºC, in DCM (Currently, I use new standard mix. See page 10 ). Compound Source Known Amino acid Pv/OiPr derivatives δ 15 N MW N(wt%) mg/10ml MW N(wt%) ng/µl ngn/µl nmol/µl L-alanine Ala CERKU-02 22.7 89 16 2.4 215 6.5 579.9 37.7 2.7 glycine Gly CERKU-03 2.2 75 19 2.1 201 7.0 562.9 39.2 2.8 L-valine Val BioGeos 3.5 117 12 3.6 243 5.8 747.8 43.1 3.1 L-leucine Leu CERKU-04 8.8 131 11 3.7 257 5.4 725.9 39.5 2.8 L-norleucine Ile BioGeos 18.0 131 11 4.4 257 5.4 863.3 47.0 3.4 L-aspartic acid Asp BioGeos 3.1 133 11 4.0 301 4.6 905.6 42.1 3.0 L-serine Ser BioGeos 2.4 105 13 3.2 315 4.4 960.2 42.7 3.0 L-glutamic acid Glu BioGeos -5.7 147 10 4.8 315 4.4 1028.9 45.7 3.3 L-phenylalanine Phe BioGeos -5.2 165 8 5.0 291 4.8 882.0 42.4 3.0 L-hydroxyproline Hyp BioGeos -7.1 131 11 4.7 341 4.1 1223.7 50.2 3.6
Procedure for δ 15 N analysis 1. Set up GC/IRMS (e.g., connect GC and IRMS, focusing) 2. Check N 2 leaks for both back-flush and straight mode 3. Check stability by On/OFF of Ref. gas 4. Check reproducibility by measuring δ 15 N-known authentic AAs (amino acid standards), as Pv/OiPr derivatives 5. Make up a sequence file 6. Measure both authentic AAs and samples of interest 7. Check baseline separation on the chromatograms 8. Normalize the measured values (i.e., δ 15 N, vs ref.gas) to international scale (i.e., δ 15 N, vs Air)
1. Focusing Check these 9 boxes To find the best (optimum) position, apply the following steps: 1. After peak center, open auto focus. 2. Enter 50 and 500 for the step width in these two boxes 3. Click OK 4. After peak center, enter 25 and 250 in these two boxes, and click OK 5. After peak center, enter 12 and 120 in these two boxes, and click OK 6. After peak center, 6 and 60 in these two boxes, and click OK 7. After peak center, enter 3 and 30 in these two boxes, and click OK 8. After peak center, enter 2 and 20 in these two boxes, and click OK 9. After peak center, enter 1 and 10 in these to boxes, and click OK (twice)
3. Check stability by On/OFF of Ref. gas Generally, 1σ < 0.03-0.05
4. Check reproducibility by δ 15 N of known authentic AAs Set the isotope value of the ref. gas to 0 on the PC software, measure the authentic AA derivatives, and check the linearity of the relationship between the measured δ 15 N (, vs ref. gas) and known δ 15 N (, vs air) values. 2500 Ref. N 2 Ref. N 2 m/z 28 Intensity (mv) 2000 1500 1000 500 Ala Gly Val Leu Nle Asp Ser Glu Phe Hyp 0 10 15 20 25 30 35 40 45 Retention time (min) 25 If you do not get a good linear relationship, please check the following points: 1. the condition of the ox./red. furnace 2. the condition of GC column 3. dirt in inlet/liner δ 15 N, vs Air 20 15 10 5 0 y = 1.0225 x 4.4523 (R 2 = 0.999) 4. deterioration of capillary connection -5-10 -5 0 5 10 15 20 25 30 δ 15 N, vs refs
5. Make up a sequence file For example, Line GC method Sample Num. Injection vol. (µl) Sample name IRMS Method 1 Ultra-2 AA 1 1 STD Pv/OiPr Ultra-2_AA_15N 2 Ultra-2 AA 2 1 Sample-A Ultra-2_AA_15N 3 Ultra-2 AA 3 1 Sample-B Ultra-2_AA_15N 4 Ultra-2 AA 4 1 Sample-C Ultra-2_AA_15N 5 Ultra-2 AA 5 1 Sample-D Ultra-2_AA_15N 6 Ultra-2 AA 1 1 STD Pv/OiPr Ultra-2_AA_15N 7 Ultra-2 AA 6 1 Sample-E Ultra-2_AA_15N 8 Ultra-2 AA 7 1 Sample-F Ultra-2_AA_15N 9 Ultra-2 AA 8 1 Sample-G Ultra-2_AA_15N 10 Ultra-2 AA 9 1 Sample-H Ultra-2_AA_15N 11 Ultra-2 AA 10 1 Sample-I Ultra-2_AA_15N 12 Ultra-2 AA 1 1 STD Pv/OiPr Ultra-2_AA_15N
8. Normalization of measured values to the international scale -1 When you set the isotope value of the ref. gas to 0 on the PC software, you have measured values ( vs ref. gas). Known δ 15 N Measured value = δ 15 N, vs ref. gas 1st 2nd 3rd 4th 5th Average 1σ Ala 22.7 26.234 26.755 26.489 26.422 26.667 26.5 0.2 Gly 2.2 6.777 6.101 6.648 6.973 6.207 6.5 0.4 Val 3.5 7.748 7.616 7.624 7.490 6.532 7.4 0.5 Leu 8.8 13.488 13.231 13.254 13.781 13.622 13.5 0.2 Nle 18.0 22.640 22.048 21.853 21.855 21.530 22.0 0.4 Asp 3.1 6.915 7.643 7.299 6.573 7.166 7.1 0.4 Ser 2.4 6.189 6.754 6.454 7.049 6.163 6.5 0.4 Glu -5.7-1.395-1.025-1.331-1.373-1.263-1.3 0.1 Phe -5.2-0.533-0.138-0.836-1.161-0.536-0.6 0.4 Hyp -7.1-2.366-1.654-2.381-2.811-2.357-2.3 0.4 δ 15 N, vs Air 25 20 15 10 5 0-5 y = 1.0225 x 4.4523 (R 2 = 0.999) -10-5 0 5 10 15 20 25 30 δ 15 N, vs refs You can normalize δ 15 N to the international scale (i.e., δ 15 N, vs Air) by using the equation: y = 1.0225x 4.4523 (in this case). The slope 1.0 means that we need this linear normalization instead of an one-point normalization. Known δ 15 N δ 15 N, vs Air 1st 2nd 3rd 4th 5th Average 1σ Δ Ala 22.7 22.4 22.9 22.6 22.6 22.8 22.7 0.2 0.0 Gly 2.2 2.5 1.8 2.3 2.7 1.9 2.2 0.4 0.0 Val 3.5 3.5 3.3 3.3 3.2 2.2 3.1 0.5-0.4 Leu 8.8 9.3 9.1 9.1 9.6 9.5 9.3 0.2 0.5 Nle 18.0 18.7 18.1 17.9 17.9 17.6 18.0 0.4 0.0 Asp 3.1 2.6 3.4 3.0 2.3 2.9 2.8 0.4-0.3 Ser 2.4 1.9 2.5 2.1 2.8 1.8 2.2 0.4-0.2 Glu -5.7-5.9-5.5-5.8-5.9-5.7-5.8 0.2-0.1 Phe -5.2-5.0-4.6-5.3-5.6-5.0-5.1 0.4 0.1 Hyp -7.1-6.9-6.1-6.9-7.3-6.9-6.8 0.4 0.3 Average 0.4 0.0 Std.dev 0.1 0.3 1σ = Precision Δ (= average known) = Trueness In this way, Accuracy (= Mean of Δ) should be Zero ± something.
8. Normalization of measured values to the international scale -2 With the equation: (δ 15 N, vs Air) = 1.0225 (measured δ 15 N, vs refs) 4.4523 (in this case), you can calculate normalized δ 15 N (, vs Air) for the samples of interest. Measured value = δ 15 N, vs ref. gas Sample-A Sample-B Sample-C Sample-D Sample-E Sample-F Sample-G Ala 8.344 10.560 6.005 8.550 13.295 12.521 10.345 Gly 7.655 11.150-2.458-3.452 4.410 5.170 9.723 Val 7.498 11.716 7.959 7.724 13.793 10.741 12.925 Leu 4.123 10.024 5.027 3.161 6.955 6.710 11.654 Ile 4.514 9.749 7.650 3.586 11.191 8.898 15.082 Pro 14.228 24.680 8.891 10.548 17.185 15.903 16.704 Ser 5.188 too small 2.288-1.058 5.942 5.843 8.308 Met too small 7.335 too small too small too small too small 2.555 Glu 11.485 15.796 6.897 7.707 14.695 14.040 18.334 Phe 11.388 15.749 13.942 15.757 13.667 15.951 10.302 δ 15 N, vs Air Sample-A Sample-B Sample-C Sample-D Sample-E Sample-F Sample-G Ala 4.1 6.3 1.7 4.3 9.1 8.3 6.1 Gly 3.4 6.9-7.0-8.0 0.1 0.8 5.5 Val 3.2 7.5 3.7 3.4 9.6 6.5 8.8 Leu -0.2 5.8 0.7-1.2 2.7 2.4 7.5 Ile 0.2 5.5 3.4-0.8 7.0 4.6 11.0 Pro 10.1 20.8 4.6 6.3 13.1 11.8 12.6 Ser 0.9 n.d. -2.1-5.5 1.6 1.5 4.0 Met n.d. 3.0 n.d. n.d. n.d. n.d. -1.8 Glu 7.3 11.7 2.6 3.4 10.6 9.9 14.3 Phe 7.2 11.6 9.8 11.7 9.5 11.9 6.1
New standard mix We currently developed wide-range (from 25.9 to +45.6 ) isotopic reference materials of amino acids, which are commercially available from Indiana University (http://php.indiana.edu/~aschimme/hc.html) and SI science (http://www.si-science.co.jp/global/en/). Compoud Source known δ 15 N Amino acid Pv/OiPr derivatives MW N(wt%) mg/10ml MW N(wt%) ng/µl ngn/µl nmol/µl L-alanine Ala Indiana Univ. 43.3 89 16 6.5 215 6.5 652 42 3.0 glycine Gly SI science -25.9 75 19 6.2 201 7.0 618 43 3.1 L-valine Val BioGeos 3.5 117 12 7.3 243 5.8 727 42 3.0 L-leucine Leu SI science 5.6 131 11 7.7 257 5.4 765 42 3.0 L-norleucine Nle SI science 18.2 131 11 7.7 257 5.4 765 42 3.0 L-aspartic acid Asp SI science 35.3 133 11 9.1 301 4.6 906 42 3.0 L-methionine Met SI science -3.9 149 9 9.0 305 4.6 901 41 3.0 L-glutamic acid Glu SI science 45.6 147 10 9.4 315 4.4 943 42 3.0 L-phenylalanine Phe Indiana Univ. 1.7 165 8 8.8 291 4.8 882 42 3.0 L-hydroxyproline Hyp SI science -9.1 131 11 10.2 341 4.1 1015 42 3.0 Known δ 15 N δ 15 N (, vs Air) by GC/IRMS δ 15 N1st δ 15 N2nd δ 15 N3rd δ 15 N4th Average 1σ Δ Ala 43.3 43.0 43.0 43.5 43.7 43.3 0.4 0.0 Gly -25.9-25.9-26.1-26.2-26.9-26.3 0.5-0.4 Val 3.5 3.2 2.7 3.2 3.3 3.1 0.2-0.4 Leu 5.6 5.5 5.8 6.2 5.5 5.7 0.3 0.1 Nle 18.2 19.0 18.4 18.4 19.3 18.8 0.4 0.6 Asp 35.3 34.4 35.1 35.1 34.6 34.8 0.3-0.5 Ser -3.9-3.6-3.8-3.8-4.1-3.8 0.2 0.1 Glu 45.6 45.7 45.4 45.2 45.9 45.6 0.3 0.0 Phe 1.7 2.2 2.3 2.2 2.1 2.2 0.1 0.5 Hyp -9.1-9.4-8.5-9.2-9.1-9.0 0.4 0.1 Average 0.3 0.0 Std.dev 0.1 0.4 δ 15 N ( vs Air) determined by GC/IRMS 50 40 30 20 10 0-10 -20 y = 1.000 R 2 = 0.9998-30 -30-20 -10 0 10 20 30 40 50 Known δ 15 N ( vs Air)