Title Page 

Determination of the δ15N of

Total Nitrogen in Solids;

RSIL Lab Code 2893

Chapter 11 of

Section C, Stable Isotope-Ratio Methods

Book 10, Methods of the Reston Stable Isotope 

Laboratory 

U.S. Department of the Interior

U.S. Geological Survey

Techniques and Methods 10–C11
This page left blank intentionally.
Determination of the δ15N of

Total Nitrogen in Solids;

RSIL Lab Code 2893

By Kinga Révész, Haiping Qi, and Tyler B. Coplen

Chapter 11 of

Section C, Stable Isotope-Ratio Methods

Book 10, Methods of the Reston Stable Isotope 

Laboratory 

Edited by Kinga Révész and Tyler B. Coplen

U.S. Department of the Interior

U.S. Geological Survey

Techniques and Methods 10–C11
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey 

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2012

Version 1.0, 2006 

Version 1.1, 2007

Version 1.2, 2012

For sale by U.S. Geological Survey, Information Services

Box 25286, Denver Federal Center

Denver, CO 80225

For more information about the USGS and its products:

Telephone: 1–888–ASK–USGS

World Wide Web: http://www.usgs.gov/

Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.

Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted material contained within this report.

Suggested citation:

Révész, Kinga, Qi, Haiping, and Coplen, T.B., 2012, Determination of the δ15N of total nitrogen in solids; RSIL lab code 

2893, chap. 11 of Stable isotope-ratio methods, sec. C of Révész, Kinga, and Coplen, T.B. eds., Methods of the Reston 

Stable Isotope Laboratory (slightly revised from version 1.1 released in 2007): U.S. Geological Survey Techniques and 

Methods, book 10, 30 p., available only at http://pubs.usgs.gov/tm/2006/tm10c11/. (Supersedes versions 1.0 and 1.1 

released in 2006 and 2007, respectively.)
iii

Foreword 

The Reston Stable Isotope Laboratory (RSIL) provides stable isotope analyses on a routine basis 

for a large user community within the U.S. Geological Survey (USGS) and elsewhere. The RSIL 

also serves the USGS National Research Program (NRP) through its project on Stable Isotope 

Fractionation in Hydrologic Processes. The NRP conducts basic and problem-oriented hydrologic 

research in support of the mission of the USGS. The stable isotope project conducts research on 

the use of isotope-ratio measurements in studies of water resources and environmental quality. 

One objective of this project is to develop new techniques for isotopic analysis of hydrogen, 

nitrogen, oxygen, carbon, and sulfur in environmental samples. New analytical techniques 

expand the range of tools available for studying the movement of those elements in hydrologic 

and biogeochemical systems. Another objective of the project is to test new applications of 

isotope measurements in specific field settings. Field studies of isotope behavior have 

contributed to understanding water-supply sustainability, groundwater/surface-water 

interactions, paleoclimate history, biologic cycling of nutrients, groundwater contamination, and 

natural remediation. This project also contributes to the improvement of measurement science 

and the development of isotope databases.

Book 10 of the Techniques and Methods Series of the USGS contains detailed descriptions of 

methods of the RSIL, including routine analytical methods called standard operating procedures 

(SOPs), along with safety guidelines, maintenance procedures, and other information about the 

operation of the RSIL. Section C of book 10 contains the SOPs for a variety of methods to 

measure stable isotope ratios, each of which constitutes a chapter. Each chapter is limited to a 

narrow field of subject matter to permit flexibility in revision as the need arises.

Pierre Glynn

Chief, Branch of Regional Research, Eastern Region
This page left blank intentionally.
v

Contents

Foreword............................................................................................................................................................ iii

Summary of Procedure.................................................................................................................................... 1

Reporting Units and Operational Range ...................................................................................................... 1

Reference Materials and Documentation ................................................................................................... 2

Reference Materials Used, Storage Requirements, and Shelf Life....................................... 2

Documentation.................................................................................................................................. 2

Labware, Instrumentation, and Reagents ................................................................................................... 3

Sample Collection, Preparation, Analysis, Retention Times, and Disposal.......................................... 4

Sample Containers, Preservation, and Handling Requirements............................................ 4

Sample Preparation and Time Requirements ............................................................................ 5

Performing the Analysis and Time Requirements..................................................................... 6

Problematic Samples....................................................................................................................... 6

Interferences..................................................................................................................................... 7

Troubleshooting and Bench Notes............................................................................................... 7

Maintenance and Maintenance Records................................................................................... 8

Sample Retention Time and Disposal .......................................................................................... 8

Data Acquisition, Processing, Evaluation, Quality Control, and Quality Assurance .......................... 8

Laboratory Information Management System for Light Stable Isotope (LIMS-LSI) .......... 8

Quality Control (QC) Samples......................................................................................................... 8

Acceptance Criteria for All QC Samples ..................................................................................... 9

Corrective Action Requirements................................................................................................... 9

Responsible Parties for All QC/QA Functions for Procedures Covered in RSIL SOPs ...... 9

Data Management and Records................................................................................................... 9

Health, Safety, and Waste-Disposal Information..................................................................................... 10

Applicable Health and (or) Safety Issues.................................................................................. 10

Personal Protection ...................................................................................................... 10

Electrical Hazards.......................................................................................................... 10

Chemical Hazards.......................................................................................................... 10

Gas Cylinder Handling .................................................................................................. 10

Specific Waste-Disposal Requirements ................................................................................... 10

Revision History............................................................................................................................................... 11

References Cited............................................................................................................................................. 13

Appendix A. Step-by-Step Procedure to Log-In Samples to LIMS-LSI............................................... 14

Appendix B. Step-by-Step Procedure to Create an Excel Sample-List Workbook and Print a 

Sample List............................................................................................................................................... 15

Excel Sample-List Workbook....................................................................................................... 15

Sample List....................................................................................................................................... 15

Worksheet 1.Weighing template................................................................................ 16

Worksheet 2. Samples to be analyzed...................................................................... 17

Appendix C. Step-by-Step Procedure for Weighing and Storing Samples........................................ 19

Appendix D. Step-by-Step Procedure of Zero Blank Autosampler Operation .................................. 20

Appendix E. Step-by-Step Procedure to Add Sample Information to Sequence Table .................. 21

Appendix F. Step-by-Step Procedure to Retrieve Data from ISODAT 2.0 for LIMS-LSI and for 

Data Back-Up .......................................................................................................................................... 22

Appendix G. Step-by-Step Procedure to Transfer Data to LIMS-LSI, to Transfer Data to 

Back-Up Computer, and to Reevaluate Old Data ........................................................................... 23

Appendix H. Step-by-Step Procedure to Determine and Apply Correction Factors and 

Evaluate Data ......................................................................................................................................... 24

Appendix I. Step-by-Step Procedure to Check Elemental Analyzer for Leaks.................................. 25

Appendix J. Daily Checklist .......................................................................................................................... 26
vi

Appendix K. Changing the Insertion Tube..................................................................................................27

Appendix L. Changing the Water Trap........................................................................................................28

Appendix M. Changing the Reaction Tubes ..............................................................................................29

Appendix N. Step-by-Step Procedure to Report Data.............................................................................30

Figures

1. Diagram of the Carlo Erba Instruments NC 2500 (CE NC 2500) Elemental Analyzer 

(used with permission of Thermo Scientific). ...........................................................................3

2. Diagram of a Finnigan MAT (now Thermo Scientific) ConFlo II Open Split (used 

with permission of Thermo Scientific). .......................................................................................4

3. Schematic of a continuous flow isotope-ratio mass spectrometer (CF-IRMS) 

(modified from Clark and Fritz, 1997)............................................................................................5
vii

Conversion Factors

Multiply By To Obtain 

Length

millimeter (mm) 0.03937 inch

centimeter (cm) 0.3937 inch

meter (m) 3.281 foot

Volume

micro liter (μL) 0.0610210–3cubic inch

milliliter (mL) 0.06102 cubic inch

cubic centimeter (cm3) 0.06102 cubic inch

Mass

nanogram (ng) = 10–3μg 3.527 × 10–11 ounce

microgram (μg) = 10–3mg 3.527 × 10–8ounce

milligram (mg) = 10–3

g 3.527 × 10–5 ounce

gram (g) = 10–3

(kg) 3.527 x 10-2ounce

kilogram = 103g; 106mg; 109μg; 1012 ng 2.205 pound

Density

gram per cubic centimeter (g/cm3) 0.5780 ounce per cubic inch

Temperature

Celsius (°C) °F = 9/5 (°C) + 32 Fahrenheit (°F)

Pressure (force/area)

kilopascal (kPa) 9.869 × 10–3 atmosphere, standard (atm)

kilopascal (kPa) 1.450 × 10–1 pound-force/square inch (psi)

kilopascal (kPa) 1.000 × 10–2

bar

kilopascal (kPa) 2.961 × 10–1 inches of mercury at 60 °F

Acronyms and Abbreviations

ANSI American National Standards Institute

CF-IRMS continuous flow isotope-ratio mass spectrometer

cm centimeter

EA elemental analyzer 

GC gas chromatograph

HDPE high-density polyethylene

IRMS isotope-ratio mass spectrometer
viii

kPa kilopascal

LIMS-LSI Laboratory Information Management System for Light Stable Isotopes

mg milligram

mg/g milligram per gram

min minute

mL milliliter

mL/min milliliter per minute

MSDS Material Safety Data Sheets

NBS National Bureau of Standards

NFPA National Fire Protection Association

NWQL National Water-Quality Laboratory (USGS)

per mil, ‰ one part in one thousand parts, with value 10

–3

pF picofarad

QA quality assurance

QC quality control

QC/QA quality control/quality assurance

RSIL Reston Stable Isotope Laboratory

SOP standard operating procedure

std international measurement standard

USGS U.S. Geological Survey

μg microgram

Symbols

Ω ohm

< less than

≤ less than or equal
1

Determination of the δ15N of Total Nitrogen in 

Solids; RSIL Lab Code 2893

By Kinga Révész, Haiping Qi, and Tyler B. Coplen

Summary of Procedure

The purpose of the Reston Stable Isotope Laboratory (RSIL) lab code 2893 is to determine the

δ(

15N/14

N), abbreviated as δ

15N, of total nitrogen in solid samples. A Carlo Erba NC 2500 elemental 

analyzer (EA) is used to convert total N in a solid sample into N2 gas. The EA is connected to a 

continuous flow isotope-ratio mass spectrometer (CF-IRMS), which determines relative difference in the

isotope-amount ratios of stable nitrogen isotopes (15N/14N) of the product N2 gas. The combustion is 

quantitative; no isotopic fractionation is involved. Samples are placed in a tin capsule and loaded into the 

Costech Zero Blank Autosampler of the EA. Under computer control, samples are dropped into a heated 

reaction tube that contains an oxidant, where the combustion takes place in a helium atmosphere 

containing an excess of oxygen gas. Combustion products are transported by a helium carrier through a 

reduction tube to remove excess oxygen and convert all nitrous oxides into N2 and through a drying tube 

to remove water. The gas-phase products, mainly CO2 and N2

, are separated by a gas chromatograph 

(GC). The gas is then introduced into the isotope-ratio mass spectrometer (IRMS) through a Finnigan 

MAT (now Thermo Scientific) ConFlo II interface, which also is used to inject N2 reference gas and 

helium for sample dilution. The IRMS is a Thermo Scientific Delta V Plus CF-IRMS. It has a universal 

triple collector, two wide cups with a narrow cup in the middle, capable of measuring mass/charge (m/z) 

28, 29, 30, simultaneously. The ion beams from N2 are as follows: m/z 28 = N2 = 14N14N; m/z 29 = N2 = 14N15N primarily; m/z 30 = NO = 14N16O primarily, which is a sign of contamination or incomplete 

reduction.

Reporting Units and Operational Range

Variations in stable isotope ratios typically are small. Stable isotope ratios commonly are measured 

and expressed as the relative difference in the ratio of the number of the less abundant isotope (usually the 

heavy isotope) to the number of the more abundant isotope (usually the light isotope) of a sample with 

respect to the measurement standard, std (Coplen, 2011). This relative difference is designated δ

i

E, which 

is a shortened form of δ(

i

E/jE) or δ(

i

E), and is defined according to equation 1 (Coplen, 2011):

( ) ( ) ( ) ( ) ( ) ( )

N( ) N( )

N N N N δ = δ = δ = i j

i j i j

i i i j

std std

P P std std

E E

E E E E E E E E − (1)

where N(

i

E)P and N(

j

E)P are the numbers of the two isotopes iE and jE of element E in specimen P and 

equivalent parameters follow for the international measurement standard, “std.” A positive δ

i

E value 

indicates that the specimen is enriched in the heavy isotope,

i

E, relative to the std. A negative δ

i

E value 

indicates that the specimen is depleted in the heavy isotope,

i

E, relative to the std. For stable nitrogen 

isotope-amount ratios, δ

15N is defined as follows:
2

( ) ( ) ( ) ( ) ( ) ( )

( ) ( )std

14

std

15

std

14

std

15

P

14

P

15

15 15 15 14

N N

N N N N N N N N

N N

N N N N δ = δ = δ = − (2)

The primary reference material for relative nitrogen isotope-ratio measurements (δ

15N) is atmospheric 

nitrogen gas (N2

), which is widespread and homogeneous and, by convention, has a consensus δ

15N value 

of 0 (Mariotti, 1983). By interspersing internationally distributed isotopic reference materials with 

accepted δ values among unknown samples, the δ values can be determined on a normalized δ

15N scale. 

The δ

15N measurements are made with a CF-IRMS, which alternately measures the isotope-number ratios 

of the sample N2 gas and one or more injections of the “working reference” N2 gas. Values of δ

15N

typically are reported in parts per thousand (‰).

The system was tested by analyzing pure inorganic samples (potassium nitrate, sodium nitrate, and 

ammonium sulfate), an array of biological tissues from vertebrates and invertebrates, and geological 

samples (Révész, 1998), such as suspended organic matter in sediment. The routine analysis requires a 

minimum of 40 μg of sample as nitrogen in a maximum of 40 milligrams (mg) of solid sample; however, 

the system is capable of analyzing samples as small as 19 μg of nitrogen in one sample aliquot. The δ

15N

values of reference materials ranged from approximately –5 to +180 ‰, which covers much of the range 

of δ

15N values of natural samples. The precision and accuracy of the results were acceptable (±0.2 ‰),

and no memory effects were observed (Qi and others, 2003).

Reference Materials and Documentation

Reference Materials Used, Storage Requirements, and Shelf Life

In this method, internationally distributed isotopic reference materials used when inorganic Nbearing samples are analyzed. These include IAEA-N-1 (NH4

)2SO4 with δ

15N of +0.43 ‰ (slightly revised 

from Böhlke and Coplen, 1995; Böhlke and others, 1993), IAEA-N-2 ((NH4

)2SO4) with δ

15N of +20.41 ‰, 

IAEA-NO-3 (KNO3

) with δ

15N of +4.7 ‰, USGS25 ((NH4

)2SO4) with δ

15N of –30.4 ‰, USGS26 

((NH4

)2SO4) with a δ

15N of +53.7 ‰, and USGS32 (KNO3

) with δ

15N of +180 ‰. When organic nitrogen 

samples or mixtures of inorganic and organic nitrogen are analyzed, USGS40 and USGS41 L-glutamic 

acid (HO2

CCH2CH2(NH2)CO2H) isotopic reference materials with δ

15N of –4.52 ‰ and +47.57 ‰, 

respectively (Qi and others, 2003), are interspersed with samples. These two L-glutamic acids were 

prepared in the RSIL (Qi and others, 2003). Detailed preparation and calibration procedures for USGS25, 

USGS26, and USGS32 nitrate salts are given by Böhlke and others (1993).

All of these reference materials are stored in glass bottles capped with Teflon-coated or cone-shaped 

caps to keep moisture out. Their shelf life is indefinite.

Documentation

All calibration results are stored in the Laboratory Information Management System for Light Stable 

Isotopes (LIMS-LSI) (Coplen, 2000) under sample identifiers N-1 (IAEA-N-1), N-2 (IAEA-N-2), N-3

(IAEA-NO-3), N-6 (USGS25), N-7 (USGS26), N-25 (USGS32), G-6740 (USGS40), and G-6741

(USGS41).
3 

Labware, Instrumentation, and Reagents

Preparatory labware and apparatus include tin capsules and a microbalance capable of measuring 

samples with 0.001-mg precision. The analytical apparatus consists of four different units: (1) EA, 

(2) ConFlo II interface, (3) IRMS, and (4) computer software.

The EA is a Carlo Erba Instruments NC 2500 (CE NC 2500) system, and it is equipped with a 

Costech “zero blank” autosampler that holds 49 samples enclosed in tin capsules (fig. 1). Each tin capsule

falls into the reaction-tube, which is kept at 1020 °C and is under constant helium flow (90 milliliters per 

minute (mL/min)). The sample immediately reacts with a measured amount of oxygen released from a 5-

milliliter (mL) loop purged at 35 mL/min. The reaction of oxygen with the tin capsule is exothermic, 

which results in localized temperatures of up to 1800 °C, thus ensuring complete and instantaneous 

sample combustion. This is called “dynamic flash combustion.” The combustion gases are first carried by 

helium through an oxidative catalyst layer (Cr2

O3) in the reaction tube, where oxidation is completed, and 

then through a reduction agent (Cu) in another reaction tube kept at 650 °C, where any nitrogen oxides 

are converted into elemental nitrogen and any excess oxygen is retained. At the outlet of the second 

reaction tube, the gas mixture (N2

, usually CO2 from carbon compound in the solid, and H2O) meets a trap 

containing Anhydrone, which absorbs water. The gas mixture, now N2

 and CO2, flows through a GC 

column (Porapak Q kept at 50 °C), which separates the gases, screens out sampling noise, and ensures 

that pure nitrogen gas pass separately through the thermal conductivity detector, through the Finnigan 

MAT ConFlo II open split, and into the IRMS.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Diagram of the Carlo Erba Instruments NC 2500 (CE NC 2500) elemental analyzer (used with permission of 

Thermo Scientific). 

The Finnigan MAT ConFlo II interface provides the means for coupling the EA to the CF-IRMS with 

an open-split arrangement (fig. 2). The principle of an open split is that it reduces the 90 mL/min helium 

flow needed to operate the EA to a 0.3 mL/min helium flow needed to operate the IRMS. This is achieved 

by placing the end of the capillary that leads to the IRMS directly in the flow of helium coming from the 

EA. The narrow diameter and length of this capillary limit the flow of gas into the IRMS. Furthermore, 

extra capillaries carrying reference gas or helium gas can be added to the open split, thereby making it 
4 

possible to inject reference gas into the IRMS or to dilute the sample with extra helium if the sample is 

too large (or to direct gases away by dilution if they are too large to ensure total separation). 

 

 

Figure 2. Diagram of a Finnigan MAT (now Thermo Scientific) ConFlo II Open Split (used with permission of Thermo 

Scientific).

The IRMS is a Thermo Scientific Delta V Plus CF-IRMS (fig. 3). The fundamental principle of the 

CF-IRMS technique is that a carrier gas transports the analyte through an initial stage of online chemistry 

for conversion to a form acceptable by the IRMS (Brenna and others, 1997). This is an automated system 

generating online, high-precision, δ values of bulk solid and nonvolatile liquid samples. In the IRMS, gas 

molecules are ionized in a source by electrons emitted from a hot filament. The ions are accelerated into 

an analyzer, separated in a magnetic field, and collected in Faraday cup collectors. The ion-beam 

intensities are measured with electrometers. This IRMS has a universal triple collector with two wide 

cups and a narrow cup in the middle, capable of measuring m/z 28, 29, and 30, simultaneously. The 

resistor-capacitor combination on the electrometer used to measure these masses are as follows: 

3 × 108

ohms (Ω) and 680 picofarads (pF) on cup 1, 3 × 1010 Ω and 5 pF on cup 2, and 3 × 1011 Ω and 2 pF 

on cup 3. 

The Thermo Scientific ISODAT 2.0 software is designed (1) to advance the autosampler carousel, (2) 

to control the Finnigan MAT ConFlo II interface to inject reference gases at the desired time and dilute 

the sample if desired, (3) to operate the IRMS, (4) to acquire data from the IRMS, and (5) to calculate 

delta values. 

Sample Collection, Preparation, Analysis, Retention Times, and 

Disposal

Sample Containers, Preservation, and Handling Requirements

Each sample is collected in a high density polyethylene (HDPE) scintillation vial with Polyseal cap 

(Wheaton 986706), available from RSIL. Sample size should be a maximum of 1 g. The sample should be 

homogenized, oven dried (90 °C), and powdered by the submitter to 100 to 200 μm. Containers are 

labeled with isotopes whose concentrations are to be determined and with the respective laboratory code 

or schedule number. A minimum of 0.15 mg of nitrogen per sample is required. The minimum sample 

concentration is 1 milligram per gram (mg/g) sample for N. Lower concentrations can be analyzed; 

contact the laboratory for further information. No treatment, preservation, or special shipping is required. 
5

Figure 3. Schematic of a continuous flow isotope-ratio mass spectrometer (CF-IRMS) (modified from Clark and Fritz, 

1997).

 

Sample Preparation and Time Requirements

Sample preparation involves logging in samples to the LIMS-LSI (see appendix A), creating a 

sample work-order sheet, and weighing samples into capsules. 

The logging-in procedure involves logging samples in batches to LIMS-LSI that could contain from 

1 to 100 samples per batch (also called a project), printing labels for each sample, and printing a summary 

project report. Each sample label contains the Field ID (the identifier by which the sample submitter 

knows each sample) and the unique RSIL Lab ID assigned by the LIMS-LSI. The summary project report 

is inserted into a three-ring binder titled “Samples in Progress.”

An Excel sample workbook is created. Step-by-step procedures and examples are shown in the 

appendix B. Sample weighing includes balance conditioning, weighing appropriate sample into capsule

(sufficient to yield 40 micrograms (μg) of nitrogen), closing capsules by folding them, and recording 

weight on template. Step-by-step procedures are shown in appendix C. The time requirement for 

performing the sample preparation described is approximately 1 to 5 days.
6

Performing the Analysis and Time Requirements

All samples are prepared and analyzed in batches of a maximum of 80 per day, and all samples are 

analyzed in duplicate. Six aliquots of two internationally distributed isotopic reference materials with 

different δ

15N values are analyzed at the beginning of a batch. Four of them are depleted in 15N, and two 

are enriched in 15N. A set of these two reference materials also is interspersed among the samples. No 

fewer than one reference material set is analyzed for every 10 unknowns. The amount of nitrogen in the 

reference materials must be in the same range as that of the samples. Therefore, one must preanalyze 

every sample to obtain the N/C concentration ratio unless it is provided by the customer. If the N/C 

concentration ratio is unknown and cannot be determined prior to isotopic analysis, a sequence of 

reference materials containing different amounts of material must be analyzed to determine a mass 

dependent correction factor. The carbon concentration is necessary to apply the correct dilution with 

helium to the CO2 to avoid its peak interference to the N2 peak.

The procedure involves loading the autosampler carousel with capsules containing an appropriate 

amount of sample in sequence according to the “Template” (appendix D), loading a new sample heading 

(including sample weights) into the “Sequence Table” in ISODAT 2.0 of the IRMS software, choosing 

the appropriate “Method,” and starting sequence acquisition (appendix E). After the analyses are 

completed, data are exported (appendix F) to a removable disk or “memory stick” from the ISODAT 2.0 

database and transferred (appendix G) to the LIMS-LSI system and Data Back-Up computer. Final daily 

correction factors are determined by LIMS-LSI (appendix H), based on the daily analyses of reference 

materials using equations 3 and 4 , which are presented with example numerical values:

–4.52 ‰ = m × δ

15NUSGS40/workingrefgas + b (3)

+47.57 ‰ = m × δ

15NUSGS41/workingrefgas + b (4)

The values –4.52 ‰ and +47.57 ‰ are the assigned values of the two international reference materials 

(USGS40 and USGS41), respectively. The δ

15Nref/workingrefgas values in the equations 3 and 4 are the mean 

daily delta values of the respective international reference materials relative to the working reference gas; 

b is the additive correction factor and m is the expansion coefficient correction factor. When two or more

reference materials with different isotopic compositions are analyzed, the correction for sample blank 

does not need to be applied if the magnitude of the blank in the samples and reference materials is 

identical. However, if only a single reference material is analyzed, only one-point calibration is possible; 

therefore, a blank correction should be applied.

If replicates do not agree within acceptable tolerances, they are analyzed until acceptable statistics are 

achieved. The computerized LIMS-LSI will not release data until the statistics are acceptable. The time 

requirement for performing the analyses described above is a minimum of two days.

The systematic procedure is listed in the “Lab Procedures” binder. The supervisor reviews suggested 

changes before they are adopted. 

Problematic Samples

Problematic samples include those that do not have nitrogen and carbon concentration ratios prior to 

isotopic analysis. In that case, the C/N amount ratio needs to be determined by a preliminary analysis to 

be able to determine the sample amounts that gives an appropriate amount of nitrogen (40 μg) and to be 

able to determine the amount of dilution of CO2 with He.

When analyzing samples containing very low nitrogen, a large amount of sample is needed to yield 

measureable N2

. This could cause poor combustion; as a consequence, a poor yield and a poor isotopic 
7

result could be observed. To solve this problem, a larger oxygen loop (10 mL) normally is used. 

However, this additional oxygen will consume the reactors quickly.

Interferences

There are no known interferences with this analytical technique. However, it should be pointed out 

that if samples contained a mixture of different nitrogen sources, reported isotope-ratio values would 

represent the isotopic composition of the total nitrogen in the sample. The δ

15N values of specific N 

species cannot be provided with this analytical technique.

Troubleshooting and Bench Notes

The most common problems with this analytical technique are (1) incomplete combustion, 

(2) leakage in the system, particularly in the EA, and (3) insufficient linearity of the IRMS. The fact that 

the melting point of the compounds being combusted are usually higher or close to the temperature of the 

reaction tube (1020 °C) could create insufficient combustion. The “flash point” has a higher temperature, 

approximately 1800 °C (because the tin oxidation reaction is exothermic); therefore, it is critical to create 

a sharp, sudden, bright, single flash. This can be done by coordinating (1) helium pressure and flow rate, 

(2) oxygen pressure and flow rate, and (3) sample “start time.” The system is so sensitive that even a 1-

second difference in the sample dropping time can make a difference in the brightness of the flash, peak 

shape, peak area, and, consequently, the δ result. The optimum set-up parameters of the RSIL EA are 

cycle = 100, oxy = 60 (O2 flow stops flowing into autosampler), sample start = 10, and sample stop = 40. 

The helium regulator is set for 150 kilopascals (kPa), and helium flow set up is 95 mL/min, which 

actually measures 120 mL/min at the vent. The oxygen regulator is set for 150 kPa, and the oxygen flow 

rate is 54 mL/min at purge. The oxygen loop is 5 mL. The oven temperature of the GC is 50 °C.

Even a tiny leak can interfere with the result; it can create double peaks, an unstable analysis, and so 

forth. The high helium flow rate makes the system more sensitive to leaks (Bernoulli principle). Leaks 

can cause uncontrollable changes in the flow rate, which could prevent quantitative combustion, affecting 

the peak shape and peak area, and consequently, the δ value. Ash accumulation is also critical. Although 

tin oxide serves as a catalyst in the reaction, it also partially “clogs” the system, interfering with the flow 

rate, although not necessarily in a measurable manner. However, this assumes a nonrandom interference 

that causes the shift in δ value. By frequently analyzing reference materials, this shift can be corrected. 

However, after approximately 50 samples (one carousel), it is advisable to remove the ashes (Révész, 

1998). The leak-checking procedure is given in appendix I. A leak is indicated in the IRMS when m/z 28 

and 29 are high and the ratios of m/z 28, 32, and 40 (N2

, O2, and argon) reflect atmospheric compositions. 

Acceptable levels for m/z 28 and 29 are approximately 0.15 and 0.015 V, respectively, with typical IRMS 

settings. (See “Daily Checklist” in appendix J.)

Adjustment of the emission current is critical. The higher it is, the higher the signal will be, but the 

poorer the linearity will be. The IRMS is temperature sensitive; therefore, it must be operated in a 

temperature-controlled environment with all of the side panels on. The resistor-capacitor combinations on 

the electrometers are as follows: 3 × 108

Ω and 680 pF for cup 1, 3 × 1010 Ω and 5 pF for cup 2, and 3 ×

1011 Ω and 2 pF for cup 3. The instrument is capable of measuring m/z 28 and 29, and m/z 30 = NO 

=14N16O primarily, which is a sign of contamination or incomplete reduction.

An internal precision check needs to be conducted using the “Zero Enrichment” method (standard gas

on/off 10 times), and it must give a standard deviation of 0.1 ‰ or better. The “Ratio Linearity” test must 

give 0.12 ‰/V or better linearity. An external precision check needs to be performed as needed, 

approximately every week or so; samples of the same amount need to be analyzed within ± 0.010 mg. 

The 1-σ standard deviation must be 0.2 ‰ or better for n = 10. If either of these criteria has not been met, 

the IRMS cannot be used and must be refocused.
8

Maintenance and Maintenance Records

Routine maintenance is necessary for the upkeep of the IRMS vacuum system, including 

(1) checking the pump performance from time to time, (2) changing the pump oil, and (3) changing the 

oil cartridge in each turbomolecular pump at 6-month intervals. Pump conditions, including date of oil 

changes, problems, and repairs to the pump, are listed in the pump database (file path: LIMS 

C:\RSIL\vacuum pumps). Maintenance of the EA includes (1) changing the insertion tube every day 

(appendix K), (2) changing the water trap approximately every 500 samples (appendix L), (3) changing 

reaction tubes approximately every 200 to 250 analyses (appendix M), and (4) cleaning the sample 

carousel every week. The IRMS also requires a daily check (appendix J). A logbook is kept for each 

IRMS, where notes of maintenance checks, history of normal settings, problems, and repairs are listed. 

Maintenance of ISODAT 2.0 includes importing (transferring) results from the ISODAT 2.0 database 

to the LIMS-LSI computer hard disk and the Data Back-Up computer hard disk (appendix G).

Sample Retention Time and Disposal

Samples are retained in the RSIL for at least 6 months after reporting data. Samples are then 

discarded unless the submitter has requested that the samples be returned. 

The ISODAT sample files from the IRMS computer are kept indefinitely on two different hard disks 

of the Data Back-Up computer. Paper reports are kept for a few weeks. Analytical results from the IRMS 

are transmitted to LIMS-LSI and kept indefinitely. No data are entered by hand, and no manual 

calculations are performed on the data.

Data Acquisition, Processing, Evaluation, Quality Control, and Quality 

Assurance

Laboratory Information Management System for Light Stable Isotope (LIMS-LSI)

In the RSIL, the LIMS-LSI (Coplen, 2000) is used for data acquisition, processing, and evaluation. 

This system is a database program capable of (1) storing information about samples, (2) storing the results 

of mass spectrometric delta values of samples after importing from the ISODAT database, (3) calculating 

analytical results using standardized algorithms stored in a database, (4) normalizing delta values using 

isotopic reference materials, and (5) generating templates for convenient sample placement to facilitate 

loading of automated IRMS sample-preparation manifolds. With this system, the following are ensured: 

(1) quality assurance (QA), (2) laboratory efficiency, (3) reduction of workload and errors owing to the 

elimination of retyping of data by laboratory personnel, and (4) a decrease of errors in data reported to 

sample submitters. This database provides a complete record of information on how laboratory reference 

materials have been analyzed and provides a record of what correction factors have been used as an audit 

trail for the RSIL.

Quality Control (QC) Samples

Samples are prepared and analyzed in batches—a maximum of 80 daily. Each batch contains 

approximately six reference materials at the beginning, four depleted in 15N and two enriched in 15N, and 

later in the batch one depleted in 15N for every ten samples. The last analysis of a batch is that of a 

reference material depleted in 15N. All of these reference materials serve as QC samples. 
9

Daily, the analyst (1) examines the computer printouts for indications of analytical problems, 

(2) determines the daily additive and expansion correction factors by the LIMS-LSI, and (3) applies 

corrections factors to isotopic data. The values of these corrections factors, along with the date and range 

of analysis numbers, are recorded manually in the laboratory “EABook” even though the data are already 

in the LIMS-LSI. After printing the list of isotopic results from the “Table of Samples in Progress,” the 

analyst reviews the results and determines which samples need to be analyzed a third time to achieve 

acceptable results (i.e., ∆δ ≤0.2 ‰). 

Acceptance Criteria for All QC Samples

Acceptance criteria for QC samples are the same as acceptance criteria for the other samples. The 

rules are as follows:

• If standard deviation is ≤0.2 ‰, use mean delta. 

• If there are three or more analyses, delete the outlier and recalculate. 

• If standard deviation of this recalculation is ≤0.2 ‰, use mean from this recalculation. 

• If none of the above is the case, the result is not accepted, and corrective action is required.

The RSIL estimates the expanded uncertainty (U = 2μc

) of δ

15N measurement results. The expanded 

uncertainty provides an envelope that represents a 95-percent probability of encompassing the true value 

that has been determined from the aggregation of measurement results over a period of time. The 

expanded uncertainty can be determined using the guide to the expression of uncertainty (Joint 

Committee for Guides in Metrology, Working Group 1 (JCGM/WG1), 2010). The application of 

expanded uncertainty to the reporting of stable isotope measurements is discussed by Coplen and others 

(2006). The estimated expanded uncertainty of δ

15N measurement results is ±0.5 ‰ unless otherwise 

specified, and this value is conservative. If any given sample were resubmitted to the RSIL for nitrogen

isotopic analysis, the measured value would fall within the uncertainty bounds of the previous result more 

than 95 percent of the time.

Corrective Action Requirements

If the analyst finds any problem with the daily reference-sample data, the analyst contacts the 

supervisor. The trouble-shooting process will require an evaluation and reanalysis of certain samples to 

ascertain the origin of the problem. 

If samples do not give satisfactory results after three or more separate analyses, the analyst averages 

all the data and reports the mean value. Such analytical results are indicated with a comment, and the 

customer will be advised by e-mail or other means.

Responsible Parties for All QC/QA Functions for Procedures Covered in RSIL SOPs

The analyst, with supervisory approval, is responsible for qualifying data and notifying customers.

Data Management and Records

In addition to evaluating daily sample analyses, every week an analyst evaluates the data project by 

project, reports results to the customers, and files final project data reports in the laboratory 

“Correspondence” binder (appendix N).
10

Health, Safety, and Waste-Disposal Information

Applicable Health and (or) Safety Issues

Personal Protection

Safety glasses and protective gloves are recommended whenever samples are handled, especially 

when the samples are of biological origin. For other precautions and safety procedures, consult Material 

Safety Data Sheets (MSDSs), which are on file in the laboratory and at the URL 

http://www.ilpi.com/msds/#Manufacturers. This URL provides links to the MSDSs of most chemical 

companies.

Electrical Hazards

Electrical systems must conform to the National Electric Code, National Fire Protection Association 

Code (NFPA 70–1971), and the American National Standards Institute (ANSI) Code (C1–1971). For 

more information, consult the U.S. Geological Survey’s Safety and Environmental Health Handbook 

(U.S. Geological Survey, 2002, sec. 4–4.1).

Shock hazards exist inside the instruments. Only an authorized service representative or an individual 

with training in electronic repair must remove panels or circuit boards where voltages are greater than 

20 volts. The instruments require a third-wire protective grounding conductor. Three-to-two wire adapters 

are unsafe for these instruments. 

Chemical Hazards

The hazardous chemicals used in the process are Cr2O3 and Co3O4, which are used in the oxidation 

reaction tube. Sometimes the reaction tube cracks, and the packing material leaks out. When this happens, 

protective gloves and a full-face mask are required during the cleanup process. All the materials, 

including gloves and any cleaning towels, need to be collected in a plastic bag and disposed of as 

hazardous waste. Upon receipt, all samples must be carefully inspected for indications of hazards. 

Gas Cylinder Handling

Compressed gas cylinders must be handled and stored according to the U.S. Geological Survey’s

Safety and Environmental Health Handbook (U.S. Geological Survey, 2002, sec. 4–4.5.1). Each cylinder 

must be (1) carefully inspected when received; (2) securely fastened at all times with an approved chain 

assembly or belt; (3) capped at all times when not in use; (4) capped when transported; (5) transported 

only by a properly designed vehicle (hand truck); and (6) stored separately with other full, empty, 

flammable, or oxidizing tanks of gas, as appropriate. 

Specific Waste-Disposal Requirements

Used reaction tubes containing Cr2O3 and CO3O4 must be collected in a closed container and must be 

given to the safety, health, and environment officer for disposal.
11

Revision History

Publication Series and Series Number: Techniques and Methods 10–C11 (Book 10, Section C, Chapter 11)

Publication Title: Determination of the δ

15N of Total Nitrogen in Solids; RSIL Lab Code 2893

Publication Authorship: Révész, Kinga, Qi, Haiping, and Coplen, Tyler B.,

Version 1.0, 2006

Version 1.1, 2007

Version 1.2, 2012

Summary of Revised Product Components

Component Description 

Last Revised 

In Publication Version 

Date of 

Last Revision 

Title Replaced δ(

15N/14N) by δ15N to be in 

accordance with recommendations of the 

Commission on Isotopic Abundances and 

Atomic Weights of the International Union 

of Pure and Applied Chemistry.

1.2 July 2012

Authorship Tyler B. Coplen added as a coauthor. 1.2 July 2012

Summary of 

procedure

Updated text to reflect new instrumentation. 1.2 July 2012

Reporting units 

and operational 

range

Updated text to reflect expression of delta 

values with number ratios.

1.2 July 2012

Labware, 

instrumentation, 

and reagents

Update text to reflect new instrumentation. 1.2 July 2012

Sample 

collection, 

preparation, 

analysis, 

retention times, 

and disposal

Updated text to reflect that samples are no 

longer submitted through the National 

Water Quality Laboratory (NWQL) and 

improved analytical techniques.

1.2 July 2012
12

Data acquisition, 

processing, 

evaluation, 

quality control, 

and quality 

assurance

Updated text to reflect that samples are no 

longer submitted through the National 

Water Quality Laboratory (NWQL).

1.2 July 2012

References cited Added three references. 1.2 July 2012

Appendix A Updated text to reflect that samples are no 

longer submitted through the National 

Water Quality Laboratory (NWQL).

1.2 July 2012

Appendix N Updated text to reflect that samples are no 

longer submitted through the National 

Water Quality Laboratory (NWQL).

1.2 July 2012

Main text footer Removed from document. 1.2 July 2012
13

References Cited

Böhlke, J. K., and Coplen, T. B., 1995, Interlaboratory comparison of reference materials for nitrogenisotope-ratio measurements, in Reference and intercomparison materials for stable isotopes of light 

elements: Vienna, Austria, International Atomic Energy Agency, IAEA-TECDOC-825, p. 51–66.

Böhlke, J. K., Gwinn, C. J. and Coplen, T. B., 1993, New reference materials for nitrogen-isotope-ratio 

measurements: Geostandards Newsletter, v. 17, p. 159–164.

Brenna, J.T., Corso, T.N., Tobias, H.J., and Caimi, R.J., 1997, High-precision continuous-flow isotoperatio mass spectrometry: Mass Spectrometry Reviews, v. 16, p. 227–258. 

Clark, Ian, and Fritz, Peter, 1997, Environmental isotopes in hydrogeology: Boca Raton, Fla., Lewis 

Publishers, 328 p.

Coplen, T.B., 2000, A guide for the laboratory information management system (LIMS) for light stable 

isotopes—Version 7 and 8: U.S. Geological Survey Open-File Report 00–345, 121 p., accessed April 

27, 2012, at http://water.usgs.gov/software/code/geochemical/lims/doc/ofr00345.pdf.

Coplen, T.B., 2011, Guidelines and recommended terms for expression of stable-isotope-ratio and gasratio measurement results: Rapid Communications in Mass Spectrometry, v. 25, p. 2538–2560,

accessed April 27, 2012, at http://onlinelibrary.wiley.com/doi/10.1002/rcm.5129/abstract.

Coplen, T.B., Brand, W.A., Gehre, Matthias, Gröning, Manfred, Meijer, H.A.J., Toman, Blaza, and 

Verkouteren, R.M., 2006, New guidelines for δ

13C measurements: Analytical Chemistry, v. 78, no. 7, 

p. 2439–2441.

Joint Committee for Guides in Metrology, Working Group 1 (JCGM/WG1), 2010, Evaluation of 

measurement data—Guide to the expression of uncertainty in measurement (known as the GUM) 

(2010 corrected version of the first edition of 2008): Joint Committee for Guides in Metrology 

[publication] JCGM 100:2008, 120 p., accessed July 16, 2012, at 

http://www.bipm.org/en/publications/guides/.

Mariotti, A., 1983, Atmospheric nitrogen is a reliable standard for natural 15N abundance measurements:

Nature, v. 303, p. 685-687.

Qi, H. P., Coplen, T. B., Geilmann, H., Brand, W. A. and Böhlke, J. K., 2003: Two new organic reference 

materials for δ

13

C and δ

15N measurements and a new value for the δ13C of NBS 22 oil: Rapid 

Communications in Mass Spectrometry, v. 17, p. 2483–2487.

Révész, Kinga, 1998, Carbon and nitrogen isotope ratios of organic and inorganic bulk samples;

Instrument performance and intercalibration [abs.], in The 5th Canadian Continuous-Flow Isotope

Ratio Mass Spectrometry Workshop, August 16–19, 1998: Ottawa, Ontario, Canada, University of 

Ottawa, not paged.

U.S. Geological Survey, 2002, USGS handbook 445–3–H, Safety and environmental health handbook, 

435 p.
14

Appendix A. Step-by-Step Procedure to Log-In Samples to LIMS-LSI

1. For sample submitter:

a. Download “Standard Submission Excel Form” from the RSIL Web site at 

http://isotopes.usgs.gov/.

b. Fill out the requested sample information.

c. Send a diskette or CD and a hard copy along with the samples or e-mail the completed RSIL 

Excel worksheet to isotopes@usgs.gov.

2. For RSIL personnel:

a. Match up information on sample bottles with the submitted “Standard Submission Excel Form.”

b. Enter all Standard Submission Excel Form information into LIMS-LSI by loading media. 

Submission date is the date samples are logged in.

c. Use “Import a New Project From Excel Submission” in LIMS-LSI to assign G#s.

d. Print out one project report and container labels (one for each sample).

e. Put a label on each sample bottle and cross-check Field IDs between bottles and Excel worksheet 

forms.

f. Punch holes in the original Excel worksheet and all the project information you have and put in 

“Samples-in-Progress” binder.
15

Appendix B. Step-by-Step Procedure to Create an Excel Sample-List

Workbook and Print a Sample List 

Excel Sample-List Workbook

1. Use default worksheet-weighing template and add samples to be analyzed.

2. Fill the appropriate amount in the cells of the rows labeled “Set weight mg.”

3. Print first worksheet in workbook.

4. Write the tray ID and the date on both the diskette and paper template.

5. Put them near the balance.

Sample List

1. Use “Create a Sample List -->” in LIMS-LSI.

2. Select appropriate template for EA and mass spectrometer.

3. Select “Create Sample List for Mass Spec” (dialog box informs user how many samples are waiting 

to be analyzed).

4. Click “OK.”

5. Click “Save and Print.”

6. Insert diskette to receive sample headings.

7. Click “OK.”

8. Write the day of the week that these samples should be analyzed on both the diskette and paper 

template.

9. Put them near the balance.

10. Exit LIMS-LSI.
16

Worksheet 1. Weighing template.

[International reference materials are in bold font.]

1 2 3 4 5 6 7 8 9 10 11 12

Our Lab 

ID

G-6740 G-6740 G-6740 G-6740 G-6741 G-6741 G9031G9031G9032G9032G-9033 G-9033

A Set 

weight 

mg

0.75 0.75 0.75 0.75 0.75 0.75 0.8 0.8 0.8 0.8 0.8 0.8

Actual 

weight, 

mg

0.75 0.69 0.73 0.766 0.751 0.764 0.804 0.798 0.799 0.802 0.805 0.803

Comment USGS40USGS40USGS40USGS40USGS41USGS41

Our Lab 

ID

G-9034 G-9034 G-9035 G-9035 G-6741 G-6741 G9036G9037G9037G9038G-9036 G-9039

B Set 

weight 

mg

0.8 0.8 0.8 0.8 0.75 0.8 0.8 0.8 0.8 0.8 0.8 0.8

Actual 

weight, 

mg

0.801 0.802 0.795 0.803 0.759 0.795 0.801 0.799 0.793 0.804 0.801 0.795

Comment USGS40USGS41

Our Lab 

ID

G-9039 G-9040 G-9040 G-6740 G-6741 G-9041 G9042G9042G9043G9043G-9044 G-9044

C Set 

weight 

mg

0.8 0.8 0.8 0.75 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8

Actual 

weight, 

mg

0.804 0.804 0.795 0.766 0.797 0.802 0.8 0.805 0.804 0.797 0.804 0.801

Comment USGS40USGS41

Our Lab 

ID

G-9045 G-9045 G-6740 G-6741 G-9046 G-9047 G9047G9048G9048G9049G-9049 G-9050

D Set 

weight 

mg

0.8 0.8 0.75 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8

Actual 

weight, 

mg

0.795 0.803 0.766 0.799 0.798 0.796 0.803 0.805 0.805 0.797 0.802 0.796

Comment USGS40USGS41

Our Lab 

ID

G-9051 G-6740 G-6741 G-9052 G-9052 G-9053 G9053G9054G9054G9055G-6740 G-6741

E Set 

weight 

mg

0.8 0.75 0.8 1.7 1.7 1.4 1.4 1.1 1.1 0.74 0.74 0.75

Actual 

weight, 

mg

0796 0.756 0.795 1.703 1.705 1.405 1.399 1.102 1.097 0.739 0.737 0.795

Comment USGS40USGS41 USGS40USGS41
17

Worksheet 2. Samples to be analyzed.

[International reference materials are in bold font]

Line Identifier Port Comment Amount Amt Unit

1 G-6740 A1 USGS40 0.75 mg

2 G-6740 A2 USGS40 0.769 mg

3 G-6740 A3 USGS40 0.763 mg

4 G-6740 A4 USGS40 0.766 mg

5 G-6741 A5 USGS41 0.751 mg

6 G-6741 A6 USGS41 0.764 mg

7 G-9031 A7 0.804 mg

8 G-9031 A8 0.798 mg

9 G-9032 A9 0.799 mg

10 G-9032 A10 0.802 mg

11 G-9033 A11 0.805 mg

12 G-9033 A12 0.803 mg

13 G-9034 B1 0.801 mg

14 G-9034 B2 0.802 mg

15 G-9035 B3 0.795 mg

16 G-9035 B4 0.803 mg

17 G-6740 B5 USGS40 0.757 mg

18 G-6741 B6 USGS41 0.795 mg

19 G-9036 B7 0.801 mg

20 G-9037 B8 0.799 mg

21 G-9037 B9 0.793 mg

22 G-9038 B10 0.804 mg

23 G-9038 B11 0.801 mg

24 G-9039 B12 0.795 mg

25 G-9039 C1 0.804 mg

26 G-9040 C2 0.804 mg

27 G-9040 C3 0.795 mg

28 G-6740 C4 USGS40 0.766 mg

29 G-6741 C5 USGS40 0.797 mg

30 G-9041 C6 0.802 mg

31 G-9042 C7 0.8 mg

32 G-9042 C8 0.805 mg

33 G-9043 C9 0.804 mg

34 G-9043 C10 0.797 mg

35 G-9044 C11 0.804 mg

36 G-9044 C12 0.801 mg

37 G-9045 D1 0.795 mg

38 G-9045 D2 0.803 mg

39 G-6740 D3 USGS40 0.766 mg

40 G-6741 D4 USGS41 0.799 mg

41 G-9046 D5 0.798 mg
18

Line Identifier Port Comment Amount Amt Unit

42 G-9047 D6 0.796 mg

43 G-9047 D7 0.803 mg

44 G-9048 D8 0.805 mg

45 G-9048 D9 0.805 mg

46 G-9049 D10 0.797 mg

47 G-9049 D11 0.802 mg

48 G-9050 D12 0.796 mg

49 G-9051 E1 0.796 mg

50 G-6740 E2 USGS40 0.756 mg

51 G-6741 E3 USGS41 0.795 mg

52 G-9052 E4 1.703 mg

53 G-9052 E5 1.705 mg

54 G-9053 E6 1.405 mg

55 G-9053 E7 1.399 mg

56 G-9054 E8 1.102 mg

57 G-9054 E9 1.097 mg

58 G-9055 E10 0.739 mg

59 G-6740 E11 USGS40 0.737 mg

60 G-6741 E12 USGS41 0.761 mg
19

Appendix C. Step-by-Step Procedure for Weighing and Storing Samples

1. Samples should be homogenized and dried.

2. Insert template diskette in the computer connected to the microbalance.

3. Condition balance (this step should be done once a day):

a. Place empty capsule on balance and close door. Wait for reading to stabilize (the “mg” on the 

display appears). Tare the balance.

b. Remove and replace capsule and make sure the stabilized weight is 0.000 mg.

c. Repeat these steps until the balance is stable.

4. Remove capsule from balance, add sample, and weigh the filled capsule. Repeat until you have the 

desired weight (which gives 0.15 μg of nitrogen).

5. Enter sample weight on the template.

6. Fold cup; secure sample in it.

7. Repeat steps 4 to 6 for every sample.

8. Note:

a. Do not cross-contaminate samples.

b. Make sure the spatula and sample area are cleaned using Kimwipes between each sample.

c. Always allow balance to stabilize before removing capsule.
20

Appendix D. Step-by-Step Procedure of Zero Blank Autosampler 

Operation

1. Before opening the lid, make sure the isolation valve (between sample chamber and reaction tube) is 

closed (arrow towards you) and make sure the helium purge isolation valve (on the left) is closed.

2. Open the purge vent on the top of the autosampler to vent the sample chamber.

3. Release the three fittings holding the lid closed and open the lid.

4. Place your samples in the autosampler and close the lid. Load 49 samples for a 50-position carousel, 

leaving the first hole empty.

5. Secure the lid with three bolts. Screw in all three bolts finger tight, and then lightly tighten two bolts

at a time, moving around the lid until they are all completely tight to minimize stress and warping of 

the lid.

6. Open the helium purge isolation valve (screw down), purge the sample chamber for 5 minutes (min)

at a helium flow rate of 298 mL/min. Make sure the purge vent valve (on top) is open. Press your 

finger on top of the vent valve for 2 s, then release; you should hear the sound of a pressure release.

7. Close the helium purge vent valve (screw down), wait for 2 min for gas pressure at sample chamber 

and a helium flow rate to stabilize. Close the helium purge isolation valve.

8. Open the isolation valve (arrow toward up). Wait for 2 min for stabilized baseline. With a helium 

carrier flow rate of 90 mL/min, one should observe m/z 28 on cup 1 at about 50 millivolts, m/z 29 on 

cup 2 at about 50 to 60 millivolts and m/z 30 on cup 3 at about 140 millivolts to 1 volt (appendix J).
21

Appendix E. Step-by-Step Procedure to Add Sample Information to 

Sequence Table

1. Insert diskette with “Weighing Template” on it.

2. Open “Sequence File” under “EA configuration.”

3. Open “Weighing Template” in Excel.

4. “Copy” and “Paste” the list of Sample IDs and sample weight from the template to sequence table.

5. Select appropriate “method” for each sample.

6. Define the reference line and blank line.

7. Start.

8. Give Folder Name: Comment

a. Select pre: “Date.”

9. Give File Name:

a. Select pre: “Analyzes #.”

b. Select post: “Identifier 1.”

c. Select: “Print Result.”

10. Click “OK.”

11. Wait for first sample to be analyzed.
22

Appendix F. Step-by-Step Procedure to Retrieve Data from ISODAT 2.0 

for LIMS-LSI and for Data Back-Up

1. For LIMS-LSI:

a. Select “Result” in ISODAT 2.0.

b. Select the result folder you wish to export data from.

c. Select all the individual analyses you want to retrieve by right clicking the selected file.

d. Select “Reprocess.”

e. Give a file name.

f. Add export template previously designed.

g. Open.

h. Click “OK.” (Reprocessing takes 1–2 min)

i. Open Excel.

j. Find the file you reprocessed (E-drive, Finnigan, ISODAT 2.0, Global, User, CONFLO II, 

Interface, Result: File name).

k. Open it in Excel.

1. Save as: “A” drive. Keep in Lotus format.

m. Define all reference peaks by adding #1 under column “Is Ref.?.”

n. Now the file can be imported to LIMS-LSI.

2. For Data Back-Up computer:

a. Go to Windows Explorer in ISODAT 2.0 computer.

b. Find the drive where the data are (D).

c. Choose: “Finnigan.”

d. Choose: “User.”

e. Choose the inlet system where you have data (Gas Bench or EA).

f. Choose “Result folder.”

g. Transfer data to memory stick.
23

Appendix G. Step-by-Step Procedure to Transfer Data to LIMS-LSI, to 

Transfer Data to Back-Up Computer, and to Reevaluate Old Data

1. Transfer data to LIMS-LSI:

a. Start LIMS.

b. Choose “Import Data From Instruments.”

c. Choose mass spectrometer, for example “H --> Hercules.”

d. Click “Import.”

e. Select the file that will be imported.

f. Depending upon the mass spectrometer, the user may be given the opportunity to select the 

columns containing the isotope data. Enter column names as appropriate for the isotope or 

isotopes to be imported.

g. Click “Import.” Note: Sample ID, sample weight, Peak Area, Analyses #s are automatically 

imported to LIMS-LSI. 

2. Transfer data to Data Back-Up computer:

a. Go to Windows Explorer in the Data Back-Up computer.

b. Find the drive where your back ups are stored.

c. Choose: “RSIL.”

d. Choose: “Mass. Spec. Analysis Back Up.”

e. Choose the mass spectrometer. For example, “H,” which stands for “Hercules,” a Thermo 

Scientific Delta V Plus IRMS.

f. Under that folder create a new folder. The name of the folder should be the date range of the 

analyses that one wishes to back up.

g. Transfer data to that folder.

h. Make back ups every 2 weeks or so, as needed.

3. Reevaluate old data:

a. Find your samples in the Data Back-Up computer.

b. Transfer data by a memory stick to the computer where a virtual version of ISODAT 2.0 is 

installed.

c. Reevaluate your data.
24

Appendix H. Step-by-Step Procedure to Determine and Apply Correction 

Factors and Evaluate Data

1. Open “Apply Data Normalization” in LIMS-LSI.

2. Select instrument and isotope, for example “H (Hercules) for 34S.”

3. Select “Query.”

4. Double click on the last sample analyzed on that day.

5. Evaluate data of the reference materials.

6. Choose “Normalize with all References.”

7. Click “Apply Normalization -->.”

8. Print out correction factor sheet.

9. Report daily reference values and correction factors along with date and range of analysis number to 

the “EA” binder.

10. Go back to LIMS-LSI main menu by closing open windows.

11. Choose “Print/Save Analyses in Progress”:

a. Choose appropriate isotope.

b. Choose appropriate prefix (W for water, N for nitrogen, S for sulfur).

c. Enter sample ID range from “Samples-to-Be-Analyzed” sheet.

d. Click Print.

12. Review results and identify samples that need to be re-analyzed.

13. Add the samples needing analyses to the Samples to Be Analyzed:

a. Go back to LIMS-LSI main menu.

b. Open “Create a Sample List -->.”

c. Select appropriate sample list for the mass spectrometer and the samples that are to be analyzed.

d. Find each sample in the “List of samples.”

e. Change entry in “Repeats” column from 0 to 1.

f. Close Windows and exit LIMS-LSI.
25

Appendix I. Step-by-Step Procedure to Check Elemental Analyzer for 

Leaks

1. Close the VENT carrier output using the proper cap (provided).

2. Adjust the helium pressure to 150 kPa (regulator is on the front central panel), wait 3 min to 

equilibrate gas in the system.

3. Close the helium inlet valve by turning the above regulator counter clockwise.

4. If the gauge needle does not move, there is no leak.

5. If the gauge needle moves, indicating the lost of pressure, there is a leak in the system. The 

decreasing pressure rate accounts for the degree of leakage.

6. Locate the leak by separating and testing the system segment by segment, using the helium detector.
26

Appendix J. Daily Checklist

Analyst in charge: ______________________Date: ______________________ 

Weekly:

1. Change the working reference gas tank and helium gas tank if the pressure is < 500 psi. Order new 

ones for a spare; reference gas is SO2 ANHY by Matheson, helium carrier is zero grade, O2 is research 

grade.

2. Change the water and CO2 trap after approximately 200 samples, or when it is necessary.

3. Check and clean the autosampler carousel.

4. Change the reaction tube after approximately 600 samples, or when it is necessary.

THESE ITEMS ARE TO BE CHECKED OFF AS YOU CHECK THEM DAILY!

1. Check the helium, O2

, and reference gas flow. [ ]

2. Check the EA reaction-tube temperature (1020 °C). [ ]

3. Change the insertion tube (ash-collector liner) in the reaction tube. [ ]

4. Check the background masses. [ ]

Typical background at N2

/CO2 configuration

At emission current: 0.54 mA/0.48 mA 

Cups Mass Intensity (V) Mass Intensity (V) Resistor (Ω) Capacity (pF) 

1 28 0.010 0 3 × 108 680

2 29 0.007 18 1.256 3 × 1010 5

3 30 0.140 18 6.818 3 × 1011 2

5. Perform the Ref on/off method three times to stabilize the IRMS. [ ]

6. Check the peak center. If it is varies from the default value by more than 50, do a mass calibration. [ ]

7. Analyze Ref on/off method 10 times to stabilize the IRMS (standard deviation should be better than 

0.1 ‰). [ ]

Optimal ConFlo II Pressure Setting N2 

(bar) 

N2 signal (V) Mass 29 on Cup 2 

0.25 0.173

0.50 0.450

0.75 0.773

1.00 1.180

1.25 1.560

1.50 Optimal setting 2.074

1.75 2.634

2.00 3.199

8. Check the paper in printer. [ ]
27

Appendix K. Changing the Insertion Tube

The insertion tube needs to be changed before a template is run.

1. Manually close needle valve near the ion source.

2. On the menu of the Model 2500 EA under “Spc. Fun.,” choose “Std By” and press “enter.” The O2

and helium gases should be turned off.

3. Unscrew the metal seals under the sample carousel to access the reaction tube.

4. Using the heat protective glove and the wire tool, remove the insertion tube from the reaction tube 

and place in the metal can. Be aware that the tube will be very hot!

5. Insert clean tube that has 0.5 centimeter (cm) of quartz wool packed at the bottom and tap it down 

with the wire tool.

6. Replace the carousel and tighten the metal seals with a wrench.

7. Turn the O2 and the helium back on by pressing “enter” and return the screen to the temperature 

readout by pressing “Spc. Fun.”

8. Open the needle valve.
28

Appendix L. Changing the Water Trap

Change the water trap after every 200 samples.

1. Retrieve quartz turnings, quartz wool, magnesium perchlorate, and a clean water trap from Delta V 

Plus supplies.

2. Under a hood, pack one end of the clean water trap with 1 cm of quartz wool.

3. On a piece of wax paper, create a mix by volume of approximately 70-percent magnesium perchlorate 

and 30-percent quartz turnings. Pack the mix into the water trap tube, leaving 1 cm of empty space.

4. Pack the open end of the water trap with quartz wool.

5. Unscrew the red plastic ends from the used water trap and replace it with the clean water trap; be sure 

to replace the rubber O-rings on the ends of the clean water trap and tighten the red ends.

6. Insert the plug into the vent and watch for any decrease in pressure to check for a leak in the system. 

If everything looks acceptable after 5 min, replace the vent line.

7. Under the “Spc. Fun.” menu of the model 2500 EA, choose “CNT” (counter) and zero “D” to set back 

the water-trap counter.

8. Clean the water trap by pushing all the mixture out of the tube into the trash can and use a clean 

cotton swab to wipe out the inside of the tube. Rinse the tube with DIW, and dry it in the oven. 

Return this tube to the water trap bag in the drawer.
29

Appendix M. Changing the Reaction Tubes

The reaction tubes should be changed approximately every 500 to 600 samples. (The new reaction tubes 

will need to equilibrate over night before they can provide accurate results; thus, change the reaction 

tubes at the end of the day.)

1. Manually close the needle valve near the ion source.

2. Turn the Model 2500 EA to “Std By” with both gases off.

3. Lower the oxidation-tube temperature to less than 900 °C; leave the reduction-tube temperature as it 

is.

4. Retrieve the reaction tubes from the cabinet.

5. Insert the insertion tube into the top of the oxidation tube; there will already be a plastic tube in the 

reaction tube that should be removed.

6. Place black O-rings on the top of the reaction tubes. Make sure that all O-ring connections are clean 

and free of any dust.

7. Loosen the top metal seal and remove the bottom metal seal. Go back and completely remove the top 

seal. It will be very hot! Use oven gloves!

8. Remove quartz reaction tubes and place them in a metal can, place clean reaction tubes in column. 

Make sure the O-rings are clean; then replace the top.

9. On the bottom, put on the metal pieces with metal O-rings first and then add the clean rubber O-rings. 

Raise the bottom portion and screw together. Then, tighten the connections with wrenches, and put 

the metal supports back in place.

10. Turn the gases back on; then, go to “CNT” in the menu under “Spc. Fun.” Set to zero the counter of 

the oxidation-reaction tube, “B,” as well as that of the reduction-reaction tube “C.”

11. Insert the plug into the vent and close the helium supply. Watch for any decrease in the pressure to 

check for a leak in the system. If everything looks okay after 3 min, open the helium supply and 

replace the vent line.

12. Open the needle valve.
30

Appendix N. Step-by-Step Procedure to Report Data

1. Open “Store Samples in Progress” in LIMS-LSI.

2. Choose the appropriate isotope.

3. Choose sample ID range from “Sample in Progress” print out.

4. Store data.

5. Go back to the main menu of LIMS-LSI.

6. Open “Project” and find the appropriate project in the list.

7. Select “Print Report” and check whether the project report contains all the results. If not, search for 

the missing results in the database.

8. Select “Results,” transfer data in Excel format or (and) text format to a diskette, and report data to 

customer through e-mail.

9. Click “Print Report” to print a project report and put it in the “Correspondence” binder along with all 

the other documents in the “Samples In Progress” binder that are related to this project.