Soil and Plant Total C and N Analysis:
Sample Preparation

1. All sample material must be dried and ground to a fine powder. For soils, the dried soil could be ground using a roller bar shaker or ground on the EPA grinder in the greenhouse. Plant tissue should be run first through the Wiley Mill and then passed through the Cyclotec grinder. In addition, the micromill may be used to even further pulverize samples that are extremely small in quantity or contain large (>2 mm) pieces of tissue following Cyclotech grinding.
2. Each CN run must begin with a tin foil blank cup (sample #1) and four acetanilide standards (samples 2 thru 5). The 1st standard should weigh about 0.2 to 0.3 mg, the 2nd standard should weigh about 0.5 to 0.6 mg, the 3rd standard should weigh about 1.0 to 1.2 mg, and the 4th standard should weigh about 2.0 to 2.8 mg. Sample #6 should be a blind standard of either soil or plant, depending on the type of samples on the run. It is important to check the printout value of this sample to ensure that the standard curve was properly set. If the values for C & N are off for this blind standard, the run should be stopped. We are attempting to initiate a new standard using the NIST tissue of citrus leaves or tomato leaves, however, the N content is not certified and the carbon value is not available from NIST.

It is recommended to run blind standards (the “soil standard” or an NIST compound) a minimum of every 15 samples to ensure confidence in the analysis. The soil standards are O519 and O559 (provided by USDA-ARS Pendleton OR) or an LTER-recommended soil NIST standard. The plant standard compounds include citrus leaves, pine needles and tomato leaves.
To Weigh a Sample or Standard

1. Turn on the Sartorius scale if the display is not lit., press tare to bring display to 0.00 mg. It is recommended that you wear gloves while weighing samples.
2. Place an empty tin foil cup on the weighing tray and close the doors.
3. Tare off the weight of the cup using the tare button.
4. Remove the cup and fill with ~ 1 spoon of tissue.
5. Crunch the cup using two sets of tweezers. Once completely closed into a ball formation, drop onto the weighing plate to ensure no sample leaks out. If the sample is leaky, discard the ball and begin again.
6. Place the closed cup onto the weighing pan and record the weight (in mg) on the provided datasheet.
7. Gently remove the sample and place into the plexiglass sample holding trays.
8. Bring the scale display back to 0.00 mg by pressing tare.
9. Using KIM wipes, clean the tweezers, the spoon, and the metal plate between each sample.

The key to the samples is a consistent weight . Soil samples should weigh between 10.0 to 15.0 mg, and plant samples from 4 to 6 mg. The Sartorius scale can’t provide an accurate weight under 0.2 mg. Plexiglass plates should be stored in a vacuum desiccator until analyzed.
Total C and N Analysis

WARNING: NEVER try to use the computer while a CN run is in progress!

Basically, the computer is used mainly as an integrator and to advance the auto sampler. Although run conditions are recorded in the methods file, all run conditions must be manually checked prior to starting a run. Each run can contain a maximum of 99 samples, including the blinds, blanks, and standards. Complete runs are stored as methods (*.ead) and contain the sample table info, chromatograms and run parameters. Stored files contain individual chromatograms (*.dat). The data should also be written to a summary file (*.sum) which may be downloaded to the VAX and run through SAS jobs to avoid any manual data entry of the results.

The return button on the mouse is the left button. Pressing the right button will bring up HELP during any application.

If the Eager software window is not showing, hit the space bar to inactivate the screen saver. If Windows95 desktop is showing, click the start button and restart in DOS mode. From the C:>WIN type cd\ and then C:> cd EAGER200 . At C:\EAGER200> EAGER. The program will boot up. User should respond YES to delete scratch files.

If the Eager software window is not showing and the screen display is a chromatogram, mouse over to the = (three lines symbol) and quit view monitor .

At the Eager software window, first load method for the appropriate template for the tissue you are working with. The template contains the correct run information for the integration parameters. Subdirectories have been established for the different investigators within the main directory EAGCHA . For example, Dr. Gross’s work is stored under the directory EAGCHA\KGROSS with subdirectories after the investigator directories. If you need to create a new directory for your samples, exit the eager software to DOS and create the directory. You will need to copy the SOIL.ead or PLANT.ead template into your new directory. To do this, start in root directory, and type: copy_soil.ead_(or plant.ead) c:\new directory name . If this is your first time doing this, please ask for assistance. Some of the current directories are (referred to as disk and directory in the LOAD option):

EAGCHA\GPR\SOIL

EAGCHA\GPR\PLANT

EAGCHA\KGROSS\

Once you are in the directory you want, for soils, load SOIL.ead ; for plants load PLANT.ead .

Bring up the Method card and change the template name ( soil.ead or plant.ead ) to the new file name (ie.L90corn.ead). Your file name should have a maximum of 8 characters, start with a letter, and be followed by the .ead suffix. It should reflect as much as possible the year, experiment, and tissue type. After you name the file, SAVE it to the correct directory.

Changes in sample table editing ; Eager Software Version 3.12 and higher

Sample table data can be entered using a spreadsheet (Cntemplate.xls) and then imported directly into the sample table. The spreadsheet format is shown below, followed by the resulting imp file that is imported into the EAGER software. The resulting file must be saved as comma delimited with a .imp filename format. If using Excel to create the spreadsheet, use the save as option, select comma delimited (*.csv) and put the filename as “file.imp”. The quotation marks are necessary to override the default .csv extension. Unfortunately, Excel seems to put in some tailing comma’s so pull the *.imp file into a word processor (Notepad, Word, etc.) to clean up any tailing commas. The tailing comma following the blank on the first line is necessary to import the file correctly.

Spreadsheet format:

blank	blk	blank
standard1	std1	standard	Acetanilide	0.25
standard2	std2	standard	Acetanilide	0.86
standard3	std3	standard	Acetanilide	1.85
standard4	std4	standard	Acetanilide	2.1
blindstdpine	g6	unk	4.821
100	a1	unk	6.77
101	A2	unk	7.93
110	A3	unk	7.61
111	A4	unk	5.35
1001	A5unk	7.97
1002	A6	unk	6.98
1011	A7	unk	6.24
1012	A8	unk	7.06
1101	A9	unk	7.73
1102	A10	unk	7.3
blindstdpine	g7	unk	4.241
1111	A11	unk	7.02
1112	A12	unk	5.92

Final document format:

blank,blk,blank,
standard1,std1,standard,Acetanilide,0.25
standard2,std2,stanard,Acetanilide,0.86
standard3,std3,standard,Acetanilide,1.85
standard4,std4,standard,Acetanilide,2.1
blindstdpine,g6,unk,4.821
100,a1,unk,6.77
101,A2,unk,7.93
110,A3,unk,7.61
111,A4,unk,5.35
1001,A5,unk,7.97
1002,A6,unk,6.98
1011,A7,unk,6.24
1012,A8,unk,7.06
1101,A9,unk,7.73
1102,A10,unk,7.3
blindstdpine,g7,unk,4.241
1111,A11,unk,7.02
1112,A12,unk,5.92

Bring up the Sample Table Card and import data from disk . The template should have the generic file and sample name codes, these must be replaced to identify the specific samples to be run. Choose the correct drive (3.5" floppy is B:/ ) and select the filename. The program imports the data directly. Check for errors and use the mouse or tab key to toggle between squares in the table. In the case of type and standard type , click the mouse within the box to reveal the appropriate choices ( blank , standard , or unknown ).

The 1st column in the sample table is for samplename and start with a letter- no punctuation (punctuation won’t go thru SAS). All of the replicate cups of each sample should have the same Samplename , and then the filename (2nd column of table) can be the same as the samplename with the suffix a , b , c , d , etc. (a different one for each cup). This will enable the *.sum files to be easily processed thru SAS. Each individual sample chromatogram will be saved under that filename specified in the sample table. Standard filenames should be sequential (ie. std5 , std6 , std7 ) in both samplename and filename boxes. Samplenames may be descriptive and reflect the experiment, year, and type of tissue as much as possible. Filenames are limited to 5 digits (ie.LTER 1990 corn grain plot 1-3 = CG13 ) = CG13a . The filename extension of *.dat will be automatically added in memory. Fill in sample 1 as a blank, specifying type as blank, and do not enter in a weight. Samples 2 through 5 are standards and should be identified as type = standard and standard type = acetanilide or phenacetin ; enter the weight.

Note: filenames (*.dat) are limited to 5 characters max and will be truncated if longer. The last of the 5 char is the analytical rep.

Sample root should be descriptive (ie: CG ) and file root same (CG), type = UNK. The number of samples should reflect the remaining number of samples (ie if 99 total samples, 94 samples remain). The blind standards should have the samplename : blind std___(whatever it is); all the blind standards should have the same samplename so SAS will integrate the values; and the blind standard filename should be an abbreviation and a , b , c , d , etc. For example, if analyzing soil, you could call the samplename: blind std 519 , and the filenames in that run: O519a , O519b , O519c , etc. Use next page to continue at the end of the chart. When finished, always UPDATE , and SAVE at the software window under the new method file name to the correct disk and directory name (ie: EAGCHA\GPR\SOIL L90corn.ead ). Say Yes when asked if want to overwrite. Be sure you are SAVING to the correct file and directory before you overwrite, to make sure you don’t overwrite a previous set of samples or someone else’s data!

The template card called CALCULATION AND REPORT PARAMETERS should be set to the specifications as shown below (only visible in hard copy version) in the printout of that table: (It is very important that these be set correctly, especially the APPEND TO SUMMARY: YES option).

Once saved, print out the sample table to double check the entered weights with those hand-recorded on the datasheet. Also carefully check that each sample has a distinct filename .

Running Samples

Load samples into the autosampler wheel,wearing gloves and using tweezers. The wheel can be turned clockwise to bring 1 back over deeply recessed hole so the blank (sample #1 on the datasheet and sample table) will go into the deeply recessed hole. Load in the remaining samples to #0 in a counterclockwise direction. If you have greater than 50 samples, put the top wheel on top of the bottom wheel (can only go on one way due to post and hole sizes) and load #51-#99 in the top wheel (also counterclockwise). The metal rod on the top wheel should be between #50 & #51 and to the left of the metal rod on the bottom wheel. Set the gray plastic cover on top of the wheel.

Run Parameters on NA1500:

Daily setup:

1. Push furnace stdby buttons to UP position (ready light should turn off
2. Turn Helium from ST BY to FLOW, and Purge to ON.
3. Check tank levels and pressures (first and secondary regulators): Primary regulator (closest to tank main stem) should be > 300 psi. Secondary regulators at:
   • Helium 200 kpa
   • Dry Air 55 psi
   • Oxygen 30 psi
4. Check temperatures on unit using the blue touch panels: (set by thumbwheels)
   • Left oven 1030 degrees C
   • Right oven 650 degrees C
   • Furnace 60 degrees C
   • Fil. Temp. 190 degrees C
   • (The Green Light below the READY indicator should be lit if the actual temperatures are the same as what the control panel thumbwheels have them set to).
5. Check pressures on instrument panel:
   • Helium (carrier M) 100 kPa
   • Reference (carrier R) 70 kPa
   • Oxygen 100 kPa
   • Dry Air 400 kPa
6. Check sample cycle parameters (thumbwheels):
   • Cycle 29 (sec X 10)
   • Start 10-15 sec
   • Stop 60 sec
   • O2 inj Stop 70 sec
   • peak enable 61 (sec X 10)
7. Manual Exclusion Buttons: should all be up (“program off normal” light should NOT be lit).

If everything looks good up to here, mouse over to RUN on the software page and Start Analysis . You may also choose to Monitor Dectector at this time.

Watch the first six samples closely. Blank areas for Carbon should ideally be <3500 units. Sample #6, your first blind standard , should be within the correct amounts for Carbon and Nitrogen for that sample. If these are within the range, continue to let the run proceed. If not, mouse to RUN and Stop at end of sample . If any problems develop during the run, mouse to RUN and Stop at end of sample . This may be done from either the Monitor Detector screen or the Blue software screen.

After Run is Completed:

Turn Flow and Purge switches to ST BY and OFF; and push furnace buttons to standby (in).

LTER Data Post-Processing

Chromatogram files are stored in a *.sum file within the proper directory for the user group. To download to a floppy disk, place the disk in the B: drive and at the eager software window pull down on the = (3 line symbol) to file organizer . At the file organizer window, select the copy file option. On the copy file line type in the correct file with the .sum suffix. Confirm , and copy the correct *.sum file to the B:\ (NOT the C:\).

Download the *.sum file to the VAX directories.

The *.sum file is a long linear file with each sample and its corresponding information written to separate lines. It is not readily imported into a spreadsheet program in this form. Use the CNDOWN.sas program (in the directories below) to transform the *.sum file into a file

with 5 columns of usable data.

LTER files should then be transferred to:

for soils: group63d:[generald.lter.baseline.chn.chnsoil]

for plants: group63d:[generald.lter.baseline.chn.ltercrop]

File transfer continues to evolve. At this time (1998) I use a PC running WIN95, Windows Explorer, and under Tools : Map Network Drive. Establish the 63d drive above as the remote drive and drag and drop the *.sum file from the floppy to the targeted VAX drive.

Meanwhile, Back in the Lab, on the VAX:

1. Log onto the VAX
2. Go to the target directory & subdirectories, based on where you send the *.sum file.
3. $ed_cndown.sas
4. change the highlighted information in the SAS file to match the appropriate file and title names for what you are working with. (see prorgram below).

data cn;
infile ' t8dom95.sum';
length fname $35
desc $20;
input sample / dummy1 /
fname / dummy2 $ /
desc / dummy3 / dummy4 / dummy5 / dummy6 /
dummy7 / dummy8 /
carbon / nitrogen/ dummy9 /;
keep sample fname desc carbon nitrogen;
proc sort; by sample fname desc carbon nitrogen;
proc print;
title 'transformed CN data file downloaded from IBM to VAX';
data cndown;
set cn;
file ' t8dom95.dat' print pagesize=500 notitles header=v1;
put sample 1-3 fname$ 5-9 desc$ 11-15 carbon 17-22 .3 nitrogen 24-29 .3;
return;
v1:
put
'File : t8dom95.dat'/
'content: transformed CN data file downloaded from IBM to VAX'/
'input : sample analyt filename %carbon %nitrogen'/;
return;

1. Hit Control Z , and at the *ex .
2. $ sasbatch_cndown.sas
3. get job completed message
4. $ p_ filename.dat This file will contain the following space delimited fields:

Sample number
filename
sample description
% Carbon
% Nitrogen
from analysis
5 digit
from sample table
elemental
elemental

1997 Changes

The filename.dat is edited at this point to decode the CN abbreviated sample and filenames to include the detailed information about the sample.

ASCII file containing the output results (plot means over analytical samples) with columns:

date plotid tissue %C %N C/Nratio stdC stdN stdC/N CVC CVN CVC/N %C, %N, %C/n are the mean (of x # samples) elemental values std is std dev

CV= coefficient of variation

QA/QC guidlines: CV <5% for analytical replicates with soil samples, CV<15% for plants

This file is transferred to the ACCESS database if samples CV’s meet QA standards.

CE Elantech reps to call

Hank Hancock (tech support) 732-370-5559

Jeff Smith (better info) 281-496-4791 fax (March 98)

Preferred Consumable Supplier

Elemental Microanalysis LTD. (Tom Jackson) 1-800-659-9885