Bam-S08-14 Total THM by GC Jan02

The South African Breweries Section : 8.
14
Page : 1 of 16
BREWING MANUAL 1st Issue : Jan 2002
Vol. X - Analytical Methods Rev. Date :
SECTION 8.14: DETERMINATION OF TOTAL

THRIHALOMETHANES IN WATER BY GAS CHROMATOGRAPHY
1. INTRODUCTION
a) Trihalomethanes are small organic compounds, similar in structure to methane, but
also containing chlorine and /or bromine. The compounds are the major by-products of
water supplies disinfected with chlorine gas before it reaches the brewery.
b) These compounds are formed when naturally occurring organic substances, such as
humic acids from decaying vegetation, reacts with chlorine used to disinfect drinking
water.
c) The following TTHM’s are measured by this method:
 Chloroform (CHCl3)
 Dichlorobromomethane (CHCl2Br)
 Dibromochloromethane (CHBr2Cl)
 Bromoform (CHBr3)
d) The levels of TTHM’s present in the sample are dependent on the amount of naturally
occurring organic material found in the water source and the amount of chlorine used
during disinfection.
e) Brewery water thus requires filtration through an activated carbon filter to remove
organic compounds and any chlorine added during disinfection.
f) It is essential for the brewing industry to remove all the chlorine because it can react
with phenol compounds during brewing to produce chlorinated phenol compounds
which have a very unpleasant medicinal taste (TCP), even at low concentrations.
These are easily formed during wort preparation if chlorine is present in the brewing
water.
g) This method is used to monitor the ability of the activated carbon filter to effectively
remove TTHM’s from the brewing water on a continuous basis. The results should be
used to determine the optimum time for regeneration and / or replacement of the
activated carbon in the filter (saving cost due to optimisation).
h) This method is applicable to any type / grade of water used in the brewery and it
makes use of a headspace sampling technique and a gas chromatograph fitted with
an ECD detector.
i) The addition of sodium thiosulphate (as stated in historical methods) is not necessary
because no significant difference in the levels of the different compounds were
detected during the development stages of the method. This is due to the headspace
sampling technique used by the method vs the more traditional liquid injection
methods.
The South African Breweries Section : 8.14
Page : 2 of 16

1. INTRODUCTION (Continued)
j) References:
i) The following manufacturer’s manuals:
- GC Reference manual
- GC Operating manual
- Headspace Sampler operating manual
- Understanding your Chemstation
- T.Wainwright, Basic Brewing Science
- H.Hachenberg and A.P.Schmidt, Gas Chromatographic Headspace Analysis
2. HEALTH AND SAFETY

2.1 HAZARDS
a) THM’s are carcinogenic and flammable.
b) Working with gases under pressure.
2.2 SAFETY REQUIREMENTS

a) Fume cupboard.
b) Surgical gloves.
c) Protective gloves (handling glass)
2.3 PRECAUTIONS
a) Keep solvents away from heat, flames and sparks.
b) Do not smoke when working with these chemicals.
c) When working with these chemicals, work in a fume cupboard.
d) Use protective gloves when breaking the seal of the ampoule.
e) Avoid breathing excess TTHM fumes.
Page : 3 of 16

3. APPARATUS
3.1 EQUIPMENT REQUIRED
a) Hewlett Packard (Agilent Technologies) Gas Chromatograph (Diacetyl Analyser)
which consists of the Hewlett Packard (HP) 5890 series II Plus or 6890 Gas
Chromatograph with Electronic Pressure Control (EPC), Electon Capture
Detector, HP 7694 Headspace Sampler and GC Chemstation software (G 2070).
Note: 1. Hewlett-Packard name has changed to Agilent Technologies,
Innovating the HP way - from November 1999 (Globally)
b) Glass funnel
c) Column, Hewlett-Packard HP-5.
d) Metal cap crimper/de-crimper.
e) Hamilton glass syringe - 2,5 ml
f) Volumetric flasks, 100, 250 ml.
g) Sample vials, 20 ml (Hewlett Packard).
h) 30 ml Amber sample bottles with air tight seal (plastic screw on cap).
i) Butyl Septa.
j) Metal crimp caps.
3.2 EQUIPMENT PREPARATION

3.2.1 HP 7694 Headspace Sampler
a) Zone Temperatures (for use with Micro ECD use temperatures [ ])
Oven 80°C [60°C]
Loop 90°C [70°C]
Transfer line 100°C [80°C]
b) Event Times
GC cycle time  8.0 min (optimise - time to cool down)
Vial equilibration time 30 min
Pressurising time0,13 min
Loop fill time 0,15 min
Loop eq. time 0,05 min
Inject time 1,0 min
c) Vial Parameters
First vial enter vial number
Last vial enter vial number
Shake high (number 2)
Page : 4 of 16

3.2 EQUIPMENT PREPARATION (Continued)

3.2.2 HP 5890 Series II plus or HP 6890 Gas Chromatograph
a) Gases
Carrier gas Nitrogen
Initial column flow 8.0 ml/min
b) Inlet
Inlet mode Split
Initial inlet temp. 90 oC
Inlet pressure 42 kPa
Split ratio 1:1
Split flow 8,0 ml/min
Total inlet flow  18,0 ml/min (automatically adjusted by instrument)
b) Detector
Detector type ECD
Temperature 150 oC
Anode purge flow 1,0 ml/min
Make-up flow 15,0 ml/min
Gas Type Nitrogen
Adjust offset 60,00
Electrometer On
c) Signal
Date rate 20 Hz
d) Vial Pressure - Auxiliary Pressure 5

Description Vial pres
Gas Type Nitrogen
Initial pressure 120 kPa (on)
e) Temperatures
Injector 90 oC
Detector 150 oC
Column:
Initial temperature 70 oC for 1 min,
Ramp to 130 oC @ 20,00 oC/min for 2,20 min
Page : 5 of 16

3.2 EQUIPMENT PREPARATION (Continued)

3.2.3 Glassware
a) Wash the sample bottles and caps with mild alkaline soap solution and rinse
with purified water.
b) Boil the bottles and caps for 30 minutes in purified water and dry in the oven.
3.3 MAINTENANCE
1. DAILY: Run the BAKE-OUT method at the end of the day or after sample
sequence has been completed.
2. MONTHLY:
a) PC housekeeping – remove all temporary files and defragment the hard
drive
b) Replace the inlet septa
c) Steam clean the headspace autosampler as per supplier’s manual.
d) Remove the headspace needle and clean by placing the needle into
ethanol and sonicate it for 10 minutes. Dry the needle and replace.
e) Remove the transfer-line needle and clean as above. Use cleaning wire
to ensure the needle is not blocked with a piece of septa.
3. 6 MONTHLY: Archive data files and remove from the hard disk.
3.4 CALIBRATION
A multilevel calibration is used (3 levels and 4 compounds).
3.4.1 Major Calibration – 1/week
a) Prepare five (5 vials) calibration standards from each of the three working
solutions (approx. 1 ppb , approx. 5 ppb and/or 10 ppb TTHM’s; additional
vials should be prepared to verify calibration i.e.:
i) Pipette 10 ml working standard into a 20 ml Hewlett Packard headspace
vial.
ii) Cap immediately with a septum and metal crimp cap.
iii) Mark each vial clearly 1 ppb, 5 ppb or 10 ppb.
b) Place the prepared standards into the auto-sampler in the following order:
i) Position 1 - 10 ppb (to quench column)
ii) Positions 2-6 - 1 ppb standards
iii) Positions 7-11 - 5 ppb standards
iv) Positions 12 – 16 - 10 ppb standards
c) Enter the vial parameters.
d) Press start on the auto-sampler keypad.
e) Enter the relevant sequence parameters in the Chemstation software – data
file name.
Page : 6 of 16

3.4 CALIBRATION (Continued)

3.4.1 Major Calibration – 1/week (Continued)
f) Enter the new sample sequence into the sequence table.
g) Start sequence.
h) After the sequence is completed determine the average area of the individual
THM standard peaks for all three levels.
i) Select one file for each standard which area is closest to the calculated
average of the four compounds present in the standard as this will ensure the
best possible correlation. (See figure 1 for example of 10 ppb standard
chromatogram).
k) Select the data analysis menu.
l) Select new calibration table (or recalibrate if table exists).
k) Load the 1 ppb standard file (as selected in Step (i) above).
l) Calibrate as level 1 in the calibration table (always replace).
m) Load the 5 ppb standard file (as selected in Step (i) above).
n) Calibrate as level 2 in calibration table (Replace).
o) Load the 10 ppb standard file, (as selected in Step (I) above).
p) In the calibration options table, select linear curve and include the origin.
q) If the correlation of the calibration curve is acceptable (with the above
parameters selected), accept the calibration by saving the method.
r) Acceptable correlation for a calibration curve – 0.999… to 1.00000.
s) If correlation is not acceptable – re-select the files used for calibration by
identifying the problematic levels using the calibration curves of each
component. Recalibrate and save the method if acceptable.
t) Verify the new calibration by analysing the 5 ppb standard as a sample.
Note: If the standard is not within the allowed control limits as calculated,
repeat the calibration (use allowed standard deviation as for Diacetyl).
u) See Figure 1 for example of calibration table.
Page : 7 of 16


Fig. 1 - Example : Calibration Table (actual amounts to be entered after calculation)
RT (min) Sig Lvl Amount Area Amt/Area NAME

(ppb)
2,796 1 1 0,996 From file From file
2 4,980 From file From file CHCl3
3 9,960 From file From file
2 5,095 From file From file CHCl2Br
3 1,.190 From file From file
2 4,890 From file From file CHClBr2
2 5,015 From file From file CHBr3
Note : Amounts in ppb as calculated in 5.3.1
RT (min ) – from chromatogram (file selected)
Note: Example 10 ppb Standard Chromatogram
Page : 8 of 16


Figure 2
3.5 STORAGE
4. CHEMICALS
4.1 CHEMICALS REQUIRED
a) Trihalomethanes Calibration mix, Supelco, Cat. No: 47904, Supplier – Sigma
Aldrich SA.
b) Nitrogen gas GC grade - Purity 99,99%.
4.2 REAGENT PREPARATION

4.2.1 Purified de-ionised water
a) Boil deionised water for 30 minutes.
b) Cover with foil and cool.
c) Check the purity on the GC (no peaks).
Note: i) Store in a refrigerator below 5°C.
ii) Use cold.
Page : 9 of 16

4.2 REAGENT PREPARATION (Continued)

4.2.2 TTHM Stock Solution – 1000 ppb
Note: i) Do not open the TTHM standard ampoule used for standard
solutions in the vicinity of the GC because it can produce peaks on
a chromatogram when using the headspace sampling technique.
ii) All chemicals, reagents and purified water are used and prepared
cold.
a) Half fill a 100 ml volumetric flask with cold pre-prepared water and place the
funnel into the flask.
b) Hold the ampoule in an upright position (over the funnel) and break the seal
of the ampoule (carefully) without spilling the content.
c) Quantitatively transfer the content of the ampoule into the funnel and rinse
the ampoule / cap of the ampoule 3 times with purified water.
d) Rinse the funnel and make up to volume with cold pre-prepared water.
e) Calculate the actual amounts of the different compounds using the
information supplied on the certificate of analysis of the ampoule (analytical
concentration (4)). See Section 5.3 (calculations).
f) Repeat the above calculation for the rest of the compounds (stock).
g) Determine the concentrations of the working solutions by using the
concentration of the individual compounds in the stock solution (calculated in
(e)), and apply dilution factor.
Note: i) Store the stock solution in a refrigerator below 5°C.
ii) Shelf life of stock solution is one month.
4.2.3 Working Solutions (Shelf life of one day)

a) 1 ppb Stock
i) Half fill 1 x 250 ml volumetric flask with cold purified water (clearly
marked as 1).
ii) Accurately transfer 0,25 ml of stock solution using a 2,5 ml Hamilton
glass syringe, into the flask
iii) Make up to volume with cold, purified water.
iv) Shake well and place in the refrigerator
Page : 10 of 16

4.2 REAGENT PREPARATION (Continued)

4.2.3 Working Solutions (Shelf life of one day) (Continued)
b) 5 ppb Stock
marked as 1).
c) 10 ppb Stock
marked as 1).
4.3 STANDARDISATION
4.4 DISPOSAL
Page : 11 of 16

5. METHOD
5.1 SAMPLING
a) Rinse 5-litre glass beaker twice with water sample.
b) Fill beaker to overflowing.
c) With lid still on, submerge sample bottle in water sample.
d) Remove lid, fill bottle with sample. Rinse sample bottle twice with sample.
e) Submerge bottle(lid is still on) in beaker, remove lid(in water) and fill bottle.
f) Cap bottle while immersed in water.
g) Ensure that no air is trapped in bottle.
h) Make sure that lid is airtight.
i) Analyse immediately or store at approx. 5 oC prior to analysis
Note: Do not store for longer than 24 hrs
5.2 TEST METHOD

a) Pipette 10 ml prepared sample into a clearly marked 20 ml vial.
Note: Only Hewlett Packard vials to be used.
b) Stopper the vial immediately with a Teflon seal and crimp a metal cap in place.
c) Transfer the samples to the auto-sampler carousel and prepare the sequence.
d) Load the auto-sampler method and set the vial parameters (see suppliers
manual).
e) Start the auto-sampler.
f) Start the sequence by clicking on <run sequence>.
Page : 12 of 16

5.3 CALCULATIONS
5.3.1 TTHM stock solution ( 1000 ppb)
Calculate the actual amounts for each of the different compounds using the
information supplied on the certificate of analysis of the ampoule (analytical
concentration (4))
i.e.: Chloroform : 99.6 g / ml (from certificate) x 1 000 = 99 600 ppb
Thus C1V1 = C2V2
C2 = C1V1
V2
= 99 600 x 1
100
= 996 ppb (Actual)
Note : Repeat th calculation for all the compounds in the calibration mix.
5.3.2 Actual TTHM concentration in the 1 ppb working solution:

996 0.25
g / l  x
1 250
= 0,996 ppb
Repeat the above calculation for each compound for all three levels.
Use this information in the calibration table (see figure 1) – amount column.
5.3.3 Samples
No manual calculation of results are required, the HP Chemstation software
loaded onto the PC calculates the results relative to the calibration standards.
5.3.4 INTERPRETATION OF RESULTS.
a) Results reported as 0,000 ppb on the print-out, should be reported as
<0,1 ppb (detection limit).
b) When compound is not detected at all by the software, verify retention time or
if RT is correct and no peak is visible, report the result as ND (not detected).
6. VERIFICATION
a) Refer to Section 3.12 Verification Routines.
Page : 13 of 16

7. COMPETENCY TEST
FORMATIVE ASSESSMENT
Date : Candidate Name :

Position : Department Name :
Unit :
Element :
Assessor 1 : Assessor 2 :
Assessment 1 2 3
CHECKLIST COMPETENT NOT YET NOT

COMPETENT ASSESSED
1.0 PRODUCT
I’d know that the end product is right if:-
1. Calibration status of the G.C. equipment is valid
and current as manualised.
2. Service and maintenance records of G.C.
equipment is available, current and maintained as
manualised.
3. Detailed records are available and up to date of
the preparation of standards (stock and working
solutions).
4. Chemicals and reagents are stored and used as
manualised.
5. At least five (5) working solutions are used for
calibration which have very similar peak heights
and are within 2 ppm from the calculated
concentration.
6. An updated record of operating parameter checks
are available.
7. Updated records of results are available.
Page : 14 of 16

7. COMPETENCY TEST (Continued)
FORMATIVE ASSESSMENT (Continued)
CHECKLIST COMPETENT NOT YET NOT

COMPETEN ASSESSED
T
2.0 PROCESS
If the candidate did the job the correct way he/she
would:-
1. Maintain and prepare samples as manualised.
2. Use ‘boiled out’ purified water where relevant as
manualised.
3. Prepare and inspect glassware as manualised.
4. Prepare reagents (standards) as manualised.
5. Identify samples and glassware for traceability.
6. Demonstrate attention to details to avoid cross
contamination.
7. Verify G.C. operating parameters.
8. Adhere to manualised Health and Safety
requirements.
9 Check gases and allow G.C. to stabilise.
10 Calibrate the G.C. as manualised.
11 Analyse the samples as manualised.
12 Adhere to manualised safety requirements.
13 Calculate concentration of working standard and
samples as manualised.
14 Verify results using the manualised Verification
Routines.
15 Record and communicate results.
Page : 15 of 16

FORMATIVE ASSESSMENT (Continued)

3.0 KNOWLEDGE
A candidate who does this job should be able
to explain or discuss:-
1. Which compounds are known as TTHM’s.
2. How TTHM’s are formed and how are they
removed.
3. Why TTHM’s are measured and the effect
they have on the product.
4. How and when a column is quenched.
5. The detector used in the determination of
TTHM’s.
6. How are the concentrated TTHM’s chemicals
stored and why?
7. Why is the temperature of the injection port,
column and detector raised at set
frequencies?
8. Why must ‘boiled-out’ purified water be used
in the analysis?
9 The principles of operation of a G.C and ECD
detector.
10 Define carrier gas and state what gas and
grade is used.
11 The term “retention time”.
12 Name the parameters which can effect
retention time.
Page : 16 of 16

Competent : Not Yet Competent:

Comments :
Action Plan Follow up Dates
Assessor 1 Candidate
Sign. Sign.
Assessor 2
Sign.

Bam-S08-14 Total THM by GC Jan02

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The South African Breweries Section : 8.

SECTION 8.14: DETERMINATION OF TOTAL

SECTION 8.14: DETERMINATION OF TOTAL

2. HEALTH AND SAFETY

2.2 SAFETY REQUIREMENTS

SECTION 8.14: DETERMINATION OF TOTAL

3.2 EQUIPMENT PREPARATION

SECTION 8.14: DETERMINATION OF TOTAL

3.2 EQUIPMENT PREPARATION (Continued)

d) Vial Pressure - Auxiliary Pressure 5

SECTION 8.14: DETERMINATION OF TOTAL

3.2 EQUIPMENT PREPARATION (Continued)

SECTION 8.14: DETERMINATION OF TOTAL

3.4 CALIBRATION (Continued)

SECTION 8.14: DETERMINATION OF TOTAL

3.4 CALIBRATION (Continued)

RT (min) Sig Lvl Amount Area Amt/Area NAME

SECTION 8.14: DETERMINATION OF TOTAL

3.4 CALIBRATION (Continued)

4.2 REAGENT PREPARATION

SECTION 8.14: DETERMINATION OF TOTAL

4.2 REAGENT PREPARATION (Continued)

4.2.3 Working Solutions (Shelf life of one day)

SECTION 8.14: DETERMINATION OF TOTAL

4.2 REAGENT PREPARATION (Continued)

SECTION 8.14: DETERMINATION OF TOTAL

5.2 TEST METHOD

SECTION 8.14: DETERMINATION OF TOTAL

5.3.2 Actual TTHM concentration in the 1 ppb working solution:

SECTION 8.14: DETERMINATION OF TOTAL

Date : Candidate Name :

CHECKLIST COMPETENT NOT YET NOT

SECTION 8.14: DETERMINATION OF TOTAL

7. COMPETENCY TEST (Continued)

FORMATIVE ASSESSMENT (Continued)

CHECKLIST COMPETENT NOT YET NOT

SECTION 8.14: DETERMINATION OF TOTAL

7. COMPETENCY TEST (Continued)

FORMATIVE ASSESSMENT (Continued)

SECTION 8.14: DETERMINATION OF TOTAL

7. COMPETENCY TEST (Continued)

Competent : Not Yet Competent:

Action Plan Follow up Dates

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