Hach Colorimetric Analysis Spectrophotometer / Colorimeter

SOP-L303 Revision 1.1
Author

Marc Los Huertos

EA Program, Pomona College

Published

2026-06-26

1 Purpose

This procedure describes the general operation, calibration, and quality control framework for colorimetric water analysis using the Hach DR3900 benchtop spectrophotometer and the Hach DR900 portable colorimeter. The SOP is designed as a modular framework: Section 6 covers instrument operation common to all analyses, while the Method Registry (Section 7) provides chemistry-specific parameters for each analyte (reagent kit, Hach method number, wavelength, range, reaction time, and interferences). New chemistries can be added to the registry as the lab’s needs evolve, without rewriting the core SOP (APHA et al., 2012; Hach Company, 2024a, 2024b).

2 Scope

  • Applies to: Colorimetric determination of nutrients (phosphorus, nitrate, nitrite, ammonia), metals (iron, manganese, copper, zinc, lead, chromium), and other water quality parameters (silica, sulfate, chloride, hardness) in surface water, groundwater, wastewater, and aqueous extracts using Hach pre-packaged reagent kits and stored programs
  • Does not apply to: Analyses requiring external standards and user-prepared calibration curves (use the DR3900 in manual wavelength mode with a separate SOP); soil KCl extracts analyzed for NO3-/NH4+ via ion chromatography or flow injection (see SOP-L403)
  • Location: EA Program laboratory, SGM 133 (DR3900); field sites (DR900)

3 Responsibilities

Role Responsibility
Lab PI Approve methods, review QC data
Lab Manager Maintain instruments, stock reagent kits, track lot numbers and expiration dates
Trained Personnel Run analyses per this SOP, record results
New Students Complete training before independent use

4 Definitions

  • Absorbance (A): The negative base-10 logarithm of transmittance: \(A = -\log_{10}(T)\); directly proportional to analyte concentration within the linear range of the instrument (Beer-Lambert law) (Hach Company, 2024a).
  • Beer-Lambert law: The relationship between absorbance, path length, and concentration: \(A = \varepsilon c l\), where \(\varepsilon\) is the molar absorptivity, \(c\) is concentration, and \(l\) is the optical path length; the physical basis for all colorimetric quantification (APHA et al., 2012).
  • Blank (reagent blank): A sample containing all reagents except the analyte; used to zero the instrument and correct for background absorbance contributed by the reagent matrix (APHA et al., 2012).
  • Continuing calibration verification (CCV): A known-concentration mid-range standard analyzed at regular intervals during a run to confirm that factory-programmed calibration curves remain accurate; acceptance criterion is ±10% of the stated value (Hach Company, 2024a).
  • Detection limit (MDL): The minimum analyte concentration reliably distinguished from a blank; method-specific and listed in the Method Registry (§7); the blank must read within the MDL of zero at the start of each session (APHA et al., 2012).
  • DR3900: The Hach benchtop spectrophotometer; wavelength range 320–1100 nm; uses stored Hach programs or user-defined methods; supports 10 mm square, 1-inch round, and 16 mm round cells (Hach Company, 2024a).
  • DR900: The Hach portable colorimeter; battery-operated; uses the same stored-program approach as the DR3900; limited to a subset of Hach methods compatible with the 1-inch round cell format (Hach Company, 2024b).
  • Hach method number: A unique numeric identifier for each Hach reagent-kit and instrument-program combination; used to select the correct stored program on the DR3900 or DR900 and to ensure that the correct reagent is paired with the correct calibration curve (Hach Company, 2024c).
  • Method Registry: The table in §7 of this SOP listing all validated Hach methods in use in the EA Program laboratory, with analyte, chemistry, program number, reagent catalog number, instrument compatibility, range, sample volume, reaction time, key interferences, and validation status.
  • Powder pillow: A heat-sealed foil packet containing a pre-weighed, factory-prepared reagent mixture; added directly to the sample to initiate the color-forming reaction; eliminates manual reagent preparation and reduces sources of error (Hach Company, 2024c).
  • RPD (relative percent difference): |A - B| / ((A + B) / 2) × 100; used to assess duplicate precision; acceptable RPD is less than 20% for concentrations above 5 × the detection limit (APHA et al., 2012).
  • TNT plus vial: A Hach proprietary pre-dosed reagent vial containing all required reagents sealed in glass; used with the DR3900 for total-nutrient digestion methods (e.g., total phosphorus, total nitrogen) (Hach Company, 2024c).
  • Zeroing: The process of inserting a reagent blank into the sample cell and pressing Zero to set the baseline absorbance to zero for the selected program; required before each analytical batch (Hach Company, 2024a).

5 Safety and Hazards

WarningHazards
  • Chemical: Hach reagent powder pillows and liquid reagents contain a variety of chemicals depending on the method. Some contain acids (e.g., sulfuric acid in TNT plus vials), heavy metal indicators (e.g., cadmium in older nitrate methods), or oxidizers. Always review the SDS for each reagent kit before first use.
  • Thermal: COD and total nutrient digestions use the DRB200 reactor block at 150 °C. Use heat-resistant gloves when handling heated vials.
  • Glassware: Glass vials can break during handling or if heated unevenly. Handle with care; wear safety glasses.

5.1 Required PPE

5.2 Emergency Procedures

For reagent splashes to skin or eyes, flush with water for at least 15 minutes. Refer to SOP-L004 Chemical Spill Response for spill cleanup. For burns from the reactor block, apply cool running water and seek first aid.

6 Equipment

Item Notes
Hach DR3900 benchtop spectrophotometer SGM 133 (Hach Company, 2024a)
Hach DR900 portable colorimeter Field use; see SOP-F208
Hach DRB200 reactor block For COD and total nutrient digestions (150 °C)
Sample cells (1-inch round, 10 mm square, 16 mm round) Matched to Hach method; keep clean and scratch-free
Lint-free wipes (Kimwipes) For cleaning cell exterior before reading
Silicone oil and applicator cloth For 1-inch round cells to minimize light scatter
Timer / stopwatch For reaction time control
DI water (squeeze bottle) For blanks and cell rinsing
Waste container (sealable) For used reagent vials and solutions

7 Procedure

7.1 Startup and Instrument Check

  1. Turn on the DR3900 and allow it to complete its self-check (approximately 30 seconds).
  2. Verify the lamp status indicator shows “Ready.”
  3. Select the stored program for your analyte by program number (see §7, Method Registry) (Hach Company, 2024a).

7.2 Zeroing

  1. Fill a clean sample cell with DI water to the fill line.
  2. Wipe the cell exterior with a lint-free cloth. Apply silicone oil for 1-inch round cells.
  3. Insert the cell, close the lid, and press Zero. The display must read 0.00 (within the method detection limit). If it does not, clean the cell and re-zero.

7.3 Sample Analysis

  1. Fill a clean cell to the fill line with the water sample.
  2. Add the reagent (powder pillow or TNT vial) per the method instructions.
  3. Cap, invert, and mix as directed. Start the timer for the required reaction time.
  4. When the reaction time is complete, wipe the cell exterior (apply silicone oil for 1-inch round cells).
  5. Insert the cell, close the lid, and press Read.
  6. Record the result, analyte, program number, sample ID, units, date, and time in the lab notebook.
  7. Rinse the cell three times with DI water before the next sample.

7.4 Shutdown

  1. Remove all samples and reagents from the instrument compartment.
  2. Wipe the cell compartment with a lint-free tissue if any liquid is present.
  3. Log the run in the instrument logbook (date, analyst, analytes run, and any instrument issues).
  4. Turn off the DR3900 if the session is complete.

8 Method Registry

The tables below list all Hach methods currently validated in the EA Program laboratory. “Active” methods have been verified with known standards; “TBD” methods are pending validation. Add new methods to the registry per §7.4 before routine use.

8.1 Nutrients

Analyte Hach Method Program No. Reagent / Cat. No. Instrument Range Sample Vol. Reaction Time Key Interferences Status
Reactive P (ortho-P) PhosVer 3 (Ascorbic Acid) 490 2106069 (pillows) DR3900, DR900 0.02–2.50 mg/L PO4-P 10 mL 2 min Silica above 200 mg/L; arsenate Active
Total Phosphorus Acid persulfate digestion + PhosVer 3 536 TNT843 DR3900 + DRB200 0.06–3.50 mg/L PO4-P 5 mL 30 min dig. + 2 min High turbidity TBD
Nitrate (NO3-) Cadmium reduction 355 2106169 (pillows) DR3900, DR900 0.1–30.0 mg/L NO3-N 10 mL 5 min NO2- above 2 mg/L; Cl- above 100 mg/L Active
Nitrate (NO3-) HR Chromotropic acid 410 TNT835/TNT836 DR3900 0.2–30.0 mg/L NO3-N 1 mL 15 min Cl- above 500 mg/L TBD
Nitrite (NO2-) Diazotization 371 2107169 (pillows) DR3900, DR900 0.002–0.300 mg/L NO2-N 10 mL 20 min Strong oxidizers TBD
Ammonia LR Salicylate 385 2668000 (pillows) DR3900, DR900 0.01–0.50 mg/L NH3-N 10 mL 20 min Ca above 1000 mg/L CaCO3 Active
Ammonia HR Salicylate HR 343 TNT832 DR3900 0.4–50.0 mg/L NH3-N 0.2 mL 15 min Same as LR TBD
Total Nitrogen Persulfate dig. + chromotropic acid 350 TNT826/TNT827 DR3900 + DRB200 1–25 mg/L N 0.5 mL 30 min dig. + 15 min – TBD

8.2 Metals

Analyte Hach Method Program No. Reagent / Cat. No. Instrument Range Sample Vol. Reaction Time Key Interferences Status
Iron (total) FerroVer 265 2105669 (pillows) DR3900, DR900 0.02–3.00 mg/L Fe 10 mL 3 min Cu above 5 mg/L Active
Iron (Fe2+) 1,10-Phenanthroline 255 2105769 (pillows) DR3900, DR900 0.02–3.00 mg/L Fe 10 mL 3 min Strong oxidizers TBD
Manganese PAN 290 Check specific kit DR3900 0.006–0.700 mg/L Mn 10 mL 2 min Fe above 5 mg/L TBD
Copper Bicinchoninate 135 2105969 (pillows) DR3900, DR900 0.04–5.00 mg/L Cu 10 mL 2 min Fe above 10 mg/L TBD
Zinc Zincon 780 Check specific kit DR3900 0.02–3.00 mg/L Zn 10 mL Immediate Cu, Pb, Co above 5 mg/L TBD
Lead LeadTrak (dithizone) 277 2877200 (AccuVac) DR3900 5–150 µg/L Pb 25 mL 1 min Cu, Hg, Bi, Sn TBD
Chromium (Cr6+) DPC 090 Check specific kit DR3900, DR900 0.010–0.700 mg/L Cr 10 mL 5 min Fe above 1 mg/L TBD

8.3 Other Parameters

Analyte Hach Method Program No. Reagent / Cat. No. Instrument Range Sample Vol. Reaction Time Key Interferences Status
Hardness (total) CalMagVer 3 105 2429800 (pillows) DR3900, DR900 10–4000 mg/L as CaCO3 2 mL Immediate Mn above 4 mg/L TBD
Chloride Mercuric thiocyanate 070 2268500 (pillows) DR3900, DR900 0.1–25.0 mg/L Cl- 10 mL Immediate High turbidity TBD
Silica Silicomolybdate 656 2429600 (pillows) DR3900 1–100 mg/L SiO2 10 mL 10 min PO4; sulfide TBD
Sulfate SulfaVer 4 680 2106769 (pillows) DR3900, DR900 2–70 mg/L SO4 10 mL 5 min Silica TBD
COD Dichromate reactor 430 TNT821/822/823 DR3900 + DRB200 3–1500 mg/L 2 mL 2 hr dig. Cl- above 2000 mg/L TBD

8.4 Adding a New Method to the Registry

  1. Obtain the Hach procedure document for the method (from the kit box or hach.com).
  2. Review the SDS for all reagents in the kit.
  3. Fill in a new row in the appropriate table above with all required fields.
  4. Run the method on at least 3 known standard concentrations spanning the method range to verify that the instrument’s stored program produces results within ±10% of the standard value.
  5. Run DI water blanks to confirm zero reading.
  6. Change the status from TBD to Active once validation is complete.
  7. Record the validation data, date, and analyst in the QC log.

9 Quality Control and Acceptance Criteria

9.1 Every Analytical Session

  • Blank: Run a DI water blank at the start of each session; must read within the method MDL of zero (APHA et al., 2012).
  • Standard check: Run one mid-range Hach standard at the start and end of each session; acceptance: ±10% of the stated value (Hach Company, 2024a).
  • Duplicates: Analyze at least 10% of samples in duplicate; acceptable RPD less than 20% above 5 × the detection limit.

9.2 Calibration Verification

The DR3900 and DR900 use factory-programmed calibration curves that do not require user calibration under normal conditions (Hach Company, 2024a, 2024b). However:

  • Run a multi-point verification using Hach-supplied standards at least annually or when the instrument is serviced.
  • If a standard check deviates by more than 10%, clean the cell, re-zero, and re-read. If the problem persists, contact Hach technical support.

10 Waste Disposal

Waste Type Disposal Method
Used powder pillow packets and caps Standard trash (verify SDS; most are non-hazardous)
Cadmium-containing reagent waste Hazardous waste; EH&S pickup
Reactor block digestion waste (COD) Collect in labeled acid waste container
Used sample cells (glass) Rinse thoroughly; reuse or recycle
Rinse water (DI) Drain

11 References

APHA, AWWA, & WEF. (2012). Standard methods for the examination of water and wastewater (22nd ed., p. 1360). American Public Health Association.
Hach Company. (2024a). DR3900 benchtop spectrophotometer: Procedures manual. https://www.hach.com/dr3900-benchtop-spectrophotometer/product?id=56440352466
Hach Company. (2024b). DR900 portable colorimeter: Procedures manual. https://www.hach.com/dr900-portable-colorimeter/product?id=56440352476
Hach Company. (2024c). Water analysis guide (7th ed.). https://www.hach.com/wag

13 Training Record

Before running analyses on the DR3900 or DR900 independently, personnel must:

  1. Read this SOP and SOP-L215 in full
  2. Complete SOP-L002 Laboratory Safety training
  3. Review the SDS for all reagent kits to be used
  4. Observe a trained operator perform startup, blank zeroing, sample analysis, and a standard check for at least two analytes
  5. Run a full session (blank, standard check, samples, duplicate) under supervision with acceptable QC results
  6. Add at least one new method to the registry and validate it before using it on real samples
  7. Be signed off by the lab manager or PI