EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
Analysis of total carbon (TC), total inorganic carbon (TIC) and total
organic carbon (TOC) in water samples
Table of content
1.
PURPOSE................................................................................................................................................ 2
2.
PRINCIPLE .............................................................................................................................................. 2
3.
REQUIREMENTS ..................................................................................................................................... 3
3.1.
EQUIPMENT AND MATERIALS ....................................................................................................... 3
3.2.
REAGENTS ...................................................................................................................................... 3
4.
PROCEDURE ........................................................................................................................................... 3
4.1.
SAMPLE PREPARATION.................................................................................................................. 3
4.2.
SAMPLE ANALYSIS ......................................................................................................................... 3
5.
CALCULATION OF RESULTS .................................................................................................................... 7
6.
QUALITY CONTROL ................................................................................................................................ 7
7.
ERRORS, CALIBRATION AND INTERFERENCES ....................................................................................... 7
8.
WASTE STREAM AND PROPER DISPOSAL .............................................................................................. 8
9.
HAZARDS AND PRECAUTIONARY STATEMENTS .................................................................................... 8
10.
REFERENCES ...................................................................................................................................... 9
11.
CONTRIBUTIONS ................................................................................................................................ 9
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EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
1. PURPOSE
The purpose of this analysis is to determine the total carbon (TC), total inorganic carbon (TIC) and total
organic carbon (TOC) in water and wastewater.
2. PRINCIPLE
The TOC test measures the total amount of non-volatile organic carbon in a sample. Total carbon (TC)
and total inorganic carbon (TIC) are converted to carbon dioxide (CO2) by persulfate oxidation (only for TC)
and acidification. The CO2 passes from the digestion cuvette through a membrane and into the indicator
cuvette (Fig. 1). The change of color of the indicator is photometrically evaluated. Total organic carbon
(TOC) is determined as the difference between the measured TC and TIC values.
𝑇𝑂𝐶 (𝑚𝑔 𝐿−1 ) = 𝑇𝐶 (𝑚𝑔 𝐿−1 ) − 𝑇𝐼𝐶 (𝑚𝑔 𝐿−1 )
Fig. 1: Reaction tube of Hach Lange kit LCK 238 to measure TOC
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EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
3. REQUIREMENTS
3.1.
EQUIPMENT AND MATERIALS





3.2.
Hach Lange kit LCK 380, 2-65 mg TOC L-1 including reaction tubes, membrane filter caps and
reagents (Hach Lange, Belgium)
a spectrophotometer (Hach Lange DR 2800, Belgium)
a thermostat (100°C) or HT 200 S machine (100°C; Hach Lange, Belgium)
a micropipette of 1-5 mL and tips
for samples with large suspended solids: a glass beaker of 500 mL, a blender or mixing device, a
magnetic stirrer, a glass beaker of 100 mL and a stirring magnet
REAGENTS

TC determination
- reagent present in the HL kit LCK 380: digestion reagent A
- raw water sample (2 mL of prepared sample is needed for the analysis; around 100 mL of
sample is needed if the sample needs to be mixed) stored at 4°C
- demineralized water

TIC determination
- raw water sample (2 mL of prepared sample is needed for the analysis; around 100 mL of
sample is needed if the sample needs to be mixed) stored at 4°C
- demineralized water
4. PROCEDURE
4.1. SAMPLE PREPARATION
 Samples which contain large (> 100 µm) suspended particles should be blended to reduce the
particle size to smaller than the micropipette tip, for example as follows:
- Add 100 mL of sample in a beaker of 100 mL.
- Mix the sample until the sample is homogenous and no large particles (not larger than the
pipette tip size) remain in the sample.
- Add a stirring magnet to the sample and put on a magnetic stirrer. The sample is then taken
from this beaker with a micropipette while the water sample is being stirred.
4.2. SAMPLE ANALYSIS
 Here under follows the procedure for samples with TOC concentration of 2-65 mg TOC L-1. For
samples with a TOC concentration above 65 mg TOC L-1, dilutions should be made with
demineralized water.
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
EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
TC determination
A. Screw powder dispenser on to digestion
reagent A. Invert so that powder dispenser is
under the reagent and shake. This causes the
dispensing chamber to be filled. Position the
centring recess of the powder dispenser above
the TC cuvette and add one dose. Close
digestion reagent A immediately with original
cap.
B. Pipette 2 mL sample into the TC cuvette. If the
sample contains suspended particles, the
sample should be taken with a micropipette
while the sample is stirring on a magnetic
stirrer.
C. Close the TC cuvette immediately with the
original cap and invert a few times.
D. Close the indicator cuvette very tightly with the
membrane double-cap. The barcode label must
be on lower half close to the indicator tube.
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EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
E. Open the TC cuvette and quickly screw the
prepared indicator cuvette tightly on to the TC
cuvette. Hold the cuvette combination
vertically. Do not invert.
The sample must not come into contact with
the membrane of the double-cap!

TIC determination
F. Pipette 2 mL sample into the TIC cuvette. If the
sample contains suspended particles, the
sample should be taken with a micropipette
while stirring on a magnetic stirrer.
G. Quickly close the TIC cuvette with the original
cap and invert a few times.
H. Close the indicator cuvette very tightly with
the membrane double-cap. The barcode label
must be on lower half close to the indicator
tube.
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EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
I.
Open the TIC tube and quickly screw the
prepared indicator cuvette tightly on to the
cuvette. Hold cuvette combination vertically.
Do not invert.
The sample must not come into contact with
the membrane of the double-cap!
J.
Heat
both
cuvette
combinations
simultaneously in the preheated thermostat
for 2 h at 100°C. The indicator tubes should not
be put directly into the heating block to avoid
heating up of the indicator solution; so a 100°C
oven cannot be used for this analysis.
K. Take the cuvettes out of the thermostat block
and allow to cool to room temperature
without inverting the tubes Clean the indicator
tubes with paper.
L. Invert the cleaned cuvette TC and TIC
combination and put successively in the
spectrophotometer. Always measure the TC
first, and the TIC second. The mode to determine
(LCK 380) will be automatically selected by the
bar code reader. The wavelength selected
should be 440 nm. The TC, TIC and TOC are
displayed on the spectrophotometer.
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EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
5. CALCULATION OF RESULTS
 The conversion of absorbance to TC, TIC, and TOC is done by the spectrophotometer.
 For diluted samples, the used dilution factor should be taken into account.
6. QUALITY CONTROL


The SOP can be screened by measuring samples with a known TIC, TC and TOC concentration.
The TIC, TOC and TC values for wastewater are strongly dependent on the wastewater type. Values
between 40 and 500 mg C L-1 have been reported (Van Den Hende, 2014).
7. ERRORS, CALIBRATION AND INTERFERENCES





The pH of the sample must be between pH 4 and pH 10.
The test reagents are stable at +15 to +25°C up to the expiry date given on the package.
Avoid contamination by ambient air. Never leave cuvette open, because carbon dioxide in the
ambient air can cause high bias. Cuvettes must only be opened when necessary (e.g. to add
sample) and must be closed or further processed immediately afterwards.
If several samples are analyzed simultaneously, label them so that the TC and TIC cuvette
combinations of the same sample can be recognized.
Measuring range: 2-65 mg TOC L-1.
Various substances can interfere with this TIC/TOC/TC measurement (Table 1, Table 2). Higher
concentrations of these ions cause high-bias results with TIC and low-bias results with TC
measurements.
Table 1. Substances which interfere with the TIC determination
Interfering substance
Methanoate (HCOO-)
Acetate (CH3COO-)
Sulfite (SO32-)
Sulfide (S2-)
Nitrite (NO2-N)
Interference level (mg L-1)
400
250
30
10
3
Table 2. Substances which interfere with the TC determination
Interfering substance
Chloride (Cl-)
Calcium (Ca2+), magnesium (Mg2+)
Ammonium (NH4+)
Interference level (mg L-1)
500
200
100
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EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
8. WASTE STREAM AND PROPER DISPOSAL
Hach Lange local agencies accept used cuvettes to ensure their proper disposal in accordance with
local and national regulations. These used cuvettes should thus be stored and sent back to Hach Lange. Do
not screw the cuvette combinations apart when the analysis has been completed (put the indicator cuvette
upwards).
9. HAZARDS AND PRECAUTIONARY STATEMENTS




Always wear a laboratory coat, eye protection and laboratory gloves.
Use only Hach Lange kit LCK 380 in well-ventilated areas.
Keep the test kit in tightly closed in a dry and well-ventilated place.
Act carefully and take appropriate measure concerning the hazardous reagents.
Reagent
Hazard statements
-
Potassium chloride
(KCI)
Sodium hydroxide
(NaOH)
-
Not a hazardous substance or mixture
according too Regulation (EC) No.
1272/2008.
Causes severe skin burns and eye
damage.
Precautionary statements
-
Store in cool place.Keep container
tighly closed in a dry and wellventilated place.
-
Wear
protective
gloves/protective clothing/ eye
protection/ face protection.
If in eye: rinse cautiously with
water for several minutes.
Remove contact lenses, if present
and easy to do. Continue rinsing.
Immediately call a Poison Center
or doctor/physician.
Wear
protective
gloves/protective clothing/ eye
protection/ face protection.
If in eye: rinse cautiously with
water for several minutes.
Remove contact lenses, if present
and easy to do. Continue rinsing.
Immediately call a Poison Center
or doctor/physician.
Keep/ Store away from clothing/
combustible materials.
Avoid breathing dust.
Wear protective gloves.
If in eyes: rinse cautiously with
water for several minutes.
Remove contact lenses, if present
and easy to do. Continue rinsing.
-
-
Lithium hydroxide
(LiOH)
-
Harmful if swallowed.
Causes severe skin burns and eye
damage.
-
-
Sodium persulfate
(Na2S2O8)
-
May intensify fire; oxidiser.
Harmful if swallowed.
Causes skin irritation.
May cause an allergic skin reaction.
Causes serious eye irritation.
-
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-
EnAlgae SOP:
UGent, Campus Kortrijk
TIC/TOC/TC in water
Version 29/06/2014
May cause allergy or asthma symptoms
or breathing difficulties if inhaled.
May cause respiratory irritation.
-
If
experiencing
respiratory
symptoms: Call a Poison Center or
doctor/ physician.
-
Obtain special instructions before
use.
If exposed or concerned: get
medical advice/ attention.
Boric acid (H3BO3)
-
May damge fertility. May damage the
unborn child.
-
10. REFERENCES










APHA, Awwa, WEF, 2005. Standard Methods for the Examination of Water and Wastewater, 21st
ed. American Public Health Association, Washington DC.
Van Den Hende S., 2014. Microalgal bacterial flocs for wastewater treatment: from concept to
pilot scale. PhD thesis, Ghent University, Ghent, 324p.
Beelen V., Van Den Hende S., 2014. Verbal communication. Ghent University, Campus Kortrijk.
Hach Lange, 2014. Safety Data Sheet LCK 380. Available on (26/06/2014):
http://www.camlab.co.uk/originalimages/sitefiles/msds/hach/hh_lck380.pdf.
Sigma-Aldrich,
2014.
Potassium
chloride.
Available
on
(26/06/2014):
http://www.sigmaaldrich.com/catalog/product/sial/p9333?lang=fr&region=FR.
Sigma-Aldrich,
2014.
Sodium
hydroxide.
Available
on
(26/06/2014):
http://www.sigmaaldrich.com/catalog/product/fluka/38210?lang=fr&region=FR.
Sigma-Aldrich,
2014.
Lithium
hydroxide.
Available
on
(26/06/2014):
http://www.sigmaaldrich.com/catalog/product/sial/442410?lang=fr&region=FR.
Sigma-Aldrich,
2014.
Sodium
persulfate.
Available
on
(26/06/2014):
http://www.sigmaaldrich.com/catalog/product/sial/216232?lang=fr&region=FR.
Sigma-Aldrich,
2014.
Boric
acid.
Available
on
(26/06/2014):
http://www.sigmaaldrich.com/catalog/product/aldrich/202878?lang=fr&region=FR.
Hach, 2009. TOC wastewater analysis. Available on (26/06/2014):
http://www.hachlange.co.uk/medias/sys_master/8852615495710/EN_20033.pdf.
11. CONTRIBUTIONS
 SOP written by: Alexandra Lefoulon (internship student EnAlgae project).
 SOP approved by: Sofie Van Den Hende (staff EnAlgae project).
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