Revision #: RV0 Revision Date: 12/2/2010

CHNS Analysis Using the Euro Vector EA

INTRODUCTION:
The analyzer operates according to the dynamics of flash combustion of the sample. The sample
is weighed in a tin or silver capsule and introduced into the combustion reactor where with the
proper amount of oxygen and combustion catalysts, the sample is combusted. After combustion,
the reaction gas products (CO2, H2O, NOx, and SO2) are carried by helium flow to a copper
reactor where excess O2 is consumed (CuO) and NOx products are converted to N2. The
products are carried through a packed GC column that provides the separation of the combustion
gases and is detected by a TCD detector.

CHNS Elemental Analysis General Schematic

To validate the system, one of two methods is generally used:

1) Least Accurate: K-Factor calibration (Single-point/Single Standard). This is performed on
one analytical standard of known high purity and composition. The K-Factor is determined
then as an area ratio of the known weight of the sample combusted. For example: 1.09 mg
of Urea was combusted as a standard (20.00 % Carbon, 6.71 % Hydrogen, 46.65 %
Nitrogen). The area of carbon (CO2) from the chromatogram was 1764 AU. The theoretical
amount of carbon in the urea sample that was combusted was 0.2180 mg (20.00% * 1.09
mg). Therefore, the ratio of area versus the theoretical weight of carbon in the sample
combusted gives the K-Factor for carbon 1.236 x 10-4 (0.2180/1764). This factor is then
multiplied to the carbon area of the unknown sample to determine the amount of carbon in
the sample, the amount of carbon is then divided into the weight of sample combusted to give
the % carbon. For example: 1.00 mg of unknown sample gave a CO2 area of 1000 AU.
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Multiplying the area by the K-Factor gave a wt. of carbon in the sample of 0.1236 mg (1000
* 1.236 x 10-4). Then dividing the wt. of carbon by the sample weight to get the % carbon of
12.36 %, (0.1236/1.00) * 100.
2) More Accurate: K-Factor calibration (Multi-point or Multiple Standards). This is
performed on several analytical standards of known high purity and composition of differing
carbon, hydrogen, and nitrogen composition. This can also be determined using one
calibration standard with multiple samples. The K-Factor is determined as the equation of
the linear regression curve of the elements area versus the elements theoretical amount for
each element in each different standard/sample. (Y-intercept can be set at 0, but does not
have to be). The variance (R2) of the linear regressions needs to be very high (0.99 ideally).

Sample K-Factor Calibration Curve using Urea (y-intercept =0)

DETERMINE INSTRUMENT CONFIGURATION (SEE ATTACHMENTS 1 & 2):

Determine if the EA or the HT-PyrOH will be used.
1) The EA is set-up as a two column (furnace) system with a maximum temperature of 1100 °C
in each furnace.
a) Front column is the oxidation/combustion column. This is where the combustion takes
place.
b) The back column is the reduction column. This where NOx is reduced to N2 gas and
excess O2 is consumed (CuO).
c) The oxidation and the reduction columns can be combined into one column, but since the
unit is set up as a two column system, a “blank” column will still need to be put in back.
2) The HT-PyrOH is a one column system with a maximum temperature of 1500 °C.
a) The column needs to be configured for oxidation and reduction.

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TRAPS (SEE ATTACHMENT 2):

Traps can be used to remove certain combustion products from the sample.
1) Magnesium perchlorate can be used to remove water (for CN analysis)
2) Traps can also be used to trap Fluorine and other halogens to protect the instrument.

PACKING COLUMNS DUAL COLUMN SYSTEM (EA) (SEE ATTACHMENTS 4 & 5):
1) Make a new column before removing the old column to minimize the time the lines are open
to atmosphere. Verify that a new column is required before packing a new column.
2) Pack a new oxidation column (CHN)
a) Stuff the end of a quartz tube with 4 cm of quartz wool.
b) Add 2-3.5 cm silvered cobalt oxide above the quartz wool in one portion and tap the tube
three times to settle the reagent and prevent dead space volumes from forming. (add
more on presence of halides and sulfur)
c) Add 2 cm quartz wool.
d) Add 8.0 cm chromium oxide in one portion and tap the tube three times to settle the
reagent and prevent dead space volumes from forming.
e) Add 1 cm quartz wool.
f) (CHNS) For Sulfur analysis or difficult to burn samples Tungsten oxide can be used in
place of the chromium oxide and the silvered cobalt oxide.
3) Pack a new reduction column (CHNS)
a) Stuff the end of a quartz tube with 1.5 cm of quartz wool.
b) Add 36.5 cm of reduced copper (2 x 100 g) above the quartz wool in two portions and tap
the tube three times to settle the reagent and prevent dead space volumes from forming
c) Add 1.5 cm quartz wool.

PACK COLUMNS SINGLE COLUMN SYSTEM (EA) (SEE ATTACHMENTS 4 & 5):
Make a new column before removing the old column to minimize the time the lines are open to
atmosphere. Verify that a new column is required before packing a new column.
1) Pack a new oxidation/Reduction column (CHN)
a) Stuff the end of a quartz tube with 0.5-1 cm of quartz wool.
b) Add 1.0-1.5 cm silvered cobalt oxide above the quartz wool in one portion and tap the
tube three times to settle the reagent and prevent dead space volumes from forming. (add
more on presence of halides and sulfur)
c) Add 1 cm quartz wool.
d) Add 8.0-8.5 cm reduced copper in two portions and tap the tube three times to settle the
reagent and prevent dead space volumes from forming.
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e) Add 1 cm quartz wool.

f) Add 3.5-4.0 cm chromium oxide in one portion and tap the tube three times to settle the
reagent and prevent dead space volumes from forming.
g) Add 1 cm quartz wool.
2) Pack a new oxidation/Reduction column (CHNS)
a) Stuff the end of a quartz tube with 0.5-1 cm of quartz wool.
b) Add 10.0 cm reduced copper in two portions and tap the tube three times to settle the
reagent and prevent dead space volumes from forming.
c) Add 2 cm quartz wool.
d) Add 3.0 cm tungsten oxide in one portion and tap the tube three times to settle the reagent
and prevent dead space volumes from forming.
e) Add 1 cm quartz wool.
3) Back “blank” column
a) Stuff the end of a quartz tube with 1.5 cm of quartz wool.
b) Add 36.5 cm of quartz chips in two portions and tap the tube three times to settle the
reagent and prevent dead space volumes from forming.
c) Add 1.5 cm quartz wool.

INSTALLING OXIDATION/REDUCTION COLUMNS EA TWO COLUMNS (SEE

ATTACHMENTS 1 & 2):
1) Front Oxidation Column
a) Open front door on EA.
b) Remove screws on cover in front of combustion chambers (right side of instrument.
c) Remove Auto Sampler from top of front column.
d) Carefully remove column from front chamber. Cover the bottom column receiver with a
kimwipe to prevent debris from falling in when installing and removing columns.
e) Insert new column through oven, remove kimwipe, and insert into holder. Wipe any
fingerprints or contamination off of column prior to installation.
f) Re-install auto sampler with 18 mm connector.
2) Back Column
a) Open front door on EA (if not already done).
b) Remove screws on cover in front of combustion chambers (right side of instrument (if not
already done).
c) Remove top connector from top of back column.

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d) Carefully remove column from front chamber. Cover the bottom column receiver with a
kimwipe to prevent debris from falling in when installing new and removing columns.
e) Insert new column through oven, remove kimwipe, and insert into holder. Wipe any
fingerprints or contamination off of column prior to installation.
f) Re-install 18 mm column connector.
g) Re-install door with screws on front of columns.

INSTALLING OXIDATION/REDUCTION COLUMN HT-PYROH ONE COLUMN (SEE

ATTACHMENTS 1 & 2):
1) Oxidation/Reduction Column
a) Open front door on HT-PyrOH.
b) Remove screws on cover in front of combustion chambers (right side of instrument.
c) Remove Auto Sampler from top of front column.
d) Carefully remove column from front chamber. Cover the bottom column receiver with a
kimwipe to prevent debris from falling in when installing new and removing columns.
e) Insert new column through oven, remove kimwipe, and insert into holder. Wipe any
fingerprints or contamination off of column prior to installation.
f) Re-install auto sampler with 17 mm connector.
g) Re-install door with screws on front of columns.

PLUMBING (SEE ATTACHMENTS 1 & 2):

1) If the EA is being used, the following plumbing connections need to be made:
a) The autosampler needs to be attached to the front combustion column of the EA. Use
special care in bending the supply lines to the autosampler when moving. NOTE: The
autosampler attachment for the EA side is 18mm.
b) The back reduction column connects to the “rear oven” port on the top of the EA.
c) Cap any open tube ends.
2) If the HT-PyrOH is being used, the following connections need to be made:
a) The autosampler needs to be attached to the combustion column of the HT-PyrOH. Use
special care in bending the supply lines to the autosampler when moving. NOTE: The
autosampler attachment for the HT-PyrOH side is 17mm.
b) The back port of the HT-PyrOH connects to the “rear oven” port on the top of the EA.
c) Cap any open tube ends.
3) GC Column:
a) Install the CHNS packed GC column in the oven on the EA.

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b) Open front door on EA.

c) Remove screw on the top of door in bottom left of the instrument.
d) Remove/Replace GC column.
e) The fittings on the GC column need to be fairly snug or they will leak. Since the column
is plastic, if the fittings are not tight enough, heating and cooling of the oven will result in
leaks. DO NOT OVER TIGHTEN AND STRIP THE FITTINGS.

WEIGHING SAMPLES:
1) Weigh the samples using the balances from Dr Minter’s lab (1.00 ± 0.01 mg) while the lab is
not busy. When the lab is busy, the accuracy of the weights can be an issue.
2) Allow all weighing samples and equipment used for weighing to equilibrate to the
temperature of the room that the balance is in for at least 30 minutes.
3) Change the weighing setting on the scale to either Fast-Reliable (~2-4 minutes per sample) or
Very-Reliable (~3-6 minutes per sample) depending on how much time you want to spend
weighing your samples.
4) Adjust the internals on the balance so that it is calibrated to the current room temperature.
5) Put a metal weighing pan on the balance inverted, so that the sides are facing up.
6) Place empty tin/silver cup on balance with tweezers and zero.
7) Remove tin/silver cup with tweezers and place on metal sample prep block.
8) Fill cup with desired sample (generally 0.5 to 1.5 mg) and crush into a ball using the
tweezers. Make sure to seal the top of the cup.
9) Grab the balled up sample with the tweezers and tap on the sample prep block with the ball
overhanging onto the table. This is to knock off any sample that may be on the outside of the
balled sample.
a) This also checks to make sure that the cup was not torn during sample preparation. It
makes it easier to see solids from the samples if performed over a black table top.
b) If there is no solid coming off of the balled sample (after the initial couple of taps) then
proceed to the next step.
c) If there is solid coming off of the balled sample (after the initial couple of taps) the cup
was torn during the balling process. Discard the sample and prepare another one.
10) Place the balled sample on the balance to get the weight.
11) Put sample in numbered sample tray and cover with parafilm after weighing all samples.

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CHOOSING CALIBRATION STANDARD, SAMPLE WEIGHTS, AND SAMPLE

ORDER:
Typical Standards Used

Sample Theor. % C Theor. % H Theor. % N Theor. % S

Tryptophane 64.69% 5.92% 13.72% 0%
Imidazole 52.93% 5.92% 41.15% 0%
Isatin 65.31% 3.43% 9.52% 0%
Alanine 40.44% 7.92% 15.72% 0%
Nicotinamide 59.01% 4.95% 22.94% 0%
Lysine 49.30% 9.65% 19.16% 0%
Cyclohexanone 73.43% 10.27% 0% 0%
Acetanilide 71.09% 6.71% 10.36% 0%
Urea 20.00% 6.71% 46.65% 0%
Atropine 70.56% 8.01% 4.84% 0%
Cystine 29.99% 5.03% 11.66% 26.69%

1) Weigh out at minimum 5 different weights of the calibration standards in the range: (0.5,
0.7, 1.0, 1.3, 1.5 mg).
2) Weigh out at minimum 3 different weights of the unknown sample in the range: (0.5-1.5
mg).
3) If possible, try to weigh multiple samples at each weight for the calibration standards to
account for variation between samples.
4) Ideally, the weights of the unknown samples needs to produce an area of C, H, N, and S that
lie in the middle of the standard curve or in the range of the K-Factor.
5) Run samples in the following order:
a) Blank (tin/silver cup only--balled up). Do not perform more blanks than necessary as it
will consumes the copper reduction catalyst faster.
b) Set of calibration standards.
c) Unknown samples.
d) Blank (15-20 samples after the last blank)
e) One calibration standard
f) Repeat steps c) through e) until done.

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EA START-UP AND OPERATION (SEE ATTACHMERNTS 1 & 2):

1) Check to make sure that there is enough gas to perform your runs. Helium and Oxygen.
a) Both the helium and the oxygen should be set at 4 bar at the tanks.
2) Close the Nupro valve leading to the mass spectrometer.
3) Perform a leak test at room temperature.
a) On EA control panel: SP-F>Test>Enter>Leak>Enter>Start.
i) Make sure Nupro valve to the MS is closed.
ii) Find and repair any leaks if test fails.
4) Select method for analysis.
a) On EA control panel: Method>

General Method

Carrier Flow (ml/min) ~ 115 ml/min

Carrier (kPA) 80
Purge (mL/min) 80
Oxygen (mL) 20 mL
ΔP O2 (kPA) 35 kPA
Sampling Delay (sec) 10 sec
Run Time (sec) (CHN) 180
TCD (baseline) 1 mV
TCD Polarity (+)
TCD Gain 10 or (1 less sensitive)
Front Furnace Temp (°C) 1000 °C
Rear Furnace Temp (°C) 600-1000 °C
GC oven Temp (°C) 85 °C

5) Once instrument is at operating temperature, perform another leak test.

a) Do not proceed until leak test passes.
6) Turn on TCD Detector:
a) Set>TCD>On>(Polarity +1, Gain 1 or 10 (10 more sensitive), 1 mV (zero level)
b) Go to zero hit start to zero TCD.

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c) Allow the TCD to stabilize for at least 30 minutes and establish a steady baseline.
d) Once the baseline is steady, re-zero the TCD.
7) On PC Start Masslynz software.
a) Select File>Open Project>Chemistry EA Analysis
b) Enter Sample Information into table
i) Name files sequentially, preferably with the date and or initials at the beginning.
8) Put sample in open hole in autosampler making sure to place to clear cover back on.
9) Highlight sample you want to run on PC
a) Press start
b) Press Ignore on error message (If it appears).
10) After run is complete, on PC type
a) View>Chromatogram, Display>Analog>Select File>(pick sample name)>enter>Chanel1-
TCD>OK
b) Set integration parameters to:
i) Apex Track Peak Detection ON
ii) Smoothing On
iii) Peak Threshold: Baseline Start Threshold: 0%
iv) Baseline End threshold: 0%
c) Integrate chromatogram.
d) Edit integrated peaks if necessary:
i) Type Edit>Integrated Peaks
e) Print chromatogram.

SHUTTING DOWN:
1) Put it in gas save mode
a) SP-F>Standby>Gas Save
b) Verify that there is enough He for gas save mode (at least 500 psi remaining). If not
perform a complete shut down after cooling all furnaces and oven.

TROUBLESHOOTING (SEE ATTACHMENTS # 1, 2, & 3):

1) Blowout: All that pressure in the machine blew the quartz wool and chemicals out the
bottom of the machine. Pull the tube back out and try to repack it if it is new. Otherwise
make another and be sure to put the support back evenly.

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2) Broken tube: This has happened before. Turn off the furnace and have someone help you
push the tube back up through the machine or may have to remove furnace. Vacuum up the
mess and count your losses.
3) Leaks: can be time consuming. Be sure to isolate the leak by not opening everything at
once. If you have a leak re-open and close all recently opened and closed bolts. You may
have a blowout or a tube may be broken (see broken tube). Do not use liquid leak detectors
on components that are not under He pressure, otherwise the liquid may enter the
system----this would be bad for the mass spectrometer. Common places for leaks are:
a) The 2 connections to the GC column (metal to plastic connection-(expansion and
contraction).
b) The bottom reactor tubes have dust in the o-ring sealing surface inside of the reactor
mount.

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