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COLOR ANALYSIS IN WATERS

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INSTRUCTIONS: IA-O-04-IT-nn

COLOR ANALYSIS IN WATERS

SPECTROPHOTOMETRIC METHOD - SINGLE WAVELENGTH

METHOD

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REVISION HISTORY
Effective
Version Description of Change Author
from
Yury Martinez
1 Initial version
Water Laboratory

CONTROLLED DOCUMENT
CONTROLLED COPY No: Original

Any printed or digital copy of the manual other than the one found in the laboratory
management system folder is considered an “Uncontrolled” copy, since the document
management procedure applies exclusively to the documents existing in this folder in digital
form.

RESPONSIBLE FOR THE MOST RECENT VERSION OF THE DOCUMENT:

Prepared Reviewed Approved

Yury Martinez Jorge Galeano P. Ricardo Maldonado

Water Laboratory Technical Direction Laboratory Management

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CONTENT

1 OBJECT 4

2 FRAME OF REFERENCE 4

3 SCOPE 4

4 ASSOCIATED DOCUMENTS 4

5 HSEQ MANAGEMENT 4

6 GLOSSARY 5

7 DESCRIPTION OF THE METHOD 5

8 INTERFERENCES 6

9 EQUIPMENT AND MATERIALS 7

10 RESPONSIBILITIES AND COMPETENCES 7

11 REAGENTS 7

12 DESCRIPTION OF ACTIVITIES 9

13 CALCULATIONS AND EXPRESSION OF RESULTS 11

14 ANALYTICAL QUALITY CONTROL 11

15 INSTRUCTION UPDATE 12

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1 OBJECT

Define the analytical procedures to determine the color concentration in aqueous samples, by
applying the color spectrophotometric method, taking into account the laboratory conditions,
the values that serve as confidence criteria of the analytical method, to ensure the quality of
the results.

2 FRAME OF REFERENCE
The following document was taken into account in the preparation of this manual:

 Standard Methods for the Examination of Water and Wastewater. American Public Health
Association, American Water Works Association, Water environment Federation, 22 ed.
Washington, 2012, SM 2120- C, Spectrophotometric method - single wavelength method,
pp. 2-7, to 2-8.

3 SCOPE
This instruction and the procedures described therein apply to the determination of color
concentration caused primarily by natural organic matter. The measure applies to surface and
groundwater; wastewater, domestic and industrial water, and especially drinking water. With
this method you can measure apparent color and real color.

4 ASSOCIATED DOCUMENTS
The manuals, instructions and forms associated with this document include:

 AI-14 Translated Manual Analytical Microbalance – (in Spanish)

 IA-O-04-FR-12 Verification of Analytical Balance and Analytical Microbalance BM-20 – (in
digital format, monthly copy in pdf, primary data CP - 04)
 Laboratory material washing instructions xxxx.
 Instructions for using the xxxx vacuum filtration system.
 Pharo 300 Spectrophotometer Manual.
 HACH HQ40d Multiparameter Manual.
 IA-O-04-FR-nn vn Color Data Capture Record Format.
 IA-O-04-FR-nn vn Pharo 300 Spectrophotometer Usage Format XXXX

5 HSEQ MANAGEMENT
Table 1. HSEQ Management.

ASPECT PURPOSES STANDARDS

Health (H) Preventing  Know the procedures established for safe work in the laboratory.
occupational  Use personal protective equipment: glasses, gloves, respirator,

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safety gown and extraction cabins.

 Avoid spilling chemicals and inhaling their vapors.
diseases
 Have prior knowledge of the nature and dangers of the
substances handled during the procedures.
Avoiding work  Use the recommended PPE for each analysis activity
accidents  Check the status of the materials, supplies, facilities, equipment
and assemblies used in the development of the analysis
techniques
Security (S)
 Recognize the location of fire extinguishers, safety showers and
eye washes
 Prevent the spillage of hazardous substances and the release of
vapors
 Collect all waste generated during analysis activities
Environment Avoid environmental  Contribute to the proper classification of hazardous waste and
(E) impacts final disposal of inputs used during analysis procedures.
 Do not dump hazardous waste into the public sewer system.
 Verify the competence of the personnel who will perform the
analysis procedures
 Verify the proper planning of analysis activities
Ensure
 Verify the correct use of equipment and materials.
representative
Quality (Q)  Verify strict application of these instructions.
samples and valid
 Verify that all relevant forms have been completed.
results
 Review and approve the quality of primary data and results
 Review and approve the results concerning the quality
assurance of the technique.

6 GLOSSARY
CU: Color Unit, equivalent to a Hazen unit and a Pt-Co unit.
MCC: Quality control sample.

7 DESCRIPTION OF THE METHOD

The color is determined spectrophotometrically at a wavelength between 456 nm, with
platinum-cobalt solutions as standards. The actual color of platinum-cobalt samples and
standards follow Beer's law.

The spectrophotometric platinum-cobalt method is applicable to natural waters, drinking water,

wastewater, and both domestic and industrial waters.

The term color is used to measure the true color, that is, the color of water whose turbidity has
been removed. Large suspended and colloidal particles scatter light, interfering with true color
determination. The term apparent color includes not only the color due to substances in
solution, but also to suspended matter. The apparent color is determined on the original
sample without filtration.

Color in groundwater and surface water results from the presence of organic matter,
particularly humic aquatic matter. Suspended particles, especially of colloidal size such as

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clays, algae, iron and manganese oxides, give water a colored appearance and must be
removed before measurement.

The minimum detectable color depends on the path length of the cell. Choose a cell size that
provides absorbance within the range that results in good accuracy and linearity of response.
This range depends on the quality of the spectrophotometer. If a 50-mm cell is used in the
wavelength range of 450 to 465 nm, it gives an absorbance of 0.005 producing a minimum
detectable color of 1 CU. With new spectrophotometers, a detection level of 2 CU method can
be obtained with a path length of 25 mm. Dilute highly colored samples to fall within the range
of the standard curve. Absorbance readings should fall in the range of 0.005 to 0.8.

COLLECTION, PRESERVATION AND HANDLING OF SAMPLES

Preferably take a sample volume of 500 mL, analyze within 48 hours of collection. The sample
must be collected in amber-colored glass containers, or failing that, in plastic containers
covered so that light does not pass through. Keep samples refrigerated at ≤ 6°C until analysis,
and bring to room temperature at the time of measurement.

CLEANING GLASS

Wash all glassware with neutral soap, rinse with tap water, then rinse with distilled water.
Refer to Material Washing Instructions xxxxx.
Use glassware that has undergone quality control.

8 INTERFERENCES

The main interference is from the presence of colloidal and suspended particles that absorb or
scatter light at the wavelength of the spectrophotometric method.

The light absorbance of organic matter is pH dependent; however, the absorbance variation is
small over the pH range of most waters. Because color measurements are made for aesthetic
reasons, it is preferable not to adjust the pH of the sample, as long as it is between 4 and 10.
If pH is adjusted, adjust to 7, and record this data. Additionally, pH can affect the solubility of
substances, which can interfere with color measurement if particulate matter forms.

9 EQUIPMENT AND MATERIALS

9.1 TEAMS

 pH meter.
 Spectroquant pharo 300 spectrophotometer.
 Glass cell with 50 mm light path.

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9.2 MATERIALS AND SUPPLIES

 Volumetric pipettes of 1, 2, 3, 4, 6, 10 and 20 mL.

 Volumetric balloons 100 mL.
 Plastic bottles for sample collection, 500 mL.
 Filter (for true color measurements): Use a cellulose membrane filter with a diameter of
0.45 µm. For the removal of colloidal particles of Mn, Fe, or others, ultrafiltration is required
in filters with diameters of 0.2 or 0.22 µm.

10 RESPONSIBILITIES AND COMPETENCES

The coordination of the water quality laboratory together with the laboratory management are
responsible for ensuring compliance with the instructions and their continuous updating.

Analysts and/or laboratory assistants are responsible for compliance with the instructions,
especially in the execution of analytical procedures and obtaining primary data. The
importance of a correct identification of the stages of the analytical procedure, materials and
reagents necessary during the analysis, as well as the complete registration of the formats
that are part of each of the analysis activities, is highlighted.

Persons responsible for analysis must have the following skills:

 Higher education as chemists, technologists or chemical and/or environmental technicians.

 Training in the IA-O-04-IT-xx instruction (this document).
 Training in the use and handling of spectrophotometers and volumetric material.
 Training in analytical techniques in the water quality laboratory.
 Minimum of 6 months experience in analysis activities and/or work in chemical analysis
laboratories.

11 REAGENTS
 Organic matter-free water: Type I water or equivalent, use for preparation of standards and
other procedures.
 Potassium chloroplatinate: K2PtCl6 analytical grade.
 Cobalt chloride: CoCl2.6H2O, analytical grade.
 Hydrochloric Acid, HCl, analytical grade.
 Sodium Hydroxide, NaOH, analytical grade.

11.1 PREPARATION OF CALIBRATION CURVE, STANDARDS AND BLANKS:

Prepare the following samples as quality control:

500 CU Color Stock Solution:

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Dissolve 1.246 g of potassium chloroplatinate and 1.00 g of cobalt(II) chloride crystals in water
with 100 mL of concentrated HCl and dilute to 1000 mL. Platinum-cobalt standards of 500 CU
are commercially available, and are suitable for use as the primary standard.

Standards for the calibration curve:

Prepare 5, 10, 15, 20, 30, 40, 50, and 100 CU standards by diluting 1.0, 2.0, 3.0, 4.0, 6.0, 8.0,
10.0, and 20.0 mL of 500 CU color standard with water in 100-mL volumetric flasks. Protect
standards from evaporation and contamination when not in use. Keep in the dark when not in
use, and keep for only 1 month.

11.2 QUALITY CONTROL STANDARDS:

 Low concentration: concentration twice the limit of quantification.

 Average concentration: concentration at the middle of the calibration curve.

 High concentration: 90% concentration of the linear range.

Note: A control standard must be chosen for each sample run; the same one should not
always be used, but rather it should be varied in order to cover the entire linear range of
the method.

 Perform repeated measurements: Use at least two portions of filtered sample.

 Perform duplicate analysis: Analyze one duplicate for every ten samples to evaluate the
precision of the method.

 Perform spectrophotometer pre-programming: For spectrophotometers with calibration

curve pre-programming, check the calibration curve regularly with the platinum-cobalt
standard, and adjust the pre-programmed curve as necessary.

12 DESCRIPTION OF ACTIVITIES
TO) CALIBRATION CURVE

Documents /
No. Activity Description Responsible
Records
1 Conditioning the Turn on the spectrophotometer 45 Laboratory IA-O-04-FR-
Spectrophotometer minutes before measurement, choose Analyst/Lab nn vn
a wavelength of 456 nm. Pt-Co at this oratory Pharo 300
wavelength has a maximum Manager Spectropho
absorbance. tometer
Usage
Before using this equipment, read the Format
instructions for use and register in the
Pharo 300 spectrophotometer usage

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form.
 Use glass cell with 50 mm light
path.

First measure a blank with distilled

water, press BLANK ZERO on the
IA-O-04-FR-
equipment.
Laboratory nn vn
Measure the Then pass the standards of 5, 10, 15,
Analyst/Lab Color Data
2 calibration 20, 30, 40, 50, and 100 CU.
oratory Capture
curve standards Record the absorbance values
Manager Record
obtained in each of them and graph
Color concentration in CU vs Format
absorbance.

B) PROCEDURE FOR BLANKS, SAMPLES AND SAMPLE DUPLICATES.

Documents /
No. Activity Description Responsible
Records
Measure the pH of the sample. If it is
outside the range of 4 to 10 adjust,
preferably the pH to 7 and record.

IA-O-04-FR-
Laboratory nn vn
Analyst/Lab Color Data
1 pH adjustment
oratory Capture
Manager Record
Format

2 True Color If it is actual color that needs to be Laboratory IA-O-04-FR-

Measurement measured, take a membrane filter and Analyst/Lab nn vn
pass at least 50 mL of distilled water oratory Color Data
through it to wash the filter. Take 25 mL Manager Capture
of the sample and filter it, then Record
immediately discard the filtered portion; Format
filter an additional 50 mL portion of

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sample, using the same filter, and use

this filtrate for analysis.

Use a cellulose membrane filter of 22-

47 mm diameter, with a pore diameter
of 0.45 µm. Wash filters before use and
watch for gaps in the filter. For the
removal of colloidal particles of Mn, Fe,
or others, ultrafiltration is required in
filters with diameters of 0.2 or 0.22 µm.
IA-O-04-FR-
Laboratory nn vn
Apparent Color Analyst/Lab Color Data
3 You should not filter the sample.
Measurement oratory Capture
Manager Record
Format
4 For the measurement in the Laboratory IA-O-04-FR-
Sample spectrophotometer, use the same Analyst/Lab nn vn
measurement wavelength that was used for the oratory Color Data
assembly of the curve, as well as the Manager Capture
cell. Record
Format
Distilled water is required for zero. Once
the zero has been adjusted, the sample
is read. At the end, with the absorbance
readings, the concentration can be

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determined with the data that was found

for the calibration curve.

13 CALCULATIONS AND EXPRESSION OF RESULTS

Calculate the color concentration in the original sample, in units of CU as follows:

Color, CU = [CU of color obtained from the curve * Dilution factor]

14 ANALYTICAL QUALITY CONTROL

Verify calibration by periodically analyzing a quality control standard and a calibration blank
during a typical run, after each batch of ten samples, and at the end of the run. The calibration
verification standard analyte or concentration parameter must be varied over the calibration
range to determine the detector response. Record the results in the method CONTROL
CHART.

Analyze an externally generated MCC blind sample, (unknown concentration) at least

annually.

For each batch of 20 samples or less, a duplicate must be read.

15 INSTRUCTION UPDATE
This instruction must be updated permanently, whenever required. The update will be
necessary, at a minimum, under the following circumstances:

 Changes in current environmental regulations or in the NTC for water quality.

 Suggestions from advisors and experts, provided they are duly justified and technically
supported.
 Result of the management of findings of the general procedure of the integrated
management system of Induanálisis.
 Detection of real or potential non-conformities as a result of internal and external audits.

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